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Characterisation of the ATP-Binding Cassette Transporters Of

Characterisation of the ATP-Binding Cassette Transporters Of

Characterisation Of The ATP-Binding Cassette

Transporters Of aeruginosa

Victoria Grace Pederick

Research Centre for Infectious Diseases,

Department of Microbiology and Immunology,

School of Molecular and Biomedical Sciences,

University of Adelaide

October 2014

TABLE OF CONTENTS

ABSTRACT ...... V DECLARATION ...... VII COPYRIGHT STATEMENT ...... VIII ABBREVIATIONS ...... IX TABLE OF TABLES ...... XII TABLE OF FIGURES ...... XIII ACKNOWLEDGEMENTS ...... XV CHAPTER 1: INTRODUCTION ...... 1 ...... 1 1.1.1. P. aeruginosa and human disease ...... 1 1.1.1.1. ...... 2 1.1.2. Virulence factors ...... 3 1.1.3. Transcriptional regulation ...... 4 1.1.3.1. ...... 4 1.1.3.2. Two-component regulatory systems ...... 5 1.1.3.3. Metalloregulatory ...... 5 1.1.4. ...... 6 1.1.5. resistance ...... 8 1.1.5.1. Low membrane permeability ...... 8 1.1.5.2. Antibiotic inactivating enzymes ...... 9 1.1.5.3. Target modifications ...... 10 1.1.5.4. Lifestyle-mediated resistance ...... 10 1.1.5.5. Antibiotic ...... 11 ABC transporters ...... 14 1.2.1. Common architecture ...... 15 1.2.1.1. Transmembrane domains ...... 15 1.2.1.2. Nucleotide-binding domains ...... 16 1.2.2. ABC exporters ...... 16 1.2.2.1. Xenobiotic efflux ...... 17 1.2.2.2. Protein ...... 19 1.2.2.2.1. and ...... 20 1.2.2.2.2. and antibiotic ...... 20 1.2.2.2.3. Heme-binding proteins and ...... 21 1.2.2.3. Glycoconjugate secretion ...... 22 1.2.3. ABC importers ...... 22 1.2.3.1. Classification of SBPs ...... 23 1.2.3.2. Roles of ABC permeases ...... 25 1.2.3.3. Metal ion acquisition ...... 26 1.2.3.3.1. ...... 26 1.2.3.3.2. Zinc ...... 27 1.2.3.3.3. Molybdenum ...... 27 ABC transporters in P. aeruginosa ...... 28 1.3.1. Aims of the study ...... 29 CHAPTER 2: METHODS AND MATERIALS ...... 30 Materials ...... 31 DNA manipulation ...... 31 2.2.1. Plasmid isolation ...... 31 2.2.2. Genomic DNA isolation ...... 31 2.2.3. DNA electrophoresis ...... 31 2.2.4. Assessment of DNA quantity and quality...... 32

II 2.2.5. DNA sequencing ...... 32 2.2.6. Polymerase Chain Reaction (PCR) ...... 32 Bacterial culturing ...... 32 Bacterial strains ...... 38 2.4.1. P. aeruginosa ...... 38 2.4.1.1. Mutagenesis ...... 38 2.4.2. E. coli ...... 41 2.4.2.1. Generation of chemically competent cells ...... 41 2.4.2.2. Transformation of E. coli ...... 41 Phenotypic analyses ...... 43 2.5.1. Growth kinetics assay ...... 43 2.5.2. Biofilm formation ...... 43 2.5.3. Whole metal accumulation ...... 44 2.5.4. Minimal inhibitory concentration (MIC) analysis ...... 44 2.5.5. Fatty acid analysis ...... 45 2.5.6. Bacterial adherence to hydrocarbons (BATH) assay ...... 45 2.5.7. Volatile compound (VOC) headspace analysis ...... 45 2.5.8. Scanning electron microscopy analysis ...... 46 ZnuA biochemical characterisation ...... 46 2.6.1. Cloning and expression of ZnuA ...... 46 2.6.2. Purification of ZnuA ...... 47 2.6.3. ZnuA zinc binding assays ...... 48 2.6.4. PAR assay ...... 48 Bioinformatic analyses ...... 48 2.7.1. Identification of transcriptional regulatory sites ...... 48 2.7.2. Generation of Weblogos ...... 49 Transcriptional analyses ...... 49 2.8.1. RNA isolation ...... 49 2.8.2. qRT-PCR ...... 49 2.8.3. RNA sequencing ...... 50 CHAPTER 3: THE ROLE OF ABC EFFLUX PROTEINS IN P. AERUGINOSA ..... 51 Introduction ...... 52 Results ...... 54 3.2.1. Identification of uncharacterised ABC transporters in P. aeruginosa PAO1 ..... 54 3.2.2. Generation of the putative ABC efflux protein deletion mutants ...... 54 3.2.3. The ABC exporters of P. aeruginosa do not significantly contribute to antibiotic resistance ...... 57 3.2.4. Predicted physiological roles of the P. aeruginosa ABC exporters ...... 61 3.2.4.1. Putative function of PA0860 ...... 61 3.2.4.2. Putative function of PA1113 ...... 63 3.2.4.3. Putative function of PA3228 ...... 63 3.2.4.4. Putative function of PA5231 ...... 66 3.2.4.5. Putative function of PA1876 ...... 68 3.2.4.5.1. Characterisation of the putative PA1875-PA1877 T1SS allocrite, PA1874 ...... 70 Discussion ...... 73 Conclusions ...... 77 CHAPTER 4: THE ACQUISITION AND ROLE OF MOLYBDATE IN P. AERUGINOSA ...... 78 Introduction ...... 79 Results ...... 83 4.2.1. ModA is required for Mo acquisition ...... 83

III 4.2.2. Characterisation of the Mo-dependent transcriptional regulon in P. aeruginosa ...... 86 4.2.3. Anaerobic respiration and nitrate reduction are Mo-dependent ...... 90 4.2.4. Nitrate reduction limits biofilm formation and alters composition ...... 93 Discussion ...... 100 Conclusions ...... 104 CHAPTER 5: ZINC HOMEOSTASIS IN P. AERUGINOSA ...... 105 Introduction ...... 106 Results ...... 109 5.2.1. ZnuA contributes to P. aeruginosa Zn2+ acquisition ...... 109 5.2.1. ZnuA is a high affinity Zn2+ binding protein ...... 114 5.2.2. Deletion of ZnuA does not result in major growth perturbation ...... 120 5.2.3. Zn2+ depletion exhibits minimal effects on cellular processes and physiology ...... 123 5.2.1. Cellular Zn2+ depletion of the ΔznuA strain results in significant transcriptional modulation ...... 130 5.2.1.1. Transport proteins ...... 137 5.2.1.2. Ribosomal proteins ...... 145 5.2.1.3. Enzymes ...... 146 5.2.1.4. Transcriptional regulators ...... 149 5.2.1.5. Putative cobalt-binding proteins ...... 150 5.2.1.6. Down-regulated genes ...... 151 Discussion ...... 151 Conclusions ...... 157 CHAPTER 6: DISCUSSION ...... 158

APPENDIX 1 ...... 167 APPENDIX 2 ...... 172 APPENDIX 3 ...... 176 APPENDIX 4 ...... 201

REFERENCES ...... 215

IV ABSTRACT

Pseudomonas aeruginosa is a ubiquitous, Gram-negative, rod-shaped, environmental bacterium. However, it is also a clinically significant, opportunistic human , responsible for life-threatening infections in immunocompromised persons, including those with cancer and significant burn wounds. Furthermore, P. aeruginosa infections are the primary cause of morbidity and mortality in individuals with the genetic disease cystic fibrosis. The success of P. aeruginosa as both an environmental organism and a human pathogen can be attributed to its 6.3 Mbp genome, which encodes an array of mechanisms that enable adaptation, persistence and propagation in diverse environments. Membrane transporters are critical to the ability of P. aeruginosa to adapt and respond to its environment. Of these proteins, the ATP-binding cassette (ABC) transporters hold a prominent role in cellular processes, facilitating the active translocation of molecules across the inner membrane. The conserved structural organisation of ABC transporters has enabled their classification based upon the vector of transport, as either efflux or import transporters. Bioinformatic analyses reveal that P. aeruginosa PAO1 is predicted to encode 289 proteins associated with ABC transporter functionality, with a subset shown to contribute to in vivo virulence.

This study presents the phenotypic characterisation of five putative ABC export proteins in P. aeruginosa PAO1, assessing their contribution to antibiotic efflux. Deletion mutants of each identified ABC exporter were assessed for changes in their antibiotic resistance profile. Transcriptional analysis of the ABC transporter genes in response to antibiotic treatment was also performed to detect drug-stimulated expression. These analyses revealed the PA0860, PA1113, PA1876, PA3228 and PA5231 ABC efflux proteins to have a negligible contribution to antibiotic resistance, with bioinformatic analyses subsequently utilized to propose alternative roles.

ABC importers, also known as ABC permeases, typically feature one, but sometimes multiple, solute-binding protein (SBP) component(s) that deliver the cargo molecule to the ABC transporter for cytoplasmic translocation. ABC permeases are central to the uptake of many essential nutrients in prokaryotes, including transition metal ions. Herein, this study characterises the acquisition and role of two crucial transition metal ions, molybdenum and zinc, in P. aeruginosa. Acquisition of either metal ion was shown to occur primarily via ABC permeases, molybdenum via ModABC, and zinc via ZnuABC. Deletion of the modA SBP abrogated molybdenum accumulation in P. aeruginosa and abolished the capacity for anaerobic growth or nitrate reduction. Unexpectedly, conditions that permitted nitrate

V reduction were shown to inhibit biofilm formation and alter membrane fatty acid composition. By contrast, although deletion of the zinc-specific SBP gene, znuA, reduced cellular zinc accumulation, the mutant strain did not exhibit a phenotype corresponding with zinc depletion. Transcriptional analyses revealed that P. aeruginosa encodes a number of additional, previously unidentified, putative mechanisms that enable it to adapt to cellular zinc deficiency, including the use of several uncharacterized zinc acquisition systems.

Collectively, this study represents a significant advance in our understanding of P. aeruginosa ABC transporters and offers detailed insight into their cellular functions. Furthermore, this work highlights the remarkable adaptability of the bacterium, which enables its survival in diverse environmental and host niches.

VI DECLARATION

I certify that this work contains no material which has been accepted for the award of any other degree or diploma in my name, in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. In addition, I certify that no part of this work will, in the future, be used in a submission in my name, for any other degree or diploma in any university or other tertiary institution without the prior approval of the University of Adelaide and where applicable, any partner institution responsible for the joint-award of this degree.

I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968.

The author acknowledges that copyright of published works contained within this thesis resides with the copyright holder(s) of those works. I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library Search and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.

Victoria G. Pederick

VII COPYRIGHT STATEMENT

Chapter 4 adapts content already published in Pederick et al. (2014), Applied and Environmental Microbiology. The published work is Copyright © American Society for Microbiology (Applied and Environmental Microbiology, Volume 80, Number 21, November 2014, pages 6843-6852, DOI: 10.1128/AEM.02465-14). The article is also found in its entirety in Appendix 4.

Appendix 3 contains Lewis et al., (2012), Protoplasma. The published work is Copyright © Springer-Verlag (Protoplasma, Volume 249, October 2012, pages 919-42, DOI 10.1007/s00709-011-0360-8).

VIII ABBREVIATIONS

C Degree celcius Å Angstroms AA ABC ATP-binding cassette ADP Adenosine diphoshpate AI Autoinducers Anr Anaerobic regulator of arginine deiminase and nitrate reductase Ap Ampicillin ApR Ampicillin resistance cassette Arch Archaea ATP Adensosine triphosphate b.d. Below detection Bap Biofilm-associated protein BATH Bacterial adherence to hydrocarbons bp Base pairs Cb Carbenicillin CbR Carbenicillin resistance cassette CDM Chemically defined media cDNA Complementary DNA CF Cystic fibrosis CFTR Cystic fibrosis transmembrane conductance regulator CFU Colony forming units Chl Chloramphenicol ChlR Chloramphenicol resistance cassette CLD C39-like domain COG Cluster of orthologous genes CPS Capsular polysaccharide CTI Cis-trans isomerase Cup Chaperone usher pathway DMSO Dimethyl sulfoxide DNA Deoxyribonucelic acid Dnr Dissimilartory nitrate reductase regulator eDNA Extracellular DNA EDTA 2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acid EPS Exopolysaccharide FA Fatty acid Fnr Fumerate and nitrate reductase regulator FRT Flp recombinase target Fur Ferric uptake regulator g Relative centrifugal force gDNA Genomic DNA GEO omnibus GI Gene identifier Gm Gentamicin GmR Gentamicin resistance cassette GPC Gel permeation chromatography h Hours His Histidine HK Histidine kinase ICP-MS Inductively coupled plasma-mass ID Identification IX IG Intergenic IM Inner membrane IMAC Immobilised ion affinity chromatography Kan Kanamycin KanR Kanamycin resistance cassette KD Dissociation constant kDa Kilodalton L Litre LB Luria bertani (Lennox) broth LIC Ligation independent cloning LPS Lipopolysaccharide M Molar MATE Multi-drug and toxic compound extrusion family Mbp Mega base pairs MCS Multiple cloning site MDR Multi-drug resistance MES 2-(N-morpholino)ethanesulfonic acid MFP Membrane fusion protein MFS Major facilitator superfamily mg Milligrams MGD Mo-bis molybdopterin guanine dinucleotide MIC Minimal inhibitory concentration min Minutes mL Millilitres mM Millimolar 2- Mo Molybdate, MoO4 MoCo Molybdenum cofactor MOPS 3-(N-morpholino)propanesulfonic acid mRNA Messenger RNA MS-SIFT Selected-ion flow mass spectrometry Myc Mycobacterium n Number of replicates NBD Nucleotide binding domain NCBI National Centre for Biotechnology Information ng Nanogram nM Nanomolar nm Nanometres nt Nucelotide OD Optical density OM Outer membrane OMP Outer membrane protein Pi Inorganic phosphate PAR 4-(2-pyridylazo)resorcinol PBP Penicillin binding protein PBS Phosphate-buffered saline PCR Polymerase chain reaction PDB Protein data bank Pht Polyhistidine triad protein PIB-1 Pseudomonas imipenem -lactamase PIPES Piperazine-N,N′-bis(2-ethanesulfonic acid) ppb Parts per billion PQS Pseudomonas quinolone signal PUM Potassium urea magnesium qPCR Quantitative PCR X qRT-PCR Quantitative reverse transcription polymerase chain reaction QS Quorum sensing RIN RNA integrity number RNA Ribonucleic acid RND Resistance-nodulation division RR Response regulator rRNA Ribosomal RNA RTX Repeat-in- s.e.m. Standard error of the mean SBP Solute binding protein SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis sec Seconds SEM Scanning electron microscopy SMR Small multi-drug resistance family SWM Putative flagellar system-associated repeat T1SS Type 1 secretion system TAE Tris acetate EDTA TCS Two-component systems TM Transmembrane TMD Transmembrane domain TPEN N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine tRNA Transfer RNA UTI Urinary tract infection V Volt VOC Volatile organic compounds 2- W Tungstate, WO4 ZIP Zrt, Irt-like protein Zur Zinc uptake regulator g Microgram M Micromolar m Micrometer

XI TABLE OF TABLES

Table 1.1: Classification of SPBs...... 24 Table 2.1: Oligonucleotides used in this study...... 33 Table 2.2: Strains used in this study...... 39 Table 2.3: Plasmids used in this study...... 42 Table 3.1: Minimal inhibitory concentration (g.mL-1) of common anti-pseudomonal ...... 59 Table 3.2: Sequences of repeating domains in PA1874...... 72 Table 4.1: Putative P. aeruginosa PAO1 ModE binding sites...... 89 Table 5.1: Minimal inhibitory concentration analysis under Zn2+ limitation...... 126 Table 5.2: Volatile organic compound analysis of P. aeruginosa...... 131 Table 5.3: Candidate Zur-binding sites...... 138 Table 5.4: Expression of genes under Zn2+ limitation...... 140

XII TABLE OF FIGURES

Figure 1.1: Structural and mechanistic diversity of drug efflux pump families...... 12 Figure 1.2: Functional diversity of ABC transporters...... 18 Figure 3.1: Protocol for P. aeruginosa mutagenesis...... 56 Figure 3.2: Mutagenesis of uncharacterised P. aeruginosa ABC exporters...... 58 Figure 3.3: Differential expression of ABC transporters in response to antibiotic treatment. 60 Figure 3.4: The putative PA0860 ABC efflux protein...... 62 Figure 3.5: The putative PA1113 ABC efflux protein...... 64 Figure 3.6: The putative PA3228 ABC efflux protein...... 65 Figure 3.7: The putative PA5230/PA5231 ABC exporter...... 67 Figure 3.8: The putative PA1876 ABC efflux protein...... 69 Figure 3.9: Bioinformatic analysis of PA1874...... 71 Figure 3.10: Biofilm formation of PA1874-PA1877 T1SS mutants...... 74 Figure 4.1: Dissimilatory nitrate reduction in P. aeruginosa...... 80 Figure 4.2: Phylogenetic analysis of cluster D-III SBPs...... 84 Figure 4.3: Deletion of modA alters P. aeruginosa Mo accumulation...... 85 Figure 4.4: Intracellular Mo deficiency causes an increase in modC expression...... 87 Figure 4.5: The P. aeruginosa ModE binding site motif...... 88 Figure 4.6: Transcriptional regulation of PA1864 by ModE...... 91 Figure 4.7: Mo depletion limits anaerobic growth...... 92 Figure 4.8: Mo availability affects nitrate reductase activity...... 94 Figure 4.9: Transcriptional response to nitrate supplementation and anaerobic conditions. ... 95 Figure 4.10: Anaerobic respiration reduces biofilm formation...... 97 Figure 4.11: Fatty acid (FA) composition of PAO1 and ΔmodA strains...... 98 Figure 4.12: Cis-trans isomerase (CTI) expression...... 99 Figure 4.13: Nitrate reduction effects Pseudomonas membrane hydrophobicity...... 101 Figure 5.1: The Zn2+ specific ZnuABC permease...... 110 Figure 5.2: Organisation of the P. aeruginosa ZnuABC regulon...... 111 Figure 5.3: Sequence alignment of ZnuA proteins...... 113 Figure 5.4: Predicted secondary structure of P. aeruginosa ZnuA...... 115 Figure 5.5: Deletion of znuA reduces P. aeruginosa Zn2+ accumulation...... 116 Figure 5.6: Purification of recombinant ZnuA...... 117 Figure 5.7: Molecular mass determination of recombinant ZnuA...... 118 Figure 5.8: Determination of the affinity of ZnuA for Zn2+ at its primary binding site...... 121 Figure 5.9: The effect of znuA deletion on growth in CDM...... 122

XIII Figure 5.10: The effect of znuA deletion on biofilm formation...... 124 Figure 5.11: Deletion of znuA alters cellular hydrophobicity...... 127 Figure 5.12: Fatty acid (FA) composition of PAO1 and ΔznuA...... 128 Figure 5.13: PAO1 and ΔznuA appear morphologically similar...... 129 Figure 5.14: Analysis of RNA for RNA sequencing...... 132 Figure 5.15: Differential expression of genes in response to the Zn2+ depletion of the ΔznuA strain...... 133 Figure 5.16: qRT-PCR validation of RNA sequencing data...... 135 Figure 5.17: Cluster of orthologous gene (COG) function analysis...... 136 Figure 5.18: The P. aeruginosa ZnuA binding site motif...... 139 Figure 5.19: Phylogenetic tree of metal binding SBPs...... 144 Figure 5.20: Sequence alignment of P. aeruginosa C- and C+ ribosomal proteins...... 147 Figure 5.21: Model of Zn2+ acquisition in P. aeruginosa PAO1...... 155

XIV ACKNOWLEDGEMENTS

My PhD had been a wonderful experience. A time rich in learning, development and friendship. I am very thankful for this opportunity and for the support I have received throughout this process.

To Christopher McDevitt, my primary supervisor. Thank you for encouragement, wisdom, teaching, support, advice and humour. I have learnt so much about being a good scientist from my time in your lab and hope to continue to develop my skills and love of ABC transporters, P. aeruginosa and metals in biology.

To James Paton, my co supervisor. Your support and wisdom thought my PhD has been invaluable. Thank you so much.

To the wonderful people of the Membrane Proteins in Bacterial Pathogenesis Lab: Bart, Miranda, Stef, Jacqueline and Bec. You have made coming to the lab each day a pleasure. Thank you for your support, encouragement, crazy antics, patience. In particular Bart, thank you for your mentoring, advice and assistance. I have learnt so much working with you. Thanks for encouraging my love of molecular microbiology.

To the Paton lab. Thank you for all of your generosity, kindness and encouragement.

To the Australian Cystic Fibrosis Research Foundation and the Channel 7 Children’s Research Foundation for their financial support of my research.

To my examiners. Thank you for giving of your time to assess my work.

To my Mum and Dad. Thank you for your care, love, encouragement and support throughout this journey. Alexander, I am so glad you have developed a love for science too.

To Marion who worked so hard on her PhD but didn't get the chance to finish.

To my husband Gerald. I could not have done this without your support, encouragement, patience and love. Thank you so much!

To my Heavenly Father who has blessed me so richly. Thank you for the wonder that is creation. Thank you that I have had the pleasure to study and come to understand the tiniest part of it. I am amazed by the works of Your hands. They are too glorious for words.

XV

CHAPTER 1:

INTRODUCTION

1 Chapter 1

Pseudomonas aeruginosa

The bacterium Pseudomonas aeruginosa was first isolated from wound bandages in 1882 by Carle Gessard (Gessard, 1882). Gessard reported the bandages to have a blue-green discolouration, later attributed to the secreted P. aeruginosa metabolite, pyocyanin. P. aeruginosa has been extensively studied since its discovery, with most work performed using the human clinical isolates PAO1 and PA14 (Rahme et al., 1995; Stover et al., 2000; He et al., 2004b). P. aeruginosa is now recognized as a ubiquitous Gram-negative, rod-shaped, member of the γ-proteobacteria. Although often described as an aerobic bacterium, P. aeruginosa also has the capacity to respire anaerobically using nitric oxides as electron donors (Yoon et al., 2002; Palmer et al., 2007). The bacterium is highly motile, by virtue of a single flagellum, and is also capable of forming surface-attached biofilm communities that facilitate persistence in a given environment (Munoz et al., 1949; Costerton, 1984; Costerton et al., 1999). P. aeruginosa is commonly found in soil and , but has also been isolated from more diverse environments, ranging from the human microflora to hospital antiseptic solutions (Lister et al., 2009). The broad survival capability of the bacterium can be attributed to the metabolic flexibility encoded in its 6.3 Mbp genome (Stover et al., 2000). The P. aeruginosa PAO1 genome encodes 5570 predicted open reading frames, with a complexity approaching that of simple eukaryotes such as Saccharomyces cerevisiae (Stover et al., 2000). It is this coding capacity which enables the ubiquity of P. aeruginosa in environmental niches while also facilitating its infection and persistence in humans where it can cause disease.

1.1.1. P. aeruginosa and human disease

Although an environmental organism, P. aeruginosa is also a clinically-relevant, opportunistic human pathogen, which infects immunocompromised persons. Individuals with significant burn wounds, human immunodeficiency virus infections, cancer, and cystic fibrosis (CF) are amongst those more commonly infected with the bacterium (Bodey et al., 1983; Williams et al., 2010). Furthermore, P. aeruginosa is the primary Gram-negative causative agent of pneumonia, responsible for 26% of ventilator-associated pneumonia (Kollef et al., 2014), in addition to 12% of catheter-associated urinary tract infections (UTIs) and 15.4% of surgical site infections (Takesue et al., 2012; Ortega et al., 2013). Most significantly, P. aeruginosa infections are the leading cause of morbidity and mortality in individuals with CF (Emerson et al., 2002; Son et al., 2007).

1 Chapter 1

1.1.1.1. Cystic fibrosis

CF is an autosomal, recessive disorder that affects 1 in every 2500 Caucasian individuals (Howard, 1944; Andersen & Hodges, 1946; Hassett et al., 2010). The disorder is characterised by a reduced number, or absence, of functional cystic fibrosis transmembrane conductance regulator (CFTR) protein (Welsh & Smith, 1993) as a result of mutations in the cftr gene. Whilst CF can arise from a range of potential cftr mutations, the deletion of phenylalanine at position 508 (ΔF508) is the most common, affecting two-thirds of CF individuals (Kerem et al., 1989; Riordan et al., 1989; Rommens et al., 1989; Gadsby et al., 2006). Mutations in the cftr gene cause mis-translation and degradation, improper insertion, or insertion of non-functional copies of the protein into epithelial cell membranes. The CFTR protein is an atypical ATP-binding cassette (ABC) transporter that secretes chloride ions from epithelial cells in the lungs, liver, pancreas, reproductive tract, small intestine, and sweat glands (Hoiby, 2011). Consequently, the disorder is associated with a broad range of symptoms throughout the body. As a result of significant advances in multidisciplinary CF healthcare since the discovery of the disorder, the nutritional consequences of CF have been minimised, allowing in most cases, survival past infancy (Cohen-Cymberknoh et al., 2011; Gaskin, 2013). Today, the principal impact of CF is associated with the lung environment, in which the abnormality in CFTR expression results in dehydrated, highly osmolar mucous that restricts cilia movement and is ideal for bacterial colonization. Species commonly isolated from CF lungs include Staphylococcus aureus, Burkholderia cepacia, Haemophilus influenzae and P. aeruginosa (George et al., 2009). P. aeruginosa is the most commonly isolated bacterium, with colonization rates between 75% and 80% observed in individuals over the age of 19 (Cystic Fibrosis Australia, 2010). P. aeruginosa undergoes significant phenotypic and genotypic alteration throughout the long-term colonization of the CF lung, making the infection more difficult to clear. Over the course of infection, strains that colonize CF individuals commonly switch from a non-mucoid to mucoid phenotype due to mutations that result in an overproduction of the polysaccharide alginate (Doggett et al., 1966; Martin et al., 1993; Govan & Deretic, 1996; Ryall et al., 2014). The resultant mucoid phenotype has been linked with increased expression of antibiotic resistance mechanisms and alterations in virulence gene expression (Hentzer et al., 2001; Ryall et al., 2014), rendering mucoid P. aeruginosa highly refractory to antibiotic treatment. Hence, once a lung infection is established, P. aeruginsa generally persists throughout life, with 57.9% of CF patient mortality attributed to pulmonary issues (Cystic Fibrosis Australia, 2010). Consequently,

2 Chapter 1 enhanced clearance or prevention of P. aeruginosa infections is seen as the next frontier for improving the standard of living and life expectancy of CF individuals.

1.1.2. Virulence factors

The ability of P. aeruginosa to cause such life threatening and diverse infections is attributed to its arsenal of virulence factors. P. aeruginosa virulence factors may be either components of the , or secreted into the extracellular milieu, where they exert their effects on the host environment. The virulence factors can also be classified according to whether they contribute to acute or chronic infections. During acute infections, adhesion of P. aeruginosa to the infection site is a key component of the bacterium’s virulence. P. aeruginosa is dependent upon type IV pili and flagellae, together with other adhesins, to facilitate persistence and enable the bacteria to cause host damage via its secreted virulence factors (Hahn, 1997; O'Toole & Kolter, 1998; Bucior et al., 2012; Haiko & Westerlund- Wikstrom, 2013). These include a range of toxins and proteases, namely elastase (LasB), LasA, phospholipase C, alkaline (AprA), ExoA, ExoU and ExoS (Wretlind & Pavlovskis, 1983; Hong & Ghebrehiwet, 1992; Cowell et al., 2003; Leidal et al., 2003; Engel & Balachandran, 2009; Howell et al., 2013). Whilst each differs in their target specificity, these virulence proteins degrade host factors and result in significant host tissue damage, allowing colonization of deeper tissue or systemic spread. During chronic stages of infection, P. aeruginosa forms structures known as , which are key to the bacterium’s virulence. Biofilms are bacterial communities within a matrix of exopolysaccharide, protein and extracellular DNA, which serves to protect the encased bacteria from the immune system, antibiotic treatment and clearance by fluid movement (Wingender et al., 2001; Whitchurch et al., 2002; Costerton et al., 2003; Ryder et al., 2007; Alhede et al., 2014). P. aeruginosa biofilms are key components of numerous chronic infections, including catheter-associated UTIs and ventilator-associated pneumonia infections (Costerton et al., 1999). Furthermore, the formation of biofilm within the mucus of the CF lung is central to the persistence of P. aeruginosa. Such infections are strongly associated with an overproduction of the polysaccharide alginate, which also acts as a (Hentzer et al., 2001). Finally, to enable survival during chronic infection, P. aeruginosa requires an efficient pathway for iron acquisition in the iron-restricted host environment (Hood & Skaar, 2012). To achieve this, P. aeruginosa secretes the extremely high-affinity iron-chelating siderophores, pyoverdine and pyochelin (Takase et al., 2000; Cornelis, 2010; Cornelis & Dingemans, 2013). Each of these factors allow for the colonization and persistence of P. aeruginosa

3 Chapter 1 within the host, resulting in significant morbidity and mortality. Some of these factors, which are critical to the scope of this study, are discussed in more detail below.

1.1.3. Transcriptional regulation

The P. aeruginosa PAO1 genome is predicted to encode 468 genes with transcriptional regulator functions (Stover et al., 2000; Schuster & Greenberg, 2006). This comprises almost 10% of the genome and highlights the ability of the bacterium to sense and respond to a multitude of environmental changes. The transcriptional regulators of P. aeruginosa are commonly found to regulate multiple genes, often operating in a hierarchical cascade to enable activation or repression of multiple targets in response to specific stimuli (Balasubramanian et al., 2013). The three transcriptional regulatory systems relevant to the work carried out herein are described below.

1.1.3.1. Quorum sensing

Cell to cell communication is critical for the co-ordinated growth and adaptation of a high-density bacterial population. Quorum sensing (QS) systems are the regulatory networks that facilitate this intercellular communication, via the use of chemical messengers known as autoinducers (AI). The three QS systems utilized by P. aeruginosa are Rhl, Las and the Pseudomonas quinolone signal (PQS) systems (Passador et al., 1993; Schuster & Greenberg, 2006). Rhl and Las dominate, with each system comprised of a synthase and transcriptional regulator. The synthases, RhlI and LasI, produce the AI acyl homoserine lactones, C4-HSL and 3OC12-HSL, respectively (Passador et al., 1993; Pearson et al., 1994; Williams & Camara, 2009). The AI molecules are then able to bind to their cognate LuxR family transcriptional regulator, RhlR or LasR, allowing transcriptional modulation and auto- regulation. Transcriptomic studies suggest that hundreds of genes are regulated by the Rhl and Las systems, including a large proportion of transcriptional regulators (Whiteley et al., 1999; Schuster et al., 2003; Wagner et al., 2003; Schuster & Greenberg, 2006). The PQS system, regulated by the LysR transcriptional regulator, PqsR (or MvfR), utilizes 2-heptyl-3- hydroxi-4-quinolone (PQS) as its signaling molecule (Cao et al., 2001) to activate the transcription of a number of targets, including Rhl, pyocyanin and the PQS synthetase genes (Diggle et al., 2003; Bredenbruch et al., 2006; Reis et al., 2011). The three QS systems compose a complex and interconnected global regulatory cascade, with the LasIR system at the top of the hierarchy (Schuster et al., 2013). Together, they are responsible for modulating a range of virulence factors, including the production of elastase, hydrogen cyanide,

4 Chapter 1 rhamnolipids, pyocyanin, biofilm formation and motility (Pearson et al., 1997; Pessi & Haas, 2000; Duan & Surette, 2007; Reis et al., 2011).

1.1.3.2. Two-component regulatory systems

Bacterial two-component regulatory systems (TCS) allow effective sensing and response to environmental stimuli, including changes in oxygen tension, and nutrient and metal ion abundance. TCS are comprised of two proteins: a histidine kinase, commonly an inner membrane protein that senses the stimuli, and a cytoplasmic response regulator that brings about the transcriptional response (Stock et al., 2000). P. aeruginosa PAO1 has the greatest number of two-component regulatory systems (TCS) of any sequenced bacterial species, encoding 63 histidine kinases (HK) and 64 response regulators (RR) (Rodrigue et al., 2000). It has been proposed that the significant number of TCS contributes to the ability of P. aeruginosa to efficiently sense and respond to its changing environment. One such example is the role the CzcRS TCS plays in the ability of P. aeruginosa to respond to increasing levels of environmental and cellular metal ion abundance. The CzcRS TCS confers resistance to zinc, cobalt and cadmium via the CzcBCA resistance-nodulation division efflux pump (Perron et al., 2004). Upon sensing the metal ion, the CzcS phosphorylates CzcR, which in turn causes the up-regulation of the CzcBCA efflux pump and results in cobalt, cadmium and zinc efflux. Another TCS, CopRS, is involved in copper resistance in a similar manner, yet at the same time also contributes to zinc resistance via increased expression of czcRS and czcBCA (Caille et al., 2007). In this way the TCS allow specific responses to the metal ion content of the cell and facilitate adaptation to environmental metal ion abundance. To date relatively few TCS in P. aeruginosa have been thoroughly characterised (Hobbs et al., 1993; Whitchurch et al., 1996; Macfarlane et al., 2000; Nishijyo et al., 2001; Zolfaghar et al., 2005; Benkert et al., 2008; Petrova & Sauer, 2010; Giraud et al., 2011; Mikkelsen et al., 2011; Sivaneson et al., 2011; Okkotsu et al., 2014; Zamorano et al., 2014), with the stimuli for the majority of systems largely unknown.

1.1.3.3. Metalloregulatory genes

First-row transition metal ions (manganese, iron, cobalt, nickel, copper and zinc) hold essential catalytic and structural roles within the bacterial cell, yet are often toxic in excess (Hood & Skaar, 2012). Consequently, efficient metal homeostasis is critical for P. aeruginosa growth and virulence. To achieve this, a number of transcriptional regulators, in addition to the CzcRS and CopRS TCS, sense and regulate the metal ion abundance within the cell. Chief amongst these in P. aeruginosa are the ferric uptake regulator (Fur) family of proteins, Fur

5 Chapter 1 and Zur. Fur is an iron-binding transcriptional regulator that controls iron acquisition (Cornelis et al., 2009), while Zur is the zinc-binding zinc uptake regulator (Zur) (Gaballa & Helmann, 1998; Patzer & Hantke, 1998; Patzer & Hantke, 2000; Fillat, 2014). Despite the differences in metal specificity, the Fur family of transcription factors act in a conserved manner, operating as dimers that bind two or more of their respective ions, and resulting in a structural rearrangement of the dimer that enables repression of its target genes (Fillat, 2014). P. aeruginosa uses multiple Fur-regulated mechanisms to acquire iron. These include the iron chelating molecules known as siderophores, heme and haemoglobin binding proteins, and iron ABC import systems (Cornelis et al., 2009). By contrast with the large transcriptional regulon of Fur, the primary regulatory target of Zur is the ZnuABC ABC import system (Gaballa & Helmann, 1998; Patzer & Hantke, 1998; Patzer & Hantke, 2000). The Zur protein of P. aeruginosa PAO1 was shown to be encoded together with the znuABC import genes, allowing auto-regulation together with repression of the uptake system (Ellison et al., 2013). ZnuABC is predicted to be the primary high affinity zinc acquisition system of P. aeruginosa. The utilization of regulated, high affinity uptake systems is balanced with metal ion efflux systems, such as the CzcRS-controlled CzcBCA pump to avoid cellular toxicity and maintain homeostasis. Together, metalloregulatory proteins such as Fur and Zur, perform critical roles in the maintenance of P. aeruginosa transition metal ion homeostasis.

1.1.4. Biofilm

P. aeruginosa can occur in either a planktonic (free-swimming) state, or form complex biofilms on biotic and abiotic surfaces (Costerton, 1984). Biofilms are sessile communities of bacteria enmeshed within a matrix composed of protein, polysaccharide and DNA (Wingender et al., 2001; Whitchurch et al., 2002; Costerton et al., 2003; Ryder et al., 2007). The biofilm matrix acts as a shield, protecting P. aeruginosa from clearance by the host immune response, in addition to reducing penetration of antibiotics, thereby providing a means of resistance. In fact, biofilm cells have been reported as having up to 1000-fold higher levels of antibiotic resistance compared with their planktonic counterparts (Hoiby et al., 2010). The formation of biofilms within the thick oxygen-depleted mucus of the CF lung is central to the persistence of P. aeruginosa (Hassett et al., 2009). Unfortunately, the subsequent immune response against the biofilm often contributes to significant lung pathology. The P. aeruginosa biofilms formed in the CF lung mucus differ in their composition and structure from those commonly found as part of catheter-associated UTIs, on surgical implants or in the laboratory setting (Hassett et al., 2009). In contrast to the mucus-

6 Chapter 1 enmeshed CF biofilms, the non-CF biofilms anchor the bacteria to an abiotic surface via their protective matrix, aiding in persistence. The diversity in P. aeruginosa biofilms is matched by the variety of components of the matrix. Such differences in composition are dependent upon a number of factors including the strain of P. aeruginosa, its mucoid or non-mucoid status and the environmental conditions. The three main polysaccharide components of P. aeruginosa biofilms are alginate (a repeating polymer of α-L-guluronate and β-D- mannuronate), Psl (a repeating pentasaccharide of D-mannose, D-glucose, L-rhamnose) and Pel (a glucose rich polysaccharide) (Friedman & Kolter, 2004a; Ramsey & Wozniak, 2005; Byrd et al., 2009). Alginate, the overproduction of which is the hallmark of mucoid strains, is the key component of the mucoid biofilm matrix (Doggett et al., 1966; Govan & Deretic, 1996). Psl also plays a key role in mucoid biofilms, and contributes to resistance against cationic peptides by virtue of its net negative charge (Ma et al., 2012; Billings et al., 2013). The matrix of non-mucoid strains is primarily composed of Pel and/or Psl polysaccharides, with minimal alginate present (Hentzer et al., 2001; Wozniak et al., 2003; Friedman & Kolter, 2004b). In addition to the polysaccharide component of the biofilm matrix, extracellular DNA (eDNA) is also a major contributor (Whitchurch et al., 2002). In catheter-associated UTIs, eDNA has been shown to be sufficient for biofilm matrix formation in the absence of alginate, Psl and Pel (Cole et al., 2014). Furthermore, on the basis of its negative charge, eDNA is capable of chelating metal ions and cationic antimicrobial peptides, providing an additional means of resisting clearance (Okshevsky & Meyer, 2013). In this way, the eDNA can prevent the antimicrobial peptides from disrupting the bacterial cell membrane, in addition to chelating metals when present at toxic concentrations. Proteinaceous appendages, such as type IV pili, Flp pili (type IVb) and the Chaperone usher pathway (Cup) fimbriae play key roles in P. aeruginosa biofilm development and architecture. The Cup fimbriae, of which five distinct types have been identified in P. aeruginosa (CupA, B, C, D, and E), act as biofilm adhesins at different stages of biofilm development (Vallet et al., 2001; He et al., 2004b; Ruer et al., 2007; Giraud et al., 2010; Giraud et al., 2011). Type IV pili, the appendages responsible for P. aeruginosa twitching motility, have been shown to aid in attachment to biotic and abiotic surfaces, in addition to facilitating the movement of bacteria to the top of the mushroom shaped biofilm structures observed in mature biofilms (Lee et al., 1994; Sheth et al., 1994; Klausen et al., 2003a; Giltner et al., 2006). P. aeruginosa is unique in that it also expresses a second type of type IV pili known as type IVb or Flp pili. These pili are required for microcolony formation during biofilm development (de Bentzmann et al., 2006). Together, this collection of P. aeruginosa appendages aids in biofilm structure and development. Secreted glycolipids known as

7 Chapter 1 rhamnolipids, are also critical components of P. aeruginosa biofilms. The quorum sensing regulated rhamnolipids exhibit important roles in modulating the architecture, development and dispersal of P. aeruginosa biofilms (Davey et al., 2003; Pamp & Tolker-Nielsen, 2007). Although much is known regarding the different components of the biofilm matrix, the biofilm structure and composition has been shown to vary depending upon a number of conditions, including media composition and carbon source (Klausen et al., 2003b; Harmsen et al., 2010). In the same way, it remains difficult to uncover the intricacies of in vivo biofilm formation due to the inability to successfully replicate the conditions in vitro. Consequently, this provides a significant impediment for research into treatment strategies that target P. aeruginosa biofilms for clearance of infection.

1.1.5. Antibiotic resistance

Clinical diagnosis of a P. aeruginosa infection in humans is typically followed by antibiotic treatment. A range of different antibiotics are used to treat P. aeruginosa: β- lactams, which target peptidoglycan synthesis; fluoroquinolones, which target DNA replication; aminoglycosides, which target protein synthesis; and cationic peptides, which disrupt the cell membrane. However, increasingly, P. aeruginosa strains are being identified as multi-drug resistant (MDR), that is, resistance to one or more antibiotics from three antimicrobial categories (Magiorakos et al., 2012). Of significant concern is the increase in resistance towards all of the commonly used antibiotic families, combined with the lack of new antibiotics in development. The major mechanisms responsible for the impressive antibiotic resistance of P. aeruginosa are reviewed below.

