INVESTIGATION OF IRON’S ROLE IN ALGINATE PRODUCTION AND

MUCOIDY BY PSEUDOMONAS AERUGINOSA

by

JACINTA ROSE SCHRAUFNAGEL

B.S., University of Colorado Denver, 2003

M.S., University of Colorado Denver, 2006

A thesis submitted to the

Faculty of the Graduate School of the

University of Colorado in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Molecular Biology Program

2013 This thesis for the Doctor of Philosophy degree by

Jacinta Rose Schraufnagel

has been approved for the

Molecular Biology Program

by

David Barton, Chair

Michael Vasil, Advisor

Mair Churchill

Jerry Schaack

Michael Schurr

Martin Voskuil

Date 10/30/13

ii Schraufnagel, Jacinta R. (Ph.D., Molecular Biology)

Investigation of Iron’s Role in Alginate Production and Mucoidy by Pseudomonas aeruginosa.

Thesis directed by Professor Michael Vasil.

ABSTRACT

Iron, an essential nutrient, influences the production of major virulence factors and the development of structured non-alginate biofilms in Pseudomonas aeruginosa. Fur, pyoverdine and various concentrations of iron sources have all been shown to affect non-alginate biofilm formation. Remarkably however, the role of iron in alginate biofilm formation and mucoidy is unknown. Because of alginate’s association with biofilms in chronic cystic fibrosis (CF) lung infections, we examined the influence of iron on alginate production and mucoidy in P. aeruginosa. Herein, we identify and characterize a novel iron-regulated protein that affects the transcription of alginate biosynthesis genes. Further, contrary to what is known about non-alginate biofilms, we discovered that biologically relevant concentrations of iron (10 µM – 300 µM), in various forms (e.g. FeCl3,

Ferric Citrate, Heme), led to the decreased expression of alginate biosynthesis genes and the dispersion of alginate biofilms in mucoid P. aeruginosa PAO1,

PA14, PAKS-1 and PS388 non-CF isolates. Examination of mucoid CF clinical isolates revealed that late-stage isolates acquired the ability to maintain the mucoid phenotype even in the presence of iron. Traditional iron acquisition (e.g.

Pyoverdine and Pyochelin) systems in these nonresponders were often dysregulated, suggesting that these isolates may be less efficient at acquiring

iii iron. Deletion of pvdS did not affect the ability of mucoid strains to produce alginate and fur mutations were not present in the nonresponders. Furthermore, in the presence of iron, mucoid non-CF isolates retained their ability to form non- alginate biofilms in a microtiter plate assay. Finally, we assessed the anti- microbial activity of gallium on mucoid strains and clinical CF isolates as well as the effects of gallium on alginate production. We found that gallium did not have any effect on the production of alginate, but did exhibit anti-microbial activity regardless of the organism’s ability to regulate alginate production in response to iron.

This dissertation represents the first in-depth examination of iron’s effect on alginate production. We demonstrate that, unlike non-alginate biofilms that require iron to form structured biofilms, alginate biofilms form in the absence of this important micronutrient.

The form and content of this abstract are approved. I recommend its publication.

Approved: Michael L. Vasil

iv DEDICATION

This dissertation is dedicated to my Grandmother, Mrs. Helen Rose

Schraufnagel, for playing such a significant and influential role throughout my life.

To my parents, Daniel and Helga Schraufnagel, for their endless love, support, encouragement and for teaching me the value of hard work and determination.

To my husband, Adam Wiens, who is the love of my life.

v ACKNOWLEDGMENTS

First and foremost, I would like to thank my parents for their love and support throughout my life. Thank you for working hard to provide a stable foundation for my life and future, you have taught me invaluable lessons about work, marriage, parenting, perseverance and integrity. I want to thank my husband for his love, understanding, for his unlimited patience and for always believing in me.

Importantly, I would like to thank my advisor, Michael L. Vasil. Dr. Vasil warmly welcomed me into his lab and gave me the independence to think for myself and develop my own project. He had confidence in my abilities, offered encouragement and advice when it was needed most and conferred enough discipline to help me develop into a strong, moral, quality scientist. His guidance and persistence have not only made me a better scientist, but also a more patient and confident person. I’d like to thank Adriana Vasil for her support and assistance over the years. Adriana’s technical expertise and dedication to making quality mutants were invaluable. Thank you to all the other members of

Dr. Vasil’s lab who made the lab an entertaining place to be. Thanks to Rhea

May, Martin Stonehouse, Art Pritchard and Zach Wilson for being great colleagues. I would also like to thank Amanda Oglesby for helping me construct my very first mutant (PA4704) during my rotation, and Sarah Parker who offered her protein expression technical expertise. I would also like to thank my thesis committee David Barton, Mair Churchill, Jerry Schaack, Michael Schurr and

vi Martin Voskuil. Thank you all for your direction, advice, encouragement, pep talks and making me think critically about science. Lastly, I would like to thank all of my friends, old and new, who have been both understanding and supportive throughout this journey. You have all been instrumental in this body of work and in my graduate career. Thank you all.

vii CONTENTS

CHAPTER

I. INTRODUCTION...... 1

Pseudomonas aeruginosa Biology and Pathogenesis...... 2

Cystic Fibrosis...... 4

Host and Bacterial Factors That Contribute to P. aeruginosa Lung Infection in Individuals With Cystic Fibrosis…………...... 7

Iron and the CF lung...... 12

P. aeruginosa Iron Acquisition...... 14

Regulation of Iron Homeostasis in P. aeruginosa...... 17

P. aeruginosa biofilms...... 19

Psl...... 22

Pel...... 23

Alginate...... 24

Iron and P. aeruginosa Biofilms...... 31

Purpose of This Study...... 34

II. MATERIALS AND METHODS...... 36

Materials and Chemicals...... 36

Bacterial Strains, Plasmids and Growth Conditions...... 36

Construction of Mutants……………………………………………………….37

P. aeruginosa Chromosomal DNA Isolation...... 41

Polymerase Chain Reaction (PCR)...... 41

Plasmid Purification...... 42

DNA Ligation...... 43

viii DNA Gel Purification...... 43

Escherichia coli Chemically Competent Cells and Transformation………………………………………………...... 44

Tri-Parental Mating...... 45

Confirmation of Mutants...... 45

RNA Isolation and qRT-PCR……….………………………………………...47

Affymetrix GeneChip Microarray……………………………………………..47

Uronic Acids Assay…………………………………………………………....48

Glycosyl Composition Analysis of Extracellular Polysaccharides...... 49

Purification of PA2384………………………………………………………...50

SDS Polyacrylamide Gel Electrophoresis...... 51

Gel Mobility Shift Assay……………………………………………………….51

Affinity Purification of Antibodies...... 52

Western Blot and Chemiluminescence...... 53

Microtiter Plate Biofilm Assay………………………………………………...54

Analysis of Siderophore Production…………………………………………54

Growth Curves…………………………………………………………………55

Minimum Inhibitory Concentration (MIC)……………………………………55

Zone of Inhibition………………………………………………………………56

III. STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF PA2384……………………………………...... 57

Structural Features of PA2384…………………………………...... 58

Iron-Regulated Expression of PA2384……………………………………..61

ix Global Gene Expression Profile of ∆PA2384 in Response to Iron-Limitation………………...……………………………………………….63

Purification of PA2384………………………………………………………..67

Does PA2384 Protein Bind to the algD Promoter?...... 70

PA2384 May Function as a Dimer…………………………………………..72

Functional Analysis of PA2384……………………………………………...74

IV. IRON-REGULATED EXPRESSION OF ALGINATE PRODUCTION AND MUCOIDY BY PSEUDOMONAS AERUGINOSA...... 78

Iron Influences Alginate Levels and the Mucoid Phenotype of P. aeruginosa Muc Mutants Constructed From Isolates of Non-CF Origin……………………...………………………………...... 79

Transcriptional Analysis of PAO1 Wild Type and PAO1 ∆mucA Under Iron-Limitation...... 86

Do CF-Pulmonary Isolates Regulate Alginate Production in Response to Iron Levels?...... 93

How Does Iron Initially Control the Increased Production of Extracellular Alginate and Mucoidy and How Can This Regulation be Lost?...... 96

Can Mucoid Strains Produce Pel and Psl Biofilms in Iron-Replete Conditions?...... 102

V. INVESTIGATION OF GALLIUM ON ALGINATE PRODUCTION AND MUCOIDY...... 104

Assessment of Growth Media for Gallium Susceptibility Testing……….106

Minimum Inhibitory Concentrations of PAO1, muc Mutants and CF Isolates in a Chemically Defined Minimal Medium...... 110

Does Iron Reverse the Bactericidal Activity of Gallium?.……………..…112

The Effects of Gallium on Alginate Production……………………………113

x VI. DISCUSSION

Iron in Bacterial Virulence and Host-Pathogen Interactions……….…....115

The Role of Iron in Biofilm Formation………………………………………118

PA2384 in Biofilm Formation..……………………………………………...120

Iron and Alginate Biofilm Formation………………………………………..125

The Inverse Relationship of P. aeruginosa Biofilms in Response to Iron…………………………………………………………...... 129

Iron Homeostasis as a Target For Therapeutic Interventions in Non-CF and CF Related Infections……………………………………...131

How Our Results May Impact the Development of Current and Future Iron-Targeted Therapies……………………………………….134

Hypotheses For Future Investigations……………………………………..135

REFERENCES...... 138

APPENDIX

A. Results of the PA2384 18 Hour GeneChip® Microarray………….…159

B. MALDI-TOF Results of Purified PA2384………………………………164

C. Results From 24 Hour GeneChip® Microarray……………….……....165

D. Table of Early Clinical Isolates………………………………………….198

E. Table of Late Clinical Isolates…………………………………………..202

F. Bacterial Media Recipes...... 206

G. Antibiotics and Concentrations…………..……………………………..208

xi TABLES

Table

1.1. Virulence factors of P. aeruginosa...... 5

1.2. Alginate biosynthesis and regulatory genes in P. aeruginosa...... 28

2.1. Strains used in this study……………………………………………………38

2.2. Plasmids used in this study…………………………………………………39

3.1. Genes with increased expression in ∆PA2384 isogenic mutant under iron-limitation……………..…………...……………………………...64

3.2. PA2384 is highly conserved in P. aeruginosa…………………………….76

3.3. Clues about the function of PA2384 in P. aeruginosa from microarray data………………………………………...…………………….77

4.1. Select genes with increased gene expression under iron- limitation vs. iron-replete conditions in PAO1 and ∆mucA…….………...88

5.1. MIC values for Ga3+ measured after 6, 24, and 48 hrs of growth for wild type PAO1, ∆mucA and three Late-CF isolates…………….…109

5.2. MIC values for Ga3+ of all strains grown in M9 + Casamino acids……………………………………….………………………………...111

xii FIGURES

Figure

1.1. Host and bacterial factors that contribute to colonization and persistent infection...... 9

1.2. The Fur regulation in P. aeruginosa...... 16

1.3. Main stages of biofilm formation in P. aeruginosa...... 20

1.4. Alginate precursor synthesis...... 25

1.5. Constitutive activation of alginate biosynthesis...... 30

3.1. Protein sequence alignment of PA2384 and Fur………….……...……...59

3.2. Model of PA2384 N-terminal region…………….……...………………….60

3.3. PA2384 is optimally transcribed under iron-limitation……………………62

3.4. qRT-PCR analysis of algD and algG in wild type PAO1 and ∆PA2384………...... ……………………………………………………..66

3.5. PA2384 insoluble and soluble fractions…………..…..…………………..68

3.6. PA2384 purified protein………………...……………….…………………..69

3.7. Gel mobility shifts of PA2384 binding to algD promoter fragments……………………………………………………………………..71

3.8. PA2384 captured in its native state using the cross-linker BS3………...73

3.9. Alginate production by PA2384…………………………………………….74

4.1. The effect of iron on alginate production by muc mutants of P. aeruginosa PAO1………………………..…………………………….81

4.2. Effects of iron levels on alginate production by ∆mucA and ∆mucB mutants of P. aeruginosa PA14, PAKS-1, and Ps388………….82

4.3. The influence of various iron sources on alginate production by P. aeruginosa PAO1 ∆mucA and ∆mucB mutants.…………………..83

4.4. The influence of assorted metals on alginate production………………..84

xiii 4.5. Alginate production and mucoidy of Late CF isolates (Late-CFI) in response to variable iron levels………………….………………………95

4.6. Investigation of alginate production in iron acquisition mutants of P. aeruginosa PAO1……………………………….……………………..98

4.7. Investigation of the role of Fur in alginate production…………………..101

4.8. Mucoid ∆mucA and ∆mucB form Pel/Psl biofilms in iron-replete conditions……………………………………………………………………103

5.1. Growth of wild type PAO1 in chemically defined and undefined media…………………………………………………………....107

5.2. Iron reversed gallium bactericidal activity…………………………….....113

5.3. The effects of gallium on alginate production…………………………...114

xiv CHAPTER I

INTRODUCTION

Despite numerous advances in our understanding of Pseudomonas aeruginosa biology and pathogenesis over the last several decades, this opportunistic pathogen remains the leading cause of morbidity and mortality in people with the recessively inherited genetic disorder cystic fibrosis (CF). P. aeruginosa does not typically harm healthy individuals, however, the high frequency of CF patients infected with P. aeruginosa results from the ubiquitous nature of this bacterium and this organism’s keen ability to thrive in the harshest environments. The airways of people with CF, beginning shortly after birth, are nearly always infected with many different microorganisms, resulting from the individual’s inability to expel inhaled foreign particles by mucociliary clearance.

The inability to eradicate P. aeruginosa from the CF airway, as well as the long- term presence of this pathogen (often times persisting for the life of the patient) is undoubtedly, at least in part, due to the intrinsic resistance of P. aeruginosa to many commonly used antibiotics, as well as this pathogen’s arsenal of virulence determinants. As a consequence, treatment is cumbersome and often results in poor patient outcomes. In recent years, new approaches have been examined to circumvent widespread antibiotic resistance. One approach that has been used to control P. aeruginosa infections in the CF airway is to interfere with bacterial iron homeostasis [2]. In some cases, patients are given iron chelators as a form

1 of treatment to sequester available iron that P. aeruginosa requires for prolific growth. Iron is not only an essential nutrient required for P. aeruginosa growth and survival, but also acts as a signaling molecule influencing the pathogen to turn on and off virulence factors, including biofilm formation. The ability of P. aeruginosa to form biofilms enhances the organism’s ability to survive in the lungs by encasing the cell with an exopolysaccharide layer that protects against antibiotics, oxidative stress and host immune responses [3, 4]. P. aeruginosa produces as many as three distinct exopolysaccharides, Polysaccharide synthesis locus (Psl), Pellicle (Pel) and alginate, each of which is associated with specific types of biofilms and conditions under which they are formed [5]. While iron augments the formation of Psl-type biofilms, the role of iron in alginate production, which is the main exopolysaccharide produced by P. aeruginosa in the CF lung, is unknown [6]. The aim of this study was to elucidate iron’s role in alginate production and the resulting mucoidy phenotype, which will aid in identifying novel therapeutic strategies for treating CF patients inflicted with P. aeruginosa infection.

Pseudomonas aeruginosa Biology and Pathogenesis

Pseudomonas aeruginosa is a gram-negative, rod-shaped, monoflagellated bacterium with broad metabolic capabilities. P. aeruginosa typically uses aerobic respiration; however, in circumstances where oxygen is limited, the organism can also thrive in hypoxic conditions and even anaerobic environments using anaerobic respiration . This pathogen has very simple growth

2 requirements and an ability to utilize a wide-range of organic material, thus this organism is endemic in many natural and artificial environments. P. aeruginosa is found in soil, water, skin flora, catheters, hot tubs, showerheads and many other human-made environments throughout the world. Additionally, the bacterium can be useful in bioremediation since it has the ability to decompose hydrocarbons and has been used to break down crude oil and tarballs from oil spills [7, 8]. However, while P. aeruginosa is ubiquitous and extremely versatile, this microbe, surprisingly, does not normally harm healthy individuals. P. aeruginosa pathogenesis is usually limited to immunocompromised individuals: patients with traumatic wounds, severe burns, cancer and people with the autosomal recessive genetic disorder cystic fibrosis (CF).

The P. aeruginosa chromosome is relatively large (6-7 Mb) and G+C-rich

(~65%), encoding around 6,000 predicted open reading frames which can vary from strain to strain by as much as 1 megabase or more [9]. According to pseudomonas.com, to date as many as 13 P. aeruginosa genomes have been completely sequenced, with greater than 5,000 genes well conserved across all

13 genomes analyzed. This large genome not only containing genes that allow this organism to be metabolically versatile, but also encompassing an abundance of genes encoding virulence factors that contribute to this pathogen’s ability to successfully cause disease in humans.

P. aeruginosa is capable of producing an arsenal of cell-associated and extracellular virulence factors that contribute to the organism’s ability to proliferate in various niches and cause disease. For example, this pathogen

3 produces flagella, type IV pili, type III secretion system, numerous toxins, phospholipases, hemolysins, pyocyanin, pyoverdine, pyochelin, and three known exopolysaccharides that aid in the organism’s survival (see Table 1.1) [10-14]. All of these virulence factors play various roles that allow this pathogen to survive, persist and adapt to changing environments, such as in the context of the CF lung.

In addition to the repertoire of virulence factors shown in Table 1.1, P. aeruginosa is innately resistant to numerous antibiotics and acquired resistance is steadily increasing [15]. The ubiquitous nature of P. aeruginosa, combined with ineffective remedies to treat infections, make this pathogen one of the leading causes of morbidity and mortality in individuals with cystic fibrosis.

Cystic Fibrosis

Cystic fibrosis (CF) is an autosomal recessive disease that mainly affects the lungs and digestive system and is the most common lethal genetic disease in the Caucasian population. There are roughly 70,000 children and adults worldwide with the disease and about 30,000 cases in the United States alone

(http://www.cff.org). CF is most common in populations of northern European descent with approximately 1,000 new cases diagnosed each year. The birth prevalence varies from country to country and with ethnicity. CF occurs in 1 in

3,000 Caucasian Americans, 1 in 4,000-10,000 Latin Americans, 1 in 15,000-

20,000 African Americans and 1 in 350,000 Japanese [16].

4 Table 1.1 Virulence factors of P. aeruginosa Virulence Factor Mechanism of Action Biological Properties References Adhesins and Motility Type IV pilus Attachment and motility Initial colonization, twitching [17] motility Flagellum Attachment and motility Adherence, invasion, [18] chemotaxis Structural LPS Endotoxin Major cell wall antigen [19] Proteases LasA Serine protease, degrades Tissue invasion and [20] elastin destruction LasB Zinc metalloprotease, Tissue invasion, degrades [20] degrades elastin innate immunity proteins such as chemokines and cytokines Alkaline Protease Degrades LasA nicked Tissue invasion, destruction [21] alastin of nonspecific host defenses PrpL (Protease IV) Endoprotease, degrades Implicated in iron scavenging [22] casein, lactoferrin, transferring, elastin and decorin Phospholipases PlcH (Hemolytic Phospholipase C) Hydrolysis of Hemolysin, nutrient [23, 24] phosphatidylcholine (PC) acquisition, protection from and sphingomyelin (SM), osmotic stress, production of SM snythase lipid signaling mediators PlcN (Non-hemolytic Hydrolysis of PC and Nutrient acquisition, [25] Phospholipase C) phosphatidylserine (PS) protection from osmotic stress, production of lipid signaling mediators Pld Hydrolysis of PC Survival in host [26] PlcB (Broad Spectrum Hydrolysis of PC, SM, PS Production of lipid signaling [25] Phospholipase C) and mediators, required for phosphatidylethanolamine chemotaxis up a PE gradient (PE) Rhamnolipids Heat stable hemolysin Hemolytic glycolipid, disrupts [27, 28] intracellular junctions, implicated in motility and biofilm formation Toxins Cytotoxin (leukocidin) Cytotoxic Disrupts leukocyte function [10, 29] Exotoxin A ADP-ribosyl transferase Inhibits protein synthesis by [30-32] activity ADP ribosylation of eukaryotic elongation factor 2 Pyocyanin (PCN) redox-active phenazine Mediates tissue damage and [33] necrosis during lung infection, blue-green pigment Cyanide toxin Prevents electron transport [34] and metabolism Type III Effectors Exoenzyme S ADP-ribosyltransferase Prevent bacterial uptake and [10, 35] and Rho family GTPase phagocytosis, Inhibition of activating protein DNA synthesis, Actin cytoskeleton disruption Exoenzyme T ADP-ribosyltransferase Prevent bacterial uptake and [10, 35] and Rho family GTPase- phagocytosis, Inhibition of activating protein cell migration and proliferation

5 Table 1.1 Virulence factors of P. aeruginosa Virulence Factor Mechanism of Action Biological References Properties Exoenzyme U Phospholipase A2/Patatin activity Cytotoxic, activates [10, 35] proapoptotic pathway Exoenzyme Y Adenylate cyclase Alteration of cell [10, 35] function and host gene expression, Actin disruption Siderophores Pyochelin High-affinity binding of iron Iron scavenging [11] Pyoverdin High-affinity binding of iron, Iron scavenging, [11] signaling of other toxins signaling other iron systems, yellow- green pigment and fluorescent Exopolysaccharides Alginate Biofilm formation 1-4’-linked β-D- [5, 36, 37] mannuronate and its C-5 α-L-guluronate residues, adherence, antiphagocytic, physical barrier, marker of chronic infection Psl Biofilm formation Galactose- and [5, 38, 39] mannose-rich exopolysaccharide Pel Biofilm formation Glucose-, rhamnose- [5, 40, 41] and mannose-rich exopolysaccharide

Cystic fibrosis is caused by a mutation in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The CFTR gene was first cloned and sequenced in 1989, since then, there have been tremendous efforts to understand the molecular mechanisms surrounding this disease [42]. The CFTR protein is expressed in many epithelial cells and blood cells. More than 1,500 CFTR mutations have been identified, but the functional importance of only a small number of these mutations are known. CFTR is a cAMP-dependent chloride channel required for normal ion transport across epithelial cell membranes. However, CFTR has been linked to many other regulatory functions such as inhibition of sodium transport through the epithelial sodium channel, regulation of ATP channels, regulation of intracellular vesicle

6 transport, acidification of intracellular organelles, and inhibition of endogenous calcium-activated chloride channels involved in bicarbonate-chloride exchange.

Advances in technology continue to allow for the identification of novel functions for this protein [43-48].

The clinical symptoms of this disease manifest in various ways: individuals with CF commonly have salty-tasting skin, poor growth/weight gain, difficulty in bowel movements, persistent coughing and phlegm, wheezing, shortness of breath, and frequent lung infections. The outlook for people diagnosed with CF has improved drastically over the past 20 years. In the 1950s, very few children born with this disease lived to attend elementary school (http://www.cff.org).

Today, the average life expectancy for children born with CF is 37 years with the potential to live to be 50 years of age or more [16]. However, even with the advent of therapies and knowledge relating to ∆CFTR mutations, 80 to 95% of patients with CF succumb to chronic P. aeruginosa infection leading to respiratory failure and death [49].

Host and Bacterial Factors That Contribute to P. aeruginosa Lung Infection in Individuals With Cystic Fibrosis

The microbial flora of a CF patient can be extremely diverse with multiple species coexisting. The airways of cystic fibrosis patients are susceptible to infection with numerous pathogens, such as Pseudomonas aeruginosa,

Burkholderia cepacia, Stenotrophomonas maltophilia, methicillin-resistant

Staphylococcus aureus (MRSA), Hemeophilus influenzae, Streptococcus

7 pneumoniae, non-tuberculous mycobacteria, various fungal species and respiratory viruses [16, 49, 50]. Despite this complex ecosystem, P. aeruginosa is the most prevalent pathogen cultured from CF sputum. Furthermore, this bacterium plays a critical role in the development and progression of pulmonary disease in CF patients. A study conducted by Burns and coworkers assessing the frequency of P. aeruginosa infection in young children with CF showed that

98% of the 40 CF patients they tested had serological or culture evidence of P. aeruginosa by 3 years of age [51]. After the onset of P. aeruginosa infection in infancy, CF patients experience episodic exacerbations throughout their lives.

There are numerous studies indicating that cross-infection (patient to patient) with P. aeruginosa is rare and that each CF patient becomes infected with a unique strain of P. aeruginosa presumably originating from the environment [49,

51, 52]. In support of this, clones have been isolated from sibling CF patients as well as unrelated patients throughout the world to understand the epidemiology of P. aeruginosa infection. It has been shown that strains infecting individuals with CF are largely unique and thought to have been acquired from the environment. While this study did show that some siblings did indeed have the same strain, it wasn’t clear if the same strain was acquired from cross-infection or a common environmental reservoir [50]. Fascinatingly, once a particular strain infects the CF lungs, that stain will typically, but not always, persist for the life of that individual.

8 CF Lung P. aeruginosa "! Genetic and metabolic flexibility "! Adhesion to epithelium !! Innate immune mechanisms •! Mucin specific adhesins (FliD) !! Mucociliary clearance "! Antibiotic resistance !! iNOS Lung Colonization and !! Expression of Flagella "!Bacterial persistence and inflammation persistent infection ! Biofilm mode of growth Permissive microhabitats •! Alginate (Mucoidy) Other unidentified factors •! Pel and Psl may be involved in one or ….more stages of infection Efficient iron acquisition systems

Figure 1.1. Host and bacterial factors that contribute to colonization and persistent infection. Host and bacterial factors that are thought to contribute to P. aeruginosa lung infection in CF patients. This Figure was adapted and modified from that in (Gomez et al., 2007).

There are several host and bacterial factors that are thought to contribute to P. aeruginosa colonization and persistent, chronic infection; however, the mechanism of infection by this pathogen is still not completely understood

(Figure 1.1). The airways of humans are highly compartmentalized with distinct microhabitats and characteristics. The human lung can generally be broken- down into the upper respiratory tract and the lower respiratory tract. The upper airways (Nasal passage, paranasal sinuses, nasopharynx, oral pharynx and trachea) are prime sites for introduction and deposition of inhaled pathogenic microbes. In normal individuals ciliated epithelium lining the airways expels inhaled microbes from the upper respiratory tract, a process referred to as mucociliary clearance, thus allowing the lower respiratory tract to remain sterile.

However, individuals with CF are largely compromised in mucociliary clearance and are unable to expel foreign invaders. In addition, P. aeruginosa produces cytotoxins, such as pyocyanin, that alter cilia function and CFTR expression and 9 trafficking to the epithelial cell surface [53, 54]. The impairment of innate immune responses, such as decreased mucociliary clearance and decreased iNOS

(component of bacterial killing), of individuals with CF reduces obstacles that would normally prevent pathogens, including P. aeruginosa, from travelling deep into the lower airways of the lungs [55]. The lower respiratory airway is a breeding ground for bacteria and in CF patients, the lower airway will become persistently colonized by microbes. Thick, viscous mucus is produced in the lower airway and acts as a major reservoir for bacterial proliferation [56]. Mucus is distributed heterogeneously throughout the lower airways along with various nutrients and oxygen levels, creating unique niches and microenvironments that support bacterial growth and survival [56, 57].

Chronic P. aeruginosa infection, defined as persistent infection lasting for at least 6 months, correlates with a decline in pulmonary function and poor outcomes for individuals with CF [49, 50, 55, 58]. However, it is not entirely clear why or how P. aeruginosa effectively colonizes the CF airways. P. aeruginosa is genetically and metabolically flexible and has an arsenal of virulence factors at it’s disposal. It has been proposed that P. aeruginosa adheres to CF airway epithelial cells better than to non-CF cells and that this interaction occurs through binding of P. aeruginosa pili and flagella to a carbohydrate sequence found in glycosphingolipids asialo-GM1 and asialo-GM2 [59-61]. Pilin-mediated adhesion is an important component of pathogenesis for this bacterium; without adhesion, injection of type III secreted toxins (e.g. ExoS and ExoT) will not occur. However, adhesion is only one attribute that this bacterium uses to cause infection. P.

10 aeruginosa has multiple regulatory pathways and virulence factors, some of which are discussed throughout the rest of this chapter, that allow it to adapt and thrive in the CF airway.

The changes that a P. aeruginosa environmental isolate undergos in the

CF lung is a remarkable example of microbial adaptation. In most cases, the originating strain goes through tremendous genotypic and phenotypic changes while surviving for decades in the CF airways. Frequently, the genotypes and phenotypes of the strains present in late stages of infection differ substantially from those that initially colonize the CF lung. The rapid adaptation of P. aeruginosa to CF airways may be partly due to the presence of hypermutator strains. It has been reported that as many as 60% of CF patients chronically infected with P. aeruginosa carry hypermutable strains, compared to less than

1% of individuals with acute infection [62-64]. Many of these hypermutators have mutations in DNA repair genes, such as mutS [62]. Normal bacteria typically have a mutation frequency of 1 x 10-8 whereas hypermutators have mutation frequencies as high as 1 x 10-6 [50, 62, 64]. Surprisingly, many genes that have been found to be mutated in late-CF isolates are virulence factors, including genes functioning in type III secretion, twitching motility, exotoxin A regulation, iron acquisition, phenazine biosynthesis, quorum sensing, multidrug efflux, osmotic balance, and O-antigen biosynthesis [65]. While hypermutators often carry mutations in genes encoding virulence factors, the ability of the organism to rapidly alter gene expression through mutagenesis may be advantageous by

11 selecting for antibiotic resistance and allowing the pathogen to form alginate biofilms (mucoidy).

Iron and the CF Lung

Iron homeostasis is of crucial importance to the biology and physiology of microbial pathogens and their mammalian hosts. Iron is a redox active metal that is required for numerous proteins (e.g. heme, iron-sulfur containing proteins, enzymes) to perform normal cellular processes, including cellular respiration,

DNA synthesis and oxygen transport. Virtually all forms of life, including most bacteria, have an absolute requirement for iron. P. aeruginosa is no exception, requiring an abundance of iron for growth and cellular respiration. The biological utility of iron comes from its ability to exist in one of two inter-convertible redox states, the reduced Fe2+ ferrous form and the oxidized Fe3+ ferric form. This property allows iron to be incorporated into proteins, act as a biocatalyst or as an electron carrier. By and large, pathogens, including P. aeruginosa, can acquire all of the nutrients they need from host tissue, blood or proteins. Carbon, nitrogen, amino acids, phosphate, sulfate, potassium, magnesium, manganese, zinc, and copper, among a plethora of others, are all freely available in fluids and tissues of the body. Iron, however, is typically exceedingly limited. In biological solutions, at physiological pH, ferrous iron is readily oxidized to the ferric form, which is highly insoluble and not biologically useful. Many mammalian and bacterial proteins bind iron in its ferric form. Ferric iron is relatively non-toxic, whereas ferrous iron can lead to the production of highly reactive oxygen species

12 that wreak havoc on cellular proteins and nucleic acids (Fenton reaction) [66].

Healthy vertebrates are virtually devoid of free iron, sequestering iron from pathogens in iron-binding proteins (e.g. hemoglobin, ferritin, transferrin, and lactoferrin) [67-69]. Therefore, pathogens encounter iron-limiting conditions upon entering their host and engage in fierce competition with the host for this important nutrient.

Historically, the CF airways have been considered to be an iron deficient environment. Hypoferremia is a common condition in people with CF, often correlating with deteriorating lung function and poor overall health [70]. However, iron homeostasis in the CF lung is not well understood and until recently, there have been little research or available techniques to accurately measure the levels of iron that exist in the lung environment. Reports within the last decade have consistently established that levels of iron in the lungs of a CF patient vary considerably, suggesting the existence of diverse microenvironments within the airways. Reid et al. suggested that the major sources of iron in the CF lung are free iron (Fe3+) and ferritin and that the levels of these iron compounds can be much higher than previously thought, 17 to 134 µM and 894 to 6,982 µg/L, respectively [71]. Moreover, numerous studies have demonstrated that there is a correlation between hypoferremia, or iron-deficiency, and increased deposition of iron and ferritin in the CF lung, as a result of redirecting iron from hemoglobin to lung tissue and ferritin (mammalian iron storage protein) [70-73]. This increase in airway iron may result from an increase in iron-regulatory cytokines (IL-1β) or an increase in the tumor necrosis factor (TNF-α) [71, 72]. In addition to increased

13 ferric iron and ferritin, there is evidence that transferrin and lactoferrin undergo proteolysis by P. aeruginosa in the CF airways, releasing stored iron into the surrounding environment [74]. Interestingly, numerous studies have shown a positive correlation between increasing CF lung iron and P. aeruginosa infection, suggesting that iron is an important factor in the establishment and persistence of chronic P. aeruginosa lung infection [69, 71, 72, 75].

P. aeruginosa Iron Acquisition

P. aeruginosa is extremely efficient at scavenging iron and has evolved a highly effective armamentarium to acquire iron from its surrounding environment.

One of the most common and efficient iron-scavenging mechanisms employed by P. aeruginosa is the production, secretion and uptake of siderophores, high- affinity Fe3+ chelating molecules that play an important role in acquiring iron for growth and pathogenesis. P. aeruginosa produces two known siderophores, pyoverdine and pyochelin [76]. Pyoverdine is the dominant siderophore, with a higher binding affinity for iron than pyochelin, between 10-24 mol/L to 10-27 mol/L, depending on the type of pyoverdine produced. P. aeruginosa is capable of synthesizing three distinct pyoverdine molecules, designated type I, II, and III [69,

76]. Each type of pyoverdine has its own specific TonB energy-transducing uptake receptor (Fpv) [77-79]. However, it is common for individual strains to only express one type of pyoverdine and its cognate receptor [77-79]. Pyoverdine is capable of sequestering iron from the host proteins transferrin and lactoferrin and has been shown to be essential for virulence [80, 81]. In a mouse model, strains

14 deficient in pyoverdine biosynthesis or transport were avirulent and were not capable of establishing infection [82]. The biosynthesis of pyoverdine is complex, involving a number of genes. The vast majority of these genes are located within the pvd locus, clustered within one region of the chromosome. Mutations within this locus can negatively impact pyoverdine biosynthesis, maturation, transport or export [83, 84]. In general, the production of pyoverdine by this bacterium increases in response to iron-limitation and is thought to be the main iron acquisition strategy used by P. aeruginosa.

Pyochelin is the secondary siderophore generated by P. aeruginosa in response to iron-limitation. Initially pyochelin was thought to have a remarkably low binding affinity for Fe3+ (5x10-5 mol/L), but more recent studies indicate that while its affinity for Fe3+ is moderate (1x10-16 mol/L), it is still capable of efficiently scavenging iron and thievery from host proteins (transferrin and lactoferrin) [85-

87]. This particular siderophore has not only been implicated in binding iron, but has also been shown to bind other metals such as nickel, molybdenum, and cobalt with appreciable affinity [88]. The genes necessary for pyochelin biosynthesis and export are located at two distinct loci on the chromosome

(pchDCBA and pchEFGHI) and the receptor (FptA) is located just downstream of pchI [89-94].

While pyoverdine and pyochelin are key players in iron acquisition by P. aeruginosa, this opportunist has various additional means of obtaining this essential nutrient. This pathogen has receptors for numerous heterologous siderophores of bacterial, fungal and plant origins (e.g. aerobactin, enterobactin,

15 ferrioxamine, cepabactin) as well as receptors for various iron chelators (e.g. citrate, deferoxamine) [76, 95]. In addition to siderophores, P. aeruginosa possesses a ferrous iron transport system (feoAB), ferric citrate (fecA) uptake system, two heme uptake systems (phu and has), and several metal-type ABC transporters [96, 97]. Further, several iron-regulated genes with unknown functions were identified by extensive microarray analysis in the Vasil lab; perhaps there are new mechanisms of iron acquisition that have yet to be discovered [84, 98].

Figure 1.2. The Fur regulon in P. aeruginosa. Fur both positively and negatively regulates the expression of numerous genes involved in iron acquisition, metabolism and virulence This Figure was adapted and modified from that in [1].

16 Regulation of Iron Homeostasis in P. aeruginosa

P. aeruginosa has evolved numerous strategies for obtaining iron from the host as well as positive and negative regulatory elements to regulate and store intracellular iron in order to avoid toxicity [1, 11, 32, 99]. While an adequate level of iron is necessary for pathogenesis and for basic cellular metabolism, the availability of iron in the cell must be tightly regulated; excessive intracellular iron can be extremely toxic causing damage to all known biological macromolecules, including DNA and proteins [66]. In P. aeruginosa and in many other bacteria, the uptake, acquisition and sensing of iron is controlled by the ferric uptake regulator

(Fur), the master iron regulator of iron homeostasis [11, 32, 98, 100]. And unlike other bacteria, Fur is an essential gene in P. aeruginosa and is known to be present at high levels (5,000-10,000 molecules) in the cell [11, 101, 102].

However, it is possible to isolate strains with mutations within the operator and open reading frame of Fur and these mutations, in some cases, limit the function of this protein (e.g. derepression of siderophore biosynthesis) [101]. However, complete removal of the Fur gene from the P. aeruginosa chromosome is lethal to this bacterium. Fur functions as a cytoplasmic regulator and when iron is readily available in the cell (iron-replete conditions), the Fur protein binds to an iron cofactor (Fe2+) resulting in an altered conformation that allows it to attach, with high affinity, to the operator regions of iron-regulated genes thereby preventing transcription [103]. Two dimers are required for the full repression of a

Fur-regulated gene in P. aeruginosa [98]. Conversely under iron-limitation, Fur regulated genes are de-repressed; these include genes involved in siderophore

17 biosynthesis, heme uptake, and extracellular virulence determinants (e.g. toxA, prpL) [11, 22, 30, 32, 83, 96, 98, 100, 104, 105]. Ochsner and Vasil developed a

SELEX-like technique in 1996 that identified twenty-five DNA fragments that bound to the Fur protein [98]. The majority of these genes were in two- component systems, extracytoplasmic sigma factors (ECF σ), siderophore receptors, two heme uptake systems, two small RNAs (PrrF1 and PrrF2), and an assortment of other genes (Figure 1.2) [98]. The direct and indirect regulation of gene expression by Fur is broad, controlling not only genes involved in iron acquisition, but also genes involved in metabolism and virulence. Interestingly, with the use of high throughput sequencing techniques (e.g. ChIP-seq) in other closely related species (i.e. P. syringae) novel Fur-regulated genes, some of which are involved in alginate biosynthesis (algJ and algF), have been identified, broadening the scope of Fur regulation [106].

One of the genes directly controlled by Fur is an extracytoplasmic sigma factor (ECFσ), PvdS. In P. aeruginosa, PvdS not only controls the transcription of genes involved in pyoverdine biosynthesis and export, but also regulates the outer membrane pyoverdine receptor (fpvA/B), as well as two virulence genes encoding exotoxin A (exoA) and the extracellular protease prpL [11, 22, 30, 31,

83, 105, 107]. Xiong et al. reported that a PvdS mutant was significantly affected in its ability to colonize the left-sided valves in an experimental endocarditis model in rabbits [107].

Fur indirectly functions as an activator through the repression of two small regulatory RNAs, PrrF1 and PrrF2 (Figure 1.2) [108]. These small sRNAs are

18 repressed by both iron and heme and control gene expression at the post- transcriptional level [96, 108]. They regulate gene expression by pairing with mRNA of their target genes followed by degradation of the sRNA:mRNA duplex by RNaseE [109]. The repression of these sRNAs in iron-replete conditions allows for the upregulation of genes involved in oxidative stress, iron storage

(e.g. bacterioferritins), intermediary metabolism (TCA cycle and Glyoxylate shunt) and quorum sensing (anthranilate) [108, 110]. The ability of Fur to regulate genes required for iron acquisition under iron-limitation as well as genes involved in iron storage, oxidative stress, metabolism and quorum sensing under iron-replete conditions shows the breadth of this regulator’s influence on global gene expression and physiology in P. aeruginosa. Further, a limited number of studies have suggested an association between iron and biofilm development, an important process associated with human disease. These findings as well as the foundation for this thesis will be set-forth in the following sections of this chapter.

