Structural and Functional Relationship of Oprp and Opro Mutants of Pseudomonas Aeruginosa and Genome Annotation of Dietzia Maris by Sonalli Ganguly

Structural and Functional relationship of OprP and OprO mutants of Pseudomonas aeruginosa and genome annotation of Dietzia maris By Sonalli Ganguly

a Thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Biochemical Engineering

Approved Dissertation Committee

Prof. Dr. Roland Benz, Jacobs University, Bremen ______Name, title and affiliation of Chair Prof. Dr. Mathias Winterhalter Jacobs University, Bremen Name title and affiliation of Committee Member

Prof. Dr. Tilman Achstetter Hoschule Bremen, Bremen Name title and affiliation of Committee Member

Prof. Dr. Ulrich Kleinekathöfer Jacobs University, Bremen Name title and affiliation of Committee Member

Date of Defense: 29.06.2016 Department of Life Sciences and Chemistry

Statutory Declaration (Declaration on Authorship of a Dissertation)

I, Sonalli Ganguly hereby declare, under penalty of perjury, that I am aware of the consequences of a deliberately or negligently wrongly submitted affidavit, in particular the punitive provisions of § 156 and § 161 of the Criminal Code (up to 1 year imprisonment or a fine at delivering a negligent or 3 years or a fine at a knowingly false affidavit). Furthermore, I declare that I have written this PhD thesis independently, unless where clearly stated otherwise. I have used only the sources, the data and the support that I have clearly mentioned. This PhD thesis has not been submitted for the conferral of a degree elsewhere.

Bremen December 6, 2016 ______

Place Date

Signature

Dedicated to my lovely aunt Meenaa Gangully (Pisi)

Table of Contents

Statutory Declaration ...... 3 ABSTRACT ...... 10 ACKNOWLEDGEMENTS ...... 11 AIM AND OBJECTIVES ...... 14 CHAPTER 1 ...... 15 INTRODUCTION ...... 15 1.1 The bacterial cell envelope ...... 15 1.2 The Gram-negative bacteria cell envelope...... 15 1.2.1 The outer membrane ...... 16 1.2.2 The peptidoglycan layer ...... 17 1.2.3 The periplasm ...... 18 1.2.4 The inner membrane ...... 18 1.3 The phylum proteobacteria ...... 19 1.3.1 Pseudomonas aeruginosa: A Bad bug ...... 20 1.3.2 Pathogenicity and drug resistance of Pseudomonas aeruginosa ...... 20 1.3.3 Porins of Pseudomonas aeruginosa ...... 20 1.4 The cell wall of Gram-positive bacteria ...... 22 1.4.1 Mycolata as members of Gram-positive bacteria ...... 23 1.5 The genus Dietzia ...... 24 1.6 The genus Rhodococcus ...... 25 1.6.1 MspA like porins in Rhodococcus species ...... 26 1.7 Prokaryotic gene sequencing and prediction ...... 26 1.8 REFERENCES: - ...... 29 CHAPTER 2 ...... 32 STUDY OF ORTHOPHOSPHATE SPECIFIC PORIN OprP MUTANTS OF PSEUDOMONAS AERUGINOSA ...... 32 2.1 Summary ...... 32 2.2 Introduction ...... 33 2.3 Materials and methods ...... 36 2.3.1 Bacterial strains and plasmid ...... 36

2.3.2 pAS27 OprP single and double mutagenesis using the oprP gene in the E. coli plasmid ...... 37 2.3.3 Growth media, enzymes and reagents ...... 38 2.3.4 Extraction of cell wall proteins of OprP wild type and mutants from E. coli38 2.3.5 Purification of cell wall proteins of wild type and mutants from E. coli ...... 39 2.3.5.1 Mono Q and Fast-Protein Liquid chromatography (FPLC) ...... 39 2.3.5.2 SDS-PAGE and Western immunoblotting (WB) ...... 41 2.3.6 Bilayer assays ...... 41 2.3.6.1 Single channel measurements ...... 42 2.3.6.2 Titration Experiments ...... 44 2.4 Results ...... 45 2.4.1 Colony PCR confirmation of mutants and sequencing ...... 45 2.4.2 Extraction and purification of cell wall proteins of wild type and mutant proteins ...... 46 2.4.3 Channel forming activity of single and double mutants ...... 50 2.4.4 Comparison studies of substrate specificity of OprP wild type with its single and double mutants ...... 51 2.5 Discussion ...... 53 2.5.1 Swapping of the specificity with the mutants from wild type OprP and OprO ...... 53 2.6 REFERENCES ...... 54 CHAPTER 3 ...... 67 MUTANTS OF DIPHOSPHATE SPECIFIC PORIN OprO OF PSEUDOMONAS AERUGINOSA ...... 67 3.1 Summary ...... 67 3.2 Introduction ...... 68 3.3 Materials and methods ...... 69 3.3.1 Bacterial strains and plasmids ...... 69 3.3.2 Development of OprO wild type gene in E.coli ...... 71 3.3.3 Transformation of pTZ19R plasmid with oprO gene into E.coli cells ...... 72 3.3.4 Site- directed mutagenesis in oprO gene using mismatch nucleotides ..... 72 3.3.5 Expression and purification of OprO and OprO mutant recombinant proteins ...... 74 3.3.6 Lipid bilayer experiments ...... 75 3.4 Results ...... 77 6

3.4.1 Confirmation of presence of OprO wild type and tyrosine mutant genes in pTZ19R vector plasmid ...... 77 3.4.2 Cell wall extraction and purification of OprO and its mutants ...... 77 3.4.3 Single channel conductance of OprO double mutant does not show similar conductance as of OprP wild type...... 79 3.4.4 Substrate specificity studies of the proteins ...... 82 3.5 Discussion ...... 84 3.5.1 OprO mutants shows similar specificity as of OprP wild type and future aspects ...... 84 3.6 REFERENCES ...... 85 CHAPTER 4 ...... 87 DIETZIA MARIS GENOME ANNOTATION ...... 87 4.1 Summary ...... 87 4.2 Introduction ...... 88 4.3 Methods and material...... 89 4.3.1 Isolation of Dietzia maris genomic DNA and sequencing ...... 89 4.3.2 RAST (rapid annotation using subsystem technology) ...... 90 4.3.3 Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) 92 4.3.4 BLAST (Basic Local Alignment Search Tool)...... 94 4.4 Results ...... 95 4.4.1 Results from RAST ...... 95 4.4.2 Automatic annotation by PGAAP ...... 98 4.4.3 Manual annotation by BLAST ...... 100 4.5 Discussion ...... 100 4.6 REFERENCES ...... 101 CHAPTER 5 ...... 106 IDENTIFICATION OF MspA PROTEIN LIKE GENES IN RHODOCOCCUS SPECIES ...... 106 5.1 Summary ...... 106 5.2 Introduction ...... 107 5.3 Materials and methods ...... 108 5.3.1 Bacterial strains and culture ...... 108 5.3.2 Amino acid alignment of Rhodococcus MspA like proteins ...... 108 5.3.2 Purification of Rhodococcus ruber cell wall...... 110

5.4 Results ...... 111 5.4.1 MALDI- tof ...... 111 5.4.2 Single channel analysis of Rhodococcus ruber ...... 111 5.5 Conclusion ...... 112 5.5 REFERENCES ...... 113 CHAPTER 6 ...... 114 6.1 Conclusion ...... 114 ABBREVIATIONS ...... 116 List of figures ...... 118 List of Tables ...... 124 Appendix ...... 126

Most people say that it is the intellect which makes a great scientist. They are wrong: it is character.

Albert Einstein

ABSTRACT

Porins are water-filled pores across the outer cell membrane of Gram-negative bacteria, Gram-positive (mycolata) bacteria, mitochondria and chloroplast which allows diffusion of hydrophilic molecules across the membrane. This thesis focuses on the study of pores from the Gram-negative bacteria, Pseudomonas aeruginosa and the Gram-positive bacteria, Dietzia maris and Rhodococcus ruber.

Pseudomonas aeruginosa can be found in a very vast environment such as soil, water, humans, animals, plants, sewage, and hospitals. Outer membrane of Pseudomonas aeruginosa consists of many specific porins. Out of these specific porins, OprP and OprO are phosphate specific porins, regulated at phosphate deficiency, which is an essential component for various biochemical activities inside the cell. For understanding the function of various amino acids in the lumen of these pores, two specific amino-acid were selected in OprP and OprO and they were interchanged. Two tyrosine at position62 and 114 positions in OprP were mutated to phenylalanine and aspartic acid of OprO. The double mutant of OprP behaved similar to OprO in single channel conductance and substrate specificity. Similarly, we also mutated phenylalanine at position 62 and aspartic acid at position 114 in OprO with two tyrosines. In this case, also OprO double mutant behaved similar to OprP in substrate specificity but the single channel varied conductance.