1.1.5.1. Low membrane permeability

The cell membrane serves as an essential permeability barrier, preventing the free diffusion of molecules, such as xenobiotics, in and out of the cell. However, all forms of biological life require that specific molecules be acquired, while others are secreted, from the cell. These import and efflux processes are reliant, in most cases, upon specific transport proteins. Bioinformatic analysis of the P. aeruginosa genome indicates that outer membrane contains a large range of putative membrane porins (102 in number), both specific and non- specific, which allow the movement of potentially diverse molecules into the (Hancock & Brinkman, 2002). Comparison of the permeability of the P. aeruginosa membrane with that of has shown that it has significantly lower permeability, despite being competent for translocation of larger molecules into the periplasm

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(Yoshimura & Nikaido, 1982; Nikaido et al., 1991; Bellido et al., 1992; Nikaido, 2003). This has since been attributed to the major P. aeruginosa outer membrane porin, OprF. OprF exists in either open or closed conformations, with the open conformation facilitating the slow movement of large molecules (2000-3000 daltons) across the outer membrane (Nestorovich et al., 2006; Sugawara et al., 2006). By contrast, the closed conformation of the protein prevents translocation of substances and consequently renders the membrane poorly permeable (Nikaido, 2003; Nestorovich et al., 2006; Sugawara et al., 2006). In this way, the low permeability of the outer membrane significantly reduces the ability of antibiotics to enter the cell, with the cell membrane serving as an intrinsic layer of antibiotic resistance. Furthermore, the zinc-responsive CzcRS TCS has been demonstrated to negatively regulate the expression of the basic amino acid and outer membrane porin, OprD (Trias & Nikaido, 1990), in response to zinc, resulting in an increase in resistance to carbapenem β-lactam antibiotics (Perron et al., 2004). This study, together with that of Conejo et al. (2003), showed that carbapenem resistance could be increased in response to extracellular zinc due to repression of oprD (Conejo et al., 2003; Perron et al., 2004). Collectively, the low permeability of the cell membrane, which confers an intrinsic resistance to antibiotics, when combined with complex regulatory pathways, illustrates the combinatorial mechanisms P. aeruginosa employs to protect itself from antibiotics, such as carpabenems.

1.1.5.2. Antibiotic inactivating enzymes

Antibiotics of the β-lactam and aminoglycoside families are susceptible to the inactivating enzymes of P. aeruginosa. β-lactams, which act to inhibit peptidoglycan synthesis in the periplasm, are, in many cases, susceptible to hydrolysis and inactivation by β- lactamase enzymes. P. aeruginosa PAO1 encodes three chromosomal β-lactamase enzymes, AmpC, OXA-50 and PIB-1 (Girlich et al., 2004; Lister et al., 2009; Fajardo et al., 2014). The AmpC β-lactamase is a class C cephalosporinase produced at low levels under basal conditions, but, when over-expressed, it confers resistance to all β-lactams apart from carbapenems (Lister et al., 2009). Over-expression of AmpC is induced by β-lactams or as a consequence of mutation in another component of the peptidoglycan biosynthesis pathway, AmpD (Lister et al., 2009). By contrast, OXA-50 is a constitutively expressed class D narrow spectrum oxacillinase (Girlich et al., 2004). However, OXA-50 does not significantly contribute to the β-lactam resistance of P. aeruginosa. Recently, a third chromosomally encoded β-lactamase was identified in the PAO1 genome. Loss of PA5542 was shown to result in an increased susceptibility of the clinical P. aeruginosa 59.20 strain to carbapenems (Fajardo et al., 2014). Subsequent characterisation of PA5542 determined the protein to

9 Chapter 1 exhibit zinc-dependent imipenemase activity, and resulted in PA5542 being renamed PIB-1 (Pseudomonas imipenem β-lactamase). As PIB-1 expression is higher in P. aeruginosa 59.20 than PAO1 at all stages of growth, the significance of PIB-1 to P. aeruginosa imipenem resistance in other strains remains unclear. In addition to the three chromosomally encoded β- lactamase genes, P. aeruginosa is also able to acquire additional β-lactamase genes on mobile genetic elements, thereby increasing its antibiotic resistance (Gupta, 2008; Zhao & Hu, 2010). It is this capacity to acquire resistance mechanisms that contributes to the significant global spread of multi-drug resistance in clinical strains.

P. aeruginosa also utilizes aminoglycoside modifying enzymes as a means of resistance. The bacterium encodes a range of enzymes which can acylate, adenylate or phosphorylate their target aminoglycosides, preventing their activity (Poole, 2005). Together, the nucleotidlytransferase, ANT(2”)-I and the 6-N-aminoglycoside acetyltransferases, AAA(6’) are the major determinants of aminoglycoside modification-based resistance in P. aeruginosa (Poole, 2005). Similar to the β-lactamase genes, genes encoding aminoglycoside modifying enzymes are able to be acquired by P. aeruginosa, adding to the bacterium’s resistance arsenal.

1.1.5.3. Target modifications

Antibiotic target modification is a key component of the P. aeruginosa resistance to fluoroquinolone antibiotics, which target the DNA gyrase and DNA Topoisomerase IV enzymes. P. aeruginosa develops resistance to fluoroquinolones, such as ciprofloxacin, via mutations in the gyrA subunit of the DNA gyrase complex, or parC of the topoisomerase IV, with strains featuring mutations in both genes exhibiting a high level of ciprofloxacin resistance (Mouneimné et al., 1999; Mesaros et al., 2007). Other P. aeruginosa strains have acquired resistance to aminoglycoside treatment via methylation of the 16S rRNA, or to β- lactams via mutation of the penicillin binding protein, PBP4 (Yokoyama et al., 2003; Mesaros et al., 2007; Moya et al., 2009; Zhou et al., 2010; Cavallari et al., 2013). Through this range of target modifications, P. aeruginosa is able to largely evade the effects of fluoroquinolones, in addition to reducing its susceptibility to β-lactams and aminoglycosides.

1.1.5.4. Lifestyle-mediated resistance

P. aeruginosa biofilm formation is strongly associated with an increase in antibiotic resistance, as briefly mentioned in Section 1.1.4. The reduced penetration of antimicrobials through the extracellular matrix is a key component of this aspect of resistance (Billings et al., 2013; Okshevsky & Meyer, 2013). P. aeruginosa PA14 has been demonstrated to produce

10 Chapter 1 cyclic glycans via NdvB, which it secretes into the biofilm matrix where they bind to aminoglycosides and thereby attenuate their impact on bacterial protein synthesis (Zhang & Mah, 2008; Sadovskaya et al., 2010). P. aeruginosa PAO1 encodes a protein with 99% identity with NdvB from PA14, strongly suggesting the potential for a similar resistance mechanism in this strain. Furthermore, P. aeruginosa in a biofilm grow more slowly than their planktonic counterparts, especially in the centre of the biofilms (Stewart & Franklin, 2008; Folsom et al., 2010). This reduces the efficacy of aminoglycoside and fluoroquinolone treatment, as these antibiotic classes target processes that occur in actively growing cells, i.e. protein synthesis and DNA replication, respectively. Consequently, this allows the existence of persister cells that, at cessation of antibiotic treatment, are able to repopulate the site of infection (Lewis, 2008; Pamp et al., 2008). Through these combined mechanisms, biofilm P. aeruginosa cells are able to be significantly more resistant to antibiotics, thereby making clearance of biofilm infections almost impossible.

1.1.5.5. Antibiotic efflux

Efflux pumps have an essential role in bacterial antibiotic resistance. There are five main protein families that have been associated with xenobiotic efflux: the resistance- nodulation division superfamily (RND), major facilitator superfamily (MFS), multidrug and toxic compound extrusion family (MATE), small multidrug resistance family (SMR), and the ABC transporter superfamily (Figure 1.1). Each of these families, apart from the ABC transporters, relies upon the use of proton motive force to move their targets across the cellular membranes, with the MATE family able to use sodium ions or protons (He et al., 2004a; Delmar et al., 2014). By contrast, ABC transporters couple the hydrolysis of ATP to actively translocate their cargo. All of these transport proteins have an inner membrane embedded transmembrane (TM) component in P. aeruginosa.

DNA sequencing and the initial annotation of the P. aeruginosa PAO1 genome indicated the bacterium encoded 11 RND-type, 20 MFS-type, 2 SMR-type, 1 MATE-type and 1 ABC-type drug efflux pumps (Stover et al., 2000). Efflux pumps have key roles in the antibiotic resistance of P. aeruginosa, enabling intrinsic, acquired and adaptive resistance. The role of the RND pumps in P. aeruginosa antibiotic resistance has been most studied. Whilst RND pumps are present in all domains of life, in Gram-negative bacteria such as P. aeruginosa, the inner membrane embedded RND pumps work in a co-operative manner with an inner membrane-anchored, periplasmic spanning, membrane fusion protein (MFP), and an outer membrane protein (OMP). In this way the antibiotic is secreted from the inner membrane or periplasm across the outer membrane into the extracellular milieu. Four of the

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Figure 1.1: Structural and mechanistic diversity of drug efflux pump families. Schematic representation of the five main drug efflux pumps predicted to contribute to xenobiotic (blue sphere) efflux in P. aeruginosa. The resistance-nodulation division (RND, red), major facilitator superfamily (MFS, orange) and small multidrug resistance (SMR, blue) proteins all rely on proton motive force to translocate drugs across the cell membrane. The multidrug and toxic extrusion (MATE, yellow) pump family are able to use either proton motive force or sodium ions to translocate their allocrites. ABC transporters (pink) are the only active drug efflux pumps, utilising the hydrolysis of ATP to energise transport. In Gram- negative organisms such as P. aeruginosa, the RND and ABC transporters commonly interact with a membrane fusion protein (green) and an outer membrane protein (purple) to transport their allocrites across the outer membrane.

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P. aeruginosa tripartite RND pumps systems have been shown to significantly contribute to the bacterium’s antibiotic resistance profile: MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM (Morita et al., 2001; Okamoto et al., 2002; Aeschlimann, 2003; Morita et al., 2012; Morita et al., 2013). The RND component of these systems (MexB, MexD, MexF and MexY), is commonly encoded adjacent to the MFP component (MexA, MexC, MexE and MexX), and the OMP component (OprM, OprJ, and OprN). However, MexXY is not encoded with an OMP, but functions with OprM. MexAB-OprM is constitutively expressed in P. aeruginosa (De Kievit et al., 2001) and contributes to the bacterium’s intrinsic antibiotic resistance profile via its capacity to transport over 25 structurally distinct compounds (Poole, 2005). The contribution of MexAB-OprM to antibiotic resistance has also been shown to be enhanced via mutations in the transcriptional regulator MexR, which result in an increase in its expression (Poole et al., 1996b; Ziha-Zarifi et al., 1999). By contrast, MexCD-OprJ and MexEF-OprN both contribute to the acquired antibiotic resistance of P. aeruginosa, in that they are expressed at low levels unless mutations are acquired that result in significant increases in their expression (Poole et al., 1996a; Kohler et al., 1997; Aeschlimann, 2003). The MexXY-OprM RND pump contributes to both the intrinsic and adaptive resistance of P. aeruginosa (Aires et al., 1999; Masuda et al., 2000a; Morita et al., 2001; Hocquet et al., 2003). However, by comparison with MexAB-OprM, MexXY-OprM exhibits a narrow substrate specificity. Despite this it is able to efflux clinically relevant anti-pseudomonal antibiotics (Aires et al., 1999; Westbrock-Wadman et al., 1999; Masuda et al., 2000a; Masuda et al., 2000b; Okamoto et al., 2002; Aeschlimann, 2003). MexXY-OprM provides P. aeruginosa with intrinsic resistance to aminoglycosides, and is also able to be up-regulated in response to low antibiotic concentrations. In this way, MexXY-OprM present a particular challenge for the clearance of P. aeruginosa infections in CF patients, who commonly undergo multiple rounds of antibiotic treatment (Poole, 2011). Furthermore, the increased expression of MexXY has been reported as a consequence of mutations, particularly in its regulator MexZ (Guenard et al., 2014). Although these four tripartite RND pumps significantly contribute to the antibiotic resistance of planktonic cells, they have not been demonstrated to do so in sessile cells (De Kievit et al., 2001). Rather, another efflux pump(s) may perform this role, but this remains to be determined. In contrast to the RND pumps, the other putative drug efflux pumps of P. aeruginosa are poorly characterised. To date only one SMR drug efflux pump has been characterised in P. aeruginosa, EmrE (PA4990). The constitutively expressed EmrE was shown to efflux cationic dyes, in addition to certain aminoglycosides (Li et al., 2003). This study also suggested that five other drug-efflux SMR proteins may be encoded in the PAO1 genome, two more than originally predicted (Stover et

13 Chapter 1 al., 2000). However, due to the paucity of further studies, the extent to which this family contributes to the antibiotic resistance of P. aeruginosa remains unclear. One protein from the MATE family of P. aeruginosa transporters, PmpM (PA1361), has also been characterised as contributing to antibiotic resistance. Increased expression of PmpM in a drug-hypersensitive E. coli strain was demonstrated to confer resistance to a range of antibiotics including the fluoroquinolone family (He et al., 2004a). PmpM was also shown to be a proton-dependent MATE pump, as opposed to the more common sodium ion-dependent MATE pumps. No other MATE drug efflux pumps have been characterised in P. aeruginosa to date. Whilst the MFS family were most highly represented amongst the initial predicted P. aeruginosa drug- efflux pumps (20 putative pumps) (Stover et al., 2000), to date there are no formally characterised examples. Therefore, it remains to be determined what role, if any, these putative transporters have in P. aeruginosa antibiotic resistance. Finally, the initial analysis of the PAO1 genome suggested it to encode a single ABC transporter that was likely to contribute to drug efflux (Stover et al., 2000). In other bacteria ABC transporters perform various roles in drug and antibiotic efflux. However to date, the contribution of ABC efflux proteins to antibiotic resistance in P. aeruginosa remains to be determined, with numerous ABC efflux proteins now identified.

ABC transporters

The ATP-binding cassette (ABC) transporter superfamily is present across all domains of life. Canonical ABC transporters translocate their cargo or allocrite in a unidirectional manner across cellular membranes via the hydrolysis of ATP. The cargo transported by ABC transporters is referred to as an allocrite as it is not altered during transport. By contrast, ATP is the transporter substrate as it is hydrolysed (to ADP + Pi) during the transport cycle (Holland & Blight, 1999). The energy dependence of this protein family enables transport against the concentration gradient, acting to effectively exclude or concentrate allocrite on one side of the cytoplasmic membrane. ABC transporters are classified as exporters or importers based on their vector of transport. Whilst ABC exporters are present across all known organisms, the ABC importers have only been identified in prokaryotes, archaea and plants (Shitan et al., 2003; Terasaka et al., 2005; Lee et al., 2008). Recently, another subset of ABC importers was identified the non-canonical energy-coupling factor importers, tentatively referred to as type III importers (Eitinger et al., 2011; Rice et al., 2014). However, despite some functional similarities to ABC importers, they also have some distinct structural differences (ter Beek et al., 2014). In addition to ABC transporters, a subset of non-transport 14 Chapter 1

ABC proteins exists, which lack transmembrane components (Hopfner & Tainer, 2003). However, these latter two subgroups are outside the scope of this study and are not discussed further. Herein, bacterial ABC exporters and ABC importers are the focus of this work.

1.2.1. Common architecture

Canonical ABC transporters share a common architecture of four domains: two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). This overall structure in conserved in both importers and exporters. The TMDs traverse the cell membrane in a series of alpha helices, with the overall structure providing a channel through which allocrites may be selectively translocated. Attached or associated with the cytosolic face of the TMDs, two NBDs bind and hydrolyse ATP, allowing the process to occur.

1.2.1.1. Transmembrane domains

Significant diversity in both size and structure are observed in the TMDs of the ABC transporter superfamily. Across taxa, ABC efflux proteins have two TMDs each, in most cases, featuring six transmembrane helices, giving a total of twelve helices per exporter. In contrast, ABC importers vary in their number of transmembrane helices per TMD, with the methionine importer MetI from E. coli having five helices per TMD (Rees et al., 2009), ranging up to ten helices per TMD in the vitamin B12 transporter BtuCD from E. coli (Locher et al., 2002). The number of helices is thought to reflect the size of the channel required for the substances transported, with larger channels supporting the movement of larger allocrites. The transport process through the TMDs remains poorly characterised, with most allocrites believed to move through the TMD channel without a binding event occurring within the TMDs. The sole known exception to this observation is the maltose import system from E. coli, MalFGK2A (Oldham et al., 2007). In this system, it has been determined that after the initial binding of malto-oligosaccharides to the maltose binding protein (MBP) SBP, the allocrite is released and subsequently bound at a secondary internal allocrite binding site within the TMDs, which facilitates transport across the membrane (Oldham et al., 2013). Together the two binding events are responsible for the high specificity of the transporter. Whether allocrite binding sites are present within a broader range of ABC transporter TMDs is yet to be determined.

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1.2.1.2. Nucleotide-binding domains

In contrast to the TMDs, the NBDs of ABC transporters are highly conserved, each featuring a characteristic ABC signature motif (LSGGQ) (Kos & Ford, 2009). NBDs are structurally divided into two subdomains: a larger, RecA-like catalytic subdomain, and a smaller helical subdomain, forming an overall L-shaped NBD (Davidson et al., 2008). For a complete ABC transporter, two NBDs dimerise in a head-to-tail formation to bind two molecules of ATP. In this way, a phosphate-binding loop (Walker A motif) on one subunit is positioned adjacent to the ABC signature sequence motif on the opposite NBD monomer such that they both bind a shared ATP molecule. This arrangement occurs twice within the dimer, allowing the binding of two molecules of ATP in a co-operative manner (Davidson et al., 1996; Hollenstein et al., 2007a). In addition to the conserved ATP-binding motifs, several other features exist at the dimer/ATP-binding interface, which facilitate ATP hydrolysis and the resulting conformational change (Hollenstein et al., 2007a). A conserved Q–loop motif in each monomer acts as a sensor for the γ-phosphate of the ATP molecule. Upon sensing the bound ATP, the glutamate of the Walker B motif facilitates a nucleophilic attack on the bound

ATP via a H2O molecule, resulting in ATP hydrolysis. The presence of the proximate Q-loop motif links the hydrolysis of ATP to the conformation changes in the TMDs during transport (Rees et al., 2009). In this way transport is energized via the NBD-dependent hydrolysis of ATP.

1.2.2. ABC exporters

ABC exporters are present in all domains of life and hold roles in the efflux of a diverse array of allocrites from the . In humans, ABC exporters are synonymous with the multidrug resistance phenotype of cancers (Gottesman et al., 2002; Sharom, 2008), in addition to serving critical roles in lipid metabolism (Li et al., 2013; Tarling et al., 2013), ion transport (Gadsby et al., 2006), and antigen processing (Parcej & Tampe, 2010). Furthermore, abnormalities in their expression and localisation result in multiple diseases including Tangier disease (Bodzioch et al., 1999; Rust et al., 1999) and Stargardt disease (Allikmets et al., 1997). In plants, ABC transporters are highly abundant (~130 in Arabidopsis thaliana) and have been shown to hold a range of roles including, xenobiotic and herbicide efflux, auxin transport and movement of excess folates (Rea, 2007; Remy & Duque, 2014). By comparison with eubacteria, ABC exporters are less common in archaea (Davidson et al., 2008). Bacterial ABC exporters are typically homodimers or heterodimers of TMD-NBD fusion proteins.

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Bacteria express a range of ABC exporters, serving diverse roles in xenobiotic efflux, and protein, toxin, glycoconjugate and lipopolysaccharide secretion. A large proportion of these have a role in bacterial virulence and have been recently reviewed (Appendix 3) (Lewis et al., 2012). The general roles of bacterial ABC efflux proteins are summarised below and in Figure 1.2A-C.

1.2.2.1. Xenobiotic efflux

ABC exporters have been characterised in a range of bacteria as contributing to xenobiotic resistance via the active extrusion of drugs and antibiotics from the cytoplasm. The first bacterial ABC exporter to be ascribed a xenobiotic efflux role was the homodimeric LmrA transporter from Lactobacillus lactis (van Veen et al., 1996). LmrA was identified on the basis of significant sequence homology with the human drug-efflux pump, ABCB1 (Juliano & Ling, 1976), and has since been shown to extrude a range of drugs from the inner leaflet of the cell membrane to the extracellular environment (van Veen et al., 1996; Poelarends et al., 2000). Furthermore, heterologous expression in an E. coli strain was shown to confer resistance to antibiotics from the tetracycline, aminoglycoside, quinolone, macrolide and - lactam families (Poelarends et al., 2002). However, a recent study has demonstrated that LmrA also exhibits H+-Na+-Cl- symport activity, suggesting the protein holds a primary role in salt tolerance, with the drug efflux activity an opportunistic function (Velamakanni et al., 2009). To date, high-resolution structural data are available for two bacterial ABC exporters: the homodimeric Sav1866 transporter from S. aureus (Dawson & Locher, 2006; Dawson & Locher, 2007) and the heterodimeric TM287/TM288 transporter from Thermotoga maritima (Hohl et al., 2012; Hohl et al., 2014). The better characterised of the two proteins, Sav1866 functions as a homodimeric ABC exporter, with each half comprising a TMD-NBD fusion of six transmembrane helices. To date, Sav1866 has only been shown to transport the fluorescent dyes Hoescht 33342 and ethidium bromide, while its physiological allocrite still remains to be determined (Velamakanni et al., 2008). Other examples of ABC exporters that exhibit xenobiotic efflux include the homodimeric transporters BmrA and HorA from and Lactobacillus brevis, respectively, and the heterodimeric LmrCD transporter from L. lactis (Sakamoto et al., 2001; Steinfels et al., 2002; Steinfels et al., 2004; Lubelski et al., 2006). Together, a large number of ABC efflux proteins have been shown to confer resistance to xenobiotic compounds and implicated in aiding bacterial survival. However, in most cases the observed xenobiotic efflux is appears to be an opportunistic or secondary function, with their primary physiological function(s) and allocrite(s) in vivo largely unknown.

17 Chapter 1

Figure 1.2: Functional diversity of ABC transporters. Adapted from Lewis et al. (2012). Schematic representation of the Gram-negative cell membranes and the roles performed by ABC transporter proteins. (A) ABC exporters provide resistance against xenobiotics and drugs (blue sphere) through their active efflux from the cytoplasm. Such proteins can operate independently or together with a membrane fusion protein and outer membrane protein as depicted in Figure 1.1. (B) Glycoconjugates (brown rectangle), including lipopolysaccharides, capsular polysaccharides and exopolysaccharides can be synthesised and secreted by ABC exporter pathways. These proteins function together with an outer membrane protein (orange) to secrete the glycoconjugates to the cell surface. (C) Type 1 secretion systems, comprised of an ABC exporter, a membrane fusion protein (green) and an outer membrane protein (purple), provide a contiguous pathway for the secretion of unfolded proteins (black line) across the inner and outer membranes. Allocrites include proteases, toxins, antimicrobial peptides and adhesion proteins. (D) ABC permeases, composed of an ABC transporter and a periplasmic solute binding protein (blue oval) are involved in the import of diverse substances (pink sphere) across the inner membrane, including metals, carbohydrates, osmoprotectants, vitamins, peptides and amino acids. ABC importers are commonly composed of four separate polypeptides that make up the transporter, whereas bacterial ABC exporters are predominantly produced as transmembrane domain- nucleotide binding domain fusion proteins that dimerise for function. The solute binding protein in Gram-negative organisms is freely diffusible within the periplasm, or lipid- anchored to the cell membrane in Gram-positive species.

18 Chapter 1

1.2.2.2. Protein secretion

Gram-negative bacteria export most of their extra-cytoplasmic proteins via the Sec- dependent Type II Secretion pathway (Economou et al., 2006; Nivaskumar & Francetic, 2014). However, a significant role is also held by the Type 1 Secretion pathway, with many of its secreted proteins contributing to bacterial virulence. Type 1 secretion systems (T1SS) are dependent on an ABC efflux protein for the Sec-independent movement of proteins across the cell membrane. Found primarily in Gram-negative bacteria, T1SS utilize three components to translocate molecules across the cytoplasmic and outer membranes: an ABC exporter, a MFP, and an OMP. The ABC exporter is responsible for the initial recognition of allocrite, and provides the energy for translocation, via hydrolysis of ATP. The MFP, anchored in the inner membrane connects the ABC transporter to the OMP, which facilitates movement of the allocrite through the outer membrane. T1SS allocrites are recognised by virtue of a poorly conserved C-terminal secretion sequence, which is bound by the ABC transporter (Benabdelhak et al., 2003). Binding of the allocrite to the ABC transporter has been proposed to trigger recruitment of the MFP and OMP, creates a channel for translocation, and prevents the allocrite folding during transport and forming a periplasmic intermediate (Delepelaire, 2004). However, the precise sequence of steps in this process remain to be definitively revealed (Delepelaire, 2004). T1SS secrete proteins and polypeptides ranging from 19 kDa (HasA) (Létoffé et al., 1994) to > 800 kDa (LapA) (Hinsa et al., 2003), with translocated proteins including toxins (Goebel & Hedgpeth, 1982), (Gilson et al., 1990), hydrolases (Masure et al., 1990), S-layer proteins (Kawai et al., 1998) and adhesion proteins (Hinsa et al., 2003). Protein allocrites are secreted post-translationally in an unfolded form, only adopting a folded conformation at the bacterial surface. Maintenance of the unfolded state has been shown, in some cases, to be achieved by chaperones or tethering to the ABC exporter. Calcium ions have also been suggested to have a role due to their low abundance in the prokaryotic cell (Holland et al., 2005) and the presence of numerous calcium binding repeats in the C-terminus of predicted T1SS allocrites (Linhartová et al., 2010). Thus, it has been suggested that the limited concentration of calcium in the cytoplasm prevents folding in the cytoplasm. Following export across the outer membrane, the high calcium levels in the surface LPS are implicated to drive folding of the protein into its native conformation (Holland et al., 2005). Hence, T1SS constitute an efficient translocation mechanism for export of proteins to the bacterial surface, particularly allowing the secretion of toxic, or very large proteins.

19 Chapter 1

1.2.2.2.1. Toxins and proteases

The repeat-in-toxin (RTX) protein family, characterised by calcium-binding glycine and aspartate-rich repeats at their C-terminus, are the best characterised of the T1SS allocrites. These RTX proteins act as proteases, lipases and pore forming toxins and are produced by a range of bacterial species. The -haemolysin (HlyA) T1SS from E. coli is the most characterised RTX secretion system, comprised of HlyB (ABC exporter), HlyD (MFP) and TolC (OMP) (Goebel & Hedgpeth, 1982; Schulein et al., 1992). HlyA is a cytolysin, which when inserted into the plasma membranes of eukaryotic cells, generates pores permitting calcium ion influx. This causes an inflammatory response, which results in cellular damage. Also worthy of mention is the P. aeruginosa AprDEF T1SS, which is required for the secretion of the zinc-dependent RTX alkaline protease, AprA (Duong et al., 1992). AprA serves as a virulence factor, associated with proteolysis of laminin, fibrin and fibronectin in the host, thereby aiding in tissue invasion and infection (Heck et al., 1986; Shibuya et al., 1991). Furthermore, the protein has been demonstrated to cleave a component of the complement system (C2), preventing phagocytosis of the P. aeruginosa by (Laarman et al., 2012), and contributing to iron acquisition by siderophores via proteolysis of human transferrin (Kim et al., 2006). A number of other T1SS have been characterised including those responsible for the secretion of EhxA from E. coli (Schmidt et al., 1995; Bauer & Welch, 1996), RtxA from Vibrio cholera (Lin et al., 1999), MbxA from Moraxella bovis (Angelos et al., 2003) and CyaA from Bordetella pertussis (Glaser et al., 1988).

1.2.2.2.2. Antimicrobials and antibiotic peptides

Bacteria that produce antibiotics and antimicrobial peptides commonly secrete these via T1SS. Gram-positive organisms use a modified T1SS, which is an ABC transporter dedicated to the secretion of produced antimicrobials and lantibiotics. Several prominent examples are the heterodimeric DrrAB ABC exporter of Streptomyces peucetius that secretes doxorubicin and daunorubicin (Guilfoile & Hutchinson, 1991); OleB and/or OleC secretion of oleandomycin in Streptomyces antibioticus (Olano et al., 1995); and PepT secretion of the lantibiotic Pep5 from Staphylococcus epidermidis (Meyer et al., 1995). The best characterized antimicrobial T1SS is CvaAB-TolC, which is responsible for the secretion of the antimicrobial toxin colicin V in the Gram-negative bacterium, E. coli (Gilson et al., 1987; Gilson et al., 1990; Fath et al., 1994). The CvaB ABC exporter component, in addition to providing the specificity and energy for transport, possesses an additional C39 catalytic domain which removes the N-terminal double glycine leader sequence from colicin V during the secretion process (Wu et al., 2012). The mature is then secreted through the 20 Chapter 1

CvaA (MFP) and TolC components to the extracellular environment. Genes homologous to colicin V and its cognate secretion system have been identified in P. aeruginosa PAO1 (PA4141-PA4144), but the cellular role of this system remains to be demonstrated (Stover et al., 2000).

1.2.2.2.3. Heme-binding proteins and siderophores

Iron is an essential transition metal for almost all forms of life (Andrews et al., 2003). However, the availability of free or uncomplexed iron is limited, particularly in the context of the host-pathogen interaction (Hood & Skaar, 2012). The challenges associated with iron recruitment, i.e. solubility and sequestration, have resulted in the evolution of multiple acquisition pathways, many of which utilize an ABC permease, for the uptake of iron in various forms. Non-ribosomally produced chelating molecules known as siderophores play a key role in iron acquisition by P. aeruginosa and a range of other bacteria (Schalk et al., 2011). P. aeruginosa produces the siderophores pyoverdine and pyochelin, with the primary pyoverdine binding iron with an affinity of 10-32 M (Albrecht-Gary et al., 1994; Hoegy et al., 2014). Pyoverdine biosynthesis is composed of two stages, both of which require ABC efflux proteins. First, a non-fluorescent precursor is produced in the cytoplasm by peptide synthetases (Visca et al., 2007). This is then secreted by the PvdE ABC efflux protein into the periplasm, where the chromophore component of the compound is cyclised, conferring to the molecule (Yeterian et al., 2010). The fully synthesised pyoverdine is then secreted from the periplasm via PvdRT-OmpQ, a tripartite complex composed of an ABC exporter (PvdT), a membrane fusion protein (PvdR) and an outer membrane protein (OmpQ) (Hannauer et al., 2010). Following binding of iron in the extracellular environment, delivery of the iron-bound siderophore to the periplasm, and release of iron, PvdRT-OmpQ serves to recycle apo-pyoverdine to the extracellular environment (Imperi et al., 2009). The use of ABC-dependent efflux systems for the secretion of siderophores is also observed in Salmonella enterica serovar Typhimurium (S. Typhimurium). S. Typhimurium utilizes the IroC ABC exporter for the secretion of the salmochelin siderophore (Crouch et al., 2008). IroC is also able to secrete another siderophore produced by the bacterium known as enterobactin (Crouch et al., 2008). However, this is a secondary transport pathway, with the EntS MFS efflux pump serving as the primary secretion system for enterobactin (Crouch et al., 2008).

T1SS also contribute to the acquisition of iron in the form of heme. Serratia marscens secretes the HasA heme-binding protein via the HasDEF TISS (Létoffé et al., 1994; Binet & Wandersman, 1996). In the extracellular environment HasA acquires free or haemoglobin-

21 Chapter 1 bound heme and delivers it to the cell for import by the TonB-dependent HasR and the TonB protein, HasB (Letoffe et al., 1994; Ghigo et al., 1997; Paquelin et al., 2001). A homologous system has also been identified in P. aeruginosa PAO1, with the HasA homolog, HasAp, shown to facilitate the acquisition of heme and hemoglobin (Letoffe et al., 1998).

1.2.2.3. Glycoconjugate secretion

ABC transporters have a significant role in glycoconjugate biosynthesis, specifically in the secretion of teichoic acids, the O-antigen component of lipopolysaccharides (LPS), capsular polysaccharide (CPS) and exopolysaccharides (EPS). In Gram-positive organisms, the is composed predominantly of peptidoglycan and anionic glycophosphate teichoic acids, the latter of which is secreted to the cell surface via an ABC transporter. The TagGH ABC transporter, present in both S. aureus and B. subtillis, is responsible for teichoic acid secretion, and has also been shown to be essential for cell viability (Lazarevic & Karamata, 1995; Schirner et al., 2011). By contrast, the surface of Gram-negative bacteria is coated with LPS. LPS is composed of two modules: a repeated O-antigen polysaccharide tail; and a lipid A core, comprised of lipid A and a core oligosaccharide. Both modules of LPS are secreted to the periplasmic face of the inner membrane via ABC exporters (Wzt/Wzm and MsbA, respectively) (Raetz et al., 2007; Cuthbertson et al., 2010), prior to assembly as a complete LPS molecule (Greenfield & Whitfield, 2012). The LPS molecule is subsequently translocated to the outer membrane in an ABC exporter-energised process (Greenfield & Whitfield, 2012). The secretion of polysaccharides that comprise the bacterial capsule and EPS, also occurs in an ABC transporter-dependent manner (Becker et al., 1995; Rosinha et al., 2002; Whitfield, 2006), with both components playing key roles in bacterial virulence (Rosinha et al., 2002; Sukupolvi-Petty et al., 2006; Peppoloni et al., 2010; Keo et al., 2011; Singh et al., 2011). Together, ABC transporters play important roles in the production and secretion of glycoconjugate molecules, although the full extent of their contribution to these processes is still being elucidated.

1.2.3. ABC importers

ABC import systems have to date, only been identified in bacteria, archaea and plants, where they associated with a broad range of functional roles. Whilst ABC importers retain the 4-domain ABC transport structure, the NBDs and TMDs are typically produced as individual polypeptides, rather than a TMD-NBD fusion as observed for most bacterial ABC exporters. Furthermore, to allow specific import of their cognate allocrite, ABC importers function with

22 Chapter 1 an additional solute binding protein (SBP) component. The specificity of import by ABC importers is determined largely via the SBP. This is demonstrated in cases where multiple SBPs deliver different cognate allocrites to the same ABC transporter (Chen et al., 2010). Together the SBP and the ABC transporter components (two NBDs and two TMDs) comprise an ABC permease (Figure 1.2D). In Gram-negative bacteria, the SBP component is commonly diffusible within the periplasm, whereas in Gram-positive bacteria, it is typically lipid anchored to the cell membrane, although in some cases it can be fused to the transporter (Biemans-Oldehinkel & Poolman, 2003; Schuurman-Wolters & Poolman, 2005).

1.2.3.1. Classification of SBPs

As more prokaryotic SBPs were identified, a classification scheme was constructed based on their secondary structure (Fukami-Kobayashi et al., 1999). Subsequently, the determination of high-resolution SBPs structures facilitated the proposal of an alternative classification scheme (Berntsson et al., 2010). Although no classification system can address the full breadth of biological diversity, the cluster system proposed by Berntsson et al. (2010), is the most comprehensive to date. This classification allocated the prokaryotic SBPs into Clusters A-F based upon alignment with the available high-resolution structures (Table 1.1). Cluster A

SBPs bind metal ions and share an overall protein fold of two (α/β)4 domains connected by a long α-helix. The allocrite binding site is located in the cleft between the two domains. Cluster A SBPs can be further subdivided as cluster A-I, that is those that bind their metal ions directly such as the manganese binding PsaA from (McDevitt et al., 2011), and the cluster A-II SBPs which bind chelated metal complexes such as the BtuF SBP from E. coli (Karpowich et al., 2003). Cluster B SBPs feature three hinges between the two domains and are involved in binding a diverse range of allocrites including carbohydrates and branched chain amino acids (Spurlino et al., 1991; Quiocho et al., 1997; Trakhanov et al., 2005). In addition to delivering their allocrite to ABC transporters, these SBPs also co-operate with peptide receptors and TCS (Berntsson et al., 2010). Cluster C SBPs are larger in size than those of the other clusters, featuring an additional domain. These SBPs bind a wide variety of molecules, namely arginine, di-peptides, oligo-peptides, nickel and cellobiose (Charon et al., 1994; Gao et al., 2012). Cluster D SBPs all feature a hinge region composed of two short strands, yet bind a wide variety of allocrites. This cluster is further sub-divided based upon their allocrite: cluster D-I SBPs bind carbohydrates, cluster D-II SBPs bind polyamines and thiamine, cluster D-III SBPs bind tetrahedral oxyanions including sulfate, molybdate, tungstate and phosphate, and cluster D-IV SBPs are ferric or ferrous iron binding

23 Chapter 1

Table 1.1: Classification of SPBs. Adapted from Berntsson et al. (2010). Cluster Structural features Allocrite A-I 2 domains of 4  helices and 4  sheets Metal ions connected by a rigid  helix of ~30 amino Chelated metal ions and A-II acids siderophores Leucine, Isoleucine, Valine, B 2 domains joined by 3 hinges carbohydrates, Peptides (di- and oligo), C 3 domains arginine, nickel, cellobiose D-I Carbohydrates D-II 2 domains joined by a hinge region Polyamine and thyamine D-III composed of 2 short strands Tetrahedral oxyanions D-IV Ferrous and ferric iron E/Extracellular Receptors -sheets connected by -helix Organic acids (ABC transport- independent) F-I Trigonal planar anions F-II 2 domains joined by a hinge region Methionine F-III composed of 2 long strands Osmoprotectants F-IV Amino acids

24 Chapter 1 proteins (Hollenstein et al., 2007b; Koropatkin et al., 2007a; Higgins et al., 2009; Hollenstein et al., 2009; Otrelo-Cardoso et al., 2014). The cluster E SBPs do not function with ABC transporters, but rather with members of the tripartite ATP-independent periplasmic (TRAP) transporter family and are comprised primarily of β-sheets connected by an α-helix (Mulligan et al., 2011). Cluster F SBPs all feature a long bi-segmented hinge region between the two domains. This cluster is also further divided into cluster F-I to F-IV, with F-I proteins binding trigonal planar anions, F-II SBPs binding methionine, F-IIIs binding choline, proline betaine and glycine betaine, and the F-IVs predominantly binding amino acids (Yao et al., 1994; Deka et al., 2004; Koropatkin et al., 2007b; Smits et al., 2008; Chen et al., 2010).

1.2.3.2. Roles of ABC permeases

Consistent with the diversity in SBPs, ABC permease systems are responsible for the import of a range of molecules, including amino acids, peptides, osmoprotectants, vitamins, antimicrobial peptides, sugars, and metals (Figure 1.2D). The transport component of ABC permeases can be classified as a type I or type II importer based on the mechanism of transport and protein topology, with a thorough review of the importer types recently published (Rice et al., 2014). One of the best characterised ABC permease systems,

MalFGK2-MBP, is responsible for the import of maltose into the bacterial cell. This type I ABC importer is atypical in that a second allocrite binding event occurs within the transmembrane domains of the MalFGK2 transporter, in addition to the binding of maltose by the cluster B SBP (Maltose Binding Protein, MBP) (Oldham et al., 2013). Significant structural and biochemical characterisation of the E. coli maltose importer has been performed to elucidate its mechanism of maltose binding and transport (Schmees et al., 1999; Daus et al., 2007; Oldham et al., 2007; Daus et al., 2009; Grote et al., 2009; Jacso et al., 2009; Oldham & Chen, 2011; Bao et al., 2013; Bohm et al., 2013; Bao & Duong, 2014), with homologs of this system now also identified in S. Typhimurium and Thermococcus litoralis (Schiefner et al., 2002; Stein et al., 2002). Another well-characterised ABC permease system, for which there is high-resolution structural data available, is the type II vitamin B12 (cobalamin) BtuCDF ABC permease from E. coli (Borths et al., 2002; Locher et al., 2002;

Hvorup et al., 2007). The acquisition of vitamin B12 (cobalamin) in E. coli first requires translocation across the outer membrane via the TonB-dependent receptor BtuB (Bassford et al., 1976; Bassford & Kadner, 1977). Once in the periplasm, the cluster A-II SBP BtuF binds vitamin B12 and delivers it to its cognate ABC importer BtuCD for transport into the cytoplasm (Van Bibber et al., 1999; Cadieux et al., 2002). Imported vitamin B12 is incorporated into various enzymes of the B12-dependent isomerase, B12-dependent

25 Chapter 1 methyltransferase and B12-dependent reductive dehalogenase families for use within the cell (Banerjee & Ragsdale, 2003). Although some organisms are able to synthesise cobalamin, it is an energy expensive process, requiring ~30 enzymes (Raux et al., 2000). Hence, acquisition from the environment, via BtuDEF, is more efficient. Although there are many other ABC permease systems that are worthy of particular mention, metal ion acquisition is a key component of this study, and consequently the metal ion ABC permeases will be addressed in greater detail below.

1.2.3.3. Metal ion acquisition

Transition metals ions, such as zinc, iron, manganese and molybdenum are essential for bacterial growth, serving structural and catalytic roles in the cell. In fact, metalloproteomic analyses indicate that at least 30% of all proteins require an interaction with a metal ion for their physiological function (Andreini et al., 2008). However in excess, metal ions also exert significant toxicity, necessitating the strict control of their acquisition (Bruins et al., 2000; Colvin et al., 2010; Botella et al., 2011; McDevitt et al., 2011; Botella et al., 2012; Braymer & Giedroc, 2014; Chaturvedi & Henderson, 2014). Due to the importance of these transition metal ions to bacteria, host limitation of their bioavailability can limit or abrogate infection (Hood & Skaar, 2012; Cerasi et al., 2013). Despite the complex array of chelating or sequestration strategies employed, bacteria can overcome these by the use of high affinity ABC permeases for acquisition of metal ions at the host-pathogen interface.

1.2.3.3.1. Iron

Iron is a critical component of numerous cellular pathways on account of its range of oxidation states. The acquisition of iron via ABC permeases occurs via two main types of SBPs. Ferric or ferrous iron is acquired by the cluster D-IV SBPs, such as hFbp from H. influenzae, nFbp from Neisseria gonorrhoeae, and FutA1 and FutA2 from Synechocystis PCC 6803 (Bruns et al., 1997; Boukhalfa et al., 2003; Guo et al., 2003; Koropatkin et al., 2007a; Badarau et al., 2008). Iron can also be acquired in a chelated form via interaction with cluster A-II SBPs. The cluster A-II SBPs bind iron chelated by siderophores, such as hydroxamate or catecholate, prior to delivery of their allocrite to the awaiting transporter. The Fhu permease system of S. aureus features two cluster A-II SBPs, FhuD1 and FhuD2, which bind the iron- bound hydroxymate siderophore, prior to delivery to the cognate FhuBGC2 ABC transporter (Sebulsky & Heinrichs, 2001; Podkowa et al., 2014). This system is of particular interest as S. aureus does not produce siderophores, but rather utilizes those made by other bacteria to acquire iron via the Fhu pathway. Through the use of multiple high affinity ABC iron

26 Chapter 1 acquisition systems, bacteria are able to meet their cellular requirement for iron, even under iron-limiting conditions.