P. aeruginosa Biofilms

Throughout the past decade, the ability of microbes to form complex communities called biofilms has become an exceedingly intense and worthy area of investigation, both in environmental and medical microbiology. In this regard,

P. aeruginosa has become a leading paradigm based on its proclivity to form biofilms in diverse environments (e.g. pipelines, heart valves, bone), yet also in highly specialized situations, such as in the bronchioles of cystic fibrosis (CF) patients [3, 4, 50, 55, 111, 112]. It is now clear that biofilms are important

19 Figure 1.3. Main stages of biofilm formation in P. aeruginosa. 1) Initial attachment of cells to a surface. 2) Production of exopolysaccharide matrix (i.e. alginate). 3) Microcolony formation. 4) Mature biofilm. 5) Cells from biofilm disperse to infect other areas of the body. biological structures present in both the environment and in human disease. The presence of bacterial biofilms, particularly in human infections, often impedes treatment resulting in increased resistance to antibiotics and chronic infection

[113-117]. Therefore, several researchers have attempted to understand the molecular genetics that govern the signaling, development and regulation of biofilms.

Even though a tremendous amount of knowledge has been garnered with regard to the molecular mechanisms of biofilm formation, there is no defining universal molecular marker that yet exists. There are however, patterns that aid in defining the various stages of development that planktonic, free-living cells undergo in order to form complex communities that we call biofilms (Figure 1.3)

[118, 119]. P. aeruginosa is thought to survive predominately in biofilms adherent to surfaces. In the mammalian host, such as the CF lung, planktonic cells enter the host and through mechanisms not fully understood develop into a community

20 life-style known as a biofilm. In order to successfully infect and colonize a mammalian host, there must be a sufficient bacterial load along with immune evasion of cellular host defenses. Once initial colonization has occurred, the infection will progress to an acute or eventually a chronic infection. In an acute infection, the bacteria produce significant amounts of extracellular virulence factors (Table 1.1) that can result in tissue damage leading to blood vessel invasion, dissemination, multiple organ failure, and ultimately death [10, 120,

121]. In contrast, chronic infections like those found in the lungs of CF patients involves the pathogen adopting a biofilm mode of growth [4, 42, 49, 50, 56, 58,

111, 122-124]. The biofilm provides a protective shield against host defenses and antibiotic treatments. The bacteria housed in these biofilms produce low levels of extracellular virulence factors and tissue damage is mainly due to chronic inflammation [125]. Periodically, planktonic cells will be released from mature biofilms into the surrounding environment to search for suitable nutrients and new colonization sites [126, 127]. The dispersed cells are capable of colonizing new areas and initiating new infections within the compromised host.

Biofilms by classic definition are complex communities of cells encased in an extracellular matrix. The matrix is essential to biofilm formation, providing structure and complexity. Exopolysaccharides (EPS) are an important component of the bacterial biofilm matrix and are fundamental for biofilm formation in many bacteria, including P. aeruginosa [41]. Exceptional research over the last decade has revealed that P. aeruginosa is able to produce as many as three distinct extracellular polysaccharide matrices, each of which is

21 associated with individual types of biofilms and the conditions under which they are formed. For example, over-expression of the alginate exopolysaccharide was first associated with P. aeruginosa mucoid strains recovered from the lungs of chronically infected CF patients, but seldom from other types of infections [49,

58, 63, 128]. The Psl (Polysaccharide Synthesis Locus) exopolysaccharide and its associated biofilm were uncovered by examining strains of P. aeruginosa that were unable to produce alginate (e.g. ∆algD mutants), yet were still able to form biofilms on glass or plastic surfaces [38, 128]. The third exopolysaccharide matrix Pel was initially associated with the ability of P. aeruginosa to produce a biofilm at an air-liquid interface (Pellicle) [40, 129, 130]. While it is not yet entirely clear what aspect of P. aeruginosa infection (colonization, survival) the Psl and

Pel associated biofilms contribute, particularly those in CF patients, the long-term presence (decades) of highly mucoid strains, deep in the airways (bronchioles) of

CF patients, continues to provide a compelling rationale for further investigation into the environmental conditions and mechanisms that govern the production of alginate and the ensuing mucoid phenotype [3, 49, 123].

Psl

The polysaccharide synthesis locus (psl) encodes one of the three known exopolysaccharides (EPS) that P. aeruginosa is known to produce. The Psl EPS is composed of a repeating pentamer containing mainly galactose, mannose and glucose with trace amounts of rhamnose, xylose, and N-Acetylglucosamine [39,

131]. Although the Psl pathway has not yet been well characterized, the Psl gene

22 cluster contains 15 genes (PA2231 – PA2245, pslA to pslO) that are cotranscribed [38, 132, 133]. These genes encode proteins predicted to synthesize the Psl EPS including hydrolases, esterases, transport, and export proteins [5]. The Psl EPS is thought to be important in the ability of this pathogen to form biofilms on surfaces. Ma et al. demonstrated that mutation of various psl genes resulted in mutants unable to adhere to and form surface-attached biofilms

[134]. Interestingly, the Psl EPS is less important in the PA14 strain. PA14 actually harbors a three-gene deletion in the Psl operon [40, 129, 132]. While the pathway involved in the synthesis of the Psl EPS varies from Pel and alginate, it is likely that the production of these various exopolysaccharides are induced by specific stresses and that P. aeruginosa may switch between the production of

EPS depending on the selective pressures the organism encounters. In support of this, the production of Psl has been shown to require the stress response regulator, RpoS, which has also been shown to regulation alginate production

[135, 136].

Pel

Pel is the dominant exopolysaccharide in wild type PA14 strains. The Pel

EPS was identified by screening P. aeruginosa PA14 transposon mutants for the lack of structured biofilms at the air-liquid interface (Pellicles) of stagnant cultures

[137]. The Pel EPS chemical structure has not yet been fully characterized.

However, Pel is a glucose-rich, cellulose-like polymer that forms a fabric-like matrix resembling a microbial mat [5, 137]. The Pel EPS is encoded by an

23 operon containing seven genes (PA3058 – PA3064; pelA to pelG). This biosynthesis operon encodes numerous proteins predicted to be involved in the synthesis of the Pel EPS including hydrolases, esterases, transport and export proteins [5]. Pel has been shown to play a protective role in biofilm formation as well as providing structure to biofilms [40, 138]. Mutants of Pel EPS produce thin, flat biofilms when compared to wild type cells that form mushroom-like structures

[40, 129]. In contrast to Psl, the Pel EPS does not appear to function in adhesion or surface attachment. Studies performed by two groups showed that pel mutants are not defective in surface attachment [40, 129]. In spite of this, it was reported that Pel mutants were defective in microcolony formation [40, 129].

While little is known about the regulation of the Pel and Psl EPS, there is evidence that they are both controlled by aspects of quorum sensing and by the intracellular second messenger cyclic-di-GMP (c-di-GMP) [139-143].

Alginate

Alginate was first discovered in P. aeruginosa over three decades ago by recovering mucoid strains overproducing alginate from the lungs of chronically infected CF patients [55, 63, 144]. The significance of alginate and mucoidy in P. aeruginosa lung infection has led to it being the best-characterized exopolysaccharide produced by this pathogen. The production of alginate by P. aeruginosa has only been associated with isolates obtained from human infections, such as CF. To date there have not been any known cases of alginate producing isolates being obtained from environmental reservoirs. The only two

24 bacterial genera known to produce alginate are Pseudomonas and Azotobacter

[36, 145].

Bacterial alginate is a linear (unbranched) heteropolymer consisting of variable amounts of β-1,4 D-mannuronic acid and C5-epimer α-L-guluronic acid residues. The structure of alginate is random with several possible arrangements. The D-mannuronate subunits are epimerized to L-guluronate and the alginate hydroxyl groups are partially acetylated at the C2’ and/or C3’ positions of mannuronic acid residues [5, 36, 37]. Biosynthesis of the alginate

EPS requires numerous proteins encoded by the alginate biosynthesis operon

(PA3540 – PA3551, algD to algA) (Table 1.2) [5, 36, 37]. The pathway for alginate biosynthesis begins with the synthesis of a sugar-nucleotide precursor

Figure 1.4. Alginate Precursor synthesis. The conversion of fructose-6- phosphate to GDP-mannuronate is an irreversible process that involves four enzymatic steps. 1) Fructose-6-phosphate is isomerized to mannose-6- phosphate by AlgA. 2) Mannose-6-phosphate is converted to mannose-1- phosphate by phosphomannomutase, AlgC. 3) Mannose-1-phosphate is converted to GDP-mannose by GDP-mannose-pyrophosphorylase AlgA. 4) Finally, the GDP-mannose dehydrogenase, AlgD irreversibly oxidizes GDP- mannose to GDP-mannuronic acid.

25 where fructose-6-phosphate is converted into GDP-mannuronate. The conversion to GDP-mannuronate occurs through a series of four enzymatic steps involving algD (PA3540), algA (PA3551) and algC (PA5322) (Figure 1.4 and

Table 2) [37, 146]. In the final step of this pathway, AlgD (GDP-mannose- dehydrogenase) irreversibly oxidizes GDP-mannose to GDP-mannuronic acid.

This final oxidation step is specific to alginate biosynthesis and is thought to be the rate-limiting step in this process.

The polymerization and cytoplasmic membrane transfer of the alginate precursor to the periplasm are not well characterized, but are thought to involve

Alg8 (PA3541) and Alg44 (PA3542) [147-149]. The proteins AlgI (PA3548), AlgJ

(PA3549), and AlgF (PA3550) form a complex at the periplasm that partially acetylates the alginate polymer [150]. AlgG, a periplasmic mannuronan C-5 epimerase converts a portion of the D-mannuronic acid to L-guluronic acid [151].

AlgX does not have a known function, but is thought to form a complex with AlgK and AlgE at the outer membrane and be involved in polymer modification [152].

AlgK is a scaffold protein and AlgE is a porin protein, the two proteins are thought to form a secretion channel for the alginate polymer [153, 154]. AlgL (PA3547) is an alginate lyase that degrades the alginate polymer and is required for the normal production of alginate [155-157]. Together, these proteins form a multi- protein complex bridging the outer membrane and inner membrane to allow the maturation and secretion of the alginate polymer.

The regulation of alginate biosynthesis is complex, mediated by a number of genotypic switching and regulatory genes scattered throughout the P.

26 aeruginosa chromosome (Table 1.2). Transcriptional activation of the algD biosynthetic operon by regulatory factors is the critical, obligatory step for the biosynthesis of the alginate exopolysaccharide [158]. algD is the first gene in the alginate biosynthetic operon and the majority of alginate transcriptional regulation occurs at the extensive 500 bp algD promoter. Although recently, two additional promoters were identified within the biosynthetic operon, one upstream of algG and the other upstream of algI [159] and (J. R. Wiens and M. L. Vasil unpublished results).

The conversion of non-mucoid isolates to mucoid, alginate producing isolates is controlled at the algU “genotypic switch” operon. This operon contains five genes algU, mucA, mucB, mucC, and mucD. This cluster of genes comprises a genetic switch that leads to the constitutive activation of alginate production and export. The algU gene encodes the alternative sigma factor AlgU, also known as AlgT. AlgU is the master regulator of alginate biosynthesis and is homologous to RpoE in Escherichia coli [160, 161]. AlgU is essential for the transcriptional activation of algD as well as the expression of several alginate regulators (e.g. AlgR, AlgB and AmrZ) [124, 160, 162-166]. Transcription initiation of algD requires both the response regulator AlgR and the alternative sigma factor AlgU [160, 167-169].

In wild type, non-mucoid P. aeruginosa isolates, AlgU is normally sequestered to the inner cytoplasmic membrane by the negative regulators

MucA, MucB, and MucC, thus not allowing the AlgU protein to interact with regulators required for the transcription of algD (Figure 1.5).

27 Table 1.2 Alginate biosynthesis and regulatory genes in P. aeruginosa

PA Number Gene Description

Biosynthesis/Structural

PA3540 algD GDP-mannose dehydrogenase

PA3541 alg8 Alginate biosynthesis

PA3542 alg44 Alginate biosynthesis

PA3543 algK Scaffold protein

PA3544 algE Outermembrane protein

PA3545 algG Alginate C5-mannuronan-epimerase

PA3546 algX Alginate biosynthesis

PA3547 algL Alginate lyase

PA3548 algI Acetylase

PA3549 algJ Acetylase

PA3550 algF Acetylase

PA3551 algA Phosphomannose isomerase/guanosine 5’-dephopho-D-

mannose

PA5322 algC Phosphomannomutase

Regulatory

PA5261 algR Two-component response regulator

PA5262 algZ Cognate sensor kinase

PA5255 algQ Transcriptional regulatory protein AlgR2

PA5253 algP Transcriptional regulator AlgR3; histone-like protein

PA5483 algB Two-component response regulator

PA5484 kinB Histidine kinase

PA4462 rpoN Sigma factor σ54

PA1754 cysB Transcriptional regulator sulfur metabolism

28 Table 1.2 Alginate biosynthesis and regulatory genes in P. aeruginosa

PA Number Gene Description

PA3385 amrZ Alginate and motility regulator

PA0652 vfr Transcriptional regulator cAMP-dependent

PA1727 mucR Diguanylate cyclase

Proteases

PA4446 algW Serine protease

PA1801 clpP ATP-dependent protease Clp subunit

PA1802 clpX ATP-dependent protease Clp subunit

PA3649 mucP Membrane-associated Zn-dependent protease

PA3257 prc Periplasmic protease; C-terminal peptidase

Genotypic Switching

PA0762 algU Sigma factor σ22

PA0763 mucA Anti-sigma factor

PA0764 mucB Protects C-terminus of MucA from degradation

PA0765 mucC Negative regulator alginate biosynthesis

PA0766 mucD Serine protease

Constitutive alginate producing isolates (mucoid) from the CF lung often harbor mutations within the mucA gene (anti-sigma factor and negative regulator of alginate biosynthesis) that interfere with MucA function [124, 170]. However, in a study investigating 115 Scandinavian CF patients, loss-of-function mutations were not only found in mucA, but also in mucB and mucD negative regulators indicating that mucoidy can be activated in situ by mutations in any number of the muc genes [171].

29 Figure 1.5. Constitutive activation of alginate biosynthesis. The proteins involved in the conversion to the mucoid phenotype are shown. Please see text for further details.

Mutations within MucA allow for AlgU to become liberated from the

AlgU/Muc sequestration complex leading to the constitutive activation of the alginate biosynthetic operon (Figure 1.5). Moreover, under various environmental stresses (e.g. oxidative stress, antibiotics, heat, phosphate starvation), MucA has protease cleavage sites on the N-terminus region of the protein, which can be degraded by proteases (AlgW, MucP, ClpXP and Prc) [172-176]. Proteolytic degradation of MucA leads to the release of AlgU and activation of algD (Figure

1.5). MucB is an inner membrane protein that dimerizes and protects the

C-terminus of MucA from proteolytic degradation [177]. MucC is a predicted inner

30 membrane protein that probably forms a complex with MucA and MucB. While initial studies of MucC revealed that inactivation of the protein was not sufficient to cause mucoidy, we demonstrated that deletion of mucC in PAO1 results in an overt mucoid phenotype (J.R. Wiens, A.I. Vasil and M.L. Vasil, unpublished results) [178]. MucD is a serine protease involved in regulating protein quality and MucA degradation. Inactivation of MucD results in conversion to mucoidy

[173, 179]. Numerous additional regulatory proteins have been implicated in influencing alginate production including AlgP, AmrZ, CysB, Vfr, AlgB, KinB,

RpoN, and MucR [180-187]. Interestingly, numerous physiological stressors have been shown to induce alginate production including phosphate limitation, oxidative stress, heat shock, decreased oxygen tension and exposure to antibiotics [36, 188-194]. Therefore, it is not surprising that a number of regulators have been linked to alginate biosynthesis.

Iron and P. aeruginosa Biofilms

There is a substantial level of knowledge relating to the biochemistry and molecular genetics of alginate production and the mucoid phenotype of P. aeruginosa. Also, during the past decade, considerable insight has been garnered relating to how iron influences the expression of an array of virulence factors of P. aeruginosa (toxins, proteases and siderophores), basic metabolic processes (quorum sensing, intermediary metabolism and resistance to redox stress) and even how this particular metal affects the formation of some types of

P. aeruginosa biofilms.

31 There is little known about the role of iron in pellicle formation at the air- liquid interface in P. aeruginosa, with the exception of one study examining the gene expression profiles of pellicle cells. This study revealed that the cells within a pellicle upregulated a plethora of iron-acquisition genes such as pyoverdine and pyochelin, and downregulated genes (nir, nar, nor, and nos) normally upregulated in low-oxygen conditions, suggesting that pellicle cells are formed under aerobic and iron-limited states [195]. Conversely, it has been shown that

Fur, pyoverdine, and iron levels can all affect biofilm formation (likely Psl biofilms) on a glass surface and that the effect of iron on biofilm formation is concentration dependent [6, 196-205].

In 2005, Banin et al. presented a comprehensive examination of molecular and biochemical processes by which known iron acquisition factors (Ferric

Uptake Regulator, Fur & the Fur-regulated σ-factor, PvdS) can affect biofilm formation of P. aeruginosa. However, the parental strain used in their study was a non-CF isolate (PAO1, a wound isolate) that does not normally express significant levels of alginate. Moreover, the biofilms in their study were formed on glass surfaces using flow-through systems, thereby most likely representing a

Psl or Pel type of biofilm. Specifically, their study demonstrated that an ECF σ- factor mutant (∆pvdS), a pyoverdine biosynthesis mutant (∆pvdA) and a pyoverdine receptor mutant (∆fpvA) all were unable to form robust biofilms compared to the wild type parent. By contrast, a Fur missense mutant (Ala10Gly mutation in the DNA binding domain of Fur) that constitutively expresses Fur- repressed iron-uptake systems was able to produce robust biofilms, both in the

32 presence and absence of lactoferrin, an iron-binding protein that inhibits biofilm formation by the wild type PAO1 [6]. While there were a number of interesting outcomes from their effort, overall it was clear that intact P. aeruginosa iron uptake systems (e.g. pyoverdine, pyochelin, ferric dicitrate) are required for the initial acquisition of sufficient levels of cytoplasmic iron needed for the formation of a robust biofilm.

Under iron-limitation, cells exhibit increased motility and when grown on glass surfaces produce thin, flat biofilms. Iron-limitation stimulates twitching motility (specialized form of bacterial movement across a solid surface mediated by pili) and it is likely that the stimulation of twitching results in biofilm dispersal

[14, 199, 200]. Yang and coworkers demonstrated that pvdA was expressed in the stalk of mushroom-like, structured biofilms [202]. They showed that pvdA mutants defective in pyoverdine biosynthesis produced thin, flat biofilms, but could be rescued if grown in conjunction with pilA mutants (motility deficient but capable of producing pyoverdine) or with an alternative iron source (ferric citrate)

[202]. This study reaffirms the essentiality of iron acquisition in Psl/Pel structured biofilm formation.

The impact of iron and iron acquisition mechanisms on alginate production has not been nearly as extensively examined. In two separate reports in the early

1980’s, Boyce and Miller presented the most clear-cut, earliest demonstration of the influence of variable levels of iron on the expression of the mucoid phenotype by P. aeruginosa. In the first of those studies they found that addition of iron

(Fe2+), but not other metals (Mg2+and Ca2+), to cultures of mucoid isolates from

33 CF patients markedly enhanced the accumulation of non-mucoid revertants, from

0.37% at 24 hours to >80% at 72 hours [206]. In a later study, these observations were extended to several additional mucoid CF isolates and these investigators showed that the mucoid phenotype of a single CF strain was more stable under iron-limited growth than under more iron-replete conditions [207]. Subsequently,

Terry et al. reported that ~2% of colonies isolated from PAO1 grown under iron- limiting conditions in a chemostat for at least 6 days became mucoid and produced increased extracellular levels of uronic acid (a surrogate measure for alginate) [208]. Since this latter study from 1992 there have not been any additional published reports that directly address how iron levels might differentially and specifically affect alginate production or the associated mucoid phenotype.

Purpose of This Study

Biofilm formation, particularly alginate biofilms, in chronic clinical infection is of great medical significance. Until this study, it was largely unknown what role iron plays in alginate production and mucoidy. This study aimed to expand our knowledge on how iron affects alginate production and mucoidy by P. aeruginosa and how it may compare or contrast to what is known about iron’s role in Psl/Pel biofilm formation. Accordingly, we began by examining a highly iron regulated gene (PA2384) that was shown to affect rhamnolipid biosynthesis and quorum sensing for its putative role in iron-regulated biofilm formation. This early study led to a deeper question which was how does iron affect alginate production and

34 mucoidy? We decided to directly examine whether iron levels known to exist in the sputa of CF patients have any influence on the biosynthesis of alginate and the resulting mucoid phenotype in P. aeruginosa strains that are able to express increased levels of alginate and demonstrate a mucoid phenotype, similar to many chronic pulmonary isolates from CF patients. Finally, we were interested in furthering our understanding of how gallium, an iron analogue that is currently undergoing clinical trial for therapeutic use, might influence mucoidy and alginate production. Taken together, these findings have provided new insights into the role of iron in biofilm formation and a deeper understanding of the complexity of the role of iron in P. aeruginosa pathogenesis.

35 CHAPTER II

MATERIALS AND METHODS

Materials and Chemicals

Unless otherwise stated, all chemicals and reagents were purchased from

Sigma Aldrich, Fisher Scientific, GE Healthcare, and Research Products

International. Enzymes were purchased from either Invitrogen or New England

Biolabs. Agar, BHI, Tryptone, Yeast Extract, Mueller-Hinton, and all other media requirements were purchased from Becton-Dickinson. Agarose was purchased from ISC BioExpress. Acrylamide and tetramethylethylenediamine (TEMED) were purchased from Bio-Rad. Western blot detection kits were purchased from

Amersham Biosciences. Microtiter plates were purchased from Nunc. All primers and probes were purchased from Integrated DNA Technologies (IDT). All qRT-

PCR reagents were purchased from Roche.

Bacterial Strains, Plasmids and Growth Conditions

The bacterial strains used in this study are listed in Table 2.1. The plasmids used in this study are listed in Table 2.2. P. aeruginosa PAO1, PA14,

PAKS-1 and Ps388 originated from various types of non-CF infections (see Table

2.1 and Chapter IV) and have been continuously maintained at -80˚C. Jane

Burns, M.D., Seattle Children’s Hospital & Department of Pediatrics, University of

Washington School of Medicine, kindly provided the Early CF isolates (Early-

36 CFI), see Appendix D. Benjamin Staudinger, M.D., Pulmonary and Critical Care

Medicine, University of Washington School of Medicine, generously provided the

Late CF isolates (Late-CFI), see Appendix E.

Unless otherwise stated, all E. coli strains were grown in Luria-Bertani

(LB) browth or on LB agar plates, supplemented with the appropriate antibiotic

(see Appendix G). Brain Heart Infusion (BHI) agar or broth were used for the general maintenance of P. aeruginosa cultures, supplemented with the appropriate antibiotic (see Appendix G). Chelex-treated and dialyzed tryptic soy broth (DTSB) or agar (DTSA) supplemented with 1% glycerol and 50 mM glutamate was used as an iron-limited, base medium [102, 104]. Iron-replete

DTSB or DTSA were supplemented with various concentrations of ferric chloride, ferric dicitrate or bovine hemin (Sigma). While the insertion mutants described herein were cultured with 30 µg/mL of tetracycline during routine growth on BHI and DTSA in order to maintain selection for the Tcr determinant carried by the pSUP203 inserted in these mutants. All strains were grown at 37˚C with vigorous shaking when appropriate.

Construction of Mutants

Marked and unmarked chromosomal deletion mutants in PAO1 described in this dissertation were constructed using genetic manipulation and conjugation methods previously described [209, 210]. The ∆PA2384, ∆mucA, ∆mucB,

∆mucC, and ∆mucD were all made using the strategy described below.

37 Table 2.1. Strains used in this study. Strain Description Reference P. aeruginosa PAO1 Wild type, wound isolate from Melbourne, Australia M. Vasil PA14 Burn isolate from Boston, MA M. Vasil PAKS-1 Urine isolate from Stockholm, Sweden M. Vasil Ps388 Blood isolate from Seattle, WA M. Vasil PAO1 ∆PA2384 Unknown function This study PAO1 ∆mucA Anti-sigma factor, alginate biosynthesis This study** PAO1 ∆mucB Protects C-terminus of MucA from degradation This study** PAO1 ∆mucC Negative regulator of alginate biosynthesis This study** PAO1 ∆mucD Serine protease This study** PAO1 ∆mucA- Anti-sigma factor, alginate biosynthesis, Tetr gene replacement This study* PAO1 ∆mucB- Protects C-terminus of MucA from degradation, Tetr gene This study* replacement PAO1 ∆pvdSmucA- Sigma factor, pvoverdine biosynthesis, Tetr gene replacement This study* PAO1 ∆pvdAmucA- Pyoverdine biosynthesis, anti-sigma factor, alginate This study* biosynthesis, Tetr gene replacement PAO1 ∆pchEFmucA- Pyoverdine biosynthesis, anti-sigma factor, alginate This study* biosynthesis, Tetr gene replacement PAO1 Pyoverdine biosynthesis, pyochelin biosynthesis, anti-sigma This study* ∆pvdA∆pchEFmucA- factor, alginate biosynthesis, Tetr gene replacement PAO1 Pyoverdine biosynthesis, pyochelin biosynthesis, anti-sigma This study* ∆pvdD∆pchEFmucA- factor, alginate biosynthesis, Tetr gene replacement PAO1 Pyoverdine biosynthesis, pyochelin biosynthesis, anti-sigma This study* ∆pvdD∆pchDAmucA- factor, alginate biosynthesis, Tetr gene replacement PAO1 Pyoverdine biosynthesis, pyochelin biosynthesis, pyoverdine This study* ∆pvdD∆pchEF∆fpvAmucA- receptor, anti-sigma factor, alginate biosynthesis, Tetr gene replacement PA14 ∆mucA- Anti-sigma factor, alginate biosynthesis, Tetr gene replacement This study* PA14 ∆mucB- Protects C-terminus of MucA from degradation, Tetr gene This study* replacement PAKS-1 ∆mucA- Anti-sigma factor, alginate biosynthesis, Tetr gene replacement This study* PAKS-1 ∆mucB- Protects C-terminus of MucA from degradation, Tetr gene This study* replacement Ps388 ∆mucA- Anti-sigma factor, alginate biosynthesis, Tetr gene replacement This study* Ps388 ∆mucB- Protects C-terminus of MucA from degradation, Tetr gene This study* replacement * Mutants created by Adriana Vasil, ** Mutants created by Urs Ochsner

38 Table 2.1. Strains used in this study. Strain Description Reference E. coli Top10 F- λ- Strepr recA1 endA1 ∆(mrr-hsdRMS-mcrBC) ∆lac74 Invitrogen Φ80lacZ∆M15 DH5α F- hsdR17 supE44 relA1 recA1 endA1 gyrA96 thi-1 ∆lacZYA Invitrogen Φ80lacZ∆M15 - BL21 hsdS gal ompT r BM S. Parker BL21 Rosetta contain codons rarely used in E. coli. S. Parker SM10 Kmr thi-1 thr leu tonA lacY supE contains λ pir conjugation system * See Appendix D and E for all clinical isolates used in this study.

Table 2.2. Plasmids used in this study. Strain Description Reference pCR2.1 Apr, TA cloning vector Invitrogen

pEX100T Apr, oriT mob sacB [210]

pEX18Tc Tcr, oriT mob sacB [210]

pPS856 Apr, GMr cassette flanked by FRT [209]

pFLP2 Apr, encodes FLP recombinase [209]

pSUP203 Tetr, mob, gene replacement vector [211]

pRK2013 Kmr, conjugation helper plasmid [212]

pGEX-4T-3 GST fusion vector GE Healthcare

In brief, PAO1 ∆PA2384, ∆mucA, ∆mucB, ∆mucC, and ∆mucD mutants have a deletion of the corresponding gene and the deleted sequences were replaced with a gentamicin resistance cassette. Internal fragments amplified by

PCR were ligated into pCR®2.1-TOPO (Invitrogen) using the TOPO-TA cloning® method and then transformed into TOP10 E. coli chemically competent cells. The cell mixture was plated on LB-Kan50 plates supplemented with X-gal. White colonies were isolated and inoculated into LB-Kan50 broth and grown overnight at

39 37˚C with vigorous shaking. Plasmid preps were performed on overnight cultures and insertion of PCR fragment was checked by restriction digest analysis.

Vectors were created by replacing the DNA sequence of the corresponding gene with a gentamicin cassette flanked by 150 – 200 bp of the corresponding gene sequence required for homologous recombination. The gentamicin cassette was excised out of the pPS856 plasmid by restriction digestion and then ligated into digested pEX100T and pEX18Tc suicide vectors.

These vectors both contain an oriT, mobilization (mob) site, and sacB (allows for negative selection on 5% sucrose). These vectors containing the deletion construct were used to transform DH5α E. coli competent cells and then transferred by conjugation into P. aeruginosa by triparental mating. Homologous recombination occurred by the addition of selection pressure and resolved mutations were validated by PCR and sequencing.

The mucA- and mucB- mutants of the non-CF isolates PA14, PAKS-1,

Ps388 were constructed by insertional mutagenesis as previously described [25].

Briefly, internal fragments from mucA or mucB genes were amplified by PCR and ligated into pCR®2.1-TOPO (Invitrogen). The fragments were excised from the cloning vector and ligated into pSUP203, which can be transferred to P. aeruginosa by conjugation, but cannot autonomously replicate in this organism

[212]. This technique results in the interruption of the mucA or mucB open reading frame by the homologous recombination of pSUP203 into these target genes. This insertional mutagenesis approach was also used to generate a mucB mutation in a non-mucoid Early-CFI as well as mucA mutations in

40 previously constructed PAO1 ∆pvdS, ∆pvdA, ∆pchEF, ∆pvdA∆pchEF,

∆pvdD∆pchED, ∆pvdD∆pchDA, and ∆pvdD∆pchEF∆fpvA mutants.

P. aeruginosa Chromosomal DNA Isolation

Overnight cultures were centrifuged and pellets were resuspended in 500

µL of TE + RNase. Cell mixture was boiled for 5 minutes and then placed on ice for 5 minutes. Genomic DNA was stored at -20˚C.

Polymerase Chain Reaction (PCR)

All oligonucleotide primers were purchased from Integrated DNA

Technologies (IDT). Upon arrival, primers were suspended in sterile DNase free ddH2O at a concentration of 20 µM and stored at –20˚C. PCR reactions were set up using a Qiagen PCR kit and contained 5-50 ng of DNA template, 400 nM – 1

µM primer, 10 µL of 5x Qiagen Q solution (novel additive that enables efficient amplification of "difficult" (e.g. GC rich) templates), 5 µL of 10x Qiagen PCR buffer, 2 mM MgCl2, 200 µM dNTPs, and 4 units of Qiagen Taq DNA polymerase.

The plasmid templates were always linearized via restriction digest prior to amplification. These conditions were sufficient for all PCRs peformed. A typical

PCR involved an initial 95˚C denaturation step followed by 30 cycles of the following: a denaturing step at 95˚C for 1 minute, an annealing step for 1 minute at an experimentally determined annealing temperature, and an extension step at

72˚C for 1 minute. The 30 cycles were followed by a 10 minute extension at 72˚C to ensure that the products were complete. The reactions were carried out in a

41 Genemate Thermal Cycler. The annealing temperature was initially determined by subtracting 5˚C from the lowest primer Tm. Based on the results with the initial annealing temperature adjustments were made to improve product purity. To

PCR amplify long DNA fragments; a high fidelity Phusion polymerase

(Finnzymes) was used per the manufacturer’s directions.

Plasmid Purification

Plasmid purifications were performed using either a boiling miniprep procedure or using a QIAprep spin Miniprep Kit (Qiagen). Small-scale boiling plasmid preparations were performed using 1 mL of overnight culture grown in

LB or BHI broth (Appendix F). The cells were harvested by centrifugation at

13,000 x g for 1 minute and fully suspended in 350 µl STET (50 mM Tris-HCl pH

8.0, 50 mM EDTA, 5% Triton X-100, 8% sucrose) with 25 µl lysozyme (10 mg/mL in 10 mM Tris-HCl pH 7.5). The mixture was boiled for 45 seconds and the resulting cell debris was pelleted by centrifugation at 13,000 x g for 5 minutes.

The cell debris was removed from the sample using a toothpick. The DNA was precipitated with 40 µL of 2.5 M sodium acetate (pH 5.2) and 420 µL of isopropanol. Tubes were inverted roughly 10 times to mix the solution and incubated at room temperature for 2 minutes. The samples were centrifuged for 5 minutes at 13,000 x g. The isopropanol was removed and the DNA pellet was allowed to air dry 5 minutes at room temperature before being suspended in 50

µL of sterile TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) with 5 µL of DNase-free

42 RNase (10 mg/mL). The sample was incubated at 37˚C for 30 minutes to insure complete resuspension of the plasmid DNA.

DNA Ligation

DNA ligation reactions were carried out in final volumes of 10-20 µL using

1 unit of T4 DNA ligase (Invitrogen or NEB) and 0.1 - 0.4 µg total DNA at a ratio of 3:1 insert to vector DNA. The ligation reactions were either incubated at 16˚C overnight (blunt ends) or for 1 hour at room temperature (sticky ends) depending on the nature of the ligation.

DNA Gel Purification

DNA was purified from agarose gels using the QIAquick Gel Extraction Kit

(Qiagen). Purification was carried out via the manufacturer’s recommended protocol. The DNA fragment of interest was excised from the agarose gel, transferred to a microfuge tube, and weighed. For every 10 mg of gel, 30 µL of

Gel Solubilization Buffer was added and the sample incubated at 50˚C with mixing every 3 minutes until the gel was completely dissolved. The dissolved gel was transferred to the outer chamber of a spin cartridge and centrifuged at

13,000 x g for 1 minute. The flow through was discarded and 700 µL of Wash

Buffer was added to the chamber. Following 5 minutes at room temperature the cartridge was centrifuged at 13,000 x g for 1 minute, the flow through discarded, and the cartridge was centrifuged again at 13,000 x g for 1 minute to remove any residual wash buffer. The cartridge was placed into a fresh tube and the DNA

43 was eluted by adding 40 µL of sterile DNase and RNase free H2O to the cartridge. The cartridge was incubated at room temperature for 1 minute before the DNA was collected via centrifugation at 13,000 x g for 1 minute.

Escherichia coli Chemically Competent Cells and Transformations

E. coli DH5α was made chemically competent in order to introduce the appropriate suicide vector into the cells. Cells from an overnight culture grown in

LB broth were subcultured (1/100) in 100 mL LB. The cells were grown at 37˚C to an OD600 = to 0.3 at which point the culture was chilled by incubating on ice for

20 minutes. The cells were harvested by centrifugation at 4˚C, 5,000 rpm and suspended in 20 mL of ice cold transformation buffer (28 mM RbCl, 11.4 mM

MnCl2, 15 mM CaCl2, 10 mM KOAc, and 3.75% sucrose). The cell pellet was gently resuspended in 2.5 mL transformation buffer and incubated on ice for 30 minutes. Following a 30 minute incubation on ice, 90 µL of DMSO was added to the 2.5 mL of cells and the cells were incubated for another 10 minutes on ice.

Finally the 100 µL of cells was aliquoted into tubes and stored at –80˚C.

For transformations, 1-10 ng of plasmid DNA was gently added to 100 µL of chemically competent DH5α cells and placed on ice for 30 minutes. The sample was heat shocked for 45 seconds in a 37˚C water bath. Following 5 minutes on ice, 900 µL of LB was added, and the cells were allowed to shake at

37˚C for 1 hour. The cells were then plated onto prewarmed LB agar plates with appropriate antibiotic selection (see Appendix G) and incubated overnight at

37˚C.

44 Tri-Parental Mating

Mutants containing deletions of corresponding genes as well as insertional mutations were generated using tri-parental matings. In order for a vector to be homologously recombined in P. aeruginosa through conjugation, the vectors must contain the following: i) a mobilization site (mob), ii) an origin that can’t replicate in P. aeruginosa, and iii) a DNA fragment for homologous recombination

[210]. The E. coli DH5α strain can conjugate with P. aeruginosa in the presence of a E. coli helper strain carrying the pRK2013 conjugation plasmid, which contains the tra genes required for pilus formation [213].

To begin the mating procedure, each strain was grown overnight. Wild type P. aeruginosa was grown in BHI at 43˚C, E. coli DH5α containing the suicide vector was grown at 37˚C in LB with the appropriate antibiotic (see Appendix G), and E. coli carrying the pRK2013 plasmid was grown at 30˚C in LB Kan50 to induce the formation of sex pili. Equal amounts of each bacterial culture were mixed and filtered onto sterile 0.2 micron filter paper. The bacteria drenched filter paper was placed onto a BHI plate with no antibiotic selection and incubated overnight at 37˚C. Cells were streaked heavily for isolation onto BHI plates containing irgasan25 and the appropriate antibiotic (see Appendix G) located on the suicide vector.

Confirmation of Mutants

Isolated colonies from the tri-parental mating antibiotic selection were streaked for isolation on BHI agar plates supplemented with 5% sucrose and 75

45 µg/mL gentamicin (Gm). Plates were incubated overnight at 37˚C, but not any longer since bacteria left on sucrose plates too long can acquire mutations.

Numerous individual colonies were picked with a sterile toothpick and replica plated onto BHI Carb750 (pEX100T) or BHI Tc150 (pEX18Tc) vs. BHI 5% sucrose

Gm75 vs. BHI Gm75. Colonies that were Carb or Tc sensitive and sucrose/Gm resistant were collected from the BHI Gm75 plates and streaked for isolation on

BHI Gm75 and incubated overnight at 37˚C. Isolated colonies were grown overnight in BHI Gm75 broth. The marked gentamicin mutants were confirmed by

PCR and sequencing. Chromosomal DNA was isolated from the overnight cultures and PCR was performed on the mutated gene to confirm mutations.

PCR fragments were checked by restriction digest as well as sequenced by the

Univeristy of Colorado, Comprehensive Cancer Center Sequencing and Analysis

Core (University of Colorado, Anschutz Medical Campus).

Unmarked mutants were generated by growing the mutant P. aeruginosa strain overnight in 3 mL of BHI Gm75 broth at 42˚C and E. coli SM10 strain carrying the pFLP2 plasmid overnight in 3 mL of LB Amp100 broth at 30˚C. The following day, the two cultures were mixed together in equal ratios and filtered onto sterile 0.2 micron filter paper. The bacteria drenched filter paper was placed onto a BHI plate without antibiotic and incubated overnight at 30˚C to allow mating conjugation to occur. The mating mixture was streaked for isolation onto

BHI irgasan25 Carb750 plates and incubated overnight at 37˚C. The following day, colonies were streaked for isolation onto BHI 5% sucrose plates and incubated overnight at 37˚C. Colonies were picked with sterile toothpicks and replica plated

46 onto BHI Carb750 vs. BHI 5% sucrose vs. BHI plates. Mutants that were sucrose resistant and Carb sensitive were grown overnight. Chromosomal DNA was isolated and PCR was performed. PCR fragments were run on a agarose gel and sent for sequencing to verify unmarked mutation.

Bacterial frozen stocks were made by adding equal portions of overnights and 50% glycerol to a cryovial®. Stocks were stored at -80˚C.

RNA Isolation and qRT-PCR

Total RNA was isolated from P. aeruginosa using a Qiagen RNeasy Mini

Kit® (Qiagen). To insure purity of RNA the columns were subjected to an additional DNase treatment using RNase-free DNaseI (NEB). After the extraction of RNA, the RNA was quantified using a nanodrop. RNA concentrations were adjusted and ImPromII Reverse Transcription System (Promega) was used for cDNA synthesis. Each reaction contained 500 ng of starting RNA as a template for cDNA synthesis. Real-time qPCR experiments were carried out in the Roche

LightCycler® 480 System using RNA Master Hydrolysis Probes Master Mix

(Roche) and gene-specific primers and probes (IDT). Relative transcript levels were determined by the comparative standard curve method. All samples were normalized to the constitutively produced omlA transcript [102].

Affymetrix GeneChip Microarray

The DNA microarray analysis was performed as previously described [84].