Dietzia maris and Rhodococcus ruber are mycolic acid containing Gram-positive bacteria. They have high G+C content. Whole genome of Dietzia maris was sequenced and annotated with automatic annotation by Rapid Annotations using Subsystems Technology (RAST) server and Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) and manual annotation by NCBI-Basic Local Alignment Search Tool (BLAST). Different species of Rhodococcus were searched to find homologous porins of MspA, which is a major porin in Mycobacterium smegmatis. This porin is used for nanopore–based DNA sequencing. Our aim of finding homologues for this porin in Rhodococcus species is to use them for sequencing technology as future aspect.

ACKNOWLEDGEMENTS

There are no secrets to success. It is the result of preparation, hard work, and learning from failure ~ by Colin Powell

My PhD degree is a combination of preparation, hard work and learning from failures. It was an awesome journey and a dream come true. The path of my PhD is a mixture of good and bad memories. And finally, I could achieve, what was meant to be achieved. This dissertation would not have been conducted without guidance and motivation of my supervisors, help from my friends, support and encouragement from my family.

Firstly, I would like to express my gratitude to my research supervisor Prof. Roland Benz for providing me a chance to work as a PhD student in his research group also for his constant encouragement, guidance and support throughout my PhD study. The numerous scientific discussions with him were a never-ending source of inspiration. He helped me enter the door of scientific life in its full depth. There are so many things I could say and for now I just want to say thanks for the endless support. I also want to thank Prof. Mathias Winterhalter for providing me with financial support and for his support and insight on my projects. I am happy to have you as my supervisor and my thesis committee member.

I would also like to thank Prof. Ulrich Kleinekathöfer for his kind advices and his collaboration in the Pseudomonas project and for being part of my thesis committee. I am thankful to Prof. Miguel Viñas for his valuable suggestion and constant help in genome annotation of Dietzia maris project.

I am extremely grateful to all the members of the present and past members of Winterhalter and Benz group for a dynamic, friendly, and enjoyable atmosphere. And, want to thank lab members of Nevoigt group for sharing the lab with us and for your co-operation and healthy atmosphere. I would like to thank Dr. Ivan Barcena Urribarri, Dr. Daniel Pletzer, Dr. Yann Barbot, Dr. Yvonne Braun, Dr. Jules Philippe, Su Hlaing Tint, Dr. Narges Abdali, Dr. Steve Swinnen, Solvejg Sevecke, Dr. Mathias Klein, Dr. Naresh Niranjan Dhanasekar, Farhan Younas, Ping-Wei Ho, Zia-Ul Islam, Miguel Angel Fernandez Niño, Dr. Tatiana Kolesnikova, Dr. Alexander Neveshkin, Dr. Harsha Bajaj, Satya Prathyusha Bhamidimarri, Usha Lamichhane, Pratik Raj Singh, Gowri Shankar, Ishan Ghai, Funda Citak, Dr. Samaneh

ACKNOWLEDGEMENTS

Mafakari, Federico Amodeo, Lorraine Benier, Alena di Primio, Al-Ghaili A. Hashem for sharing their enthusiasm, for their scientific advices and helpful discussions. I also want to thank Sabine Meier Team assistant of Research II and MOLIFE Center. And, I want to thank Svenja Frischholz.

I would like to thank Dr. Niraj Modi, Karunakar Potula and Anusha Kesi Reddy from Kleinekathöfer group for their collaboration in the Pseudomonas project. I would also like to thanks M. Guadalupe Jimenez-Galisteo for her constant help for during Dietzia maris annotation project.

My heartfelt gratitude goes to the person without whom my stay in Bremen would not have been so pleasant and wonderful without her support, my lovely grandma Marlene Georgi. I would also like to thank my friends in India, without whom I wouldn’t have achieved this success Pratibha, Geeta, my sweet baacha Ashwin, Shiva, and also my friends here in Germany, Savan Bhatt, Aniket, Sankalita mandal and many more.

I would like to thank my family back home for their blessings have helped me become the person I am today. I am grateful to my mom and dad especially my aunt for their constant love and care and their support at the darkest of times. I would like to thank my sister Shofale, and brother Ronit who has been like a friend and a support to me throughout my life. At last, I thank the Almighty for all the wonderful things that he has done to me so far.

ACKNOWLEDGEMENTS

Funding

This PhD thesis has received support from the Innovative Medicines Initiatives Joint Undertaking under Grant Agreement No. 115525, resources that are composed of financial contributions from the European Union’s seventh framework programme (FP7/2007-2013) and European Federation of Pharmaceutical Industries and EFPIA companies’ in-kind contribution.

The research PhD stipend was provided by the work group budget of Prof. Dr. Roland Benz / Prof. Dr. Mathias Winterhalter in Jacobs University, Bremen.

AIMS AND OBJECTIVES

AIM AND OBJECTIVES

The aim of this PhD thesis was to get an insight on structure and functioning of various outer membrane proteins present in bacteria which helps in uptake of different nutrients from the environment for their growth. Characterization of these porins gives an insight into the physiology of various organisms in their habitat as well as transport of different molecules through it which can be later exploited for different antibiotic approaches.

The first part of the project is focused on gram-negative bacteria Pseudomonas aeruginosa, which is an opportunistic pathogen and multidrug resistant, which has become concern for many research projects. This bacterium has a permeability barrier for general undefined molecules and contains mostly specific porins in its outer membrane. Out of different types of specific porins, OprP and OprO are phosphate specific porins. Both porins are expressed during phosphate deficiency. The main aim of the project is to identify and study the role of few amino acids that differ in both porins near the constriction zone and to mutate and to study these mutants in bilayer. These amino acids were found to be important in binding of phosphate and diphosphate to the porin and also for its translocation. Changing of the amino acids of OprP to OprO and OprO to OprP can reverse completely the behavior of the channel porin. This gives an idea of how an evolution would have evolved and how it affects the characteristics of the porin.

The second part of the project is focused on Gram-positive bacteria, actinomycetes such as Dietzia maris and Rhodococcus species. The whole genome sequence of Dietzia maris was annotated. Different genes and proteins were had to be predicted. Each sequence is annotated individually with different annotating softwares. In previous studies, Dietzia maris showed presence of porin in cell wall extracts, but the sequence of the porin is unknown. So, our goal is to find the sequence of this porin.

Different porin proteins having sequence similarity to MspA protein was found in Rhodococcus species. These protein sequences of Rhodococcus were aligned with reference to MspA protein and Rhodococcus Ruber was found to be having sequence similar to MspA porin. Rhodococcus Ruber showed four genes which has similar sequence to MspA. The objective of this project was to clone and characterize the porins of Rhodococcus Ruber.

INTRODUCTION

CHAPTER 1

INTRODUCTION

1.1 The bacterial cell envelope

Unlike the cells of higher organisms, the bacteria have to face an unpredictable, dilute and hostile environment. To survive and to protect it against these challenges of environment, bacteria have evolved a sophisticated and complex cell envelope. This cell envelope protects the cell from osmotic lysis by retaining water and creating a stable osmotic environment with in the cell. These cell envelopes also mediate the uptake of nutrients into the cell and the export of waste and toxins to the outside of the cells. The main components of the cell envelopes of Gram-positive and Gram-negative bacteria are plasma membrane, the periplasmic space (especially in Gram-negative bacteria) containing the peptidoglycan layer and the outer membrane.

1.2 The Gram-negative bacteria cell envelope

During the Gram-staining procedure, these cells lose their primary stain, also called as Gram- stain i.e crystal violet but retain secondary stain i.e safranin and appear pink in color. Thus, these cells are known as Gram-negative bacteria. Gram-negative bacterial cell envelope is made up of outer membrane, periplasm and inner membrane starting from outside towards inside. Gram-negative bacteria consist of a thin peptidoglycan layer when compared to Gram- positive bacteria (Figure 1.1).

INTRODUCTION

1.2.1 The outer membrane

Outer membrane (OM) is the foremost layer in Gram-negative bacteria. It is formed by lipid bilayer membrane and composed of mainly proteins, phospholipid and lipopolysaccharides (LPS) (1). LPS plays an important role in assembly and maintenance of the OM as permeability barrier (2). LPS is also a unique molecule which represents an endotoxin, which can stimulate mammalian immune response. LPS consists of three components: - Lipid A, an R-polysaccharide and an O polysaccharide. It is also referred as a cell-associated toxin. Toxicity of LPS is associated with the lipid component (lipid A) and its immunogenicity factor is associated with the polysaccharide component (O-antigen) of LPS.