1.2.3.3.2. Zinc

Zinc plays an essential role within the bacterial cell, serving as a structural or catalytic component of 5-6% of prokaryotic proteins (Hantke, 2005; Andreini et al., 2006). The utilization of zinc across a range of different proteins is attributed to its non- nature and the ability to act as a Lewis acid and an electrophile (Andreini et al., 2008). Specific, high affinity zinc acquisition in bacteria is carried out via the cluster A-I SBP ZnuA and its homologs (AdcA and AdcAII) (Lewis et al., 1999; Chen & Morse, 2001; Campoy et al., 2002; Davis et al., 2009; Dahiya & Stevenson, 2010; Desrosiers et al., 2010; Murphy et al., 2013). ZnuA proteins are unique amongst the cluster A-I SBPs in that they all feature a histidine and acidic amino acid rich loop of varying lengths, which is proposed to aid in zinc binding and delivery to the ZnuBC ABC transporter (Banerjee et al., 2003; Yatsunyk et al., 2008). Zinc is also proposed to be acquired via the manganese/iron/zinc cluster A-I TroA SBPs (Desrosiers et al., 2007), but there is no direct evidence that TroA-like SBPs are involved in zinc acquisition. In addition to the high affinity ABC permeases, lower affinity zinc uptake pathways have been identified in some bacterial species, such as the ZupT Zrt, Irt-like Protein (ZIP) family transporter in E. coli (Grass et al., 2002; Grass et al., 2005). Despite this, Znu permeases, and homologs, appear to be the primary zinc acquisition pathway throughout the prokarya.

1.2.3.3.3. Molybdenum

The transition metal molybdenum is essential to most forms of life. Although it exists in a range of oxidation states, cellular uptake of the metal ion occurs as the tetrahedral oxyanion, 2- molybdate (MoO4 ). Molybdate serves as an enzymatic cofactor in a range of enzymes that contribute to carbon, nitrogen and sulphur metabolism (Hernandez et al., 1991; Iobbi-Nivol & Leimkuhler, 2013). Molybdate acquisition occurs primarily via the high affinity ModABC ABC permease, in which molybdate is bound by the cluster D-III SBP, ModA. The ModA SBP has been characterised in a range of prokaryotes, including E. coli, Xanthamonas citri and the archeon Methanosarcina acetivorans (Rech et al., 1996; Hu et al., 1997; Balan et al., 2006; Balan et al., 2008; Chan et al., 2010). Recently, a second molybdate ABC permease system, MolABC, has been identified in H. influenzae (Tirado-Lee et al., 2011). The SBP component MolA, has a lower affinity for molybdate than ModA (~100 μM vs <10 nM for E. coli ModA) (Rech et al., 1996; Tirado-Lee et al., 2011), and differs in its structure, sharing

27 Chapter 1 greater similarity with the cluster A-I SBPs than the cluster D-III SBPs (Vigonsky et al., 2013). The presence of multiple systems in certain bacterial species has been suggest to arise from a need to ‘fine tune’ the molybdate acquisition and thereby ensure the requisite cellular abundance of the ion.

ABC transporters in P. aeruginosa

Analysis of the P. aeruginosa genome indicates the presence of 289 genes that are associated with ABC transporter function, that is, with homology to either ABC transporter TMDs, NBDs or SBPs (Ren et al., 2007). Of these, the majority are uncharacterised. P. aeruginosa PAO1 encodes 13 ABC efflux proteins, of which eight have been assigned a function or characterised, albeit to varying extents. These are (i) MsbA (PA4997), an essential lipid flippase (Ghanei et al., 2007); (ii) HasD (PA3406), a putative component of the heme acquisition pathway (Letoffe et al., 1998); (iii) PvdE (PA2937), a pyoverdine secretion protein (Yeterian et al., 2010); (iv) PvdT (PA2390), a pyoverdine siderophore secretion and recycling transporter (Hannauer et al., 2010; Hannauer et al., 2012); (v) CvaB (PA4143), a probable toxin transporter; (vi) AprD (PA1246), an alkaline protease transporter (Guzzo et al., 1991; Duong et al., 1996; Duong et al., 2001); (vii) PchI/PchH (PA4222/PA4223), a putative transporter associated with pyochelin biosynthesis (Reimmann et al., 2001); and (viii) TagT/TagS (PA0072/PA0073), an ABC transporter associated with a type VI secretion system (Casabona et al., 2013). There are five remaining putative ABC transporters (PA0860, PA1113, PA1876, PA3228 and PA5231) for which a function has not been proposed or demonstrated. Each putative ABC efflux protein shares > 24% identity with the bacterial MDR pump LmrA, a homodimeric ABC efflux protein from L. lactis (van Veen et al., 1996). Whilst ABC efflux proteins are associated with drug and antibiotic resistance in a range of bacterial species, none of the characterised P. aeruginosa PAO1 ABC efflux proteins have been shown to have roles in drug efflux. Despite this, P. aeruginosa exhibits significant antibiotic resistance towards antibiotics from many classes. On this basis it could be suggested that these five remaining putative ABC efflux proteins contribute to the MDR phenotype of P. aeruginosa. Alternatively, these proteins, like many of the ABC exporters already characterized in P. aeruginosa, may contribute to virulence. The exact role and importance of these proteins in P. aeruginosa remains to be determined. Such work will provide critical insight into the mechanisms that aid in making P. aeruginosa such a successful bacterium.

28 Chapter 1

ABC permease proteins comprise the majority of ABC transporter-associated proteins in P. aeruginosa. These proteins are proposed to have diverse roles, aiding in P. aeruginosa growth and survival. Part of the success of P. aeruginosa as both an environmental organism and opportunistic pathogen is its ability to acquire metal ions from its environment. The many iron acquisition pathways of P. aeruginosa have been well-characterised and studies continue to reveal their precise molecular details (Andrews et al., 2003; Imperi et al., 2009; Schalk et al., 2011; Brandel et al., 2012; Brillet et al., 2012; Hannauer et al., 2012; Konings et al., 2013; Schalk & Guillon, 2013). However, there is a paucity of data surrounding the ability of P. aeruginosa to acquire other essential metals ions that facilitate its ubiquitous survival. Many of these acquisition pathways are likely to be ABC permease-dependent due to their high affinity. The importance of zinc, particularly at the host-pathogen interface is becoming increasingly evident for a range of bacteria. P. aeruginosa, as a opportunistic human pathogen which infects a range of host niches with accompanying variations in zinc abundance, requires the ability to sense, respond to, and acquire sufficient zinc. This necessitates the use of efficient high affinity zinc uptake system(s). P. aeruginosa is also able to survive in environments of varying oxygen tension. The ability of the bacterium to grow under anaerobic conditions via the reduction of nitrate reduction is dependent upon the metal ion molybdenum, which acts as a cofactor for the nitrate reductase complex. Whilst the molybdenum acquisition pathways of other bacterial species have been elucidated, a homologous system is yet to be characterised in P. aeruginosa.

1.3.1. Aims of the study

ABC transporters hold key roles in the growth and virulence of the opportunistic human pathogen P. aeruginosa. This study provides a detailed characterisation of the P. aeruginosa ABC transport proteins, examining their contribution to drug resistance, and the acquisition of the essential metal ions, zinc and molybdenum. The specific aims of this study are:

1. To ascertain the contribution, if any, of the uncharacterised P. aeruginosa ABC efflux proteins to antibiotic resistance.

2. To elucidate the molybdate acquisition pathway of P. aeruginosa and characterise the role of the metal ion within the bacterium.

3. To characterise the mechanism(s) of zinc acquisition and assess the effect of zinc limitation on cellular physiology.

29

CHAPTER 2:

METHODS AND MATERIALS

30 Chapter 2

Materials

Chemicals and reagents were purchased from Sigma-Aldrich (Australia) unless otherwise stated. Stock solutions of antibiotics and metals were prepared in MilliQ water or appropriate solvent according to the manufacturer’s instructions for the individual compounds, filter sterilized (0.22 m) and stored at 4 C or -20 C. Buffers were filtered with 0.45 m filter to remove particulate matter.

DNA manipulation

2.2.1. Plasmid isolation

Five millilitres of mid-log bacterial culture was used for isolation of plasmid DNA from E. coli. Cells were harvested by centrifugation at 2250 x g, 22 °C for 15 min, and the plasmid isolated using the Spin MiniPrep Kit (Qiagen, Australia) as per the manufacturer’s instructions.

2.2.2. Genomic DNA isolation

Ten millilitres of mid-log bacterial culture was used for isolation of genomic P. aeruingosa DNA. Cells were harvested by centrifugation at 2250 x g, 22 °C for 15 min, and the DNA isolated using the DNeasy Blood and Tissue Kit (Qiagen) as per the manufacturer’s instructions.

2.2.3. DNA electrophoresis

DNA was electrophoresed according to standard protocols (Sambrook & Russell, 2001). Agarose (Life Technologies, Australia) was dissolved in TAE buffer containing 40 mM Tris, 20 mM acetic acid and 1 mM 2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) at an agarose concentration appropriate to the size of the DNA fragments (0.7-2%), prior to the addition of RedSafe Nucleic Acid Solution (Intron Biotecnology, Korea) at 1:10,000. DNA samples were mixed with 5 X GelPilot DNA loading dye (Qiagen) prior to electrophoresis in TAE buffer. Electrophoresis was performed at 160 V until the dye front reached the bottom of the gel. DNA was visualised and photographed using Quantity One 1-D analysis software on a Gel Doc XR+ system (Biorad, Australia). DNA fragments were subsequently purified using the Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions.

31 Chapter 2

2.2.4. Assessment of DNA quantity and quality

DNA was assessed for abundance and purity on a NanoDrop spectrophotometer

(ThemoFisher Scientific, Australia). The A260/A280 ratio was used to assess protein contamination, and the A260/A230 ratio indicating sample contamination with salts and/or detergent. Samples with A260/A280 and A260/A230 ratios of >1.8 were considered of high quality and were used for subsequent applications.

2.2.5. DNA sequencing

DNA sequencing was performed using 150 ng of plasmid or 300 ng of genomic DNA. The sequencing reaction was performed using BigDye Terminator 3.1 Cycle sequencing kit (Applied Biosystems, Australia) according to the manufacturer’s instructions and oligonucleotides listed in Table 2.1 on a MasterCycler Nexus thermal cycler (Eppendorf, Australia) with the following parameters for 25 cycles: 90 °C for 30 sec, 52 °C for 45 sec, 60 °C for 4 min. Clean-up of sequencing reactions were subsequently performed with BigDye Xterminator Purification Kit (Applied Biosystems) according to the manufacturer’s instructions. Samples were then analysed at SA Pathology, Adelaide.

2.2.6. Polymerase Chain Reaction (PCR)

Standard PCRs were performed on a MasterCycler Nexus thermal cycler (Eppendorf) using the High Fidelity DNA polymerase (Roche, Australia). Oligonucleotides used for PCR (Table 2.1) were purchased from Sigma-Aldrich or Integrated DNA Technologies (USA). Standard cycling conditions were 94 C for 5 min, followed by 35 cycles of 94 C for 30 sec, 52 C for 35 sec and 68 C for 1 min 40 sec.

Bacterial culturing

P. aeruginosa and E. coli were maintained on Luria Bertani (Lennox) (LB) agar plates, containing 1% tryptone, 0.5% yeast extract, 0.5% NaCl and 1.5% agar. Plates were supplemented with 50 g.mL-1 kanamycin (Km), 100 g.mL-1 ampicillin (Ap) or 30 g.mL-1 chloramphenicol (Chl) for plasmid maintenance. Supplementation with 30 g.mL-1 gentamicin (Gm), 30 g.mL-1 Gm + 5% sucrose, or 200 g.mL-1 carbenicillin (Cb) was used for marker selection and isolation of P. aeruginosa mutants.

32 Chapter 2

Table 2.1: Oligonucleotides used in this study.

Name Target Sequence (5’  3’) Source

(Choi & Gm-F GmR CGAATTAGCTTCAAAAGCGCTCTGA Schweizer, 2005) (Choi & Gm-R GmR CGAATTGGGGATCTTGAAGTTCCT Schweizer, 2005) (Choi & GW-attB1 attB1 GGGGACAAGTTTGTACAAAAAAGCAGGCT Schweizer, 2005) (Choi & GW-attB2 attB2 GGGGACCACTTTGTACAAGAAAGCTGGGT Schweizer, 2005) PA0860-UpF- PA0860 TACAAAAAAGCAGGCTGTGAGCGCGGAGACCGCCTG This study GWL PA0860-UpR- PA0860 TCAGAGCGCTTTTGAAGCTAATTCGGGTCGGTGACCAGGTGGGC This study GW PA0860-DnF- PA0860 AGGAACTTCAAGATCCCCAATTCGAGGAAATCGGCCTGGACTGC This study GW PA0860-DnR- PA0860 TACAAGAAAGCTGGGTGCCGATGAGCCACGATCAAT This study GWR PA1113-UpF- PA1113 TACAAAAAAGCAGGCTCAGCCATTCTCTCCTCCC This study GWL PA1113-UpR- PA1113 TCAGAGCGCTTTTGAAGCTAATTCGTTCGTAGAAACCGGGATG This study GW PA1113-DnF- PA1113 AGGAACTTCAAGATCCCCAATTCGTCGGCAGCGTCGAGGAAA This study GW PA1113-DnR- PA1113 TACAAGAAAGCTGGGTCCAGCCGCCCCTGGTCGA This study GWR PA1863-UpF- CA modA TACAAAAAAGCAGGCTCCTGCCCCAGCTATTGC GWL McDevitt PA1863-UpR- CA modA TCAGAGCGCTTTTGAAGCTAATTCGACATAGCCGGCCTTGG GW McDevitt PA1863-DnF- CA modA AGGAACTTCAAGATCCCCAATTCGCCGAAGCTGGTGGAAGG GW McDevitt PA1863-DnR- CA modA TACAAGAAAGCTGGGTGGACTTGATCAGCGCG GWR McDevitt PA1874-UpF- PA1874 TACAAAAAAGCAGGCTGCTGCCGTCGAGGTTC This study GWL PA1874-UpR- PA1874 TCAGAGCGCTTTTGAAGCTAATTCGGCTCATGCCACCGAGC This study GW PA1874-DnF- PA1874 AGGAACTTCAAGATCCCCAATTCGGCCACGCCAGTGATCA This study GW PA1874-DnR- PA1874 TACAAGAAAGCTGGGTGCCGTCGGTGAGGATCA This study GWR PA1876-UpF- PA1876 TACAAAAAAGCAGGCTACCCCGGATAACGTCGT This study GWL PA1876-UpR- PA1876 TCAGAGCGCTTTTGAAGCTAATTCGGCCGAAGAACCAGTGTT This study GW PA1876-DnF- PA1876 AGGAACTTCAAGATCCCCAATTCGCGGCAAGTCGACCCTG This study GW PA1876-DnR- PA1876 TACAAGAAAGCTGGGTCAGGGCGAGCATGCTTT This study GWR PA1875-UpF- PA1875 TACAAAAAAGCAGGCTGCGGGCGCAGGCAGTACGCG This study GWL PA1875-UpR- PA1875 TCAGAGCGCTTTTGAAGCTAATTCGCGCGCTGGCGCTGTCGAC This study GW

33 Chapter 2

PA1877-DnF- PA1877 AGGAACTTCAAGATCCCCAATTCGGATCCCGGTAGAGGAGCG This study GW PA1877-DnR- PA1877 TACAAGAAAGCTGGGTGGATGTCCACCTCGGCGAC This study GWR PA3228-UpF- PA3228 TACAAAAAAGCAGGCTTTTATCGTCGTTTCGAA This study GWL PA3228-UpR- PA3228 TCAGAGCGCTTTTGAAGCTAATTCGAGCGGTGGTTCTGCCAGC This study GW PA3228-DnF- PA3228 AGGAACTTCAAGATCCCCAATTCGCTCATGGAAGCGGTGCGC This study GW PA3228-DnR- PA3228 TACAAGAAAGCTGGGTCGAGCAACTCGGCATGCG This study GWR PA5231-UpF- PA5231 TACAAAAAAGCAGGCTACCGCTGGCGGGAGCGGC This study GWL PA5231-UpR- PA5231 TCAGAGCGCTTTTGAAGCTAATTCGGCTCGGCCTTGTCGTGG This study GW PA5231-DnF- PA5231 AGGAACTTCAAGATCCCCAATTCGCAAGCAGCTGCCCTACGT This study GW PA5231-DnR- PA5231 TACAAGAAAGCTGGGTAGGCCCAGAGATCGGAC This study GWR PA5498-UpF- PA5498 TACAAAAAAGCAGGCTCCCTGCGTCCCTTTGCCC This study GWL PA5498-UpR- PA5498 TCAGAGCGCTTTTGAAGCTAATTCGGAAATTGACGAACTTGCG This study GW PA5498-DnF- PA5498 AGGAACTTCAAGATCCCCAATTCGCTTGCCGGCAAGCCTTTC This study GW PA5498-DnR- PA5498 TACAAGAAAGCTGGGTGCGTCCACGCTCACGTTC This study GWR BA qPA16S_F 16S GGAGAAAGTGGGGGATCTTC Eijkelkamp BA qPA16S_R 16S CGTAGGAGTCTGGACCGTGT Eijkelkamp BA qPA0576_F rpoD CGATATAGCCGCTGAGGA Eijkelkamp BA qPA0576_R rpoD GAGATCGAAATCGCCAAG Eijkelkamp BA qPA0487_F modE TGTTCAGTTCGTCGATGG Eijkelkamp BA qPA0487_R modE ATGTCCGCACTCTCCACT Eijkelkamp BA qPA1846_F cti GTCCAGCTACCCGAACTTCA Eijkelkamp BA qPA1846_R cti TAGCTCCAGAACTGCGGATT Eijkelkamp BA qPA1861_F modC GTTGGCATCCTGGAACAC Eijkelkamp BA qPA1861_R modC ACTTGCCTGCGCTGTATC Eijkelkamp BA qPA1863_F modA CTTCCTGCTCCAGTTTCG Eijkelkamp BA qPA1863_R modA GCCAAGGAATTCGAGAAA Eijkelkamp BA qPA1864_F PA1864 GTAGAGTTGCGCCTTGGA Eijkelkamp BA qPA1864_R PA1864 GCAACCTGCAACTGATCC Eijkelkamp BA qPA3393_F nosD AGGCGCTGTTCATCTACAA Eijkelkamp qPA3393_R nosD TGGCGACGTACTTGACCT This study qPA3877_F narK1 CCATGTTGCTCGGGTAGT This study

34 Chapter 2 qPA3877_R narK1 ACCACCATGACCATCCAC This study qPA3918_F moaC GAGCTTGACGCTGGTGA This study qPA3918_R moaC AACCCTGCAACTGATCCA This study qPA0042_F PA0042 CAGGCAGAAGTACGTGTGGA This study qPA0042_R PA0042 GTAATAGCCGCTCTGGTTGG This study qPA0237_F PA0237 AATGTTGCTGACCAACCTGA This study qPA0237_R PA0237 GCGATGTTGTCTTCGTTGG This study qPA0534_F PA0534 ACAACCAGTTCGTCCTCGAC This study qPA0534_R PA0534 GGGAACACCCTTTCGAGATA This study qPA1502_F PA1502 GCAAGACCCACTTCGACAAC This study qPA1502_R PA1502 GGCTGGTAGACATGGAGGAA This study qPA1503_F PA1503 CGGCCTTCGTTCATTTCC This study qPA1503_R PA1503 GCGCATTGCTCGTCGATA This study qPA1569_F PA1569 GCAAGAACATCGGCAGTTTC This study qPA1569_R PA1569 CAGGTACCGAACACGCTGTA This study qPA1570_F PA1570 GTTGAAGGCTTTCCATGCAG This study qPA1570_R PA1570 TTGTTACGGTGGAACAGCAG This study qPA1738_F PA1738 GAAGGTTTCGCCAATATCGT This study qPA1738_R PA1738 CCTTGCCGTCGGAAGTAAG This study qPA1739_F PA1739 AACAGCGGCCTGAAAGTCT This study qPA1739_R PA1739 ATCGGCACTGTCGAAGAAGT This study qPA1939_F PA1939 GCCATCGGACAAGGACTATG This study qPA1939_R PA1939 GGTCGAGATCTTGAGCTTGG This study qPA2522_F PA2522 AAGCGGTACAGGCCTACGA This study qPA2522_R PA2522 AGTTGAACTTGCCCATCTCG This study qPA2523_F PA2523 GGATCAACCTGACGACCAAG This study qPA2523_R PA2523 ACTTCCACCACGTTGGAGTC This study qPA3067_F PA3067 ATGCGCTCGTCGAATCTG This study qPA3067_R PA3067 GCGGTTGCCGTAGAGATTT This study qPA4523_F PA4523 GCCTCAGCCTGGTACAGAAC This study qPA4523_R PA4523 TTGCTCTTCTCGTCGACCTT This study qPA4524_F PA4524 CCTCTACGATGCCTTCCTGA This study qPA4524_R PA4524 CATCGAGGTTCTCCACTTCC This study qPA4581_F PA4581 TGGGACTTCGAGGAGGTCTA This study qPA4581_R PA4581 GGTAAGGAGGAACCAGCAGA This study qPA4623_F PA4623 CGGTGCCCAACAACTGAT This study qPA4623_R PA4623 AAGCGATCGATGGTCTGC This study qPA4648_F PA4648 CGGCAATATCCAGATCCAGT This study qPA4648_R PA4648 GGCGTCCGAATAGATGTTGT This study

35 Chapter 2 qPA4881_F PA4881 TGAAAGCTTCCCTGATCCTC This study qPA4881_R PA4881 CAGTTCGACGCTGGACATA This study qPA5029_F PA5029 AACAACTCCACCCAATGCAC This study qPA5029_R PA5029 AGGCCTATCCGGACGTACAC This study qPA5030_F PA5030 CGATGACCTATCTCAGCGAAG This study qPA5030_R PA5030 GGAGATGAAATCGACCAGGA This study qPA5105_F PA5105 ATGTTGTGCACCTCGAACAG This study qPA5105_R PA5105 TCAGTCGCATGACCATCAAT This study qPA5106_F PA5106 GAGCTGATGTACCGGATGGT This study qPA5106_R PA5106 TCGTGATGGACGTAGTGGAA This study qPA5170_F PA5170 CTGTGGCTGACCAACATCTG This study qPA5170_R PA5170 GGACCAGAAGTACGGGATCA This study qPA5295_F PA5295 CTTCGTCCACCTCAATAGCC This study qPA5295_R PA5295 CAACCACTTCCAGGTTGAGG This study qPA5296_F PA5296 ATGAGCAACGTCTGGTTCCT This study qPA5296_R PA5296 GCCTTCCTCTTCTTCCTGCT This study qPA0860_F PA0860 TTGCTGATCCTGCTGTTC This study qPA0860_R PA0860 GCTTCTTGAGATGCTTGAC This study qPA1113_F PA1113 AATTGCTCAATCGCTACAT This study qPA1113_R PA1113 CAGGACACCAGGTAGAAG This study qPA1876_F PA1876 CAGCGAGAAACAGGTCAT This study qPA1876_R PA1876 TTCTTGTGGGTGGTGATG This study qPA3228_F PA3228 ATGCTTTATCGTCGTTTC This study qPA3228_R PA3228 GCAGGAAGTAGACATAGA This study qPA5231_F PA5231 TTTCCTCTATGTGGTCACC This study qPA5231_R PA5231 GAATACAGCGGCGATCTG This study qPA0781_F znuD GAACGCCGCTGTAGAGGTAT This study qPA0781_R znuD ACAGCCGCTTCTCCAACTAC This study qPA4063_F PA4063 CTGTTAGCCCTCGTCTTGCT This study qPA4063_R PA4063 AGCTCCAGTTCGAGGGTCTT This study qPA2435_F hmtA GATCAGCAACACCAGCAACA This study qPA2435_R hmtA GCATCGAGATAACCCGTACC This study qPA0779_F asrA GTCCAGGTAGTGGTCGAGGA This study qPA0779_R asrA GGAAGTCGAGGTGATGAACC This study qPA1927_F metE ACCCAGATCCATACGCACAT This study qPA1927_R metE GTTCGGGTAGTCGAACTGCT This study qPA3527_F pyrC GTCTTCGAGCACATCACCAC This study

36 Chapter 2 qPA3527_R pyrC TAGAAGTGCGGACGGATACC This study qPA3690_F zntA GAGTTGATCGAGGCGAAGTC This study qPA3690_R zntA GCTTCCAGTTCCACTTGCTT This study qPA5230_F PA5230 GCGACGATGTTCTGGATGTA This study qPA5230_R PA5230 TCGGCCTGTATACCTTCACC This study qPA0277_F PA0277 GTACGCCTTCGATCTCGTTG This study qPA0277_R PA0277 GTGCCGGTGAACTACAAGGT This study qPA5539_F folE2 GGAAGACTGCATCGGACT This study qPA5539_R folE2 CCTCGCAGAACATCAGGT This study qPA2911_F PA2911 GCAACCTGATCACCCTCA This study qPA2911_R PA2911 CGCTGAGCAGCTTCTTCT This study qPA1544_F anr CGATGACCAGCAGATGAT This study qPA1544_R anr GACAGGTTGACCAGGAAG This study LIC PAT7F MCS TAATACGACTCACTATAGG This study LIC PAT7R MCS GCTAGTTATTGCTCAGCGG This study

PA5498LIC1F PA5498 TGGGTGGTGGATTTCCTGAGGTCAGCGTGCTGACC This study PA5498LIC1R PA5498 TTGGAAGTATAAATTTCCGAGCTTTTCCAGACAGCC This study

37 Chapter 2

P. aeruginosa was routinely grown in a semi-synthetic cation-defined media (CDM) containing 8.45 mM Na2HPO4, 4.41 mM KH2PO4, 1.71 mM NaCl and 3.74 mM NH4Cl, supplemented with 0.5% yeast extract (Difco, Becton Dickinson, USA) and vitamins (0.2 μM biotin, 0.4 μM nicotinic acid, 0.24 μM pyridoxine-HCl, 0.15 μM thiamine-HCl, 66.4 μM riboflavin-HCl, and 0.63 μM calcium pathothenate) and Chelex-100 treated. CaCl2 and

MgSO4 were subsequently added to 0.1 mM and 2 mM, respectively. Metal concentrations of the CDM were ascertained by inductively coupled plasma-mass spectroscopy (ICP-MS), with zinc present at 800 nM, and molybdenum and tungsten both present at ≤ 10 nM. When required, cultures were supplemented with 100 mM KNO3, and 10 mM Na2MoO4 and/or

Na2WO4.

For routine P. aeruginosa growth, media with nitrate and metal supplementation, where appropriate, was inoculated to OD600 of 0.05 using overnight culture. Cells were grown to an

OD600 of 0.6 on an Innova 40R shaking incubator (Eppendorf, Germany) at 240 rpm, 37 °C. If required for anaerobic shift, culture was then transferred to a smaller sterile tube, completely filling it to minimize the amount of oxygen available. Cultures were then statically incubated with tightened lids at 37 °C for 2 h, allowing oxygen depletion and a switch to anaerobic -1 respiration. To confirm cell viability, colony forming units (CFU).mL and OD600 were determined before and after the anaerobic shift experiment, with no change observed. 24 h cultures were inoculated similarly, however lids of tubes were kept loose followed by static incubation at 37 °C for 24 h. Those for anaerobic growth were placed in a GasPak EZ anaerobic chamber with a single GasPak EZ anaerobic chamber sachet and oxygen indicator (Becton Dickinson, USA). Chamber was closed securely and placed at 37 °C for 24 h.

Bacterial strains

2.4.1. P. aeruginosa

The wild-type P. aeruginosa strain used in this study was PAO1 ATCC 15692 obtained from Prof. Herbert Schweizer (University of Colorado) (Table 2.2).

2.4.1.1. Mutagenesis

The PAO1 strain was used to generate the ΔPA0860, ΔPA1113, ΔPA1863 (ΔmodA), ΔPA1874 (bapA), ΔPA1875-7 (bapBCD), ΔPA1876 (bapC), ΔPA3228, ΔPA5231 and ΔPA5498 (ΔznuA) deletion mutants (Table 2.2) according to the protocol of Choi and Schweizer (2005) with primers listed in Table 2.1. Briefly, 300 – 500 bp regions from 38 Chapter 2

Table 2.2: Strains used in this study.

Strain Description Source/Reference P. aeruginosa Wild type, ATCC 15692 HP Schweizer PAO1 PA0860::Gm PAO1 with Gm interrupted PA0860 This study PA1113::Gm PAO1 with Gm interrupted PA1113 This study PA1874::Gm PAO1 with Gm interrupted PA1874 This study PA1876::Gm PAO1 with Gm interrupted PA1876 This study PA1875-7::Gm PAO1 with Gm interrupted PA1875-7 This study PA3228::Gm PAO1 with Gm interrupted PA3228 This study PA5231::Gm PAO1 with Gm interrupted PA5231 This study PA1863::Gm PAO1 with Gm interrupted PA1863 This study PA5498::Gm PAO1 with Gm interrupted PA5498 This study PA0860::FRT PAO1 ΔPA0860 This study (ΔPA0860) PA1113::FRT PAO1 ΔPA1113 This study (ΔPA1113) PA1874::FRT PAO1 ΔPA1874 This study (ΔPA1874) PA1876::FRT PAO1 ΔPA1876 This study (ΔPA1876) PA1875-7::FRT PAO1 ΔPA1875-7 This study (ΔPA1875-7) PA3228::FRT PAO1 ΔPA3228 This study (ΔPA3228) PA5231::FRT PAO1 ΔPA5231 This study (ΔPA5231) PA1863::FRT PAO1 ΔPA1863 or PAO1 ΔmodA This study (ΔmodA) PA5498::FRT PAO1 ΔPA5498 or PAO1 ΔznuA This study (ΔznuA) XL-10 Gold Tetr Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 ultracompetent supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F´ proAB E. coli lacIqZDM15 Tn10 (Tetr) Amy Camr]. Agilent fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS/ pLemo(CamR) Lemo21(DE3) λ DE3 = λ sBamHIo ∆EcoRI- New England E. coli B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5 Biolabs, (Wagner et pLemo = pACYC184-PrhaBAD-lysY al., 2008) F′{proAB+lacIq lacZ∆M15 Tn10(TetR) ∆(ccdAB)} OmniMAX 2-T1 mcrA ∆(mrr-hsdRMS-mcrBC) φ80(lacZ)∆M15 E. coli ∆(lacZYA-argF) U169 endA1 recA1 supE44 thi-1 Life Technologies gyrA96 relA1 tonA panD

39 Chapter 2 the 3’ and 5’ ends of the target genes were PCR amplified using primers complementary to both the target sequence and either the gentamicin resistance cassette (GmR) flanked by Flp recombinase target (FRT) sites, or a Gateway cloning compatible regions. The FRT-flanked Gm cassette was also amplified using the Gm-F and Gm-R primers (Table 2.1). An overlap extension PCR (94 C for 2 min, followed by 3 cycles of 94 C for 30 sec, 55 C for 30 sec and 68 C for 1 min, then 30 cycles of 94 C for 30 sec, 56 C for 30 sec and 68C for 5 min) was used to combine the gene-specific regions and the Gm cassette, and cap the PCR product with attB1 and attB2 sites. The overlap extension PCR product was then combined with a pDONR221 vector in a Gateway LR clonase reaction to generate the entry clone vector pDONR221-Gene::Gm. This was then added to a Gateway BP clonase reaction together with pEx18ApGW vector to produce the gene specific pEX18ApGW-Gene::Gm suicide plasmid for electroporation into P. aeruginosa PAO1.

LB broth was inoculated from a LB agar plate of PAO1, and grown to an OD600 of 1.2 on an Innova 40R shaking incubator (Eppendorf) at 240 rpm, 37 °C. Cells were pelleted by centrifugation and washed three times with 300 mM sucrose, prior to resuspension to a final

OD600 of 100. One hundred microliters of cell suspension was added to a sterile electroporation cuvette with 600 ng of gene specific pEX18APGw::gene plasmid. Cells were electroporated at 25 F, 200 Ohm, 2.5 kV on a Gene Pulser electroporation system (Biorad, USA), prior to the addition of 1 mL warm LB and transfer of the cell suspension to a 15 mL tube. Cells were placed on orbital shaker at 37 C for 1 h to recover, prior to harvest by centrifugation, resuspension in 100 L warm LB and plating on warm LB agar plates containing 30 g.mL-1 Gm. Plates were incubated at 37 C until colonies appear. Colonies were selected and streaked for single colonies on plates containing 30 g.mL-1 Gm and 5% sucrose. Colonies were subsequently patched on plates containing 30 g.mL-1 Gm and 5% sucrose, in addition to plates with 200 g.mL-1 Cb. Strains that grew on the Gm plus sucrose plates, but did not grow on the carbeniciilin plates were taken to be deletion mutants. This was confirmed by PCR of the region using isolated gDNA and the gene-specific UpF-GWL and DnR-GWR primers (Table 2.1).

To remove the gentamicin resistance cassette, deletion mutant cultures were grown during day to OD600 = 1. Following harvest and sucrose washes as described above, 100 L of

OD600 = 100 culture was added to an electroporation cuvette with 20 ng pFLP2 plasmid. Electroporation and cell recovery was performed as described above, with cells diluted 1:1000 and 1:10,000 prior to plating on LB agar plates containing 200 g.mL-1 Cb, and

40 Chapter 2 subsequently streaked on LB agar plates containing 200 g.mL-1 Cb for isolation of a single clonal population.

Genomic DNA was isolated from the unmarked deletion mutants, and the deletion confirmed by PCR and DNA sequencing across the target region using the gene-specific UpF- GWL and DnR-GWR primers (Table 2.1).

2.4.2. E. coli

Routine DNA manipulation was performed in the XL-10 GOLD ultracompetent E. coli strain (Agilent, Australia) (Table 2.2). ZnuA protein expression was performed using the T7 RNA polymerase based pCAMcLIC01-znuA expression vector (Table 2.3) in Lemo21(DE3) competent cells (New England Biolabs, USA) (Table 2.2).

2.4.2.1. Generation of chemically competent cells

Lemo21(DE3) and XL-10 GOLD chemically competent cells were generated for bacterial transformation. A single colony of XL-10 GOLD cells was inoculated in 20 mL LB broth with 20 mM MgSO4 and grown for 8 h in an Innova 40R shaking incubator (Eppendorf) at 180 rpm, 37 °C. Culture was then diluted into 500 mL LB with 20 mM MgSO4 and grown overnight in an Innova 40R shaking incubator (Eppendorf) at 180 rpm, 20 °C to an OD600 of 0.5-0.7. Culture was incubated on ice for 10 min, prior to harvest in a pre-chilled centrifuge at 2250 x g, 4 °C for 10 min. Cells were gently resuspended by swirling in 80 mL of cold, sterile transformation buffer containing 10 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),

15 mM CaCl2, 250 mM KCl and 55 mM MnCl2. Cells were incubated on ice for a further 10 min, prior to harvest and resuspension in 20 mL transformation buffer plus 2.5 mL dimethyl sulfoxide (DMSO). Cells were incubated for a further 10 min on ice prior to aliquotting, flash freezing of cells and storage at -80 C. Generation of Lemo21(DE3) cells were performed essentially as described above, with the exception that 30 g.mL-1 chloramphenicol was present in all cultures to maintain the pLemo plasmid, and the 500 mL of LB was inoculated with an overnight 10 mL seed culture and grown throughout the day at 22 C in an Innova 40R shaking incubator (Eppendorf) at 180 rpm.

2.4.2.2. Transformation of E. coli

Approximately 200 ng of plasmid DNA was added to 100 L E. coli chemically competent cells and incubated on ice for 30 min. Cells were heat-shocked at 42 C for 20 sec on a dry heat block (Labtek, Australia). Cells were replaced on ice, prior to the addition of 1 mL warm LB broth and placement in an Innova 40R shaking incubator (Eppendorf) at 37 C, 41 Chapter 2

Table 2.3: Plasmids used in this study.

Plasmid Description Source pCAMcLIC01 KanR, ligation independent cloning CA McDevitt expression vector encoding a C- terminal dodecahisitidine tag pCAMcLIC01-ZnuA KanR, znuA gene lacking the signal This study sequence cloned in pCAMcLIC01 pEX18ApGw AmpR, ChlR ccdB, sacB Gateway HP Schweizer (Accession number compatible suicide vector AY928469) pDONR 221 KanR, ccdB, Gateway donor vector Life Technologies KanR, confers resistance to 50 g.mL-1 kanamycin; AmpR, confers resistance to 100 g.mL-1 ampicillin; ChlR confers resistance to 30 g.mL-1 chloramphenicol.

42 Chapter 2

240 rpm for 1 h to recover. One hundred microliters of culture was then spread on an 1.5% agar LB plate containing 50 g.mL-1 Km, 100 L.mL-1 Ap or 30 g.mL-1 Chl where required, and incubated at 37 C overnight. Colonies were selected and streaked for a clonal population.

Phenotypic analyses

2.5.1. Growth kinetics assay

Growth analysis of PAO1 and the ΔznuA strain was performed in the absence or presence of different concentrations of the preferential zinc chelator, N,N,N′,N′-Tetrakis(2- pyridylmethyl)ethylenediamine (TPEN). Overnight cultures were grown in biological duplicate in CDM or CDM supplemented with 10 M TPEN. For assay, cultures were diluted to a final OD600 of 0.05 in 150 L fresh CDM without supplementation, or with 5 M or 10 M TPEN. The flat bottom 96-well microtitre plate (Costar) was covered with a breathable seal and incubated in a FluoSTAR spectrophotometer (BMG Labtech, Germany) at 37 C, for

9 h. OD600 well-scanning measurements were taken even 30 min, with double orbital shaking of the plate at 500 rpm between each read to ensure good aeration.

2.5.2. Biofilm formation

Planktonic cells and media were discarded from 24 h static cultures grown in 96-well trays (Costar, Corning, USA) from an initial OD600 of 0.05. Biofilm cells were washed twice in tap water, and stained with 0.1% crystal violet for 10 min. Crystal violet was then discarded, followed by four washes of the stained cells in tap water, and then left to dry. To determine biofilm formation 30% acetic acid was added and allowed to solubilize the biofilm for 10 min after which 150 μL was transferred to another 96-well microtitre plate (Costar,

Corning, USA) for analysis by well scanning at OD595 using a PHERAstar FS Spectrophotometer (BMG Labtech).

Biofilm analysis for the ModA study was performed with some variation on the above protocol. Planktonic cells and media were discarded from 5 mL 24 h static cultures, which were prepared as described above. Biofilm cells were washed twice in tap water, and stained with 0.1% crystal violet for 10 min. Crystal violet was then discarded, followed by four washes of the stained cells in tap water, and then left to dry. To determine relative biofilm formation, with the exclusion of the pellicle or air-liquid interface biofilm, 3 mL 30% acetic

43 Chapter 2 acid was carefully added and allowed to solubilize the biofilm for 10 min, after which 200 μL was transferred to a 96-well microtitre plate (Costar) for analysis by well scanning at OD595 using a PHERAstar FS Spectrophotometer (BMG Labtech).

2.5.3. Whole cell metal accumulation

20 mL of P. aeruginosa was grown in a 50 mL tube to an OD600 of 0.6 at 37 °C, on an Innova 40R shaking incubator at 240 rpm. The bacteria were then washed, by resuspension and centrifugation at 7,000 × g for 8 min, three times with phosphate-buffered saline (PBS) containing 5 mM ethylenediaminetetraacetic acid (EDTA), and then six times with PBS. Bacterial pellets were desiccated at 95 °C overnight. The dry cell weight was measured and the pellets resuspended in 35% HNO3 and boiled at 95 °C for 1 h prior to removal of debris by centrifugation. Samples were diluted to a final concentration of 3.5% HNO3 and analyzed by ICP-MS on an Agilent 7500cx ICP-MS (Adelaide Microscopy, University of Adelaide) (McDevitt et al., 2011)

2.5.4. Minimal inhibitory concentration (MIC) analysis

The antibiotic resistance profile of the ABC efflux protein mutants was assessed via the broth micro-dilution method in Luria Bertani and cation-adjusted Mueller Hinton media. Ten serial 1 in 2 dilutions of antibiotics were setup across a 96-well plate. Cell stocks stored at -

80C were diluted and added to the well at a final OD600 of 0.05. Plates were covered with a breathable seal, placed in a humid box and incubated at 37 C for 22 h. Growth inhibition was determined via absorbance at 600 nm using a Spectramax M2 spectrophotmeter (Molecular Devices, USA). The assay was performed on ≥ 2 separate occasions using biological duplicates.

MIC analysis of the wild-type and ΔznuA strain was assessed essentially as described in Wiegand et al. (2008), with the assay performed in a total volume of 100 L using a final inoculum of 0.5 x 105 cells per well. As the assay sought to determine the effect of zinc deficiency on the MIC values through use of the znuA strain, MIC assay was performed in CDM rather than the more metal-replete cation-adjusted Mueller-Hinton broth. To ensure appropriate MIC values for aminoglycosides, the media was checked for sufficient concentrations of Mg2+ and Ca2+, and consequently MIC analysis for the aminoglycosides was also performed in CDM supplemented with Ca2+ at a final concentration of 20 mg.mL-1.

Growth inhibition was determined by both visual inspection and OD600 well-scanning using a

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SpectroSTAR spectrophotometer (BMG Labtech). The assay was performed on three separate occasions using biological duplicates.