In this dissertation, RNA was harvested from the strains indicated in Chapter III

47 and Chapter IV after growth for either 18 or 24 hours. Cells producing alginate were washed with 0.85% saline before RNA isolation. Cells were immediately placed in either RNALater® (Invitrogen) or RNAprotect Bacteria Reagent (Qiagen) to stabilize the RNA. Total RNA was extracted from cells using an RNeasy Mini

Kit® (Qiagen). RNA was quantified using a nanodrop and submitted to the

Microarray Core. Microarray analysis including end-labeling, hybridization, washing and scanning were performed by the Microarray Core Laboratory at the

University of Colorado Anschutz Medical Campus. To ensure reproducibility, three separate biological replicate experiments were performed. Analysis of global gene expression was performed using Microarray Suite software

(Affymetrix).

Uronic Acids Assay

P. aeruginosa cultures were grown at 37˚C on DTSA +/- supplementation.

Biological material was removed from the surface of each agar plate with a sterile petri dish cell scraper and suspended in 0.85% NaCl. The suspension was spun at 10,000 x g to pellet cells and the supernatant fraction removed. Levels of the uronic acids were measured using a carbazole assay previously described by

Knutson & Jeanes [69], with slight modifications. Alginate was quantified using a standard curve made from Macrocystis pyrifera alginate (Sigma) and reported as

µg uronic acid/mg wet cell weight.

48 Glycosyl Composition Analysis of Extracellular Polysaccharides

P. aeruginosa PAO1 wildtype, ∆mucA and ∆mucB mutants were grown at

37˚C for 24 hours on DTSA +/- 100 µM FeCl3 on top of a 12-14,000 MWCO membrane (Life Science Products, Inc.). Total extracellular exopolysaccharides were isolated as previously described by Read and Costerton [70], with slight modifications.

Glycosyl composition analysis was performed at the Complex

Carbohydrate Research Center, University of Georgia, under the supervision of

Parastoo Azadi, Ph.D. This analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. The samples were placed into test tubes and 20 µg of inositol was added. Methyl glycosides were then prepared from the dry samples by methanolysis in 1 M HCl in methanol at 80˚C for 18 hours, followed by re-N- acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples were then per-O-trimethylsilylated by treatment with Tri-Sil

(Pierce) at 80˚C for 0.5 hour. These procedures were carried out as previously described by Merkle and Poppe [71]. GC/MS analysis of the TMS methyl glycosides was performed on an Agilent 6890N GC interfaced to a 5975B MSD, using an Agilent DB-1 fused silica capillary column (30m x 0.25 mm ID).

49 Purification of PA2384

The E. coli BL21 Rosetta-PA2384-GST strain was constructed using the methods described above. To overexpress PA2384, this strain was grown overnight in LB Amp100 broth. The following day, 1 L flasks of LB Amp100 + 100 mg/mL chloramphenicol were inoculated with 1 mL of overnight culture and placed in the 37˚C incubator. Bacteria was grown to an OD590 0.5 – 0.7 (~ 2 – 3 hours) and then induced by the addition of 0.5 mL of 1 mM IPTG. Bacteria were induced for 4 hours at 37˚C. After induction, the cells were harvested by centrifugation at 6,000 x g for 10 minutes at 4˚C. The cells were stored overnight at -80˚C. The following day, cell pellets were resuspended in 50 mL of 1X PBS and 0.5 mL of 100 mg/mL lysozyme was added. The suspension was allowed to stir at room temperature for 20 minutes. The beaker with the cell mixture was placed on ice and sonicated 4 times for 2 minute intervals (50% 10 seconds on/off). After sonication, the cell debris was pelleted by centrifugation at 6,000 x g for 30 minutes at 4˚C to separate the soluble and insoluble fractions. The soluble fraction was transferred to a 50 mL conical flask. 2 mL of glutathione sepharose

4B beads (GE healthcare) were equilibrated with 20 mL of 1X PBS. The beads were spun down in a centrifuge for 5 minutes at 500 rpm, PBS poured off, and another 20 mL was added – this was repeated five times. After equilibration, the beads were added to the 50 mL conical flask. The bead-protein slurry was placed on a rotating platform for 3 hours at 4˚C to allow the GST-tagged protein to bind to the beads. After the 3 hour incubation, the bead-protein slurry was added to an equilibrated filter column and allowed to run dry. The beads were washed 5 times

50 with 10 mL aliquots of 1X PBS to wash unbound protein and cell debris from the beads. After all of the solution was removed from the beads, the column was plugged and 950 µL of 1X PBS + 50 µL of 1 U/µL thrombin was added to the beads. The thrombin cleaves the protein from the bound GST. The thrombin was incubated for 16 hours at 4˚C. The 1 mL of PBS was eluted into a tube and then

1 mL fractions were collected after additional of 1X PBS. Protein was quantified using the nanodrop and concentrated using 10 kDa concentration filters

(Millipore).

SDS Polyacrylamide Gel Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli [214]. 15% polyacrylamide gels were prepared. Protein samples were prepared by adding 2x loading buffer to the sample and boiling for 5 minutes. Samples were run on the SDS-PAGE using Tris-glycine electrophoresis buffer. After separation, proteins were visualized by staining with Coomassie Brilliant Blue.

Gel Mobility Shift Assay

Biotinylated probes were amplified by PCR and gel purified for use in the gel shift assays. Probes were diluted to 500 pg/µL and 2 µL (50 pg) was used in each reaction. The LightShift® Chemiluminescent EMSA Kit (Thermo Scientific) was used for both protein-DNA binding assays and detection. Each reaction was optimized with the ideal amount of reagents for the best protein-nucleic acid

51 binding. In general the following components were added to each reaction 1.5 µL of 10X binding buffer, 0.75 µL of 50% glycerol, 0.75 µL of 100 mM MgCl2, 0.75 µL poly (dIdC), 0.75 µL of 1% NP-40. The binding reactions were incubated at room temperature for 20 minutes. 5 µL of 5X loading buffer was added to each reaction and then loaded into a 5% non-denaturing gel (40% acrylamide-Bis

37.5:1). The gel was run for 55 minutes at 180 V. The binding reactions were transferred to nylon membrane in a electrophoretic transfer unit. 0.5X TBE was cooled to ~10˚C with a circulating waterbath and transfer was run at 380 mA for

30 minutes. After transfer, the membrane was cross-linked using a UV-light cross-linker with 254 nm bulb for 60 seconds. The membrane was blocked and the biotin-labeled DNA was detected by chemiluminescence following the manufacturer’s instructions.

Affinity Purification of Antibodies

Affinity purification of polyclonal antibodies was performed via a small- scale method using antigen immobilized on PVDF membrane [215]. Small strips of PVDF (0.5 x 2.5 cm) were wet in methanol and then equilibrated in TBS (25 mM Tris-HCl, 150 mM NaCl, pH 8.0). A wicking system was set up by placing a wet piece of 3 mm Whatman paper on top of dry paper towels. The equilibrated strip of PVDF was placed on top of the wet Whatman and 10 µg of antigen was spotted onto each side of the PVDF. After allowing the membrane to dry, it was wet in methanol, equilibrated in TBS, and blocked 2 hours in TBS, 2% BSA at

4˚C. The membrane was then washed twice with 50 ml PBS (pH 7.4) for 5

52 minutes at room temperature. The antigen strip and 1 ml of antiserum were added to a microfuge tube and incubated at 4˚C for 4-10 hours with agitation.

Following the binding step the strip was washed 4 times with TBS-0.1% Triton X-

100 (50 ml/wash, 5 min each) and then twice with TBS, 1 mM EDTA (50 mL/wash, 5 minutes each). The strip was transferred to a microfuge tube and 1 mL of ice cold 0.2 M glycine (pH 3.0) was added. The glycine was pipetted over both sides of the PVDF membrane for 2 minutes to elute the bound antibodies.

The 1 mL of glycine was transferred to a new tube and 2 M NaOH was added until the pH was between 7.5-8.0. The elution was repeated with another milliliter of glycine as before. Following the second elution the eluted antibody along with

5 mL PBS was transferred to an Ultrafree-15 centrifugal filter devise (Millipore,

Billerica, MA) with a 10 kDa MWCO. The antibodies were concentrated until a suitable volume was reached.

Western Blot and Chemiluminescence

Western Blots were performed on whole cell extracts of bacteria grown for

24 hours at 37˚C on DTSA medium with either 0 µM or 100 µM FeCl3. Roughly

9 3x10 cells (1.0 OD590) were harvested from plates and washed with 0.85% NaCl and then pelleted by centrifugation. Cell pellets were resuspended in 500 µL of

PBS and sonicated 3x, 30 seconds at an output of 0.5. Protein concentration was determined by UV absorption using a Nanodrop spectrophotometer. Protein was separated by SDS-PAGE followed by transfer to polyvinylidene difluoride (PVDF) membrane. The membrane was probed with a 1:500 dilution of anti-Fur and a

53 1:1000 dilution of anti-OmlA antibodies, followed by a 1:25000 dilution of HRP- conjugated goat anti-rabbit IgG antibody. The anti-Fur and anti-OmlA antibodies were generated previously in the lab [17]. The antibody-protein complex bound to membrane were detected using Amersham™ ECL™ Prime Western Blotting

Detection Reagent (GE Healthcare). The intensity of the bands was determined using Image Lab Software™ (BioRad) and then normalized to the OmlA protein.

The values are the averages obtained from three independent blots.

Microtiter Plate Biofilm Assay

The microtiter dish assay was performed as described by O’Toole, with slight modifications [56]. In brief, overnight bacterial cultures were sub-cultured into fresh DTSB +/- FeCl3 in the wells of 96 well microtiter flat bottom plates

(Corning), 100 µL total volume. The plates were incubated in a shaker set at 150 rpm for 24 hours at 37˚C. Surface-attached cells were stained with 0.1% crystal violet; the stain was solubilized in ethanol and measured at OD590.

Analysis of Siderophore Production

Siderophore production was measured using Chrome azurol S (CAS) agar

(see Appendix F) as previously described [17]. Bacteria were grown on CAS agar

+/- 100 µM FeCl3 for 24 hr. The production of siderophores causes an orange halo to appear around the colonies on the CAS agar plate. Bacteria that exhibit normal siderophore production will form a strong halo on CAS agar plates with no

FeCl3 supplementation. In contrast, bacteria exhibiting normal siderophore

54 production should not typically show a halo when grown on CAS agar plates supplemented with 100 µM FeCl3.

Growth Curves

Growth curves were conducted using a 100-well honeycomb plate and the

Bioscreen C growth instrument. Wells containing 200 µL of media were inoculated with 0.1 or 0.05 OD600 overnight culture. The plates were placed into the Bioscreen C machine and incubated at 37˚C with continuous shaking. The

OD600 for each well was read every 15 minutes for 48 hours. Data generated were exported to Excel and graphed. Each media type and condition was tested in biological replicates of three.

Minimum Inhibitory Concentration (MIC)

Minimum inhibitory concentration assays were performed to assess the bacterial susceptibility of gallium in several different media types (see Chapter V and Appendix F). MICs were conducted using a 100-well honeycomb plate and the Bioscreen C growth instrument. Bacteria were grown overnight in the media being used to test gallium activity. The overnight cultures were diluted to a final

OD600 of ~.05. Bacterial dilutions were dispensed into the 100-well plate containing increasing gallium concentrations (0 – 512 µM). Iron-gallium competition assays were performed in the same manner as described above with the exception of the addition of FeCl3. All assays were incubated for up to 48 hours at 37˚C and growth (OD600) was measured every 15 minutes. Bacterial

55 susceptibility was called when bacterial growth was inhibited at least 90% as determined by OD600.

Zone of Inhibition

The zone of inhibition experiments were performed to assess gallium susceptibility. These tests were performed using the Kirby-Bauer disc diffusion method as previously described [216]. Bacteria were grown overnight in broth culture of the same media that was being used to test zone of inhibition at 37˚C.

6 mm paper discs (Becton Dickinson) were autoclaved and loaded with various concentrations of gallium (0 – 10 mM) and allowed to dry for one hour. The bacterial overnight cultures were diluted to OD600 0.132 and streaked onto the agar plates in three directions with a sterile cotton swab dipped into the culture.

This technique ensures that there is equal distribution of the bacteria over the entire surface of the plate. Using aseptic technique the loaded paper discs were placed on top of the agar surface and the plates were incubated overnight upright at 37˚C. The zones of bacterial growth were observed and zones of inhibition were measured.

56 CHAPTER III

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF PA2384

Several studies have shown that P. aeruginosa needs to acquire iron in order to establish infection. In addition, it is known that P. aeruginosa commonly forms biofilms throughout the course of infection, and particularly alginate biofilms in the later stages of chronic disease. However, little is known about how these two essential physiological processes, iron homeostasis and biofilm formation, interconnect at the molecular level. Previous microarray analysis conducted more than a decade ago by U. Ochsner, a former post-doctoral fellow in the Vasil Lab, identified a highly iron-regulated gene, PA2384 [84]. This gene was greatly expressed under iron-limitation and was initially thought to be involved in pyoverdine biosynthesis, based on the gene’s proximity to the pyoverdine biosynthetic locus. Further analysis into the function of this gene however, revealed that it has a limited role in the biosynthesis of pyoverdine [84].

Several years later, in 2007, a study was published characterizing the transcriptome of PA2384, under what the authors referred to as iron-limitation (2

µM FeSO4) as compared to wild type under the same conditions [217]. The authors of this report engineered a ∆PA2384 mutant and analyzed the transcriptional profile of this mutant by microarray analysis with RNA isolated from ∆PA2384 cells grown in a modified glucose minimal media supplemented with FeSO4. The authors reported that greater than 350 genes were affected by the ∆PA2384 mutant including virulence factors, basic physiological processes,

57 rhamnolipids and quorum sensing. Interestingly, rhamnolipids, quorum sensing and iron (see introduction for a review on iron and P. aeruginosa biofilms) have all been linked to Pel/Psl biofilm formation. Rhamnolipids are surfactants that are regulated by quorum sensing and are thought to play a role in swarming motility as well as Pel/Psl biofilm formation [218, 219]. Mutants defective in rhlA

(rhamnolipid biosynthetic enzyme) form flat biofilms on glass surfaces [27]. P. aeruginosa has three quorum sensing systems that are all coregulated and interconnected. Mutants of quorum sensing systems affect biofilm structure, producing flat, uniform biofilms [140, 143, 199, 219-222]. Therefore, this 2007 study revealed some confusing data that warranted our further investigation into the structure and function of this gene. In the present study, we investigated

PA2384 and its putative iron-regulated function in P. aeruginosa biofilm formation.

Structural Features of PA2384

The gene PA2384 is located directly downstream of the pyoverdine biosynthesis operon. The gene is 321 bp in length and encodes an 11.6 kDa hypothetical protein, which based on the pseudomonas.com website is homologous to the ferric uptake regulator protein Fur. In order to determine the structural similarities between PA2384 and other iron-regulated proteins we utilized bioinformatics analysis. These analyses revealed that PA2384 has sequence homology to the P. aeruginosa Fur protein as well as Fur in other bacteria (Figure 3.1). The crystal structure of the P. aeruginosa PAO1 Fur protein

58 was published in 2003, revealing in-depth architectural detail of this important

global regulator [103]. To further characterize PA2384, ClustalW was used to

align the PA2384 protein sequence to P. aeruginosa PAO1 Fur and E. coli Fur

(Figure 3.1). The aligned sequence was then compared to the known P.

aeruginosa PAO1 Fur structure to gain an understanding of the function of

PA2384 (Figure 3.1). The known iron and zinc binding sites, structural motifs and

areas of conservation were mapped on the sequence alignment (Figure 3.1).

This comparison revealed that PA2384 is 33.4% identical to the P. aeruginosa

Fur protein sequence in the N-terminal, DNA-binding region of the protein.

However, the C-terminal region, previously identified as the control region of P.

aeruginosa Fur, is vastly different between the two proteins. P. aeruginosa Fur

contains both an iron-binding domain and dimerization domain in the C-terminal

portion of the protein, both of which appear to be absent in PA2384.

Figure 3.1. Protein sequence alignment of PA2384 and Fur. Alignment of the 105 amino acid sequence of PA2384 with the Fur protein sequence from the organisms shown above. The alignment is annotated with conserved iron and zinc binding sites as well as structural motifs derived from the known Fur crystal structure.

59 Figure 3.2. Model of PA2384 N-terminal region. Using Phyre2, we generated a predicted structure of the N-terminal region of the PA2384 protein.

The structure prediction tool, Phyre2 (http://www.sbg.bio.ic.ac.uk

/phyre/index.cgi), validated with 100% precision (E-value 1.1e-9) that the PA2384

N-terminal DNA binding region was structurally related to P. aeruginosa Fur

(Figure 3.2) [223]. The DNA-binding domain of Fur is composed of four helices.

This winged helix is made up of a three-helix bundle with an additional fourth helix that is predicted to recognize DNA. Based on both the sequence alignment and the predicted PA2384 structure, this region in PA2384 is very similar to Fur, comprising four helices at the N-terminal region (Figure 3.2). Additionally, this

DNA-binding, winged helix-turn-helix domain is present in most

60 metalloregulators. The fifth helix in the Fur structure is involved in dimerization, while PA2384 does have this fifth helix, it does not have the extensive beta sheet that is important for Fur dimerization. Several residues are conserved in the

PAO1 Fur structure including Asp 103. This residue is thought to form a salt bridge that assists in dimerization of the protein and when mutated negatively affects DNA binding [101, 103]. PA2384 does contain this residue and may still form a dimer like PAO1 Fur. Together, these analyses suggest that PA2384 may act as a regulatory DNA binding protein and may be controlled in a way that is distinct from P. aeruginosa Fur.

Iron-Regulated Expression of PA2384

Transcriptional profiling of genes affected by iron indicated that the transcription of PA2384 was greatly induced under iron-limitation [84]. Most known iron-regulated genes either contain a Fur or PvdS (the two main iron regulators) binding sequence in their operator region. Interestingly, PA2384 does not appear to have either a Fur or PvdS regulatory sequence in its promoter.

Peculiarly, while the PA2384 promoter does not contain a PvdS binding site, the expression of PA2384 was dramatically decreased in a ∆pvdS mutant, as shown in the study published in 2002 by the Vasil lab, suggesting that the expression of

PA2384 is somewhat dependent on PvdS or its downstream genes [84]. These results contrast with the 2007 study by Zheng and coworkers that reported that the transcription of pvdS was decreased 7.25 fold in a ∆PA2384 mutant [217].

Therefore, we wanted to verify the iron-regulated expression of PA2384. In order

61 to characterize the response of PA2384 to iron, we performed Affymetrix

GeneChip® microarray analysis on wild type P. aeruginosa PAO1 in iron-limiting and iron-replete conditions. The microarray analysis was confirmed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) on wild type RNA isolated from the same conditions as described for the microarray analysis (see Materials and Methods). Both the microarray results and qRT-PCR analysis confirmed the study performed by U. Ochsner in 2002 that indicated that the PA2384 transcript was highly expressed under iron-limitation (Figure 3.3).

Figure 3.3. PA2384 is optimally transcribed under iron-limitation. A. Wild type P. aeruginosa was grown for 18 hrs in DTSB +/- FeCl3. RNA was isolated at 18 hrs and microarray analysis was performed. Results indicate 3 biological replicates. B. Using the same conditions described above, RNA was isolated at 18 hrs and analyzed by qRT-PCR. Results indicate 3 biological replicates.

62 Global Gene Expression Profile of ∆PA2384 in Response to Iron-limitation

In-depth review and comparison of the 2002 study from the Vasil lab and the

2007 publication by Zheng and coworkers revealed a number of disparities between these two studies [84, 217]. As mentioned above, the 2002 study reported that the expression of the PA2384 transcript was dramatically decreased in a ∆pvdS mutant, whereas the 2007 study indicated that the expression of pvdS was decreased 7.25 fold in a ∆PA2384 mutant. The authors of the later study suggest that PA2384 may have a positive effect on the transcription of pyoverdine biosynthesis genes through the control of PvdS. More notably, the 2002 study conducted in the Vasil lab not only examined the transcriptional profile of genes expressed under iron-limitation as compared to iron-replete conditions, but also analyzed putative genes involved in pyoverdine biosynthesis, including PA2384. In this study, a deletion mutant of PA2384 was constructed and analyzed for its putative role in pyoverdine biosynthesis. In this

2002 study, the ∆PA2384 did not have any negative, or positive effects on pyoverdine biosynthesis. Curiously however, the report by Zheng and coworkers state that the majority of pyoverdine biosynthesis genes were decreased in the

∆PA2384 mutant grown under iron-limitation. Since there was a number of differences between the study done in the Vasil lab in 2002 and the study by

Zheng and coworkers published in 2007, we constructed a mutant containing a chromosomal deletion of PA2384 and conducted Affymetrix GeneChip® microarray analysis to assess the gobal gene expression profile of ∆PA2384 under iron-limitation and iron-replete conditions as compared to wild type. In

63 brief, wild type PAO1 and the ∆PA2384 isogenic mutant were grown in DTSB +/-

100 µM FeCl3 for 18 hrs. RNA was isolated and microarray analysis performed

(for more details see Materials and Methods). In vast contrast to the study conducted by Zheng et al., who identified greater than 350 genes affected by

∆PA2384, we identified just over 50 genes that had either increased or decreased expression in the mutant under iron-limitation (Appendix A). In our study, none of the pyoverdine genes were affected, nor was pvdS. Moreover, the most significantly affected genes were those involved in alginate production showing an increase in the majority of genes in the alginate biosysnthesis operon

(Table 3.1).

Table 3.1. Genes with increased expression in ∆PA2384 isogenic mutant under iron-limitation PA Number Gene Function or Class Fold Change PA0572 Hypothetical protein 4.4 PA1137 Probable oxidoreductase 5.8 PA1202 Probable hydrolase, Isochorismatase, 7.1 Siderophore biosynthesis, Iron uptake PA1203 Conserved hyp. protein 2.3 PA1347 Probable transcriptional regulator 1.9 PA2432 Probable transcriptional regulator 11.3 PA2696 Probable transcriptional regulator 2.8 PA2697 Hypothetical protein 12.5 PA2698 Probable hydrolase, Isochorismatase, 8.6 Siderophore biosynthesis, Iron uptake PA3540 algD Alginate biosynthesis 8.8 PA3541 Alginate biosynthesis 2.4 PA3542 Alginate biosynthesis 2.1 PA3543 algK Alginate biosynthesis 4.7 PA3544 algE Alginate biosynthesis 1.3 PA3545 algG Alginate biosynthesis 10.7 PA3546 algX Alginate biosynthesis 1.1 PA3547 algL Alginate biosynthesis 2.4 PA3548 algI Alginate biosynthesis 1.6 PA3549 algJ Alginate biosynthesis 0.8 PA3550 algF Alginate biosynthesis 1.4 PA3551 algA Alginate biosynthesis 1.3

64 These results were compelling since up until this point there were minimal data on how iron affected alginate production and mucoidy. The alginate biosynthesis genes have historically been considered to be one long operon.

However, careful examination of our microarray gene expression data suggested that there might be more than one transcriptional start site, the first site at algD, the second at algG, and possibly a third beginning at algL (Table 3.1). These three transcriptional start sites were confirmed years later in 2012 in a published study by Paletta and Ohman [159]. To confirm our microarray results, we performed qRT-PCR on algD, the first gene in the biosynthesis operon and obligatory gene for alginate production, and on algG, which was also highly expressed in our studies. In brief, the wild type PAO1 parental strain and the isogenic ∆PA2384 mutant were grown for 18 hrs in DTSB +/- 100 µM FeCl3. RNA was isolated and analyzed by qRT-PCR. The data obtained were normalized to the constitutively expressed omlA gene that encodes an outer membrane protein

[102]. Our results from this analysis correlate with the data we obtained from our microarray analysis indicating that under iron-limitation the expression of genes within the alginate biosynthesis operon (algD and algG) are increased in

∆PA2384 (Figure 3.4). This increase was only observed under iron-limitation and not seen under iron-replete conditions (Figure 3.4). Taken together, our results suggest that PA2384 is a novel iron-regulated protein that may affect, either directly or indirectly, alginate biosynthesis genes under iron-limitation.

65

Figure 3.4. qRT-PCR analysis of algD and algG in wild type PAO1 and ∆PA2384. A. Cells were grown in DTSB +/- 100 µM FeCl3 for 18 hrs. RNA was isolated and processed for qRT-PCR analysis on the algD transcript. Results indicate three biological replicates. B. Cells were grown and harvested as described above. qRT-PCR analysis was performed on the algG transcript. Results indicate three biological replicates.

66 Purification of PA2384

In order to further characterize PA2384 we needed to overexpress and purify the protein. The PA2384 protein was purified using a glutathione S- transferase (GST) fusion system. For this particular protein, the GST system has advantages over other systems, such as the histidine-tag system. The GST system will not strip metals that are putatively bound to PA2384, which can have denaturing effects on proteins. This can be a problem when using nickel columns commonly used for purifying his-tagged proteins. Additionally, the GST system results in greater than 90% purity, can increase protein stability, and the GST-tag can be removed using thrombin protease in one easy step after purification. To fuse GST to the PA2384 translational open reading frame (ORF), the entire

PA2384 ORF was cloned into the pGEX-4T-3 fusion vector (see materials and methods). The pGEX-4T-3 vector contains a 26 kDa GST tag and a strong T7 promoter that is inducible with IPTG. The pGEX-4T-3 fusion vector containing the

PA2384 ORF was used to transform BL21 Rosetta E. coli cells. The BL21

Rosetta E. coli cells were used to enhance the expression of proteins containing codons rarely used in E. coli. Since the genome of P. aeruginosa is extremely

(~65%) GC rich, Rosetta cells will reduce unforeseen complications in regard to rare codon usage. Each step of the cloning and transformation was verified by sequencing to ensure that the integrity of the PA2384 ORF was maintained (data not shown). After verification, the BL21 Rosetta E. coli cells containing the pGEX-

4t-3-PA2384 vector were grown at 37˚C for 4 hrs and then induced for 2 hours at various temperatures with varying amounts of IPTG. After induction the cells

67 were pelleted by centrifugation and lysed by sonication. The soluble and

insoluble fractions were separated by SDS-PAGE (Figure 3.5). Induction at 37˚C

did not allow the GST-PA2384 protein to localize to the soluble fraction.

However, induction at 18˚C solved this problem (Figure 3.5). Finally, the purified

PA2384 protein was obtained by using glutathione sepharose™ 4B beads. The

protein was incubated with the beads at 4˚C for 4 hours followed with on-column

purification. After several washes of the beads to remove unbound GST-tagged

protein, the protein was cleaved from GST-bound beads using Thrombin

protease. High quantities of pure PA2384 were obtained from this procedure

(Figure 3.6).

Figure 3.5. PA2384 insoluble and soluble fractions. Cells were grown in LB medium, induced with varying amounts of IPTG at 18˚C, and then harvested. Cell pellets were sonicated and then centrifuged to separate the insoluble and soluble fractions. The samples were separated on a 12% SDS-PAGE and stained with Coomassie blue. EV: empty vector; NI: no induction.

68

Figure 3.6. PA2384 purified protein. Purified PA2384 was recovered from on- column purification and thrombin cleavage. Concentrated protein was run on a 12% SDS-PAGE gel and stained with Coomassie blue. PA2384 is 11.7 kDa and GST is 26 kDa. The higher molecular weight bands could be multimers or nonspecific protein that was not removed during purification.

69 Does PA2384 protein bind to the algD promoter?

Our results suggest that PA2384 might act as a novel iron-regulated protein that may affect (either directly or indirectly) alginate biosynthesis genes under iron-limitation. To determine if PA2384 directly regulates algD transcription, Electrophoretic Mobility Shift Assays (EMSA) were performed using the purified PA2384 protein. The EMSA assay will determine if there is direct interaction between the PA2384 protein and DNA within the promoter region of algD. The promoter region of algD is where the majority of alginate biosynthesis regulation occurs therefore, we decided to test the algD promoter region over the other alginate biosynthesis genes upregulated in our microarray study. To this end, starting at the algD translational start site and walking up the entire promoter region, DNA fragments between 115 and 150 bp were generated. The

DNA fragments were labeled with biotin and used in our assay to test for PA2384 binding. Figure 3.7 demonstrates that PA2384 bound to three different regions within the algD promoter. The first region was upstream of the +1 transcriptional start site and the two others were downstream of the +1 transcriptional start site

(Figure 3.7). The downstream binding sites are indicative of where you might expect to see the binding of a repressor protein. Further, the DNA fragment of the promoter region of fimU was used as a control to illustrate that PA2384 does not bind random DNA fragments (Figure 3.7). The absence of binding to the fimU

DNA fragment suggests that PA2384 binds to the three algD DNA fragments with specificity, thereby suggesting that PA2384 may have a direct regulatory role in regulating algD transcription.

70 ! ~ ~~

' · I I h ~ .. i 1 ~ ~ 1: i ! - i ~ r :, ~ g ; ~ ~N ~ ~ a i .. .. ~ .. !~ ~ w olio co 4250 425 212.5 2125 850 4250 2125 0 850 8500 8500 0 S::: :::SN ...... l ~ -=- " ~ ~ """ £ I ( r r . £ F ~ : N C" 0 (!) "tl ., C" 0 (!) "tl ...... , ~ 425 4250 4250 212.5 2125 2125 850 850 8500 8500 0 0 3:w :::SN I -~ ...... I ( r ,. r - - :: r · ...... ~ ' (_ ( [ ( ~ - -=c k; ~ E 1 I : olio "tl ., 0 C" (!) w 0 (!) ""C C" ., ~ w "tl i:~ ~ ...... 21250 850 85400 10675 4250 212.5 2125 21350 0 0 8500 8500 4250 42700 425 I . .. • ' ra I II • • II · ~ &:: ~ 1 ~ c:: C" 0 (!) "tl U1 .,

Figure 3.7. Gel mobility shifts of PA2384 binding to algD promoter fragments. Purified PA2384 bound to the algD promoter in three locations. One location was upstream of the +1 transcriptional start site and the other two were downstream of the +1 start site.

71 PA2384 May Function as a Dimer

There are several similarities between the PA2384 protein and the PAFur protein. PAFur is known to function as a repressor, binding to DNA in iron-replete conditions as a dimer. PA2384 lacks several residues involved in PAFur dimerization. However, in PAFur the residue Asp 103 is involved in dimerization and DNA-binding, this residue is also conserved in PA2384 [101, 103]. Our data suggest that PA2384 may act as a regulatory DNA-binding protein and may be controlled in a way that is distinct from PAFur. To get a better understanding of how PA2384 functions in its native state, we initially tried to perform Native-

PAGE on purified PA2384. This method was unsuccessful presumably because

PA2384 is positively charged (4.47) and has a high isoelectric point, 10.66.

Native-PAGE is generally useful for negatively charged proteins or proteins that have a lower isoelectric point. The buffering conditions can be adjusted for native-PAGE assays, within limits. Extreme pH changes can have denaturing effects on proteins or cause proteins to lose their physiological activity. Proteins with a very high isoelectric point require extreme buffering conditions for this type of assay. Therefore, in order to understand how PA2384 acts in its native state we used two methods, i) chemical cross-linking and ii) matrix-assisted laster desorption/ionization time of flight (MALDI-TOF) mass spectrometry.

Bis(sulfosuccinimidyl)suberate (BS3) is a noncleavable, membrane-impermeable, water-soluble crosslinking agent. This agent is a useful chemical for identifying protein interactions and fixes proteins in their native state so the protein interactions can be visualized by SDS-PAGE and coomassie staining [224].

72 Cross-linking studies demonstrated that PA2384 might function as a dimer in its native state. The PA2384 monomer is 11.7 kDa, PA2384 dimer is 23.2 kDa, trimer is 35.1 kDa, and tetramer is 46.8 kDa. Figure 3.8, shows PA2384 in a dimeric state after the addition of BS3. However, it is also possible that PA2384 forms a trimer. While there is a significant amount of monomer still present in the

SDS-PAGE, it is possible that PA2384 has weak protein-protein interactions.

Further, we submitted purified PA2384 to the Proteomics Core facility at the

University of Colorado Anschutz Medical Campus where MALDI-TOF analysis was performed. Just as our cross-linking studies indicate, the MALDI-TOF results suggest that while PA2384 is predominantly a monomer, it does form a dimer in solution (Appendix B).

Figure 3.8. PA2384 captured in its native state using the cross-linker BS3. Bis(sulfosuccinimidyl)suberate was used to cross-link purified PA2384 in solution. The monomer is 11.7 kDa, dimer 23.2 kDa, trimer 35.1 kDa and tetremer 46.8 kDa.

73 Functional Analysis of PA2384

While there were remarkable structural similarities between PA2384 and

PAFur, at this point the biological function of PA2384 still wasn’t clear. We saw an increase in alginate biosythesis genes in a ∆PA2384 mutant and demonstrated PA2384 binding to the algD promoter. However, it still wasn’t apparent how PA2384 impacted alginate production. We further investigated the function of PA2384 by performing uronic acid analysis on wild type PAO1 and the

∆PA2384 mutant grown on iron-limiting DTSA. The PA2384 mutant grown on iron-limiting DTSA did not produce a mucoid (overproduction of alginate) appearance. Nevertheless, the uronic acid analysis detected a 2.5 fold increase in the amount of alginate produced by the mutant (Figure 3.9). One major caveat

Figure 3.9. Alginate production by PA2384. Alginate was measured by uronic acid analysis in triplicate from bacteria grown on iron-limited DTSA after 24 hours.

74 to this experiment was that the alginate assays were performed on bacteria grown on a solid surface whereas the microarray and qRT-PCR studies were performed on cultures grown in liquid media. This difference may account for the large transcriptional increase of alginate biosynthesis genes seen in our study and only a small increase in the measured alginate production. It is possible that

PA2384 might have a more definitive role in liquid culture, perhaps in a pellicle, at the air-liquid interface (see Discussion).

Clinical isolates from the CF lung have a tendency to be severely mutated, often containing mutations in alginate regulatory, iron acquisition and quorum sensing genes. We decided to investigate the level of PA2384 conservation among numerous CF clinical isolates to gain more of an understanding of

PA2384s role in alginate production and mucoidy. The PA2384 promoter region and open reading frame (ORF) were amplified by polymerase chain reaction

(PCR) and sequenced to identify any potential mutations (Table 3.2).

Interestingly, we did not find any mutations in either the promoter region or ORF of PA2384 in any of the CF clinical isolates examined. These results suggest that

PA2384 is highly conserved in clinical isolates and may imply that maintaining the function of PA2384 is important. Further, it suggests that mutation of PA2384 is not related to the mucoid phenotype commonly observed in chronic CF isolates.

75 Table 3.2. PA2384 is highly conserved in P. aeruginosa Mucoid (M) or Clinical isolate Nonmucoid Mutations in PA2384 promoter or ORF (NM) V209 alg+ M NO V209 alg- Slightly M NO PA107 alg+ M NO PA107 alg- Slightly M NO 8830 mucoid M NO 8802 nonmucoid M NO PA111 alg+ Slightly M NO PA111-2 alg- M NO QP589 NM NO QP590 NM NO

To further examine the role of PA2384 in alginate production and mucoidy, a detailed review of past microarray data generated in the Vasil lab along with a comprehensive literature review was conducted. Table 3.3 represents the data garnered from this exhaustive review. While we gathered many interesting pieces of data that are noted in Table 3.3, the most noteworthy observations were 1)

PA2384 is most highly expressed when bacteria are exposed to epithelial cells and 2) PA2384 is highly expressed under aerobic conditions and repressed in microaerobic and anaerobic conditions. The latter, suggests that PA2384 functions in highly oxygenated environments. From the data described throughout this Chapter as well as the data obtained from several microarray analyses, it appears that PA2384 might be important during early infection, in highly oxygenated, iron-limited conditions. It is unclear what role Pel and Psl biofilms might have in infection. However, it is known that alginate is not a major component of Pel or Psl biofilms. Therefore, it might be possible that PA2384 acts as a repressor of alginate when Pel biofilms are present (see Discussion).

76

Table 3.3. Clues about the function of PA2384 in P. aeruginosa from microarray data PA2384 data from various microarrays conducted in the Vasil lab PA2384 Fold Change

Fur C6 (Ala10Gly) mutant high Fe D-TSB vs. PAO1 WT high Fe D-TSB 6.5 (log2) Fur C6 (Ala10Gly) mutant high Fe M9 (glucose) vs. PAO1 WT high Fe M9 No Change (glucose) PAO1 WT low Fe M9 (glucose) vs. PAO1 WT high Fe M9 (glucose) 71 PAO1 WT low Fe D-TSB vs. PAO1 WT high Fe D-TSB 148 PAO1 WT low Fe D-TSB vs. PAO1 ∆pvdS low Fe D-TSB 12

3% O2 vs. air -81.2

13% O2 vs. 3% O2 44.4

3% O2 vs. 21% O2 -57.4 PA2384 data from various microarrays conducted by others PA2384 Fold Change UCBPP-PA14 WT MOPS-Sputum vs. UCBPP-PA14 WT MOPS-Glucose 12 UCBPP-PA14 WT in vivo (rat implant) vs. UCBPP-PA14 WT MOPS- 41 Glucose UCBPP-PA14 WT in vivo (rat implant) vs. UCBPP-PA14 WT & Staph 46 aureus in vivo rat implant PAO1 WT exposed to epithelial cells vs. PAO1 WT grown in TSB 27.7 PAO-SC11 mutant (TSS & rhamnolipid deficient) exposed to epithelia cells 23.5 vs. PAO1 WT grown in TSB PAO1 WT supplemented with 40 µM PQS vs. PAO1 WT grown without 2.5 supplement * No affects seen in LasR, RhlR, AlgR, Vfr, CyaAB microarray studies * References: [225-228]

77 CHAPTER IV

IRON-REGULATED EXPRESSION OF ALGINATE PRODUCTION AND

MUCOIDY BY PSEUDOMONAS AERUGINOSA

Bacterial exopolysaccharides are an essential component of the extracellular matrix of biofilms and understanding the regulation of these exopolysaccharides is critical in developing novel therapies to combat P. aeruginosa infection. P. aeruginosa is a model organism for biofilm studies and can produce three distinct exopolysaccharides (alginate, Psl, and Pel) that are associated with unique biofilms and conditions under which they are formed (see

Introduction). Over the last decade, the impact of iron on Pel and Psl biofilms has been extensively studied in P. aeruginosa [6, 198, 200, 201, 203-205, 229].

However, little is known regarding the role of iron in alginate biofilms and mucoidy. In this study, we examined whether biologically relevant levels of iron might have any influence on the production of alginate biofilms. Data presented in this Chapter indicate that as little as 10 µM FeCl3 results in the inhibition of alginate production in PAO1 mucA, mucB, mucC, and mucD isogenic mutants.

Similarly, iron-regulated alginate production was observed in newly constructed muc mutants of other non-cystic fibrosis (CF) isolates (PA14, PAKS-1, Ps388).

Further, we investigated if iron regulated the production of alginate and mucoidy in CF isolates collected at an early stage and late stage of infection. The majority of the early stage CF isolates regulated alginate production in response to iron levels present in the medium. Conversely, the vast majority of mucoid late clinical

78 isolates tested had lost their ability to control alginate production in response to iron. Finally, alteration of known iron regulatory factors or acquisition systems

(e.g. siderophore biosynthesis or receptors) altered the regulated expression of alginate biofilms and some mutants with more than one altered iron acquisition system exhibited enhanced production of extracellular alginate and mucoidy, even under iron-replete conditions, as was observed with more than half of the

Late-CFI examined. Taken together, these observations suggest that during early infection iron tightly controls the production of alginate biofilms and that this regulation can be lost after years or decades of colonization in the harsh CF lung environment.