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Figure 1.1:- A typical gram negative cell wall consists of an outer membrane, a periplasmic space composed of peptidoglycan layer and an inner membrane. The outer leaflet of the outer membrane is made up of lipopolysaccharides (LPS). LPS is composed of Lipid A, an R-polysaccharide, and an O polysaccharide (3).

In Gram-negative bacteria, the OM proteins are divided into two classes, lipoproteins and ß- barrel proteins. Lipoproteins contain lipid moieties that are attached to an amino-terminal of cysteine residue embedded in the OM (3). ß-barrel proteins are trans-membrane proteins that have the structure of a ß-barrel folded into cylinders and are known as outer membrane proteins (OMPs). Some of the OMPs such as the porins, are responsible for passage of small

INTRODUCTION hydrophilic molecules across the outer membrane. OmpF is one of the well-studied passive diffusion porins in E. coli (4).

1.2.2 The peptidoglycan layer

Gram-negative bacteria contain a thin layer of peptidoglycan layer mainly consisting of one or two layers, outside the cytoplasmic membrane. Peptidoglycan acts as support and gives strength to the cell wall. The rigidity of the peptidoglycan provides the shape and controls the size of the bacteria. It also protects the cells from osmolysis because it withstands the turgor pressure of the cell. Peptidoglycan is made of a copolymer of disaccharides (glycan- moiety) backbone cross-linked by ß 1, 4 linked N-actylglucosamine (NAG) and N-actylmuramic acid (NAM) (Figure 1.2). Rows of NAG and NAM are linked by polypeptides (peptide- moiety). The structure of the polypeptide cross-bridges may vary in numbers but they always have a tetrapeptide side chain, which consists of 4 amino acids attached to NAMs. These amino acids may occur in alternating D and L forms.

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Figure 1.2:- Structure of peptidoglycan in cell wall and N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) joined as in peptidoglycan. Alternate molecules of NAG and NAM form a carbohydrate backbone (the glycan portion). Rows of NAG and NAM are linked by polypeptides (the peptido- portion). (copyright © 2010 Pearson Education, Inc.).

Peptidoglycan in bacteria is also called murein as they contain N-acetylmuramic acid, which is a defined component of murein. Except in archaea bacteria where it contains proteins,

INTRODUCTION polysaccharide, or peptidoglycan-like structures but not murein (5). The OM of bacteria is attached to peptidoglycan by these murein lipoproteins (6)).

1.2.3 The periplasm

Periplasmic space is present underneath the OM layer and above cytoplasm of bacterial cell wall. It is a sort of gel-like matrix located in Gram-negative bacteria, due to presence of high concentration of proteins and peptidoglycan found within it. It contains precursors of mono- and oligosaccharides, amino acids, peptidoglycans and also proteins that may function as binding proteins for small solutes and also for reception of extracellular signals. These binding proteins in combination with other cytoplasmic membrane proteins, can release the bound compounds, which can then be transported into the cytoplasm of the bacteria. In the cytoplasm, secreted proteins are synthesized containing N-terminal signal peptides, which are directed through plasma membrane with the help of various protein transporting machineries, later they are processed and targeted to the site of functioning (7).

1.2.4 The inner membrane

Below the periplasmic space is the inner membrane, known as cell membrane or plasma membrane. It acts as a real barrier between the external environment and the cell interior. This membrane contains the receptors, which can sense the external environment and controls the import of nutrients and the export transport of toxic substances and waste products (3). It has many membrane-associated functions like energy production, lipid biosynthesis etc. The inner membrane is a phospholipid bilayer and is composed mainly of phosphatidyl ethanolamine and phosphatidyl glycerol.

INTRODUCTION

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Figure 1.3:- Fluid-mosaic model. The plasma membrane is composed of phospholipid molecules, integral proteins, carbohydrates bound to proteins forming glycoproteins or lipids forming glycolipids. The lipids in the plasma membrane are arranged in such a way that their polar heads are facing outward while their hydrophobic side chains face inward. ( http://johnfredycastro.blogspot.de/2011_07_01_archive.html).

The structure of plasma membrane can be explained with different structural models. One of the well-established models is the Fluid-mosaic model (8) (Figure 1.3), where it suggested that the plasma membrane is made up of phospholipids and proteins. The phospholipids are arranged in such a way that their polar heads form the outermost and hydrophobic, non-polar side chains which are insoluble in water forms the inner core of the membrane.

1.3 The phylum proteobacteria

Proteobacteria got their taxonomical origin as ‘purple bacteria’ which contained four bacterial sub groups (alpha, beta, gamma, and delta) which were classified depending on their 16S rRNA gene sequence. The phylum was divided into five constitutive classes, all containing Gram-negative bacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria (9).

INTRODUCTION

Out of these proteobacteria, Pseudomonas belongs to Gammaproteobacteria. The Gammaproteobacteria are a diverse group that comprise free-living, pathogenic and endosymbiotic members. Morphologically they appear as rods, curved rods, cocci, spirilla and filamentous. This phylum includes bacteria like Escherichia coli, the well-known pathogens Salmonella, Yersinia, Vibrio and Pseudomonas, and few other species such as Coxiella and Franscisella (10).

1.3.1 Pseudomonas aeruginosa: A Bad bug

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative, rod shaped bacterium. It is an important opportunistic human pathogen, which causes a wide range of diseases, including some life-threatening diseases (10). The OM of P. aeruginosa plays an important role as permeability barrier, and uptake molecules depending on the size of the solute. In the OM, certain class of proteins forms water-filled pores (porins) which allow the passage of hydrophilic solutes under a certain molecular mass.

1.3.2 Pathogenicity and drug resistance of Pseudomonas aeruginosa

P. aeruginosa affects persons having impaired immune response such as patients suffering from cystic fibrosis, cancer or AIDS (11, 12) and causes nosocomical (hospital-acquired) infections. It forms biofilms on different types of surfaces, niches, water pipes, drinking sources, and other equipments in hospitals (12). Its genome carries many chromosome- mobilizing plasmids, which represents a specific characteristic of the organism, and may be involved in its pathogenicity and antibacterial resistance. Another reason for showing antibiotic resistance is presence of specific porins.

1.3.3 Porins of Pseudomonas aeruginosa

In general, porins are divided into two categories, non-specific or general diffusion and specific porins (13). General diffusion porins or non-specific porins are those which allow

INTRODUCTION passage of any hydrophilic molecule smaller than the size compared to the exclusion limit of the channel. The major non-specific porin of P. aeruginosa outer membrane is OprF (outer membrane protein F), which forms a wide and water-filled outer membrane channel and has a low pore-forming activity because only about 5% of OprF in the outer membrane are in a pore-forming configuration, i.e. in a 16-stranded ß-barrel cylinder (14), which corresponds to the 3D-structure of many general diffusion porins of Gram-negative bacteria. The ß-strands of these porins are not perpendicular to the membrane plane but tilted at an angle from the plane of the membrane and are connected by loops exposed to the cell surface and by short turns at the side of the periplasmic space (13).

Another type of porins family is specific porins. These porins contain binding-site for classes of solute inside lumen of their pore. Only certain substrates can bind to these sites compared to other molecules of same size. Regarding the structure of specific porin, relative little information is known compared to general porins. P. aeruginosa has evolved many more specific porins than general diffusion porins for the uptake of different molecules needed for nutrition and survival.

P. aeruginosa genome contains a major family of genes encoding 19 specific porins belonging to the OprD porin family. The OprD family is the largest family of substrate- specific outer membrane channels. OprD itself is a specific channel-forming protein and uptakes basic amino acids and peptides. The OprD homologues are likely to be specific for a wide range of nutrients, which highly contributes to the limited outer membrane permeability of P. aeruginosa (15). This can be one of the reasons why this organism is resistance towards many antibiotics.

Another specific porin of P. aeruginosa is OprB, which is specific for glucose and is induced in the outer membrane when cells are grown in media containing glucose as the carbon source. Substrates regulate it such as glucose, fructose, glycerol, and mannitol (16). Another substrate specific porin is OprE, which is induced when P. aeruginosa is grown anaerobically with nitrate as electron receptor. OprP and OprO also belong to OprD family; they are specific for phosphate and pyrophosphate respectively. OprP is a true specific channel, as it has a higher affinity for phosphate than for any other molecule of same size (17). Beside these porins, also other porins are present in P. aeruginosa. In addition, it contains gene coding for Ton-B dependent receptors or gated porins (FpvA, FptA, PfeA, PirA HasR, PhuR) and efflux pumps (OprM, OprJ, OprN, OpmG, OpmH, OpmI, AprF) (2).