2.5.5. Fatty acid analysis

Strains for fatty acid analysis were grown statically for 24 h as described above. Total cellular lipids were extracted using chloroform/isopropanol and were subsequently methylated using a 1% sulfuric acid solution in methanol. The fatty acid methyl esters were analyzed by gas chromatography at the School of Agriculture, Food and Wine, University of Adelaide, as previously described (Eijkelkamp et al., 2013).

2.5.6. Bacterial adherence to hydrocarbons (BATH) assay

Twenty-four hour or OD600 of 0.6 BATH assay cultures were prepared for the ModA and ZnuA studies, respectively. Cells were harvested by centrifugation at 2250 x g, 25 °C for 20 min. Cell pellets were resuspended in potassium urea magnesium (PUM) buffer (127 mM

K2HPO4, 53 mM KH2PO4, 29.97 mM urea, 0.8 mM MgSO4.7H2O) for 2 x 5 mL washes.

Cells were resuspended in 5 mL PUM buffer prior to dilution to 6 mL of OD600 = 0.25. Three millilitres xylene was added to each sample, vortexed for 30 sec and allowed to separate over the course of 5 min. Glass Pasteur pipettes were used to extract 1 mL of the hydrophilic layer to determine OD600. Hydrophobicity (association with xylene) was expressed as a percentage, as calculated using the following formula:

푂퐷 표푓 ℎ푦푑푟표푝ℎ𝑖푙𝑖푐 푝ℎ푎푠푒 % 퐻푦푑푟표푝ℎ표푏𝑖푐𝑖푡푦 = 100 − ( 600 ) 푇표푡푎푙 푂퐷600

2.5.7. Volatile compound (VOC) headspace analysis

100 μL of overnight culture grown in LB broth was spread on 1.5% agar LB plates. Plates were placed in 3 L Tedlar bags (company) and heat-sealed. Air was removed from bags, prior to the addition of ~ 600 mL of laminar flow air. Bags were placed at 37 °C and incubated for 24 h. After incubation, a bacterial lawn was present on plates. A new 1 L Tedlar bag was connected to each Tedlar bag containing the plates and air was moved from the growth bag into the collection bag and the valve sealed. Headspace samples were analysed on a MS-Syft selected-ion flow mass spectrometry (Syft Technologies, New Zealand) at the University of South Australia. Sequential 15 sec injections were analysed for volatile compounds according to scan parameters.

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2.5.8. Scanning electron microscopy analysis

Following routine bacterial growth, P. aeruginosa were then washed once, by resuspension and centrifugation at 7,000 × g for 8 min with sterile phosphate-buffered saline (PBS). Cells were resuspended in 5 mL sterile PBS. Cells were transferred to a 0.2 μm nitrocellulose filter membrane using a 5 mL syringe. The membrane was subsequently placed with the cell-side up into 2 mL of electron microscopy fixative (4% paraformaldehyde, 1.25% glutaraldehyde in PBS and 4% sucrose, pH 7.2). Cells were fixed for 30 min. Fixed cells were incubated in washing buffer (PBS, 4% sucrose) for 5 min to remove excess fixative, prior to incubation in 2% OsO4 prepared in MQ for 30 min. Samples were then dehydrated with sequential ethanol washes: 10 min in 70% ethanol, 10 min in 90% ethanol and three times 10 min in 100% ethanol. Membrane-fixed cells were then dried using a Tousimis 931 series Critical Point Dryer (Tousimis, USA), and subsequently mounted on an aluminium stub with conductive graphite paint. Samples were then graphite coated prior to analysis on a Philips XL30 field emission guns SEM (Philips, The Netherlands) with energy dispersive X-ray spectroscopy (EDAX, USA), cryogenic stage (Oxford Cryosystems, UK), and electron backscatter detection (Oxford Instruments, UK).

ZnuA biochemical characterisation

2.6.1. Cloning and expression of ZnuA

Recombinant ZnuA was generated by PCR amplification of P. aeruginosa PAO1 znuA (PA5498) gene with the exclusion of the region encoding the signal sequence, using ligation- independent cloning. The region of interest was PCR amplified using the PA5498LIC1F and PA5498LIC1R primers listed in Table 2.1 according to the following cycling parameters: 95 C for 2 min followed by 20 cycles of 95 C for 30 sec, 52 C for 30 sec and 68 C for 2 min. The amplified fragment was then purified by gel electrophoresis prior to insertion into a C- terminal dodecahistidine tag-containing vector, pCAMcLIC01, to generate pCAMcLIC01- ZnuA (Table 2.3). The integrity of the construct was confirmed by DNA sequencing across the region of interest. Protein expression was performed in E. coli LEMO21(DE3), by growing the cells in an autoinducing TB medium (Overnight Express, Merck, USA) using UltraYield Flasks (Thomson Instrument Company, USA) for 18 h at 27 °C, on an Innova 44R shaking incubator at 215 rpm. Cells were harvested and pellets frozen at -80 °C until required.

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2.6.2. Purification of ZnuA

Expression cell pellets were defrosted, then resuspended in 50 mM 3-(N- morpholino)propanesulfonic acid (MOPS) (pH 7.3), 400 mM NaCl, 20% glycerol buffer, and 15 mM imidazole, and disrupted at 30 kPSI by a Constant Systems cell disruptor, and the soluble supernatant isolated by centrifugation at 4 °C for 60 min at 120,000 × g. Purification of ZnuA was achieved by a Histrap HP column (GE Healthcare, UK) on an ÄKTA Purifier (GE Healthcare) using a binding buffer containing 50 mM MOPS (pH 7.3), 400 mM NaCl, 20% glycerol, and 15 mM imidazole, and an elution buffer containing 50 mM MOPS (pH 6.6), 400 mM NaCl, 20% glycerol and 1 M imidazole. ZnuA was then desalted on a HiPrep 26/10 Desalting column (GE Healthcare) into the binding buffer, and the oligomeric state determined by gel permeation chromatography in a buffer containing 50 mM MOPS (pH 7.3), 100 mM NaCl and 20% glycerol on a Superdex 200 10/30 column (GE Healthcare) using an ÄKTA Purifier (GE Healthcare). Thryoglobulin (669 kDa), Apoferritin (443 kDa), -amylase (200 kDa), alcohol dehydrogenase (150 kDa) albumin (66 kDa), and carbonic anhydrase (29 kDa) were used as molecular weight standards, with a blue dextran standard (2 MDa) used to determine the void volume (Sigma-Aldrich). ZnuA purity was assessed via analysis of IMAC and GPC fractions on a 4-12% Bis-Tris polyacrylamide gel (Life Technologies), in a 50 mM MES (pH 7.3), 50 mM Tris Base, 0.1% SDS, and 1 mM EDTA running buffer at 200 V for 35 min. Protein was visualized via staining with Page Blue Protein stain (ThermoFisher Scientific, Australia).

ZnuA yield was determined using the ZnuA molar extinction coefficient (21555 M-1 cm-1), pathlength correction of 0.588, and the sample absorbance at 280 nm as measured on the PHERAstar FS Spectrophotometer (BMG Labtech):

A (sample) − A (blank) Concentration (M) = ( 280 280 ) Molar extinction coefficient (ε) × pathlength correction

Prior to biochemical characterization of ZnuA, the dodecahistidine tag was removed. Purified ZnuA was mixed with purified 3C HRV protease at a ratio of 5:1 for 1 h at 4 °C. Untagged ZnuA was then purified by binding of the 3C protease and uncleaved ZnuA to a Histrap HP column (GE Healthcare) on an ÄKTA Purifier (GE Healthcare), and collection of the untagged ZnuA in a binding buffer containing 50 mM MOPS (pH 7.3), 400 mM NaCl, 20% glycerol, and 15 mM imidazole.

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2.6.3. ZnuA zinc binding assays

Zinc loading assays were performed on 3C cleaved ZnuA (20 μM) by mixing with 10- 2+ fold molar excess Zn (200 μM ZnSO4) in a total volume of 2 ml into the binding assay buffer (50 mM MOPS (pH 7.3), 400 mM NaCl, 20% glycerol, and 15 mM imidazole) for 2 h at 4 °C. Unbound metal was removed by desalting on a PD10 column (GE Healthcare) into the binding assay buffer and the protein concentration was determined. 1.4-2 μM solutions of metal-loaded protein were prepared in 3.5% HNO3 and boiled for 15 min at 95 °C. Samples were then cooled and centrifuged for 20 min at 14,000 x g. The supernatant was then analyzed by ICP-MS (Adelaide Microscopy, University of Adelaide) and the protein-to-metal ratio determined.

2.6.4. PAR assay

The affinity of ZnuA for zinc was determined via a competitive assay with 4-(2- pyridylazo)resorcinol (PAR) (Hunt et al., 1985). Each replicate contained 128 M PAR, 8 M zinc, 50 mM MOPS (pH 7.3), 100 mM NaCl, and 20% glycerol, with the excess of PAR allowing the exclusive formation of the Zn(PAR)2 complex. Sequential additions of EDTA- treated ZnuA resulted in the of zinc from PAR, and a corresponding with a change in absorbance at 500 nm. The affinity of ZnuA for zinc (10 nM) was determined in GraphPad Prism 6 using a dose-response inhibition curve of non-linear fit.

Bioinformatic analyses

2.7.1. Identification of transcriptional regulatory sites

The 15 putative γ-proteobacterial ModE binding sites, as determined in (Studholme & Pau, 2003), and the sequences of the P. protegens Pf-5 Zur motif (Lim et al., 2013) were separately aligned using ClustalW2 (Larkin et al., 2007) and subsequent weight matrices were generated using HMMER 2.0 as an integral tool in UGENE (Okonechnikov et al., 2012). The ModE binding sites identified in the genome of P. aeruginosa strain PAO1 with an E-value <0.01 (n = 15) underwent iterative HMMER 2.0 analysis. Optimization of the P. aeruginosa PAO1 ModE binding site motif was completed when no new sites with an E-value <0.001 were identified. Similarly, iterative HMMER 2.0 analysis was performed to identify the putative P. aeruginosa PAO1 Zur-binding sites. Zur-binding sites were identified on the basis

48 Chapter 2 of their intergenic nature, up-regulation ≥ 2-fold in response to zinc limitation and E-value of ≤ 0.002.

2.7.2. Generation of Weblogos

WebLogos were generated using the WebLogo application (Version 3) (Crooks et al., 2004). The ModE logo was generated using the 20 predicted PAO1 ModE binding sites with an E-value of <0.01. The Zur logo was generated using the sequences which had an E-value of ≤ 0.002. Sequence alignment of the 18 repeats of 82 amino acids in PA1874 was also used to generate a WebLogo depicting sequence repetition within the protein.

Transcriptional analyses

2.8.1. RNA isolation

Cells were grown aerobically or statically for 24 h as detailed above, then harvested at 7000 x g, for 8 min, 4 °C and lysed in 1 mL Trizol reagent (Life Technologies, USA) and 200 L chloroform. Following phase separation by centrifugation at 16,000 x g for 30 min, 4 °C, RNA was isolated from the aqueous phase using a PureLink RNA Mini Kit (Life Technologies). DNaseI treatment was performed on 1 μg total RNA using 100 units of recombinant RNase-free DNaseI (Roche, Germany) at 37 °C for 15 min, prior to inactivation of the enzyme by the addition of ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, pH 8.0) to a final concentration of 2 mM, and incubation at 65 °C for 10 min. Samples were analyzed by qRT-PCR or stored at -80 °C until required.

For analysis of P. aeruginosa cultures that underwent a shock antibiotic treatment, overnight cultures of PAO1 were diluted in fresh CDM to OD600 of 0.05, and then grown to 0.6 as detailed above. Samples were taken to determine CFU per milliliter prior to the addition of antibiotics at a 1 in 1000 dilution to give a final concentration of 25% of the MIC. Cells were grown for a further 30 min as before. Samples were taken to determine the CFU prior to harvest of cells by centrifugation and isolation of RNA as described above. Comparison of the CFU before and after antibiotic treatment showed negligable cell death occurred as a result of the shock antibiotic treatment.

2.8.2. qRT-PCR

For transcriptional analysis of OD600 0.6 aerobic, antibiotic shock treatment cultures or anaerobic shift cultures, qRT-PCR was performed using a two-step method as previously described (Eijkelkamp et al., 2014; Plumptre et al., 2014a). Briefly, cDNA was synthesized

49 Chapter 2 using random hexamers (Sigma-Aldrich) and Moloney murine leukaemia virus RNaseH minus point mutant (M-MLV, RNaseH minus) reverse transcriptase (Promega, USA), as per the manufacturer’s protocol. Quantitative PCR was performed on a LightCycler 480 (Roche) using DyNAmo ColorFlash SYBR Green qPCR mix (ThermoFisher Scientific). For analysis of RNA from 24 h cultures, qRT-PCR was performed on a LightCycler 480 (Roche) using SuperScript III Platinum SYBR One-Step qRT-PCR Kit (Life Technologies). Oligonucleotides used in this study were designed using Primer3 integrated within UGENE v1.11.4 (Unipro) (Okonechnikov et al., 2012) and are listed in Table 2.1. 16S rRNA and the constitutively expressed sigma factor gene rpoD (PA0576) were used as controls to normalize gene expression.

2.8.3. RNA sequencing

RNA isolated from biological triplicates of wild-type PAO1 and ΔznuA strains was pooled and submitted to the Adelaide Microarray Centre (University of Adelaide) for sequencing. Briefly, the Epicentre Bacterial Ribozero Kit (Illumina, USA) was used to reduce the ribosomal RNA content of the total RNA pool, followed by use of the Ultra Directional RNA kit (New England Biolabs, USA) to generate the barcoded libraries. Prepared libraries were then sequenced using the Illumina HiSeq2500 with Version 3 SBS reagents using 2x100 bp paired-end chemistry. Reads were aligned to the P. aeruginosa PAO1 genome (GenBank accession number AE004091.2) (Stover et al., 2000) using BOWTIE2 version 2.2.3 (Langmead & Salzberg, 2012). Counts for each gene were obtained with the aid of Samtools (v 0.1.18) (Li et al., 2009) and Bedtools (Quinlan & Hall, 2010), and the differential gene expression was examined using DESeq (Anders & Huber, 2010); the data have been submitted to GEO (accession number GSE60177).

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CHAPTER 3:

THE ROLE OF ABC EFFLUX PROTEINS

IN P. AERUGINOSA

51 Chapter 3

Introduction

ABC transporters are present in all forms of life. The ABC efflux proteins are central to the extrusion of allocrite (or cargo) molecules across cellular membranes. This process is coupled to the hydrolysis of ATP, which enables the efficient translocation of allocrites against their concentration gradient. In this way, ABC exporters provide an efficient means to extrude compounds that are toxic, unwanted, or in excess from a cell. In addition, ABC efflux proteins also serve an essential role in the secretion of specific proteins, compounds and metabolites for their extra-cytoplasmic function. As one component of their cellular role, ABC efflux transporters provide resistance to drug and antibiotic challenge via active efflux. The contribution of ABC exporters to multidrug resistance is well established in humans, wherein they significantly contribute to chemotherapeutic and drug resistance (Kim et al., 1998; Lee et al., 1998; Gottesman et al., 2002; Sharom, 2008). However, their relative contribution to prokaryotic drug resistance has been studied to a lesser extent.

Analysis of prokaryotic genomes reveals a high proportion of genes that are predicted to encode proteins with a canonical ABC exporter architecture. Although ABC exporters have many potential functions, it was predicted that one or more of these prokaryotic ABC exporters could also facilitate drug efflux. Eubacterial ABC transporter-mediated drug efflux was first demonstrated by LmrA, a homodimeric ABC efflux protein from Lactococcus lactis (van Veen et al., 1996). Since then several bacterial ABC exporters have shown drug efflux capabilities in in vitro settings, including BmrA (Steinfels et al., 2004), LmrCD (Lubelski et al., 2006) and Sav1866 (Velamakanni et al., 2008). This drug efflux capacity exhibited by certain bacterial ABC export proteins, together with their functional similarity to the multi- drug resistance transporters in humans, has led to ABC exporters being associated with the ever-increasing rates of bacterial antibiotic resistance. However, to date, only a relatively small proportion of bacterial ABC exporters have been shown to actively efflux antimicrobial compounds in physiologically relevant settings. These include the heterodimeric ABC efflux proteins SatAB from S. suis, PatAB from S. pneumoniae, and EfrAB from Enterococcus faecelis (Lee et al., 2003; Escudero et al., 2011; Boncoeur et al., 2012). Other examples include the heterodimeric ABC exporters YheIH, and DrrAB. Expression of the YheIH transporter in B. subtillis was shown to be induced following exposure of the bacterium to a range of structurally diverse antibiotics, implicating a role in xenobiotic efflux (Torres et al., 2009). By contrast, the DrrAB transporter, from the soil bacterium Streptomyces peucetius, was demonstrated to export the antibiotic rifampicin, in addition to its native allocrites, doxorubicin and danorubicin, which are used as chemotherapeutic agents (Li et al., 2014). 52 Chapter 3

The majority of ABC exporters associated with antibiotic efflux, to date, are present in Gram- positive bacteria. As a consequence, the contribution of ABC exporters to antibiotic resistance in Gram-negative organisms remains poorly defined, with only two examples available: the homodimeric ABC exporter VcaM from an environmental non-O1 strain, and MacB from E. coli (Kobayashi et al., 2001; Huda et al., 2003). MacB is an atypical ABC efflux protein, possessing only four transmembrane helices per transmembrane domain (rather than six) (Kobayashi et al., 2003) and functioning together with the membrane-fusion protein MacA, and the general outer membrane protein, TolC, thereby facilitating secretion across the outer membrane (Kobayashi et al., 2001). Together the MacAB-TolC complex confers resistance to macrolide antibiotics. However, more recently it has been assigned a physiological role in secretion of rough-LPS and similar glycolipids to the outer membrane (Lu & Zgurskaya, 2013). This suggests that whilst ABC efflux proteins may exhibit efflux of multiple drugs or antibiotics, this role may be opportunistic, rather than their primary physiological function. However, the contribution, if any, of ABC efflux transporters in P. aeruginosa PAO1 to antibiotic resistance remains unknown.

P. aeruginosa infections of immune-compromised individuals are notoriously difficult and often impossible to clear. This is despite intensive antibiotic treatment with antimicrobials from a range of structural and functional families. The barrage of antibiotic resistance mechanisms employed by P. aeruginosa is diverse, with active efflux having a crucial role in this defence (Mesaros et al., 2007). To date, the Mex RND tripartite pump complexes (MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM) have been characterised as the major contributors to P. aeruginosa antibiotic efflux (Morita et al., 2001; Okamoto et al., 2002; Aeschlimann, 2003; Morita et al., 2012; Morita et al., 2013). However, based on the ever-increasing contribution of ABC exporters to drug resistance in a range of bacteria, together with the number of uncharacterised ABC exporters in the PAO1 genome which share homology with MDR-associated ABC efflux proteins, it is possible that that one or more of these proteins may contribute to antibiotic resistance. Whether this proposed efflux would be a primary role for the transporters, or secondary to a physiological function, remains to be determined. Herein, the contribution of the five putative uncharacterised ABC exporters (PA0860, PA1113, PA1876, PA3228 and PA5231) to antibiotic resistance is determined. Furthermore, bioinformatic analysis provides substantial insight into the physiological function and importance of these putative ABC efflux proteins to P. aeruginosa.

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Results

3.2.1. Identification of uncharacterised ABC transporters in P. aeruginosa PAO1

The general architecture of bacterial ABC efflux proteins is well conserved. Typically, bacterial ABC efflux proteins are encoded as fusion proteins of two domains: a NBD containing an ABC signature motif, and a TMD of up to six TM helices (Davidson et al., 2008). Bioinformatic analysis of the P. aeruginosa PAO1 genome indicated the presence of five uncharacterised genes that encode proteins with an architecture homologous with ABC exporters: PA0860, PA1113, PA1876, PA3228 and PA5231 (Appendix 1). Primary sequence analysis of each gene product using the BLAST algorithm, PSORTb 3.0 and manual annotation, revealed the presence of a NBD, which included the ABC signature motif, Walker A, Walker B and Q-, H- and D-loops, in addition to a TMD of up to six predicted TM helices (Altschul et al., 1990; Yu et al., 2010). Intriguingly, PA5231 featured two N-terminal NBDs and a C-terminal TMD. To exhibit ABC efflux protein functionality, it would be predicted that PA5231 would interact with a protein comprised of a lone TMD, thereby achieving the four domains required for export. PA1876 is also atypical in that it features an additional N- terminal domain homologous to the C39-like domain of the HlyB ABC transporter (Lecher et al., 2012). Subsequent analysis using the PSORTb 3.0 and LipoP 2.0 algorithms predicted the five putative ABC efflux proteins to localise to the inner membrane for their respective function(s) (Juncker et al., 2003; Yu et al., 2010). The fused NBD-TMD arrangement within a single polypeptide, together with the lack of proximal SBP gene, indicates each of the genes of interest to encode an ABC efflux protein (Davidson et al., 2008). Although, it cannot be completely excluded that these ABC transporters could be uptake pathways, to date there are no known ABC permeases with this gene organisation.

3.2.2. Generation of the putative ABC efflux protein deletion mutants

To investigate the potential capacity of these ABC transporters to contribute to antibiotic efflux, unmarked deletion mutants (gene::FRT) in the P. aeruginosa PAO1 background were generated. This was performed using the method established by Choi et al. (2005) (Figure 3.1). Briefly, for each gene of interest, a 300 - 500 bp region from the 5’ and 3’ regions of the gene was amplified, prior to overlap extension PCR and the use of Gateway cloning technology (Hartley et al., 2000) to produce the gene-specific pEX18ApGW::Gm suicide plasmids. The gene-specific pEX18ApGW::Gm plasmids were then electroporated

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Figure 3.1: Protocol for P. aeruginosa mutagenesis. This schematic diagram, adapted from Choi et al. (2005), outlines the protocol for generation of unmarked deletion mutants in P. aeruginosa PAO1. Initially, a mutant PCR product is generated by overlap extension PCR. In the first round of PCR (PCR 1), a gentamicin (Gm) resistance cassette flanked by FLP recombinase target (FRT) sites is amplified using GmF and GmR primers. Simultaneously, 5’ and 3’ regions of the gene of interest are amplified using gene-specific UpF-GWL, UpR-Gm, DnF-Gm, and DnR-GWR primers that feature extensions to allow overlap assembly with the amplified Gm fragment. In the second round of PCR (PCR 2), the three products of PCR 1 are combined and undergo three cycles in the absence of primers for annealing of overlapping regions. GW-attB1 and GW-attB2 primers are then added to amplify the assembled fragments over 25 cycles, generating the final overlap extension PCR product capped at each end by attB sites. Following purification, the overlap extension PCR product is combined with the pDONR 221 Gateway plasmid in an LR clonase reaction. The resulting pDONR 221-Gene::Gm is then combined with pEx18ApGW in a BP clonase reaction to yield the gene-specific suicide plasmid, pEx18ApGW-Gene::Gm. Electroporation of pEx18ApGW-Gene::Gm into P. aeruginosa, allows homologous recombination with the wild-type gene. Following isolation of the deletion mutant, a pFLP2 plasmid is electroporated into the mutant strain, allowing excision of the Gm cassette. This results in an unmarked deletion mutant with a neutral 85 bp scar.

56 Chapter 3 into PAO1, allowing homologous recombination with the target gene and insertion into the chromosome. Replacement of the wild-type gene with the Gm-interrupted copy (gene::Gm) was confirmed by PCR, prior to electroporation of the deletion strain with a pFLP2 plasmid, facilitating Flp recombinase-mediated excision of the Gm resistance cassette and generation of an unmarked deletion mutant (gene::FRT). The sequential interruption of the ABC exporter genes of interest, and the removal of the Gm resistance cassette to generate the unmarked deletion mutants was validated at each step by PCR (Figure 3.2), with the final mutants also confirmed by DNA sequencing across the region of interest. In contrast to the deletion of P. aeruginosa msbA (Ghanei et al., 2007), deletion of the predicted ABC exporter genes was non-lethal, indicating the genes do not hold roles essential to cell viability.

3.2.3. The ABC exporters of P. aeruginosa do not significantly contribute to antibiotic

resistance

Bacterial ABC efflux proteins have been widely implicated in contributing to antibiotic resistance, whether as their primary function or as an ancillary role. However, the capacity of bacterial ABC transporters to efflux antibiotics has predominantly been shown in Gram- positive bacteria (Lee et al., 2003; Escudero et al., 2011; Boncoeur et al., 2012). To assess whether the uncharacterised P. aeruginosa ABC exporters contribute to antibiotic resistance, analysis of the minimal inhibitory concentration (MIC) of a range of common anti- pseudomonal antibiotics was performed (Table 3.1). Analysis was carried out in LB broth using a broth micro-dilution method based on the Clinical and Laboratory Standards Institute guidelines (Wikler & Clinical Laboratory Standards Institute, 2006). No significant changes (>2-fold) in the MICs were observed for any of the ABC exporter deletion mutants relative to the wild-type PAO1 strain. Due to the requirement of Mg2+ and Ca2+ ions for Pseudomonas aminoglycoside MIC testing (D'Amato R et al., 1975; Wiegand et al., 2008), tobramycin and gentamycin were also tested against each of the strains in cation-adjusted Mueller Hinton-II broth, with no alteration in MIC observed between the PAO1 and mutant strains.

To complement the MIC analysis, the relative expression of each transporter was quantified in response to a shock antibiotic treatment. Wild-type P. aeruginosa PAO1 underwent a 30 min shock treatment with 25% of the antibiotic MIC as determined above. Relative mRNA levels of the predicted ABC exporters as assessed by qRT-PCR revealed a lack of antibiotic-induced ABC transporter gene expression, with none of the antibiotics tested stimulating an increase in expression greater than 2-fold (Figure 3.3). Taken together,

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Figure 3.2: Mutagenesis of uncharacterised P. aeruginosa ABC exporters. Agarose gel of the PCR amplification of the wild type ABC exporter genes (PA0860, 1791 bp; PA1113, 1767 bp; PA1876, 2172 bp; PA3228, 1833 bp; PA5231, 2751 bp) gentamicin- interrupted genes (PA0860::Gm, 1956 bp; PA1113::Gm, 1860 bp; PA1876::Gm, 2086 bp; PA3228::Gm, 1870 bp; PA5231::Gm, 1860 bp), and final unmarked ABC exporter deletion mutants (PA0860::FRT, 883 bp; PA1113::FRT, 892 bp; PA1876::FRT, 1118 bp; PA3228::FRT, 865 bp bp; PA5231::FRT, 892 bp). The Gm cassette is 1053 bp. Molecular weight marker was used to determine PCR product size (bp).

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Table 3.1: Minimal inhibitory concentration (g.mL-1) of common anti-pseudomonal antibiotics.

Antibiotic Antibiotic class Cellular target PAO1 PA0860::FRT PA1113::FRT PA1876::FRT PA3228::FRT PA5231::FRT Tobramycin aminoglycoside protein synthesis 3.125 3.125 1.563 3.125 1.563 1.563 Gentamicin aminoglycoside protein synthesis 3.125 6.250 1.563 3.125 3.125 1.563 Ciprofloxacin fluoroquinalone DNA replication 0.098 0.098 0.098 0.195 0.195 0.098 Ceftazidime peptidoglycan β-lactam 50.000 50.000 50.000 50.000 50.000 50.000 hydrate synthesis Colistin sulfate polymyxin cell wall 3.125 3.125 6.250 3.125 1.563 3.125 peptidoglycan Piperacillin β-lactam 20.000 10.000 10.000 10.000 10.000 20.000 synthesis

Chapter 3

Figure 3.3: Differential expression of ABC transporters in response to antibiotic treatment. Relative mRNA levels, corrected for rpoD of (A) PA0860, (B) PA1113, (C) PA1876, (D) PA3228 and (E) PA5231 ABC exporters were analysed by qRT-PCR following a shock treatment with 25% of the MIC of common anti-pseudomonal antibiotics. Data are the mean ± s.e.m., where n ≥ 2. Broken line indicates threshold of 2-fold change in expression. 60 Chapter 3 these data suggest that the uncharacterised ABC exporters of P. aeruginosa do not contribute significantly to the bacterium’s antibiotic resistance. Although they may perform non-specific efflux in the presence of high antibiotic concentrations, or confer significant resistance to antimicrobials when over-expressed, the transporters are likely to exhibit other primary functions.

3.2.4. Predicted physiological roles of the P. aeruginosa ABC exporters

To aid in co-ordinated transcriptional control, proteins secreted by ABC transporters or those that are required under similar conditions are commonly co-localised with the transporter genes. This has been observed with the previously characterised ABC exporters of P. aeruginosa (Letoffe et al., 1998; Duong et al., 2001; Imperi et al., 2009). Here, analysis of the ABC exporter genes and their genomic context was undertaken to provide an indication of their putative functional roles.

3.2.4.1. Putative function of PA0860

PA0860 is present in a putative with the uncharacterised genes, PA0858 and PA0859 (Figure 3.4). Sequence analysis indicated the co-transcribed PA0858 gene encoded a homolog of a cytoplasmic protein with rhodanese-related sulfurtransferase activity (Altschul et al., 1990). Sulfurtransferases have been speculated to hold a range of roles, with the rhodaneses being a specific type of sulfurtransferase that mobilise the sulfane sulfur from thiosulfate to cyanide, thereby forming thiocyanate and sulfite (Ray et al., 2000). PA0859, 39 bp downstream of PA0858, is a putative DsbA oxidoreductase responsible for isomerisation of disulfide bonds. While DsbA proteins are commonly localised to the periplasm (Denoncin & Collet, 2013), bioinformatic analysis using PSORTb 3.0 did not predict the cellular localisation of PA0859, nor was a signal peptide for secretion identified (Yu et al., 2010). The PA0859 gene overlaps with PA0860 by 8 bp, further indicating co-ordinated expression. PA0860 (1791 bp) encodes a protein with an N-terminal TMD of five predicted TM helices, and a C-terminal NBD. The organisation of both PA0858 and PA0859 within a putative operon with the predicted PA0860 ABC efflux protein suggests a co-ordinated role for the cluster in sulfur metabolism, and protein folding. This cluster is conserved across other environmental Pseudomonas strains, supporting the prediction that the cluster of genes is required under specific conditions, or as part of a co-ordinated process. In the γ- proteobacterium Azotobacter vineladii DJ, a putative DsbA oxidoreducatase protein 61 Chapter 3

Figure 3.4: The putative PA0860 ABC efflux protein. (A) A representation of the PA0860 genomic context, with PA0860 (1791 bp) indicated in red. The neighbouring genes PA0858 (putative sulfurtransferase) and PA0859 (putative DsbA oxidoreductase) are predicted to be co-transcribed with PA0860. (B) A schematic diagram of the homodimeric PA0860 ABC efflux protein, comprised of two NBD-TMD fusion proteins.

62 Chapter 3

(Avin_34850) with significant homology (51% identity, E-value = 5 x 10-78) to PA0859 is also encoded adjacent to a putative ABC efflux protein, further suggesting a functional basis for their co-localisation.

3.2.4.2. Putative function of PA1113

PA1113 differs from each of the other characterised and uncharacterised ABC exporters of PAO1, in that it is not present within an operon (Figure 3.5). PA1113, with an N-terminal TMD of five predicted TM helices and a C-terminal NBD, shares 35% identity and 55% similarity with Sav1866, the proposed multidrug resistance ABC transporter from S. aureus (Dawson & Locher, 2006). However, MIC analysis demonstrated that PA1113 does not have a major contribution to resistance against common anti-pseudomonal antibiotics. Consequently, in the absence of an interaction network of genes, PA1113 holds an alternative, but yet to be determined role in P. aeruginosa.

3.2.4.3. Putative function of PA3228

The putative ABC exporter, PA3228, shares significant homology with the characterised VcaM ABC exporter from Vibrio cholerae (53% identity, E-value = 0), suggesting that it could potentially contribute to antibiotic and drug efflux (Huda et al., 2003). However, expression of PA3228 was not stimulated in response to antibiotic treatment, nor was a change in MIC observed upon deletion of the PA3228 gene (Table 3.1 and Figure 3.3). Consequently, the balance of evidence does not support a primary physiological role for PA3228 in drug efflux. Primary sequence analysis indicates that PA3228 encodes a protein with an N-terminal TMD of six predicted helices and a C-terminal NBD. PA3228 is present in a putative operon with three other genes: PA3225, PA3226, and PA3227 (Figure 3.6). The first gene of the putative operon, PA3225, shares 80% identity with an uncharacterised LysR family transcriptional regulator from Azotobacter vinelandii (Avin_19100). The adjacent PA3226 gene was predicted to encode an enzyme with 3-oxoadipate enol-lactonase activity, responsible for conversion of 3-oxoadipate enol-lactone into β-ketoadipate (Bains et al., 2011). Taken together, this suggests a role for PA3225 and possibly PA3226 in P. aeruginosa lactone metabolism. Lactones have diverse functions in many organisms, including signalling as part of the quorum sensing systems of P. aeruginosa (Rutherford & Bassler, 2012). PA3227 is a putative periplasmic peptidyl-prolyl cis-trans isomerase A (Yu et al., 2010), also known as a cyclophilin. Cyclophilins have been suggested to hold roles in protein folding (Obi et al., 2011). However, the periplasmic PpiA in E. coli was previously shown to be unimportant to the peptidyl-prolyl cis-trans isomerase activity of the

63 Chapter 3

Figure 3.5: The putative PA1113 ABC efflux protein. (A) The 1767 bp PA1113 gene is predicted to be transcribed independently from adjacent genes. (B) A schematic diagram of the homodimeric PA1113 ABC efflux protein, comprised of two NBD-TMD fusion proteins.

64 Chapter 3

Figure 3.6: The putative PA3228 ABC efflux protein. (A) A representation of the PA3228 genomic context, with PA3228 (1833 bp) indicated in pink. The neighbouring genes, PA3225 (putative LysR transcriptional regulator), PA3226 (putative 3-oxoadipate enol-lactonase) and PA3227 (putative peptidyl-prolyl isomerase) are predicted to be co-transcribed with PA3228. (B) A schematic diagram of the homodimeric PA3228 ABC efflux protein, comprised of two NBD-TMD fusion proteins.

65 Chapter 3 periplasm (Kleerebezem et al., 1995). The activity of the putative periplasmic PpiA of P. aeruginosa has not, to date, been examined. Intriguingly, a putative PpiA (Avin_19090) with which PA3227 shares significant homology (68% identity, E-value = 1 x 10-87) is encoded adjacent to the identified Avin_19100 LysR transcriptional regulator, which was identified as sharing significant homology with PA3225. However, in A. vinelandii, an ABC efflux protein or a protein with 3-oxoadipate enol-lactonase activity are not components of the cluster. Nevertheless, PA3228 is more likely to exhibit a physiological role in line with its putatively co-transcribed genes, than fulfilling a drug efflux function as has been indicated for the MDR- associated VcaM exporter.

3.2.4.4. Putative function of PA5231

Analysis of PA5231 indicates it to be a non-canonical ABC efflux protein, in that it comprises two N-terminal NBDs and one TMD with six predicted helices at its C-terminus (Appendix 1). This second NBD domain accounts for the protein’s high predicted molecular mass (99.9 kDa), more than 20 kDa larger than the next largest P. aeruginosa ABC efflux protein, PA4143. Due to this unusual architecture, a second protein that contains a TMD without an NBD would most likely dimerise with PA5231 to provide the functional ABC efflux architecture (Figure 3.7). Analysis of the adjacent gene, PA5230, using PSORTb 3.0 suggested it to encode a TMD protein of six helices (Yu et al., 2010). Based on the organisation of the PA5231 gene adjacent to TMD-encoding PA5230, an interaction between the two gene products could be predicted, forming a complete ABC exporter. PA5230 and PA5231 are present in a putative operon with PA5232. Primary sequence analysis of PA5232 indicated it to share homology with haemolysin D family proteins and contain a single transmembrane helix, anchoring it into the cytoplasmic membrane (Yu et al., 2010). Haemolysin D family proteins are membrane fusion proteins, which interact with ABC and RND transporters to extend the transport complex into periplasm (Schulein et al., 1992; Lee et al., 2012). Consequently, PA5232 may act together with the proposed PA5231/PA5230 ABC efflux system to export an allocrite from the cytoplasm. This provides a basis for their localisation in a single operon. PA5230-PA5232 may also associate with an OM porin to facilitate movement of the allocrite from the cytoplasm to the supernatant without a periplasmic intermediate, effectively forming a T1SS. Although no such porin gene is present in the region surrounding the transporter genes, another OM porin may be utilized for this role, as it is the ABC exporter that governs the transport specificity. This role would likely be held by proteins of the OprM-family of outer membrane porins, such as OprM, which is used

66 Chapter 3

Figure 3.7: The putative PA5230/PA5231 ABC exporter. (A) A representation of the PA5231 genomic context, with PA5231 (2751 bp) indicated in blue. The neighbouring gene PA5230 possibly acts as an additional TMD component for PA5231 to achieve a complete ABC efflux protein architecture. PA5232 is a putative MFP protein and may act together with the PA5230/PA5231 transporter for secretion. (B) A schematic diagram of the proposed non-cannonical ABC exporter PA5230/PA5231, with PA5231 featuring two NBDs and a TMD and PA5230 featuring a single TMD.

67 Chapter 3 by both the MexAB and MexXY RND pumps for xenobiotic secretion, or PA4592, PA4144, or PA1875 (Hancock & Brinkman, 2002). However, further investigation is required to determine whether an OMP associates with the putative PA5230-PA5232 T1SS. Analysis of the adjacent gene, PA5233, using PSORTb 3.0, reveals the presence of a cleavable N-terminal signal sequence in the adjacent PA5233 gene product (Yu et al., 2010). Thus, it is unlikely that PA5233 is the allocrite of the proposed secretion complex. Whilst this PA5230-PA5232 transporter model could be inferred based on the known general architecture of ABC export proteins, an alternate functional role can be proposed for PA5231. PA5231 shares significant sequence identity (69%, E-value = 0) with the -Bound ATPase (RbbA), from E. coli (Kiel et al., 1999; Babu et al., 2011). Furthermore, the rbbA gene is present between a gene encoding a protein of six TM helices, yhhJ (69% identity to PA5230), and yhiI, a putative membrane fusion protein (58% identity to PA5232), mirroring the organisation of the PA5230-PA5232 cluster in P. aeruginosa. This organisation is also present in the archaeon Geobacter sulfureducens, indicating a basis for the conserved gene arrangement across genomes. The E. coli RbbA acts as an elongation factor, responsible for the ATP-dependent release of deacyl-tRNA from the ribosome, thereby enabling continued polypeptide elongation (Babu et al., 2011). However, this study also suggested RbbA to operate with membrane proteins involved in LPS production and export. On the basis of significant homology with RbbA, PA5231 may hold similar roles in P. aeruginosa.

3.2.4.5. Putative function of PA1876

The putative ABC exporter PA1876 is present in an operon with PA1874, PA1875 and PA1877 (Figure 3.8). Similar to PvdT, HasD, AprD and PA4143, PA1876 is predicted be the ABC exporter component of a T1SS with PA1877 encoding a putative MFP, and PA1875 encoding a putative OMP (Guzzo et al., 1991; Duong et al., 1996; Letoffe et al., 1998; Duong et al., 2001; Hannauer et al., 2012). Furthermore, primary sequence analysis of PA1876 indicated it to encode an N-terminal TMD of five predicted TM helices and C-terminal NBD domain, in addition to the presence of a putative C39-like peptidase family domain at the N- terminus of the protein (Appendix 1). This C39-like peptidase domain is present in some ABC exporters of T1SS, including the well-characterised HlyB of the -hemolysin (HlyA) T1SS, HlyBD-TolC (Lecher et al., 2012). The C39-like domain (CLD) of HlyB is non- catalytic, instead holding a role in binding the unfolded HlyA allocrite and acting as a chaperone. A similar role is likely to be played by the CLD of PA1876, binding its unfolded allocrite, ready for secretion. The positioning of the PA1874 gene with the putative PA1875- 68 Chapter 3

Figure 3.8: The putative PA1876 ABC efflux protein. (A) A representation of the PA1876 genomic context, with PA1876 (2172 bp) indicated in yellow. The neighbouring genes, PA1874 (putative large adhesion, pink), PA1875 (putative OMP, purple) and PA1877 (putative MFP, green) are predicted to be co-transcribed with PA1876 and form a T1SS. (B) A schematic diagram of the proposed PA1875-PA1877 T1SS, with PA1876 acting as a homodimeric ABC efflux protein comprised of two NBD-TMD fusion proteins to energise transport via hydrolysis of ATP. PA1876 is predicted to work together with PA1877 and PA1875 to form a contiguous translocation pathway for the secretion of PA1874. The C39-like domains are not indicated, but likely function near the NBDs to aid in the secretion of PA1874 via the T1SS.

69 Chapter 3

PA1877 T1SS, and its predicted extracellular localisation, suggests PA1874 may be the allocrite secreted by the PA1875-PA1877 complex. As the PA1876 ABC exporter does not appear to contribute to antibiotic resistance (Table 3.1 and Figure 3.3) it can be suggested that PA1876, as part of the PA1875-PA1877 T1SS contributes to the secretion of PA1874.