Iron Influences Alginate Levels and the Mucoid Phenotype of P. aeruginosa

Muc Mutants Constructed From Isolates of Non-CF Origin

Human lungs, including those of CF patients, historically have been considered to be iron-limited environments. However, recent studies indicate that there can be diverse microenvironments in the lung, and that iron levels may be considerably variable and not necessarily iron-limited as previously thought. Reid et al. suggested that Fe3+ ions (e.g. 13-134 µM) and ferritin (15-300 µg/L) are the major sources of iron in the CF lung and that patients with chronic obstructive pulmonary disease (COPD) commonly have much higher levels of elemental iron and ferritin [69, 71, 72, 75]. In order to directly investigate the impact of differing iron levels on alginate biosynthesis, we constructed ∆mucA, ∆mucB, ∆mucC or

∆mucD isogenic mutants of P. aeruginosa PAO1, or mucA and mucB insertion mutants of PA14, PAKS-1 or Ps388, and examined their production of

79 extracellular alginate and mucoid phenotypes in response to variable iron levels

(≤5 µM – ≥100 µM). These strains are all non-CF related isolates originating from widely differing types of infections and geographic regions; PAO1 is a wound isolate from Melbourne, Australia, PA14 is a burn isolate from Boston, MA, USA,

PAKS-1 is a urine isolate from Stockholm, Sweden and Ps388 is a blood isolate from Seattle, WA, USA. The strains were grown on varying concentrations of iron reported to be present in the sputa of CF patients (<1 µM to >100 µM) and were evaluated for alginate production by measurement of extracellular uronic acid levels (see Materials and Methods). As shown in Figure 4.1 and Figure 4.2, all of the aforementioned strains, which carry either single deletions in mucA, mucB, mucC, or mucD (PAO1), or insertion mutations in mucA or mucB (PA14, PAKS-1 or Ps388), produced substantially increased levels of extracellular alginate when grown under iron-limiting (≤5 µM) conditions as compared to when they were grown under more iron-replete conditions (≥10 µM) (Figure 4.1 and Figure 4.2).

Moreover, when PAO1 ∆mucA or ∆mucB mutants were cultured in the presence of alternative iron sources, such as ferric dicitrate or hemin, significant decreases in extracellular alginate levels were observed relative to these mutants grown without the addition of these iron sources (Figure 4.3). It should also be noted that corresponding increases in the concentrations other metals (Zn2+, Cu2+,

Ca2+, Mg2+), equivalent to those used for iron (≤5 µM – 300 µM), failed to cause parallel decreases in alginate levels and loss of mucoidy by any of these muc mutants (Figure 4.4 and data not shown).

80

Figure 4.1. The effect of iron on alginate production by muc mutants of P. aeruginosa PAO1. (A) P. aeruginosa PAO1 ∆mucA was grown on DTSA with +/- 100 µM FeCl3 and imaged after 48 hr. (B) PAO1 ∆mucA, ∆mucB, ∆mucC and ∆mucD were grown on DTSA containing +/- 5, 10, 25 or 100 µM FeCl3 for 48 hr and the levels of extracellular alginate were evaluated using a uronic acid assay (see Materials and Methods). (C) Relative expression of algD and algG in PAO1 wild type and PAO1 ∆mucA was analyzed using qRT-PCR after 24 hr of growth on DTSA with +/- 100 µM of FeCl3.

81

Figure 4.2. Effects of iron levels on alginate production by ∆mucA and ∆mucB mutants of P. aeruginosa PA14, PAKS-1 and Ps388. Alginate production was evaluated by measurement of extracellular uronic acid levels produced by PA14, PAKS-1 and Ps388 with insertion mutations in either mucA or mucB after 48 hr of growth on DTSA -/+ 100 µM FeCl3. ****P <0.0001; **P <0.01

82 Figure 4.3. The influence of various iron sources on alginate production by P. aeruginosa PAO1 ∆mucA and ∆mucB mutants. The strains were grown on DTSA supplemented with various sources and concentrations of iron carrying compounds. Extracellular alginate production was measured by uronic acid assay after 48 hr of growth.

83 Figure 4.4. The influence of assorted metals on alginate production. PAO1 wild-type and the PAO1 ∆mucD mutant were grown on DTSA supplemented with 100 µM of ZnCl2, CuCl2, CaCl2, MgCl2 or FeCl3. Extracellular alginate was measured by uronic acid assay after 48 hr of growth.

84 To ensure that the increased levels of uronic acids, produced under iron- limiting conditions by the PAO1 ∆mucA and ∆mucB mutants, accurately mirror those of alginate, a (1-4)-linked β-D-mannuronate and α-L-guluronate polymer, extracellular polysaccharides extracted from these mutants were evaluated by size exclusion chromatography and glycosyl composition analysis with combined gas chromatography/mass spectrometry (GC/MS) (see Material and Methods).

Because it was possible that other biofilm-associated polysaccharides (Psl or

Pel) could be produced by these strains, particularly those from cells cultured in iron-replete environments, polysaccharides extracted from the PAO1 wild type parent and the ∆mucA/B mutants grown both under iron-limited, and iron-replete conditions, were also evaluated by these methods at the University of Georgia

Complex Carbohydrate Research Center.

The levels of alginate detected from wild type PAO1, ∆mucA and ∆mucB in the total extracellular fraction of 1.0 x 107 CFU mL-1 after 24 hr of growth were as follows: PAO1 wild type, iron-limited growth – none detected; PAO1, iron- replete growth – none detected; PAO1 ∆mucA, iron-limited growth – 88.2 µg/107

CFU mL-1; PAO1 ∆mucA, iron-replete growth – 12.1 µg/107 CFU mL-1; PAO1

∆mucB, iron-limited growth – 176 µg/107 CFU mL-1; PAO1 ∆mucB, iron-replete growth – 33.7 µg/107 CFU mL-1. The most plentiful extracellular carbohydrate produced under iron-limited growth by the ∆mucA and ∆mucB mutants was alginate. By contrast, carbohydrates detected from PAO1 wild type samples were glucose and rhamnose, with a small amount of KDO, which was not detected in the ∆mucA or ∆mucB samples. The samples from the ∆mucA and ∆mucB

85 mutants grown under iron-limitation contained significantly lower levels of glucose and rhamnose (18.6 µg/107 CFU mL-1 glucose; 6.6 µg/107 CFU mL-1 rhamnose for iron-limited ∆mucA), than those from the PAO1 iron-limited parent

(274.8 µg/107 CFU mL-1 glucose; 54 µg/107 CFU mL-1 rhamnose). The extracellular polysaccharide composition of pel biofilms is mainly glucose, rhamnose, and mannose whereas the extracellular polysaccharide composition of psl biofilms is mainly galactose and mannose [39, 128]. These results are consistent with previous findings that mannuronic acid is the predominant extracellular polysaccharide of alginate biofilms and mucoidy [128].

Taken together, the above data provide the first evidence that biologically significant levels of iron can have substantial effects on the ability of newly derived muc mutants from a very diverse group of non-CF P. aeruginosa strains to express high levels of extracellular alginate and manifest mucoid phenotypes, which are most typically associated with a large proportion of P. aeruginosa isolates from chronic pulmonary infections in CF victims.

Transcriptional Analysis of PAO1 Wild Type and PAO1 ∆mucA Under Iron-

Limitation

It is perhaps a bit surprising that, while there has been a considerable number of transcriptional studies (microarray and PCR based analyses) of P. aeruginosa grown under differing levels of iron, there have not been any comparable assessments of transcriptional responses to variable levels of this biologically consequential metal for isogenic mucoid variants and their

86 nonmucoid parents [84, 98]. Based on the observations described above, we determined it to be worthwhile to examine the global transcriptional responses of

PAO1 wild type compared to those of a ∆mucA mutant under iron-limited and iron-replete conditions.

Duplicate cultures of P. aeruginosa wild type PAO1 and ∆mucA isogenic mutant were grown on DTSA solid medium +/- 100 µM FeCl3 and cells were harvested after 24 hr of growth. The global gene expression profiles were examined using GeneChip® microarray analysis and results were cross- compared and averaged. Table 4.1 provides a complete list of the genes and operons of the PAO1 wild type parent and its isogenic ∆mucA mutant, which exhibited the most robust responses to iron-limited as compared to iron-replete levels. As with most microarray-based approaches, a surfeit of data was generated. Nevertheless, there are several interesting outcomes based on these experiments that warrant specific comments.

First, it should be noted that more than ten years ago (2002) the Vasil lab published a similar microarray-based analysis where gene transcription, in the same PAO1 wild type parent, in response to differing iron levels (iron-limited vs. iron-replete) was examined. In that study however, the iron responses of the

PAO1 parent were evaluated during its growth in a well-aerated liquid medium, whereas in the present effort, the cells were grown on solid medium. Otherwise, the nutritional composition of both media was identical. The liquid media (DTSB) used in the earlier study was converted to a solid medium by the addition of 1.5% agar (DTSA). Other more subtle differences between the 2002 experiments and

87 Table 4.1. Select genes with increased gene expression under iron- limitation vs. iron-replete conditions in PAO1 and ∆mucA. PAO1 Gene PAO1 ∆mucA Description/Function Number Fold Fold Change Change PA0509 nirN NC 3 Probable c-type cytochrome; Energy metabolism; Biosynthesis of cofactors, prosthetic groups and carriers PA0510 NC 3 Probable uroporphyrin-III c-methyltransferase; Energy metabolism; Biosynthesis of cofactors, prosthetic groups and carriers PA0511 nirJ NC 4 Heme d1 biosynthesis protein; Biosynthesis of cofactors, prosthetic groups and carriers; Energy metabolism PA0512 NC 3 Conserved hypothetical protein; Energy metabolism; Hypothetical, unclassified, unknown; Biosynthesis of cofactors, prosthetic groups and carriers PA0513 NC 3 Probable transcriptional regulator; Energy metabolism; Transcriptional regulators; Biosynthesis of cofactors, prosthetic groups and carriers PA0514 nirL NC 4 Heme d1 biosynthesis protein; Energy metabolism; Biosynthesis of cofactors, prosthetic groups and carriers PA0515 NC 4 Probable transcriptional regulator; Energy metabolism; Biosynthesis of cofactors, prosthetic groups and carriers PA0516 nirF NC 4 Heme d1 biosynthesis protein; Biosynthesis of cofactors, prosthetic groups and carriers; Energy metabolism PA0517 nirC NC 5 Probable c-type cytochrome precursor; Biosynthesis of cofactors, prosthetic groups and carriers; Energy metabolism PA0518 nirM NC 6 Cytochrome c-551 precursor; Biosynthesis of cofactors, prosthetic groups and carriers; Energy metabolism PA0519 nirS NC 7 Nitrite reductase precursor; Energy metabolism PA0520 nirQ NC 3 Regulatory protein; Central intermediary metabolism; Energy metabolism PA0523 norC NC 11 Nitric-oxide reductase subunit C; Energy metabolism PA0524 norB 2 11 Nitric-oxide reductase subunit B; Energy metabolism PA0525 norD 4 7 Probable dinitrification protein; Energy metabolism PA2147 katE NC 13 Heme-binding catalase PA2185 katN 4 2 Mn-containing catalase PA3540 algD NC 23 GDP-mannose 6-dehydrogenase PA3541 alg8 NC 7 Alginate biosynthesis

88 Table 4.1. Select genes with increased gene expression under iron- limitation vs. iron-replete conditions in PAO1 and ∆mucA. PAO1 Gene PAO1 ∆mucA Description/Function Number Fold Fold Change Change PA3542 alg44 NC 4 Alginate biosynthesis; long-chain fatty acid-CoA ligase activity PA3543 algK NC 60 Scafold protein PA3544 algE NC 6 Outermembrane protein PA3545 algG NC 11 Alginate-C5-mannuronan-epimerase PA3546 algX NC 4 Alginate biosynthesis PA3547 algL NC 4 Alginate lyase; L-threonine ammonia-lyase activity PA3548 algI NC 8 Acetylase; predicted membrane protein involved in D-alanine export PA3549 algJ D 5 Alginate o-acetyltransferase PA3550 algF NC 5 Alginate o-acetyltransferase PA3551 algA 2 11 Phosphomannose isomerase/guanosine 5’- diphospho-D-mannose PA5097 HutT 3 6 Gamma-aminobutyrate permease; down regulates Type III cytotoxicity PA5098 hutH 2 14 Histidine ammonia-lyase; down regulates Type III cytotoxicity PA5099 NC 47 Nucleoside transporter family PA5100 hutU 5 23 Urocanase Increased Gene Expression 0 µM vs. 100 µM FeCl3; D Decreased; NC No change

the present ones were: the mRNA samples for the earlier study were harvested at 18 hr, as compared to 24 hr in the present experiments, and 200 µM FeCl3 was used for the iron-replete conditions in the 2002 experiments, rather than 100

µM of FeCl3 for the current efforts. No iron was added to the already iron-limited media, which was <5 µM in either case.

Despite the differences noted above between these two studies, performed more than a decade apart, there were several genes that were substantially increased in expression under iron-limitation in both studies

(Appendix C). These include a large number of genes encoding biosynthetic enzymes and regulatory factors (e.g. σ-factors – PvdS, and AraC regulator –

PchR), relating to the production of the two major siderophores of P. aeruginosa,

89 pyoverdine and pyochelin, as well to those involved in heme acquisition. In any case, it is remarkable that, of those genes showing an increased response to iron-limitation reported in the 2002 study (78 genes/operons), only 10 (~13%) were not detected as such in our present experiments in the PAO1 wild type parent. The failure to detect an iron-limitation response for these 10 genes/operons could simply be a consequence of the difference between growth in liquid versus solid media, time variation (18 hr vs. 24 hr), or technical and experimental variation.

Yet, the function of one of these operons could perhaps provide some insight about possible differences in the availability of different forms of iron (i.e.

Fe2+ vs. Fe3+) in liquid versus solid growth medium (or perhaps within a biofilm).

That is, the genes encoding FeoA (ferrous transport core domain), FeoB (ferrous transporter) and FeoC (transcriptional regulator) were highly expressed: 32X increased under iron-limitation in the 2002 study, but none of these genes showed any significantly increased expression in the non-mucoid PAO1 parent, nor in the ∆mucA mutant, grown under iron-limitation on the agar-based medium in our present study.

Another interesting aspect of the present study is the increased expression of a notable number of genes/operons under iron-limitation in the

∆mucA mutant, but significantly lower increased expression, or no increased expression, in the PAO1 parent under iron-limitation (Table 4.1 and Appendix C).

Such results could provide insights into genes that may play an important role in the processes that impact on the basic metabolism of mucoid strains and the

90 stresses related to increased production and secretion of extracellular alginate under iron-limitation.

Indeed, the most striking and obvious examples in this regard are the increased transcriptional responses of genes involved in alginate biosynthesis and export (algD – algA) in the ∆mucA mutant, but not the PAO1 parent, under iron-limitation, in either the former (2002) or present study (Table 4.1). These results, and the fact that we did not observe corresponding increases in the expression of the sets of genes involved in the biosynthesis of the Psl (pslA-pslO;

PA2231-PA2245), nor the Pel (pelA-pelG; PA3058-PA3064) exopolysaccharides, strongly reinforces the idea that iron-limitation selectively influences the increased production of alginate, in contrast to either of the other, biofilm associated, extracellular polysaccharides (Psl or Pel).

It is likewise worth mentioning that there are some genes/operons that show increased expression under iron-limitation, particularly in the ∆mucA mutant, but not in the PAO1 parent grown either on solid or liquid media. These include nirN-nirQ (PA0509-0520) and norCBD (PA0523-5025), which encode enzymes (nitric oxide reductases), cytochromes and regulatory factors involved in denitrification, thereby perhaps reflecting a shift toward the use of nitrates for anaerobic respiration during increased alginate biosynthesis (Table 4.1). Along a similar line, there was a substantial increase (13X) in the expression of the katE gene in the ∆mucA mutant, but not in PAO1 wild type under iron-limitation, in either solid or liquid media (Table 4.1). While there was increased expression of katN, encoding a Mn-catalase under iron-limitation in the ∆mucA mutant, as well

91 as the PAO1 wild type, an additional increase in KatE (heme-binding catalase) levels may reflect enhanced redox stresses resulting from increased alginate exopolysaccharide production.

Finally, one additional salient outcome from these experiments is the considerably increased expression of PA5097 (hutT), PA5098 (hutH) and

PA5099 (Table 4.1). Transcription of these genes, which comprise an operon, was significantly increased in the PAO1 ∆mucA mutant under iron-limitation, but to a substantially lesser extent, or not at all in PAO1 wild type under iron- limitation, in either solid or liquid media. In this regard, it was reported that the increased expression of these genes in P. aeruginosa PAO1, which are involved in the utilization of histidine, led to a decreased cytotoxicity, concomitant with the down regulation of the expression of at least one of the Type III secreted cytotoxins (e.g. ExoS) [29, 125, 230, 231]. Taken together, such results could explain why a substantial number of mucoid CF isolates show a considerable tendency toward a diminished Type III mediated cytotoxicity phenotype.

The regulation, biosynthesis and export of alginate exopolysaccharide are complex processes involving numerous adjacently located structural genes

(PA3540 to PA3551), as well as an assortment of genes encoding regulatory factors (e.g. algR, algC, algB, kinB) scattered throughout the P. aeruginosa genome [36, 158, 160, 176, 232]. The biosynthesis and export of this exopolysaccharide is initially determined at the algD (PA3540) promoter, by regulatory proteins that tightly control the transcription of the biosythesis and export genes at this locus. In addition to direct regulation of the algD promoter,

92 various proteases (AlgW, MucP, ClpXP and Prc) function in posttranscriptional regulation by liberating AlgU/T from the MucA/B/C/D sequestration complex at the membrane, thereby leading to the constitutive transcription of algD, which encodes the first enzyme (GDP-mannose 6-dehydrogenase) in the pathway leading to alginate biosynthesis (see introduction for details) [5]. Real-time qRT-

PCR was used to validate the expression of algD (encoding GDP-mannose 6- dehydrogenase) and algG (encoding alginate-c5-mannuronan-epimerase) in the

∆mucA mutant grown under iron-limitation. Triplicate cultures of P. aeruginosa wild type PAO1 and the ∆mucA mutant were grown on DTSA solid medium -/+

100 µM FeCl3 and cells were harvested after 24 hrs (see Materials and Methods).

Real-time qRT-PCR analysis showed that the transcript levels of both algD and algG in the mucoid mucA mutant were greatly increased in comparison to wild type levels under iron starvation (Figure 4.1C). As predicted, expression of both algD and algG transcripts in the ∆mucA mutant was correspondingly decreased under iron-replete conditions (Figure 4.1C). Microarray analysis and real-time qRT-PCR, correlated extremely well in terms of the observed corresponding alginate-related phenotypes.

Do CF-Pulmonary Isolates Regulate Alginate Production in Response to

Iron Levels?

In light of the above observations about the iron-regulated mucoid phenotype of muc mutants of non-CF isolates, it was of interest to ascertain whether those variable iron levels (≤5 µM – 100 µM), known to exist in sputa of

93 CF patients, can similarly influence the expression of alginate and mucoidy in an assortment of isolates from CF-associated infections. For this purpose, two groups of CF-pulmonary isolates (CFI) were examined. One group (Early-CFI) consists of 8 isolates from individual children with CF, who were less than 3 years of age (yoa) at the time of sputum collection (Appendix D). The other group

(17 isolates) originated from older CF patients (>10 yoa), who had been colonized with P. aeruginosa for at least 5 years (Late-CFI) (Appendix E). While both the Early-CFI and the Late-CFI groups contain isolates with mucoid phenotypes, there are some appreciable differences between these groups in relation to the number of isolates having a mucoid phenotype, yet there is an even more conspicuous difference between the number of mucoid isolates in each group that show constitutive alginate levels and mucoid phenotypes, irrespective of whether they are grown in an iron-limiting, or an iron-replete environment.

Of the Early-CFI group, only 50% (4 of 8) of the isolates intrinsically produced increased alginate levels along with an obvious mucoid phenotype, while most (16/17, 94%) of the Late-CFI produced significantly increased levels of extracellular alginate, along with an obvious mucoid phenotype (Appendices D and E). However, only one single mucoid isolate (1/4), 25% from the Early-CFI group showed constitutive expression of increased alginate levels and a mucoid phenotype both when grown under iron-limited and iron-replete growth conditions, while 56% (9/16) of the Late-CFI were constitutive for increased alginate expression and mucoidy, even with the highest level of iron tested

94 (Appendices D and E and Figure 4.5). What is more, when a mucB insertional mutation was introduced into one of the non-mucoid isolates from the Early-CFI group, it clearly displayed a mucoid phenotype and regulated it’s alginate production in response to iron, as did the non-CF isolates (PAO1, PA14, PAKS-

1, Ps388), where either a mucA or mucB mutation was freshly introduced.

Figure 4.5 illustrates the differences between a select group of

Figure 4.5. Alginate production and mucoidy of Late CF isolates (Late-CFI) in response to variable iron levels. (A) Late-CFI were grown on DSTA -/+ 100 µM FeCl3 and imaged at 48 hr. (B) The level of extracellular alginate produced by various Late-CFI grown on DSTA -/+ 100 µM FeCl3 was measured by uronic acid assay at 48 hr. (C) Relative expression of algD and algG of Late-CFI-1 and Late- CFI-4 was analyzed using qRT-PCR after 24 hr of growth on DSTA -/+ 100 µM FeCl3. *P ≤ 0.05

95 constitutively producing alginate isolates and those that regulate alginate production in response to iron levels. As shown by uronic acid analysis of extracellular alginate and real time qRT-PCR, two of the Late-CFI (Late-CFI-4,

Late-CFI-7) have an iron-regulated phenotype, while two others (Late-CFI-1,

Late-CFI-2) distinctly exemplify constitutive phenotypes (Figure 4.5). These data demonstrate the robust variations that occur in the expression of a highly relevant virulence determinant (alginate production and mucoidy) as the consequence of fluctuations in a relatively narrow range of iron levels that are known to occur during CF pulmonary infections.

Although the number of CF isolates examined in this study was relatively small, our results support the concept that over time (years to decades) an initially vigorous regulatory influence of iron on the expression of alginate and mucoidy, in some manner, may be lost, resulting in alginate production and mucoidy that are essentially indifferent to the variable levels of iron known to exist in the lungs of CF victims.

How Does Iron Initially Control the Increased Production of Extracellular

Alginate and Mucoidy and How Can This Regulation Be Lost?

Chrome azurol S (CAS) agar plate assay is a common technique used to study siderophore production in bacteria [101]. Based on the high numbers of

Late-CFI that had lost iron-regulated alginate production, it was of interest to examine whether these Late-CFI were capable of acquiring iron. Reduced siderophore production or regulation was seen with several strains exhibiting

96 constitutive alginate production and mucoidy (Appendices D and E). For example, see the Late CF isolates, 1, 2, 12 & 16, which exhibit an iron- constitutive increase in alginate production and mucoidy, yet produce very low, if any, levels of siderophores (e.g. pyoverdine, pyochelin) on either iron-limited or iron-replete CAS agar plates (Appendix E). Nonetheless, no compelling correlation could be discerned between this and the loss of iron-regulated alginate production and mucoidy. Perhaps, there is a variety of ways that a constitutive mucoid phenotype, more often associated with Late-CFI, can occur.

In the Banin et al. 2005 study, where the impact of iron and iron-limitation on the formation of a Psl/Pel type of biofilms were considered, an assortment of mutants altered in their ability to: (i) express key iron related regulatory factors

(Fur, PvdS), (ii) produce enzymes involved in siderophore biosynthesis (PvdA) or

(iii) express receptors for specific siderophores (FpvA), was examined [6]. Based on those data, it was clear that interfering with such iron regulatory or iron acquisitions systems resulted in a significantly diminished ability of PAO1 to produce robust Psl-type biofilms (those with mushroom-like structures vs. thin, flat biofilms) under the conditions used in that study.

In the present study, a similar approach was used. However, it was necessary to first introduce either mucA or mucB mutations into a variety of single, double, and triple mutants of PAO1 carrying deletions in genes encoding: an iron regulatory factor (pvdS), assorted siderophore biosynthesis enzymes

(pvdA, pvdD, pchEF, pchDA) or the pyoverdine receptor, (fpvA). The single, double, and triple mutants containing mucA and mucB insertional mutations were

97 grown on DTSA +/- 50 µM FeCl3 for 24 hr and 48 hr, and then analyzed for

alginate production using uronic acids assay and real time qRT-PCR analysis.

None of the above single, double or even triple iron-related mutations resulted in

any significant decrease in the expression of alginate or the algD transcript under

iron-limitation, and some of the mutants (e.g. ΔpvdD, pchEF, fpvA) even show a

tendency to express increased levels of alginate and algD transcript under iron-

replete conditions, as compared to mutants with only mucA or mucB mutations

Figure 4.6. Investigation of alginate production in iron acquisition mutants of P. aeruginosa PAO1. (A) P. aeruginosa PAO1 and several isogenic iron acquisition mutants were grown on DTSA with +/- 50 µM FeCl3 and imaged after 48 hr. (B) Relative expression of algD in PAO1 wild type and several isogenic iron acquisition mutants were analyzed using qRT-PCR after 24 hr of growth on DTSA with +/- 50 µM of FeCl3.

98 (Figure 4.6).

PvdS is a positive regulator (sigma factor) required for the increased expression of genes encoding proteins involved in virulence (exotoxin A and

PrpL protease) and iron acquisition (pyoverdine biosynthesis and uptake) [22, 30,

32, 84, 100, 233]. Its expression is not normally detected under iron-replete conditions because pvdS transcription is virtually abrogated by Fe2+-Fur in iron- replete environments [84]. Consequently, if PvdS were to be required for, or associated with, the constitutive expression of alginate, as seen in many of the

Late-CFI, it would have to be expressed under iron-replete, as well as iron- limiting, conditions. Western blots and real time qRT-PCR analysis of cells from

CF isolates of P. aeruginosa exhibiting both iron-regulated and iron-constitutive expression of alginate and mucoidy demonstrated that PvdS could only be detected under iron-limitation (<5 µM) in either of these types of isolates (data not shown). Therefore, alterations in PvdS levels can, in no manner, account for increased alginate expression under iron-replete conditions as observed for most

Late-CFI and a single Early-CFI. Even more compelling evidence however, is provided by the fact that when a mucA insertional mutation is introduced into a

ΔpvdS mutant of PAO1, despite the loss of this iron-regulated sigma factor and the ensuing down-regulation of PvdS controlled genes (pyoverdine biosynthesis, toxA, prpL) in ∆pvdAmucA- mutant, it still expressed alginate and mucoidy in a completely iron-dependent manner, as did new mucA mutants of PAO1, or other non-CF related isolates with mutations in either mucA or mucB (Figure 4.6B).

99 An alternative scenario where an iron-regulated phenotype could become a constitutive phenotype, in the context of the iron levels used in this study, would be where a mutation in the fur gene, encoding the master regulator of iron homeostasis, had occurred. A mutation in Fur would result in the de-repression of a large number of regulatory and structural genes, including those that might be involved in alginate production and mucoidy [11, 32]. Such a mutation could be hypothesized to lead to constitutive expression of alginate and mucoidy. While

Fur is essential in P. aeruginosa, point mutations that partially affect Fur function, or reduce Fur levels, can be isolated and have been demonstrated to show a constitutive phenotype in terms of the expression of a plethora of iron-regulated factors, including two siderophores (pyoverdine & pyochelin) and exotoxin A

[101, 105]. As a consequence, it is possible that CF isolates, which exhibit a constitutive phenotype in terms of an increased expression of alginate and mucoidy under both iron limited and iron-replete conditions, could have acquired point mutations that either affect Fur function or reduce its levels [101].

These possible scenarios regarding Fur were examined in two ways. First, the sequence of the fur gene, including its regulatory region, was examined for both Early-CFI and Late-CFI isolates including those showing an iron-regulated and an iron-constitutive phenotype with regard to the production of increased levels of alginate and mucoidy. In all cases there were no detected base changes in the structural and regulatory region of fur in any of these isolates (data not shown). Secondly, the levels of Fur protein and fur transcripts, as detected by western blot and real time qRT-PCR respectively, were examined in CFI that

100 exhibit both an iron-regulated and an iron-constitutive phenotype with regard to alginate levels and mucoidy (Figure 4.7). There were no differences in the levels of Fur or fur transcripts produced under iron-limited or iron-replete conditions that could be associated with either an iron-regulated or iron-constitutive phenotype in terms of increased alginate production or mucoidy.

Figure 4.7. Investigation of the role of Fur in alginate production. (A) PAO1, ∆mucA, Early-CFI-1, Late-CFI-1, and Late-CFI-4 were grown on DTSA +/- 100 µM FeCl3. The levels of fur gene expression were measured by qRT-PCR after 24 hr of growth. (B) PAO1, ∆mucA, Early-CFI-1, Late-CFI-1, Late-CFI-2, and Late-CFI-4 were grown on DTSA +/- 100 µM FeCl3. Fur protein levels were analyzed by Western blotting and quantified as described in materials and methods.

101 Can Mucoid Strains Produce Pel and Psl Biofilms?

Banin et al. demonstrated that intracellular levels of iron serve as a signal for the development of P. aeruginosa PAO1 structured, non-alginate biofilms grown in a flow-through system on a glass surface [6]. They also discovered that active pyoverdine iron acquisition is required for the formation of mushroom-like structures and that iron signaling in biofilm development is mediated by Fur.

Unlike alginate biofilms where alginate is the predominant exopolysaccharide, wild type biofilms do not contain significant amounts of alginate [128]. Our discovery that iron-limitation causes the expression of copious amounts of alginate is in stark contrast to what is known about Pel and Psl biofilms. The detailed observations of iron’s impact on alginate production and mucoidy described throughout this Chapter led us to hypothesize that alginate and Pel/Psl biofilms might be inversely regulated by iron. To test this hypothesis, P. aeruginosa PAO1 mucA, mucB, mucC, and mucD isogenic mutants were grown in a microtiter dish assay in DTSB supplemented with or without 100 µM FeCl3.

The microtiter plate assay is useful in measuring Pel and Psl biofilms, particularly

Pel biofilms in solution. Bacteria are grown in wells within a 96-well plate for either 24 or 48 hours, washed, and then stained with crystal violet. The result of this assay is a measurement of bacterial cells that adhered to the wells forming a biofilm. This assay does not measure alginate biofilms because alginate biofilms will not adhere to the sides of the wells; alginate collects in the center of the wells and can be washed away. A recent study compared the adhesive properties of a mucoid mucA mutant to PAO1 wild type in a flow-cell system [234]. This study

102 revealed that the mucoid strain had reduced adherence properties as compared to wild type bacteria. The authors suggest that alginate masks adhesins and surface features that would normally allow P. aeruginosa to attach to surfaces.

We performed this microtiter assay, knowing that iron-limitation enhances alginate production and iron-replete conditions repress alginate expression in muc strains. In iron-rich medium, mucoid P. aeruginosa formed biofilms similar to the wild type PAO1 (Fig 4.8). These results suggest that iron-replete conditions allow muc strains to form Pel and Psl biofilms while repressing the expression of alginate.

Figure 4.8. Mucoid ∆mucA and ∆mucB form Pel/Psl biofilms in iron- replete conditions. Biofilm formation was assayed by microtiter plate assay after 24 hr of growth in DTSB +/- 100 µM FeCl3. Surface-attached cells were stained with 0.1% crystal violet, the stain was solubilized in ethanol and measured at OD590.

103 CHAPTER V

INVESTIGATION OF GALLIUM ON ALGINATE PRODUCTION AND MUCOIDY

Gallium (Gallium(III) or Ga3+) is an iron-analog that has a very similar chemistry to ferric iron (iron(III) or Fe3+) in solution. The chemical behaviors of

Ga3+ and Fe3+ are largely attributed to their similarities in nuclear radius and coordination chemistry [235]. These similarities allow Ga3+ to compete with Fe3+ for binding to iron-containing enzymes, siderophores, and the host iron-binding proteins transferrin and lactoferrin [235]. While Ga3+ has many similarities to Fe3+ and can act as a competitor for many essential bacterial proteins, it is the significant differences between the two metals that are intriguing, both in the context of novel therapeutics and as a tool for studying iron acquisition and homeostasis in P. aeruginosa. There are two major differences between the two metals 1) Ga3+ is not capable of redox-cycling under physiological conditions, whereas Fe3+ can be readily reduced and reoxidized, and 2) Ga3+ is extremely soluble in aqueous solutions at neutral pH, whereas Fe3+ precipitates out of solution at neutral pH and can only exist bound to proteins or iron-chelators (i.e. siderophores). Most bacteria depend upon the redox-cycling capabilities of iron; consequently the replacement of Ga3+ for Fe3+ inhibits a number of essential biological reactions including DNA and protein synthesis, energy production and metabolism. Further, since Ga3+ cannot be reduced to Ga2+, it can’t be incorporated into many iron-binding compounds such as hemoglobin.

104 Numerous studies have shown the Ga3+ inhibits the growth of P. aeruginosa as well as the ability of the organism to form biofilms [197, 236-238].

The bactericidal properties of Ga3+ are likely due to the inhibition of important physiological properties of the organism. However, at this point little is known about Ga3+ transport into bacterial cells or the mechanism of killing in the cell.

There is some evidence that indicates that Ga3+ decreases iron uptake and interferes with pyoverdine-mediated signaling through the activity of PvdS [197].

Nevertheless, more studies need to be conducted to understand how Ga3+ affects pyoverdine biosynthesis and PvdS function. The clinical appeal of Ga3+ is that this metal is already FDA approved and large concentrations are used to treat hypercalcemia and bone metastasis [239]. In fact, clinical trials are currently underway to test the feasibility of using Ga3+ as a therapeutic for P. aeruginosa infection (see Discussion).

In this study, we were interested in using Ga3+ as a tool to dissect the mechanism by which iron affects the expression of alginate production and mucoidy as presented in Chapter IV. Since Ga3+ is not capable of redox-cycling, we thought this metal could be used to examine which type of iron source, either

Fe3+ or Fe2+, might contribute to the iron-regulated expression of alginate.

Several of the Late-CFI examined in Chapter IV that displayed constitutive alginate production regardless of the iron concentration were also dysregulated in siderophore production (see Appendix E). Therefore, we hypothesized that those isolates might be inefficient at transporting Ga3+ into the cell and might be more resistant to Ga3+ bactericidal effects. If the isolates were indeed more

105 resistant to Ga3+, this might indicate that the isolates have a deficiency in their ability to transport Fe3+, which is thought to be the dominant form of iron transported by P. aeruginosa.

Assessment of Growth Media for Gallium Susceptibility Testing

The antibacterial properties of Ga3+ are attributed to the ability of this metal to substitute for iron and disrupt iron homeostasis. A number of previous reports has indicated that a surplus of iron as well as ligand complexation and carbon source will affect the bactericidal activity of Ga3+ [197, 236, 238].

Therefore, in order to test the anti-bacterial affects of this metal we needed to define the conditions under which Ga3+ could be efficiently tested. A preliminary investigation was performed using wild type PAO1, ∆mucA and three Late-CF isolates (Late-CFI-1, Late-CFI-2 and Late, CF1-3) to test growth of the bacteria and Ga3+ activity. These strains were grown in several chemically defined

(M9+Succinate, M9+Glucose, M9+Casamino Acids) and undefined (MH, DTSB,

DTSB + No Supplement, 1% DTSB, 10% DTSB) media to assess suitable bacterial growth (Figure 5.1 and data not shown). DTSB is typically supplemented with glycerol and monosodium glutamate (MSG). In this study we tested DTSB with (DTSB) and without supplementation (DTSB + No Supp.) as well as diluted DTSB. Diluted DTSB was tested since diluted TSB was used in a study conducted by Kaneko et al. and was shown to be an effective growth medium for testing Ga3+ sensitivity. Growth of wild type PAO1, ∆mucA and the

Late-CF isolates tested was extremely poor in both 1% and 10% DTSB (Figure

106 5.1 and data not shown). Since the strains tested in our study showed little to no growth in this medium, this medium was not considered suitable for our studies

[197]. The addition of 100 µM of iron (FeCl3) to the majority of both the defined and undefined media resulted in increased growth of the bacteria tested, suggesting that several of the media used are limited in iron (Figure 5.1).

Therefore, with the exception of 1% and 10% DTSB, all of the media were considered suitable to test for gallium activity.

Figure 5.1. Growth of wild type PAO1 in chemically defined and undefined media. Wild type P. aeruginosa PAO1 was grown in various defined and undefined media for up to 24 hours. Results represent three biological replicates.

107 Accordingly, the wild type PAO1, ∆mucA and Late-CF isolates were used to test for Ga3+ susceptibility in all of the media types listed above (Table 5.1).

The strains were grown in a microtiter plate assay and MICs were determined after 6, 24 and 48 hours of growth (Table 5.1). The effect of gallium on the growth of wild type PAO1, ∆mucA and three Late-CF isolates is reported in µM concentrations in Table 5.1. Gallium inhibited the growth of all strains in a dose- dependent and strain-dependent manner. As a general trend, the MIC values were lower at earlier time points, for example the MIC for Late-CFI-1 was 16 µM of Ga3+ at 6 hours, 32 µM of Ga3+ at 24 hours, and 64 µM Ga3+ at 48 hours. The

MIC values were greatly affected by the medium, suggesting that the carbon source in the medium was a significant determinant in gallium anti-bacterial activity. Based on our assessment, the complex media (MH and DTSB) were not suitable for testing the bactericidal activity of gallium. The minimal, defined medium were substantially more useful in assessing gallium activity, particularly the M9 supplemented with casamino acids. Interestingly, the growth and the

MICs of P. aeruginosa were poorer in glucose and succinate media as compared to the casamino acids. These results may suggest that the metabolism of the bacteria is important for gallium toxicity and bactericidal effects may be more evident when the organism is utilizing specific carbon sources and metabolic pathways. We decided to move forward with our MIC testing using the M9 supplemented with casamino acids. This medium was chosen over the other two chemically defined media due to the better growth of the clinical isolates in the casamino acids over glucose or succinate (data not shown).

108

h M) CF " 512 512 512 48 - >512 >512 ( 3 ) 3

h MH 512 512 512 256 512 24

h MIC Ga(NO 6 256 256 256 256 256

and three Late h M) 256 48 >512 >512 >512 >512 " ( 3

)

3 mucA h

! 128 256 24 >512 >512 >512 DTSB

h MIC Ga(NO 6 128 256 >512 >512 >512

h ND M) 256 256 256 48 >512 " ( 3 )

3 h

256 256 256 256 24 >512

h MIC Ga(NO DTSB + No Supp. 6 ; ND= Not detected 256 256 256 >512 >512

h 8 8 4 M) 32 32 " 48 ( 3 )

3 cinate

h

4 4 4 32 32 24

h M9 + Suc 4 4 4 MIC Ga(NO 32 32 6

h M) 4 4 4 " 16 16 48 ( 3 ) 3

constitutive alginate production

-

h 4 4 4 16 16 24

M9 + Glucose h 4 4 4 16 16 MIC Ga(NO 6

measured after 6, 24, and 48 hrs of growth for wild type PAO1, h

M) 3+ 32 32 64 16 " 128 48 ( 3 )

3

h

16 16 32 16 16 24

M9 + CasAA

h 4 16 16 16 16 MIC Ga(NO 6

1 2 3 - - -

MIC values for Ga

A CFI CFI CFI

- - - regulated alginate production; IC= Iron

- muc ate ate ate n PAO1 ! L L L Strain

Table 5.1. isolates. IR IC IC IC IR= Iro 109 Minimum Inhibitory Concentrations of PAO1, muc Mutants and CF Clinical

Isolates in a Chemically Defined Minimal Medium

Gallium may serve as a useful tool in deciphering which type of iron source is contributing to the iron-regulation of alginate production as discussed in

Chapter IV. We hypothesized that strains that are still capable of expressing high levels of alginate under iron-replete conditions would be more resistant to the bactericidal activity of gallium. This increased resistance to gallium could be attributed to the organism’s reduced ability to transport Fe3+ through siderophores, the main mechanism of iron acquisition in P. aeruginosa. To determine the relative susceptibility to various concentrations of gallium, MIC testing was conducted on all of the strains used in Chapter IV. The strains were grown in a microtiter plate assay and MICs, reported in µM concentrations, were determined after 6, 24 and 48 hours of growth (Table 5.2). As seen in the preliminary susceptibility assay, for all of the strains tested, gallium inhibited growth in a concentration-dependent and strain-dependent manner. The MIC values were lower at earlier time points for all strains and there was no correlation between increased resistance and the strain’s ability to constitutively express alginate. The strain’s ability to produce alginate or regulate alginate in response to iron had no bearing on whether or not the strains were susceptible to gallium. Furthermore, there was no correlation between the strain’s gallium susceptibility and dysregulation of siderophore production (Table 5.2,

Appendices E & F). In fact, Late-CFI-5, which produced very little, if any, siderophores in a CAS assay, was more susceptible to gallium at 6 hours and

110 Table 5.2. MIC Values for Ga3+ of all strains grown in M9 + Casamino Acids.

M9 + CasAA Strain

MIC Ga(NO3)3 (µM) 6 h 24 h 48 h PAO1 16 16 32 IR ∆mucA 16 16 32 IR ∆mucB 32 32 32 IR ∆mucC 32 32 64 IR ∆mucD 64 64 64 IR Early-CFI-1 8 8 16 0037-10 IC Early-CFI-2 16 16 16 0205-1 IR Early-CFI-3 16 16 32 006-11 IR Early-CFI-4 32 32 64 0026-1 IC Early-CFI-5 16 32 32 0103-1 IR Early-CFI-6 32 32 32 0020-1 IR Early-CFI-7 16 32 32 0023-8 IR Early-CFI-8 16 16 16 0199-1 IR Early-CFI-3 16 16 32 ∆mucB IC Late-CFI-1 16 32 64 IC Late-CFI-2 4 16 128 IC Late-CFI-3 16 16 16 IR Late-CFI-4 32 32 64 IC Late-CFI-5 4 32 64 IR Late-CFI-6 16 16 16 IR Late-CFI-7 512 >512 >512 IR Late-CFI-8 256 >512 >512 IR Late-CFI-9 16 16 16 IR Late-CFI-10 16 16 32 IR= Iron-regulated alginate production; IC= Iron- constitutive alginate production

111 only slightly more resistant to gallium at 48 hours when compared to wild type

PAO1 (Table 5.2, Appendices E & F). Taken together, these results suggest that gallium may affect P. aeruginosa growth in a way that is independent from siderophore mediated iron uptake.