INTRODUCTION

1.4 The cell wall of Gram-positive bacteria

In the Gram-staining procedure, these cells appear violet in color due to presence of thick layer of peptidoglycan layer on their outer surface. These layers take up the dye and appear violet in color under microscope during the four-step staining procedure. This peptidoglycan layer forms a barrier for the dye such that it is not washed off after application of destaining solution. That is the reason, why these cells are called Gram-positive because they retain the crystal violet stain or Gram-stain. Peptidoglycan in Gram-positive microorganisms is densely functionalized with anionic glycopolymers called wall teichoic acids. These glycopolymers are made of glycerol phosphate, glucosyl phosphate or ribitol phosphate repeats. As seen in Figure 1.4, there are two major teichoic acid derivatives – wall teichoic acids (WTAs), which are attached to peptidoglycan, and lipoteichoic acids (LTAs), which are attached to the cell membrane (18). Wall teichoic acids are composed of a disaccharide linkage unit and are linked via a phosphodiester bond to N-actylmuramic acid residue in peptidoglycan. Lipoteichoic acids are similar to wall teichoic acids and are composed of polyGroP polymers. They extend from the plasma membrane into the peptidoglycan layer. Teichoic acids count for a significant fraction of the cell wall mass in the organism and their functions may vary depending on the species.

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Figure 1.4:- Scheme of a typical gram positive cell wall. It has a thick peptidoglycan layer functionalized with teichoic acid. Teichoic acids occur in two types- cell wall teichoic acids and lipoteichoic acids. The latter ones extend into the cytoplasmic membrane (19)

INTRODUCTION

In addition to teichoic acids, the surfaces of Gram-positive bacteria consist of a variety of proteins, some of which are similar to proteins found in the periplasm of Gram-negative bacteria (20). These proteins retain themselves near or in the membrane. Some contain membrane-spanning helices and some are attached to the lipid submerged in the membrane. Other proteins are covalently attached or tightly associated with peptidoglycan (21). Others bind to teichoic acids. In Gram-positive bacteria, the peptidoglycan layer does not represent a diffusion barrier for the passage of nutrients and for export of wastes.

1.4.1 Mycolata as members of Gram-positive bacteria

The cell wall structure of mycobacteria and related organism is more complex than that of “classical” Gram-positive bacteria. Mycolata contain layer of mycolic acids on the surface of the cells. These mycolic acids are covalently attached via arabinogalactan to the peptidoglycan- skeleton of the cell wall of Gram-positive bacteria and form a thick waxy coating around it (Figure 1.5). These mycolic acids layer represents a secondary permeability barrier besides the cytoplasmic membrane which is similar to the OM of Gram-negative bacteria which means that the mycolic acid layer has a similar function as the OM of Gram- negative bacteria. Bacteria of the order Mycolata (Corynebacteriales) are generally rich in G+C contents.

INTRODUCTION

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Figure 1.5:- Structure of outer membrane of Mycolata (Corynebacteriales) (22). In mycolata, the mycolic acids are attached via an arabinogalactan skeleton to the peptidoglycan.

The members of mycolata (Corynebacteriales) are Corynebacteria, Dietziaceae, Gordonaceae, Mycobacterium, Nocardiaceae, and Tsukamurellaceae. The length of the mycolic acid varies in different genera of mycolata. The smallest ones are found in corynebacteria with 22-38 total carbons and the longer and complex ones are found in mycobacterium with a size of 60-90 carbons. The first mycolic acid containing structure was studied in detail in the pathogenic organisms Mycobacterium tuberculosis and Mycobacterium leprae.

On the surface of mycolata, certain water-filled channels are present which allows the permeation of hydrophilic solutes called porins similar to the situation in Gram-negative bacteria (Figure 1.5). MspA porin was found in M. smegmatis and was characterized in detail.

1.5 The genus Dietzia

The morphology of Dietzia species was initially confused with Rhodococcus species due to the presence of similar morphological characteristics and chemotaxonomic characteristics. Later on in1995 Reiney et al (23) based on comparison of 16S ribosomal RNA (rRNA) sequences and unusual structure of polar lipids and presence of short chain mycolic acids classified it as a separate genus Dietzia. Phenotypically this genus forms a part of mycolic

INTRODUCTION acid containing group consisting of genera Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, Tsukamurella and Gordonia. Dietzia species are generally found in soil or intestinal tracts of carp. Dietzia species are morphologically Gram-positive, aerobic, short, rod or coccoid like non-motile, non-endospore forming, non-acid fast, oxidase positive and catalase positive. The colonies of Dietzia species appear as circular, raised or convex, glistening, orange to coral red colonies with entire edges sticking to the agar media as could be seen in Figure 1.6 (24).

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Figure 1.6:- Colonies of different Dietzia species on iso sensitest agar after 48 hours of incubation at 36°C (24).

These Dietzia species are mainly used in biodegradation, bioremediation, industrial fermentation, and carotenoid pigmentation as they possess the enzymes for these activities. At the beginning of 21st century, the Dietzia species such as D. cinnamea, D. maris, and D. papillomatosis emerged as new pathogens. They are found to effect human beings with weak immune systems.

1.6 The genus Rhodococcus

Rhodococci are generally aerobic, gram positive, non-motile with filamentous rods, mycolate-containing nocardioforms. They are isolated from a large variety of sources such as

INTRODUCTION soils, rocks, boreholes, ground water, marine sediments, animal dung, gut of insects and also from healthy and diseased animals and plants. They have the ability to chemically transform or degrade many chemical compounds. Due to which they are used in environment and industrial biotechnology. One of the species of Rhodococci, Rhodococcus equi is a known pathogen since 70 years. It causes lung and respiratory infections in a wide range of animals and birds (25). In humans, Rhodococcus equi affects immunosuppressed individuals especially infected with human immunodeficiency virus (HIV), tumors, leukemia, lymphoma and alcoholism, people undergoing organ transplants.

1.6.1 MspA like porins in Rhodococcus species

On the cell wall of Rhodococci many porin proteins are reported to be present. Cation selective porin channels are isolated from many Rhodococcus species and Rhodococcus equi. Some of these porins are prototype of MspA proteins from Mycobacterium smegmatis with significant homology. Other novel channel forming proteins are also present on the surface of Rhodococcal cell envelope (26).

1.7 Prokaryotic gene sequencing and prediction

After the whole genome of an organism is sequenced, annotation of genes are required. Genome annotation is an important step for extraction of the useful information from the genomes (27). Annotation of genome often consists of running an automatic pipeline followed by manual correction of the results. High-quality annotation is not only using gene predicting software and annotating the gene from its closest relative, but also to include of coding sites (CDS), ribosomal-binding sites (RBSs), termination sites and conserved motifs/domains. This annotation method not only gives full features of annotation but also rectify the errors from earlier parts of the annotation process (27).

INTRODUCTION

Note: - This figure is hidden due to copyright issues

Figure 1.7:- Flow chart of annotation of prokaryote genome (27). The nucleotide sequences are present in FASTA format. Different genes are predicted with different software. Then each gene is taken and its homologues or any conserved domain is tried to find. Depending on the predicted domains, annotation is added to the gene. Later, any other information related to its tRNA etc are added to it.

A diagrammatic presentation for annotation of bacterial gene has been show in Figure 1.7. The general process is to look for closely related strain/serovar which has already been sequenced and annotated. Next step is to use the gene prediction software to predict coding regions in the genome of interest. Once the coding regions have been identified, they are aligned with a reference genome annotation or with an entry of UniProt using fast sequence alignment tools, FASTA or Basic Local Alignment search tool (BLAST). Top hits are accepted as homologs and the annotation is transferred across for genes showing high similarity with the gene of interest. The last step of annotation is adding of other features like tRNA`s and rRNA`s with the help of different prediction software (28).

INTRODUCTION

Along with advantages of this annotating software, there are many limitations associated with it. Many times the genes of the organism are annotate depending on the presence of close relative but this is paradoxical because we are trying to find the differences between these strains but using a similarity based method to annotate it and sometimes genes of interest may not be annotated because they are not in the reference genome. Gene prediction software sometimes assigns the wrong start/terminator sites. Along with it misannotation and errors may occur as many genomes which are currently available may have been annotated using old or out of date techniques. Another limitation of annotation is to produce inconsistence annotation due to presence of different gene prediction software, which may produce split/fuses and false conserved domains genes or coding regions. Other limitations include spelling mistakes, same gene name but different product name, producing of many hypotheticals named proteins (28).