3.2.4.5.1. Characterisation of the putative PA1875-PA1877 T1SS allocrite, PA1874

PA1874, the putative allocrite of the PA1875-PA1877 T1SS, has previously been implicated in P. aeruginosa virulence in a rat lung infection model (Potvin et al., 2003). However, the molecular mechanism behind this finding was not elucidated. To gain insight into the function of PA1874, extensive bioinformatic analyses were performed (Figure 3.9). PA1874 is an unusually large, 2468 amino acid protein that is predicted to be embedded in the outer membrane by virtue of a single N-terminal TM helix, as well as being secreted into the culture supernatant (PSORTb 3.0) (Yu et al., 2010). Following the predicted TM helix are a large number of imperfect repeating domains: three domains of 60-86 amino acids, then 18 repeats of 82 amino acids. Alignment of the second series of 18 repeats (Table 3.2) revealed a high degree of sequence conservation (Figure 3.9). Each repeat shares significant homology with immunoglobulin fold-like domains, a common feature of proteins that hold roles in bacterial adherence (Berisio et al., 2012). At the C-terminus of the protein sequence is a 307 amino acid repeat, designated SWM, which is a putative flagella associated region (PFAM 13753) often found in outer membrane proteins. The function of this region is currently unknown. PA1874 also features a nine amino acid Repeat-in-Toxin (RTX) motif (GGxGxDxLx) at its C-terminus. RTX sequences bind Ca2+ ions and are found at the C- terminus of proteins targeted to T1SS (Chenal et al., 2009). The motif is usually present 5 to 50 times within an RTX-containing protein and precedes a secretion sequence. However, in PA1874 only one RTX motif precedes the L, D, A, V, T, S, I, and F residue-rich T1SS secretion sequence in the final 43 amino acids of the protein (Delepelaire, 2004).

In recent years, a number of large adhesin proteins, rich in extensive repeats have been characterised. These proteins, such as LapA from and Pseudomonas putida (Hinsa et al., 2003), LapF from Pseudomonas putida (Martinez-Gil et al., 2010), Bap from S. aureus (Cucarella et al., 2001), and BapA from Salmonella enterica serovar Enteritidis (S. Enteritidis) (Latasa et al., 2005) each contribute to biofilm formation and structure. As predicted for PA1874, LapA, LapF, Bap, and BapA are secreted via the T1SSs with which they are co-transcribed. Sequence alignment of PA1874 with LapA and LapF reveals a low level of sequence homology (29-30%, over 2% of the protein sequence). However, Bap and BapA share 38% and 34% identity, respectively (≥62% sequence

70

Figure 3.9: Bioinformatic analysis of PA1874. Schematic representation of PA1874 depicting predicted repeating domains (A-U), transmembrane (TM) helix region, repeat-in toxin (RTX) motif (GGxGxDxLx, yellow), SWM repeat domain and C-terminal secretion sequence. An amino acid logo prepared using Weblogo (Version 3.4) (Crooks et al., 2004) as outlined in Section 2.7.2 was used to represent the sequence conservation across the 18 repeats (D-U) of 82 amino acids.

Table 3.2: Sequences of repeating domains in PA1874.

Repeat Sequence (5’  3’)

A DGSSISGQAEAGASVGIDTNGDGKPDLTVIADANGNFTAPLNPPLTNGQTVTVVVTDPAG B TAPAPATDVQVAPDGSSVTGKAEPGSTVGVDTDGDGQPDTTVVVGPGGSFEVPLNPPLTNGETVTVIVTDPAGNNSTPVTVEAPDT C TAPAPATDVQVAPDGSSVTGNAEPGATVGVDTDGDGQPDTTVVVGPGGSFEVPLNPPLTNGETVTVIVTDPAGNSSTPVTAE D APDFPDAPQVNASNGSVLSGTAEAGVTIVITDGNGNPIGQTSADANGNWSFTPGSQLPDGTVVNVVARDAAGNSSPATSITV E DGVAPNAPVVEPSNGSELSGTAEPGSSVTLTDGNGNPIGQTTADANGNWSFTPSTPLPDGTVVNVVARDAAGNSSPPASVTV F DAVAPATPTVDPSNGTTLSGTAEPGSSVTLTDGNGNPIGQVTADGSGNWTFTPSTPLPNGTVVNATATDPSGNASSPASVTV G DAVAPATPVVNPSNGTTLSGTAEPGATVTLTDGNGNPIGQVTADGSGNWSFTPTTPLPNGTVVNATATDASGNTSAGSSVTV H DSVAPATPVINPSNGTTLSGTAEPGSSVTLTDGNGNPIGQVTADGSGNWSFTPSTPLADGTVVNATATDPAGNTSGQGSTTV I DGVAPTTPTVNLSNGSSLSGTAEPGSTVILTDGNGNPIAEVTADGSGNWTYTPSTPIANGTVVNVVAQDAAGNSSPGASVTV J DSQAPAAPVVNPSNGTTLSGTAEPGATVTLTDGNGNPIGQVTADGSGNWSFTPGTPLANGTVVNATASDPTGNTSAPASTTV K DSVAPAAPVVNPSNGAEISGTAEPGATVTLTDGSGNPIGQVTADGSGNWSFTPSTPLADGTVVNATATDPAGNTGGQGSTTV L DAIAPATPTVNLSNGSSLSGTAEPGSTVILTDGNGNPIAEVTADGSGNWTYTPSTPIANGTVVNVVAQDASGNSSPPATVTV M DSSAPPAPVINPSNGVVISGTAEAGATVTLTDAGGNPIGQVTADGSGNWSFTPGTPLANGTVIVATATDPTGNTGPQAATTV N DAVAPPAPVIDPSNGTTISGTAEAGAKVILTDGNGNPIGETTADGSGNWSFTPGTPLANGTVVNAVAQDPAGNTGPQGSTTV O DAVAPNTPVVNPSNGNLLNGTAEPGSTVTLTDGNGNPIGQTTADGSGNWSFTPGSQLPNGTVVNVTASDAAGNTSLPATTTV P DSSLPSIPQVDPSNGSVISGTADAGNTIIITDGNGNPIGQVTADGSGNWSFTPGIPLPDGTVVNVVARSPSNVDSAPAVITV Q DGVAPAAPVIDPSNGTEISGTAEAGATVILTDGGGNPIGQATADGSGNWTFTPSTPLANGTVINAVAQDPAGNTSGPASVTV R DAIAPPAPVINPSNGVVISGTAEAGATVILTDGNGNPIGQVTADGSGNWSFTPGTPLANGSVINALAQDAAGNNSSPTSATV S DSLAPAAPVIDPSNGSVIAGTAEAGATVILTDGNGNPIGQVTADGSGNWSFTPGTPLSNGTVVNAVAQDAAGNTSGPVSTTV T DAVAPATPVIDPSNGVELSGTAEPGVRVILTDGNGNPIGQTLADGSGNWSFTPGTPLANGTVVNAVAQDPAGNTSGPASTTV U DTVAPATPVINPSNGSVITGTAEVGAKVILTDGNGNPIGETTADGSGNWTFTPGTPLANGTVINAVAEDAAGNASGPASTTV Sequence of repeats A-U identified in PA1874 as depicted in Figure 3.9. Although conserved, repeats A-C differ from the highly conserved D-U repeats.

Chapter 3 coverage), with PA1874 from P. aeruginosa. Bap from S. aureus has been shown to contribute to biofilm formation, and aid in pathogenesis (Cucarella et al., 2001). Bap transposon mutants exhibited a decrease in biofilm formation on microtitre plates after 24 h, compared with the isogenic wild-type strain. Similarly, a BapA mutant of S. Enteritidis resulted in biofilm deficiency and a reduction in ability to invade host tissue (Latasa et al., 2005). Based on homology of PA1874 to Bap and BapA, the biofilm formation capacity of strains that lacked component(s) of the PA1874-PA1877 operon were examined. The deletion of the PA1876 ABC exporter (PA1876::FRT) and the entire PA1875-PA1877 T1SS (PA1875-7::FRT) from PAO1 would be expected to prevent secretion of PA1874, with the PA1874 C-terminal secretion sequence predicted to be specific for the PA1875-PA1877 T1SS. Similarly, a PA1874 mutant (PA1874::FRT) would also be expected to exhibit reduced biofilm formation capacity. However, quantification of biofilm formation in a 96-well microtitre plate assay revealed no difference in the ability of the mutant strains to form a biofilm, relative to the isogenic wild-type strain, PAO1 (Figure 3.10). This indicated that in a static, in vitro scenario, PA1874 does not contribute to biofilm formation, as deletion of its dedicated transport system, or part of PA1874 itself, did not affect the ability of P. aeruginosa to form a biofilm. However, based on its homology with the biofilm-contributing proteins, Bap and BapA, PA1874 may contribute to biofilm formation in vivo, under specific environmental conditions or in particular host niches. Further characterisation of the T1SS and its contribution to biofilm formation was not performed as a publication examining the PA1874-PA1877 T1SS was released during the course of this study (de Bentzmann et al., 2012).

Discussion

ABC exporters have an essential role in facilitating the active extrusion of molecules and compounds from the bacterial cytosol. Here, bioinformatic and primary sequence analysis was used to identify five uncharacterised ABC efflux proteins in P. aeruginosa PAO1: PA0860, PA1113, PA1876, PA3228 and PA5231. On the basis of the homology of these putative ABC transporters to MDR-associated efflux proteins from other organisms, the transporters were then investigated for their capacity to contribute to the antibiotic resistance of P. aeruginosa. This study showed, by using MIC analysis of deletion mutants and the transcriptional response to a shock antibiotic treatment, that the five ABC exporters did not significantly contribute to the antibiotic resistance profile of PAO1. However, while this does not preclude a potential contribution to antibiotic resistance, it does suggest that these 73 Chapter 3

Figure 3.10: Biofilm formation of PA1874-PA1877 T1SS mutants. Static biofilm formation after 22 h was assessed for the wild-type PAO1, PA1876 deletion mutant (PA1876::FRT), PA1875-7 T1SS mutant (PA1875-7::FRT) and the PA1874 deletion mutant (PA1874::FRT) by 0.1% crystal violet staining and measuring absorbance at OD595. Data are the mean ± s.e.m., from three independent experiments.

74 Chapter 3 transporters have a distinct primary function. In the context of other studies, it should be noted that in many cases bacterial ABC exporters do not often confer significant levels of antibiotic resistance in the host organism. Rather, the assignment of an antibiotic resistance capability has most commonly arisen from studies of drug efflux undertaken by heterologous expression in drug-sensitive organisms. An additional consideration is that drug-stimulated expression of the ABC exporters is not commonly observed in the native organism, although the YheIH exporter from B. subtilis is a notable exception (Torres et al., 2009). Overall, the observations here, and in the context of the literature, would suggest that while a considerable number of bacterial ABC transporters have been ascribed with drug resistance functionality, it is in many cases likely to be opportunistic, with their physiological roles remaining unknown. Hence, in the context of the current work, the five ABC exporters examined herein may still have an opportunistic contribution to antibiotic efflux in addition to their physiological role(s), but this contribution is relatively minor and masked by the numerous exporter systems and resistance mechanisms of P. aeruginosa.

To address the possibility of an opportunistic contribution to antibiotic efflux, the ABC exporters would have to be individually over-expressed in E. coli, followed by an assessment of changes in the antibiotic resistance profile. The masking of an antibiotic resistance contribution on the part of ABC exporters is particularly likely in Gram-negative organisms, such as P. aeruginosa, that makes extensive use of tripartite RND efflux pumps. RND efflux pumps, such as MexAB-OprM and MexXY-OprM, enable extrusion of structurally diverse compounds across both membranes in a proton-coupled manner (Morita et al., 2001; Okamoto et al., 2002; Aeschlimann, 2003; Morita et al., 2012). Furthermore, P. aeruginosa RND efflux pumps undergo significant up-regulation in response to sub-inhibitory antibiotic treatments, consistent with their roles as major drug efflux pathways (Aeschlimann, 2003). This, together with the low permeability of the membrane, β-lactamase enzymes and the frequency of mutation in antibiotic targets, confers P. aeruginosa with significant antibiotic resistance independent of ABC exporters (Poole, 2011). Consequently, the primary physiological roles of the identified ABC exporters remain cryptic.

In the absence of obvious phenotypic impacts associated with the deletion mutants, the genomic context and sequence analyses of the five ABC exporters were used to identify potential physiological roles. Based on the predicted function of its adjacent genes, PA0860 may play a role under conditions requiring sulfur metabolism and disulfide bond isomerisation. Intriguingly, expression of PA0860 has been shown to be up-regulated by P. aeruginosa in the CF lung (Son et al., 2007). This indicates a potential role for the ABC

75 Chapter 3 exporter in virulence and colonization by P. aeruginosa. On this basis, further characterisation of the PA0858-PA0860 cluster is warranted, with the aim of providing key insight into the precise roles of these proteins in P. aeruginosa colonization and virulence. By contrast, PA1113 was not present within an operon and, consequently, the genomic context did not allow for the assignment of a putative function. The genomic contexts of PA3228 and PA5231 allowed for speculation on the possible roles for these transporters and their associated proteins. Further work will be required to confirm these inferred roles, and determine their contribution, if any, to P. aeruginosa virulence. As the PA3225-PA3228 and PA5230-PA5233 clusters are conserved across the plant P. putida, P. fluorescens and P. protogens, this indicates any such role may be applicable to diverse environmental and human niches.

The ABC transporter PA1876 was identified as the ABC component of the PA1874- PA1877 T1SS. Although considerable efforts to biochemically characterise PA1876 and the surrounding gene cluster (PA1874-PA1877) were made, during the course of this work a study characterising PA1874-PA1877 T1SS was published (de Bentzmann et al., 2012). That work showed the PA1874-PA1877 T1SS to be a polycistronic operon under the strict positive regulation of the PprAB two-component regulatory system (de Bentzmann et al., 2012). Further, this publication outlined that PA1874-PA1877 were not expressed in PAO1 unless PprAB was activated, which the authors achieved via a constitutive PprB overexpression strain. Under conditions of PprB overexpression, a hyper-biofilm phenotype was observed in which PA1874, eDNA, CupE fimbriae and Flp (type IVb) pili predominated the biofilm matrix, with the notable absence of Pel or Psl polysaccharide. This indicated that the lack of biofilm inhibition expected for the PA1874, PA1876 and PA1875-PA1877::FRT mutants (Figure 3.10) may be attributed to PprB repression of the locus, masking the effect of the deletions. In contrast to the previous finding that PA1875-PA1877 contributes to biofilm- specific antibiotic resistance (Zhang & Mah, 2008), the PprB overexpression strain in which PA1875-PA1877 is expressed, had an increased sensitivity to tobramycin. Consequently, the role of this cluster in antibiotic resistance seems unlikely, correlating with the MIC analysis of the PA1876::FRT strain. Further, consistent with the bioinformatic predictions, de Bentzmann et al. (2012) demonstrated PA1874 to be the physiological allocrite of the PA1875-PA1877 T1SS, with deletion of PA1877 resulting in a lack of secreted PA1874. Based on the role of PA1874-PA1877 in biofilm formation, and the greater homology between BapA and PA1874 as compared to LapA and LapF, PA1874-PA1877 was renamed bapABCD, for biofilm- associated protein (bap). The molecular mechanisms through which this increasingly growing

76 Chapter 3 family of large, repeat-rich proteins contribute to biofilm formation have not yet been well defined. Together with cellulose and curli fimbriae, BapA is a major component of S. Enteritidis biofilm matrix (Zogaj et al., 2001; Solano et al., 2002; Latasa et al., 2005). Similarly, BapA, CupE fimbriae and Flp (type IVb) pili may co-operatively act to provide structure and adhesion within P. aeruginosa biofilms through the transcriptional control of PprB. PprB overexpression strains lacking BapA, CupE fimbriae or Flp (type IVb) pili all showed similar biofilm defects (de Bentzmann et al., 2012). Based on the PprAB-dependent expression of the CupE fimbriae and Flp (type IVb) pili biofilm appendages, which are proposed to aid in surface and cell to cell attachment during initial biofilm formation (Mikkelsen et al., 2011), it is likely that BapA also plays a role during the biofilm developmental stage. Consequently, this work, together with that by de Bentzmann et al. (2012) has provided insight into P. aeruginosa biofilm formation mechanisms. As biofilm formation significantly contributes to persistent infections of the CF lung, it is of interest to note that PprB activation was observed in early P. aeruginosa strains isolated from the CF lung (Yang et al., 2011). This suggests a role for BapA in the development of biofilms within the CF mucus, warranting further investigation of the structure and molecular function of this large secreted protein.

Conclusions

Taken together, these data demonstrate that the uncharacterised ABC exporters of P. aeruginosa PAO1 do not have a primary role in antibiotic resistance. Herein, a range of predicted physiological functions for the ABC exporters are presented, including protein secretion. Of these exporters, PA1876 (BapC) has recently been identified as the ABC efflux protein of the PA1875-PA1877 (BapBCD) T1SS. BapBCD was predicted and confirmed (de Bentzmann et al., 2012) to have a physiological role in the secretion of the large, repeat-rich, biofilm associated protein, PA1874 (BapA). Examination of the five ABC exporters of interest has provided a foundation for further investigation of their predicted roles within P. aeruginosa PAO1, enabling future confirmation of their physiological functions and contribution to virulence.

77 Chapter 4

CHAPTER 4:

THE ACQUISITION AND ROLE OF

MOLYBDATE IN P. AERUGINOSA

78 Chapter 4

Introduction

P. aeruginosa is a ubiquitous environmental organism, capable of survival and proliferation under diverse conditions. This ability to thrive in such a range of environments is attributed to the coding capacity of its large 6.3 Mbp genome. Whilst P. aeruginosa is commonly classified as an aerobic bacterium, its genome provides the ability to survive and grow in anaerobic and microaerophilic environments (Yoon et al., 2002; Alvarez-Ortega & Harwood, 2007; Arai, 2011). Growth of P. aeruginosa under anaerobic conditions requires the use of nitric oxides as terminal electron acceptors. In the absence of nitrate, P. aeruginosa is forced to reply upon arginine deimination or pyruvate fermentation (Vander Wauven et al., 1984; Eschbach et al., 2004). However, these pathways do not provide sufficient energy to support significant bacterial growth. Consequently, in anaerobic environments, P. aeruginosa preferentially utilizes the dissimilatory reduction of nitrate for energy production (Yoon et al., 2002; Palmer et al., 2007). The major dissimilatory nitrate reduction pathway employs four enzymatic complexes to reduce nitrate to nitrite (NarGHI), nitrite to nitric oxide (NirS), nitric oxide to nitrous oxide (NorCB) and finally, nitrous oxide to dinitrogen (NosZ) (Figure 4.1) (Schreiber et al., 2007). P. aeruginosa also possesses a periplasmic nitrate reductase complex (NapAB), although this is not believed to play a major role in anaerobic growth (Noriega et al., 2005; Van Alst et al., 2009). The anaerobic regulator of arginine deiminase and nitrate reductase, Anr, of the fumarate and nitrate reductase (Fnr) family of transcriptional regulators, controls transcription of the dissimilatory nitrate reduction pathway (Sawers, 1991; Trunk et al., 2010). Anr senses oxygen tension via its [4Fe-4S]2+ cluster (Yoon et al., 2007). Under low oxygen tension, Anr up-regulates expression of the nar nitrate reductase operon and the dissimilatory nitrate reductase pathway regulator, dnr, which further modulates expression of the nar operon, the nitrite reductase and nitric oxide reductase regulatory gene nirQ, and the nitric oxide reductase genes norCB (Schreiber et al., 2007; Giardina et al., 2008; Trunk et al., 2010; Giardina et al., 2011). As a consequence, in response to low oxygen tension, the cell is able to reduce nitrate to dinitrogen and generate energy for growth.

Each enzyme of the dissimilatory nitrate reductase pathway utilizes a transition metal cofactor for its activity, namely iron, copper, or molybdenum (Figure 4.1) (Kraft et al., 2011). The initial membrane-embedded enzymatic complex NarGHI, which reduces nitrate to nitrite, requires molybdenum incorporated in a modified molybdenum cofactor, Mo-bis molybdopterin guanine dinucleotide (MGD) (Bertero et al., 2003). Cellular molybdenum 2- uptake occurs in the form of its oxyanion, molybdate (MoO4 ), referred to herein as Mo. Upon uptake, Mo is introduced into a complex molybdopterin molecule to generate the

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Figure 4.1: Dissimilatory nitrate reduction in P. aeruginosa. A schematic representation of the dissimilatory nitrate reductase pathway, with the proteins clusters responsible for each enzymatic step indicated. The transition metal ion required for each step is also indicated in square brackets. The dissimilatory nitrate reduction pathway contributes to energy production in P. aeruginosa via the coupled efflux of protons from the - - cytoplasm into the periplasm, generating proton motive force. Nitrate, NO3 ; nitrite, NO2 ; nitric oxide, NO; nitrogen dioxide, N2O; dinitrogen, N2.

80 Chapter 4 molybdenum cofactor (MoCo), which may then be further modified prior to insertion into MoCo-dependent proteins (Iobbi-Nivol & Leimkuhler, 2013). Such proteins include the DMSO reductase, xanthine oxidase, and sulfite oxidase families, all of which have broad roles in nitrogen, carbon, and sulfur metabolism (Hernandez et al., 1991; Iobbi-Nivol & Leimkuhler, 2013).

Molybdate is present at trace levels in humans and most terrestrial environments (Rodushkin et al., 1999; He et al., 2005; Burguera & Burguera, 2007). Consequently, high affinity transport systems are required for Mo uptake. Acquisition of Mo by prokaryotes occurs primarily via a high affinity ATP-binding cassette (ABC) permease, ModABC. Secondary transport has been reported via sulfate/thiosulfate ABC permeases and a non- specific anion importer in E. coli (Rosentel et al., 1995), as well as a low affinity Mo ABC permease, MolABC, in H. influenzae (Tirado-Lee et al., 2011). However, no such MolABC ortholog has been identified in P. aeruginosa (Stover et al., 2000). The high affinity Mo ABC permease, ModABC, comprises a solute binding protein (ModA), responsible for binding Mo; a dimer of nucleotide binding domains (ModC) which hydrolyze ATP in the cytoplasm to energize the transporter; and two transmembrane domains (ModB) which traverse the membrane, and to which ModA delivers its cargo for transport (Self et al., 2001). In Gram- negative organisms, ModA is a soluble, freely diffusible, periplasmic solute binding protein

(SBP) that delivers Mo to the assembled ModB2C2 transporter (Rech et al., 1996), whereas in Gram-positive bacteria, ModA is lipid-anchored to the cell membrane (Neubauer et al., 1999). Structural analysis of ModA from E. coli and Azotobacter vinlandeii has facilitated the classification of ModA as a cluster D-III SBP, sharing a conserved two-domain architecture, linked by a hinge region of two short strands (Berntsson et al., 2010). Together the cluster D- III SBPs bind oxyanions, such as molybdate, sulfate and phosphate, and deliver these allocrite molecules to the cognate ABC importers for transport into the cytoplasm.

The ModABC system has been extensively characterized in E. coli where its expression is under the control of the Mo-binding LysR transcriptional regulator, ModE (Anderson et al., 1997; Grunden et al., 1999; Self et al., 1999). In addition to binding Mo, E. coli ModE has 2- been shown to bind its structurally similar oxyanion, tungstate (WO4 ) (Grunden et al., 1999), likely exerting regulatory effects on its target genes. Similarly, substitution of W for Mo has also been observed in ModA (Imperial et al., 1998). However, the capability of E. coli ModA or ModE to interact with W has no physiological basis, as E. coli does not utilize W-cofactor containing enzymes and, in many instances, W-substitution for Mo results in enzyme inactivation (Kletzin & Adams, 1996). Only a single exception has been reported,

81 Chapter 4 with the in vivo substitution of W in the molybdoenzyme, trimethylamine N-oxide reductase (Buc et al., 1999). The underlying molecular basis of W interaction with E. coli ModA and ModE is most likely a result of the amino acid side-chains at the metal-binding site retaining the -binding chemistry permissive for both Mo and W interaction. This limitation in ligand discrimination was recently exemplified in the manganese-recruiting cluster A SBP of S. pneumoniae, which was shown to have the capacity to interact with any first-row transition metal, despite its sole physiological role in manganese acquisition (Counago et al., 2014). Organisms that specifically utilize W, such as the archaea Pyrococcus furiosus and Archaeoglobus fulgidus, and the bacterium Campylobacter jejuni, encode W-specific (TupABC) or Mo/W (WtpABC) ABC permeases in their genomes (Bevers et al., 2006; Hollenstein et al., 2007b; Smart et al., 2009). No orthologs of these systems have been identified in P. aeruginosa (Stover et al., 2000). Whether, similar to E. coli, the P. aeruginosa Mod permease is capable of W import remains to be determined.

Studies from a number of research groups have demonstrated the Mo-requiring NarGH nitrate reductase complex to contribute to P. aeruginosa virulence. A mutant strain lacking narG was shown to exhibit a severe anaerobic growth defect when grown in the presence of physiologically relevant concentrations of nitrate (0 – 5 mM) (Palmer et al., 2007). Another study reported a PAO1 narGH mutant to exhibit altered biofilm formation and motility (Van Alst et al., 2007). The same study also examined the virulence of a narGH mutant in the closely related P. aeruginosa PA14 strain using a slow killing assay. Deletion of the membrane-bound nitrate reductase rendered the ΔnarGH mutant strain avirulent, in contrast to the wild-type P. aeruginosa (Van Alst et al., 2007). Taken together, these data indicate the importance of the Mo-dependent membrane-bound nitrate reductase in P. aeruginosa. On this basis, the ability of P. aeruginosa to effectively acquire Mo from its environment is predicted to contribute to the virulence of the bacterium. The work presented herein provides a detailed analysis of the P. aeruginosa PAO1 high-affinity Mo acquisition pathway, ModABC, with a particular focus on the Mo-recruiting SBP, ModA. Further, this study highlights the importance of high-affinity Mo acquisition to nitrate reduction, biofilm formation, and cellular physiology under oxygen limiting or anaerobic conditions. Additionally, these findings provide new insight into the ability of W to substitute for Mo in transport, regulation, and other functional roles in P. aeruginosa.

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Results

4.2.1. ModA is required for Mo acquisition

Molybdate acquisition in prokaryotes, such as E. coli, occurs primarily through a high affinity ModABC ABC permease (Maupin-Furlow et al., 1995; Imperial et al., 1998). In P. aeruginosa, PA1863 encodes a 251 amino acid protein that shares ~30% identity with the cluster D-III SBP ModA from E. coli (Berntsson et al., 2010) (Figure 4.2). The proposed P. aeruginosa modA is clustered with two other genes in a putative operon that includes PA1861 (modC) and PA1862 (modB). Overall, the genetic organization is indicative of a canonical ABC import pathway (Davidson et al., 2008) and orthologous can be found in many other Pseudomonas species including P. fluorescens, P. putita and P. syringae. Based upon its similarity with ModA homologs, the putative P. aeruginosa modA (PA1863) was assessed for its contribution to the high affinity acquisition of Mo. A modA deletion strain (ΔmodA) was constructed in P. aeruginosa PAO1 (as described in Section 2.4.1.1), and grown in CDM with a Mo concentration of ≤ 10 nM. Analysis by ICP-MS showed that loss of the SBP abrogated Mo accumulation (Figure 4.3A), whilst the concentration of other transition metal ions (iron, zinc, copper, nickel, manganese, cobalt) remained unchanged (data not shown). Molybdate uptake could be restored by supplementation of the ΔmodA strain with 10 mM

Na2MoO4. Collectively, these data indicate that ModA has a crucial role in Mo uptake at nanomolar concentrations, but at elevated, non-physiological Mo concentrations, other unidentified low affinity, or non-specific uptake pathway(s) could acquire Mo.

Molybdate and W are highly similar in their structural characteristics and redox capabilities (Arai, 2011). Prior studies of the Mo ABC permease from E. coli (ModABC) have shown that although its physiological role was in Mo uptake, E. coli ModA was also able to bind W (Imperial et al., 1998). Consequently, the impact of W supplementation upon Mo accumulation in the P. aeruginosa wild-type and ΔmodA strains was assessed. Upon supplementation with 10 mM Na2WO4, whole cell accumulation of Mo by wild-type P. aeruginosa decreased by more than 90% (P = 0.006), consistent with the two ions competing for ModA-facilitated import (Figure 4.3B). Supplementation with equimolar concentrations of Mo and W (10 mM) relieved the competition of W for ModA, allowing restoration of Mo accumulation (P = 0.0009, Figure 4.3B). However, when the ΔmodA strain was supplemented with equimolar concentrations of Mo and W, ModA-independent Mo acquisition was reduced (P = 0.0156, Figure 4.3B). This suggests that the ModA-independent Mo acquisition pathway(s) have limited capacity to discriminate between Mo and W oxyanions. Collectively,

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Figure 4.2: Phylogenetic analysis of cluster D-III SBPs. The phylogenetic relationship between P. aeruginosa ModA and characterised cluster D-III SBPs was assessed. Amino acid sequences were aligned using ClustalW2 and a phylogenetic tree constructed in CLC Sequence Viewer (Version 6.7.1) using the Neighbour Joining method. Proteins clustered according to their cognate high affinity ligand(s): molybdate (blue), sulfate (pink), phosphate (green), tungstate (orange), and tungstate and molybdate (brown). Organisms were classified as – for Gram-negative bacteria, + for Gram-positive bacteria, Myc for Mycobacteria, and Arch for Archaea. ModA, Xax (GI:21109708, Xanthomonas axonopodis); ModA, Eco (GI:341917273, E. coli); ModA, Cdi (GI:491668812, Clostridium difficile); ModA, Avi (GI:157830075, Azotobacter vinelandii); ModA, Pae (GI:15597060, P. aeruginosa); ModA, Cje (GI:315057724, Campylobacter jejuni); SBP, Eco (GI:190906999, E. coli); SBP, Sty (GI:332990892, S. Typhimurium); CysP, Eco (GI:190906818, E. coli); Psts, Eco (GI:147256, E. coli); PstS, Ype (GI:115349704, Yersinia pestis); Psts-1, Mtu (GI:15840351, Mycobacterium tuberculosis); TupA, Cje (GI:218563133, Campylobacter jejuni); TupA, Gsu (GI:298506735, Geobacter sulfurreducens); TupA, Eac (GI:14330288, Eubacterium acidaminophilum); WtpA, Pho (GI:499186722, Pyrococcus horikoshii); WtpA, Pfu (GI:46397233, Pyrococcus furiosus); WtpA, Afu (GI:224983468, Archeoglobus fulgidus).

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Figure 4.3: Deletion of modA alters P. aeruginosa Mo accumulation. (A and B) In vitro accumulation of Mo by wild-type PAO1 and ΔmodA cultures was assessed via growth in CDM with and without 10 mM Mo and/or 10 mM W supplementation. Metal content was expressed as μg of Mo per gram of dry cells, as determined by ICP-MS. Data are the mean ± s.e.m., with duplicate readings taken from each biological replicate grown on 3 separate days. Statistical significance was determined using the two-tailed unpaired Student’s t-test, where * represents P < 0.05 and ** represents P < 0.01; b.d. represents values below the limit of detection.

85 Chapter 4 these data indicate that ModA has a crucial role in Mo acquisition and that W can, at high extracellular concentrations, compete for binding to the SBP, reducing Mo uptake.

4.2.2. Characterisation of the Mo-dependent transcriptional regulon in P. aeruginosa

Molybdate acquisition in E. coli is under the control of the transcriptional regulator ModE (Anderson et al., 1997; Grunden et al., 1999; Self et al., 1999). Accumulation of free Mo in the cytoplasm allows homodimeric ModE to bind two Mo ions and interact with its cognate DNA-binding site, thereby repressing transcription of the E. coli modABCD genes (Anderson et al., 1997; Gourley et al., 2001). The PAO1 genome contains PA0487 (designated modE), a gene that encodes a protein with 41% identity and 52% similarity to ModE from E. coli. Unlike E. coli modE, which is divergently transcribed from modABCD, the putative PAO1 modE is located elsewhere in the genome. The transcriptional responsiveness of the modC gene, as a surrogate for the mod gene cluster, to Mo deficiency was examined by use of not only the wild-type and ΔmodA strains, but also the wild-type supplemented with Mo and the structurally similar oxyanion, W (Figure 4.4). Intriguingly, modC was found to be up-regulated in response to Mo-deficiency in the ΔmodA strain only. By contrast, modC was repressed to wild-type levels when PAO1 was supplemented with Mo, Mo + W or W, with the latter resulting in a W-induced Mo-deficiency (Figure 4.3B). Taken together, these results show that the presence of either Mo or W can repress expression of modC, thus revealing the oxyanion dependency of modABC transcriptional regulation, likely via the putative ModE.

As observed in E. coli and indicated above, the putative P. aeruginosa ModE is predicted to regulate the modABC locus. However, the localization of the putative P. aeruginosa modE at a distal location to the modABC locus suggested it may exhibit broader transcriptional control than that reported for E. coli ModE (Grunden et al., 1996). Thus, analysis was performed to identify the P. aeruginosa genes regulated by ModE. To do so, a scoring matrix was generated using previously characterized γ-proteobacterial ModE binding sites (Studholme & Pau, 2003) to yield a PAO1 optimized ModE motif (Figure 4.5). Similar to other ModE binding motifs, the PAO1 ModE motif was palindromic and featured a number of highly conserved resides (Studholme & Pau, 2003). The PAO1 genome was then analysed using this motif, and 20 intergenic putative ModE-binding sites with E-values ≤ 0.001 were identified (Table 4.1). This highlighted numerous genes as being potentially responsive to Mo availability via ModE, including the mod locus. Consequently, each putative ModE target was assessed by qRT-PCR for responsiveness to Mo deficiency by use of the ΔmodA strain (Table 4.1). Transcriptional analysis revealed that despite a range of putative ModE sites being

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Figure 4.4: Intracellular Mo deficiency causes an increase in modC expression. Relative expression of modC, corrected for rpoD, was assessed for PAO1 and ΔmodA strain grown in CDM ([Mo] ≤10 nM). Where indicated, Mo (Na2MoO4) and W (Na2WO4) were each supplemented at 10 mM. Data are the mean ± s.e.m., with n ≥ 3 biological replicates. Statistical significance was determined using the two-tailed unpaired Student’s t-test, where * represents P < 0.05.

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Figure 4.5: The P. aeruginosa ModE binding site motif. Nucleotide logo prepared using Weblogo (Version 3) as outlined in Section 2.7.2. * indicates highly conserved residue of interest.

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Table 4.1: Putative P. aeruginosa PAO1 ModE binding sites.

Fold Change in Distance from Locus-tag1 Predicted ModE binding site E-value2 mRNA levels Predicted product start codon (ΔmodA vs. wild-type) PA1864 TTCGTTATATAAAAATATTAACAACGAA 0.00013 21 35.90 TetR transcriptional regulator PA1863 AGCGCTATATTCCAAAAGCCATAGCGAA 0.00017 58 3.553 ModA IG: PA5029/ LysR-family transcriptional regulator, putative arginine ATCAATATATTGGAAATCATATAAACCT 0.00021 42/105 0.88 / 0.80 PA5030 sensor/ MFS PA4648 CACGTTATTTTCAAAAGCATGTAGCCAA 0.00023 259 1.04 Spore-coat protein (type I fimbrial cluster) PA0042 AGAGTTATTTGGATGACTACATAAAAAA 0.00030 1239 1.15 Highly variable region across genomes PA4623 TTCGATTTCTTCGATTTTTCATATCGAT 0.00032 176 0.90 Hypothetical protein RtcR, σ54-dependent transcription regulator containing an PA4581 AAAGATCGATATGAAAATATATAGAAAT 0.00037 20 0.97 AAA-type ATPase domain and a DNA-binding domain PA1939 ATCGTCGTGTTTTTATCTAAATCGAGAT 0.00058 37 1.09 DNA helicase IG: PA1738/ CGAGTTATTTTAATAATCCAATGAAGAA 0.00330 -3/131 1.15 / 0.97 Transcriptional regulator/oxidoreductase PA1739 PA5170 AAAATTATTTTTACTTATAAATAAAAAA 0.00420 399 0.90 ArcD, arginine/ornithine antiporter PA0534 AGTGTTATTTTTCATGAAAAATAACCAA 0.00520 21 1.14 Oxidoreductase IG: PA5295/ TTCGTTCTTTACGAAGATTCTTAAAACT 0.00550 11/148 1.10 / 0.65 Diguanylate cyclase/ATP-dependent DNA helicase PA5296 IG: PA1569/ ATCGATTTATCAAATTTTAAATATAAGT 0.00610 229/120 1.15 / 1.07 MFS/transcriptional regulator PA1570 PA4881 TACGAAATCTTCGAAATCACAAAAATAA 0.00630 142 0.93 Hypothetical protein IG: PA5105/ TCTGTTTTATTTGTATATACATATACAG 0.00640 55/17 0.96 / 0.84 HutC/N-formimino-L-glutamate deiminase PA5106 IG: PA2522/ AACGCCCGAAATGTAACTTTATAGCAAT 0.00800 433/51 1.12 /0.98 czcC/Two-component response regulator PA2523 PA3067 ATAATCATAGCTATATTTTCATAGCAAA 0.00860 10 0.95 Transcriptional regulator IG: PA1502/ AACAATATGAACATAATTTTGTATACAT 0.00870 183/117 1.113/1.57 Glyoxylate carboligase/glycolate utilization PA1503 IG: PA4523/ AGAGATACTTTCAAATCTAGATAGAGGG 0.00880 123/134 0.90 / 1.06 Thymidine phosphorylase/nadC (modD) PA4524 PA0237 ATCGTTGTTTTCTTGTTCGCTTCAAGAT 0.00970 11 1.07 Oxidoreductase 1. IG, intergenic region, 2. E-values ≤ 0.01, 3. Expression assessed for PA1861 (modC) as PA1863 gene deleted in ΔmodA strain

Chapter 4 associated with low E-values, only minimal changes in transcription levels were observed for the majority of genes. In addition to the transcriptional up-regulation observed for modC modest differences were recorded for PA5296 and PA1503 in the ΔmodA strain (approximately 1.5-fold down- and up-regulation relative to wild-type levels, respectively; Table 4.1). PA1864, a putative TetR transcriptional regulator, exhibited the greatest transcriptional response to Mo depletion, with a 35.9-fold up-regulation in the ΔmodA strain (Table 4.1). This is consistent with the putative ModE-binding site adjacent to PA1864 featuring the lowest E-value (0.00013). To further elucidate the oxyanion-dependent nature of PA1864 expression, additional transcriptional analyses were performed not only for the wild- type and ΔmodA strains, but also the wild-type supplemented with Mo and the structurally similar oxyanion, W (Figure 4.6). Consistent with the observed repression of PA1864 in the Mo-replete PAO1 strain, PA1864 was also repressed in the PAO1 strain supplemented with Mo. Repression was also observed for PA1864 under W-induced Mo depletion (PAO1 + W) and in the presence of equimolar concentrations of W and Mo (PAO1 + Mo + W). This demonstrated that the proposed ModE-dependent repression of PA1864 was induced by the presence of either Mo or W. This, together with the Mo or W-dependent repression of the mod locus, indicated ModE to be unable to distinguish between the oxyanions.

4.2.3. Anaerobic respiration and nitrate reduction are Mo-dependent

Molybdate plays an essential role in anaerobic respiration where it serves as a component of the molybdenum cofactor in the first step of the nitrate reduction pathway, that - - is, the reduction of NO3 to NO2 by NarGHI (Philippot & Hojberg, 1999; Arai, 2011; Iobbi- Nivol & Leimkuhler, 2013). Consistent with our predictions, the ΔmodA strain, which lacks Mo, did not exhibit significant anaerobic growth over 24 h when supplemented with 100 mM

KNO3 (Figure 4.7). Whilst some growth was observed for the ΔmodA strain (OD600 of 0.25), this was likely to have occurred during the ~2.5 h period required for the growth chamber to become anaerobic. Anaerobic growth in the presence of 100 mM KNO3 was also inhibited for the wild-type strain when supplemented with 10 mM W, and for the ΔmodA strain under similar conditions (10 mM W; Figure 4.7), due to both strains being deficient in Mo accumulation (Figure 4.3B). The inhibited anaerobic growth of PAO1 + W could be restored by supplementation with Mo (PAO1 + W + Mo; Figure 4.7), indicating that it was a lack of Mo accumulation, rather than an inhibitory effect of W that was preventing anaerobic growth. To confirm this, nitrate reductase assays, which measure nitrite production, were performed on 24 h static cultures of ΔmodA, ΔmodA + W, or PAO1 + W. These demonstrated that when Mo accumulation was impaired, nitrate reductase activity was

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Figure 4.6: Transcriptional regulation of PA1864 by ModE. Transcriptional response of putative ModE controlled PA1864 gene to Mo and W availability. Relative expression, corrected for rpoD, was assessed for PAO1 and ΔmodA strain grown in

CDM. Where indicated, Mo (Na2MoO4) and W (Na2WO4) were each supplemented at 10 mM. Data are the mean ± s.e.m., with n ≥ 3 biological replicates. Statistical significant was determined using the two-tailed unpaired Student’s t-test, where ** represents P < 0.01.

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Figure 4.7: Mo depletion limits anaerobic growth. Optical density (600 nm) of static cultures grown anaerobically in CDM supplemented with

100 mM KNO3 for 24 h. Where indicated, Mo and W were supplied at 10 mM as Na2MoO4 and Na2WO4, respectively. Mean ± s.e.m. shown for at least biological triplicates. Statistical significance was determined using the two-tailed unpaired Student’s t-test, where * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.005, and **** represents P < 0.0001.

92 Chapter 4 essentially abrogated (Figure 4.8A). Furthermore, when sufficient intracellular Mo was present, as was the case for PAO1, ΔmodA + Mo, and PAO1 + Mo + W, nitrate reduction was facilitated. Taken together, these data indicate that ModA has an essential role in recruitment of Mo for nitrate reduction, a process prevented by W competing for import by ModA. The ability of P. aeruginosa to reduce nitrate in the presence of W was then examined by performing an anaerobic shift experiment. The wild-type and the ΔmodA strains were grown initially to an OD600 of 0.6, and then shifted to static anaerobic conditions for 2 h. During this transition to anaerobiosis, the cells were forced to switch to nitrate reduction for energy production. Molybdenum limitation in the ΔmodA and ΔmodA + W strains similarly resulted in impaired nitrate reductase activity (Figure 4.8B). The inhibition of nitrate reduction activity in the ΔmodA strain supplemented with W also suggests that W was unable to functionally substitute for Mo within the Mo-bis MGD-dependent NarGHI nitrate reductase complex. Interestingly, the wild-type strain supplemented with W did not exhibit a significant impairment in nitrate reductase activity, with levels recorded similar to PAO1 and PAO1 + Mo + W (Figure 4.8B). When considering the high concentration of W present in the cell (91.57 and 129.4 μg W.g cells-1 for PAO1 + W and PAO1 + Mo + W, respectively), this result indicated that W is not inherently toxic to the nitrate reductase enzymes.