Does Iron Reverse the Bactericidal Activity of Gallium?

The addition of iron to gallium-containing medium has been shown to reverse the anti-bacterial effects of this metal [197]. Gallium has been proposed to interfere with bacterial iron homeostasis through pyoverdine signaling and the sigma factor PvdS [197]. Therefore, the effect of iron on gallium growth- dependent inhibition was investigated in all of the strains examined above

(Figure 5.2 and data not shown). Kaneko and coworkers demonstrated that the addition of iron in a concentration five times that of Ga3+ present in the medium would fully restore bacterial growth, overriding the anti-bacterial activity of gallium

[197]. To investigate whether the addition of iron had any affect on gallium susceptibility, increasing amounts of iron were added to inhibitory concentrations of Ga3+ in the media used for MIC studies. As expected, all of the strains tested recovered growth in the presence of at least five times the amount of iron as compared to gallium present in the medium (Figure 5.2 and data not shown). The addition of lesser amounts of iron was sufficient to increase gallium susceptibility however, five times the amount of iron was necessary to restore growth to normal levels (Figure 5.2 and data not shown).

112 Figure 5.2. Iron reversed gallium bactericidal activity. In PAO1, the addition 3+ of iron (FeCl3) to the growth media diminishes Ga growth-inhibitory effects.

The Effects of Gallium on Alginate Production

As shown in Chapter IV, iron has a significant influence on the expression

of alginate and the mucoid phenotype in P. aeruginosa. Since gallium is an iron

analogue, we were curious as to whether gallium would influence the production

of alginate in mucoid strains of P. aeruginosa. To test this, we grew PAO1,

∆mucA and Late CFI-1 (iron-constitutive alginate producer) in DTSB containing

various concentrations of gallium (Figure 5.3). We hypothesized that gallium

would decrease alginate production as did iron; yet, to our surprise, gallium did

not have any effect on the expression of alginate in any of the strains tested. The

bactericidal activity of gallium was observed at 25 µM and 100 µM Ga3+ though,

the production of the alginate exopolysaccharide was unaffected and the mucoid

113 strains continued to produce copious amounts of the alginate polymer. Taken together, these results raise even more questions about the mechanism of gallium anti-bacterial activity as well as the source of iron required to elicit an iron-regulated alginate response in P. aeruginosa (see Discussion). Based on our studies, it appears that gallium might act through mechanisms that do not involve siderophore production.

Figure 5.3. The effects of Gallium on alginate production. Wild type PAO1, ∆mucA and Late-CFI-1 were grown on DSTB supplemented with varying concentrations of Gallium. Uronic acid was measured after 24 hours of growth.

114 CHAPTER VI

DISCUSSION

Iron in Bacterial Virulence and Host-Pathogen Interactions

For more than three decades, the regulation of gene expression, virulence and pathogenesis by iron has been a constant theme in the study of bacterial infectious diseases. Iron, probably more so than any other metal has been shown to have profound effects on the expression of bacterial virulence determinants, host-pathogen interactions, and the ensuing outcomes of bacterial infectious diseases. Entire books, a surfeit of reviews and publications that are too numerous to cite have been written on the role of iron in both gram-positive and gram-negative virulence. There is no question that iron is an important, possibly the most important, micronutrient and cofactor for biological processes for nearly every living organism, particularly prokaryotes. The extent of iron regulation in bacteria is broad, including the regulation of global regulators (e.g. Fur), toxin production (e.g. ToxA, Shiga toxin, diphtheria toxin), adherence and invasion, oxidative stress, intracellular survival, acid tolerance, biofilm development, and metabolism. This widespread regulation by iron occurs not just in P. aeruginosa, but in most bacteria studied to date. It is not surprising that for many decades to come new investigations into the breadth and complexity of iron-regulation will continue to uncover novel details and control mechanisms of bacterial pathogenesis.

115 While free, unbound iron (Fe3+ or Fe2+) in uninfected mammalian hosts is ordinarily limiting for the rapid in vivo growth of bacterial pathogens, most prokaryotes have at their disposal multiple strategies (e.g. production of siderophores, uptake of foreign siderophores, heme uptake systems, degradation of host iron-binding proteins) that can overcome the scarcity of iron they might encounter upon colonization of a eukaryotic host [99, 240-242]. Yet, an abnormal increase in the levels of free iron in sera or tissues of mammalian hosts, can lead to rapidly fulminating infections [58, 69, 71, 75, 122]. Patients with elevated iron levels may experience exacerbated or chronic infections, whereas iron deficiency often leads to more moderate disease [70, 71, 122, 243-246]. Host-mediated iron sequestration from pathogens causes a dramatic shift in the expression of bacterial virulence factors and iron-acquisition proteins. This natural struggle between host and pathogen for this precious commodity is at the center of understanding bacterial growth and survival within the host environment, since during infection pathogens rely solely on their host for nutrients.

During an infectious disease, the levels of free iron can vary from one that is extremely restrictive, to one where free iron, or a readily available source of iron (e.g. heme), may become more accessible as a consequence of infection- related pathology (hemorrhage, inflammation, proteolysis of ferritin, lactoferrin or transferrin) [69, 71, 75, 240]. For instance, the levels of free iron in the airway mucus (sputa) of CF patients during acute exacerbations are known to fluctuate between very low, growth-limiting levels (<1 µM), to more than 100 times such concentrations (>100 µM) [71, 72, 75]. Notably however, such low levels of free

116 iron (<1 µM) are those that are typically responsible for an increase of the in vitro expression of iron-regulated genes encoding virulence determinants (e.g. exotoxins, proteases, siderophores) of P. aeruginosa and other pathogens, while higher levels (25 µM to >100 µM) most often suppress their expression, but favor increased growth rates and in certain cases, the eventual formation of particular types of biofilms [6, 11, 32, 84].

This variation in iron and nutrient availability leads to diverse and complex niches within the host that can have very different and profound effects on the microbial evolution in the human body. In the CF lung, microhabitats can develop due to the differences in resource partitioning, immune response and oxygen tension. P. aeruginosa strains isolated from the CF lung after years or decades of infection have often undergone severe genetic and phenotypic changes, with the goal of improving the ability to survive and proliferate in the host. Many of these phenotypic changes are observed repeatedly in isolates sampled from different patients and clinical settings. For example, CF isolates from chronic infection often display slow growth, antibiotic resistance, lack of motility, loss of quorum sensing, loss of iron acquisition (e.g. pyoverdine), changes in cell envelope and the over-production of alginate. The most common reported phenotypes in CF isolates are the appearance of the mucoid phenotype and the appearance of rough small-colony variants (RSCV), both of which are discussed in more detail below. However, it is important to note that when multiple isolates from the same sputum sample are evaluated, there is a high level of phenotypic diversity within that one sample. It is critical to keep this natural diversity in mind when

117 evaluating P. aeruginosa pathogenesis, even in the context of iron-regulation and biofilm development.

The Role of Iron in Biofilm Formation

Microorganisms in the natural world do not live as pure cultures as often exist in the laboratory. Instead, the microbial world is made up of complex ecosystems of organisms that live communally in aggregates, films, and mats known as biofilms. Bacterial biofilms have been studied since the advent of microbiology; Anton van Leeuwenhoek first described single cell organisms using his own handcrafted microscope to observe biofilm plaques scraped from his teeth. However, it was not until greater than three centuries later (1970s – 1980s) that scientists would begin to appreciate the importance of bacterial biofilms in both environmental and medical microbiology [247, 248]. Today it is known that an impressive number of chronic bacterial infections involve bacterial biofilms, which are not easily eradicated by conventional antibiotic therapies [4].

P. aeruginosa is a model organism for studying biofilm formation due to the organism’s ability to form several distinct types of biofilms and the occurrence of P. aeruginosa biofilms in chronic infection (see Introduction). The appearance of P. aeruginosa biofilms in infections, particularly in CF lung infections, causes devastating chronic disease that often results in the death of the patient. Biofilms constitute a protective mode of growth that allows for bacterial survival in harsh, hostile environments. In the context of the host environment, bacterial biofilms protect the bacteria from the immune response, oxidative stress, nutrient

118 scarcity, and antibiotics. Because of the biofilm’s protective properties, it comes as no surprise that P. aeruginosa can form multiple types of biofilms that are as diverse as the environments in which the organism may encounter (see

Introduction).

As discussed in the Introduction, Banin et al. demonstrated that the robust formation of a Psl-type biofilm on glass slides by P. aeruginosa requires either the biosynthesis of pyoverdine (an intact pvdA or pvdS gene) or the expression of its cognate receptor (an intact fpvA gene) [6]. The biofilms formed by mutants having disrupted pyoverdine biosynthesis or pyoverdine receptor formed flat, unstructured biofilms. The attenuation of biofilm formation by these mutants could be overcome by providing an alternative source of iron for P. aeruginosa (1

µM ferric dicitrate or 1.5 µM desferrioxamine) [6]. Furthermore, the formation of

Psl, surface grown biofilm by the wild type nonmucoid PAO1 parent was significantly inhibited by addition of lactoferrin (20 µg/mL) to the media. Taken together, these and other results from that study provided strong support for the view that the normal ability to form a robust Psl-type biofilm by P. aeruginosa can be affected merely by disruption of only one of its iron acquisition systems

(pyoverdine biosynthesis or uptake) or, by exposing it to an exogenous competitor for iron (lactoferrin). Further, these results suggested that it is probably the ability of P. aeruginosa to acquire sufficient levels of cytoplasmic iron, not necessarily a specific uptake system (i.e. pyoverdine, pychelin, or ferric citrate), that is crucial for the formation of structured Psl-type biofilms. It should be noted however, that the study by Banin et al. did not directly examine,

119 quantitatively, the impact of iron levels on the actual production of a biofilm- associated exopolysaccharide (Psl, Pel). Importantly, the study by Banin et al. was the first comprehensive study on how iron levels and iron acquisition might affect the formation of P. aeruginosa biofilms and led to the paradigm that iron

(within limits) is required for biofilm formation. However, it is wrong to assume that all types of biofilms produced by P. aeruginosa require iron or iron acquisition. In fact, the requirement of iron in Pel and alginate biofilm formation has only been loosely examined at best, and there are multiple lines of evidence that suggest these particular types of biofilms, particularly alginate biofilms, may not require iron. In light of this, the underlying basis for this dissertation was to examine the role of iron in biofilm formation, particularly alginate biofilms and mucoidy in P. aeruginosa.

PA2384 in Biofilm Formation

We began our studies by investigating a highly iron-regulated gene

(PA2384) that had putative functional implications involving rhamnolipids and quorum sensing (see Chapter III). Rhamnolipids, quorum sensing and iron (see introduction for a review on iron and P. aeruginosa biofilms) have all been linked to Pel- and Psl-type biofilm formation in P. aeruginosa. Therefore, we had some compelling reasons that warranted our further investigation into the structure and function of this gene. Before our investigation, very little was known about

PA2384, except that it was iron-regulated and that a mutant of PA2384

120 potentially affected the expression of numerous genes under iron limitation including rhamnolipid biosynthesis and quorum sensing [84, 217].

Therefore, as described in Chapter III, we investigated PA2384 and its putative iron-regulated function in P. aeruginosa biofilm formation. We first examined the amino acid sequence of PA2384 using bioinformatics to try and understand the function of this protein. Analysis of the amino acid sequence revealed that PA2384 is 34% similar to the P. aeruginosa Fur protein. Moreover, analysis of the secondary structure of PA2384 protein

(www.sbg.cio.ic.ac.uk/phyre/) indicated that it contains a winged-helix structure, similar to the DNA-binding region of P. aeruginosa Fur protein. Additionally, cross-linking experiments, along with MALDI-TOF, with purified PA2384 suggested that this protein forms a dimer in its native state. Our extensive analysis of PA2384’s predicted structure led us to hypothesize that this protein might act as a repressor similar to the Fur protein, but since it appeared to lack the iron-regulatory region of Fur, we hypothesized that this putative repressor may function in a way that is distinct from Fur. Many bacteria not only have a Fur protein, but also possess several Fur homologues that each have distinct functions in the cell [249]. These homologues are recognized as members of a superfamily of transcriptional regulators responsive to zinc (Zur), manganese

(Mur), peroxides (PerR), and nickel (Nur) [250, 251]. While many of these Fur- type regulators are responsive to very different stresses or metals, all of the members of this family act as transcriptional repressors, with very similar N- terminal, DNA-binding regions.

121 Microarray analysis, corroborated by qRT-PCR, revealed that deletion of

PA2384 (∆PA2384) resulted in a significant increase in the expression of multiple genes in the alginate biosynthetic operon (e.g. algD, algK, algG) as compared to wild type PAO1 grown under iron-limitation. Furthermore, we demonstrated that purified PA2384 bound to several regions of the algD promoter region. These results suggested direct regulation of the transcription of algD by PA2384.

However, when the mutant PA2384 was grown on DTSA plates under iron- limitation, there was only a 2.5 fold increase in the amount of detectable alginate exopolysaccharide produced. The regulation of alginate production and export is extremely complex, involving transcriptional regulation, post-transcriptional regulation and post-translational modifications. Yet, these results were puzzling since the drastic increase (~10 fold in our studies) in algD typically corresponds with an increase in the production of the alginate exopolysaccharide.

To further understand the role of PA2384 in alginate production and mucoidy, a detailed review of past microarray data generated in the Vasil lab along with a comprehensive literature review were undertaken, revealing many interesting pieces of data that are noted in Table 3.3. The most noteworthy observations were i) PA2384 is highly expressed when exposed to epithelial cells and ii) PA2384 is highly expressed under aerobic conditions and repressed in microaerobic and anaerobic conditions. The latter finding, suggests that PA2384 functions in highly oxygenated environment. While, at the present time we do not fully understand PA2384’s role in alginate production and mucoidy in P.

122 aeruginosa, there are some basic hypotheses that can be garnered from our studies of this interesting gene.

To our knowledge, negative regulators that directly bind to and repress algD transcription have, thus far, not been identified. Yet, the production of alginate is an intense, high-energy process that diverts oxaloacetate from the

TCA cycle towards alginate biosynthesis through gluconeogenesis (see

Introduction and [37]. A study conducted in 2003 by Wozniak et al. demonstrated that wild type nonmucoid PAO1 and PA14 biofilms do not contain significant amounts of alginate (uronic acid) [128]. Their results clearly show that alginate biosynthetic genes are not expressed during the formation of nonmucoid biofilms

(Pel or Psl biofilms). These results were novel since it had been, and still is, wrongfully assumed that wild type P. aeruginosa biofilms contain substantial amounts of the alginate exopolysaccharide, as commonly seen in isolates from

CF patients. Further, the essential regulators required for the transcription of algD and the production of alginate (see Introduction) have additional functions in

P. aeruginosa, separate from alginate production, which makes it unlikely that all alginate regulators are completely inactive in wild type nonmucoid strains. For example, a recent study by Wood and Ohman identified numerous (293) genes in the AlgU regulon [252]. While most of the genes identified were associated with cell wall stress and induced algD transcription, many were involved in maintaining cell envelope homeostasis under nonstressed conditions in planktonic cells that do not express high levels of the algD transcript. Moreover the essential alginate regulator, algR, has many functions in P. aeruginosa

123 outside of alginate production. AlgR has been shown to act as a repressor of the

Rhl quorum sensing system as well as an activator of Type IV Pilus biosynthesis, indicating the importance of this regulator outside of alginate production [28, 34,

253, 254]. Therefore, it is not unlikely that PA2384 acts as a direct repressor of alginate under conditions where this exopolysaccharide would not normally be expressed. One of the caveats to our study was that our alginate assays were performed on the ∆PA2384 mutant grown on a solid surface whereas the microarray and qRT-PCR analyses were performed on liquid cultures (Figure

3.9). It is possible that PA2384 acts as a repressor of alginate in liquid media, or within a pellicle (Pel biofilm), and not on a solid surface as we measured. Further supporting this theory, a transcriptome analysis was conducted on cells grown in a pellicle (Pel-type biofilm) [195]. This study not only reported a 13 fold increase in the expression of PA2384 in a pellicle, but also reported the increased expression of pyoverdine and pyochelin as well as a decrease in expression of genes involved in anaerobic respiration. These results suggest that the Pel-type biofilm, which is known to form at the air-liquid interface, is highly oxygenated and iron-limited. PA2384 is expressed under highly oxygenated, iron-limited conditions and seems to act as a repressor of algD transcription. Therefore, it is possible that PA2384 acts as a repressor of alginate within Pel-type biofilms, where alginate would normally not be expressed. More studies, of course, would need to be performed to test this hypothesis.

124 Iron and Alginate Biofilm Formation

Our studies on PA2384 led to a much broader question about iron and alginate production. While the effects of iron on Psl-type biofilms had been examined, there were minimal studies on how iron influenced the production of alginate. In Chapter IV, we examined whether biologically relevant levels of elemental iron, as those described in the Introduction and in Chapter IV (≤5 µM to ≥100 µM), might influence the production of alginate and mucoidy by P. aeruginosa. In the present study we did not specifically assess whether iron had any control over nonmucoid biofilm formation on a glass slide in a flow-through system, as in the Banin et al. report [6]. However, it could be argued that that the striking mucoid presentation on a solid agar surface as shown in the Figure 4.1 could, at least in some manner, be akin to an alginate-type biofilm as they exist in mucus-plugged CF airways.

Due to the fact that wild type PAO1 is not normally mucoid and does not express notable levels of alginate, it was first necessary to construct PAO1 muc mutants (mucA, mucB, mucC or mucD) and then determine whether biologically relevant iron levels had any affect on the production of alginate and the mucoidy phenotype. Additionally, we introduced insertion mutations into mucA and mucB in a variety of nonmucoid, non-CF isolates from diverse geographic regions in order to determine whether other freshly constructed muc mutants showed the same iron-regulated phenotypes as did the mucA, mucB, mucC and mucD isogenic variants of PAO1. Further, a non-mucoid isolate from a CF patient

(Early-CFI) was converted to mucoidy by introduction of an insertion mutation in

125 mucB. All of these newly constructed muc mutants produced increased levels of alginate under iron-limiting (≤5 µM) growth and significantly reduced levels of alginate under iron-replete (≥10 µM) growth. At this point we were curious as to whether or not mucoid CF isolates regulated alginate production in response to iron. An assortment of Early and Late CF isolates was examined for production of alginate and mucoidy under both iron-limiting and iron-replete levels. In contrast to what was observed with newly constructed muc mutants, a significant proportion (56%) of the Late-CFI’s had, at some time in the past, lost iron- regulated control over the production of alginate and mucoidy. That is, these isolates presently express constitutively increased levels of alginate and were mucoid irrespective of the biologically significant levels of iron in which they are grown.

These observations posed additional salient questions about iron, the expression of alginate and the overtly mucoid phenotype, (i) how does iron initially exhibit its tight control over alginate production, and (ii) how can this regulatory influence be ostensibly nullified during the course of chronic pulmonary disease? To address these questions an approach similar to that of

Banin et al. was taken. The Banin et al. study revealed that mutation in one or more iron acquisition systems in wild type PAO1, led to a significant reduction in the organism’s ability to produce robust, structured Psl-type biofilms [6]. We considered whether alteration of one of more known iron regulatory factors (Fur and PvdS) or iron acquisition systems (siderophore biosynthesis or receptors) could specifically affect the iron-regulated expression of alginate production and

126 mucoidy, as they did in the production of a Psl-type biofilm in the Banin study.

Our research demonstrated in a variety of ways that, in contrast to the Psl-type biofilm studies, abrogation of the pyoverdine biosynthesis or alternative iron acquisition mechanisms, pyochelin biosynthesis failed to diminish the increased expression of alginate under iron-limitation. Additionally, some mutants with more than one mutated key iron acquisition system actually exhibited an enhanced production of extracellular alginate even under iron-replete conditions, as was observed with more than half (56%) of the mucoid Late-CFI examined.

Although we do not presently know whether any of the iron-constitutive,

Late-CFI display such a phenotype as a consequence of mutations that affect the same iron uptake systems that we altered in PAO1 muc mutants, several of these Late-CFI do exhibit significantly reduced levels of siderophores as revealed by their very weak CAS reactions (see Appendix E, Late-CFI 1, 2, 12 and 16). In support of our observation, a significant number of P. aeruginosa isolates from

CF patients are unable to produce detectable levels of pyoverdine [255]. On the other hand, not all of the Late-CFI with an iron-constitutive mucoidy phenotype

(see Appendix E, Late-CFI 3, 4, 5, 11, and 13) exhibited significant decreases in their CAS reactions. Perhaps, as suggested in the review by Poole and McKay entitled Iron acquisition and its control in Pseudomonas aeruginosa: many roads lead to Rome, there are multiple ways for this pathogen to strategically acquire this nutrient [76]. Further, a very recent report by Konings et al. suggests that there are various processes that might lead to a diminished capacity of P.

127 aeruginosa to acquire iron, especially after years or decades in the stressful, highly oxidative, mutagenic environment of the CF airways [256].

At this time, it has not yet been discerned whether alginate expression is transcriptionally controlled by specific iron-responsive regulatory factors (e.g. sigma factors, two-component regulators, sRNAs), or whether generally low cytoplasmic iron levels from a variety of causes (e.g. auxotrophy, reduced quorum sensing), generating further nonspecific stresses, result in enhanced alginate biosynthesis. Nevertheless, it was shown that none of the iron- constitutive alginate producers thus far evaluated carry mutations that could alter the protein level or function of the Fur protein, the master regulator of iron homeostasis. Likewise, a possible role for the Fur-regulated sigma factor, PvdS, was eliminated. But, these observations do not exclude the possibility that a mutation may exist in an unidentified Fur-regulated gene that controls the expression of one or more alginate biosynthesis genes (e.g. algD, algR). A mutation in a Fur-regulated gene could in some cases be the cause of the iron- constitutive mucoidy phenotype seen in many of the Late-CFI. Along these lines, it is of particular interest that recently two genes of Pseudomonas syringae

(closely related plant pathogen) known to be associated with alginate biosynthesis (algJ and algF) were found to be Fur-regulated [106]. While in P. aeruginosa, AlgJ and AlgF seem to be involved in the acetylation of alginate rather than in its biosynthesis, these genes should be further examined [150].

Ultimately, it is also possible that some of the CF isolates (Late-CFI) identified in our study that express increased levels of extracellular alginate, both

128 under iron-limiting and iron-replete conditions, may do so because the mutations responsible for increased alginate expression are not in the muc genes

(mucABCD) but in other alginate regulatory genes that induce the mucoid phenotype [257, 258]. We report a novel link between iron and alginate production, and in contrast to Psl-type biofilms that require iron acquisition for the formation of structured mushroom-like biofilms, we demonstrate that iron- limitation is required for the expression of alginate-type biofilms.

The Inverse Relationship of P. aeruginosa Biofilms in Response to Iron

Since our observations discussed above and in Chapter IV, indicate that alginate production and mucoidy are negatively regulated in response to iron, which is in stark contrast to what is known about the iron requirements of wild type PAO1 biofilm formation, we were curious as to whether mucoid strains of P. aeruginosa retained their ability to form Psl- or Pel-type biofilms under iron- replete conditions. As presented in Chapter IV, we grew wild type PAO1, ∆mucA and ∆mucB in a microtiter plate assay under both iron-limitation and iron-replete conditions to assess these strain’s ability to form wild type nonmucoid biofilms.

As expected, the wild type PAO1, ∆mucA and ∆mucB mutants formed robust biofilms in the microtiter plate assay only under iron-replete conditions (Figure

4.8). Our results provide a very significant finding where, in contrast to Pel and

Psl biofilms that require iron (within limits), alginate biofilm formation is prevented with levels of iron ≥10 µM. Additionally, we show that mucoidy strains, while unable to form alginate biofilms in iron-replete conditions, retain their ability to

129 form Pel and Psl biofilms in a microtiter plate. Overall, these findings suggest that alginate and Pel/Psl biofilms may be reciprocally regulated in response to iron.

It is not yet known what role, if any, Pel- or Psl-type biofilms play throughout the course of infection. However, based on the current state of knowledge we might propose that Pel and/or Psl biofilms might predominate during colonization and early-stage infection (acute), whereas alginate-type biofilms predominate in late-stage infection (chronic). There is some evidence to suggest that Psl and alginate biofilms are both regulated by common transcriptional regulators. AmrZ acts as an activator of algD, while at the same time repressing pslA [259]. RpoS has also been shown to regulate Psl biofilm formation and in a study conducted by Suh et al. alginate production was decreased by 70% in a mucoid FRD1 strain containing an RpoS mutation [135,

136, 260]. Further, there are many overlapping factors that influence all three

(alginate, Pel and Psl) of the types of biofilms formed by P. aeruginosa including motility, quorum sensing, c-di-GMP, iron, carbon sources, and metabolic precursors. It seems likely that the type of biofilm that P. aeruginosa forms might largely be determined by the stressors encountered by the bacterium, including nutrient and iron availability. It is also likely that while one type of biofilm may become dominant, as in the case of mucoidy in CF chronic infection, mixed- populations would remain to exist that form Pel- or Psl-type biofilms. For example, a study published in Nature in 2002 demonstrated that rough small- colony variants (RSCV) that exhibit a rough colony phenotype (nonmucoid) are common variants that exist in the lungs of CF patients [261]. These RSCVs when

130 compared to wild type were highly antibiotic resistant, exhibited increased attachment to glass and polyvinylchloride plastic (PVC), and formed better biofilms in a flow-cell (Pel or Psl biofilms). In our examination of late-stage CF isolates, we too identified nonmucoid variants, although the prevalence was low

(~23%). Either way, it is not difficult to imagine that each of these different types of biofilms can be produced in response to very different environmental stresses that the bacterium encounters. And while many different factors can elicit biofilm formation, iron is emerging as a key determinant in the type of biofilm produced by this pathogen.

Iron Homeostasis as a Target For Therapeutic Intervention in Non-CF and

CF Related Infections

Iron chelators, iron acquisition mechanisms, and iron-dependent metabolic processes, particularly in the context of biofilm formation, have in the past few years been increasingly considered to be attractive targets for therapeutic interventions in treating P. aeruginosa non-CF (e.g. sepsis, eye infections) and

CF infections [2, 236, 262]. In some cases, they are even the focus of current ongoing clinical trials.

One of these approaches is based on the therapeutic administration of

FDA-approved Gallium (Ga3+), a Fe3+ analog, see Chapter V (clinical trials.gov #

NCT01093521) [197, 236]. Although Ga3+ is not an iron chelator, it is thought to interfere, in a manner that is not completely understood, with how iron is processed by P. aeruginosa [235]. Even though Ga3+ can kill P. aeruginosa in

131 vitro, including in mucoid strains and those growing in biofilms, there is a significant proportion of organisms that show resistance to this agent. In one

Ga3+-related study, Banin et al. investigated the use of Ga3+ linked to desferrioxamine (DFO-Ga3+), which is a siderophore that can be taken up by receptors of P. aeruginosa [236]. These investigators examined the efficacy of this metallo-complex (DFO-Ga3+) against planktonic and biofilm grown wild type

PAO1, as well as in an eye infection model with this non-mucoid strain. This effort was based on the rationale that DFO-Ga3+ would be more efficiently transported into the cell than Gallium alone. It was also thought that because P. aeruginosa has DFO receptors the likelihood of resistance developing to DFO-

Ga3+ might be assuaged. However, as indicated in their report, they did not measure DFO-Ga3+ transport in P. aeruginosa, and they opined that:

“experimental details about how this molecule interacts with P. aeruginosa, to kill it, await further studies”. They further stated: “there is also little known about transport of the gallium ion into bacterial cells”. In this regard, it should be noted there are some extremely pertinent features of Gallium, relating to its potential effect on bacteria, that are clearly worthy of consideration in terms of its presently unknown effect on iron homeostasis, especially pertaining to P. aeruginosa.

There are some key basic properties of Gallium that, very likely, would not allow it to affect all the iron scavenging systems that P. aeruginosa possesses for maintaining iron homeostasis. For example, unlike iron, Ga3+ cannot be reduced and therefore it cannot substitute for ferrous iron (Fe2+), which P. aeruginosa can acquire via its ferrous (Feo) uptake system [84]. What is more, Gallium cannot

132 interfere with heme uptake by P. aeruginosa, because iron is incorporated into heme in the ferrous form.

Another approach with the aim to interfere with biofilm formation was to combine the use of FDA-approved iron chelators deferoxamine (DFO) and deferasirox (DSX, Exjade®), with tobramycin, a primary antibiotic used to treat

CF lung infections [113, 229]. While these investigators examined P. aeruginosa biofilm formation on human CF airway epithelial cells, they used wild type PAO1 as their model organism. They reported that tobramycin and DFO together did not reduce biofilm formation to a greater extent than tobramycin alone. However, tobramycin and DSX together significantly decreased the ability of wild type

PAO1 to form biofilms on human CF airway epithelial cells as compared with tobramycin alone. A major caveat to this study however, is that, as mentioned above, P. aeruginosa has a receptor for DFO and it is not yet known whether

PAO1 has a receptor for DSX.

Consequently, thus far, the only studies that have investigated the use of iron chelators or other compounds that may interfere with iron acquisition mechanisms in order to affect biofilm formation have used wild type P. aeruginosa strains, that while capable of biofilm formation, do not express high levels of extracellular alginate, nor exhibit a notable mucoid phenotype. Biofilm formation has been a frequent focus for the development of therapeutics and whenever examined, the ability of P. aeruginosa to gather iron has been the intended target.

133 How Our Results May Impact the Development of Current and Future Iron-

Targeted Therapies

It would be predicted, based on the observations presented in this dissertation, that diminution of iron acquisition in P. aeruginosa would actually lead to increased alginate production and mucoidy. As discussed above, this approach to limit iron availability is often the focus of mainstream treatment strategies for P. aeruginosa infection. Such a strategy could be potentially detrimental in some cases, especially in the context of P. aeruginosa CF lung infections. Treatment efforts focused on reducing iron, by iron chelation or other agents, could very well lead to constitutive expression of alginate and mucoidy, which is closely associated with increased chronic P. aeruginosa infections. On the other hand, administering iron supplements to patients to increase local iron levels could also prove to be problematic, since the formation of Psl biofilms requires some level of iron. This begs the question, is iron homeostasis really the best target for the development of therapeutics? P. aeruginosa has clearly evolved sophisticated protection barriers to deal with environmental stresses including iron-limitation and iron-abundance.

A recent publication in PNAS describes the use of the FDA-approved drug flucytosine to suppress P. aeruginosa infection [262]. This report states that this

FDA-approved drug is an iron-uptake inhibitor, acting by inhibiting the expression of the iron-starvation sigma-factor PvdS and pyoverdine biosynthesis. The investigators demonstrated the clinical promise of this drug using an in vivo mouse model for P. aeruginosa infection. At first glance, this study seems

134 promising and the authors suggest moving forward with clinical trials. However, these investigators did not infect the mice with a mucoid strain of P. aeruginosa, nor did they look at the inhibitory properties of this drug on alginate biofilms. The mice were infected with wild type PAO1, which is only capable of producing Pel or Psl biofilms. For that reason, the realistic application of this drug has yet to be seen, since the vast majority of CF isolates are alginate producing, mucoid isolates. In spite of all this, this drug could be useful at very early-stages of infection, before P. aeruginosa mucoid isolates accumulate in the CF lung.

However, an important question is whether limiting the pathogen’s ability to take up iron during early infection would add selective pressure to the organism that would encourage it to genotypically switch to an alginate producing, mucoid phenotype. More research will need to be conducted to assess the feasibility of drugs targeting iron homeostasis.

Hypotheses for Future Investigations

This dissertation touches upon several very novel findings in regard to iron and biofilm formation, as well as brings up numerous interesting questions about how these very different biofilms produced by this organism are coordinately regulated. There are several key hypotheses that can be drawn from this work, some of which have been touched upon throughout this dissertation, which would be relevant to future investigations. However, in this summary I will point out only a few that would yield significant insight as to how these biofilms might be regulated.

135 First, based on our analysis, its seems that PA2384 acts as a repressor of alginate production in highly oxygenated, iron-limited conditions similar to those required for Pel biofilm formation. It would be interesting to further characterize

PA2384 in the context of Pel biofilms. A few very simple experiments could be conducted to further understand this correlation between PA2384 and biofilm formation i) measure the amount of alginate produced in liquid culture during Pel biofilm formation in wild type PAO1 and ∆PA2384, ii) conduct microtiter or flow- cell biofilm experiments with a ∆PA2384 mutant and wild type PAO1 under iron limitation to understand how PA2384 functions in a wild type biofilm, and iii) perform carbohydrate analyses on the exopolysaccharides produced in the

∆PA2384 mutant as compared to wild type PAO1.

Second, early after P. aeruginosa genotypically converts to the mucoid phenotype, we have shown that available levels of iron in the organism’s surroundings regulate the production of alginate. However, throughout the course of CF infection, after years or decades of colonization in the CF lung, the pathogen loses its ability to control alginate production in response to iron. It would be worthwhile to understand the mechanism by which iron controls the levels of alginate produced by this opportunist. While microarray analysis didn’t produce any straightforward regulatory candidates that would be involved in directly controlling alginate in response to iron, it might be more valuable to directly investigate the Fur regulon for candidate Fur-regulated genes. Fur is the global regulator of iron-homeostasis. If a Fur-regulated gene activated alginate production under iron-limitation, it would be logical that alginate production would

136 be repressed under iron-replete conditions. Therefore, chromatin immunoprecipitation followed by sequencing (ChIP-seq) would be a very straightforward approach that could be conducted to assess the role of Fur in alginate production. An alternative approach for identifying novel alginate regulatory genes would be to conduct whole genome sequencing on several clinical CF isolates that regulate alginate production and compare the sequences to several clinical CF isolates that have lost their ability to regulate alginate in response to iron. This approach would allow for the identification of candidate genes containing mutations that might be involved in the iron-regulated expression of alginate.

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254. Belete, B., H. Lu, and D.J. Wozniak, Pseudomonas aeruginosa AlgR regulates type IV pilus biosynthesis by activating transcription of the fimU- pilVWXY1Y2E operon. J Bacteriol, 2008. 190(6): p. 2023-30.