INTRODUCTION

1.8 REFERENCES: -

1. Kamio Y, Nikaido H (1976). Outer membrane of Salmonella typhimurium: accessibility of phospholipid head groups to phospholipase c and cyanogen bromide activated dextran in the external medium. Biochemistry, 15(12): p.2561-70. 2. Benz, R. (2003). Porins - structure and function. In Microbial transport systems. Edited by G. Winkelmann. Weinheim: Wiley-VCH, p. 227- 246. 3. Silhavy. T.J; Kahne.D; and Walker.S (2010). The Bacterial Cell Envelope. Cold Spring Harb Perspect Biol; 2: a000414, p. 1-16. 4. Bauer.K, Schmid I.A, Boos. W, Benz.R and Tommassen. J (1988). Pore formation by pho-controlled outer-membrane proteins of various Enterobacteriaceae in lipid bilayers. Eur. J. Biochem, 174 (1), p.199-205. 5. Vollmer.W and Bertsche.U (2008). Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli. Biochim Biophys Acta, 1778 (9), p. 1714–34. 6. Braun.V (1975). Covalent lipoprotein from the outer membrane of Escherichia coli. Biochim Biophys Acta, 415, p.335 –377. 7. Goemans C, Denoncin K, Collet JF (2014). Folding mechanisms of periplasmic proteins. Biochim Biophys Acta, 1843(8), p.1517-28. 8. Singer SJ, Nicolson GL (1972). The fluid mosaic model of the structure of cell membranes. Science, 175 (4023).p.720–31. 9. Williams.K.P and Kelly.D.P (2013). Proposal for a new class within the phylum Proteobacteria, Acidithiobacillia classis nov., with the type order Acidithiobacillales, and emended description of the class Gammaproteobacteria. Int J Syst Evol Microbiol, 63 (Pt 8), p.2901-6. 10. Hancock R. E. W (1987). Role of Porins in Outer Membrane Permeability. J Bacteriol, 169(3).p. 929–933. 11. Govan.JR, Deretic.V (1996). Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev, 60(3).p.539-74. 12. Botzenhardt, K., and Doring, G. (1993). Ecology and epidemiology of Pseudomonas aeruginosa. Pseudomonas aeruginosa as an Opportunistic Pathogen. Edited by Mario Campa, Mauro Bendinelli, Herman Friedman. Springer US, p. 1-18.

INTRODUCTION

13. Sukan.A, Hancock R. E. W (1995). Insertion mutagenesis of the Pseudomonas aeruginosa phosphate specific porin OprP. J Bacteriol,177(17), p. 4914-20. 14. Rawling EG, Martin NL, Hancock RE (1995). Epitope mapping of the Pseudomonas aeruginosa major outer membrane porin protein OprF. Infect Immun; 63(1), p. 38-42. 15. Tamber S, Ochs MM, Hancock RE (2006) Role of the novel OprD family of porins in nutrient uptake in Pseudomonas aeruginosa. J Bacteriol, 188 (1), p.45-54. 16. Adewoye.L.O, Worobec.E.A. (1999). Multiple environmental factors regulate the expression of the carbohydrate-selective OprB porin of Pseudomonas aeruginosa. Can J.Microbiol, 45, p.1033–42. 17. Hancock.R.E.W and Benz R, (1986). Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa. Biochim. Biophy.Acta, 860(3).p. 699- 707. 18. Neuhaus FC, Baddiley J (2003). A continuum of anionic charge: structures and functions of D-alanyl-teichoic acids in gram-positive bacteria. Microbiol Mol Biol Rev, 67(4), p. 686-723. 19. Wang. G, Mishra. B, Epand R. F, Epand R. M (2014). High-quality 3D structures shine light on antibacterial, anti-biofilm and antiviral activities of human cathelicidin LL-37 and its fragments. Biochimica et Biophysica Acta 1838 p.2160–2172. 20. Dramsi S, Magnet S, Davison S, Arthur M (2008). Covalent attachment of proteins to peptidoglycan. FEMS Microbiol Rev, 32(2), p. 307-20. 21. Scott JR, Barnett TC (2006). Surface proteins of gram-positive bacteria and how they get there. Annu Rev Microbiol, 60, p. 397-423. 22. Brown.L, Wolf.J.M, Prados-Rosales.R and Casadevall.A (2015). Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi. Nat Rev Microbiol, 13(10), p. 620-30. 23. Rainey FA, Klatte S, Kroppenstedt RM, Stackebrandt E (1995). Dietzia, a new genus including Dietzia maris comb. nov., formerly Rhodococcus maris. Int J Syst Bacteriol, 45, p. 32–36. 24. Koerner RJ, Goodfellow M, Jones AL (2009). The genus Dietzia: a new home for some known and emerging opportunist pathogens. FEMS Immunol Med Microbiol, 55(3), p.296-305.

INTRODUCTION

25. Bell.K. S, Philp.J.C, Aw. D.W.J, Christofi.N (1998). The genus Rhodococcus. J Appl Microbiol, 85(2), p.195-210. 26. Sutcliffe. I.C, Brown.K. A, and Dover.L. G (2010). The Rhodococcal Cell Envelope: Composition, Organisation and Biosynthesis. Biology of Rhodococcus. Edited by H.M. Alvarez. Springer Berlin Heidelberg, 16, p. 29-71. 27. Warren.A. S, Archuleta.J, Feng.W and Setubal.J.C (2010). Missing genes in the annotation of prokaryotic genomes. BMC Bioinformatics, 11, p.131. 28. Richardson.E. J and Watson.M (2012). The automatic annotation of bacterial genomes. Brief Bioinform,14(1), p.1-12.

CHAPTER 2

STUDY OF ORTHOPHOSPHATE SPECIFIC PORIN OprP MUTANTS OF PSEUDOMONAS AERUGINOSA

(NOTE: - This chapter is derived from the content of the following publication, which is attached at the end of the chapter.

Modi N, Ganguly S, Bárcena-Uribarri I, Benz R, van den Berg B, Kleinekathöfer U (2015) Structure, Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa. Biophysical Journal; Vol. 109; p.1429–1438.)

2.1 Summary

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the outer membrane channels OprP and OprO from Pseudomonas aeruginosa show specificity for phosphate and diphosphate, respectively. In this study we use electrophysiology experiments to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residues in generating the different substrate specificities, the double mutants were generated in silico and in vitro. Electrophysiological experiments demonstrated that the double mutations interchange the phosphate and diphosphate specificities of OprP and OprO. Our findings outline a possible

CHAPTER 2 strategy to rationally design channel specificity by modification of a small number of residues that may be applicable to other pores as well.

2.2 Introduction

Earlier in 1976 Nakae invented the word “porin” for a class of OM proteins forming nonspecific diffusion channels in Salmonella. Later it was found that porins were found in every species of Gram-negative bacteria and in few species of Gram-positive bacteria (1). The OM of Gram-negative bacteria acts as adaptive barrier and as molecular sieve because of the presence of channel forming proteins (1, 2). Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen and is a major cause of nosocomial infections. The OM of P. aeruginosa contains only one general diffusion porin OprF, which is rarely active and many substrate selective channels (e.g., channels of the OprD/Occ-family). The lack of wide pores is likely the reason for the poor permeation of antibiotics through the P. aeruginosa OM. Such substrate specificities are achieved by the virtue of having a defined substrate binding site in the channels. Understanding the specificity and transport properties of OM channels is an active area of research with implications ranging from nanoanalytics (e.g., nanopore-based DNA sequencing) (3-5) to research on how to improve antibiotic translocation through outer membrane channels (6, 7).

Among these substrate-selective channels is OprP, a phosphate-selective porin that is induced under phosphate starvation conditions, it facilitates the high-affinity acquisition of phosphate ions that are important for growth of Pseudomonas species under the conditions of phosphate deficiency (8). OprP is found to be an anion- specific channel. At molecular level, the regulation of OprP was found to be mediated by the PhoB regulator which also controls the expression of a periplasmic phosphate binding protein. Experiments related to transposon lacking studies of OprP gene has confirmed that OprP is involved in phosphate uptake in P. aeruginosa (2) (Figure 2.1). A crystal structure of OprP is available (9) (Figure 2.2) and detailed investigations have been carried out to understand the phosphate specificity of this interesting channel using bilayer electrophysiological measurements (10-12) and computational molecular dynamics (MD) simulations (13).

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Note: - This figure is hidden due to copyright issues

Figure 2.1:- Up-regulation of OprP gene by PhoB regulator which controls the expression of a periplasmic phosphate binding protein and which is involved in phosphate uptake in Pseudomonas aeruginosa (2).

Another homologous channel to OprP was discovered in P. aeruginosa, named OprO, which shares a high sequence identity and similarity with OprP of 74% and 86%, respectively (14,15). Measurements with lipid bilayers have demonstrated that OprO is selective for polyphosphate, e.g., pyrophosphate, whereas OprP is selective for phosphate (12, 15). To know the difference in the specificity of the highly analogous channels is intriguing, and therefore further structural and mechanistic investigations were performed. The details obtained from such experiments can answer many questions related to the understanding between the differences in the specificity between these two particular channels. In addition, they could potentially provide a template to fine-tune the specificity and transport properties of various ion channels. A study was carried out with electrophysiological measurements on lipid bilayers to understand the polyphosphate specificity of this pore. Going one step further, we also could show that with the help of mutagenesis studies one can interchange the specificity of OprP and OprO. In the framework of this thesis we have experimentally demonstrated that an engineered double mutant channel of OprP has OprO-like properties and becomes selective for pyrophosphate.