It was then necessary to confirm that the inability of the ΔmodA strain to reduce nitrate was due to Mo deficiency, rather than other effects, such as a lower abundance of nitrate reductase enzyme. Transcriptional analyses of the wild-type and ΔmodA P. aeruginosa strains revealed that the narK1K2GHJI operon was equally up-regulated in both strains during the 2 h anaerobic shift, as indicated by narK1 mRNA levels (Figure 4.9A). Transcription of moaC and nosD (Figure 4.9B and C), two other components of the nitrate reduction pathway, was also assessed, with up-regulation observed in both strains during the nitrate supplemented anaerobic shift (+ NO3 – O2). However, this up-regulation was significantly lower in the ΔmodA strain compared with the wild-type strain, most likely due to the lack of nitrate reduction, which is crucial for activating additional downstream transcription regulators via the production of nitric oxide (Arai et al., 2003). Taken together, these transcriptional analyses further support the crucial role of Mo acquisition in the nitrate reduction pathway and the ability of P. aeruginosa to grow anaerobically.

4.2.4. Nitrate reduction limits biofilm formation and alters cell membrane composition

Biofilm formation by P. aeruginosa is of major significance for the colonization of both biotic and abiotic surfaces (Costerton et al., 1999; Parsek & Singh, 2003). The biofilm matrix provides P. aeruginosa with significant protection from the host immune system, antibiotics,

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Figure 4.8: Mo availability affects nitrate reductase activity. Nitrate reductase activity of (A) 24 h static cultures and (B) 2 h anaerobic shift cultures grown in CDM + 100 mM KNO3. Nitrate reductase expressed in arbitrary units, as the mean ± s.e.m., with experiment performed in biological duplicate on 3 separate occasions. Where indicated, Mo and W were supplied at 10 mM as Na2MoO4 and Na2WO4, respectively. In each figure, statistical significance was determined using the two-tailed unpaired Student’s t- test, where * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.005, and **** represents P < 0.0001.

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Figure 4.9: Transcriptional response to nitrate supplementation and anaerobic conditions. Relative expression levels of (A) PA3877, (B) PA3918 and (C) PA3393, corrected for rpoD, were determined for the PAO1 and the ΔmodA strain grown to

OD600 of 0.6 in CDM. + NO3 indicates cultures were supplemented with 100 mM KNO3 throughout growth, with – O2 denoting 2 h anaerobic shift cultures. Data are mean ± s.e.m., with n ≥ 3 biological replicates. Statistical significance was determined using the two-tailed unpaired Student’s t-test, where * represents P < 0.05, ** represents P < 0.01 and **** represents P < 0.0001.

Chapter 4 shear stress and cationic antimicrobial peptides (Anderson et al., 2008; Mulcahy et al., 2008; Hoiby et al., 2010), thereby facilitating persistence. Consequently, the in vitro biofilm formation capacity of the wild-type and ΔmodA strain was assessed on polycarbonate tubes, in the absence and presence of nitrate (Figure 4.10). Analysis revealed that the inability to acquire Mo did not alter the production of biofilm by the ΔmodA strain in comparison with the wild-type. However, upon supplementation with 100 mM KNO3, thereby facilitating nitrate reduction, the wild-type strain showed a significant 84% reduction in biofilm formation (P = 0.0139), whereas the ΔmodA strain showed no significant change. The reduction in biofilm was also achieved by the ΔmodA strain supplemented with 100 mM

KNO3 and 10 mM Na2MoO4 (P = 0.0012). Taken together these data demonstrate that biofilm formation does not require ModA and hence, by extension, Mo. Furthermore, these data also show that nitrate reduction inhibits biofilm formation and may have a role in driving dispersal, although this requires further investigation.

Transition from the planktonic state to biofilm lifestyle has previously been shown to result in an alteration of the bacterial cell membrane lipid content (Benamara et al., 2011). Consequently, the fatty acid (FA) composition of the wild-type and ΔmodA strains grown with and without KNO3 was assessed (Figure 4.11). These analyses indicated that the P. aeruginosa cell membranes consist predominantly of 16 and 18 carbon FAs, with the saturated 16:0 (16 carbon acyl chain) and monounsaturated 18:1n-7 (18 carbon acyl chain with a double bond in the Δ7 position) FAs each comprising ~30-40% of the total FA pool (Figure 4.11A and E). The FA composition of the wild-type and ΔmodA strains were essentially the same when grown in the absence of nitrate. Interestingly, the nitrate reducing strain (PAO1 + KNO3) exhibited a higher proportion of 16:0, 16:1n-7, and 18:0 compared to the ΔmodA strain supplemented with nitrate (Figure 4.11 A, B and D). Strikingly, major differences in the presence of trans monounsaturated FAs were seen. Culturing of PAO1 +

KNO3 resulted in significantly lower levels (>30-fold) of t16:1 (one trans double bond) and t18:1n-7 (one trans double bond in the Δ7 position) (Figure 4.11 C and F), an effect likely due to differences in cis-trans isomerization. Consequently, the transcription levels of the cis- trans isomerase gene (CTI; PA1846) were assessed. This demonstrated CTI to be up- regulated in strains that do not reduce nitrate (>3-fold; Figure 4.12), corresponding with the higher levels of trans fatty acids (Figure 4.11 C and F). Whilst the presence of unsaturated FAs leads to a more fluid cell membrane, the acyl chains of trans unsaturated FAs are straight, allowing tighter packing and providing increased rigidity. The benefits of a more rigid cell membrane under these conditions are currently unknown.

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Figure 4.10: Anaerobic respiration reduces biofilm formation. Static biofilm formation after 24 h was assessed by 0.1 % crystal violet staining and measuring absorbance at OD595. Mean ± s.e.m. shown for biological duplicates on three separate occasions. Statistical significant was determined using the two-tailed unpaired Student’s t-test, where * represents P < 0.05, and ** represents P < 0.01.

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Figure 4.11: Fatty acid (FA) composition of PAO1 and ΔmodA strains. The major constituents of cellular fatty acids are depicted, indicating differences in relative abundance between PAO1 and the ΔmodA strain grown in CDM with (+NO3) and without

100 mM KNO3. (A) 16:0, saturated FA of 16 carbon chains. (B) 16:1n-7, a monounsaturated FA of 16 carbons, with a cis double bond at the 7th carbon. (C) t16:1, a trans monounsaturated fatty acid with 16 carbons. The position of the double bond is unable to be determined. (D) 18:0, saturated FA of 18 carbon chains. (E) 18:1n-7, a monounsaturated FA of 18 carbons with a cis double bond at the 7th carbon. (F) t18:1n-7, a trans monounsaturated fatty acid of 18 carbons, with the double bond at the 7th carbon. The data are the mean ± s.e.m., with n ≥ 3 biological replicates. Statistical significance was determined using the two- tailed unpaired Student’s t-test, where * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.005, and **** represents P < 0.0001.

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Figure 4.12: Cis-trans isomerase (CTI) expression. Relative mRNA expression levels of CTI (PA1846) were assessed for 24 h static cultures of

PAO1 and ΔmodA, corrected for 16S rRNA abundance. The + NO3 notation indicates cultures were grown with 100 mM KNO3. Data are the mean ± s.e.m., where n ≥ 3 biological duplicates. Statistical significance was determined using the two-tailed unpaired Student’s t- test, where * represents P < 0.05.

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In contrast to the observed variations in CTI expression, most studies to date have suggested that pseudomonal CTIs are constitutively expressed (Kiran et al., 2005). However, resistance to hydrocarbons, in particular toluene, has previously been linked with the isomerization of cis to trans FAs through an up-regulation of CTI (up to 1.4-fold) in P. putida (Bernal et al., 2007). On this basis, P. aeruginosa strains unable to reduce nitrate were analysed to determine whether a lack of nitrate reduction, and hence up-regulation of CTI and trans FA abundance, provided P. aeruginosa with the ability to associate with hydrocarbons via increased hydrophobicity. To do so, P. aeruginosa was incubated with xylene in a bacterial adhesion to hydrocarbons (BATH) assay. In agreement with previous observations made by Bernal et al. (2007), a greater abundance of trans FAs correlated with the ability of cells to associate with the hydrophobic xylene phase, in contrast to cells containing very low levels of trans FAs (Figure 4.13), which associated with the aqueous/hydrophilic buffer. Collectively, these data show that cells unable to reduce nitrate have a distinct FA composition compared to those capable of nitrate reduction, with this difference attributable to the differential expression of CTI. Consequently, this may facilitate an enhanced tolerance to hydrocarbons and assist with the persistence of the bacterium in diverse environments.

Discussion

Molybdate is an essential transition element for almost all forms of life. This study has characterized the PA1861-PA1863 operon, and provided direct evidence for its role as the Mo ABC import system, modABC, of P. aeruginosa. Deletion of the SBP component of the ABC permease, ModA, abrogated Mo accumulation. This impairment was consistent with characterization of other ABC permeases, in which transport is dependent on the SBP to deliver the cargo for cellular uptake. Detailed primary sequence analysis of ModA also supported its classification within the cluster D-III subgroup of ABC transporter associated SBPs, together with most other Mo-recruiting SBPs, the one exception being the MolA cluster A SBP (Tirado-Lee et al., 2011). The limited bioavailability of Mo in some environmental niches has led to certain organisms also utilizing W due to its capacity for similar redox chemistry. Biochemical characterization of P. aeruginosa ModA, performed by Dr. Miranda Ween (Research Centre for Infectious Diseases, University of Adelaide), also revealed that, similar to ModA from E. coli (Imperial et al., 1998), the SBP was also competent for interaction with W (Pederick et al., 2014) (Appendix 4). As W has no known role in Pseudomonas, and the relative environmental abundance of W is of the order of 100- 100 Chapter 4

Figure 4.13: Nitrate reduction effects Pseudomonas membrane hydrophobicity. The bacterial adherence to hydrocarbons (BATH) assay was performed using xylene. Changes in membrane lipid hydrophobicity are reflected in altered adherence to xylene. The +

NO3 notation indicates cultures were grown with 100 mM KNO3. Data represents the mean ± s.e.m., where n ≥ 3 biological duplicates grown on 2 separate days. Statistical significance was determined using the two-tailed unpaired Student’s t-test, where *** represents P < 0.005.

101 Chapter 4 fold lower in most environments, this interaction is most likely non-physiological (Schwarz et al., 2009). Furthermore, as P. aeruginosa is unlikely to encounter significant concentrations of W in its native environments, the capacity of ModA to interact with W does not appear to exert a fitness cost on the bacterium. Thus, the findings herein, together with the biochemical characterisation of ModA in Pederick et al. (2014), highlight an apparent limitation in how ModA, and related cluster D SBPs, discriminate between structurally similar oxyanions. Overall, this observation is consistent with a growing body of evidence, which indicates that the selectivity of the SBPs is ultimately constrained by the composition of the ligand-binding site. That is, the chemical properties of the side chains required to co-ordinate the cognate ligand, e.g. Mo, also permit other with similar structural or chemical characteristics, e.g. W, to interact with the SBP. The work detailed here and the biochemical characterization of ModA in Pederick et al. (2014) shows that W is capable of competing with Mo for P. aeruginosa ModA and results in cellular Mo depletion.

Although W is capable of similar redox chemistry, it does not appear to be able to functionally replace Mo in the NarGHI nitrate reductase complex. Whether this arises from issues in the formation of the oxyanion-derived cofactor or due to the inability of NarG to utilize a W-cofactor remains to be determined. Rather this study suggests that W exerts toxicity towards P. aeruginosa via competition with Mo for binding to ModA, and repression of modABC transcription via potentially substituting for Mo in ModE. Together, these behaviours lead to depletion of intracellular Mo and impairment of the organism’s capacity to further acquire the oxyanion.

This study has shown that Mo acquisition is directly linked to anaerobic nitrate reduction for in vitro growth. Interestingly, biofilm formation was observed to be inhibited when P. aeruginosa employed anaerobic nitrate respiration. Furthermore, anaerobic cultures grown in vitro in the presence of nitrate did not form biofilms after 24 h (data not shown). Nitric oxide, a product of nitrate reduction, has been shown to act as a signaling molecule when present at sub-lethal concentrations, driving dispersal of biofilms (Barraud et al., 2006). Consequently, this by-product of nitrate respiration could be responsible for the inhibition of biofilm formation observed.

The effect of Mo accumulation and nitrate reduction upon the fatty acid composition of Pseudomonas cell membranes has not, on the basis of literature searches, been previously investigated. Consequently, the impact of these environmental effects on trans fatty acid content was investigated. Cis-trans isomerization has previously been reported in association with environmental stresses such as elevated temperatures or the presence of toxic substances

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(Heipieper et al., 2003). Similar to saturated FAs, trans FAs are capable of more dense packing within the membrane, making it more rigid and less permeable. In this study, it was observed that the inability to reduce nitrate was associated with a higher trans FA content. The isomerization of pre-existing cis to trans FAs may be a stress response of cells unable to reduce nitrate due to a lack of Mo. Accordingly, transcriptional analysis demonstrated the gene encoding CTI, the enzyme responsible for converting cis FAs to trans FAs, to be up- regulated in strains unable to reduce nitrate, providing an explanation for the higher trans FA content. Although this finding contradicts a previous report, which indicated that CTI was constitutively expressed (von Wallbrunn et al., 2003), these results indicate that cis-trans isomerization of the P. aeruginosa FAs may be induced as a component of cellular stress response mechanisms. However, the consequence of such changes in the fatty acid composition, and consequently membrane fluidity and hydrophobicity, requires further investigation.

In addition to elucidating the consequences of Mo depletion upon nitrate reduction, cell membrane properties and biofilm formation, the wider impact of Mo depletion on the previously undefined P. aeruginosa ModE regulon was explored. Based on the generated ModE binding site motif, 28 potential target loci were identified as part of the PAO1 ModE regulon. However, subsequent transcriptional analysis revealed that only four of these loci were differential transcribed (≥2-fold) under Mo-depleted conditions (modC, PA1864, PA5296 and PA1503). The disparity in transcriptional response of genes with putative ModE binding sites was not anticipated, given the strong E-values of the 28 targets tested. However, examination of the ModE motif and putative binding sites provides a possible explanation. The motif has a highly conserved thymine nucleotide at position 22 of the sequence. In the ModE binding site adjacent to PA1864 (E-value = 0.00013), a cytosine was substituted for the thymine nucleotide of the motif. This variation was associated with PA1864 showing the strongest response to Mo depletion, exhibiting up to 35-fold up-regulation. In contrast, the ModE binding site adjacent to the modABC operon, which had a similar E-value (0.00017), yet contained a thymine nucleotide at position 22, exhibited only a ~3.5-fold level of up- regulation. Whether the substitution of cytosine for the conserved thymine nucleotide is a contributing factor to the amplitude of differential expression via ModE remains to be determined. The ModE binding sites of the differentially expressed genes (PA5496 and PA1503) maintained the thymine at position 22. By contrast, another putative ModE target gene, PA4881, has an adenine substituted for thymine at position 22 of its ModE binding site, although no differential expression was observed in response to Mo depletion. Consequently,

103 Chapter 4 the ModE motif contains a hitherto unrecognized level of complexity with respect to how variation in nucleotide composition of the binding site sequence exerts an effect on the strength of transcriptional response. A more detailed analysis would be required to confirm the putative targets of the proposed PAO1 ModE regulon and to elucidate how sequence variations, for example at nucleotide 22, correlate with transcriptional activation or repression. Bioinformatic analysis of PA1864 suggests it belongs to the TetR family of transcriptional regulators, of which there are 37 other members in P. aeruginosa PAO1 (Ramos et al., 2005). This family has a broad range of functions, including regulation of drug efflux and quorum sensing (Ramos et al., 2005). PA1864 shares significant homology with the putative transcriptional regulator PA1504 and the RutR TetR-family transcriptional regulator from E. coli (53% and 40% identity, respectively). RutR is involved in the regulation of pyrimidine transport and catabolism, facilitating use of pyrimidines as a nitrogen source (Loh et al., 2006; Shimada et al., 2007). A deletion mutant of rutR has been shown to result in a decrease in expression of the car operon encoding critical enzymes of the pyrimidine and arginine biosynthesis pathways, tRNA synthetases, and amino acid transporters (Gupta et al., 2012). Intriguingly, the rutR deletion mutant also exhibited a hypersecretion phenotype (Gupta et al., 2012). Whether PA1864 holds a regulatory role in P. aeruginosa, similar to that of RutR, remains to be determined. However, PA1864 has been shown to be up-regulated in response to exposure to epithelial cells, suggesting a possible role in host colonization (Chugani & Greenberg, 2007). Further genetic and phenotypic studies will be required to determine the role of PA1864 in P. aeruginosa and to elucidate its contribution to Mo-dependent cellular processes.

Conclusions

This study provides a detailed characterization of ModABC, the primary Mo uptake pathway of P. aeruginosa. Phenotypic analysis has indicated that the toxicity of W is multifaceted, and is mediated via the limitation of Mo import and likely via the substitution for Mo in ModE transcriptional regulation. Analysis of the putative ModE operon has also identified a novel Mo-responsive transcriptional regulator, PA1864, the role of which remains intriguing and is worthy of further investigation. Finally, this work has shown anaerobic nitrate respiration to modulate both biofilm formation and the FA composition of the bacterial cell membrane. These properties, both of which facilitate the survival of P. aeruginosa in diverse and dynamic environments, have major implications for understanding how this bacterium adapts to environmental stimuli and may be applicable to other Gram-negative environmental organisms and opportunistic pathogens.

104

CHAPTER 5:

ZINC HOMEOSTASIS IN

P. AERUGINOSA

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Introduction

Zinc is the second most abundant transition metal ion in biological organisms, present as the divalent ion, Zn2+ (Hantke, 2005). Recent bioinformatic studies have predicted that 5- 6% of all prokaryotic proteins are predicted to bind Zn2+, although it is likely that the physiological distribution is much higher (Andreini et al., 2006). The prevalent utilization of Zn2+ in biological systems can be attributed to the ability of the metal ion to serve structural and catalytic roles, where, in the latter, it can act as both a Lewis acid and an electrophile (Hantke, 2005; Andreini et al., 2008). By contrast with other first-row transition metal ions, i.e. iron and copper, Zn2+ lacks redox activity due to its completely filled d-shell. Despite this, Zn2+ is still able to mediate significant toxicity by inappropriately binding to the metal binding sites of proteins or DNA, thereby perturbing or inhibiting their function (Bruins et al., 2000; Colvin et al., 2010; Botella et al., 2011; McDevitt et al., 2011; Botella et al., 2012).

Efficient Zn2+ homeostasis is critical for survival in environments of changing Zn2+ abundance. As an environmental , P. aeruginosa is exposed to niches that have widely varying Zn2+ content and bioavailability, such as different soils, plants and waterways. The Zn2+ content of soils and plants is highly variable depending on numerous geological and weather factors. Typical Zn2+ abundance in soils and plants ranges, on average, between 15 and 200 mg Zn2+ per kg (dry weight) (Lepp, 1981; Sillanpää, 1990; Markert, 1996). However, more extreme variations have been reported. Variation in Zn2+ environmental abundance and bioavailability necessitates adaptability on the part of P. aeruginosa to cope with fluctuations in extracellular Zn2+ abundance. However, this adaptability is also of great importance in host colonization and disease pathogenesis. Hosts, including humans, employ nutritional immunity as part of their innate defence, wherein they restrict the bioavailability of Zn2+ during the initial stages of infection (Hood & Skaar, 2012; Cerasi et al., 2013). This is achieved through the use of chelating proteins such as calprotectin and psoriasin, which sequester Zn2+ and limit its availability to the bacteria, hampering colonization (Glaser et al., 2005; Kehl-Fie & Skaar, 2010). By contrast, at later stages of infection, significant fluxes in Zn2+ occur, which have been associated with Zn2+-toxicity towards the pathogen (Cerasi et al., 2013). To combat these fluctuations, P. aeruginosa utilizes an intricate homeostatic network to modulate cellular responses to the varying Zn2+ abundance in the extracellular environment and ensure precise intracellular Zn2+ management.

Survival, colonization and pathogenesis require the acquisition of an appropriate concentration of Zn2+. As the bacterial cell membrane serves as an impermeable barrier to

106 Chapter 5 metal ions, the acquisition or efflux of Zn2+ is achieved through the use of membrane transporters. The specific, high affinity acquisition of Zn2+ was first characterised in the Gram-negative bacterium, E. coli in 1998 (Patzer & Hantke, 1998). E. coli was shown to import Zn2+ using a Zn2+-specific ABC permease, ZnuABC, which consists of the ZnuA solute-binding protein (SBP) and an ABC transporter, comprising two ZnuB transmembrane proteins and two ZnuC nucleotide-binding domains. The ZnuA SBP was shown to be Zn2+- 2+ specific, delivering bound Zn ions to its cognate ZnuB2C2 transporter embedded in the inner membrane. High-resolution structural analysis of ZnuA resulted in its classification as a cluster A-I SBP, sharing a two domain globular architecture of (α/β)4 folds linked by a long alpha helix (Li & Jogl, 2007; Berntsson et al., 2010). Cluster A-I SBPs bind their cognate ligand in the cleft between the two domains, resulting in a structural rearrangement. As sequenced genomes of other eubacteria became available, ZnuABC homologs were identified in numerous Gram-positive and Gram-negative organisms, with ZnuA being lipid-anchored in Gram-positive organisms, as opposed to freely diffusible in the periplasm of Gram-negative bacteria (Lewis et al., 1999; Chen & Morse, 2001; Campoy et al., 2002; Davis et al., 2009; Dahiya & Stevenson, 2010; Desrosiers et al., 2010; Murphy et al., 2013). Whilst ZnuABC serves as the primary, high-affinity Zn2+ acquisition pathway, other transporters aid in Zn2+ recruitment and uptake, including the low affinity ZupT transporter from the ZIP family of divalent cation transporters (Grass et al., 2002; Grass et al., 2005), and a related ABC permease that utilizes the TroA cluster A-I SBP (Desrosiers et al., 2007). Intriguingly, although TroA-like cluster A-I SBPs show high sequence similarity to Zn2+-specific SBPs, i.e. ZnuA, they have the capacity to interact with a broader range of divalent cations in vitro. As a consequence, their precise physiological role remains to be elucidated.

Zinc-specific cluster A-I SBPs from Gram-positive organisms have a number of structural differences from Gram-negative SBPs, despite fulfilling an identical functional role. The best characterised Gram-positive Zn2+ recruiting cluster A-I SBPs belong to the Streptococcus genus. In S. pneumoniae the ZnuBC homolog, AdcBC, operates with two cluster A-I SBPs, AdcA, which is encoded in the same operon as AdcCB, and AdcAII, which is encoded elsewhere in the genome (Loisel et al., 2008; Bayle et al., 2011; Plumptre et al., 2014a). The ‘orphan’ Zn2+-recruiting SBP, AdcAII, has a canonical cluster A-I fold, although it lacks the histidine-rich loop present in Gram-negative Zn2+-specific SBPs (Loisel et al., 2008). By contrast, the primary SBP, AdcA, has a two-domain organisation, comprising the cluster A-I SBP domain at its N-terminus and a domain with homology to the Gram-negative protein ZinT, at the C-terminus (Plumptre et al., 2014a). In Gram-negative organisms, ZinT

107 Chapter 5 has been proposed to aid in delivery of Zn2+ to ZnuA (Petrarca et al., 2010; Ilari et al., 2014). Hence, from a functional perspective, AdcA appears to be a fusion of two separate periplasmic proteins, ZnuA and ZinT, which presumably allows their cooperative role in Gram-negative organisms to be fulfilled at the outer face of the cell membrane in S. pneumoniae. Although either Zn2+-recruiting SBP is sufficient for viability, together AdcA and AdcAII serve complementary roles in Zn2+ acquisition with the assistance of the cell surface-associated polyhistidine triad (Pht) proteins (Loisel et al., 2011; Bersch et al., 2013; Plumptre et al., 2014b), which aid in Zn2+ delivery to the SBPs. Finally, an alternative Zn2+ acquisition pathway has recently been elucidated in Yersinia pestis. In addition to its canonical ZnuABC permease, Y. pestis utilizes the siderophore yersiniabactin to import chelated Zn2+ through the major facilitator superfamily transporter, YbtX (Bobrov et al., 2014). The two systems were shown to be functionally redundant in a septicaemic plague model, both contributing to the high affinity acquisition of Zn2+ in Y. pestis. Collectively, Zn2+ can be acquired by a variety of uptake mechanisms. However, to date, the Znu type- permeases are the most prevalent and high-affinity Zn2+ import systems in prokaryotes.

Zinc homeostasis is controlled by metalloregulatory proteins, such as Zur from E. coli, which is a Zn2+-specific regulatory protein, belonging to the ferric uptake regulator (Fur) family of transcriptional regulators (Patzer & Hantke, 2000). Zur is a homodimeric transcriptional repressor, which upon binding at least two Zn2+ ions, binds cognate Zur sites in promoters and represses transcription (Patzer & Hantke, 2000). Key amongst Zur targets, are 2+ 2+ znuABC, with the presence of available Zn within the cell resulting in Zn -Zur2 repression of the znuABC genes. In this way the cell prevents further uptake of Zn2+. Conversely, during conditions of Zn2+ limitation, znuABC is de-repressed and the produced ZnuABC is available for Zn2+ acquisition. In some species, the zur gene is localised adjacent to the znuABC genes (Stover et al., 2000; Ellison et al., 2013; Lim et al., 2013; Mortensen et al., 2014). To complement Zur-repression, the cell also utilizes Zn2+ efflux to prevent Zn2+ toxicity. Efflux of Zn2+ occurs through a range of different systems, including the CzcCBA RND pump, ZitB and CzcD cation diffusion facilitator pumps, and the ZntA P-type ATPase transporter (Silver, 1996; Rensing et al., 1997; Guffanti et al., 2002; Legatzki et al., 2003). Each efflux system is tightly controlled by a cognate transcriptional regulator (Singh et al., 1999; Kloosterman et al., 2007; Wang et al., 2012). Although the absolute accumulation of Zn2+ varies depending on the organism, studies of E. coli indicated that the concentration of ‘free’ or labile Zn2+ present in the cytoplasm was extraordinarily low (in femtomolar range). In the context of E. coli, this is ~6 orders of magnitude less than a single Zn2+ atom and suggests that

108 Chapter 5 intracellularly, all Zn2+ ions are buffered or sequestered (Outten & O'Halloran, 2001). Through the co-ordinated and intricate acquisition and efflux of Zn2+, bacteria are capable of maintaining the required intracellular level of Zn2+ to support growth, colonization and pathogenesis.

Previous studies examining the uptake and role of metal ions in P. aeruginosa have focused primarily on iron, with a wealth of literature now available (Andrews et al., 2003; Imperi et al., 2009; Schalk et al., 2011; Brandel et al., 2012; Brillet et al., 2012; Hannauer et al., 2012; Konings et al., 2013; Schalk & Guillon, 2013). However, there is a paucity of data regarding the acquisition and roles of Zn2+ in the bacterium. Herein, Zn2+ is determined to comprise approximately 10% of the total cellular transition metal content of P. aeruginosa, highlighting its importance within the cell. This, together with the effort on the part of the innate immune system to restrict or flood invading bacteria with Zn2+ during infection, suggests the role of Zn2+ in P. aeruginosa requires further investigation. The work presented provides a thorough characterisation of the P. aeruginosa Zn2+ acquisition pathways, and offers key insight into the effect of Zn2+ limitation upon cellular physiology.

Results

5.2.1. ZnuA contributes to P. aeruginosa Zn2+ acquisition

Bacterial Zn2+ uptake occurs primarily via high affinity ABC permeases, such as ZnuABC from E. coli (Figure 5.1), with homologs now characterised in numerous bacterial species (Lewis et al., 1999; Chen & Morse, 2001; Campoy et al., 2002; Yang et al., 2006; Desrosiers et al., 2007; Davis et al., 2009; Dahiya & Stevenson, 2010; Murphy et al., 2013). Analysis of the P. aeuringosa PAO1 genome revealed the presence of three genes homologous to znuABC: PA5498 (znuA), PA5500 (znuB), and PA5501 (znuC) (Figure 5.2) (Stover et al., 2000). The znuA gene is present in a separate operon to the transporter components (znuB and znuC), under the control of the Zur transcriptional regulator (Ellison et al., 2013). Zur is encoded immediately upstream of znuB and znuC, with the three genes forming a Zur-regulated operon (Ellison et al., 2013). PA5502, encoding a putative lipoprotein, is present following znuC, but this gene is transcribed independently of zur and znuBC (Ellison et al., 2013), suggesting it does not play a role in Zn2+ acquisition. A primary sequence alignment of the putative P. aeruginosa ZnuA with characterised ZnuA proteins revealed a high degree of homology (Figure 5.3). Additionally, comparison of the putative P. aeruginosa ZnuA sequence with a high-resolution structure of E. coli ZnuA

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Figure 5.1: The Zn2+ specific ZnuABC permease. A schematic representation of ZnuA binding Zn2+ in the periplasm, and delivering the metal ion to the ZnuBC transporter. The ZnuBC transporter is comprised of two copies of the ZnuB, embedded in the inner membrane (IM), and two associated cytoplasmic ZnuC proteins. ATP hydrolysis by the ZnuC proteins provides the energy for transport of Zn2+ into the cytoplasm. The outer membrane is indicated by OM.

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Figure 5.2: Organisation of the P. aeruginosa ZnuABC regulon. A schematic representation of the zur transcriptional regulator and znuABC permease genes from P. aeruginosa PAO1. Components of the ABC permease complex are indicated in shades of blue. The Zn2+ uptake regulator (zur) gene is in red. The putative Zur-binding site that facilitates transcriptional repression in both orientations is indicated (black). Genes are to scale.

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Figure 5.3: Sequence alignment of ZnuA proteins. Primary sequence alignment of P. aeruginosa ZnuA, and ZnuA orthologs from other bacterial species. Zinc binding residues are indicated in red, based on structural and/or biochemical analyses (Banerjee et al., 2003; Chandra et al., 2007; Li & Jogl, 2007; Yatsunyk et al., 2008; Ilari et al., 2011), with the His- rich loop region indicated in blue. The presence of a signal sequence cleavage site (as determined by SignalP 4.0) is indicated by a green line (Petersen et al., 2011). The aligned ZnuA proteins are from: Pae, P. aeruginosa (GI:15600691); Hin, H. influenzae (GI:491963406); Yru, Y. ruckeri (GI:490857750); Sen, S. enterica (GI:541470409); Eco, E. coli (GI:635897169); Tpa, T. palladium (GI:504108253); Cje, C. jejuni (GI:504330062); Syn, Synechocystis 6803 (GI:499174152)

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(PDB: 2OGW.1.A) using SWISS-MODEL (Biasini et al., 2014), indicated the putative ZnuA

to possess the characteristic cluster A-I bi-lobed (α/β)4 fold secondary structure (Figure 5.4), further implicating its role in Zn2+ recruitment. To assess the function of the putative znuA in P. aeruginosa, a znuA (PA5498) deletion mutant (ΔznuA) was constructed. Whole cell ICP- MS analysis of the P. aeruginosa wild-type and ΔznuA strain revealed a significant 61.9% decrease in cellular Zn2+ through loss of the SBP (Figure 5.5). This indicated the P. aeruginosa znuA gene was associated with Zn2+ recruitment and, based on the primary sequence and secondary structure analyses, was predicted to encode a Zn2+-specific cluster A- I SBP. Disruption of the Znu permease had no impact on the cellular accumulation of other transition metal ions, apart from cobalt, which increased in cellular abundance by 420% (P < 0.0001; Figure 5.5). These data suggest that the Zn2+ regulatory and/or homeostatic mechanisms are also linked with P. aeruginosa cobalt homeostasis. However, overall cobalt is a minor constituent of the intracellular transition metal ion content (<1%). Collectively, deletion of znuA and the resulting decrease in intracellular Zn2+ abundance indicates ZnuA, and by inference the ZnuABC permease, to be the primary Zn2+ acquisition pathway in P. aeruginosa. However, as P. aeruginosa is still able to acquire Zn2+ in the absence of znuA, the observed Zn2+ accumulation can be attributed to alternative uncharacterised Zn2+ acquisition pathways.

5.2.1. ZnuA is a high affinity Zn2+ binding protein

To ascertain whether P. aeruginosa ZnuA was a high affinity Zn2+ binding protein, biochemical characterisation was undertaken. Primary sequence analysis of znuA revealed the presence of a putative Sec-type signal peptide, which would be cleaved at residue 24 during its secretion into the periplasm (Petersen et al., 2011) (Figure 5.3). To prevent recombinant ZnuA from being secreted, or causing toxicity in E. coli via obstruction of the Sec translocon, the znuA gene was PCR amplified without the signal sequence. This facilitated expression of a recombinant mature ZnuA isoform that was incompetent for secretion. The PCR product was cloned into a C-terminal dodecahistidine ligation independent cloning vector (pCAM- cLIC01) to produce pCAM-cLIC01-ZnuA (Table 2.3). Recombinant ZnuA was expressed in E. coli and purified by use of a C-terminal dodecahistidine tag using immobilised metal ion affinity chromatography (IMAC) (Figure 5.6). Homogeneity and monodispersity were assessed by gel permeation chromatography (Figure 5.7A and B), and comparison with calibration standards of known molecular mass (Figure 5.7C) provided a molecular size of 34.5 kDa, which matched closely with the predicted molecular mass of monomeric

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Figure 5.4: Predicted secondary structure of P. aeruginosa ZnuA. The predicted secondary structure of P. aeruginosa ZnuA was determined using SWISS-MODEL (Biasini et al., 2014) and an alignment with a high-resolution structure of E. coli ZnuA (PDB: 2OGW.1.A). Alpha helices are represented in purple and beta sheets are represented in yellow. Unstructured regions are in grey. The putative Zn2+ co-ordinating resides of the primary binding site and the His-rich loop of P. aeruginosa PAO1, based on the sequence alignment (Figure 5.3) are indicated in orange.

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Figure 5.5: Deletion of znuA reduces P. aeruginosa Zn2+ accumulation.

In vitro accumulation of metals by wild-type PAO1 (black) and ΔznuA cultures (red) was assessed via growth in CDM. Metal content was expressed as g of metal per gram of dry cells, as determined by ICP-MS. Data correspond to mean ± s.e.m., where n ≥ 6. Statistical significance was determined using the two-tailed unpaired Student’s t-test, where **** represents P < 0.0001.

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Figure 5.6: Purification of recombinant ZnuA. Immobilised metal ion affinity chromatography (IMAC) was used to purify recombinant ZnuA by virtue of its C-terminal dodecahistidine tag. (A) The elution profile shows ZnuA to elute with 600 mM imidazole. Protein concentration is represented by absorbance at 280 nm (blue), with imidazole concentration also indicated (red). (B) Coomassie-stained 4-12% SDS- PAGE analysis of IMAC purification of ZnuA (indicated by arrow) by comparison with soluble molecular mass standards. Fraction volumes are indicated above the lanes. 117 Chapter 5

Figure 5.7: Molecular mass determination of recombinant ZnuA. (A) Absorbance (280 nm) trace of gel permeation chromatography of purified recombinant ZnuA. (B) Coomassie-stained 4-12% SDS-PAGE analysis of monomeric ZnuA (indicated by arrow) by comparison with soluble molecular mass standards. Fractions analysed were 0.5 mL, with elution volume at start of each fraction indicated above the lane. (C) Linear regression of soluble molecular mass standards used to determine the apparent molecular mass of ZnuA. ZnuA was determined to be monomeric with a molecular mass of 34.5 kDa.

118 Chapter 5 dodecahistidine-tagged ZnuA (34.4 kDa). Together, these data indicated that the recombinant mature ZnuA was expressed and occurred as a monomeric species in solution.

Canonical ZnuA proteins contain one high affinity Zn2+-binding site, in addition to a flexible histidine-rich loop, which can potentially bind one or more Zn2+ atoms, but with much lower (~3-4 orders of magnitude) affinity. This region is variable in length, ranging from 12 amino acids (T. pallidum ZnuA) to 50 amino acids (H. influenzae ZnuA) (Lu et al., 1997; Desrosiers et al., 2007). The high affinity metal binding site is composed of three histidine residues that provide the co-ordinating nitrogen atoms, and an oxygen atom provided by either a carboxylate residue or water molecule (Banerjee et al., 2003; Chandra et al., 2007; Li & Jogl, 2007; Yatsunyk et al., 2008; Ilari et al., 2011). Sequence analysis of the P. aeruginosa ZnuA protein identified the presence of the three conserved His residues (His70, His140 and His204) predicted to be required for Zn2+ co-ordination at the primary binding site (Figure 5.3). As the P. aeruginosa ZnuA does not feature an oxygen-contributing residue adjacent to the first histidine as observed in some E. coli structures (Li & Jogl, 2007), the co-ordinating oxygen is likely to be supplied by a water molecule. Furthermore, P. aeruginosa ZnuA also features a 15 residue histidine-rich loop resembling that observed in other canonical ZnuA proteins (Figure 5.3). Based on the combination of structural features, ZnuA would be predicted to bind one Zn2+ atom with high affinity and potentially more Zn2+ atoms with lower affinity. Due to the Zn2+-co-ordinating capacity of histidine residues, the dodecahistidine tag of the recombinant ZnuA was removed prior to Zn2+ binding analyses of the protein. Purified 3C Human Rhinovirus protease was incubated with ZnuA to cleave off the histidine tag by virtue of an engineered 3C site, prior to purification of the cleaved ZnuA by IMAC. Cleaved recombinant ZnuA was then assessed for its ability to interact with Zn2+ in an equilibrium metal binding assay, wherein the protein was mixed with a 10-fold molar excess of ZnSO4 to permit binding, followed by removal of unbound Zn2+. Metal binding to the protein was assessed by ICP-MS and revealed that ZnuA bound 1.8 ± 0.1 moles of Zn2+ per mole of ZnuA. As the high affinity site is competent for interaction with only a single Zn2+ atom, these data suggest the presence of an additional Zn2+-binding region. This is entirely consistent with studies of the His-rich loop, which has previously been suggested to be involved in lower-affinity Zn2+ interactions (Yatsunyk et al., 2008), with possible roles in delivery of Zn2+ to the primary binding site or in release to ZnuB (Banerjee et al., 2003). Similar metal binding studies of E. coli ZnuA, with an approximately 5-fold molar excess of Zn2+, found that the protein bound ~1.85 moles of Zn2+ per mole of ZnuA (Yatsunyk et al., 2008), while in H. influenzae the ZnuA homolog, Pzp1, bound 1.6-1.9

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Zn2+ atoms per protein molecule (Lu et al., 1997). Collectively, analysis of P. aeruginosa ZnuA revealed the protein to be competent for Zn2+ binding at a ratio of 1.8 moles of Zn2+ per mole of ZnuA, suggesting binding in addition to the predicted high affinity site.

Consequently, the affinity of the primary ZnuA binding site for Zn2+ was assessed using the 4-(2-pyridylazo)resorcinol (PAR) assay (Hunt et al., 1985) (Section 2.6.4). In solution (pH 7.4), PAR is calculated to form 1:1 and 2:1 complexes with Zn2+ with stepwise affinity constants of 9.7 × 106 and 6.1 × 105 M−1, respectively. Therefore, an excess of PAR was used 2+ to ensure that Zn(PAR)2 was the only Zn complex in solution prior the addition of protein. 2+ PAR exhibits absorbance at 500 nm when it is bound to Zn as Zn(PAR)2. As ZnuA is added 2+ to Zn(PAR)2, it sequesters the Zn , decreasing the absorbance at 500 nm and providing an affinity of ZnuA for Zn2+. ZnuA exhibited a high affinity for Zn2+ with a dissociation constant of 10 nM (Figure 5.8). This is consistent with the high affinity of other ZnuA homologs for Zn2+ (Wei et al., 2007; Yatsunyk et al., 2008) (≤10 nM). Taken together, these analyses demonstrate the P. aeruginosa ZnuA to be a high affinity Zn2+ binding SBP, which together with ZnuBC forms the primary Zn2+ acquisition pathway.

5.2.2. Deletion of ZnuA does not result in major growth perturbation

The efficient acquisition of transition metal ions such as manganese, zinc and iron, is essential for cellular viability and propagation. Zinc ions act as metal cofactors in a large proportion of bacterial proteins, including RNA polymerases, ribosomal proteins, transcription factors and enzymes (Vallee & Auld, 1990; Coleman, 1992; Laity et al., 2001; Makarova et al., 2001; Hensley et al., 2011). Furthermore, deletion of components of the ZnuABC permease from S. enterica, C. jejuni, Moraxella catarrhalis, B. abortus and Y. pestis, resulted in a significant growth defect in Zn2+-limited media (Campoy et al., 2002; Yang et al., 2006; Ammendola et al., 2007; Davis et al., 2009; Desrosiers et al., 2010; Murphy et al., 2013). Deletion of the P. aeruginosa high affinity SBP, znuA, resulted in a 61.9% reduction in cellular Zn2+ and was predicted to be accompanied by an alteration in the growth phenotype. Prior growth analysis of a PAO1 ΔznuA strain in ethylenediaminetetraacetic acid (EDTA)-treated media revealed a small perturbation in cell density following overnight growth, likely as a result of overall transition metal deficiency (Ellison et al., 2013). However, rigorous P. aeruginosa growth analyses of wild-type and ΔznuA strains in Zn2+-limiting Chelex-100 chemically defined media (CDM; 800 nM Zn2+) revealed no apparent differences (Figure 5.9A). Further analyses were carried out using

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Figure 5.8: Determination of the affinity of ZnuA for Zn2+ at its primary binding site.