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159 APPENDIX A

RESULTS OF THE ∆PA2384 18 HOUR GeneChip® MICROARRAY

Iron-Limitation Increases PA Number Fold Description change ig_3051348 2 Intergenic region between PA2729 and PA2730, _3051956 3051348-3051956, (+) strand PA0062 2 PA0062 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA0372 1 PA0372 /DEF=probable zinc protease /FUNCTION=Putative enzymes PA0399 1 PA0399 /DEF=cystathionine beta-synthase /FUNCTION=Amino acid biosynthesis and metabolism PA0569 2 PA0569 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA0572 4 PA0572 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA0931 1 PA0931 /DEF=siderophore receptor protein /FUNCTION=Transport of small molecules PA0970_tol 1 PA0970 /GENE=tolR /DEF=TolR protein R /FUNCTION=Transport of small molecules PA1202 6 PA1202 /DEF=probable hydrolase /FUNCTION=Putative enzymes PA1203 3 PA1203 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA1204 2 PA1204 /DEF=conserved hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA1205 2 PA1205 /DEF=conserved hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA1347 3 PA1347 /DEF=probable transcriptional regulator /FUNCTION=Transcriptional regulators PA1363 2 PA1363 /DEF=probable sigma-70 factor, ECF subfamily /FUNCTION=Transcriptional regulators PA1364 2 PA1364 /DEF=probable transmembrane sensor /FUNCTION=Transcriptional regulators; Membrane proteins PA1471 1 PA1471 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA1493_cys 2 PA1493 /GENE=cysP /DEF=sulfate-binding protein of P ABC transporter /FUNCTION=Transport of small molecules PA1494 2 PA1494 /DEF=conserved hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA1800_tig 2 PA1800 /GENE=tig /DEF=trigger factor /FUNCTION=Cell division; Chaperones & heat shock proteins PA2067 1 PA2067 /DEF=probable hydrolase /FUNCTION=Putative enzymes PA2068 2 PA2068 /DEF=probable MFS transporter

160 /FUNCTION=Membrane proteins; Transport of small molecules PA2281 1 PA2281 /DEF=probable transcriptional regulator /FUNCTION=Transcriptional regulators PA2377 2 PA2377 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA2398_fpv 1 PA2398 /GENE=fpvA /DEF=ferripyoverdine receptor A /FUNCTION=Transport of small molecules PA2413 1 PA2413 /DEF=probable class III aminotransferase /FUNCTION=Putative enzymes PA2430 2 PA2430 /DEF=conserved hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA2432 10 PA2432 /DEF=probable transcriptional regulator /FUNCTION=Transcriptional regulators PA2452 1 PA2452 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA2531 2 PA2531 /DEF=probable aminotransferase /FUNCTION=Amino acid biosynthesis and metabolism PA2588 1 PA2588 /DEF=probable transcriptional regulator /FUNCTION=Transcriptional regulators PA2667 1 PA2667 /DEF=conserved hypothetical protein /FUNCTION=Transcriptional regulators PA2688_pfe 2 PA2688 /GENE=pfeA /DEF=ferric enterobactin receptor A precursor PfeA /FUNCTION=Membrane proteins; Transport of small molecules PA2696 3 PA2696 /DEF=probable transcriptional regulator /FUNCTION=Transcriptional regulators PA2697 12 PA2697 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown; Membrane proteins PA2698 8 PA2698 /DEF=probable hydrolase /FUNCTION=Putative enzymes PA2699 2 PA2699 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA3148_wb 2 PA3148 /GENE=wbpI /DEF=probable UDP-N- pI acetylglucosamine 2-epimerase WbpI /FUNCTION=Cell wall / LPS / capsule; Putative enzymes PA3149_wb 2 PA3149 /GENE=wbpH /DEF=probable glycosyltransferase pH WbpH /FUNCTION=Cell wall / LPS / capsule; Putative enzymes PA3151_his 2 PA3151 /GENE=hisF2 /DEF=imidazoleglycerol-phosphate F2 synthase, cyclase subunit /FUNCTION=Amino acid biosynthesis and metabolism PA3540_alg 8 PA3540 /GENE=algD /DEF=GDP-mannose 6- D dehydrogenase AlgD /FUNCTION=Adaptation, protection; Cell wall / LPS / capsule; Secreted Factors (toxins, enzymes, alginate) PA3542 3 PA3542 /DEF=alginate biosynthesis protein Alg44 /FUNCTION=Adaptation, protection; Cell wall / LPS / capsule; Secreted Factors (toxins, enzymes, alginate) PA3545_alg 10 PA3545 /GENE=algG /DEF=alginate-c5-mannuronan- G epimerase AlgG /FUNCTION=Adaptation, protection; Cell wall / LPS / capsule; Secreted Factors (toxins, enzymes, 161 alginate) PA3547_alg 3 PA3547 /GENE=algL /DEF=poly(beta-d-mannuronate) L lyase precursor AlgL /FUNCTION=Adaptation, protection; Cell wall / LPS / capsule; Secreted Factors (toxins, enzymes, alginate) PA3548_alg 2 PA3548 /GENE=algI /DEF=alginate o-acetyltransferase I AlgI /FUNCTION=Adaptation, protection; Cell wall / LPS / capsule; Secreted Factors (toxins, enzymes, alginate) PA3550_alg 2 PA3550 /GENE=algF /DEF=alginate o-acetyltransferase F AlgF /FUNCTION=Adaptation, protection; Cell wall / LPS / capsule; Secreted Factors (toxins, enzymes, alginate) PA3551_alg 2 PA3551 /GENE=algA /DEF=phosphomannose isomerase A / guanosine 5 -diphospho-D-mannose pyrophosphorylase /FUNCTION=Adaptation, protection; Cell wall / LPS / capsule; Secreted Factors (toxins, enzymes, alginate) PA4142 2 PA4142 /DEF=probable secretion protein /FUNCTION=Protein secretion/export apparatus PA4143 2 PA4143 /DEF=probable toxin transporter /FUNCTION=Membrane proteins; Protein secretion/export apparatus PA4144 1 PA4144 /DEF=probable secretion protein /FUNCTION=Protein secretion/export apparatus PA4261_rpl 1 PA4261 /GENE=rplW /DEF=50S ribosomal protein L23 W /FUNCTION=Translation, post-translational modification, degradation PA4639 1 PA4639 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA4661 1 PA4661 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA5182 1 PA5182 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown; Membrane proteins PA5183 2 PA5183 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown; Membrane proteins PA5192_pck 2 PA5192 /GENE=pckA /DEF=phosphoenolpyruvate A carboxykinase /FUNCTION=Carbon compound catabolism; Energy metabolism PA5460 1 PA5460 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA5568 1 PA5568 /DEF=conserved hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown; Membrane proteins Decreases PA0208_md 2 PA0208 /GENE=mdcA /DEF=malonate decarboxylase cA alpha subunit /FUNCTION=Carbon compound catabolism PA0209 2 PA0209 /DEF=conserved hypothetical protein /FUNCTION=Putative enzymes PA1137 5 PA1139 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA1180_pho 2 PA1180 /GENE=phoQ /DEF=two-component sensor PhoQ Q /FUNCTION=Two-component regulatory systems PA2003_bdh 2 PA2003 /GENE=bdhA /DEF=3-hydroxybutyrate 162 A dehydrogenase /FUNCTION=Carbon compound catabolism PA2007_mai 2 PA2007 /GENE=maiA /DEF=maleylacetoacetate A_i isomerase /FUNCTION=Carbon compound catabolism PA2008_fah 2 PA2008 /GENE=fahA /DEF=fumarylacetoacetase A /FUNCTION=Carbon compound catabolism PA2383 4 PA2383 /DEF=probable transcriptional regulator /FUNCTION=Transcriptional regulators PA2384 233 PA2384 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA2820 2 PA2820 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA3278 2 PA3278 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown; Membrane proteins PA3431 2 PA3431 /DEF=conserved hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown; Membrane proteins PA3721 2 PA3721 /DEF=probable transcriptional regulator /FUNCTION=Transcriptional regulators PA4226_pch 2 PA4226 /GENE=pchE /DEF=dihydroaeruginoic acid E synthetase /FUNCTION=Transport of small molecules; Secreted Factors (toxins, enzymes, alginate)

Iron-Replete Increases ig_721556_ 1 Intergenic region between PA0701 and PA0702, 721556- 727608_s_a 727608, (+) strand t ig_727608_ 2 Intergenic region between PA0701 and PA0702, 721556- 721556_s_a 727608, (-) strand t PA1202_at 12 PA1202 /DEF=probable hydrolase /FUNCTION=Putative enzymes PA1203_at 3 PA1203 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA1205_at 1 PA1205 /DEF=conserved hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA1557_at 1 PA1557 /DEF=probable cytochrome oxidase subunit (cbb3-type) /FUNCTION=Energy metabolism PA2380_at 2 PA2380 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA2382_lldA 7 PA2382 /GENE=lldA /DEF=L-lactate dehydrogenase _at /FUNCTION=Energy metabolism PA2383_at 6 PA2383 /DEF=probable transcriptional regulator /FUNCTION=Transcriptional regulators PA2385_at 6 PA2385 /DEF=probable acylase /FUNCTION=Putative enzymes PA2388_at 2 PA2388 /DEF=probable transmembrane sensor /FUNCTION=Membrane proteins; Transcriptional regulators PA2389_at 2 PA2389 /DEF=conserved hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown 163 PA2390_at 2 PA2390 /DEF=probable ATP-binding/permease fusion ABC transporter /FUNCTION=Membrane proteins; Transport of small molecules PA2432_at 9 PA2432 /DEF=probable transcriptional regulator /FUNCTION=Transcriptional regulators PA2696_at 2 PA2696 /DEF=probable transcriptional regulator /FUNCTION=Transcriptional regulators PA2698_at 4 PA2698 /DEF=probable hydrolase /FUNCTION=Putative enzymes PA2699_at 2 PA2699 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown PA4525_pilA 2 PA4525 /GENE=pilA /DEF=type 4 fimbrial precursor PilA _at /FUNCTION=Motility & Attachment PA4661_at 1 PA4661 /DEF=hypothetical protein /FUNCTION=Hypothetical, unclassified, unknown Decreases None

164 APPENDIX B

MALDI-TOF RESULTS OF PURIFIED PA2384 II 0 6.6E+ 60001.0 - · 41000.6 64514] 32000.2 -· 11680.8, = (mlz) Mass #1[BP 91 Spec 23379 Dimer L 22999.8 Voyager -· . . 22 . 13999.4 70 . 6 11903 11681.45 Monomer 6 . 10 9573 64 . 5846 10 - 40 20 70 90 30 60 60 80 100 t: iii E ~ ~ .2! " "

165 APPENDIX C

RESULTS FROM 24 HOUR GeneChip® MICROARRAY

Global gene expression changes in PAO1, ∆mucAi and ∆mucDi under iron limitation.

Increased Gene Expression (0 µM vs. 100 µM FeCl3)

PA Number Gene PAO1 ∆mucAi ∆mucDi Description/Function Fold Fold Fold Change Change Change ig_1064555_1063544 3 2 NC Intergenic region between PA1003 and PA1004, 1063544-1064555, (-) strand ig_1427453_1428080 9 9 9 Intergenic region between PA1372 and PA1373, 1427453-1428080, (+) strand ig_1428080_1427453 2 2 5 Intergenic region between PA1372 and PA1373, 1427453-1428080, (-) strand ig_2116265_2117030 NC 3 5 Intergenic region between PA1978 and PA1979, 2116265-2117030, (+) strand ig_224101_223454 NC 6 NC Intergenic region between PA0263 and PA0264, 223454- 224101, (-) strand ig_2282480_2281578 2 3 2 Intergenic region between PA2127 and PA2128, 2281578-2282480, (-) strand ig_2721530_2722174 3 5 3 Intergenic region between PA2439 and PA2440, 2721530-2722174, (+) strand ig_2722174_2721530 23 4 23 Intergenic region between PA2439 and PA2440, 2721530-2722174, (-) strand ig_2902217_2901558 NC 4 NC Intergenic region between PA2570 and PA2571, 2901558-2902217, (-) strand ig_2928538_2927920 NC 5 NC Intergenic region between PA2698 and PA2699, 2927920-2928538, (-) strand ig_3051348_3051956 5 NC NC Intergenic region between PA2729 and PA2730, 3051348-3051956, (+) strand ig_3206914_3206252 3 2 NC Intergenic region between PA2910 and PA2911, 3206252-3206914, (-) strand ig_3514564_3515415 Dec 4 NC Intergenic region between PA3141 and PA3142, 3514564-3515415, (+) strand ig_3677080_3677719 NC 3 Dec Intergenic region between PA3305 and PA3306, 3677080-3677719, (+) strand ig_3705160_3705889 2 3 NC Intergenic region between PA3463 and PA3464, 3705160-3705889, (+) strand ig_3961922_3962824 2 14 3 Intergenic region between PA3781 and PA3782, 3961922-3962824, (+) strand ig_4509996_4510970 NC 3 2 Intergenic region between PA4080 and PA4081, 4509996-4510970, (+) strand ig_5086695_5087407 2 NC 5 Intergenic region between PA4581 and PA4582, 5086695-5087407, (+) strand ig_518083_517462 NC 3 NC Intergenic region between PA0484 and PA0485, 517462- 518083, (-) strand ig_545644_546334 NC 2 8 Intergenic region between PA0574 and PA0575, 545644- 546334, (+) strand ig_546334_545644 3 NC Dec Intergenic region between PA0574 and PA0575, 545644- 546334, (-) strand ig_774416_773696 NC 3 NC Intergenic region between PA0714 and PA0715, 773696- 774416, (-) strand ig_785174_785969 2 NC 3 Intergenic region between PA0716 and PA0717, 785174- 785969, (+) strand PA0030 NC 4 NC Hypothetical protein; ABC-type proline/glycine betaine transport systems, periplasmic components

PA0058 3 NC NC Hypothetical protein; Predicted protein-disulfide isomerase; thioredoxin (TRX)-like superfamily

PA0059 osmC 2 4 2 Osmotically inducible protein

PA0062 2 4 2 Hypothetical protein; homologue Azotobacter

PA0105 coxB 4 4 2 Cytochrome c oxidase, subunit II; Energy metabolism

166 PA0106 coxA 3 4 NC Cytochrome c oxidase, subunit I; Energy metabolism

PA0107 2 3 NC Conserved hypothetical protein; probable cytochrome c assembly protein; Oxidative phosphorylation

PA0108 coIII 2 5 NC Cytochrome c oxidase, subunit III; Energy metabolism

PA0111 3 5 2 Hypothetical protein; homologue Azotobacter

PA0135 3 NC 5 Hypothetical protein; putative toxin

PA0136 Dec 3 NC Probable ATP-binding component of ABC transporter

PA0149 3 2 4 Probable sigma-70 factor

PA0166 Dec 2 3 Probable transporter; Xanthine/uracil permeases; homologue Azotobacter PA0173 5 2 NC Probable methylesterase; two-component system, chemotaxis family, response regulator CheB

PA0184 3 2 NC Probable ATP-binding component of ABC transporter; nitrate/sulfonate/bicarbonate transport

PA0187 3 2 2 Hypothetical protein; Probable taurine catabolism dioxygenase PA0188 3 3 2 Hypothetical protein; Membrane proteins; Predicted permeases; auxin efflux carrier (AEC) family protein

PA0191 5 NC NC Probable transcriptional regulator; LysR family transcriptional regulator; homologue Azotobacter

PA0198 exbB 3 2 2 Transport protein

PA0199 exbD 5 2 3 Transport protein

PA0208 mdcA 3 2 2 Malonate decarboxylase alpha subunit; Carbon compound catabolism PA0210 mdcC 4 2 NC Malonate decarboxylase delta subunit; Carbon compound catabolism PA0213 3 NC 2 Hypothetical protein; malonate decarboxylase holo-[acyl- carrier-protein] synthase PA0216 NC 6 2 Probable transporter; malonate transporter

PA0229 pcaT 2 3 NC Dicarboxylic acid transporter; metabolite-proton symporter PA0244 NC 3 2 Hypothetical protein; Putative enzymes; shikimate 5- dehydrogenase; homologue Azotobacter

PA0251 2 NC 4 Hypothetical protein; Suppressor of fused protein

PA0252 NC 2 7 Hypothetical protein; putative membrane protein

PA0266 gabT 3 2 2 4-aminobutyrate aminotransferase; Amino acid biosynthesis and metabolism; Carbon compound catabolism; Central intermediary metabolism PA0270 3 2 2 Hypothetical protein; Cupin 2 conserved barrel domain protein PA0271 3 2 2 Hypothetical protein; antibiotic biosynthesis monooxygenase PA0283 sbp 3 NC NC Sulfate-binding protein precursor; sulfate transport system PA0284 4 NC 2 Hypothetical protein; homologue Azotobacter

PA0323 4 5 3 Probable binding protein component of ABC transporter

PA0346 2 4 4 Hypothetical protein; outer membrane protein; homologue Acetobacter PA0355 pfpL 2 4 2 Intracellular protease; cysteine-type peptidase activity

PA0433 3 2 3 Hypothetical protein

PA0435 NC 3 NC Hypothetical protein

PA0441 dht 3 NC Dec Dihydropyrimidinase; Nucleotide biosynthesis and metabolism

167 PA0446 3 3 3 Conserved hypothetical protein; oxalate catabolic process

PA0447 gcdH 3 4 3 Glutaryl-CoA dehydrogenase; Amino acid biosynthesis and metabolism; Carbon compound catabolism; Fatty acid and phospholipid metabolism

PA0452 2 4 2 Probable stomatin-like protein; Membrane proteins

PA0471 fiuR 7 3 6 Probable transmembrane sensor; Membrane proteins; Transcriptional regulators; Two-component regulatory systems; Fe2+-dicitrate sensor, PA0472 fiuI 5 5 6 Probable sigma-70 factor; Transcriptional regulators

PA0474 NC NC 3 Hypothetical protein; phenylacetate catabolic process

PA0475 NC NC 3 Probable transcriptional regulator

PA0476 NC NC 3 Probable permease; cytosine/purines uracil thiamine allantoin permease PA0480 3 NC NC Probable hydrolase

PA0499 NC 3 Dec Probable pili assembly chaperone; Motility & Attachment; Chaperones & heat shock proteins

PA0505 3 2 2 Hypothetical protein

PA0509 nirN NC 3 NC Probable c-type cytochrome; Energy metabolism; Biosynthesis of cofactors, prosthetic groups and carriers

PA0510 NC 3 NC Probable uroporphyrin-III c-methyltransferase; Energy metabolism; Biosynthesis of cofactors, prosthetic groups and carriers

PA0511 nirJ NC 4 NC Heme d1 biosynthesis protein; Biosynthesis of cofactors, prosthetic groups and carriers; Energy metabolism

PA0512 NC 3 2 Conserved hypothetical protein; Energy metabolism; Hypothetical, unclassified, unknown; Biosynthesis of cofactors, prosthetic groups and carriers

PA0513 NC 3 NC Probable transcriptional regulator; Energy metabolism; Transcriptional regulators; Biosynthesis of cofactors, prosthetic groups and carriers

PA0514 nirL NC 4 2 Heme d1 biosynthesis protein; Energy metabolism; Biosynthesis of cofactors, prosthetic groups and carriers

PA0515 NC 4 NC Probable transcriptional regulator; Energy metabolism; Biosynthesis of cofactors, prosthetic groups and carriers

PA0516 nirF NC 4 NC Heme d1 biosynthesis protein; Biosynthesis of cofactors, prosthetic groups and carriers; Energy metabolism

PA0517 nirC NC 5 NC Probable c-type cytochrome precursor; Biosynthesis of cofactors, prosthetic groups and carriers; Energy metabolism

PA0518 nirM NC 6 2 Cytochrome c-551 precursor; Biosynthesis of cofactors, prosthetic groups and carriers; Energy metabolism

PA0519 nirS NC 7 3 Nitrite reductase precursor; Energy metabolism

PA0520 nirQ NC 3 NC Regulatory protein; Central intermediary metabolism; Energy metabolism PA0523 norC NC 11 2 Nitric-oxide reductase subunit C; Energy metabolism

PA0524 norB 2 11 2 Nitric-oxide reductase subunit B; Energy metabolism

PA0525 4 7 2 Probable dinitrification protein; Energy metabolism

PA0529 2 7 3 Conserved hypothetical protein; MOSC domain is predicted to be a sulfur-carrier domain

168 PA0530 NC NC 3 Probable class III pyridoxal phosphate-dependent aminotransferase; Putative enzymes

PA0531 3 2 14 Probable glutamine amidotransferase; Putative enzymes

PA0534 pauB1 10 7 8 Conserved hypothetical protein; FAD-dependent oxidoreductase PA0535 2 3 3 Probable transcriptional regulator

PA0539 3 NC 2 Hypothetical protein

PA0557 6 NC 2 Hypothetical protein; Ketosteroid isomerase homolog

PA0572 NC 2 3 Hypothetical protein

PA0672 hemO 10 9 17 Heme oxygenase

PA0676 vreR 3 3 6 Probable transmembrane sensor; Virulence regulator; Fe2+-dicitrate sensor PA0679 3 2 Dec Hypothetical protein; General secretion system

PA0680 hxcV Dec 3 2 Probable type II secretion system protein; Protein secretion/export apparatus; HxcV putative pseudopilin

PA0684 NC 3 3 Probable type II secretion system protein; Protein secretion/export apparatus PA0692 NC 3 3 Hypothetical protein; Hemolysin activation/secretion protein; OMP85 family outer membrane protein

PA0697 2 3 NC Hypothetical protein

PA0702 NC 4 NC Hypothetical protein; fatty acid hydroxylase

PA0707 toxR 8 6 14 Transcriptional regulator

PA0724 Dec 2 3 Probable coat protein A of bacteriophage Pf1

PA0737 2 5 NC Hypothetical protein

PA0738 2 2 3 Conserved hypothetical protein; Predicted membrane protein PA0748 NC 2 3 Still frameshift probable transcriptional regulator

PA0752 NC 3 2 Conserved hypothetical protein; tripartite tricarboxylate transporter; homologue Azotobacter

PA0755 opdH NC 5 3 Probable porin; outer membrane porin

PA0763 mucA NC 3 NC Anti-sigma factor MucA

PA0792 prpD 5 3 5 Propionate catabolic protein

PA0818 2 3 5 Hypothetical protein

PA0828 NC 6 NC Probable transcriptional regulator

PA0844 plcH NC NC 3 Hemolytic phospholipase C precursor

PA0865 hpd NC 3 4 4-hydroxyphenylpyruvate dioxygenase

PA0870 phhC NC 3 3 Aromatic amino acid aminotransferase

PA0871 phhB 2 3 3 Pterin-4-alpha-carbinolamine dehydratase

PA0872 phhA 3 5 5 Phenylalanine-4-hydroxylase

PA0873 phhR 2 NC 3 Transcriptional regulator

PA0883 NC 3 NC Probable acyl-CoA lyase beta chain; Putative enzymes

PA0886 NC NC 3 Probable C4-dicarboxylate transporter; Transport of small molecules PA0894 NC 3 2 Hypothetical protein

PA0929 5 NC 3 Two-component response regulator; phosphate regulon; homologue Azotobacter PA0931 pirA 3 3 3 Siderophore receptor protein; ferric enterobactin receptor

PA0939 2 3 6 Hypothetical protein; transcriptional antiterminator

169 PA0941 2 3 3 Hypothetical protein; small redox-active disulfide protein 2

PA0957 3 NC NC Hypothetical protein; HGG motif-containing thioesterase, possibly involved in aromatic compounds catabolism

PA0984 2 11 2 Colicin immunity protein; Membrane proteins; Secreted Factors (toxins, enzymes, alginate)

PA0985 pyoS5 2 4 2 Probable colicin-like toxin; Membrane proteins; Secreted Factors (toxins, enzymes, alginate)

PA0987 NC NC 3 Conserved hypothetical protein; Transposase and inactivated derivatives PA0993 cupC2 NC 4 Dec Probable pili assembly chaperone

PA0994 cupC3 NC 3 NC Probable fimbrial biogenesis usher protein

PA0996 pqsA NC 4 2 Probable coenzyme A ligase; Putative enzymes

PA0997 pqsB 2 3 NC Hypothetical protein; HHQ/PQS synthesis

PA0998 pqsC NC 4 2 Putative enzymes; HHW/PQS synthesis

PA0999 pqsD NC 3 2 3-oxoacyl-[acyl-carrier-protein] synthase III; Fatty acid and phospholipid metabolism; HHQ/PQS synthesis

PA1000 pqsE NC 3 NC Hypothetical, unclassified, unknown; HHQ/PQS synthesis

PA1001 phnA NC 4 2 Anthranilate synthase component I

PA1002 phnB NC 3 2 Anthranilate synthase component II

PA1106 3 2 2 Hypothetical protein; Predicted redox protein, regulator of disulfide bond formation PA1130 3 2 2 Hypothetical protein

PA1131 3 NC 2 Probable MFS transporter; Antibiotic resistance and susceptibility; Membrane proteins; Transport of small molecules

PA1134 7 5 8 Hypothetical protein; Predicted thiol-disulfide oxidoreductase PA1135 3 2 2 Conserved hypothetical protein; Putative intracellular protease/amidase PA1136 3 NC NC Probable transcriptional regulator, LuxR family

PA1137 2 4 2 Probable oxidoreductase

PA1148 toxA 3 4 26 Exotoxin A

PA1153 NC 3 NC Hypothetical protein; Prophage antirepressor

PA1190 3 4 2 Conserved hypothetical protein; putative inner membrane protein PA1210 3 2 NC Conserved hypothetical protein; Pirin-related protein

PA1212 NC 3 NC Probable MFS transporter; Na+/melibiose symporter

PA1228 Dec 4 NC Hypothetical protein

PA1230 NC Dec 5 Hypothetical protein

PA1231 NC 3 NC Conserved hypothetical protein; Multidrug resistance efflux pump PA1239 3 NC 3 Hypothetical protein; putative secretion protein

PA1245 aprX 16 7 13 Apr type I secretion system

PA1246 aprD 16 6 7 Alkaline protease secretion protein; Secreted Factors (toxins, enzymes, alginate); Protein secretion/export apparatus

PA1247 aprE 5 3 4 Alkaline protease secretion protein; Protein secretion/export apparatus; Secreted Factors (toxins, enzymes, alginate)

PA1248 aprF 4 3 3 Alkaline protease secretion protein; Protein secretion/export apparatus; Secreted Factors (toxins, enzymes, alginate)

170 PA1249 aprA 6 5 6 Alkaline metalloproteinase precursor; Secreted Factors (toxins, enzymes, alginate) PA1250 aprL 3 NC 2 Alkaline proteinase inhibitor; Secreted Factors (toxins, enzymes, alginate) PA1254 NC 3 NC Probable dihydrodipicolinate synthetase; Putative enzymes PA1256 4 6 3 Probable ATP-binding component of ABC transporter; Transport of small molecules PA1300 33 15 33 Probable sigma-70 factor

PA1301 11 10 22 Probable transmembrane sensor

PA1302 2 3 3 Probable heme utilization protein precursor; Membrane proteins; Transport of small molecules

PA1310 phnW Dec 3 NC 2-aminoethylphosphonate:pyruvate aminotransferase; phosphonotase pathway PA1313 2 NC 4 Probable MFS transporter; Membrane proteins; Transport of small molecules PA1317 cyoA 11 11 17 Cytochrome o ubiquinol oxidase subunit II

PA1318 cyoB 8 12 11 Cytochrome o ubiquinol oxidase subunit I

PA1319 cyoC 7 6 12 Cytochrome o ubiquinol oxidase subunit III

PA1320 cyoD 24 13 23 Cytochrome o ubiquinol oxidase subunit IV

PA1321 cyoE NC 6 3 Cytochrome o ubiquinol oxidase protein

PA1323 NC 4 NC Hypothetical protein; putative stress response protein

PA1324 NC 4 NC Hypothetical protein; putative lipoprotein

PA1344 3 2 NC Probable short-chain dehydrogenase

PA1347 Dec NC 3 Probable transcriptional regulator; LuxR family transcriptional regulatory protein PA1349 3 2 2 Conserved hypothetical protein

PA1355 3 3 3 Hypothetical protein; thiamine protein

PA1356 3 2 2 Hypothetical protein; putative glycosyl hydrolase

PA1386 4 NC NC Probable ATP-binding component of ABC transporter

PA1393 cysC NC 3 4 Adenosine 5 -phosphosulfate (APS) kinase; Central intermediary metabolism; Nucleotide biosynthesis and metabolism; Amino acid biosynthesis and metabolism

PA1403 3 2 6 Probable transcriptional regulator

PA1404 2 3 2 Hypothetical protein

PA1406 3 NC NC Hypothetical protein; putative nucleotidyltransferase

PA1409 aphA NC 5 3 Acetylpolyamine aminohydrolase; Histone deacetylase family PA1419 2 3 NC Probable transporter; cytosine/purine, uracil, thiamine, allantoin transporter PA1426 3 2 NC Hypothetical protein;

PA1434 2 3 NC Hypothetical protein; Predicted transglutaminase-like cysteine proteinase PA1471 3 4 3 Hypothetical protein

PA1494 2 5 2 Conserved hypothetical protein; putative mucoidy inhibitor PA1498 pykF NC 4 NC Pyruvate kinase I

PA1502 gcl Dec 3 NC Glyoxylate carboligase

PA1523 xdhB 2 3 NC Xanthine dehydrogenase

PA1524 xdhA NC 3 NC Xanthine dehydrogenase

PA1540 4 2 2 Conserved hypothetical protein; multidrug efflux protein

PA1541 11 3 5 Probable drug efflux transporter

171 PA1559 2 2 5 Hypothetical protein; Nucleoside-diphosphate-sugar epimerases; GDP-mannose 4,6 dehydratase

PA1560 NC NC 3 Hypothetical protein

PA1569 NC NC 3 Probable MFS transporter

PA1592 NC 3 NC Hypothetical protein; cytochrome c class I

PA1596 htpG NC NC 3 Heat shock protein

PA1635 kdpC NC 2 6 Potassium-transporting ATPase, C chain

PA1672 11 3 3 Hypothetical protein; Predicted enzyme related to lactoylglutathione lyase PA1696 pscO NC 3 NC Translocation protein in type III secretion

PA1707 pcrH NC 3 2 Regulatory protein PcrH

PA1708 popB NC 3 2 Translocator protein

PA1709 popD NC 3 2 Translocator protein

PA1730 4 3 2 Conserved hypothetical protein

PA1732 3 3 2 Conserved hypothetical protein; Transglutaminase-like enzymes, putative cysteine proteases

PA1738 NC 3 2 Probable transcriptional regulator; LysR family

PA1739 3 NC NC Probable oxidoreductase

PA1742 3 4 3 Probable amidotransferase

PA1744 NC 3 2 Hypothetical protein

PA1762 3 4 NC Hypothetical protein; Predicted double-glycine peptidase

PA1779 3 2 NC Assimilatory nitrate reductase

PA1784 3 3 2 Hypothetical protein; putative alginate lyase

PA1785 Dec NC 4 Conserved hypothetical protein; similar to Azotobacter vinelandii NasT protein; Assimilatory nitrate reductase antiterminator

PA1848 5 Dec NC Probable MFS transporter

PA1852 3 2 NC Hypothetical protein

PA1856 NC Dec 4 Probable cytochrome oxidase subunit; Oxidative phosphorylation PA1870 3 2 2 Hypothetical protein

PA1871 lasA 4 3 2 Protease

PA1872 3 2 NC Hypothetical protein; putative protease similar to LasA

PA1887 5 NC NC Hypothetical protein

PA1907 NC 3 2 Hypothetical protein; Predicted dienelactone hydrolase; Platelet-activating factor acetylhydrolase

PA1908 2 3 4 Probable MFS transporter

PA1909 NC 4 3 Hypothetical protein; Uncharacterized iron-regulated membrane protein PA1910 femA 2 2 3 Probable tonB-dependent receptor protein; ferric- mycobactin receptor PA1911 femR 12 18 15 Probable transmembrane sensor; Fe2+-dicitrate sensor, membrane component PA1912 femI 11 18 12 Probable sigma-70 factor

PA1916 NC 3 4 Probable amino acid permease

PA1930 3 3 2 Probable chemotaxis transducer

PA1934 NC 4 8 Hypothetical protein

PA1951 3 NC NC Hypothetical protein; Putative MetA-pathway of phenol degradation 172 PA1955 Dec 5 NC Hypothetical protein

PA1962 azoR2 2 NC 8 Conserved hypothetical protein; FMN-dependent NADH- azoreductase 2; Acyl carrier protein phosphodiesterase

PA1974 2 NC 3 Hypothetical protein; outer membrane protein (Porin)

PA1984 exaC NC 3 NC Probable aldehyde dehydrogenase

PA1985 pqqA 3 NC 2 Pyrroloquinoline quinone biosynthesis protein A

PA1999 dhcA 5 7 9 Probable CoA transferase, subunit A

PA2000 dhcB 5 6 7 Probable CoA transferase, subunit B

PA2001 atoB 5 6 6 Acetyl-CoA acetyltransferase

PA2002 2 3 3 Conserved hypothetical protein; Short chain fatty acids transporter PA2007 maiA 2 2 3 Maleylacetoacetate isomerase

PA2008 fahA NC 2 3 Fumarylacetoacetase

PA2009 hmgA NC 2 3 Homogentisate 1,2-dioxygenase

PA2011 liuE NC NC 3 Hydroxymethylglutaryl-CoA lyase

PA2012 liuD NC NC 3 Probable acyl-CoA carboxylase alpha chain

PA2013 liuC NC NC 3 Probable enoyl-CoA hydratase/isomerase

PA2014 liuB NC NC 3 Probable acyl-CoA carboxyltransferase beta chain

PA2015 liuA NC 2 3 Probable acyl-CoA dehydrogenase

PA2016 liuR 2 2 4 Probable transcriptional regulator; Cu(I)-responsive transcriptional regulator PA2021 2 3 2 Hypothetical protein

PA2024 2 3 2 Probable ring-cleaving dioxygenase; putative lyase

PA2030 4 2 2 Hypothetical protein

PA2031 4 2 2 Hypothetical protein

PA2032 3 NC 4 Probable transcriptional regulator

PA2033 44 38 52 Hypothetical protein; Siderophore-interacting protein

PA2034 49 20 119 Hypothetical protein; magnesium protoporphyrin O- methyltransferase PA2046 NC 6 NC Hypothetical protein

PA2050 NC NC 3 Probable sigma-70 factor

PA2054 cynR 2 NC 3 Transcriptional regulator

PA2061 NC NC 7 Probable ATP-binding component of ABC transporter

PA2062 7 3 3 Probable pyridoxal-phosphate dependent enzyme

PA2070 NC 2 4 Hypothetical protein; Outer membrane receptor proteins, TonB-dependent receptor PA2071 fusA2 3 3 3 Elongation factor G

PA2073 NC 3 NC Probable transporter (membrane subunit)

PA2079 Dec NC 4 Probable amino acid permease

PA2080 kynU NC 2 3 Kynureninase

PA2081 kynB NC 2 3 Kynurenine formamidase

PA2083 3 4 2 Probable ring-hydroxylating dioxygenase subunit

PA2084 2 6 NC Probable asparagine synthetase

PA2085 2 7 NC Probable ring-hydroxylating dioxygenase small subunit; anthranilate 1,2-dioxygenase, small subunit

PA2087 4 6 NC Hypothetical protein

173 PA2088 19 11 NC Hypothetical protein; Trans-aconitate methyltransferase

PA2090 3 3 2 Hypothetical protein; alkanesulfonate monooxygenase, FMNH[263]-dependent PA2091 5 6 NC Hypothetical protein; oxalate/formate antiporter family transporter PA2092 8 10 3 Probable MFS transporter

PA2094 NC 3 NC Probable transmembrane sensor; Fe2+-dicitrate sensor, membrane component PA2095 5 4 4 Hypothetical protein

PA2108 3 NC 2 Probable decarboxylase; pyruvate oxidase activity

PA2109 NC 3 2 Hypothetical, unclassified, unknown; Probable Sialic acid transporter PA2110 NC 3 2 Hypothetical, unclassified, unknown; Probable Sialic acid transporter PA2111 NC 3 2 Hypothetical, unclassified, unknown; Probable Sialic acid transporter PA2112 NC 3 2 Hypothetical, unclassified, unknown; Probable Sialic acid transporter PA2113 NC 3 2 Probable porin; Probable Sialic acid transporter

PA2114 2 3 3 Probable MFS transporter; Probable Sialic acid transporter PA2116 8 8 6 Conserved hypothetical protein

PA2124 NC NC 4 Probable dehydrogenase; Choline dehydrogenase

PA2125 NC 4 NC Probable aldehyde dehydrogenase; glycine betaine aldehyde dehydrogenase PA2131 cupA4 NC 4 NC Fimbrial subunit CupA4

PA2132 cupA5 NC 3 NC Probable pili assembly chaperone; chaperone CupA5

PA2134 4 4 3 Predicted outer membrane protein

PA2135 6 3 NC Probable transporter; sodium:hydrogen antiporter activity

PA2136 6 NC 3 Hypothetical protein

PA2137 3 3 NC Hypothetical protein; Putative histidine kinase

PA2138 3 3 NC Probable ATP-dependent DNA ligase; DNA replication, recombination, modification and repair

PA2140 5 4 3 Probable metallothionein

PA2141 6 3 9 Hypothetical protein; Competence/damage-inducible protein PA2142 5 3 2 Probable short-chain dehydrogenase; fatty acid biosynthetic PA2143 3 21 2 Hypothetical protein

PA2144 glgP 3 3 2 Glycogen phosphorylase; glycogen degradation

PA2145 4 2 3 Hypothetical protein; BCCT family betaine/carnitine/choline transporter PA2146 5 7 5 Conserved hypothetical protein; stress-induced protein

PA2147 katE NC 13 3 Catalase HPII

PA2148 NC 5 NC Conserved hypothetical protein; Mg(2+) transporter

PA2150 NC 4 2 Conserved hypothetical protein; Non-homologous end- joining; Ku-homolog [Replication, recombination, and repair] PA2151 3 7 3 Conserved hypothetical protein; glycosyl hydrolase; homologue Azotobacter PA2152 3 3 2 Probable trehalose synthase

PA2153 glgB 2 5 2 1,4-alpha-glucan branching enzyme

PA2155 2 3 6 Probable phospholipase; phosphatidylserine/phosphatidylglycerophosphate/cardioli pin synthase

174 PA2156 3 2 6 Conserved hypothetical protein; Metal-dependent hydrolase; Endonuclease/exonuclease/phosphatase

PA2157 3 2 2 Hypothetical protein

PA2158 3 16 2 Probable alcohol dehydrogenase (Zn-dependent)

PA2159 3 9 2 Conserved hypothetical protein; inosine-5'- monophosphate dehydrogenase PA2160 2 3 2 Probable glycosyl hydrolase

PA2161 2 9 2 Hypothetical protein

PA2162 2 4 2 Probable glycosyl hydrolase; Maltooligosyl trehalose synthase PA2163 2 6 2 4-alpha-glucanotransferase

PA2164 3 3 2 Probable glycosyl hydrolase; malto-oligosyltrehalose trehalohydrolase PA2165 3 6 2 Probable glycogen synthase; glycogen/starch synthases, ADP-glucose type PA2167 3 4 4 Hypothetical protein; biotin carboxylase; homologue Azotobacter PA2169 3 4 3 Hypothetical protein; Ferritin-like superfamily of diiron- containing four-helix-bundle proteins

PA2170 3 3 2 Hypothetical protein; phospholipase_D-nuclease family

PA2171 2 10 NC Hypothetical protein; iron-sulfur cluster repair di-iron protein PA2172 2 4 2 Hypothetical protein; glutamyl aminopeptidase

PA2173 3 4 2 Hypothetical protein; cyclase/dehydrase

PA2174 4 3 2 Hypothetical protein

PA2176 2 20 5 Hypothetical protein; Putative NADH-flavin reductase

PA2178 4 7 2 Hypothetical protein; putative membrane protein

PA2179 3 3 4 Hypothetical protein; Methylase of polypeptide chain release factors; HemK-like protein methyltransferase-like protein

PA2180 3 2 2 Hypothetical protein

PA2182 4 2 3 Hypothetical protein; adenylate/guanylate cyclase family protein PA2183 3 11 2 Hypothetical protein; Predicted membrane protein

PA2184 4 4 3 Conserved hypothetical protein; Ferritin-like superfamily of diiron-containing four-helix-bundle proteins

PA2185 katN 4 2 4 Mn-containing catalase

PA2187 4 4 2 Predicted small integral membrane protein; Low affinity iron permease PA2188 4 2 2 Threonine dehydrogenase and related Zn-dependent dehydrogenases PA2190 3 8 2 general stress protein

PA2191 exoY NC 3 2 Adenylate cyclase, toxin

PA2192 5 6 3 Conserved hypothetical protein; inosine-5'- monophosphate dehydrogenase PA2198 7 Dec Dec Hypothetical protein; Antibiotic biosynthesis monooxygenase protein; homologue Azotobacter

PA2208 3 Dec 2 Hypothetical protein

PA2211 4 4 NC Conserved hypothetical protein; Predicted metal- dependent hydrolase of the TIM-barrel fold

PA2221 NC 4 NC Conserved hypothetical protein; integrase catalytic subunit

175 PA2243 pslM NC 5 NC Hypothetical protein; Succinate dehydrogenase/fumarate reductase, flavoprotein subunit

PA2244 pslN 2 3 NC Hypothetical protein; Topoisomerase IB

PA2251 2 2 3 Hypothetical protein; putative transcriptional regulator

PA2255 pvcB NC 3 3 Pyoverdine biosynthesis protein; paerucumarin biosynthesis protein PA2259 ptxS 4 3 7 Transcriptional regulator

PA2260 5 4 8 Possible Sugar Phosphate Isomerase

PA2261 15 3 17 Probable 2-ketogluconate kinase; Pentose phosphate pathway PA2262 3 NC 4 Probable 2-ketogluconate transporter; Sugar phosphate permease PA2263 5 NC 7 Probable 2-hydroxyacid dehydrogenase; Lactate dehydrogenase and related dehydrogenases

PA2275 3 NC NC Probable alcohol dehydrogenase (Zn-dependent)

PA2292 NC 4 NC Hypothetical protein

PA2295 NC 4 NC Probable permease of ABC transporter

PA2296 2 3 2 Hypothetical protein; ABC-type nitrate/sulfonate/bicarbonate transport systems, periplasmic components

PA2300 chiC 4 2 3 chitinase activity

PA2303 ambD 3 2 NC Hypothetical protein; L-2-amino-4-methoxy-trans-3- butenoic acid (AMB) biosynthesis; Probable taurine catabolism dioxygenase

PA2307 3 2 3 Probable permease of ABC transporter

PA2308 3 3 3 Probable ATP-binding component of ABC transporter

PA2309 3 4 2 Hypothetical protein; ABC-type nitrate/sulfonate/bicarbonate transport systems, periplasmic components

PA2310 4 3 2 Hypothetical protein; Probable taurine catabolism dioxygenase PA2314 pvdH NC NC 3 Probable MFS transporter

PA2320 gntR 6 3 7 Transcriptional regulator; gluconate repressor

PA2321 14 17 28 Gluconokinase

PA2322 9 4 26 Gluconate permease

PA2323 2 2 3 Probable glyceraldehyde-3-phosphate dehydrogenase

PA2324 4 4 6 Hypothetical protein; Acyl-CoA dehydrogenases

PA2334 5 NC NC Probable transcriptional regulator; homologue Azotobacter PA2356 msuD 3 NC NC Methanesulfonate sulfonatase

PA2357 msuE NC NC 3 NADH-dependent FMN reductase

PA2358 NC 2 3 Hypothetical protein

PA2359 5 NC 2 Probable transcriptional regulator; sigma-54 dependent transcriptional regulator PA2362 3 NC NC Hypothetical protein; Type VI protein secretion system component VasF; homologue Azotobacter

PA2366 puuD 3 2 NC Conserved hypothetical protein; Predicted component of the type VI protein secretion system; homologue Azotobacter PA2369 5 2 NC Hypothetical protein; Type VI protein secretion system component VasA; homologue Azotobacter

PA2375 3 2 2 Hypothetical protein

176 PA2377 15 23 71 Hypothetical protein; ABC-type Fe3+ transport system, periplasmic component PA2382 lldA 3 7 2 L-lactate dehydrogenase