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Note: - This figure is hidden due to copyright issues

Figure 2.2:- Cartoon representation of crystal structure of OprP porin. a) An OprP trimer is shown embedded in a lipid membrane. Important loops - L3, L5 and T7 - which are responsible for the formation of narrow regions inside the pore are shown. EC and PC denote the extracellular and periplasmic sides of the pore respectively. b) The monomeric pore is shown together with an arginine ladder. c) Top view of lysine ladder of OprP monomer (13).

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2.3 Materials and methods

2.3.1 Bacterial strains and plasmid

Porin-deficient mutant bacterial stain Escherichia coli (E. coli) CE1248 cells (16) were used for overexpression of the proteins. This strain lacks the genes coding for expression of E. coli general porins OmpC, OmpF, and PhoE, and allows overexpression of the cloned outer membrane proteins from other bacteria. pAS27 plasmid was used for overexpression of OprP and OprP mutants. It is a precursor of pTZ19U phagemid vector and OprP wild type gene from P. aeruginosa was cloned into this vector in the Hancock lab (17). This vector has an ampicillin resistance gene, origin of replication, T7 promoter and a multiple cloning site (Figure 2.3). OprP gene was subcloned into multiple cloning sites by using a series of steps.

Figure 2.3:- Restriction digest map of plasmid pAS27. The fragment containing the oprP gene (grey color) was cloned into the multiple cloning site of phagemid pTZ19U downstream of the T7 promoter. ori, origin of replication (yellow color).

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2.3.2 pAS27 OprP single and double mutagenesis using the oprP gene in the E. coli plasmid

The oprP wild type gene in the plasmid pAS27 was mutated using Quick-Change site- directed mutagenesis kit (Agilent technologies, Germany). OprP single mutant are created by point mutation at 62 position and 114 position by mutating tyrosine to phenylalanine and aspartic acid respectively Double mutations are created by causing two-point mutation simultaneously in the gene. Mutagenesis primers are designed such that the mutant site is at the center of the primer. The primers are designed according to the standard protocols (see Table 2.1) and are obtained from MWG eurofins (Ebersberg, Germany). PCR (polymerase chain reaction) reactions are carried out as per the protocol of Quick change lighting mutagenesis kit (Agilent technologies, Germany). The primers used for mutagenesis are listed in Table 2.1. The newly mutated plasmids are digested with Dpn I enzyme such that all the parental plasmids and strands are degraded and only mutated plasmid was left. Then 30µl of PCR reaction is transformed into XL-1 blue cells and incubated for 16 hours at 37°C for growth. Transformed colonies are picked and grown in 50 ml LB medium +antibiotic for miniculture. Plasmids are extracted and send for sequencing to GATC Biotech sequencing center (Köln, Germany) to confirm the presence of wild type and mutated gene sequence.

OprP Codon Primer 5´-3´

Y62 and Y114 TAC Non-mutated

Y62F TTC CTTCCGCCGCGCCTTCCTGGAGTTCGGCG

Y114D GAC CTTCCGCCGCGCCGACCTGGAGTTCGGCG

Table 2.1:- Primers designed to mutate OprP to OprO and to change the mutant specificity towards phosphate and diphosphate. Mutants are indicated by the residue and its position followed by the mutated residue, using the one letter amino acid code. The codons of OprP wild type and its mutants are indicated by the forward primers used for mutation.

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The results confirmed that the mutated genes contained correct mutations and did not contain any other undesired mutations.

2.3.3 Growth media, enzymes and reagents

E. coli DH5α cells were used for maintaining the OprP wild type and mutant plasmids. Plasmids are extracted and transformed into CE1248 E. coli cells for overexpression and purification of the protein. Few colonies are picked from the agar plates for preculture in Luria-Bertani (LB) medium at 37°C with shaking at 200 rpm. For this, the media is supplemented with 100µg/ml ampicillin.

On the consecutive day, 250 ml of cell culture starting with OD600= 0.1 in LB medium supplemented with ampicillin and 0.4 % of glucose for suppression of expression of LamB porin is inoculated and cultured OD600 reaches a value of 0.5-0.8. It takes approximately 6-8 hours.

Later, the cells are induced with 0.8mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Applichem, Germany) (200µl of 1 M IPTG stock in 250ml culture) for overexpression and the cells were left for overnight growth at 37°C with shaking at 220 rpm. Later, the cells are harvested at 5,000 × g for 30 minutes at 4°C. Cell pellet is washed with 10mM ice-cold Tris- HCl (Tris-Hydrochloride), pH 8.0. Weight of the cell pellets were noted down and store at - 20°Cfor further steps.

2.3.4 Extraction of cell wall proteins of OprP wild type and mutants from E. coli

Frozen E. Coli CE1248 cells are thawed and resuspended in 10mM ice-cold Tris-HCl pH 8.0 including 50 µg/ml of pancreatic DNase I (Applichem, Germany) and proteases. The cells are incubated on ice and sonicated for 10 minutes at 50 % duty cycle. The unbroken cells are removed by centrifugation at 5,000 × g for 30 minutes at 4°C. The supernatant is placed in ultracentrifuge tubes for 70.1Ti Beckman ultracentrifuge (Beckman, Germany) rotor. The pellet of unbroken cells is discarded.

CHAPTER 2

The tubes are ultracentrifuged at 100,000 × g (40,000 rpm) for 1hour at 4°C. The protein pellet represents the membrane fraction. Supernatants were separated from pellet and stored for further analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE). Protein pellet is solubilized with 8mL of 0.15% octyl-POE (N-octylpolyoxyethylene), 10mM Tris-HCl pH 8.0 using the potter homogenizer for 3-4 minutes until the whole pellet is dissolved in the detergent. The protein fraction is ultracentrifuged again at 100,000 × g for 1hour at 4°C. Supernatants are again separated from pellets and were kept for further analysis. With increasing concentration of detergent protein pellets were again solubilized with 8mL of 3% octyl-POE, 10mM Tris-HCl pH 8.0 using the potter homogenizer. The liquid was incubated at 37°C for 1 hour and centrifuged again at 100,000 × g for 1 hour at 20°C. This solubilization step is repeated and the supernatant is stored at 4°C. Samples are taken from all washing and extractions steps and are kept for further analyzed by SDS-PAGE. OprP single and double mutant proteins are extracted in the same way as wild type protein.

2.3.5 Purification of cell wall proteins of wild type and mutants from E. coli

The samples which showed desired protein band on SDS-PAGE for wild type and mutants are collected and concentrated with 50 KD Amicon filters (Merck Millipore, Darmstadt, Germany). Where chemicals or proteins below 50 KD molecular mass run down as flow- through.

2.3.5.1 Mono Q and Fast-Protein Liquid chromatography (FPLC)

For further purification of OprP and its mutant proteins from concentrated samples, these s samples were passed through MonoQ 5\50 ® GL tricorn column. These Mono Q columns are prepacked monobeads columns with a strong anion exchanger (Figure 2.4) and is attached to FPLC (Figure 2.4) (Fast-Protein Liquid chromatography) (Biorad; Germany).

CHAPTER 2

Note:- This figure is hidden due to copyright issues

Figure 2.4:- An example of MonoQ column and FPLC (Biorad). It consists of various parts such as pumps, injection valve, column, monitor/recorder, fraction collector. For our purification purpose we used MonoQ column which is a strong anionic column. (https://aswebsaxs.synchrotron.org.au/saxswiki/index.php/SR13_ID01_Size_Exclusion_Chromatography)

FPLC is a form of liquid chromatography having different affinities for two materials, a moving fluid called the "mobile phase" and a porous solid material called “the stationary phase”. Generally, in FPLC, the mobile phase is an aqueous solution, or "buffer" and the stationary phase is a resin composed of beads packed in columns as shown in Figure 2.4. The protein of interest will bind to the resin by exchange of charge present in buffer A (the running buffer) but become dissociated and return to solution, in buffer B (the elution buffer).

When a mixture of proteins is dissolved in 100% buffer A (the “binding buffer”) and pumped into the column. The proteins of interest bind to the resin while other components which are not specific for the resin are flowing through the column with the buffer. When Buffer B (the "elution" buffer) is pumped gradually increasing from 0% to 100% gradient, bound proteins dissociate and appear in the effluent. In the FPLC, there are two detectors which measure salt concentration (by conductivity) and protein concentration (by absorption of ultraviolet light at a wavelength of 280nm). Since all proteins from the effluent pass these detectors, they appear as peaks and can be collected for further analysis (18). Different parts of FPLC are- Pumps, Injection loop, Injection valve, Column, Flow Cells, Monitor/Recorder, Fraction collector. The pressure of FPLC is kept constant at 580psi or 4MPa and a flow rate of 1-3 ml\min for the buffer.