The absorbance of Zn(PAR)2 at 500 nm was monitored in response to the addition of increasing concentrations of apo-ZnuA. Assay was performed in triplicate, with a representative curve shown.

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Figure 5.9: The effect of znuA deletion on growth in CDM. Wild-type PAO1 (black) and the isogeneic ΔznuA strain (red) were compared for growth by absorbance at 600 nm. (A) CDM cultures without TPEN supplementation. (B) CDM overnight seed cultures were supplemented with 10 μM TPEN, prior to growth analyses in unsupplemented CDM. (C) CDM overnight seed cultures were supplemented with 10 μM TPEN, prior to growth analyses in CDM supplemented with 5 μM TPEN. (D) CDM overnight seed cultures were supplemented with 10 μM TPEN, prior to growth analyses in CDM supplemented with 10 μM TPEN. Data are the mean ± s.e.m., with n ≥ 3.

122 Chapter 5 overnight CDM cultures supplemented with the chelating agent N,N,N′,N′-tetrakis(2- pyridylmethyl)ethylenediamine (TPEN), which has a preference for Zn2+ over other divalent cations (Arslan et al., 1985; Shumaker et al., 1998), then subsequently grown in CDM without further TPEN supplementation or with 5 or 10 μM TPEN (Figure 5.9B-D). No significant differences in growth were observed between the two strains when subsequently cultured without TPEN supplementation or with 10 μM TPEN-treated CDM. However, a slight growth perturbation of the ΔznuA strain was observed in the exponential phase when cells were subsequently cultured in the 5 μM TPEN-treated CDM (Figure 5.9C). Taken together, these data show that P. aeruginosa PAO1 is able to acquire adequate Zn2+ for cellular processes despite the lack of ZnuA, even when Zn2+ is present at low, sub-micromolar extracellular concentrations. This suggests the presence of at least one alternative high affinity Zn2+ uptake system(s) to ZnuABC that is able to support normal growth under Zn2+-limiting conditions.

5.2.3. Zn2+ depletion exhibits minimal effects on cellular processes and physiology

Although deletion of znuA did not perturb the growth phenotype of PAO1 in Zn2+- limited media, other cellular processes may be impacted by the reduced Zn2+ accumulation. P. aeruginosa PAO1 readily forms biofilms on surfaces, enhancing persistent colonization (Costerton et al., 1999; Parsek & Singh, 2003). The capacity of the ΔznuA strain and wild- type PAO1 strain to form a biofilm was assessed in Zn2+-depleted media, under conditions that were essentially identical to the growth analyses (Figure 5.10). Overall, biofilm formation decreased with decreasing Zn2+ concentrations in the media (via addition of TPEN). A difference in biofilm formation capacity between the two strains was measured when seed cultures were treated with 10 μM TPEN overnight, then assayed without further supplementation or when supplemented with a further 10 μM TPEN. Under these conditions the ΔznuA strain exhibited a reduction in biofilm formation relative to the wild-type strain. This indicates a key role for intracellular Zn2+ in biofilm formation by P. aeruginosa, with higher intracellular Zn2+ concentrations contributing to the biofilm formation process.

The capacity of P. aeruginosa to resist antibiotics is a critical factor in the success of the bacterium to persist within the human host. As one arm of its resistance, P. aeruginosa employs Zn2+-dependent metallo-β-lactamase enzymes to degrade β-lactam antibiotics (Murphy et al., 2006; Garcia-Saez et al., 2008; Fajardo et al., 2014). Furthermore, many of the efflux protein families that contribute to drug and antibiotic efflux also exhibit roles in tolerance to a range of compounds including metal ions (Hinchliffe et al., 2013). Consequently, under metal-limiting conditions, the expression of these systems may be

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Figure 5.10: The effect of znuA deletion on biofilm formation. Wild-type PAO1 (black) and the isogeneic ΔznuA strain (red) were compared for static biofilm formation on polycarbonate microtitre plates at 37 °C. Biofilm was stained with 0.1% crystal violet and dissolved in 30% acetic acid, prior to quantification of absorbance at 595 nm. Cultures were grown without or without supplementation with TPEN as indicated. Data are the mean ± s.e.m., with n ≥ 3. Statistical significance was determined using the two-tailed unpaired Student’s t-test, where * represents P < 0.05.

124 Chapter 5 altered, thereby affecting the antibiotic susceptibility of the bacterium. To address this, the impact of Zn2+ deficiency upon the antibiotic resistance phenotype of P. aeruginosa was examined using the Zn2+-deficient ΔznuA strain. The minimal inhibitory concentration of commonly administered anti-pseudomonal antibiotics were determined in CDM, allowing the effects of the znuA deletion to be more pronounced. MIC analyses were performed using antibiotics from the aminoglycoside, β-lactam, fluoroquinalone, and polymyxin families (Table 5.1). However, no significant differences of more than 2-fold were observed in the MIC values of the ΔznuA strain. This indicates that the antibiotic resistance profile of P. aeruginosa is not significantly altered in response to Zn2+ depletion.

Zinc also has roles in cell membrane remodelling, acting as a cofactor in peptidoglycan modifying enzymes, such as the N-acetylmuramyl-l-alanine amidase, AmiA (Rocaboy et al., 2013; Buttner et al., 2014). Furthermore, the Zn2+-requiring MucR from Brucella melitensis was shown to be responsible for modification of lipid A and the modification of the cell membrane surface properties (Mirabella et al., 2013). Therefore, the impact of the znuA deletion upon cell membrane hydrophobicity was investigated using a bacterial adhesion to hydrocarbon (BATH) assay. Wild-type P. aeruginosa and the ΔznuA strain were assessed for their ability to associate with the hydrocarbon, xylene (Figure 5.11). The ΔznuA strain exhibited a significant 5.2% increase in association with xylene (P = 0.0207), indicating the strain to be more hydrophobic than the wild-type strain. To determine if the change in hydrophobicity could be attributed to alterations in the fatty acid (FA) composition, the FA profiles of the ΔznuA and wild-type strains were assessed by gas chromatography-mass spectroscopy (Figure 5.12). The saturated 16:0, and monounsaturated 16:1n-7, and 18:1n-7 FAs comprised > 90% of the fatty acid content, with minimal trans FAs measured. Overall, no significant differences in FA composition were observed between the wild-type and ΔznuA strain. Taken together, these data show that the change in xylene association was independent of cell membrane FA composition. Therefore, another cell membrane component, such as LPS, may be responsible for the change in hydrophobicity and further investigations would be required to elucidate this factor. The ΔznuA strain was also examined using scanning electron microscopy (SEM) to ascertain whether Zn2+ depletion altered cellular architecture. Mid-logarithmic phase wild-type and ΔznuA cultures were imaged using a Philips XL-30 SEM. Both strains showed cells with a typical rod-shape, with many cells featuring a polarised flagellum. Despite the Zn2+ depletion, no apparent differences in cellular morphology were observed between the wild-type and ΔznuA strains (Figure 5.13).

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Table 5.1: Minimal inhibitory concentration analysis under Zn2+ limitation.

MIC (μg.mL-1) Antibiotic PAO1 ΔznuA Gentamicin1 12.50 12.50 Tobramycin1 6.25 6.25 Ciprofloxacin 0.78 0.78 Ceftazidime Hydrate 100.00 50.00 Colistin Sulphate 6.25 6.25 Piperacillin 20.00 20.00 Imipenem 2.00 2.00 Assay performed in triplicate. 1 MIC testing for aminoglycosides was performed in CDM supplemented with 20 mg.L-1 Ca2+.

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Figure 5.11: Deletion of znuA alters cellular hydrophobicity. The bacterial adherence to hydrocarbons (BATH) assay was performed using xylene, with the wild-type PAO1 (black) and the Δ znuA strain (red). Changes in membrane lipid hydrophobicity are reflected in altered adherence to xylene. Data are the mean ± s.e.m., where n ≥ 3 biological duplicates grown on at least 2 different days. Statistical significance was determined using the two-tailed unpaired Student’s t-test, where * represents P < 0.05.

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Figure 5.12: Fatty acid (FA) composition of PAO1 and ΔznuA.

The major constituents of cellular fatty acids are depicted, indicating differences in relative abundance between PAO1 (black) and the ΔznuA strain (red) grown in CDM. 16:0, saturated FA of 16 carbon chains. 16:1n-7, a monounsaturated FA of 16 carbons, with a cis double bond at the 7th carbon. t16:1, a trans monounsaturated fatty acid with 16 carbons. The position of the double bond in t16:1 is unable to be determined. 18:0, saturated FA of 18 carbon chains. 18:1n-7, a monounsaturated FA of 18 carbons with a cis double bond at the 7th carbon. 18:3n-6, a polyunsaturated FA of 18 carbons, with a cis double bond at the 6th, 9th, and 12th carbons. t18:1n-7, a trans monounsaturated fatty acid of 18 carbons, with the double bond at the 7th carbon. Data are the mean ± s.e.m. of biological duplicates.

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Figure 5.13: PAO1 and ΔznuA appear morphologically similar.

SEM analysis of mid-logarithmic CDM cultures of PAO1 and the ΔznuA was performed using a Philips XL-30 SEM. (A and C) Wild-type PAO1. (B and D) ΔznuA strain.

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Analysis of the volatile organic compounds (VOC) produced by P. aeruginosa in the wild-type and ΔznuA strains was also performed (Section 2.5.7). Monitoring of VOC profiles has been postulated as a non-invasive method for screening for infections in the CF lung (Robroeks et al., 2010; Bos et al., 2013), with the expectation that different P. aeruginosa strains may generate a unique VOC profile, allowing their identification. Headspace analysis of the wild-type PAO1 and the ΔznuA strains was performed, with the VOC profile analysed by selected-ion flow tube mass spectrometry (SIFT-MS) on an MS-Syft. Both strains produced a range of VOCs (Table 5.2), with dimethyl sulphide and hydrogen cyanide the most abundant of detectable compounds. By comparison with the wild-type strain, ΔznuA showed a trend towards an increase in dimethyl sulfide, and a reduction in hydrogen cyanide. Intriguingly, the analysis also revealed that the ethanol and butanol released from the LB agar plates during the experiment were metabolized by P. aeruginosa. However, overall no significant differences in the VOC profile were observed between the wild-type and ΔznuA strains, indicating that Zn2+ depletion does not have a phenotypic impact on VOC production. Collectively, these data show that while biofilm production was dependent on the cellular acquisition of Zn2+, cell architecture, FA membrane composition, VOC production and antibiotic sensitivity showed negligible perturbation in response to Zn2+ depletion.

5.2.1. Cellular Zn2+ depletion of the ΔznuA strain results in significant transcriptional

modulation

Despite the significant reduction in Zn2+ content, the ΔznuA strain did not exhibit any major phenotypic defects. To account for this, it was expected that significant alterations in gene transcription were occurring. Consequently, the genome-wide effects of Zn2+ depletion were assessed using Illumina HiSeq RNA sequencing of the wild-type P. aeruginosa PAO1 and ΔznuA strains. Each PAO1 and ΔznuA sample, grown in biological triplicate to mid-log phase, was assessed for RNA purity and quality using an Agilent Bioanalyzer Nano RNA chip (Figure 5.14). All samples had a high RNA integrity number (RIN) (≥ 9.4) and were submitted to the Adelaide Microarray Centre for ribosomal RNA removal and library assembly prior to Illumina HiSeq analysis. Following initial data processing in collaboration with Dr. Bart Eijkelkamp and Dr. Lauren McAllister (Research Centre for Infectious Diseases, University of Adelaide), RNA sequencing data were expressed as the fold change in the ΔznuA strain relative to wild-type P. aeruginosa (Figure 5.15). Overall 88 genes were

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Table 5.2: Volatile organic compound analysis of P. aeruginosa.

Concentration (ppb) Hydrogen Dimethyl 2-amino Butanone Methanol Ethanol Acetone Isoprene Pentanone Butanol cyanide sulfide acetophenone LB agar 20 104 0 280 1730 355 2 1 10 584 PAO1 1290 427 6820 748 488 462 52 2 42 37 ΔznuA 1230 417 6920 730 441 449 49 2 41 38

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Figure 5.14: Analysis of RNA for RNA sequencing.

Representative (A) Wild-type P. aeruginosa PAO1 and (B) ΔznuA strain RNA samples assessed for purity and integrity using an Agilent Bioanalyzer Nano RNA chip. Values represent relative fluorescence units, with peaks representing 16S and 23S rRNA indicated. The majority of intact mRNA is ~4000 nt in length. Separation of RNA by electrophoresis on the Bioanalyzer is represented on the right of each figure.

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Figure 5.15: Differential expression of genes in response to the Zn2+ depletion of the ΔznuA strain.

RNA sequencing of P. aeruginosa PAO1 and the isogenic ΔznuA deletion strain was used to determine relative gene expression (expressed as log2-fold change). Each dot represents a gene, with each gene distributed on the x-axis in accordance with locus tag numbering for PAO1. Genes more highly expressed in the ΔznuA strain are present above the x-axis, with those below the x-axis expressed at a lower level in the ΔznuA strain. Genes of interest are annotated with their putative or characterized functions.

133 Chapter 5 up-regulated ≥ 2-fold (1.63%) in response to the Zn2+ limitation of the ΔznuA strain, with 43 genes up-regulated ≥ 4-fold (Appendix 2). Surprisingly, only 22 genes were down-regulated ≥ 2-fold in the ΔznuA strain (Appendix 2). Quantitative reverse transcription-PCR was used to confirm the changes in gene expression with a strong correlation observed between the qRT- PCR and RNA sequencing results (Figure 5.16, R2 value = 0.996).

Cluster of orthologous gene (COG) function analysis, performed in collaboration with Dr. Bart Eijkelkamp (RCID, University of Adelaide) was used to provide putative roles for the identified genes. Analysis of the P. aeruginosa PAO1 COG annotations revealed that genes from a number of different COG categories were differentially regulated ≥ 2-fold in response to znuA deletion (Figure 5.17). The ‘inorganic ion transport and metabolism’ COG function (296 genes) featured the largest percentage of genes up-regulated in response to Zn2+ limitation (~6%). This would be consistent with the expectation that the ΔznuA strain would seek to acquire additional Zn2+ via alternative pathways in the absence of znuA. Of note, the ‘defence mechanisms’ (74 genes), and ‘posttranslational modification, protein turnover and chaperones’ (192 genes) categories also featured a number of genes that were up-regulated (4%, respectively). From the ‘defence mechanisms’ COG, PA4064-PA4065 and PA5542 were found to be up-regulated in the ΔznuA strain. PA4064-PA4065 encode components of an ABC-transport system that may be contribute to Zn2+ acquisition, placing into question their classification as genes that contribute to defence. Conversely, PA5542 encodes the Pseudomonas Imipenem -lactamase, PIB-1, which has been demonstrated to contribute to imipenem and carbapenem resistance (Fajardo et al., 2014), thereby aiding in cellular defence. Of the genes up-regulated from the ‘posttranslational modification, protein turnover and chaperones’ COG category, many were involved in protein turnover. As Zn2+ serves as a cofactor in a range of proteins, the up-regulation of protein turnover genes may represent an attempt on the part of the cell to redistribute Zn2+ for use in essential proteins. However, this requires further investigation. Overall, COG function analysis indicates the importance of Zn2+ to a range of different aspects of cellular physiology and the reveals the complexity of the Zn2+-limitation response in P. aeruginosa.

P. aeruginosa Zn2+ acquisition and utilization is controlled by the Zn2+ uptake regulator protein, Zur (formerly Np20) (Ellison et al., 2013). Analysis of the promoter regions of genes up-regulated by ≥ 4-fold using the Zur-binding motif of P. protegens Pf-5 (Lim et al., 2013) indicated a number of P. aeruginosa clusters to be potentially Zur-controlled (featuring an adjacent putative Zur-binding site). To generate a P. aeruginosa PAO1 optimised Zur-binding motif, candidate sites were required to be intergenic, up-regulated ≥ 2-fold and possess an E-

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Figure 5.16: qRT-PCR validation of RNA sequencing data. qPCR analysis of PAO1 and ΔznuA samples grown in biological triplicate was used to validate the relative expression as determined by RNA sequencing. Relative expression is 2 expressed as log2-fold change, with a strong correlation coefficient (R = 0.9957) recorded.

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Figure 5.17: Cluster of orthologous gene (COG) function analysis.

Genes differentially expressed in the PAO1 and ΔznuA strains were categorized according to their annotated COG function. Bars represent the percentage of genes in each COG function which were up-regulated (blue) or down-regulated (orange) > 2-fold.

136 Chapter 5 value ≤ 0.002. Compilation of the 9 PAO1 candidate Zur-binding sites detected on the basis of these parameters (Table 5.3) permitted generation of a Zur-binding motif (Figure 5.18) (Crooks et al., 2004). The candidate binding sites then allowed refined HMMER2 analysis of the P. aeruginosa genome, revealing the presence of a total of 11 putative Zur-binding sites that were adjacent to genes that were up-regulated ≥ 4-fold. Bioinformatic analyses were performed for all genes up-regulated ≥ 4-fold, with the predicted gene function, putative Zur- binding sites and their E-values indicated (Table 5.4). The putative functions of genes up- regulated ≥ 4-fold were diverse, and included transport proteins, ribosomal proteins, enzymes, transcription factors and hypothetical proteins. Furthermore, as observed in the P. protegens Pf-5 Zn2+-limitation transcriptome, a number of P. aeruginosa genes, which include Zn2+- independent paralogs of Zn2+-dependent proteins, and those with a particular requirement for Zn2+ were clustered together (PA5532-PA5543). This may aid in co-ordinated transcriptional de-repression by Zur upon sensing Zn2+ limitation.

5.2.1.1. Transport proteins

Although deletion of znuA reduced cellular Zn2+ abundance, the ΔznuA strain was still capable of acquiring adequate Zn2+ to prevent significant phenotypic alterations. Hence, it was likely that P. aeruginosa PAO1 possessed multiple high affinity Zn2+ acquisition mechanisms to ensure the cellular Zn2+ requirement was met. Consistent with this expectation, analysis of the RNA-sequencing data identified four putative inner membrane transport pathways up-regulated in response to the Zn2+ limitation of the ΔznuA strain: PA2912-PA2914, hmtA, PA4063-PA4066 and PA4834. Each of these clusters featured a putative Zur regulatory site (Table 5.4). The PA2912-PA2914 and PA4063-PA4066 clusters both share homology with ABC permease systems. PA2912-PA2914 is predicted to encode an iron ABC transport system, with PA2913 sharing 61% identity with a putative iron- binding SBP from Pseudogulbenkiania ferrooxidans. However, study of the homologous cluster in P. protegens Pf-5 indicated that it did not have a role in iron acquisition (Lim et al., 2012), but rather under conditions of Zn2+ limitation (Lim et al., 2013). Furthermore, the putative regulation of the cluster by Zur (Table 5.4) supports the role of PA2912-PA2914 in Zn2+ acquisition. However, comparison of the putative PA2913 SBP amino acid sequence with ZnuA proteins from a range of organisms indicated that PA2913 is not a Zn2+-specific cluster A-I SBP, as it lacks the conserved histidine residues required for Zn2+ binding (Banerjee et al., 2003; Chandra et al., 2007; Li & Jogl, 2007; Yatsunyk et al., 2008; Ilari et al., 2011). Rather, PA2913 may be a cluster A-II SBP (Berntsson et al., 2010), which binds a chelated form of Zn2+, thereby negating the need for the Zn2+-co-ordinating histidine residues.

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Table 5.3: Candidate Zur-binding sites.

Adjacent gene(s) Putative Zur-binding site PA0781 AAGAAATAATATAACAT PA1921/22 AAGTTATAACATAACAT PA2911 ATGTTATACTATAACAA PA3601 ATGTTATATAGTTACAT PA4063 TCGTTATAACGTAACAA PA4837/38 ATGATATACTATAACGC PA5498/99 ACGTTATTATATTACCC PA5536/37 ACGTTATACTATAACAA PA5539 ACGTTATATTATAACAT

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Figure 5.18: The P. aeruginosa ZnuA binding site motif. Nucleotide logo prepared using Weblogo (Version 3) as outlined in Section 2.7.2.

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Table 5.4: Expression of genes under Zn2+ limitation.

Zur- Fold binding Locus ID Predicted functions (BLAST E-value) Reference change site (E-value) PA0781 172.2 homologous to ZnuD from Neisseria meningiditis (1x10-66) 0.0012 (Stork et al., 2010) PA1921 15.1 homologous to methyltransferase (4x10-107) 0.00027 PA1922 147.3 homologous to TonB-dependent receptor (0) 0.00027 PA1923 122.2 homologous to cobaltochelatase subunit, CobN (0) PA1924 44.7 homologous to ExbD from Hyphomicrobium nitrativorans (2x10-75) PA1925 56.4 hypothetical PA2434 6.5 hypothetical PA2435 5.6 HmtA, P-type ATPase importer (Lewinson et al., 2009) PA2437 14.4 homologous to membrane protease subunit of HflC family from Rahnella aquatilis (1x10-134) PA2438 10.6 homologous to HflC membrane protease subunit from Enterobacter cloacae (1x10-127) PA2439 9.0 homologous to membrane protease subunitof HflK family from Rhizobium mongolense (0) 0.11 PA2911 8.1 homologous to TonB dependent receptor from Cupriavidus sp. WS (0) 0.00027 PA2912 8.8 homologous to nucleotide binding domain of ABC transporter (6 x 10-100) PA2913 9.1 homologous to iron periplamic binding protein from Pseudogulbenkiania ferrooxidans (3x10-133) -117 PA2914 8.0 homologous to transmembrane domain of Vitamin B12 ABC permease (7x10 ) PA2915 5.2 homologous to metallo beta lactamase from Azotobacter vinelandii (1x10-145) PA3282 4.1 hypothetical PA3283 5.0 hypothetical PA3284 5.0 hypothetical PA3600 89.2 homologous to 50S ribosomal protein L36 from Kushneria aurantia (1x10-18) PA3601 109.0 homologous to 50S ribosomal protein L31 from (7x10-36) 0.0013 PA4063 45.1 homologous to Zn2+ periplasmic binding protein from Phaeobacter gallaeciensis (8x10-24) 0.0011 PA4064 17.2 homologous to ABC transporter nucleotide binding protein from Azotobacter vinelandii DJ (3 x 10-114)

PA4065 22.1 homologous to lipoprotein release ABC transporter permease from Thioflavicoccus mobilis 8321 (0) PA4066 10.6 homologous to lipoprotein from Alteromonas macleodii AltDE1 (4x10-36) PA4833 5.0 homologous to hemolysin III family proteins PA4834 27.4 homologous to a membrane transporter from Clostridium tetanomorphum DSM 665 (2x10-123) PA4835 35.2 hypothetical PA4836 72.9 hypothetical PA4837 110.0 homologous to TonB-dependent siderophore receptor from Azotobacter vinelandii DJ (0) 0.0014 PA4838 6.7 hypothetical membrane protein 0.0014 PA5498 5.2 znuA, Zn2+ ABC transporter SBP 0.0012 PA5532 8.9 homologous to the cobalamin biosynthesis protein CobW from Marinobacter sp. EN3 (1x10-45) PA5534 57.5 hypothetical PA5535 46.1 homologous to cobalamin synthesis protein from Azotobacter vinelandii DJ (0) PA5536 134.6 DksA2, Zn2+-independent transcription regulator 0.00021 (Blaby-Haas et al., 2011) PA5537 6.7 hypothetical / homologous to glutamine synthetase in other Pseudomonas species 0.00021 PA5538 18.6 homologous to AmiA, N-acetylmuramoyl-L-alanine amidase from Azotobacter vinelandii DJ 0.00017 (2x10-145) PA5539 93.6 homologous to GTP cyclohydrolase FolE2 from Halomonas sp. A3H3 (6x10-112) 0.00017 PA5540 37.6 homologous to carbonate dehydratase from Thiocapsa marina (3x10-111) PA5541 37.9 PyrC2, dihydroorotase (Brichta et al., 2004) PA5542 6.6 PIB-1 Pseudomonas imipenem β-lactamase (Fajardo et al., 2014) PA5543 5.4 hypothetical

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Consistent with this, PA2913 groups with other cluster A-II SBPs in a phylogenetic tree of cluster A-I, A-II and iron-binding cluster D proteins (Figure 5.19). However, the exact binding and transport capability of the PA2912-PA2914 system, and specifically the PA2913 SBP, requires further investigation. Up-regulation of PA4063-PA4066, another putative ABC import system was also observed in response to Zn2+ limitation (>10.5-fold). This is consistent with the identification of an adjacent Zur site that is predicted to control its expression (E-value = 0.0011). In the P. protegens Pf-5 Zn2+-limitation transcriptome, a homologous cluster (PFL_5528-PFL_5531) was also up-regulated, albeit to a slightly lesser extent (Lim et al., 2013). The PA4063-PA4066 cluster differs from typical metal ABC import systems through the presence of two putative SBP components clustered with the transporter. Primary sequence analysis of PA4063 and PA4066 shows that their size (196 and 172 amino acids, respectively) is inadequate to produce full cluster A-I SBPs (minimum of 300 amino acids for a complete SBP fold). The high-resolution structure of PA4066, solved to 2.45 Å, showed that the protein was a homotetramer (PDB ID: 4HSP), although, in the absence of any functional characterisation, the physiological relevance of this oligomerisation remains unknown. Based on the size of the protein, and its lack of conserved Zn2+ binding determinants, it is unlikely that PA4066 directly binds Zn2+ ions as a cluster A-I SBP. Analysis of the PA4063 protein sequence reveals the presence of two histidine and acidic amino acid-rich regions and one of the conserved His residues (His161, E. coli numbering), both features of Zn2+-specific ZnuA proteins. This suggests a potential role in Zn2+ interaction, but, as stated for PA4066, PA4063 is too small to be a cluster A-I SBP. Whether PA4063 and PA4066 may act co-operatively to bind Zn2+ remains highly speculative, but is a possibility. Interactions between SBPs to deliver an allocrite to an ABC importer have previously been reported for the FpvCDEF ABC import system in P. aeruginosa (Brillet et al., 2012). Consequently, the possibility of co-operative Zn2+ binding by PA4063 and PA4066 is not infeasible. The structure and mechanism of any such interaction would be of great interest to the field and further characterisation may uncover homologous systems in other organisms. However, the role of the PA4063-PA4066 cluster in Zn2+ accumulation remains speculative in the absence of any biochemical characterisation. Zinc depletion also resulted in the up-regulation of hmtA (PA2435; 5.6-fold), an atypical P-type ATPase importer shown to transport zinc and copper (Lewinson et al., 2009). Together, these transport systems are likely to substitute for the non-functional Znu permease and import Zn2+ into the cytoplasm.

Analysis of the ΔznuA transcriptome also revealed four putative TonB dependent outer membrane proteins that were up-regulated in response to Zn2+ depletion (PA0781, PA1922, PA2911, and PA4837). The putative TonB-dependent receptor, PA0781, was the

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Figure 5.19: Phylogenetic tree of metal binding SBPs. The phylogenetic relationship between P. aeruginosa PA2913 and characterised cluster A-I, A-II and D-IV iron binding SBPs was assessed. Amino acid sequences were aligned using ClustalW2 (Larkin et al., 2007) and a phylogenetic tree constructed in CLC Sequence Viewer (Version 6.7.1) using the Neighbour Joining method. Proteins clustered according to their SBP classification. All cluster A SBPs feature a long α-helical region that joins the 2 domains of the protein. They are further subdivided into cluster A-I SBPs that bind their metal ligand directly, and cluster A-II SBPs that bind a chelated for of their metal ligand. The cluster D-IV SBPs are an iron specific sub-cluster of cluster D protein, binding ferric or ferrous iron. BtuF_Eco (GI: 215485321, E. coli); PA2913_Pae (GI: 15598109, P. aeruginosa PAO1) FitE_Eco (GI: 190907145, E. coli); Ceu_Cje (GI: 112360670, C. jejuni); Feu_Bsu (GI: 2632430, B. subtilis); FutA2_Syn (GI: 170785083, Synechocystis PCC6803); FutA1_Syn (GI: 152149369, Synechocystis PCC6803); nFBP_Ngo (GI: 28948320, Neiserria gonorrhoeae); hFBP_Hin (GI: 157832046, H. influenzae); SitA_Sen (GI: 16761638, S. enterica); SitA_Eco (GI: 84060846, E. coli); YfeA_Ype (GI: 51596671, Y. pestis); MntC_Syn (GI: 16330511, Synechocystis PCC6803); SitC_Sep (GI: 27467323, S. epidermis); MntC_Sau (GI: 88194402, S. aureus); MntA_Ban (GI: 30263122, B. anthracis); ScaA_Sgo (GI: 1168357, Streptococcus gordonii); PsaA_Spn (GI: 182684593, S. pneumoniae); MtsA_Spy (GI: 489075835, S. pyrogenes); TroA_Ssu (GI: 330832169, S. suis); Lbp_Spy (GI: 15675794, S. pyrogenes); AdcAII_Spn (GI: 15902950, S. pneumoniae); YcdH_Bsu (GI: 668752994, B. subtilis); AdcA_Spn (GI: 15904016, S. pneumoniae);ZnuA_Hin (GI: 491963406, H. influenzae); ZnuA_Yru (GI: 490857750, Y. ruckeri); ZnuA_Eco (GI: 635897169, E. coli); ZnuA_Sen (GI: 541470409, S. enterica); ZnuA_Pae (GI:15600691, P. aeruginosa PAO1); ZnuA_Cje (GI: 504330062, C. jejuni), ZnuA_Syn (GI: 499174152, Synechocystis PCC6803).

Chapter 5 most highly up-regulated gene (172.2-fold) in the ΔznuA strain. PA0781 shares 29% identity with the TonB-dependent Zn2+ binding protein ZnuD from Neisseria meningiditis. ZnuD has been shown to aid in recruitment of Zn2+ to the periplasm under conditions of low Zn2+, facilitating import into the cytoplasm (Stork et al., 2010), and it is predicted that PA0781 (designated ZnuD) serves a similar function in P. aeruginosa PAO1. PA2911, which is also homologous with TonB-dependent receptors is clustered with the putative cluster A-II PA2912-PA2914 ABC permease. Hence, PA2911 could be predicted to act as an outer membrane receptor for PA2913 and its chelated Zn2+ allocrite, transporting it into the periplasm. The role of the remaining Zur-regulated TonB-dependent receptors, PA1922 and PA4837, up-regulated in response to the Zn2+ limitation, is less clear. PA1922 is present in an operon with a putative cobaltochelatase (cobN), which is involved in cobalamin biosynthesis, whereas PA4837 shares an operon with a putative nicotianamine synthase (PA4836). The exact role of these four TonB-dependent receptors under Zn2+ limitation remains to be determined.

As TonB-dependent receptors, each of these proteins would require the use of TonB, ExbB and ExbD to energise the transport of the metal into the periplasm (Noinaj et al., 2010). P. aeruginosa PAO1 features two identified exbB and exbD genes (exbB1 and exbB2, and exbD1 and exbD2), and three tonB genes (tonB1, tonB2 and tonB3). However, these genes were not significantly up-regulated (>2-fold) under Zn2+ limitation in the ΔznuA strain. By contrast, PA1924, a gene up-regulated 44.7-fold under Zn2+ deficiency encodes a protein that shares homology with ExbD from Hyphomicrobium nitrativorans (E-value = 2x10-75). Therefore, it is not unreasonable to speculate that there are unidentified exbB and exbD homologs in PAO1, such as PA1924, that may facilitate Zn2+ uptake by ZnuD and the other TonB-dependent proteins. Taken together, these data indicate the presence of several Zn2+ acquisition pathways that could account for the observed accumulation of Zn2+ in the ΔznuA strain.

5.2.1.2. Ribosomal proteins

Ribosomal proteins utilize the major proportion of cellular Zn2+ in bacteria (Scrutton et al., 1971; Hensley et al., 2011). As a consequence, Zn2+ depletion would be anticipated to have a significant impact on protein translation, via impaired ribosomal protein activity. To circumvent this issue, Zn2+-independent ribosomal paralogs, known as C- copies, which lack the Zn2+-binding ribbon, commonly substitute for their Zn2+-dependent C+ ribosomal proteins under Zn2+ limitation (Makarova et al., 2001). Such C- genes are typically controlled by Zur. Analysis of the P. aeruginosa genome indicates the presence of genes encoding both C+ and

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C- paralogs of the 50s ribosomal proteins L36 and L31 (Makarova et al., 2001). The C+ copies of L36 and L31 (RpmJ/PA4242 and RpmE/PA5049, respectively) each feature canonical Zn2+ binding resides (either His or Cys) (Figure 5.20). Some or all of these residues are lacking in the C- copies of the L36 and L31 proteins (PA3600 and PA3601). The C- genes were predicted to be co-transcribed under the control of a putative Zur site (E-value = 0.0013). In response to the Zn2+ limitation of the ΔznuA strain, the Zn2+-independent (C-) 50s ribosomal proteins L36 (PA3600) and L31 (PA3601) were significantly up-regulated (89.2- and 109-fold, respectively; Table 5.4). This would enable redeployment of intracellular Zn2+ for utilization in other Zn2+ requiring proteins, for which the requirement for Zn2+ cannot be substituted.

5.2.1.3. Enzymes

Zinc commonly serves as a structural or catalytic cofactor in enzymes, due to its ability to act as a Lewis acid or as an electrophile (Hantke, 2005; Andreini et al., 2008). Consistent with expectations, several enzyme-encoding genes were up-regulated in response to Zn2+ depletion, including those commonly clustered together in a Zur-regulated region (PA5532- PA5543). Within this region, the PA5538 gene was up-regulated 18.6-fold in response to the Zn2+ depletion of the ΔznuA strain. PA5538 has homology to the Zn2+ metallo-enzymes, AmiA, AmiB and AmiC from E. coli that have N-acetylmuramoyl-L- alanine amidase activity (Heidrich et al., 2001). These enzymes are involved in peptidoglycan cleavage and membrane remodelling in the periplasm during the final stages of cell division (Priyadarshini et al., 2007; Yang et al., 2012). PA4947 has been previously designated AmiB on the basis of its N- acetylmuramoyl-L-alanine amidase activity and homology with AmiB from E. coli (Scheurwater et al., 2007). In agreement with our analysis, this study identified PA5538 as most homologous to the E. coli amiC. Despite this, the PA5538 gene has been annotated as amiA, with the amiC annotation belonging to a ligand-sensitive negative regulator (PA3364) of the aliphatic amidase operon (Wilson & Drew, 1991). Similar to its E. coli homologues, the protein encoded by PA5538 is predicted to be Zn2+-requiring, providing a basis for its up- regulation under Zn2+-limiting conditions. Of interest, the amiB gene from P. aeruginosa was down-regulated in the ΔznuA strain (1.2-fold). Hence, AmiB may be expressed under Zn2+ replete conditions, but under Zn2+ limiting conditions, the expression of AmiA (PA5538) is increased via Zur de-repression and AmiA becomes the dominant N-acetylmuramoyl-L- alanine amidase. Presumably the AmiA metal binding site has a higher affinity for Zn2+ than AmiB, allowing cell division under conditions of Zn2+ depletion.

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Figure 5.20: Sequence alignment of P. aeruginosa C- and C+ ribosomal proteins. Primary sequence alignment of the C- and C+ paralogs of the (A) L36, and (B) L31 ribosomal proteins. The Zn2+ co-ordinating residues are highlighted in red, with the residues partially or completely absent from the C- copies (PA3600 and PA3601).

Chapter 5

The adjacent operon is predicted to comprise of PA5539, PA5540 and PA5541. PA5539 was strongly up-regulated (93.6-fold) in response to Zn2+ limitation of the ΔznuA strain. PA5539 is homologous to the GTP cyclohydrolases, FolE and FolE2. The Zn2+-requiring enzyme, FolE, is responsible for catalysing the first step of the de novo tetrahydrofolate biosynthesis pathway (Sankaran et al., 2009) as well as the production of modified ribonucleosides found in tRNA molecules (Phillips et al., 2008). Unusually, two folE-type genes are present in the P. aeruginosa PAO1 genome (PA1674 and PA3438) (El Yacoubi et al., 2006). In species that possess both the Zn2+ dependent (folE) and independent forms (folE2), folE2 is Zur regulated (Sankaran et al., 2009). The putative Zur-binding site adjacent to the PA5539 gene features the lowest E-value = 0.00017, indicating its regulation by Zur to be highly likely. Furthermore, analysis of the primary sequence reveals the PA5539 gene product to share 63% of its identity with FolE2 from Halomonas sp. A3H3, indicating PA5539 to be the P. aeruginosa folE2 gene. The expression of a Zn2+-independent, FolE2, would enable GTP cyclohydrolase I activity under the Zn2+ limiting conditions of the ΔznuA strain, reducing the effects of Zn2+ deprivation on cellular processes.

PA5540 was predicted to be encoded in the same Zur-regulated operon as PA5539, and was also up-regulated in response to Zn2+ depletion (37.6-fold). The PA5540 gene product shares homology with the γ-carbonic dehydratase family, which is responsible for the Zn2+- dependent conversion of carbon dioxide to bicarbonate (Capasso & Supuran, 2014). Bacterial γ-carbonic dehydratases are predicted to be involved in maintenance of cellular pH and contribute to metabolism, but only a limited number have been characterised and it is likely that they serve in other roles as well (Alber & Ferry, 1994; Zimmerman et al., 2010; Del Prete et al., 2013). PA5541 was also up-regulated in response the Zn2+ depletion of the ΔznuA strain, consistent with its location within a Zur-regulated operon. PA5541 has previously been annotated as a dihydroorotase from the amino hydrolase superfamily (Brichta et al., 2004). Dihydroorotase enzymes are responsible for the conversion of carbamoyl L-aspartate to L- dihydroorotate in the third step of the pyrimidine biosynthesis pathway. As the second such enzyme identified in P. aeruginosa, PA5541 was annotated as PyrC2, the other being the previously characterised, PyrC (PA3527) (Brichta et al., 2004). While functionally redundant in their dihydroorotase activity, the Zn2+-dependent PyrC was shown to be constitutively expressed, with PyrC2 only expressed in the absence of PyrC (Brichta et al., 2004). Larger than PyrC by 10 kDa, the 48 kDa PyrC2 protein lacks one of the conserved histidine residues predicted to contribute to the co-ordination of Zn2+ within the functional homodimer. Based on the Zur-controlled expression of PyrC2, and the lack of required residues in the Zn2+-

148 Chapter 5 binding pocket, PyrC2 is most likely to be a Zn2+-independent dihyroorotase that is expressed and active under Zn2+-limiting conditions. In this way, the requirement of pyrimidine synthesis for cellular growth can be met despite Zn2+ limitation. A small subset of up- regulated enzymes were linked with resistance to antibiotic treatment (Kindrachuk et al., 2011). The PA2437-PA2439 cluster, encoding putative members of the HflC and HflK protease families were up-regulated in the ΔznuA strain. In E. coli, HflC and HflK are accessory factors for the membrane embedded FtsH protease (Akiyama, 2009). Inactivation of these factors in E. coli and P. aeruginosa results in an increase in sensitivity to tobramycin (Hinz et al., 2011). Hence, it can be presumed that this protease, in addition to others, degrades membrane embedded proteins that are mis-folded in response to aminoglycoside treatment. Whether PA2437, PA2438, and PA2439 act as accessory factors for FtsH (PA4751) in P. aeruginosa and contribute to proteolysis of mis-folded proteins produced under conditions of Zn2+ depletion, remains to be determined.

PA5542, the Pseudomonas imipenem β-lactamase (PIB-1), and PA2915, a putative Zn2+-containing metallo β-lactamase were also up-regulated in response to the Zn2+ limitation of the ΔznuA strain. However, the MIC analysis of the wild-type and ΔznuA strain did not reveal a difference in profiles in response to Zn2+ limitation (Table 5.1). The MIC of imipenem for both PAO1 and ΔznuA strain (2 g.mL-1) was similar to that previously reported for PAO1 (3 g.mL-1), with the contribution of PIB-1 to imipenem resistance only described previously for the clinical P. aeruginosa 59.20 strain (Fajardo et al., 2014). As PIB-1 was up-regulated in both P. aeruingosa 59.20 and the ΔznuA strain (~3-fold and 6.6-fold, respectively), this suggests other strain specific factors may be affecting the resistance profile. Whether, like PIB-1, PA2915 is a narrow spectrum β-lactamase, the target of which was not tested in the MIC analysis, remains to be determined, but may provide a basis for the lack of alteration in β-lactam resistance observed for the ΔznuA strain.

5.2.1.4. Transcriptional regulators

Whilst the transcriptional response to Zn2+ depletion is widely attributed to the Zn2+ uptake regulator, Zur, another transcriptional regulator, PA5536, was also up-regulated in response to Zn2+ depletion. PA5536 encodes DksA2, a Zn2+-independent transcriptional regulator that is expressed under Zn2+ limiting conditions and can, in most cases, functionally replace its Zn2+-dependent paralog, DksA (PA4723), which is expressed under Zn2+ replete conditions (Blaby-Haas et al., 2011). DksA2 act as a global transcriptional regulator, binding the RNA polymerase and altering transcription of target genes, particularly the ribosomal RNA genes and those involved in the starvation response. The consequences of enhanced 1 49 Chapter 5

DksA2 expression in the ΔznuA strain remains to be determined. However, the up-regulation of DksA2 may aid in the adaptation to Zn2+ limitation of the P. aeruginosa cell.