PA2383 4 4 4 Probable transcriptional regulator; putative glycine cleavage system transcriptional activator

PA2384 14 12 24 Hypothetical protein; Fe2+/Zn2+ uptake regulation proteins; homologue ferric uptake regulation protein

PA2385 pvdQ 7 9 15 Probable acylase; 3-oxo-C12-homoserine lactone acylase PA2386 pvdA 13 11 27 L-ornithine N5-oxygenase

PA2389 pvdR 7 7 10 Conserved hypothetical protein; RND family efflux transporter, MFP subunit PA2390 pvdT 4 5 7 Probable ATP-binding/permease fusion ABC transporter

PA2391 opmQ 5 5 6 Probable outer membrane protein; porin activity

PA2392 pvdP 16 12 23 Hypothetical protein; Pyoverdine synthesis

PA2393 14 11 23 Probable dipeptidase precursor

PA2394 pvdN 10 10 20 Probable aminotransferase; Selenocysteine lyase/Cysteine desulfurase PA2395 pvdO 9 10 16 Hypothetical protein; kinase

PA2396 pvdF 7 9 15 Hypothetical protein; pyoverdine synthetase F

PA2397 pvdE 13 11 20 Pyoverdine biosynthesis protein

PA2398 fpvA 6 5 8 Ferripyoverdine receptor

PA2399 pvdD 14 14 25 Pyoverdine synthetase D

PA2400 pvdJ 14 13 28 Probable non-ribosomal peptide synthetase

PA2401 13 15 31 Probable non-ribosomal peptide synthetase

PA2402 13 15 30 Probable non-ribosomal peptide synthetase

PA2405 2 2 3 Hypothetical protein

PA2411 14 11 31 Probable thioesterase

PA2412 11 8 20 Conserved hypothetical protein

PA2413 pvdH 23 23 33 Probable class III aminotransferase

PA2414 5 4 3 L-sorbosone dehydrogenase

PA2415 5 3 3 Hypothetical protein; Predicted membrane protein

PA2418 3 NC NC Hypothetical protein; Pirin-related protein

PA2419 2 NC 4 Probable hydrolase; isochorismatase hydrolase

PA2424 pvdL 22 14 28 Probable non-ribosomal peptide synthetase

PA2425 pvdG 13 9 39 Probable thioesterase

PA2426 pvdS 16 12 36 Sigma factor

PA2427 30 24 39 Hypothetical protein; putative acetylase

PA2430 NC 5 NC Conserved hypothetical protein; Multidrug resistance efflux pump PA2433 2 3 2 Hypothetical protein

PA2441 4 3 NC Hypothetical protein

PA2442 gcvT2 2 3 2 Glycine cleavage system protein T2

PA2443 sdaA 2 4 2 L-serine dehydratase

PA2444 glyA2 6 34 2 Serine hydroxymethyltransferase

PA2445 gcvP2 3 10 2 Glycine cleavage system protein P2

PA2446 gcvH2 3 9 3 Glycine cleavage system protein H2

177 PA2451 6 5 6 Hypothetical protein; Enterochelin esterase and related enzymes PA2452 12 16 31 Hypothetical protein

PA2466 foxA 2 3 3 Probable TonB-dependent receptor

PA2467 foxR 3 2 3 Probable transmembrane sensor; Anti-sigma factor; Fe2+-dicitrate sensor PA2468 foxI 5 5 6 Probable sigma-70 factor

PA2476 dsbG 2 NC 3 Thiol:disulfide interchange protein; Protein-disulfide isomerase PA2478 3 2 3 Probable thiol:disulfide interchange protein; oxidation- reduction process PA2479 2 2 3 Probable two-component response regulator; QseB homologue PA2483 3 3 3 Conserved hypothetical protein; oxidoreductase activity

PA2485 2 8 5 Hypothetical protein

PA2486 2 6 3 Hypothetical protein

PA2504 3 2 2 Hypothetical protein

PA2506 2 2 3 Hypothetical protein

PA2531 11 7 9 Probable aminotransferase

PA2552 NC NC 3 Probable acyl-CoA dehydrogenase

PA2553 NC 2 3 Probable acyl-CoA thiolase; Fatty acid metabolism

PA2554 NC 2 3 Probable short-chain dehydrogenase; Fatty acid metabolism PA2555 NC 2 3 Probable AMP-binding enzyme; AMP-dependent synthetase/ligase PA2562 2 3 2 Hypothetical protein; Putative inner membrane protein

PA2566 3 2 NC Conserved hypothetical protein; NAD(FAD)-dependent dehydrogenases PA2570 lecA 2 3 2 PA-I galactophilic lectin

PA2594 6 NC 2 Conserved hypothetical protein; aliphatic sulfonates family ABC transporter, periplasmic ligand-binding protein

PA2598 3 NC NC Hypothetical protein; alkanesulfonate monooxygenase, FMNH[263]-dependent PA2665 fhpR NC 3 2 Probable transcriptional regulator; Transcriptional activator of flavohemoglobin PA2673 NC NC 4 Probable type II secretion system protein

PA2676 NC NC 3 Probable type II secretion system protein

PA2679 NC NC 3 Hypothetical protein; putative methyltransferase

PA2681 NC NC 3 Probable transcriptional regulator

PA2686 pfeR 2 2 4 Two-component response regulator

PA2687 pfeS 2 3 3 Two-component sensor

PA2688 pfeA 6 NC 7 Ferric enterobactin receptor precursor

PA2694 NC 4 NC Probable thioredoxin

PA2700 opdB NC 3 NC Probable porin

PA2746 7 3 4 Hypothetical protein

PA2747 3 2 NC Hypothetical protein

PA2751 NC 3 NC Conserved hypothetical protein; Ribonuclease BN protein, homologue Azotobacter PA2761 NC 2 4 Hypothetical protein; membrane associated protein

PA2764 3 2 NC Hypothetical protein

PA2776 pauB3 5 2 4 Conserved hypothetical protein; FAD-dependent oxidoreductase PA2779 NC 3 NC Hypothetical protein

178 PA2786 6 3 5 Hypothetical protein; diguanylate cyclase (GGDEF) domain protein PA2803 Dec 3 Dec Hypothetical protein; Predicted phosphatase/phosphohexomutase PA2808 4 NC 2 Hypothetical protein; phosphonoacetaldehyde hydrolase

PA2814 3 5 2 Hypothetical protein; Transglutaminase-like enzymes, putative cysteine proteases PA2819 NC 3 NC Hypothetical protein

PA2835 Dec 4 NC Probable MFS transporter

PA2846 NC NC 3 Probable transcriptional regulator

PA2847 NC NC 4 Conserved hypothetical protein; Putative permease

PA2861 ligT 2 5 NC 2 -5 RNA ligase

PA2895 NC 3 2 Hypothetical protein

PA2896 2 3 2 Probable sigma-70 factor

PA2919 NC 3 NC Hypothetical protein

PA2922 3 3 NC Probable hydrolase

PA2927 3 NC NC Hypothetical protein; phospholipase

PA2933 NC 3 4 Probable MFS transporter

PA2937 3 2 2 Hypothetical protein

PA3044 rocsS2 NC 5 NC Probable two-component sensor

PA3049 rnf 3 5 3 Ribosome modulation factor

PA3057 NC 3 NC Hypothetical protein; DNA binding domain protein, excisionase PA3062 pelC Dec 2 7 Hypothetical protein; putative lipoprotein; Pel exopolysaccharide biosynthesis PA3064 pelA NC 4 NC Hypothetical protein; extracellular protein; Pel exopolysaccharide biosynthesis PA3065 NC 3 NC Hypothetical protein

PA3066 NC 3 NC Hypothetical protein; Amidases related to nicotinamidase

PA3126 ibpA 3 6 3 Heat-shock protein

PA3175 NC 3 NC Probable arginase family protein, HutG Histidine metabolism PA3219 NC 3 NC Hypothetical protein; UDP-2,3-diacylglucosamine hydrolase PA3268 NC 3 NC Probable TonB-dependent receptor; Outer membrane receptor for Fe3+-dicitrate PA3273 2 3 2 Hypothetical protein

PA3274 3 4 3 Hypothetical protein

PA3283 NC 3 NC Conserved hypothetical protein

PA3284 NC 3 NC Hypothetical protein

PA3319 plcN NC 4 2 Non-hemolytic phospholipase C precursor

PA3320 NC 4 2 Hypothetical protein

PA3361 lecB 5 5 5 Fucose-binding lectin PA-IIL

PA3366 amiE 2 3 2 Aliphatic amidase

PA3369 3 3 NC Hypothetical protein

PA3370 3 3 NC Hypothetical protein

PA3371 3 3 NC Hypothetical protein

PA3382 phnE Dec 3 2 Phosphonate transport protein

PA3391 nosR 2 6 3 Regulatory protein; Regulator of nitric oxide reductase transcription PA3392 nosZ 2 5 NC Nitrous-oxide reductase precursor

179 PA3394 nosF NC 4 NC NosF protein

PA3396 nosL NC 3 3 NosL protein

PA3397 fpr 4 2 3 NADP+-dependent ferredoxin reductase

PA3404 NC 9 3 Outer membrane protein; porin activity

PA3405 hasE 3 5 7 Metalloprotease secretion protein

PA3406 hasD 3 10 7 Transport protein HasD

PA3407 hasAp 231 362 461 Heme acquisition protein

PA3408 hasR 13 62 17 Heme acquisition protein

PA3409 3 11 10 Probable transmembrane sensor; Fe2+-dicitrate sensor; putative antisigma PA3410 6 7 9 Probable sigma-70 factor, ECF subfamily

PA3415 2 4 2 Probable dihydrolipoamide acetyltransferase

PA3416 2 4 3 Probable pyruvate dehydrogenase E1 component, beta chain PA3417 2 3 2 Probable pyruvate dehydrogenase E1 component, alpha subunit PA3418 ldh 3 5 2 Leucine dehydrogenase; Glutamate dehydrogenase

PA3441 7 3 3 Probable molybdopterin-binding protein

PA3442 4 2 NC Probable ATP-binding component of ABC transporter

PA3444 9 2 2 Conserved hypothetical protein; alkanesulfonate monooxygenase, FMNH[263]-dependent

PA3445 11 2 2 Conserved hypothetical protein; ABC transporter, substrate-binding protein, aliphatic sulfonates family

PA3446 7 NC 2 Conserved hypothetical protein; FMN reductase, SsuE family; oxidation-reduction process

PA3447 7 5 3 Probable ATP-binding component of ABC transporter

PA3448 6 2 7 Probable permease of ABC transporter

PA3449 6 13 9 Conserved hypothetical protein; ABC transporter, substrate-binding protein, aliphatic sulfonates family

PA3450 6 2 2 Probable antioxidant protein; putative peroxidase

PA3451 3 2 2 Hypothetical protein; Putative thiolase

PA3452 mqoA 3 4 5 Malate:quinone oxidoreductase

PA3460 2 3 2 Probable acetyltransferase

PA3461 2 3 2 Conserved hypothetical protein; glutamyl aminopeptidase family protein PA3462 3 NC NC Probable sensor/response regulator hybrid; integral membrane hybrid histidine protein kinase, homologue Azotobacter

PA3477 rhlR 3 2 2 Transcriptional regulator

PA3478 rhlB 7 3 6 Rhamnosyltransferase chain B

PA3479 rhlA 5 3 4 Rhamnosyltransferase chain A

PA3498 Dec 5 NC Probable oxidoreductase

PA3512 Dec 2 NC Probable permease of ABC transporter

PA3513 3 NC NC Hypothetical protein; ABC transporter, substrate-binding protein, aliphatic sulfonates family

PA3520 6 4 5 Hypothetical protein; Copper chaperone

PA3530 bfd 8 6 11 Conserved hypothetical protein; bacterioferritin- associated ferredoxin

180 PA3540 algD NC 23 9 GDP-mannose 6-dehydrogenase

PA3541 alg8 NC 7 8 alginate biosynthesis

PA3542 alg44 NC 4 2 alginate biosynthesis

PA3543 algK NC 60 6 Scafold protein

PA3544 algE NC 6 2 Outermembrane protein

PA3545 algG NC 11 2 alginate-c5-mannuronan-epimerase

PA3546 algX NC 4 2 alginate biosynthesis

PA3547 algL NC 4 2 alginate lyase

PA3548 algI NC 8 2 acetylase

PA3549 algJ Dec 5 2 acetylase

PA3550 algF NC 5 3 acetylase

PA3551 algA 2 11 3 phosphomannose isomerase / guanosine 5'-diphospho- D-mannose pyrophosphorylase PA3560 fruA NC 4 NC Phosphotransferase system, fructose-specific IIBC component PA3561 fruK NC 4 NC 1-phosphofructokinase; Fructose and mannose metabolism PA3562 NC 5 NC Probable phosphotransferase system enzyme I; Fructose and mannose metabolism PA3590 NC 4 3 Probable hydroxyacyl-CoA dehydrogenase

PA3597 NC NC 3 Probable amino acid permease

PA3598 NC NC 3 Conserved hypothetical protein; Predicted amidohydrolase; nitrilase/cyanide hydratase and apolipoprotein N-acyltransferase

PA3599 NC NC 3 Probable transcriptional regulator; luxR family signature

PA3600 2 NC 3 Conserved hypothetical protein; ribosomal protein L36, cytosolic large ribosomal subunit

PA3601 2 NC 3 Conserved hypothetical protein; ribosomal protein L31, cytosolic large ribosomal subunit

PA3608 potB NC 4 NC Polyamine transport protein

PA3662 3 2 3 Hypothetical protein

PA3691 NC 3 NC Hypothetical protein; putative lipoprotein

PA3692 lptX NC 4 NC Outer membrane protein and related peptidoglycan- associated [264]proteins PA3734 3 2 2 Hypothetical protein; putative hydrolase

PA3740 3 NC 2 Hypothetical protein; Type II secretory pathway, ATPase PulE/Tfp pilus assembly pathway, ATPase PilB

PA3779 2 3 3 Hypothetical protein; tripartite ATP-independent periplasmic transporter complex PA3783 3 NC NC Hypothetical protein; Amidases related to nicotinamidase; isochorismatase hydrolase PA3815 lscR 2 2 3 Iron-sulfur cluster assembly transcription factor IscR

PA3819 2 6 2 Conserved hypothetical protein; Predicted outer membrane lipoprotein; surface antigen

PA3841 exoS 2 4 2 Exoenzyme S

PA3866 3 2 NC Pyocin protein

PA3870 moaA1 NC 3 Dec Molybdopterin biosynthetic protein A1

PA3871 Dec 3 NC Probable peptidyl-prolyl cis-trans isomerase, PpiC-type

PA3872 narI NC 3 NC Respiratory nitrate reductase gamma chain

PA3873 narJ NC 3 NC Respiratory nitrate reductase delta chain

181 PA3874 narH Dec 3 Dec Respiratory nitrate reductase beta chain

PA3875 narG Dec 3 Dec Respiratory nitrate reductase alpha chain

PA3876 narK2 Dec 3 NC Nitrite extrusion protein 2

PA3877 narK1 Dec 3 Dec Nitrite extrusion protein 1

PA3890 NC 5 2 Probable permease of ABC transporter

PA3899 9 4 8 Probable sigma-70 factor, ECF subfamily

PA3900 2 2 3 Probable transmembrane sensor; Fe2+-dicitrate sensor,

PA3901 fecA 6 NC 4 Fe(III) dicitrate transport protein FecA

PA3919 NC 3 2 Conserved hypothetical protein; NYN ribonuclease and ATPase of PhoH family domains; Conserved hypothetical protein; Phosphate starvation; also in Azotobacter

PA3926 Dec 3 NC Probable MFS transporter; phosphoglycerate transporter family protein PA3933 3 3 6 Probable choline transporter

PA3935 tauD 3 NC 2 Taurine dioxygenase

PA3936 2 NC 2 Probable permease of ABC taurine transporter

PA3937 4 NC 2 Probable ATP-binding component of ABC taurine transporter PA3938 3 NC 2 Probable periplasmic taurine-binding protein precursor

PA3939 3 2 5 Hypothetical protein; acyl-CoA dehydrogenase

PA3952 2 4 2 Hypothetical protein; putative periplasmic ligand-binding sensor protein PA3957 2 2 3 Probable short-chain dehydrogenase

PA3986 3 3 3 Hypothetical protein

PA4008 NC NC 3 Probable hydrolase; Predicted hydrolases or acyltransferases PA4033 mucE 4 3 NC Hypothetical protein

PA4037 NC 4 NC Probable ATP-binding component of ABC transporter

PA4041 3 NC 2 Hypothetical protein; Mandelate racemase/muconate lactonizing protein PA4070 3 NC NC Probable transcriptional regulator; DNA-binding transcriptional activator FeaR PA4078 3 2 2 Probable nonribosomal peptide synthetase

PA4082 3 Dec 3 Probable adhesin

PA4090 3 3 4 Hypothetical protein

PA4105 3 3 Dec Hypothetical protein

PA4142 4 6 5 Probable secretion protein

PA4143 3 5 3 Probable toxin transporter

PA4144 2 4 2 Probable secretion protein

PA4146 2 3 NC Hypothetical protein

PA4155 2 3 5 Hypothetical protein; oxidoreductase activity

PA4156 8 4 11 Probable TonB-dependent receptor

PA4158 fepC 6 12 7 Ferric enterobactin transport protein

PA4159 fepB 2 2 3 Ferrienterobactin-binding periplasmic protein precursor

PA4160 fepD 4 3 6 Ferric enterobactin transport protein

PA4161 fepG 2 2 5 Ferric enterobactin transport protein

PA4167 2 NC 3 Probable oxidoreductase

PA4168 fpvB 10 6 11 Probable TonB-dependent receptor; second ferric pyoverdine receptor

182 PA4171 3 NC 2 Probable protease; Putative intracellular protease/amidase; cysteine-type peptidase activity

PA4172 3 NC 2 Probable nuclease; Exonuclease III

PA4175 piv 6 11 13 Probable endoproteinase Arg-C precursor; Pvds- regulated endoprotease, lysyl class PA4179 Dec Dec 5 Probable porin

PA4187 NC 3 Dec Probable MFS transporter

PA4218 ampP 97 104 495 Probable transporter; siderophore transporter, RhtX/FptX family PA4219 ampO 187 431 512 Hypothetical protein; Uncharacterized iron-regulated membrane protein PA4220 362 530 402 Hypothetical protein

PA4221 fptA 284 446 338 Fe(III)-pyochelin receptor precursor

PA4222 47 82 82 Probable ATP-binding component of ABC transporter

PA4223 44 97 108 Probable ATP-binding component of ABC transporter

PA4224 pchG 215 115 100 Hypothetical protein; pyochelin biosynthetic protein PchG

PA4225 pchF 416 431 326 Pyochelin synthetase

PA4226 pchE 512 653 175 Dihydroaeruginoic acid synthetase

PA4227 pchR 15 7 23 Transcriptional regulator

PA4228 pchD 495 588 776 Pyochelin biosynthesis protein

PA4229 pchC 294 923 478 Pyochelin biosynthetic protein

PA4230 pchB 208 215 187 Salicylate biosynthesis protein

PA4231 pchA 530 530 653 Salicylate biosynthesis isochorismate synthase

PA4297 tadG 5 NC NC Predicted membrane protein

PA4304 rcpA 3 2 NC Probable type II secretion system protein

PA4364 2 3 NC Hypothetical protein

PA4365 NC 3 NC Probable transporter; Lysine efflux permease

PA4390 2 3 3 Hypothetical protein

PA4467 82 28 36 Hypothetical protein; Predicted divalent heavy-metal cations transporter PA4468 sodM 69 87 108 Superoxide dismutase

PA4469 62 82 91 Hypothetical protein

PA4470 fumC1 56 62 84 Fumarate hydratase

PA4471 fagA 94 69 111 Fur-associated gene FagA

PA4495 NC 3 2 Hypothetical protein; Predicted periplasmic/secreted protein; signal peptide PA4508 NC 3 NC Probable transcriptional regulator

PA4515 3 3 3 Conserved hypothetical protein; Fe(II)-dependent oxygenase superfamily protein PA4516 2 2 3 Hypothetical protein

PA4542 clpB 2 3 3 ClpB protein

PA4570 45 42 66 Hypothetical protein, Sel1/TPR repeat-containing protein, homologue Azotobacter PA4573 5 3 3 Hypothetical protein

PA4591 NC 3 NC Hypothetical protein; Multidrug resistance efflux pump

PA4610 NC 3 NC Hypothetical protein; putative copper export protein

PA4657 3 4 3 Hypothetical protein; Predicted NAD/FAD-dependent oxidoreductase PA4685 2 3 2 Hypothetical protein

183 PA4706 2 2 3 Probable ATP-binding component of ABC transporter; ABC-type hemin transport system, ATPase component

PA4708 phuT 4 4 6 Heme-transport protein, PhuT

PA4709 5 5 6 Probable hemin degrading factor

PA4710 phuR 4 5 6 Heme/Hemoglobin uptake outer membrane receptor PhuR precursor PA4759 dapB 2 3 3 Dihydrodipicolinate reductase

PA4760 dnaJ 2 3 3 DnaJ protein

PA4761 dnaK 3 4 3 DnaK protein

PA4762 grpE 2 3 3 Heat shock protein

PA4772 Dec 3 NC Probable ferredoxin

PA4792 3 2 3 Conserved hypothetical protein; Glycerophosphoryl diester phosphodiesterase PA4813 lipC NC NC 3 Llipase

PA4819 3 NC NC Probable glycosyl transferase

PA4822 Dec 3 NC Hypothetical protein; Na+/phosphate symporter

PA4824 Dec 4 Dec Hypothetical protein

PA4833 3 2 2 Conserved hypothetical protein; Predicted membrane protein, hemolysin III homolog PA4844 NC 7 NC Probable chemotaxis transducer

PA4861 NC NC 3 Probable ATP-binding component of ABC transporter

PA4876 osmE NC 3 NC Osmotically inducible lipoprotein

PA4877 2 3 NC Hypothetical protein; Rho termination factor Superfamily

PA4880 Dec 3 NC Probable bacterioferritin

PA4895 3 5 6 Probable transmembrane sensor; Fe2+-dicitrate sensor

PA4896 8 5 9 Probable sigma-70 factor, ECF subfamily

PA4897 3 2 3 hypothetical protein; TonB-dependent heme/hemoglobin receptor family protein PA4898 opdK NC 3 NC Probable porin; histidine porin

PA4899 3 2 2 Probable aldehyde dehydrogenase

PA4900 3 NC NC Probable MFS transporter

PA4902 NC 5 NC Probable transcriptional regulator

PA4903 NC 2 3 Probable MFS transporter

PA4905 vanB 3 NC 5 Vanillate O-demethylase oxidoreductase

PA4908 NC 3 NC Hypothetical protein; Predicted ornithine cyclodeaminase, mu-crystallin homolog PA4909 3 NC NC Probable ATP-binding component of ABC transporter

PA4912 NC 4 NC Probable permease of ABC branched chain amino acid transporter PA4925 3 2 2 Conserved hypothetical protein; Small-conductance mechanosensitive channel PA5030 3 2 2 Probable MFS transporter

PA5031 NC 4 NC Probable short chain dehydrogenase; protochlorophyllide reductase activity PA5032 NC 3 NC Probable transcriptional regulator

PA5054 hslU NC 2 3 Heat shock protein

PA5061 3 2 2 Conserved hypothetical protein; poly(hydroxyalkanoate) granule-associated protein PA5084 NC 2 5 Probable oxidoreductase

PA5092 hutI NC 4 NC Imidazolone-5-propionate hydrolase

PA5093 3 6 NC Probable histidine/phenylalanine ammonia-lyase

184 PA5094 2 6 2 Probable ATP-binding component of ABC transporter

PA5095 NC 6 NC Probable permease of ABC transporter

PA5096 2 3 NC Probable binding protein component of ABC transporter

PA5097 3 4 3 Probable amino acid permease

PA5098_hutH hutH 2 14 10 Histidine ammonia-lyase

PA5099 NC 47 15 Probable transporter

PA5100_hutU hutU 5 23 6 Urocanase

PA5105_hutC hutC 2 3 2 Histidine utilization repressor

PA5106 4 7 5 Conserved hypothetical protein; formiminoglutamate deiminase PA5150 8 5 7 Probable short-chain dehydrogenase

PA5180 NC 3 2 Conserved hypothetical protein; formate dehydrogenase family accessory protein PA5181 NC 3 NC Probable oxidoreductase

PA5182 p34 NC 3 NC ncRNA

PA5212 NC 5 2 Hypothetical protein

PA5218 3 3 NC Probable transcriptional regulator; positive regulation of transcription PA5282 NC 3 NC Probable MFS transporter

PA5284 3 2 NC Hypothetical protein; fimbrial-like adhesin protein

PA5294 NC NC 3 Hypothetical protein; Na+-driven multidrug efflux pump

PA5302 dadX 3 4 2 Catabolic alanine racemase

PA5303 3 5 2 Conserved hypothetical protein; Putative translation initiation inhibitor PA5304 dadA 4 6 4 D-amino acid dehydrogenase, small subunit

PA5312 3 2 3 Probable aldehyde dehydrogenase

PA5313 gabT2 6 2 4 Transaminase

PA5314 4 2 3 Hypothetical protein; Predicted enzyme of the cupin superfamily PA5341 NC 3 NC Hypothetical protein; Putative threonine efflux protein

PA5372 betA NC 4 NC Choline dehydrogenase

PA5375 betT1 2 2 3 Choline transporter

PA5383 2 2 4 Conserved hypothetical integral membrane protein

PA5385 cdhB Dec 6 NC Carnitine dehydrogenase-related gene B

PA5386 cdhA NC 3 NC Carnitine dehydrogenase

PA5405 NC 3 Dec Hypothetical protein

PA5415_glyA1 glyA1 NC 5 2 Serine hydroxymethyltransferase

PA5446 2 6 4 Hypothetical protein

PA5460 7 9 4 Hypothetical protein

PA5480 NC NC 4 Hypothetical protein

PA5486 NC 2 3 Conserved hypothetical protein; Multiple antibiotic transporter PA5506 2 4 2 Hypothetical protein; Transcriptional regulator; SIS domain protein; regulate the expression of genes involved in synthesis of phosphosugars PA5507 NC 4 2 Hypothetical protein; Amidases related to nicotinamidase; isochorismatase hydrolase PA5508 pauA7 NC 3 2 Glutamylpolyamine synthetase homologue

PA5509 NC 5 NC Hypothetical protein; Predicted N-formylglutamate amidohydrolase; catalyzes the terminal reaction in the five-step pathway for histidine utilisation

185 PA5531 tonB1 3 2 3 TonB protein

PA5566 Dec 4 Dec Hypothetical protein

Decreased Gene Expression (0 µM vs. 100 µM FeCl3)

PA Number Gene PAO1 ∆mucAi ∆mucDi Description/Function Fold Fold Fold Change Change Change ig_1087843_1087095 -3 NC NC Intergenic region between PA1030 and PA1031, 1087095-1087843, (-) strand ig_1247932_1246788 NC -4 NC Intergenic region between PA1156 and PA1157, 1246788-1247932, (-) strand ig_1553675_1554415 -3 -4 NC Intergenic region between PA1507 and PA1508, 1553675-1554415, (+) strand ig_1638379_1637696 NC -8 NC Intergenic region between PA1655 and PA1656, 1637696-1638379, (-) strand ig_2069490_2068728 -3 NC -6 Intergenic region between PA1934 and PA1935, 2068728-2069490, (-) strand ig_2226144_2226879 -4 -4 NC Intergenic region between PA2046 and PA2047, 2226144-2226879, (+) strand ig_2341640_2342493 -3 NC NC Intergenic region between PA2228 and PA2229, 2341640-2342493, (+) strand ig_2450765_2451707 NC -6 NC Intergenic region between PA2319 and PA2320, 2450765-2451707, (+) strand ig_2737881_2737193 NC -2 -3 Intergenic region between PA2559 and PA2560, 2737193-2737881, (-) strand ig_2918211_2918966 NC -3 NC Intergenic region between PA2583 and PA2584, 2918211-2918966, (+) strand ig_2922568_2923366 NC -5 -6 Intergenic region between PA2587 and PA2588, 2922568-2923366, (+) strand ig_2927920_2928538 NC -3 NC Intergenic region between PA2698 and PA2699, 2927920-2928538, (+) strand ig_3051956_3051348 -2 -3 NC Intergenic region between PA2729 and PA2730, 3051348-3051956, (-) strand ig_3088659_3087490 NC -3 -3 Intergenic region between PA2750 and PA2751, 3087490-3088659, (-) strand ig_3265210_3265847 -3 -4 -2 Intergenic region between PA3094 and PA3095, 3265210-3265847, (+) strand ig_3265847_3265210 -2 -4 -2 Intergenic region between PA3094 and PA3095, 3265210-3265847, (-) strand ig_3962824_3961922 -3 -2 NC Intergenic region between PA3781 and PA3782, 3961922-3962824, (-) strand ig_4560294_4559649 NC -3 -2 Intergenic region between PA4100 and PA4101, 4559649-4560294, (-) strand ig_4585148_4584530 -3 NC NC Intergenic region between PA4138 and PA4139, 4584530-4585148, (-) strand ig_4629943_4629184 -3 -2 NC Intergenic region between PA4204 and PA4205, 4629184-4629943, (-) strand ig_4713098_4713795 -3 -2 -3 Intergenic region between PA4280 and PA4281, 4713098-4713795, (+) strand ig_546334_545644 Inc NC -3 Intergenic region between PA0574 and PA0575, 545644- 546334, (-) strand ig_6090705_6092045 -7 Inc NC Intergenic region between PA5440 and PA5441, 6090705-6092045, (+) strand ig_6092045_6090705 -3 NC NC Intergenic region between PA5440 and PA5441, 6090705-6092045, (-) strand PA0007 NC NC -3 Hypothetical protein

PA0022 yrdC NC -4 -2 Conserved hypothetical protein; Putative translation factor; tRNA modification PA0035 trpA -2 -3 -2 Tryptophan synthase alpha chain

PA0036 trpB -2 -4 -2 Tryptophan synthase beta chain

PA0040 -3 -2 NC Conserved hypothetical protein; Hemolysin activation/secretion protein PA0045 -4 NC NC Hypothetical protein; Uncharacterized protein involved in formation of curli polymers PA0046 -5 NC NC Hypothetical protein; putative lipoprotein

PA0056 -3 Inc -2 Probable transcriptional regulator

PA0070 tagQ1 -4 -2 -2 Hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system

186 PA0072 tagS1 -9 -2 -3 Hypothetical protein; ABC-type transport system, involved in lipoprotein release, permease component; Hcp secretion island I (HSI-I) type VI secretion system

PA0074 ppkA -3 NC -2 Serine/threonine protein kinase

PA0075 pppA -3 NC -2 Probable phosphoprotein phosphatase

PA0076 tagF1 -3 -2 -2 Hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system PA0077 icmF1 -3 -2 -2 Hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system PA0078 tssL1 -3 NC -2 Hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system PA0079 tssK1 -3 NC -2 Hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system PA0080 tssJ1 -3 NC -2 Hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system PA0083 tssB1 -3 -2 -2 Conserved hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system

PA0084 tssC1 -3 -2 -2 Conserved hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system

PA0085 hcp1 -5 -2 -2 Conserved hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system

PA0086 tagJ1 -4 -2 -2 Hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system PA0087 tssE1 -3 -2 -2 Hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system PA0088 tssF1 -4 -2 -2 Hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system PA0089 tssG1 -6 -2 -2 Hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system PA0090 clpV1 -5 -2 -2 Probable ClpA/B-type chaperone

PA0091 vgrG1 -3 -2 -2 Conserved hypothetical protein; Hcp secretion island I (HSI-I) type VI secretion system

PA0093 -3 NC NC Hypothetical protein

PA0097 -3 Inc -2 Hypothetical protein

PA0126 -3 -2 -3 Hypothetical protein

PA0128 -2 -3 -2 Conserved hypothetical protein; Uncharacterized Zn- ribbon-containing protein involved in phosphonate metabolism

PA0136 -3 Inc NC Probable ATP-binding component of ABC transporter

PA0140 ahpF -3 -2 -3 Alkyl hydroperoxide reductase subunit F

PA0153 pcaH NC NC -3 Protocatechuate 3,4-dioxygenase, beta subunit

PA0166 -3 NC Inc Probable transporter

PA0205 NC -4 NC Probable permease of ABC transporter

PA0206 -4 NC NC Probable ATP-binding component of ABC transporter

PA0211 mdcD -3 NC NC Malonate decarboxylase beta subunit

PA0226 -2 -3 -2 Probable CoA transferase, subunit A

PA0227 -2 NC -4 Probable CoA transferase, subunit B

PA0239 -3 -2 -8 Hypothetical protein; Threonine/homoserine efflux transporter PA0242 Inc -5 -3 Hypothetical protein; 4-hydroxyphenylpyruvate dioxygenase and related hemolysins PA0258 NC -3 NC Hypothetical protein

PA0260 -2 -3 NC Hypothetical protein

PA0261 -2 -4 NC Hypothetical protein

187 PA0262 -2 -3 -2 Conserved hypothetical protein

PA0263 hcpC -12 -3 -6 Secreted protein Hcp

PA0277 -7 -2 -3 Conserved hypothetical protein; Zn-dependent protease with chaperone function PA0288 gpuA NC NC -3 3-guanidinopropionase

PA0316 serA -4 -5 -5 D-3-phosphoglycerate dehydrogenase

PA0364 -6 -2 -2 Probable oxidoreductase

PA0441 dht Inc NC -7 Dihydropyrimidinase

PA0444 hyuC -2 NC -3 N-carbamoyl-beta-alanine amidohydrolase; peptidase activity PA0492 ycsF -6 NC NC Conserved hypothetical protein; homologs of lactam utilization protein B PA0493 -5 NC NC Probable biotin-requiring enzyme

PA0499 -2 Inc -3 Probable pili assembly chaperone

PA0527 dnr -2 -2 -4 Transcriptional regulator

PA0528 -3 NC NC Probable transcriptional regulator

PA0573 NC -5 NC Hypothetical protein

PA0600 NC -3 NC Probable two-component sensor

PA0602 -3 -2 NC Probable binding protein component of ABC transporter

PA0612 ptrB -3 -2 -2 Hypothetical protein; DnaK suppressor protein

PA0613 -3 -2 -2 Hypothetical protein

PA0616 -3 -2 -2 Hypothetical protein; Phage P2 baseplate assembly protein gpV PA0617 -9 -2 -2 Probable bacteriophage protein

PA0618 -3 NC -2 Probable bacteriophage protein

PA0619 -3 -2 -2 Probable bacteriophage protein

PA0621 -2 -2 -3 Conserved hypothetical protein

PA0622 -3 -2 -2 Probable bacteriophage protein

PA0623 -3 -2 -2 Probable bacteriophage protein

PA0624 -3 -2 -2 Hypothetical protein

PA0625 -3 -2 -2 Hypothetical protein; Mu-like prophage protein

PA0627 -3 -2 -2 Conserved hypothetical protein; P2-like prophage tail protein X PA0632 -3 -2 -2 Hypothetical protein

PA0633 -3 NC -2 Hypothetical protein

PA0637 -3 -2 -2 Conserved hypothetical protein; Phage-related protein

PA0638 -3 -2 -2 Probable bacteriophage protein

PA0639 -3 -2 -2 Conserved hypothetical protein; Predicted metal- dependent protease of the PAD1/JAB1 superfamily

PA0640 -3 -2 -3 Probable bacteriophage protein

PA0641 -6 -2 -3 Probable bacteriophage protein

PA0642 -2 -2 -3 Hypothetical protein

PA0654 speD -3 -2 -2 S-adenosylmethionine decarboxylase proenzyme

PA0678 hxcU NC -2 -6 Probable type II secretion system protein; HxcU putative pseudopilin PA0681 hxcT NC -3 NC Probable type II secretion system protein; HxcT pseudopilin PA0682 hxcX -3 NC NC Probable type II secretion system protein; HxcX atypical pseudopilin PA0696 NC NC -3 Hypothetical protein; putative outer membrane TonB- dependent receptor 188 PA0700 -4 NC NC Hypothetical protein

PA0710 gloA2 NC -3 NC Lactoylglutathione lyase

PA0715 NC -4 NC Hypothetical protein; Retron-type reverse transcriptase

PA0716 NC -3 -2 Hypothetical protein; ATPase components of various ABC-type transport systems, contain duplicated ATPase

PA0717 -4 NC NC Hypothetical protein of bacteriophage Pf1

PA0724 -4 NC Inc Probable coat protein A of bacteriophage Pf1

PA0740 sdsA1 -3 -2 NC SDS hydrolase SdsA1 (changed from probable beta- lactamase) PA0758 -3 NC NC Hypothetical protein; Predicted signal transduction protein

PA0824 NC NC -3 Hypothetical protein

PA0825 NC -3 -2 Hypothetical protein; Predicted membrane protein

PA0842 -3 Inc -2 Probable glycosyl transferase

PA0850 NC -4 -2 Hypothetical protein

PA0885 dctQ NC -3 NC Probable C4-dicarboxylate transporter

PA0887 acsA -3 -5 NC Acetyl-coenzyme A synthetase

PA0908 -7 NC NC Hypothetical protein

PA0909 -3 -2 -2 Hypothetical protein

PA0910 -3 -3 NC Hypothetical protein

PA0914 -2 NC -3 Hypothetical protein

PA0915 yehS -2 NC -3 Conserved hypothetical protein

PA0958 oprD -3 NC -2 Outer membrane porin protein OprD precursor

PA0975 -3 -3 -4 Probable radical activating enzyme

PA0977 -3 NC NC Hypothetical protein

PA0981 NC -3 -3 Hypothetical protein

PA0986 -3 -5 NC Conserved hypothetical protein

PA0993 cupC2 NC Inc -4 Probable pili assembly chaperone

PA1018 NC -3 NC Hypothetical protein; Acyl dehydratase

PA1025 opdD -3 NC NC Probable porin

PA1070 braG -3 -2 -2 Branched-chain amino acid transport protein BraG

PA1071 braF -3 -2 -2 Branched-chain amino acid transport protein BraF

PA1073 braD -3 -2 -2 Branched-chain amino acid transport protein BraD

PA1143 -3 NC NC Hypothetical protein; Phosphoserine phosphatase

PA1144 -2 -3 NC Probable MFS transporter; Sugar phosphate permease

PA1168 -3 NC -5 Hypothetical protein

PA1173 napB -2 NC -3 Cytochrome c-type protein NapB precursor; Nitrogen metabolism PA1178 oprH -2 -2 -3 Outer membrane protein H1 precursor

PA1195 -3 NC -2 Hypothetical protein; arginine deiminase pathway

PA1213 -4 -2 NC Hypothetical protein; putative clavaminic acid synthetase

PA1223 NC -5 -2 Probable transcriptional regulator

PA1225 -2 -3 -2 Probable NAD(P)H dehydrogenase

PA1228 -5 Inc NC Hypothetical protein

PA1230 NC -5 Inc Hypothetical protein

189 PA1238 ompJ -3 NC NC Probable outer membrane component of multidrug efflux pump PA1260 -3 NC NC Probable binding protein component of ABC transporter

PA1261 -2 NC -3 Probable transcriptional regulator

PA1266 NC NC -3 Probable oxidoreductase

PA1273 cobB -3 -2 -2 Cobyrinic acid a,c-diamide synthase

PA1276 cobC -3 NC -2 Cobalamin biosynthetic protein CobC

PA1284 NC -3 NC Probable acyl-CoA dehydrogenase

PA1286 NC -3 NC Probable MFS transporter; oxalate/formate antiporter family transporter PA1298 NC -3 -4 Conserved hypothetical protein

PA1310 phnW -3 Inc -2 2-aminoethylphosphonate:pyruvate aminotransferase

PA1325 -5 -8 -2 Conserved hypothetical protein; Ketosteroid isomerase homolog PA1338 ggt -5 -2 -3 Gamma-glutamyltranspeptidase precursor