CHAPTER 2

2.3.5.2 SDS-PAGE and Western immunoblotting (WB)

Samples are taken from whole cell lysates, from different purification steps from ultracentrifugation to the different FPLC fractions of wild type and mutant proteins from E. coli CE1248. These samples are loaded on to SDS-PAGE containing 10% polyacrylamide (19). The samples are heated for 95°C for 2×10 minutes and 10µg of samples are loaded per well. The gels are stained with silver staining (20). For Western blots the proteins are run on 10% SDS gels and then transferred on to nitrocellulose membrane and incubated with monomeric specific OprP antibody (17) and anti-OprP rabbit serum (Sigma-Aldrich, Germany), the bands are developed with BCIP® pills (Sigma Germany). E. coli CE1248 containing pAS27 plasmid was taken as positive controls.

2.3.6 Bilayer assays

Black lipid membrane (BLM) method represents a model system which was introduced almost 40 years ago, and since then it has had a great impact on the characterization of membrane active molecules, particularly porins (21). Planar lipid bilayers allow to detect size and functional characteristic of channel-forming proteins and physico-chemical properties of lipid membranes.

BLM set up consists of a Teflon cuvette divided into two compartments connected by a small aperture with a diameter of about 0.5 mm. A thin film of lipid-dissolved (DiphPC- diphytanoyl phosphatidylcholine) in an organic solvent (mostly n-decane) is applied on the aperture, and quality of the membrane is controlled by illuminating the film and following the reflected light through a microscope. Both sides of the compartments are filled with buffered electrolytic solution (usually 1M KCl or other salt solutions). For the electrical measurements, the membrane cell is connected to the external circuit through Ag/AgCl electrodes (Figure 2.5). The electrode at the cis-side is connected to a voltage source to allow the application of defined membrane potentials to the cis-side of the membrane. The electrode at the trans-side is connected to a current-to-voltage converter made on the basis a Burr-Brown operational amplifier to measure the current through the membrane. The

CHAPTER 2 amplified signal is monitored by a digital oscilloscope and recorded by a strip chart recorder (22).

Lipid membrane is a perfect insulator, it is non-permeable to ions in the presence of voltage. When channel protein from a stock solution, dissolved in detergents is added in small quantities to the aqueous solutions bathing the membrane, the membrane conductance starts to increase in a stepwise fashion. Reconstitution of approximately 100 porin channels is consider for derivation of a meaningful statistics. The occurrence of these conductance steps is specific to the porin and is not seen when a detergent alone is added.

Note:- This figure is hidden due to copyright issues

Figure 2.5:- Schema of a set up for conductance recording with planar lipid bilayer. On one side, usually called cis-side, a voltage Um is applied. The current across the membrane or through the channel is detected by a current–voltage amplifier. Rf is the feed-back resistance and determines the amplification. The amplified signal V(t) is further processed, either by an AD converter and an adequate computer program, or an advanced oscilloscope (21).

2.3.6.1 Single channel measurements

When a single porin molecule is inserted into the bilayer, depending upon its size, charges and oligomeric configuration it allows flow of ions through the fully open channel, allowing the calculation of the porin conductance. Each step represents a single porin insertion and the single channel conductance is described as the most commonly occurring conductance value.

CHAPTER 2

The conductance steps are not uniform in size but distributed over a certain range. Histograms are formed by considering these conductance steps.

Conductance of a porin can be calculated with the following equations:

I G= Eq.1 Where G = Conductance (S) Ue I = Current (A) Ue = Voltage (V) The amplifier transforms the current of the system (I) into voltage. This current I can be calculated using the following:

U I = a Eq.2 Ua = output voltage (V) Vf Vf = feed-back resistor (amplification factor) of the current to voltage converter given in (V A-1)

If we substitute the equation (2) in the equation (1) we obtain the conductance G:

U G= a Eq.3 Vf. Ue

The chart recorder is divided into 100 units. The conductance Ua for each unit will be divided by 100 (Uv):

Uv Gk= Eq.4 Where Gk = conductance per unit (S) Vf.Ue.100 Uv = the applied full scale of the recorder (V)

The single channel conductance of a channel protein can be easily determined by multiplying

퐺푘 by the number of units the insertion of a porin channel has covered during the reconstitution process.

CHAPTER 2

2.3.6.2 Titration Experiments

Passage of substrates through pores is estimated by calculating the number of ions passing through the pore in a certain time. To estimate the transport of phosphate and diphosphate ions through OprP wild type and its mutant proteins titration experiments were performed. The membrane cell was bathed with 0.1M KCl, 10mM 2-(N-morpholino) ethanesilfonate (MES)-KOH adjusted at pH 6. Concentrated amount of proteins samples are added on both sides under stirringconditions. Conductance is allowed to increase for about 30 minutes or longer due to reconstitution of OprP and its mutants in to the membrane until it reaches saturation. Once the conductance of the membrane is stationary and did not increase further, increasing amounts of concentrated phosphate or diphosphate solutions are added on both sides of the membrane cell under stirring, to allow equilibration of the KCl solution. If the protein has affinity towards that particular substrate, decrease in the total conductance of the membrane can be observed because phosphate or diphosphate bind to the active site inside the channels and block the passage of chloride. Stability constants were calculated for binding of these substrates for OprP and its mutants from the titration experiments using Michaelis-Menten equation or the Langmuir adsorption isotherm (23) as it is given below.

G G(c) 퐾. 푐 max- = Gmax - G∞ 퐾 . 푐 + 1

Where, G(c) = the membrane conductance in the presence of the substrate concentration “c”

G max = the membrane conductance before addition of the substrate

K = the stability constant for binding

c = the phosphate or diphosphate concentration

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2.4 Results

2.4.1 Colony PCR confirmation of mutants and sequencing

To obtain mutants, mutagenesis PCR reaction is performed. pAS27 plasmid with OprP gene subcloned into it, was subjected to mutagenesis. XL-1 blue cells are transformed with mutant plasmids of OprP Y62F or Y114D or Y62FY114D by electroporation method. They are plated on to the agar plates supplemented with 100 µg\ml ampicillin. The plates are left for 16 hours of incubation at 37°C. Later, colonies were picked and screened for the presence of the gene of interest before sending it for sequencing with colony PCR.

A B

Figure 2.6:- Agarose gel pictures A) Gel picture showing PCR products after mutagenesis PCR, the plasmid size is of approx. 4500 b.p for the wild type and mutant plasmids. All the four lanes show the same band size; Lane 2 – pAS27 plasmid (control); Lane 3- Y62F mutagenesis PCR product; Lane 4- Y114D mutagenesis; Lane 5- Y62FY114D mutagenesis PCR product. PCR products show only a single amplified band, representing the presence of only amplified product without any contamination. B) Gel picture showing amplified bands of OprP gene in colony PCR. Colony PCR was performed with Forward and reverse primers of OprP gene wild type or OprP mutants to confirm the presence of insert. The size of OprP gene or OprP mutant genes is 1305 b.ps.

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The appearance of a band at around 4000-5000 base pairs provided confirmation of the presence of gene/ or insert. Once the colony PCR shows the presences of plasmids with a desired insert, then these plasmids were send for sequencing. Sequencing results confirmed the presence of the mutated gene without any unwanted mutations. Figure 2.7 shows an example of sequencing result where the replacement of single base pairs can be observed at the desired position.

Figure 2.7:- A sequencing file which shows the presence of two mutations together representing double mutation in a single gene. Letters in blue show the points of mutation. The 649 position show A to T mutation indicating Y62F point mutation and 804 position show T to G mutation indicating Y114D point mutation. These mutations were created with the designed primer for mutagenesis as given in Table 2.1Error! Reference source not found..

2.4.2 Extraction and purification of cell wall proteins of wild type and mutant proteins

OprP wild type gene from P. aeruginosa is cloned without its signal peptide. It is optimized to be produced in E. coli having its own outer membrane signal such that it is expressed in the OM and not in inclusion bodies. For the extraction of OM protein from E. coli cells, high speed ultracentrifugation method is used along with solubilizing the proteins with different concentrations of octyl-POE detergent. As mentioned in materials and method section 2.3.5,

CHAPTER 2 in each step, the cells and the detergents solutions were centrifuged at high speed and the supernatants are stored.