5.2.1.5. Putative cobalt-binding proteins

The Zn2+ depletion in the ΔznuA strain resulted in up-regulation of a number of proteins with predicted roles in cobalamin biosynthesis: PA1923, homologous to cobaltochelatase subunit CobN; PA5532, a homolog of the cobalamin biosynthesis protein, CobW; and PA5535, a homolog of a cobalamin biosynthesis protein. Cobalamin is a modified terrapyrole with a cobalt ion at its centre (Heldt et al., 2005). P. aeruginosa is able to synthesise cobalamin in a 30-enzymatic step pathway (Raux et al., 2000). However, the two cobalamin biosynthesis genes, cobN and cobW, with which the PA1923, PA5532 and PA5535 genes share homology, have already been identified in P. aeruginosa (PA2944 and PA2945, respectively), with multiple copies not expected to be present in the genome. This calls into question the role of PA1923, PA5532 and PA5535 in P. aeruginosa. Both PA5532 and PA5535 are members of the COG0523 annotation family. A recent study concluded that many proteins annotated as being part of the cobalamin biosynthesis pathway and belonging to the COG0523 family were mis-annotated, with only 12.5% actually holding such a role in cobalamin biosynthesis (Haas et al., 2009). Analysis of COG0523 proteins by Hass et al. (2009) revealed ~30% to be associated with Zn2+ homeostasis, with ~8% of these predicted to be regulated by Zur. The COG0523 family exhibit GTPase and metal binding capacity, and putative COG0523 proteins have been up-regulated in a number of bacteria in response to Zn2+-limitation (Gabriel et al., 2008; Lim et al., 2013; Mortensen et al., 2014). Consequently, it could be proposed that the three Zur de-repressed proteins, PA1923, PA5532 and PA5535, may have roles in Zn2+ chelation, possibly acting as Zn2+ chaperones. Future characterisation of the function of PA1923, PA5532 and PA5535 in P. aeruginosa Zn2+ homeostasis would provide significant insight into the roles held by this poorly characterised protein family.

Intriguingly, transcriptomic analysis did not identify a cobalt transporter in P. aeruginosa PAO1 to which the increased cobalt accumulation of the ΔznuA strain could be attributed (Figure 5.5). Rather, the putative cluster A-II cobalamin SBP, BtuF (PA4045), was not differentially expressed under Zn2+ limitation. It could instead by speculated that the Zn2+- limitation responsive putative cluster A-II PA2913 SBP may, in addition to binding a chelated form of Zn2+, also exhibit the capacity to bind and deliver cobalamin to the putative PA2912/PA2914 ABC transporter for import. However, this requires further investigation.

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5.2.1.6. Down-regulated genes

Only a small proportion of genes (22 in number) were down-regulated by ≥ 2-fold in the ΔznuA strain. A large proportion of these were tRNA genes (45.5%). Unexpectedly, most of the other genes that are down-regulated by more than 2-fold, are members of the nitrite reductase cluster (nirCFGHJL). As the nitrite reductase enzyme (NirS) utilizes a haem cofactor (Nicke et al., 2013), connection between this cluster and Zn2+-depletion remains to be elucidated. Interestingly, the Zn2+ efflux pathways were not significantly down-regulated in the ΔznuA strain, with PA2522 (czcC) down-regulated by 1.3-fold, and the E. coli zntA homolog, PA3690, down-regulated by only 1.6-fold. This indicates minimal Zn2+ efflux was required by the wild-type PAO1 strain in the CDM media used, with intracellular Zn2+ concentrations attributable to high affinity uptake pathways.

Discussion

In environments of changing Zn2+ abundance, bacteria require efficient mechanisms of Zn2+ acquisition and efflux to maintain homeostasis and meet the cellular requirement for Zn2+. The acquisition of Zn2+ is achieved primarily via the high affinity Zn2+ ABC permease system, ZnuABC. Analysis of the P. aeruginosa PAO1 genome revealed the presence of three genes encoding homologs to the ZnuABC proteins characterised in other bacteria (PA5498, PA550 and PA5501). Furthermore, the solute-binding protein ZnuA was shown to share homology with Zn2+-specific cluster A-I SBPs, featuring the three Zn2+-co-ordinating histidine residues (His70, His140 and His204), and the histidine rich loop between the two

(α/β)4 domains. The classification of the P. aeruginosa ZnuA protein as a cluster A-I SBP was strengthened by the identification of a long α-helix joining the two domains. This helix provides cluster A-I SBPs with the rigidity associated with the structural re-arrangements upon metal-ion binding. Metal-binding assays showed that ZnuA was a high-affinity Zn2+- binding protein. Similar to other ZnuA homologs, the stoichiometry of metal binding was higher than would anticipated based on the expectation that the high-affinity site co-ordinates only a single metal-ion. The His-rich loop, which is present in all known Gram-negative Zn2+- specific SBPs characterised to date, has also been shown to interact with Zn2+, although as a lower affinity Zn2+-binding site. Analysis of the Synechocystis ZnuA revealed that deletion of the His-rich loop in part or in full, did not affect Zn2+ binding at the high-affinity site (Wei et al., 2007). However, the additional Zn2+ binding capacity of Synechocystis ZnuA was lost. Considerable speculation surrounds the physiological role of the His-rich loop in ZnuA. It has

151 Chapter 5 previously been proposed to supply the high affinity site with Zn2+ through its increased mobility (Yatsunyk et al., 2008), or aid in the release of the bound Zn2+ ion to the ZnuBC transporter (Banerjee et al., 2003) for import. Further molecular characterisation is required to unravel the role of this region in the import of Zn2+. This is of particular interest as P. aeruginosa lacks a ZinT homolog, which has been shown in E. coli and S. Typhimurium, to aid in delivery of Zn2+ to ZnuA (Graham et al., 2009; Petrarca et al., 2010; Gabbianelli et al., 2011; Ilari et al., 2014). Whether this role is fulfilled by a homoplastic protein in P. aeruginosa remains to be determined. However, by use of a logical set of criteria, that is, the protein would likely be Zur-regulated and up-regulated in the Zn2+ deficient ΔznuA strain, no clear candidates amongst genes up-regulated by ≥ 4-fold could be identified. Hence, it is highly probable that ZnuA in P. aeruginosa operates independently of ZinT, and instead possibly relies on the His-rich loop to aid in delivery of Zn2+ to the primary binding site.

As the primary high-affinity Zn2+ acquisition pathway in many bacteria, deletion of components of the ZnuABC permease often results in a phenotypic growth defect in Zn2+- limited media (Lewis et al., 1999; Yang et al., 2006; Ammendola et al., 2007; Desrosiers et al., 2010). However, deletion of znuA from P. aeruginosa did not show any growth perturbation, despite a 61.9% reduction in the intracellular Zn2+ concentration. This suggested that additional Zn2+ acquisition pathways were present in P. aeruginosa, but that they were less efficient than the Znu permease due to their inability to restore the wild-type Zn2+ accumulation. Analysis of the transcriptional response to Zn2+ depletion identified a number of putative Zn2+ acquisition systems, which could account for the Zn2+ uptake observed in the ΔznuA strain (Figure 5.21). Under Zn2+-limitation, P. aeruginosa would be expected to transcriptionally activate genes for the specific import of Zn2+ across its poorly permeable outer membrane. Consistent with this prediction, four TonB-dependent outer membrane receptors were significantly up-regulated; PA0781, a homolog of the Zn2+ outer membrane receptor from Neisseria meningitidis, ZnuD, in addition to PA1922 and PA2911 as components of ABC importers, and PA4837. Through up-regulation of these systems, the periplasmic Zn2+ concentration would be raised sufficiently such that less efficient uptake systems could transport the Zn2+ into the cytoplasm. Accordingly, transcriptomic analysis identified two novel ABC import systems (PA2912-PA2914 and PA4063-PA4066), in addition to the P-type ATPase, HmtA, to be up-regulated under Zn2+ limitation. Although the affinities of these putative Zn2+ import systems have not been determined, their functional capacity to acquire Zn2+ is clearly less efficient than the Znu permease. Of the identified pathways, only HmtA has previously been studied and shown to serve as a Zn2+ and copper P-

152 Chapter 5 type ATPase uptake pathway (Lewinson et al., 2009). Consequently, HmtA likely contributes to the acquisition of Zn2+ in the absence of ZnuA. Intriguingly, no increase in copper was observed in the ΔznuA strain, despite the increase in HmtA expression. This suggests that HmtA may have a higher affinity for Zn2+, relative to copper, and thus, preferentially mediate Zn2+ transport. Alternatively, this may be reflective of the ~9-fold higher concentration of Zn2+ in the media (800 nM Zn2+ versus 86.5 nM copper), the increased periplasmic Zn2+ concentration arising from enhanced PA0781 Zn2+ import, and the impaired transport into the cytoplasm in the absence of ZnuA. Further studies of the function of HmtA will be required to ascertain its metal-ion preference and, hence, its physiological function. PA2913 was identified as a putative cluster A-II SBP as part of an ABC permease and TonB-dependent receptor cluster (PA2911-PA2914). As a cluster A-II SBP, PA2913 would be expected to bind a chelated form of Zn2+ in both the periplasm and extracellular environment for its import. However, no such chelating molecule has been identified as yet. Zinc chelation by the P. aeruginosa iron siderophore, pyochelin has been previously reported (Brandel et al., 2012), although uptake was not assessed. A Zn2+-chelate ABC-dependent uptake system has not been previously characterised. However, recently a Zn2+-chelating molecule known as yersiniabactin, was identified in Y. pestis (Bobrov et al., 2014). Yersiniabactin Zn2+ uptake was shown to be dependent upon the major facilitator family transporter, YbtX. This finding, together with the identification of the PA2911-PA2914 putative uptake system, indicates that the diversity of metal-chelate import systems is likely to be greater than previously thought. Further work is necessary to identify the Zn2+ chelating molecule that is acquired by the PA2911-PA2912-PA2914 import system. Whether in P. aeruginosa this is pyochelin, remains to be determined. However, analysis of the P. aeruginosa genome revealed the yersiniabactin synthetase gene, irp2, to share homology with the pyochelin synthesis genes pchE and pchF. Despite this, the pyochelin synthesis genes were not up-regulated in the Zn2+-limited ΔznuA strain, suggesting an alternative siderophore or chelating molecule may be responsible for Zn2+ uptake via PA2911-PA2914. PA4063-PA4066 was also up-regulated under Zn2+ deficiency in the ΔznuA strain, suggesting a possible role in Zn2+ uptake across the inner membrane. However, the putative SBP components of this cluster are atyptical, with both PA4063 and PA4066 being independently too small to comprise an SBP. Whilst the role of this cluster in Zn2+ acquisition appears significant and is supported through the presence of two His-rich loops in PA4063, the mechanisms through which this cluster contributes to Zn2+ uptake in P. aeruginosa requires further investigation. Collectively, the transcriptome analyses have identified multiple pathways that potentially contribute to Zn2+ acquisition under P. aeruginosa Zn2+-limitation (Figure 5.21). Further mutagenesis, growth and

153

Figure 5.21: Model of Zn2+ acquisition in P. aeruginosa PAO1. Schematic representation of the multiple Zn2+ import pathways in P. aeruginosa under the control of the transcriptional regulator Zur. Under Zn2+ replete conditions, dimeric Zur binds Zn2+, repressing transcription of the Zn2+ import pathways. Upon Zn2+ limitation conditions, Zn2+ is removed from Zur, allowing de-repression of the Zn2+ uptake pathway genes. To increase the intracellular concentration of Zn2+ four TonB-dependent outer membrane proteins are produced, ZnuD (PA0781), PA2911, PA1922 and PA4837, bringing Zn2+ into the periplasm. Within the periplasm Zn2+ specific SBPs (ZnuA, PA2913 and PA4063/PA4066) bind Zn2+ ions and deliver them to their cognate ABC import systems (ZnuBC, PA2912/PA2914 and PA4064/PA4065) for active transport into the cytoplasm. HmtA, a P-type ATPase is also able to import periplasmic Zn2+ ions into the cytoplasm. Upon meeting the intracellular Zn2+ requirement, the transcription of the Zn2+ import systems is once again repressed by Zur. Zinc imported by the PA2911-PA2914 ABC permease system is likely to be in a chelated form, consistent with the identification of PA2913 as a cluster A-II SBP

Chapter 5 transcriptional analyses of the individual systems are required to determine the hierarchical nature of the P. aeruginosa Zn2+ acquisition systems, and the external Zn2+ concentrations that result in their Zur-dependent repression.

The acquisition of Zn2+ by P. aeruginosa is under the control of the Zur transcriptional regulator (Ellison et al., 2013). As observed in other bacteria, including Acinetobacter baumanii, and P. protegens Pf-5, P. aeruginosa zur is found together with the znuABC genes (Stover et al., 2000; Lim et al., 2013; Mortensen et al., 2014). Zur auto-regulates its expression, together with the expression of znuABC through a shared Zur-binding site. Based on the Zur-binding sites identified from the P. protogens Pf-5 Zn2+ limitation transcriptome, refinement was carried out to generate a P. aeruginosa PAO1 Zur consensus sequence, with 11 putative Zur-binding sites present adjacent to genes up-regulated by ≥4-fold in the ΔznuA strain. Indeed, only one cluster up-regulated ≥4-fold in the ΔznuA strain lacked a predicted Zur-binding site (PA3282-PA3284). The P. aeruginosa Zur-binding motif is roughly palindromic, allowing bi-directional transcriptional repression. Transcriptional de-repression has been shown previously to correlate with the E-value of putative transcriptional sites (Eijkelkamp et al., 2011). However, analysis of the putative Zur-regulated genes up-regulated by ≥4-fold in the ΔznuA strain does not indicate that to be the case in P. aeruginosa, with some additional factors likely affecting the magnitude of up-regulation. Whilst overall, the presence of an adjacent Zur-binding site correlated with gene up-regulation in the ΔznuA strain, the extent of up-regulation could not be predicted based on the E-value of the putative site. A recent publication by Gilston et al., (2014) examined the structural basis for Zur transcriptional control in E. coli, revealing the regulator to operate primarily as a dimer of dimers. However, this study also identified the gene pliG, encoding a periplasmic inhibitor of p-type lysozyme, as being regulated by a single Zur dimer (Gilston et al., 2014). This variation in operator binding by Zur in E. coli may provide some basis for the complexity and magnitude of Zur transcriptional control observed for P. aeuringosa Zur target genes. Further genetic analysis is required to confirm whether this variation in mode of regulation is observed for the Zur regulon in P. aeruginosa.

Deletion of the ZnuABC permease in a range of bacteria, including S. enterica, C. jejuni, Moraxella catarrhalis, and B. abortus, has resulted in a reduction of virulence (Campoy et al., 2002; Yang et al., 2006; Ammendola et al., 2007; Davis et al., 2009; Murphy et al., 2013). Similarly, interruption of znuB in a transposon mutant of PAO1 resulted in a significant 60% reduction in paralytic killing of C. elegans (Gallagher & Manoil, 2001). This result is perhaps unexpected in light of the lack of growth perturbation observed in the ΔznuA

156 Chapter 5 strain and the number of putative Zn2+ import systems shown to be up-regulated in response to Zn2+ limitation. Together, this indicates that whilst the Zn2+ imported via the identified Zn2+ uptake systems is adequate to maintain wild-type growth characteristics, it is insufficient for full pathogenesis within C. elegans. Whether perturbation of Zn2+ homeostasis would impact P. aeruginosa virulence in other animal models of infection remains to be determined.

Conclusions

This study significantly advances our understanding of the role and importance of Zn2+ in P. aeuringosa. The P. aeruginosa requirement for Zn2+ is emphasised by the numerous Zn2+ acquisition pathways identified through transcriptomic analysis of the bacterium under Zn2+ limitation. Furthermore, this work provides detailed characterisation of the high-affinity Zn2+ acquisition pathway ZnuABC, with a particular focus on the role of the cluster A-I SBP, ZnuA. Together, this study reveals the complex transcriptional response by P. aeruginosa to changes in cellular Zn2+ abundance, and provides key insight into the mechanisms utilized by the bacterium to survive in environments of varying Zn2+ abundance.

157

CHAPTER 6:

DISCUSSION

158 Chapter 6

P. aeruginosa is a clinically significant opportunistic human pathogen which is responsible for significant morbidity and mortality in immunocompromised individuals. The capacity of this environmental bacterium to persist in diverse niches and cause disease is attributed to the metabolic flexibility encoded by its large 6.3 Mbp genome. The cell membrane and its embedded proteins are key to the ability of the bacterium to monitor and adapt to its surroundings. Membrane proteins play diverse roles, acting to sense environmental changes, translocate proteins, lipids, metal ions, polysaccharides and nucleic acids, both in and out of the cell, and provide adhesion or motility. As a family of active membrane transport proteins that exhibit high allocrite specificity, ABC transporters have been shown to serve critical roles in P. aeruginosa growth and infection. Although only a handful have been characterised to date, they perform roles critical to iron acquisition via siderophores and haemophores, secretion of toxins, adhesins and proteases, translocation of lipopolysaccharides, and acquisition of metal ions, amino acids, osmoprotectants and carbohydrates (Guzzo et al., 1991; Duong et al., 1996; McMorran et al., 1996; Rocchetta & Lam, 1997; Letoffe et al., 1998; Nishijyo et al., 1998; Adewoye & Worobec, 2000; Duong et al., 2001; Reimmann et al., 2001; Ghanei et al., 2007; Hannauer et al., 2010; Malek et al., 2011; Brillet et al., 2012; de Bentzmann et al., 2012; Hannauer et al., 2012; Lewis et al., 2012; Casabona et al., 2013; Chou et al., 2014). Accordingly, the expanding contribution of ABC transporters to P. aeruginosa growth and infection required further detailed investigation.

Due to the immunocompromised nature of individuals most susceptible to P. aeruginosa infections, clearance of the bacterium is commonly dependent on antibiotic treatment. However, as observed for many human pathogens, antibiotic resistance rates are continuing to rise (Gales et al., 2001; National Nosocomial Infections Surveillance System, 2004; Talbot et al., 2006). The extensive antibiotic resistance of P. aeruginosa is a major obstacle to the clearance of infections, with antibiotic efflux deemed to be a key component (Poole, 2011). In this study, one hypothesis that was addressed examined the contribution of the uncharacterised ABC efflux proteins to P. aeruginosa antibiotic resistance. The data showed that PA0860, PA1113, PA1876, PA3228 and PA5231 do not have a major contribution to antibiotic efflux. However, they may have some potential capacity to efflux certain drug molecules at a low level, or be involved in efflux of drugs outside of those examined in this study. Hence, an unequivocal exclusion from drug resistance processes cannot be concluded. Over-expression of the individual transporters in an antibiotic sensitive E. coli strain may reveal some ability to efflux drug compounds. However, the available data

159 Chapter 6 indicate that these ABC exporters do not contribute to active antibiotic efflux. Instead, the majority of antibiotic efflux may be attributed to the highly efficient RND efflux systems, MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM. The RND Mex-type efflux pumps have classically been reported to efflux xenobiotics from the periplasm or periplasmic leaflet of the inner membrane (Nikaido, 2011). However, this assumption was recently overturned by a study that showed MexAB-OprM to be able to efflux xenobiotics from the cytoplasm, expanding the contribution of this group of proteins to antibiotic resistance (Ohene-Agyei et al., 2012). This functionality, together with their broad efflux specificity, and significant up-regulation in response to xenobiotics, highlights the prominent role of the RND pumps as the prime mechanism of xenobiotic efflux in P. aeruginosa.

In the absence of a contribution to drug efflux, bioinformatic analyses were used to predict likely physiological functions for the ABC efflux proteins. These insights provide the basis for future studies to directly test these predictions and determine the roles of these ABC transporters in P. aeruginosa. Based on the bioinformatic analysis, PA0860, and its co- transcribed proteins (PA0858 and PA0859), are of considerable interest. PA0860 has been shown to be up-regulated in the CF lung (Son et al., 2007). Although there are limited animal models available to mimic human CF, C. elegans killing assays are commonly employed to screen for virulence-attenuated P. aeruginosa mutants. This assay makes use of cyanide- dependent paralytic killing of C. elegans by P. aeruingosa PAO1 (Gallagher & Manoil, 2001). As PA0858 is a putative rhodanese enzyme involved in cyanide metabolism, analysis of a PAO1 ΔPA0858 mutant in such an assay would be highly informative. Analysis of PAO1 mutants in C. elegans models could also be extended to PA0859, PA0860, or the other ABC transporters and their co-transcribed genes. Such assays will reveal the contribution of these proteins to virulence, highlighting those worthy of further investigation. The functional role of the PA5231/PA5230 ABC transporter is also of particular interest. Analyses to determine the interaction of PA5231 with proteins such as PA5230, or ribosomal components, are required to provide evidential insight into the role of this atypical ABC protein.

During the course of this study, a publication was released characterising the BapABCD (PA1874-PA1877) T1SS and showing its contribution to biofilm formation (de Bentzmann et al., 2012). Despite this, there remains a paucity of data regarding the mechanism of action of the P. aeurignosa BapA (PA1874) and other Bap related proteins. Further studies are required to determine the molecular details of the adhesion mechanism, and the spatial and temporal roles of BapA in the biofilm formation process. These molecular analyses will have implications not only for P. aeruginosa, but also other bacterial species that use large

160 Chapter 6 adhesion proteins in the transition to a sessile lifestyle. Together, this study has provided insight into the various roles ABC exporters hold, beyond the typical drug-efflux paradigm, and hasprovided a platform for further investigation of their function in P. aeruginosa.

Transition metal ion homeostasis is critical to cellular survival. Biological processes are dependent on the fine-balance between acquisition of essential metal ions to sufficient concentrations, while avoiding metal toxicity due to excessive accumulation. Cellular homeostasis is tightly controlled through transcriptional regulators and, in many cases, uses multiple pathways for the transport of each metal ion. This study sought to characterise the role and contribution of ZnuABC to Zn2+ acquisition in P. aeruginosa, revealing that although it was the primary Zn2+ uptake pathway, it was clearly not the sole pathway. Thus, while the Znu permease may be sufficient for Zn2+ acquisition, it is not essential. This is consistent with analysis of znu deletion mutants in the Oncorhynchus mykiss (rainbow trout) pathogen, Yersinia ruckeri, and the human UTI causative agent, Proteus mirabilis, wherein a growth perturbation was not observed, likely due to the presence of additional unidentified Zn2+ acquisition pathways (Dahiya & Stevenson, 2010; Nielubowicz et al., 2010). Transcriptomic analysis here revealed that this inference as correct for P. aeruginosa as the ΔznuA strain up- regulated a number of putative metal ion associated (i.e. Zn2+) acquisition systems. These systems were quite diverse, including an atypical P-type ATPase Zn2+ and copper importer (HmtA), TonB-dependent outer membrane proteins (PA0781, PA1922, PA2911 and PA4837), a putative cluster A-II dependent ABC import pathway (PA2912-2914), and an atypical ABC import pathway with two putative SBP components (PA4063-4066), which, in themselves are not of adequate size to function as SBPs. The identification of these novel putative Zn2+ import pathways poses a number of questions about the contribution of each system to P. aeruginosa Zn2+ acquisition and the hierarchical and/or plastic nature of these systems as a supplement to the Zn2+ uptake by ZnuABC. The most highly up-regulated gene, PA0781, is homologous to the TonB-dependent outer membrane Zn2+ transporter, ZnuD, from N. meningiditis (Stork et al., 2010). Two Zur-controlled ZnuD homologs are also present in A. baumainii, indicating these proteins may be part of a conserved mechanism to transport Zn2+ across the outer membrane (Hood et al., 2012). P. aeruginosa mutagenesis studies will be required to determine the contribution of PA0781, PA1922, PA2911 and PA4837 to Zn2+ accumulation, both in the absence and presence of the Znu permease. Under Zn2+-limiting conditions, it could be expected that deletion of these outer membrane proteins, in particular ZnuD, would result in an abrogation of intracellular Zn2+ accumulation. Such effects are likely to be even more severe in the absence of the high affinity Zn2+ ZnuABC

161 Chapter 6 system. While ZnuD, and possibly PA1922 and PA4837, are predicted to import Zn2+ ions into the periplasm, transcriptional and bioinformatic analyses suggests that Zn2+ may also be imported into the periplasm and subsequently the cytoplasm in a chelated form. The cluster A-II ABC permease system (PA2912-PA2914), which is predicted to import a chelated form of Zn2+, is co-transcribed with a putative TonB-dependent receptor, PA2911. Together, these proteins are predicted to encode a Zn2+-siderophore uptake system, in which PA2911 translocates the Zn2+-siderophore into the periplasm, where it is bound by the cluster A-II SBP, PA2913, for delivery to the PA2912/PA2914 ABC transporter and transport into the cytoplasm.

The possibility of a Zn2+-siderophore ABC import system is particularly intriguing. Recently, the Zn2+ siderophore, yersiniabactin from Y. pestis was characterised, with the Zn2+- yersiniabactin complex being imported via the MFS transporter, YbtX (Bobrov et al., 2014). Similarly, P. aeruginosa iron acquisition via the siderophore pyochelin relies first on a pyochelin-iron specific TonB-dependent outer membrane protein (FptA) for transport into the periplasm, and subsequently, an inner membrane MFS transporter (FptX) for translocation into the cytoplasm (Cunrath et al., 2015). However, to date no Zn2+-siderophore ABC permease systems have been characterised. If in fact PA2913 does bind a Zn2+-siderophore, further analysis is required to determine the identity of the siderophore, and the genes necessary for its synthesis. It could be assumed that such genes would be significantly up- regulated under Zn2+-limitation, but no clear candidates were observed in the transcriptome analysis carried out in this study. Hence, further investigation is required to elucidate the molecular details of this potentially novel Zn2+ acquisition pathway. P. aeruginosa can thus be proposed to have two distinct mechanisms to ensure it receives adequate Zn2+ from the environment, in either a chelated or labile form, enabling survival under Zn2+ deficiency. Whether PA1922 and PA4837 are capable of importing free or chelated Zn2+ remains to be determined.

The complexity of P. aeruginosa Zn2+ acquisition revealed by this study was not anticipated on the basis of prior studies of bacterial Zn2+ homeostasis. While a number of bacteria have more than one Zn2+ uptake pathway, the possibility that P. aeruginosa utilizes up to four high affinity pathways to transport Zn2+ into the cytoplasm sheds new light on the ability of the bacterium to survive under Zn2+ limiting conditions. This is of particular importance at the host-pathogen interface, wherein the host significantly restricts the labile Zn2+ availability in an attempt to hamper colonization (Hood & Skaar, 2012; Cerasi et al., 2013). Previous analysis of a ΔznuB mutant in a C. elegans paralytic killing assay revealed

162 Chapter 6 the strain to be attenuated in virulence (39 ± 20% killing compared with PAO1), highlighting the importance of Zn2+ for infection (Gallagher & Manoil, 2001). Whilst significant, this attenuation in virulence would likely be more severe, were it not for the presence of the additional Zn2+ acquisition systems identified in the transcriptomic analysis. In particular, the proposed PA2911-PA2914 Zn2+-siderophore system, may be capable of stealing bound Zn2+ from host proteins in a similar manner to iron acquisition by pyoverdine and pyochlein. The variety of acquisition systems present in P. aeruginosa may also be advantageous in an environmental setting, enabling the acquisition of Zn2+ from a range of different compounds or states. Whilst the identified Zn2+ acquisition systems are well conserved throughout P. aeruginosa strains (data not shown), further analysis of other Pseudomonas strains lacking the identified systems will provide greater insight into their role in colonization of human as opposed to environmental niches.

Bacterial ABC permeases enable the specific and efficient acquisition of nutrients and metals. This specificity is conferred largely by the SBP component which co-ordinates the allocrite in a binding pocket between the two lobes of the protein. The P. aeruginosa ZnuA SBP is predicted to co-ordinate divalent Zn2+ tetrahedrally, via three Nε2 atoms contributed by histidine residues, and an oxygen ligand from a water molecule, or glutamate or aspartate residue. However, future structural studies will be required to determine the nature of this additional O-ligand. The long rigid alpha helix between the two lobes, characteristic of cluster A-I SBPs (Berntsson et al., 2010) provides a rigid backbone for structural rearrangements upon binding of the metal ion (Counago et al., 2014). The histidine and acidic amino acid rich loop also contributes to Zn2+ binding, although whether its role is to deliver the Zn2+ to the high affinity site buried deep within the ZnuA protein or to aid in release of the Zn2+ to the ZnuBC transporter remains to be determined (Banerjee et al., 2003). A role for the ZnuA loop in the delivery of Zn2+ to ZnuBC is likely, particularly due to the notable absence of a ZinT homolog in P. aeruginosa, which acts to deliver Zn2+ to ZnuA in other bacterial species (Petrarca et al., 2010; Ilari et al., 2014). However, the precise function of the ZnuA loop region requires further investigation.

The high affinity nature of ABC permeases positions these systems as ideal candidates for the acquisition of trace elements such as molybdenum. In its oxyanionic form, molybdate (Mo), the metal ion is acquired from the environment via the P. aeruginosa ModABC permease system. In contrast to ZnuA, the Mo binding component, ModA, is a cluster D-III SBP, and is predicted to feature two short hinge regions between the two lobes of the protein (Berntsson et al., 2010). Similar to ZnuA, P. aeruginosa ModA is predicted to co-ordinate the

163 Chapter 6

Mo ion tetrahedrally. By contrast with ZnuA, ModA has been shown to co-ordinate Mo via a series of hydrogen bonds (Hu et al., 1997; Lawson et al., 1998; Balan et al., 2008). However, the co-ordinating amino acid side chains are, similar to those in cluster A-I SBPs, unable to discriminate between similar transition row metal ions, such Mo or W, thereby revealing the molecular basis of potential mis-metallation in ModA. Intriguingly, in the Mo/W or W SBPs (WtpA or TupA, respectively) for which high-resolution structural data are available, the oxyanion is co-ordinated octahedrally (Hollenstein et al., 2009; Chan et al., 2010), and is dependent upon hydrogen and ionic bonds, with an additional arginine salt bridge proposed in TupA (Hagen, 2011). This is predicted to aid in the specific acquisition of W from environments with greater Mo abundance to enable its insertion into W-requiring proteins (Hagen, 2011). Further structural and functional characterisation of both P. aeruginosa ModA and ModE proteins would be required to extend the metal binding analyses described in Pederick et al. (2014) and provide further insight into the oxyanion preference of these proteins and the co-ordination of their respective ligands.

Deletion of modA resulted in complete abrogation of Mo accumulation, indicating no other high-affinity Mo transporters were present in P. aeruginosa. Consistent with the known requirement of Mo for dissimilatory nitrate reduction, the abrogation of Mo accumulation resulted in P. aeruginosa being unable to respire anaerobically in the presence of KNO3. Of particular interest, an unanticipated alteration in cell membrane composition and a lack of biofilm formation was observed in cultures undergoing dissimilatory nitrate reduction. The - sequential reduction of NO3 to produce N2 generates NO in an intermediate step, which, while toxic in excess, also acts as a messenger molecule and has been shown to modulate P. aeruginosa biofilm dispersal (Barraud et al., 2006). In both humans and plants, NO has been associated with alterations in membrane fatty acid composition and fluidity (McLauren Dorrance et al., 2000; Zhu & Zhou, 2006). On this basis, NO may be responsible for the alterations observed in P. aeruginosa cell membrane composition under nitrate reducing conditions. Cellular changes, including the promotion of rhamnolipid production are also - stimulated by NO3 (Déziel et al., 2003), with NO similarly speculated to increase rhamnolipid synthesis (Van Alst et al., 2007). Rhamnolipids play key roles in the modulation of biofilm architecture and dispersal (Davey et al., 2003; Boles et al., 2005), and may contribute to the lack of biofilm formation observed in nitrate reducing strains. Growth of P. aeruginosa anaerobically in the presence of NO, together with analysis of a strain incapable of producing NO (nir mutant) would help clarify the effects of NO on both biofilm formation and membrane composition. The findings of this study, that nitrate reduction inhibits biofilm

164 Chapter 6 formation, are in disagreement with that of Van Alst et al. (2007), in which a ΔnarGH strain unable to dissimilate nitrate exhibited reduced biofilm formation. Hence, further work is required to reconcile these findings and elucidate the mechanisms by which the cell membrane composition and biofilm formation of P. aeruginosa is altered in response to nitrate reduction.

The dissimilatory nitrate reduction phenotypes observed in this study have significant implications for human P. aeruginosa infections. In the CF lung, P. aeruginosa has been shown to undergo anaerobic and microaerophilic respiration, with both processes utilizing different terminal electron acceptors for energy generation (Yoon et al., 2002; Alvarez-Ortega & Harwood, 2007; Arai, 2011). The consequences of anaerobic nitrate reduction by P. aeruginosa likely modulate the colonization lifestyle of the bacterium in the CF lung. However, much remains unclear about the precise nature of P. aeruginosa CF lung infections. Furthermore, catheter-associated UTI infections, of which P. aeruginosa is a significant causative agent, are also reported to have varying levels of oxygen tension (Giannakopoulos et al., 1997; Narten et al., 2012). Hence, the impact of these findings upon clinical outcomes remains to be determined, with the significant challenges associated with in vitro characterisation of in vivo processes providing added complexity to data interpretation. Alternatively, the observed phenotypes may have greater relevance to the environmental lifestyle of P. aeruginosa and its colonization of habitats of varying oxygen tension.

The role of metalloregulatory proteins in P. aeruginosa, specifically ModE and Zur, was also investigated in this study. Whilst Zur regulatory sites were identified for all but one of the gene clusters up-regulated ≥4-fold in response to Zn2+ limitation, the P. aeruginosa ModE regulon was more elusive. Twenty putative ModE regulatory sites were identified in the PAO1 genome on the basis of iterative refinements using a γ-proteobacterial ModE motif (Studholme & Pau, 2003). However unexpectedly, little alteration in transcription of these target genes was observed in response to the Mo-deficiency of the ΔmodA strain. Most significantly, Mo-limitation was demonstrated to up-regulate the putative ModE target, PA1864. However, DNA electrophoretic mobility gel shift, immunoprecipitation or DNA pulldown assays are required to confirm the transcriptional regulation of PA1864 by ModE. The uncharacterised PA1864 gene is present adjacent to the modABC permease genes and is predicted to encode a LysR-type transcriptional regulator of unknown target(s). Bioinformatic analyses suggest that PA1864 may have a role in regulating pyrimidine transport and catabolism. However, biochemical and phenotypic analyses will be required to determine its physiological role under Mo-deficiency. The lack of differential expression of putative ModE

165 Chapter 6 target genes in response to Mo-deficiency also raises a new research direction, to understand how such variation in transcriptional response arises from well-conserved putative ModE binding sites with similar E-values. Further analysis would be required to determine if other regulatory proteins provide additional transcription control of the ModE targets, preventing up-regulation under the Mo-limiting conditions assayed.

Collectively, this study provides a detailed description of the roles held by an uncharacterised subset of ABC transporters in P. aeruginosa. It offers significant insight into bacterial metal ion homeostasis in Gram-negative bacteria, highlighting the complexity of the cellular response, and the versatility required on the part of the bacterium to survive in environments of varying metal ion abundance. Furthermore, this work provides a platform for the continued characterisation of P. aeruginosa ABC transporters, exploring both their contribution to virulence and colonization of both humans and environmental niches, in addition to improving the current mechanistic understanding of these proteins, with the observations applicable to the entire ABC transporter superfamily. Together, this study enhances our understanding of P. aeruginosa as both an environmental organism and an opportunistic human pathogen, and highlights the importance of ABC transporters to its complex and versatile lifestyle.

166

APPENDIX 1

167

Sequence alignment of the putative uncharacterised ABC efflux proteins from P. aeruginosa PAO1. The primary sequences of PA0860, PA1113,

PA1876, PA3228 and PA5231 were aligned using Clustal Omega (Sievers et al., 2011) and analysed for ABC efflux protein features. The transmembrane domain and nucleotide binding domain(s) of each protein are indicated in purple and green, respectively. Within the nucleotide binding domains, the characteristic ABC nucleotide binding domain motifs are indicated (Walker A, Q-loop, ABC signature motif, Walker B, D-loop and H-loop). Red asterisks are present above nucleotide binding domain residues that comprise the ATP-binding site. The C39-like domain of PA1876 is indicated in blue. Atypically,

PA5231 features two N-terminal nucleotide binding domains and a C-terminal transmembrane domain. The features of the additional nucleotide binding domain are annotated as Walker A2, Q-loop 2, ABC signature 2, Walker B2, D-loop 2 and H-loop 2.

APPENDIX 2

172 Appendix 2

Genes up-regulated ≥2-fold in ΔznuA vs PAO1

Locus Tag Fold Genome Annotationa Change PA0781 172.20 hypothetical protein PA1922 147.28 TonB-dependent receptor PA5536 134.62 hypothetical protein PA1923 122.23 cobaltochelatase subunit CobN PA4837 109.98 hypothetical protein PA3601 108.96 50S ribosomal protein L31 PA5539 93.65 GTP cyclohydrolase PA3600 89.17 50S ribosomal protein L36 PA4836 72.94 hypothetical protein PA5534 57.55 hypothetical protein PA1925 56.35 hypothetical protein PA5535 46.09 hypothetical protein PA4063 45.09 hypothetical protein PA1924 44.71 hypothetical protein PA5541 37.90 dihydroorotase PA5540 37.63 hypothetical protein PA4835 35.20 hypothetical protein PA4834 27.45 hypothetical protein PA4065 22.06 hypothetical protein PA5538 18.57 N-acetylmuramoyl-L-alanine amidase PA4064 17.20 ABC transporter ATP-binding protein PA1921 15.15 hypothetical protein PA2437 14.40 hypothetical protein PA4066 10.61 hypothetical protein PA2438 10.58 hypothetical protein PA2913 9.06 hypothetical protein PA2439 9.04 hypothetical protein PA5532 8.89 hypothetical protein PA2912 8.76 ABC transporter ATP-binding protein PA2911 8.12 TonB-dependent receptor PA2914 7.97 ABC transporter permease PA4838 6.73 hypothetical protein PA5537 6.66 hypothetical protein PA5542 6.57 hypothetical protein PA2434 6.50 hypothetical protein PA2435 5.57 cation-transporting P-type ATPase PA5543 5.35 hypothetical protein PA5498 5.22 adhesin PA2915 5.21 hypothetical protein PA4833 5.05 hypothetical protein PA3284 5.04 hypothetical protein PA3283 5.00 hypothetical protein PA3282 4.07 hypothetical protein PA2433 3.99 hypothetical protein

173 Appendix 2

Locus Tag Fold Genome Annotation Change PA5499 3.60 transcriptional regulator np20 PA5533 3.44 hypothetical protein PA2664 2.96 NADH dehydrogenase subunit I PA1983 2.68 cytochrome C550 PA2498 2.63 hypothetical protein PA4170 2.61 hypothetical protein PA3597 2.61 amino acid permease PA2663 2.56 psl and pyoverdine operon regulator PpyR PA2436 2.54 hypothetical protein PA3205 2.52 hypothetical protein PA3380 2.51 hypothetical protein PA4577 2.47 hypothetical protein PA4918 2.46 hypothetical protein PA5170 2.43 arginine/ornithine antiporter PA1111 2.42 hypothetical protein PA2357 2.42 NADH-dependent FMN reductase MsuE PA2343 2.38 xylulose kinase PA3281 2.38 hypothetical protein PA5500 2.38 Zn2+ transporter ZnuC PA3444 2.37 alkanesulfonate monooxygenase PA4832 2.35 short-chain dehydrogenase PA3448 2.31 ABC transporter permease PA3126 2.25 heat-shock protein IbpA PA1920 2.23 anaerobic ribonucleoside triphosphate reductase PA2375 2.23 hypothetical protein PA1919 2.20 class III anaerobic ribonucleoside-triphosphate reductase activating protein NrdG PA4761 2.20 molecular chaperone DnaK PA1597 2.20 hypothetical protein PA0998 2.19 PqsC protein PA0673 2.19 hypothetical protein PA4759 2.16 dihydrodipicolinate reductase PA0997 2.15 PqsB protein PA2937 2.14 hypothetical protein PA4917 2.12 hypothetical protein PA2753 2.08 hypothetical protein PA1952 2.07 hypothetical protein PA3900 2.07 transmembrane sensor PA0200 2.06 hypothetical protein PA5501 2.05 Zn2+ ABC transporter permease PA4708 2.03 Heme-transporter PhuT PA1323 2.02 hypothetical protein PA4709 2.02 hemin degrading factor PA3404 2.02 hypothetical protein PA1911 2.01 sigma factor regulator FemR

174 Appendix 2

Genes down-regulated ≥2-fold in ΔznuA vs PAO1

Locus Tag Fold Genome Annotationa Change PA4817 0.50 hypothetical protein PA0618 0.50 bacteriophage protein PA0905.2 0.49 tRNA-Arg PA3792 0.47 2-isopropylmalate synthase PA1555 0.47 cytochrome C oxidase cbb3-type subunit CcoP PA4541.1 0.47 tRNA-Lys PA0517 0.46 cytochrome C PA0013 0.46 hypothetical protein PA3133.2 0.45 tRNA-Ala PA0516 0.45 heme d1 biosynthesis protein NirF PA3133.3 0.44 tRNA-Glu PA0277 0.44 hypothetical protein PA0511 0.43 heme d1 biosynthesis protein NirJ PA0512 0.42 hypothetical protein PA3133.4 0.42 tRNA-Ala PA2819.3 0.42 tRNA-Glu PA2819.2 0.41 tRNA-Gly PA2819.1 0.40 tRNA-Gly PA4937.2 0.39 tRNA-Leu PA0513 0.37 transcriptional regulator PA4937.1 0.37 tRNA-Leu PA0514 0.32 heme d1 biosynthesis protein NirL

a Functional annotation as recorded in GenBank database.

175

APPENDIX 3

176

Lewis [Pederick], V.G., Ween, M.P. & McDevitt, C.A. (2012). The role of ATP- binding cassette transporters in bacterial pathogenicity. Protoplasma, 249(4), 919-942.

NOTE:

This publication is included on pages 177 - 200 in the print

copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1007/s00709-011-0360-8

APPENDIX 4

201 Appendix 4

202 Appendix 4

203 Appendix 4

204 Appendix 4

205 Appendix 4

206 Appendix 4

207 Appendix 4

208 Appendix 4

209 Appendix 4

210 Appendix 4

211 Appendix 4

212 Appendix 4

213 Appendix 4

214

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