PA1340 -3 -3 -2 Probable permease of ABC transporter

PA1341 -3 -2 -3 Probable permease of ABC transporter

PA1347 -3 NC Inc Probable transcriptional regulator; luxR family signature

PA1379 NC -3 NC Probable short-chain dehydrogenase

PA1380 -5 -3 NC Probable transcriptional regulator

PA1384 galE -5 -2 -2 UDP-glucose 4-epimerase

PA1385 -3 -4 Inc Probable glycosyl transferase

PA1391 -2 -3 NC Probable glycosyl transferase

PA1394 -2 -5 -2 Hypothetical protein

PA1395 NC -4 -6 Hypothetical protein

PA1396 -3 NC -2 Probable two-component sensor

PA1425 -3 NC NC Probable ATP-binding component of ABC transporter

PA1428 NC -2 -4 Conserved hypothetical protein; ribosome biogenesis

PA1478 -2 -2 -3 Hypothetical protein; Heme exporter protein D

PA1481 ccmG -2 -2 -3 Cytochrome C biogenesis protein CcmG

PA1483 cycH -2 -2 -3 Cytochrome c-type biogenesis protein

PA1501 glp -2 NC -3 Conserved hypothetical protein; Hydroxypyruvate isomerase PA1502 gcl -3 Inc NC Glyoxylate carboligase

PA1507 -2 -6 NC Probable transporter

PA1509 NC -3 NC Hypothetical protein

PA1510 -6 -3 NC Hypothetical protein

PA1511 -3 -5 -2 Conserved hypothetical protein; protein secretion by the type VI secretion system PA1553 ccoO1 -3 -2 -3 Probable cytochrome c oxidase subunit

PA1565 pauB2 -5 -2 -2 Probable oxidoreductase; FAD-dependent oxidoreductase PA1583 sdhA -3 -2 -2 Succinate dehydrogenase (A subunit)

PA1598 NC -3 NC Conserved hypothetical protein; Ketopantoate hydroxymethyltransferase PA1656 hsiA2 -3 -2 -2 Hypothetical protein; protein secretion by the type VI secretion system PA1657 hsiB2 -5 -3 -4 Conserved hypothetical protein; protein secretion by the type VI secretion system PA1658 hsiC2 -4 -4 -4 Conserved hypothetical protein; protein secretion by the type VI secretion system

190 PA1659 hsiF2 -5 -3 -3 Hypothetical protein; protein secretion by the type VI secretion system PA1660 hsiG2 -3 -3 -2 Hypothetical protein; protein secretion by the type VI secretion system PA1661 hsiH2 -3 -3 -2 Hypothetical protein; protein secretion by the type VI secretion system PA1662 clpV2 -3 -3 -2 Probable ClpA/B-type protease

PA1663 sfa2 -3 -3 -2 Probable transcriptional regulator

PA1664 orfX -8 -4 -3 Hypothetical protein; protein secretion by the type VI secretion system PA1665 fha2 -13 -3 -4 Hypothetical protein; protein secretion by the type VI secretion system PA1666 lip2 -5 -3 -2 Hypothetical protein; protein secretion by the type VI secretion system PA1667 hsiJ2 -5 -3 -3 Hypothetical protein; protein secretion by the type VI secretion system PA1668 dotU2 -2 -3 NC Hypothetical protein; protein secretion by the type VI secretion system PA1669 icmF2 -4 -4 -3 Hypothetical protein; protein secretion by the type VI secretion system PA1670 stp1 -2 -3 -2 Serine/threonine phosphoprotein phosphatase; protein secretion by the type VI secretion system

PA1671 stk1 -8 -11 NC Serine-threonine kinase; protein secretion by the type VI secretion system PA1690 pscU -5 NC NC Translocation protein in type III secretion

PA1720 pscG -3 Inc NC Type III export protein PscG

PA1735 NC -3 NC Hypothetical protein; Predicted permeases

PA1740 -4 -10 NC Hypothetical protein; Gluconolactonase

PA1764 -2 -2 -3 Hypothetical protein; Long-chain fatty acid transport protein PA1771 estX -4 -2 NC Probable esterase/lipase; Lysophospholipase

PA1781 nirB -3 NC -2 Assimilatory nitrite reductase large subunit

PA1824 -7 NC -2 Conserved hypothetical protein; Predicted permeases

PA1842 -2 -3 -3 Hypothetical protein

PA1843 metH -3 -3 -3 Methionine synthase

PA1845 -4 -2 -2 Hypothetical protein

PA1848 Inc -3 NC Probable MFS transporter

PA1856 NC -3 Inc Probable cytochrome oxidase subunit; oxidation- reduction process PA1867 xphA -3 NC NC Hypothetical protein; Protein secretion/export apparatus

PA1868 xqhA -4 NC NC Secretion protein

PA1891 -3 NC -2 Hypothetical protein; putative membrane protein

PA1892 -3 -2 -5 Hypothetical protein

PA1894 -5 -5 -4 Hypothetical protein; 2OG-Fe(II) oxygenase oxidoreductase PA1895 -3 -3 -2 Hypothetical protein; fatty acid desaturase

PA1896 -4 -4 -3 Hypothetical protein; putative methionine aminopeptidase

PA1897 -5 -6 -10 Hypothetical protein; Sterol desaturase

PA1921 NC -4 NC Hypothetical protein; Trans-aconitate methyltransferase

PA1922 -2 -5 -2 Probable TonB-dependent receptor

PA1923 -2 -6 -2 Hypothetical protein; Cobalamin biosynthesis protein CobN and related Mg-chelatases

PA1924 -2 -6 -2 Hypothetical protein; Biopolymer transport proteins

PA1925 -2 -7 -2 Hypothetical protein

191 PA1927 metE -50 -47 -17 5-methyltetrahydropteroyltriglutamate-homocysteine S- methyltransferase PA1955 -4 Inc -2 Hypothetical protein

PA1956 NC NC -4 Hypothetical protein

PA1971 braZ -3 -2 -2 Branched chain amino acid transporter BraZ

PA1979 eraS NC -13 -2 Probable two-component sensor; ethanol oxidation pathway PA1980 eraR -4 -3 -3 Probable two-component response regulator; ethanol oxidation pathway PA2079 -4 NC Inc Probable amino acid permease

PA2097 -3 NC NC Probable flavin-binding monooxygenase

PA2099 -2 -3 -2 Probable short-chain dehydrogenase

PA2130 cupA3 -3 NC NC Probable fimbrial biogenesis usher protein

PA2198 Inc -8 -5 Hypothetical protein; antibiotic biosynthesis monooxygenase PA2228 NC -3 -2 Hypothetical protein; Beta-lactamase class C and other penicillin binding proteins; 6-aminohexanoate-dimer hydrolase

PA2328 -2 -2 -3 Hypothetical protein; ABC-type nitrate/sulfonate/bicarbonate transport systems, periplasmic components

PA2329 -2 -2 -4 Probable ATP-binding component of ABC transporter

PA2330 -2 -3 -4 Hypothetical protein; Acyl-CoA dehydrogenases; sulfur acquisition oxidoreductase PA2331 -2 -3 -4 Hypothetical protein; alkylhydroperoxidase like protein

PA2338 -4 -2 -3 Probable binding protein component of ABC maltose/mannitol transporter PA2340 -3 -2 -3 Probable binding-protein-dependent maltose/mannitol transport protein PA2342 mtlD -6 -2 -2 Mannitol dehydrogenase

PA2343 mtlY -4 -5 -3 Xylulose kinase

PA2352 -3 NC NC Probable glycerophosphoryl diester phosphodiesterase

PA2374 Inc NC -3 Hypothetical protein

PA2378 -2 -2 -3 Probable aldehyde dehydrogenase; Aerobic-type carbon monoxide dehydrogenase PA2379 -2 -2 -3 Probable oxidoreductase

PA2473 -3 NC -2 Probable glutathione S-transferase

PA2490 -4 -4 -3 Conserved hypothetical protein; Mannose-6-phosphate isomerase PA2496 -8 NC NC Hypothetical protein; putative DNA damage-inducible gene PA2507 catA -3 NC -3 Catechol 1,2-dioxygenase

PA2508 catC -5 -3 -4 Muconolactone delta-isomerase

PA2509 catB -8 -2 -4 Muconate cycloisomerase I

PA2512 antA -8 -4 -3 Anthranilate dioxygenase large subunit

PA2513 antB -10 -8 -4 Anthranilate dioxygenase small subunit

PA2514 antC -10 -9 -4 Anthranilate dioxygenase reductase

PA2515 xylL -4 NC -4 Cis-1,2-dihydroxycyclohexa-3,4-diene carboxylate dehydrogenase PA2518 xylX -3 NC -2 Toluate 1,2-dioxygenase alpha subunit

PA2522 czcC -3 -2 NC Outer membrane protein precursor CzcC

PA2539 -5 -2 -2 Conserved hypothetical protein; Predicted protein- tyrosine phosphatase PA2548 -3 NC NC Hypothetical protein; ABC-type phosphate transport system, periplasmic component PA2644 nuoI -2 -3 -2 NADH Dehydrogenase I chain I; oxidation-reduction process

192 PA2645 nuoJ -3 -2 -2 NADH dehydrogenase I chain J

PA2648 nuoM -3 -2 -2 NADH dehydrogenase I chain M

PA2649 nuoN -3 -2 -2 NADH dehydrogenase I chain N

PA2682 -4 -2 -3 Conserved hypothetical protein; Dienelactone hydrolase and related enzymes PA2684 -3 NC -2 Conserved hypothetical protein; Rhs family protein

PA2697 NC -3 NC Hypothetical protein

PA2712 -2 -2 -3 Hypothetical protein; Carboxylate/Amino Acid/Amine Transporte PA2715 -4 -2 -4 Probable ferredoxin

PA2727 -3 NC -2 Hypothetical protein; Serine/threonine protein kinase; DNA repair PA2753 -3 NC NC Hypothetical protein

PA2760 oprQ -3 NC -2 Probable outer membrane protein

PA2765 -4 -2 -3 Hypothetical protein; Predicted iron-dependent peroxidase PA2782 -6 NC -2 Hypothetical protein

PA2783 -7 NC -2 Hypothetical protein; putative Ni,Fe-hydrogenase I small subunit PA2784 -3 NC NC Hypothetical protein

PA2789 -2 -4 NC Hypothetical protein

PA2792 -3 -2 -2 Hypothetical protein

PA2803 -3 Inc -3 Hypothetical protein; Predicted phosphatase/phosphohexomutase PA2835 -3 Inc -2 Probable MFS transporter; drug resistance transporter

PA2836 -3 NC -2 Probable secretion protein; Multidrug resistance efflux pump PA2905 cobH -2 -3 NC Precorrin isomerase CobH

PA2911 -6 -5 -3 Probable TonB-dependent receptor

PA2912 -5 -4 -3 Probable ATP-binding component of ABC transporter; ABC-type cobalamin/Fe3+-siderophores transport systems, ATPase components

PA2913 -4 -4 -2 Hypothetical protein; ABC-type Fe3+-hydroxamate transport system, periplasmic component

PA2916 -14 -7 -11 Hypothetical protein; Putative threonine efflux protein; The Resistance to Homoserine/Threonine (RhtB) Family protein

PA2917 -5 -4 -6 Probable transcriptional regulator

PA2924 hisQ -3 -2 NC Histidine transport system permease HisQ

PA2935 NC NC -3 Hypothetical protein

PA2953 -3 -2 -3 Electron transfer flavoprotein-ubiquinone oxidoreductase

PA3018 -3 -2 -4 Hypothetical protein

PA3038 opdQ -4 -5 -3 Probable porin

PA3081 -3 NC NC Conserved hypothetical protein

PA3082 gbt -3 NC -2 Hypothetical protein; glycine betaine transmethylase

PA3137 NC -3 NC Probable MFS transporter; drug resistance transporter

PA3146 wbpK -3 -2 NC Probable NAD-dependent epimerase/dehydratase WbpK

PA3147 wbpJ -3 -2 -2 Probable glycosyl transferase WbpJ

PA3186 oprB -4 NC NC Outer membrane porin OprB precursor

PA3187 gltK -7 NC NC Probable ATP-binding component of ABC transporter

PA3188 gltG -8 NC NC Probable permease of ABC sugar transporter

193 PA3189 gltF -7 NC NC Probable permease of ABC sugar transporter

PA3190 gltB -3 NC NC Probable binding protein component of ABC sugar transporter PA3191 gtrS -3 -2 -2 Probable two-component sensor; glucose transport sensor PA3192 gltR -3 -2 -2 Two-component response regulator GltR

PA3193 glk -3 -2 -2 Glucokinase; homoserine O-acetyltransferase activity

PA3221 csaA -6 -6 -5 Transcriptional regulators; export-related chaperone

PA3222 -6 -5 -4 Hypothetical protein; Predicted permeases

PA3233 -3 -7 -2 Hypothetical protein; Predicted signal-transduction protein containing cAMP-binding and CBS domains

PA3234 -2 -5 -2 Probable sodium:solute symporter

PA3235 -2 -7 -2 Conserved hypothetical protein; Predicted membrane protein PA3280 oprO -3 NC -2 Outer membrane porin OprO precursor

PA3291 -4 -4 -2 Hypothetical protein; putative lipoprotein

PA3292 -3 -3 -2 Hypothetical protein

PA3293 -3 -3 NC Hypothetical protein

PA3294 -4 -3 -3 Hypothetical protein; protein secretion by the type VI secretion system PA3327 -3 -2 -2 Probable non-ribosomal peptide synthetase

PA3328 -3 -2 -2 Probable FAD-dependent monooxygenase

PA3329 -4 -2 -5 Hypothetical protein

PA3330 -3 -2 -3 Probable short chain dehydrogenase

PA3331 -4 -2 -4 Cytochrome P450

PA3332 -3 -2 -3 Conserved hypothetical protein; Ketosteroid isomerase- related protein PA3333 fabH2 -4 -2 -4 3-oxoacyl-[acyl-carrier-protein] synthase III

PA3334 -3 -2 -3 Probable acyl carrier protein

PA3335 -3 -2 -2 Hypothetical protein

PA3359 -2 -3 NC Hypothetical protein

PA3379 phnH -4 NC -2 Conserved hypothetical protein; Phosphonate utilization

PA3380 phnG -6 NC -4 Conserved hypothetical protein; Phosphonate utilization

PA3383 phnD -10 NC -4 Binding protein component of ABC phosphonate transporter PA3384 phnC -3 NC NC ATP-binding component of ABC phosphonate transporter

PA3433 -7 -2 NC Probable transcriptional regulator

PA3486 -2 -4 -7 Conserved hypothetical protein; protein secretion by the type VI secretion system PA3487 pldA -3 -4 -3 Probable phospholipase; phospholipase D

PA3488 -3 -4 -2 Hypothetical protein

PA3498 -3 Inc -2 Probable oxidoreductase; phenylacetate catabolic process PA3507 -2 -4 -3 Probable short-chain dehydrogenase; fatty acid biosynthetic process PA3516 -4 Inc NC Probable lyase

PA3518 -7 -2 -3 Hypothetical protein; coenzyme PQQ biosynthesis protein C PA3522 -4 NC NC Probable RND efflux transporter

PA3531 bfrB -2 NC -5 Bacterioferritin

PA3575 -3 -2 -3 Hypothetical protein; Cytochrome B561

194 PA3591 -3 NC -2 Probable enoyl-CoA hydratase/isomerase

PA3595 -6 Inc Inc Probable MFS transporter

PA3602 -2 -3 -3 Conserved hypothetical protein; Glutamate synthase domain 2 PA3661 -2 -3 -10 Hypothetical protein

PA3669 NC NC -3 Hypothetical protein

PA3671 -3 NC -2 Probable permease of ABC transporter; involved in multi- copper enzyme maturation, permease component

PA3727 -3 NC -2 Hypothetical protein; Phosphatidylserine/phosphatidylglycerophosphate/cardiol ipin synthases and related enzymes

PA3728 -4 -2 -2 Hypothetical protein; Chromosome segregation ATPases

PA3729 -3 -2 -3 Conserved hypothetical protein; putative membrane protein PA3730 -4 -2 -3 Hypothetical protein

PA3761 nagE -2 -4 -2 N-Acetyl-D-Glucosamine phosphotransferase system transporter PA3775 -6 NC NC Hypothetical protein; Predicted permeases

PA3837 -3 -2 -2 Probable permease of ABC transporter

PA3838 -3 -2 -2 Probable ATP-binding component of ABC transporter; type I secretion system ATPase, LssB family

PA3870 moaA1 NC Inc -3 Molybdopterin biosynthetic protein A1

PA3871 -3 Inc NC probable peptidyl-prolyl cis-trans isomerase, PpiC-type

PA3874 narH -2 Inc -4 Respiratory nitrate reductase beta chain

PA3875 narG -2 Inc -3 Respiratory nitrate reductase alpha chain

PA3877 narK1 -3 Inc -2 Nitrite extrusion protein 1

PA3897 -5 NC -2 Hypothetical protein; Threonine/homoserine efflux transporter PA3905 -4 -2 -2 Hypothetical protein

PA3906 -4 -2 -2 Hypothetical protein

PA3907 -3 -2 -2 Hypothetical protein

PA3908 -3 -2 -2 Hypothetical protein

PA3926 -4 Inc -2 Probable MFS transporter; Sugar phosphate permease

PA3959 -4 -2 -2 Hypothetical protein

PA3967 -4 -2 -2 Hypothetical protein; Putative globin, Globins are heme proteins, which bind and transport oxygen

PA3991 -2 -4 NC Hypothetical protein; putative transporter

PA3994 NC NC -3 Probable epoxide hydrolase

PA4064 NC -3 NC Probable ATP-binding component of ABC transporter

PA4072 -5 NC Inc Probable amino acid permease

PA4074 NC -3 NC Probable transcriptional regulator

PA4082 cupB5 Inc -3 Inc Probable adhesin

PA4093 -4 NC NC Hypothetical protein; HGG motif-containing thioesterase, possibly involved in aromatic compounds catabolism

PA4103 -3 NC NC Hypothetical protein; ferric reductase domain-containing protein PA4128 -2 -3 -2 Conserved hypothetical protein; 2,4-dihydroxyhept-2-ene- 1,7-dioic acid aldolase PA4131 -3 -2 -3 Probable iron-sulfur protein; Polyferredoxin; respiratory chain complex IV assembly 195 PA4132 -2 -2 -3 Conserved hypothetical protein; GntR transcriptional regulator PA4133 -3 -2 -2 Cytochrome c oxidase subunit (cbb3-type); oxidation- reduction process PA4134 -3 -2 -2 Hypothetical protein

PA4137 -6 -4 -6 Probable porin

PA4138 tyrS -3 NC -3 Tyrosyl-tRNA synthetase

PA4148 -2 -4 -4 Probable short-chain dehydrogenase

PA4150 -3 -3 -2 Probable dehydrogenase E1 component

PA4169 NC -4 NC Conserved hypothetical protein; Predicted transcriptional regulator PA4170 -2 -6 NC Hypothetical protein; Thioredoxin reductase

PA4179 -2 -3 Inc Probable porin

PA4205 mexG -2 -2 -3 Hypothetical protein

PA4206 mexH -2 -2 -4 Probable RND efflux membrane fusion protein precursor

PA4207 mexI -3 -2 -4 Probable RND efflux transporter

PA4208 opmD -3 -2 -3 Probable outer membrane efflux protein precursor

PA4217 phzS -6 NC -5 Probable FAD-dependent monooxygenase; phenazine biosynthetic process PA4236 katA -3 NC -4 Catalase

PA4292 -3 NC -2 Probable phosphate transporter; Phosphate/sulphate permeases PA4295 fppA -2 -2 -4 Hypothetical protein; Flp prepilin peptidase A, Flp pilus assembly protein, protease CpaA

PA4307 pctC -3 -2 -2 Chemotactic transducer PctC

PA4317 -3 NC -2 Hypothetical protein; integral membrane protein

PA4318 -3 -2 NC Hypothetical protein; membrane protein, RDD domain- containing protein PA4330 NC -3 -2 Probable enoyl-CoA hydratase/isomerase; phenylacetate catabolic process PA4333 -3 -3 -3 Probable fumarase

PA4430 -3 -2 -2 Probable cytochrome b

PA4501 opdD -3 NC NC Probable porin; Glycine-glutamate dipeptide porin

PA4525 pilA -3 NC -2 Type 4 fimbrial precursor PilA

PA4526 pilB -3 -2 -3 Type 4 fimbrial biogenesis protein PilB

PA4550 fimU -3 NC NC Type 4 fimbrial biogenesis protein FimU

PA4551 pilV -3 NC NC Type 4 fimbrial biogenesis protein PilV

PA4552 pilW -3 NC NC Type 4 fimbrial biogenesis protein PilW

PA4553 pilX -3 NC NC Type 4 fimbrial biogenesis protein PilX

PA4554 pilY1 -4 NC NC Type 4 fimbrial biogenesis protein PilY1

PA4555 pilY2 -3 NC NC Type 4 fimbrial biogenesis protein PilY2

PA4556 pilE -3 NC NC Type 4 fimbrial biogenesis protein PilE

PA4571 -3 NC -2 Probable cytochrome c

PA4582 NC -3 NC Conserved hypothetical protein; Membrane protease subunits, negative regulation of proteolysis

PA4583 NC -3 NC Conserved hypothetical protein; translational termination

PA4587 ccpR -3 NC -3 Cytochrome c551 peroxidase precursor

PA4599 mexC -5 NC NC RND multidrug efflux membrane fusion protein MexC precursor PA4602 glyA3 -4 -4 -4 Serine hydroxymethyltransferase

196 PA4613 katB -4 -5 -2 Catalase

PA4616 -3 NC NC Probable c4-dicarboxylate-binding protein; tripartite ATP- independent periplasmic transporter solute receptor

PA4621 -5 -2 NC Probable oxidoreductase; Aerobic-type carbon monoxide dehydrogenase, large subunit CoxL/CutL homologs

PA4632 -2 -2 -3 Hypothetical protein; Zn-dependent protease with chaperone function PA4635 mgtC -4 NC -2 Conserved hypothetical protein; Mg2+ transport ATPase

PA4683 -4 NC -2 Hypothetical protein

PA4812 fdnG -2 NC -3 Formate dehydrogenase-O, major subunit

PA4822 -3 Inc -2 Hypothetical protein; Na+/phosphate symporter

PA4823 -4 Inc -10 Hypothetical protein

PA4824 -6 Inc -6 Hypothetical protein

PA4825 mgtA -16 NC -31 Mg(2+) transport ATPase, P-type 2

PA4826 -16 -2 -7 Hypothetical protein

PA4834 NC -3 NC Hypothetical protein; Threonine/homoserine efflux transporter PA4835 NC -3 NC Hypothetical protein

PA4849 -4 NC NC Hypothetical protein

PA4858 NC -3 NC Conserved hypothetical protein; urea ABC transporter, urea binding protein PA4860 -5 -3 -4 Probable permease of ABC transporter

PA4888 desB -3 -2 -2 Acyl-CoA delta-9-desaturase; Fatty acid desaturase

PA4889 -4 -2 -2 Probable oxidoreductase; phenylacetate catabolic process PA4894 -3 -2 NC Hypothetical protein; Hydrogenase/urease accessory protein PA4901 mdlC -2 NC -4 Benzoylformate decarboxylase

PA4973 thiC -4 -5 -5 Thiamin biosynthesis protein ThiC

PA4986 -3 -2 -2 Probable oxidoreductase

PA5088 NC -5 -3 Hypothetical protein; TPR repeat, SEL1 subfamily

PA5089 -2 -3 -2 Hypothetical protein; Phosphatidylserine/phosphatidylglycerophosphate/cardiol ipin synthases and related enzymes; Phospholipase D. Active site motif (weak) PA5114 -10 -3 -5 Hypothetical protein; Predicted membrane protein

PA5136 -3 NC -2 Hypothetical protein

PA5137 -7 NC -2 Hypothetical protein; ABC-type amino acid transport/signal transduction systems, periplasmic component/domain

PA5138 -4 -2 -2 Hypothetical protein; ABC-type amino acid transport/signal transduction systems, periplasmic component/domain

PA5139 -4 NC -2 Hypothetical protein; putative exported protein

PA5144 -2 -2 -6 Hypothetical protein; putative acetyltransferase

PA5154 -3 NC -2 Probable permease of ABC transporter

PA5207 -3 NC -2 Probable phosphate transporter

PA5256 dsbH -3 NC NC Disulfide bond formation protein

PA5264 -4 -2 -2 Hypothetical protein

PA5265 -6 -4 -3 Hypothetical protein

PA5266 -6 -3 -3 Conserved hypothetical protein; protein secretion by the type VI secretion system 197 PA5283 NC -4 -2 Probable transcriptional regulator

PA5300 cycB -2 -2 -3 Cytochrome c5

PA5351 rubA1 -3 -2 -2 Rubredoxin; electron carrier activity

PA5353 glcF NC NC -5 Glycolate oxidase subunit GlcF; iron-sulfur cluster binding

PA5354 glcE NC Inc -3 Glycolate oxidase subunit GlcE

PA5361 phoR -5 -8 -3 Two-component sensor PhoR

PA5368 pstC -3 -2 -2 Membrane protein component of ABC phosphate transporter PA5369 pstS -3 -2 -3 Hypothetical protein; phosphate ABC transporter, periplasmic phosphate-binding protein

PA5385 cdhB -3 Inc NC Hypothetical protein; Carnitine dehydrogenase-related gene B PA5388 -3 NC NC Hypothetical protein; choline ABC transporter, periplasmic binding protein PA5405 -2 Inc -4 Hypothetical protein

PA5417 soxD NC NC -6 Sarcosine oxidase delta subunit

PA5440 NC -2 -3 Probable peptidase; Collagenase and related proteases

PA5442 -2 -2 -4 Conserved hypothetical protein; diguanylate cyclase

PA5445 -2 -5 NC Probable coenzyme A transferase

PA5479 gltP -3 -2 -2 Proton-glutamate symporter

PA5514 -2 -5 NC Probable beta-lactamase

PA5532 -2 -7 -2 Hypothetical protein; Putative GTPases (G3E family); cobalamin synthesis protein PA5533 -2 -3 -2 Hypothetical protein

PA5534 -2 -9 -3 Hypothetical protein

PA5535 -2 -9 -2 Conserved hypothetical protein; Putative GTPases (G3E family); cobalamin biosynthetic process

PA5536 dksA2 NC -7 -2 Conserved hypothetical protein; DnaK suppressor protein; TraR/DksA family transcriptional regulator

PA5538 amiA NC -4 NC N-acetylmuramoyl-L-alanine amidase

PA5539 NC -4 NC Hypothetical protein; cofactor biosynthetic process; putative GTP cyclohydrolase PA5540 NC -4 -2 Hypothetical protein; Carbonic anhydrases/acetyltransferases, isoleucine patch superfamily PA5541 pyrQ NC -4 -2 Probable dihydroorotase

PA5542 -2 NC -3 Hypothetical protein; Beta-lactamase class C and other penicillin binding proteins PA5543 -3 NC -2 Hypothetical protein

PA5544 -3 NC -2 Conserved hypothetical protein; TRAP-type uncharacterized transport system, fused permease components PA5566 -3 Inc -4 Hypothetical protein

198 APPENDIX D

TABLE OF EARLY CLINICAL ISOLATES

Strain ID BHI Plates DTSB Low [Fe3+] DTSB High [Fe3+] (Jane Burns- phenotype) 24 Hours 48 hours 24 Hours 48 Hours 24 Hours 48 Hours

AMT0037-10 Non- Mucoid (±) Mucoid (±) Mucoid Non- Non-mucoid (mucoid) mucoid (± Mucoid (2+) mucoid (Brown) @ 3-4 (Yellow) days)

NC-AMT0205- Mucoid (4+) Mucoid Mucoid Mucoid Mucoid (4+) Mucoid (4+) 1 (4+) (4+) (4+) (White) (White) (mucoid) (White) (White)

AMT006-11 NM-Rough NM-Rough NM-Rough NM- NM-Rough NM-Rough (non-mucoid) (green) (green) Rough (no color) (Lt. Brown) (green)

AMT006-11 Rough to Smooth Mucoid Mucoid Non- Non-Mucoid – Smooth mucB mutant Smooth (green) (green) mucoid – to Rough Smooth to Rough

AMT0026-1 Non- Non- Non- Non- Non- Non-mucoid (non-mucoid) mucoid mucoid mucoid mucoid mucoid (Brown) (green) (green) (green) (green) (Lt. brown)

NC-AMT0103- Mucoid (4+) Mucoid Mucoid Mucoid Non- Mucoid (2+) 1 (4+) (4+) (4+) mucoid (Brown) (mucoid) (Green) (Green) (Lt. brown)

AMT0020-1 Non- Non- Non- Non- Non- Non-mucoid (non-mucoid) mucoid mucoid mucoid mucoid mucoid (Brown) (green) (green) (Lt. brown)

AMT0023-8 Non- Non- Non- Mucoid Non- Non-mucoid (mucoid) mucoid mucoid mucoid (2+) mucoid (Brown) (green) (green) (Lt. brown)

AMT0199-1 Non- Non- Non- Non- Non- Non-mucoid (non-mucoid) mucoid mucoid mucoid mucoid mucoid (Brown & Green) (green) (Yellow) (Brown)

Analysis of CF Isolates (Jane Burns (early isolates) – Univ. Washington)

Strain ID CAS (Culture) CAS (Supernatant) (Jane Burns-phenotype) # Zone Size (Halo/Growth) Zone Size OD

Iron Added (µM) → Low High Low High 199

AMT0037-10 (mucoid) 9/10, 9/10 11/9, 11/9 17,19 0,0 3.6 5.6

NC-AMT0205-1 13/10, 13/10 10/10,10/10 20,20 0,0 (mucoid) 4.4 5.7

AMT006-11 20/11,20/11 10/10,10/10 19,19 (non-mucoid) 3.8 0,0 6.8

AMT006-11 mucB mutant 17/12,17/12 12/12,12/12 19,20 0,0 3.7 6.3

AMT0026-1 (non-mucoid) 19/11, 19/11 10/10,10/10 19,20 0,0 3.6 6.7

NC-AMT0103-1 16/12,17/13) 10/10,10/10 20, 20 (mucoid) 2.7 0,0 7.2

AMT0020-1 18/12,18/11 10/10,10/10 18,18 0,0 (non-mucoid) 4.2 6.4

AMT0023-8 18/12, 17,12 10/10,10/10 23, 23 0,0 (mucoid) 3.9 7.3

AMT0199-1) 18/13,19/13 10/10,10/10 18, 17 0,0 (non-mucoid) 4.0 5.7

Analysis of PAO1 WT & MucABCD Mutants

Strain ID CAS (Culture) CAS (Supernatant)

Zone Size Zone Size OD

Iron Added (µM) → Low Fe Hi Fe Low Fe Hi Fe

PAO1 Wild Type (non-mucoid) 19/13,19/12 10/10,10/10 21, 20 0,0 3.9 5.8

MucA (mucoid) 18/12,18/12 10/10,10/10 20,20 0,0 3.7 6.1

MucB (mucoid) 17/13, 17/12 12/11,12/11 20, 20 0,0 3.7 5.3

MucC (mucoid) 17/11, 17,12 10/10,10,10 18,18 0,0 2.4 4.3

200 MucD (mucoid) 18/13, 18,13 10/10,10/10 22, 21 0,0 4.1 5.9

PAO1 Siderophore Mutants

PAO1 ∆pvdS 25/12, 25,12 12/12,12,12 Weak clearing 0,0 13,12 5.8 3.9 Weak clearing PAO1 ∆pvdA 25/12,25/12 12/12,12/12 13,13 0,0 3.7 5.3

Weak clearing PAO1 ∆pvdD 25/12,25/12 12/12,12/12 13,13 0,0 3.9 5.0

PAO1 ∆pchEF 18/11,18/11 11/11,11/11 20,20 0,0 3.6 5.4

No Reaction PAO1 ∆pvdD, ∆pchEF 14/12,13/12 13/13,13/13 0,0 0,0 3.7 5.2

201 APPENDIX E

TABLE OF LATE CLINICAL ISOLATES

Analysis of CF Isolates (Pradeep Singh – Univ. Washington)

Singh # Comments Brain Heart Infusion Agar PIA (Irgasan) (BHI) 24 Hours 48 Hours 24 Hours 48 Hours From CF patient mediastinum 3-4 Non-Mucoid Mucoid (4+) ND Mucoid (1) weeks after Lung Transplant (4+) Brown Mucoid Mucoid (4+) ND Mucoid [263] (3+)

Mucoid Mucoid (4+) ND Mucoid (3) (4+) Green Mucoid Mucoid (4+) ND Mucoid (4) (4+) Purple Mucoid (±) Non-Mucoid ND Non- (5) Mucoid

Non-Mucoid Non-Mucoid ND Mucoid (6) Green coloration on Agar (2+)

Non-Mucoid Mucoid (1+) ND Non- (7) From lingula of explanted lung Mucoid

Non-Mucoid Mucoid (1+) ND Mucoid (8) Sibling of #7 (RAPD) from same (2+) location Mucoid Mucoid (2+) ND Mucoid From CF patient with normal lung (±) (9) function – different colonial morphology than (10)

Mucoid Mucoid (4+) ND Mucoid (10) From CF patient with normal lung & Non- function – different colonial mucoid morphology than (9) Purple

Analysis of CF Isolates (Pradeep Singh – Univ. Washington)

Singh # Comments Dialyzed Trypticase Soy Dialysed Trypticase Soy (Chelexed) 24 hours (Chelexed) 48 hours

Iron Added (µM) → 0 5 100 0 5 100

(1) Very sparse growth, Mucoid Mucoid Mucoid Mucoid Mucoid Mucoid microcolonies (4+) (4+) (4+) (4+) (4+) (4+) Purple Brown color on all iron levels

[263] Tiny Colonies but uniform Mucoid Mucoid Mucoid Mucoid Mucoid Mucoid morphology (4+) (4+) (4+) (4+) (4+) (4+)

202 (3) Small colonies, uniform Mucoid Mucoid Mucoid Mucoid Mucoid Mucoid morphology (4+) (4+) (4+) (4+) (4+) (4+)

(4) Average to small Mucoid Mucoid Mucoid Mucoid Mucoid Non- colonies, some tiny (4+) (4+) (4+) (4+) (4+) mucoid colonies mixed with Mucoid (3+)

(5) Tiny colonies High Mucoid Mucoid Mucoid Mucoid Mucoid Mucoid pyoverdine production (1+) (1+) (1+) (1-2+) (1-2+) (1-2+) (yellow) in low Fe, brown in high Fe

(6) Very tiny colonies – NM NM NM NM NM NM difficult to phenotype @ 24 hrs.

(7) Small colonies, mixed Mucoid Mucoid NM Mucoid Mucoid NM rough & small (3+) (3+) (3+) (3+)

(8) Tiny colonies, Mucoid Mucoid NM Mucoid NM NM mixed rough & small (2+) (1+) (1-2+)

Small & Tiny colonies Mucoid Mucoid NM Mucoid Mucoid NM (9) (2+) (2+) (1+) (1+)

Tiny colonies yellow in Mucoid Mucoid NM Mucoid Mucoid NM (10) low iron plates (1+) (1+) (1+) (1+)

Pt3(B12) Mucoid Mucoid Mucoid Mucoid (11)

Ex9-R3(E8) Mucoid Mucoid Mucoid Mucoid (12)

Pt6(D8) Mucoid NM Mucoid NM (13)

Pt37(c6) Mucoid NM Mucoid NM (14)

Pt18(A3) Mucoid NM Mucoid NM (15)

Pt22(D2) Mucoid Mucoid Mucoid Mucoid (16)

Pt10(B3) Mucoid Mucoid Mucoid Mucoid (17)

Analysis of CF Isolates (Pradeep Singh – Univ. Washington)

Singh # Comments CAS (Culture) CAS (Supernatant)

Zone Size/Growth Size Zone Size OD

Iron Added (µM) → Low High Low High

9,9 (1) Very sparse growth, 10/9,10/9 No reaction 2.5 microcolonies Very weak, 0,0

203 Purple Brown color on all diffuse 2.1 iron levels 10, 10 [263] Tiny Colonies but uniform 11/10,11/10 No reaction 2.2 0,0 morphology Very weak, 4.2 diffuse

(3) Small colonies, uniform 15/10, 15/10 No reaction 15, 14 0,0 morphology 2.9 6.0 Clear

(4) Average to small colonies, some tiny 20/10, 20/10 11/10,11/10 13,13 0,0 colonies 4.1 6.3 Diffuse

(5) Tiny colonies High pyoverdine production 19/11,19/10 No reaction 0.0 0,0 (yellow) in low Fe, brown 3.0 2.0 in high Fe (no reaction)

(6) Tiny colonies 16/11, 15,11 No reaction 7,7 2.0 0,0 (very weak 3.1 diffuse)

(7) Small colonies, mixed 20/11,19/11 No reaction 17, 17 0,0 rough & small 4.0 4.4 Clear

(8) Tiny colonies, 19/11, 20/11 No reaction 18, 18 mixed rough & small 3.0 0,0 Clear 3.3

Small & Tiny colonies 20/11,20/11 11/10,11,10 13, 12 0,0 (9) 3.0 2.4 Diffuse Tiny colonies yellow in 21/11, 22/11 No reaction 18, 18 0,0 (10) low iron plates 3.2 4.9 Clear

204 APPENDIX F

BACTERIAL MEDIA RECIPES

LB Media

The following components were dissolved in a final volume of 1-liter ddHsO and sterilized by autoclaving. For agar plates, 15 g/liter of Bacto-agar was added before autoclaving. 10 g Bacto-tryptone 5 g Bacto-yeast extract 10 g NaCl

Brain Heart Infusion Media

Dissolve 37 g brain heart infusion in a final volume of 1-liter ddH2O and sterilize by autoclaving. For agar plates, 15 g/liter of Bacto-agar was added before autoclaving.

M9 Minimal Medium The following components were dissolved in a final volume of 500 mL sterile ddH2O. 500 µL 1 M MgSO4 final concentration: 1 mM 200 µL 250 mM CaCl2 final concentration: 0.1 mM 250 µL 50 mM FeCl2 final concentration: 0.025 mM 5 mL 20% glucose final concentration: 11 mM 100 mL 5x M9 salts

5x M9 salts The following components were dissolved in a final volume of 1-liter ddH2O and sterilized by autoclaving. 28.4 g Na2HPO4 (F.W. 142) final concentration: 40 mM 15 g KH2PO4 (F.W. 136) final concentration: 20 mM 2.5 g NaCl (F.W. 58.14) final concentration: 8 mM 5.0 g NH4Cl (F.W. 53.5) final concentration: 19 mM

1 M MgSO4 (F.W. 120.04) Dissolve 6.002 g in 50 ml ddHsO and filter sterilize.

250 mM CaCl2 (F.W. 147.02) Dissolve 1.838 g in 50 ml ddHsO and filter sterilize.

50 mM FeCl2 (F.W. 270.3) Dissolve 0.1352 g in 10 ml ddHsO and filter sterilize.

205 DTSB

The following components were dissolved in 90 mL of H2O:

30 g Trypticase Soy Broth 10 g Sodium Chelex-100 resin

The mix was stirred for 6 hours at room temperature and then poured into mwt 12-14,000 cut off membrane dialysis tubing. The mix was then dialysed against 900 mL dH2O overnight at 4˚C. If making solid media, 15 g/liter of Bacto-agar was added before autoclaving. The media was allowed to cool before the addition of the following filter sterilized components:

50 mL 1 M MSG 40 mL 25% Glycerol

For iron supplementation, various concentrations of FeCl3 can be added to the media.

206 APPENDIX G

ANTIBIOTICS AND CONCENTRATIONS

Working Concentration Antibiotic Grams per 500 mL E. coli P. aeruginosa Ampicillin 0.05 g 100 µg/mL - Kanamycin 0.025 g 50 µg/mL - Carbenicillin 0.375 g - 750 µg/mL Gentamicin 0.0075 g 15 µg/mL - Gentamicin 0.0375 g - 75 µg/mL Streptomycin 0.05 g 100 µg/mL - Streptomycin 0.250 g - 500 µg/mL Tetracyclin 0.0075 g 15 µg/mL - Tetracyclin 0.075 g - 150 µg/mL Irgisan 0.012 g - 25 µg/mL

207