Samples from each step of cell culture, from the time of induction to the final step of ultracentrifugation was checked on 10% SDS-PAGE gels to have a track and also to confirm the presence of the desired protein, in our case OprP wild type and its mutant proteins. Proteins were dissolved in 10mM Tris-HCl, 0.15% octyl-POE, or 3 % octyl-POE. This can be seen in figure given below (Figure 2.8).

Figure 2.8:- SDS-PAGE gel picture of different purification steps of OprP wild type. Lane 1- Extraction with 10mM Tris-HCl; Lane 2- Extraction with 0.15% octyl-POE; Lane 3 -1st extraction with Octyl-POE; Lane 4 -2nd extraction with 3%Octyl-POE; , Lane L- Protein Ladder. The protein band of OprP can be observed below the 55KDa in lane 3 and 4. But the band in lane 4 is more near to the molecular weight of OprP. Protein samples representing lane 4 were taken for further purification steps.

The monomeric size of OprP protein is 48 kDa, but it always runs a little higher than its molecular mass (17). A corresponding band could be observed below 55 KDa in SDS-PAGE of the 4th purification step representing pure OprP protein. The trimeric form of OprP can be seen at 148 kDa, but this band is not seen in the above Figure 2.8, as the samples are heated before loading onto the gels. Due to heating, the trimeric form is lost and only monomeric form can be seen in the gel. The above gel can be considered for all OprP mutant proteins purification, because the change of only one single amino acid did not affect the molecular mass of the whole protein. 47

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A band corresponding to OprP protein was observed in lane 3 (is represented with blue square) i.e. first extraction with 3% Octyl-POE and a faint band is observed in lane 4 i.e. the second extraction with 3%Octyl-POE. As OprP protein band was observed in both lanes, so both the fractions are concentrated and ran again on gel. Out of both fractions, samples from first extraction with 3% octyl-POE are used for further purification by FPLC.

Figure 2.9:- Purification protocol of OprP protein by FPLC. The chromatogram shows various peaks and each peak represent some protein that is eluted out. OprP protein in eluted out at 39th fraction.

OprP is an anionic protein, as judged from its PI of 4.93. For its purification, a MonoQ column is chosen. A sample containing 6 to 8 mg of protein was loaded onto the column and the proteins are eluted gradually with a linear NaCl gradient increasing from 0 to 1 M. The OprP wild type and mutant proteins were eluted at a salt concentration of about 350-400mM NaCl.

Different fractions of FPLC are taken and analyzed on 10% SDS gels. Out of different fractions of FPLC the 39th fraction showed a clear band of interest on the gel. Same fractions showed activity in the bilayer.

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Figure 2.10:- SDS-PAGE is showing all the OprP wild type and mutant pure proteins. Lane 1- protein ladder; Lane 2- OprP Y62F single mutant; Lane 3- OprP Y114D single mutant; Lane 4- OprP Y62FY114D double mutant; Lane 5- OprP wild type.

To confirm that the protein produced and purified is OprP or its mutant proteins, we performed western blots with anti-OprP protein (Figure 2.11). Similarly, protein samples from different purification steps are tested with dot blot to know presence and expression of OprP protein in different steps (Figure 2.12). Dot blot experiments show that amount of OprP protein was higher in steps where the proteins are solubilized with higher concentration of detergents.

Figure 2.11:- Western blot of OprP WT Lane 1- Protein ladder; Lane 2-OprP Wild type FPLC fraction 39 ; Lane 3- OprP Wild type from FPLC fraction 38; Lane 4- OprP Wild type FPLC fraction 37; Lane 5- OprP Wild type from FPLC fraction 36.

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Figure 2.12:- Dot blot for different purification steps. Number 5 indicates the sample from washing step with 10mM Tris-HCl, 6 indicates the extraction step with 10mM Tris-HCl, 7 indicates the sample from the solubilizing step with 0.15% octyl-POE, 8 and 9 indicate solubilizing steps with 3% octyl-POE.

2.4.3 Channel forming activity of single and double mutants

We investigated the conductance of the wild-type OprP and OprO channels as well as their mutants after purification to know if these mutants have the same pore forming activity as wild type OprP in BLM. Purified proteins were added to the bilayer membrane and the increase in the conductance is studied. Previous studies showed that OprO has an about two times higher conductance than OprP (14). Table 2.2 represents the single channel conductance of OprP and the OprP mutants in 0.1 M KCl and 1 M KCl. Single channel measurements of OprP single mutants showed similar pore forming activity as OprP wild type in both 0.1 M KCl and 1 M KCl. However, the OprP double mutant (Y62FY114D) had a channel conductance similar to wildtype OprO as expected.

Porins Electrolytic solution Conductance (pS)

OprO (wildtype) 1 M KCl 440 0.1 M KCl 240 OprP (wildtype) 1 M KCl 260 0.1 M KCl 160 OprP (Y62F) 1 M KCl 380 0.1 M KCl 160 OprP (Y114D 1 M KCl 230 0.1 M KCl 180 OprP (Y62F Y114D) (PO) 1 M KCl 400

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0.1 M KCl 240

Table 2.2:- Single-channel conductance of OprP, OprO, and three OprP mutant’s channels in 0.1 M and 1 M KCl solutions buffered with 10 mM MES, pH 6, T=20 °C. The applied voltage was 50 mV.

2.4.4 Comparison studies of substrate specificity of OprP wild type with its single and double mutants

When the amino acid sequences of these two proteins OprP and OprO is aligned with each other, they showed a sequence similarity of 84% and sequence identity of 74%. To understand the functional difference between these two porins in detail, the not identical amino acids were inspected in detail. Out of these amino acids, two amino acids which are present in the constriction region of the channel were found to be important for showing different binding specificity of these porin. In OprP, bulkier tyrosine groups are present in Y114 and in Y62 in the constriction zone whereas residues D114 and F62 are present in the same region of OprO. It can be predicted that the radius of the OprP porin due to the presence of bulkier groups near the binding site is reduced, whereas OprO has less bulky groups near the binding site resulting in a larger radius of the pore than OprP. This is presumably the reason OprP is specific for phosphate and OprO is specific for diphosphate as it was also reported in previous studies (14).

CHAPTER 2

Porin Phosphate Diphosphate

Half saturation Stability constants Half saturation Stability constants constant in mM (K in 1/M) for constant in mM (K in 1/M) for inhibition inhibition

OprO (wildtype) 4.5 220 ± 50 0.6 1450 ± 120

OprP (wildtype) 1.3 770 ± 150 3.2 310 ± 35

OprP (Y62F) 1.6 600 ± 75 1.7 590 ± 50

OprP (Y114D) 3.1 320 ± 40 3.0 330 ± 25

OprP (Y62F Y114D) (PO) 8.3 120 ± 50 0.7 1260± 120

Table 2.3:- The single channel conductance was measured in 0.1 M KCl, 10 mM MES, pH 6, T=20 °C and applying 50 mV voltage. At least 100 single events were used to calculate the average value of conductance. The half saturation and stability constants for inhibition of Cl- conductance by phosphate or diphosphate were obtained from titration experiments using either the Michaelis- Menten equation or the Langmuir equation as described elsewhere. The phosphate and diphosphate solutions used for the titration experiments had also a pH of 6. Mean values (± SD) of at least three individual titration experiments are shown.

Our main idea for these whole experiments was to swap the specificity between the two phosphate specific porins OprP and OprO by changing these bulkier tyrosine groups to phenylalanine and to aspartic acid. To put our theory into practical we generated single and double mutants of OprP, replacing the OprP amino acids with OprO experimentally and investigated the mutant for its phosphate/diphosphate specificity using titration experiments to block chloride conductance with phosphate and diphosphate.

Titration experiments carried out with OprP single mutants (Y62F and Y114D) showed similar results as that of its OprP wild type with stability constant of 690 1/M and 500 1/M for phosphate and 490 1/M and 630 1/M for diphosphate respectively, which represents a strong binding affinity towards phosphate and less binding affinity towards diphosphate 52

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(Table 2.3). Interestingly, the OprP double mutant (Y62FY114D) displayed a stronger binding affinity for diphosphate with stability constant of 343 1/M compared to phosphate with 720 1/M in bilayer measurements.

2.5 Discussion

2.5.1 Swapping of the specificity with the mutants from wild type OprP and OprO

Previous electrophysiological studies on lipid bilayer membranes indicated that phosphate binds more strongly to OprP whereas diphosphate binds more strongly to OprO. These results were also found in this study, when previous experiments were repeated so, based on the experimental veriﬁcation, we can conclude that the OprP double mutant (Y62F Y114D) has become diphosphate selective and that by changing the two residues in the central constriction region, it now behaved like OprO in terms of substrate speciﬁcity and conductance.

By MD simulations also it has been proved that OprP has only a single binding site and OprO has two binding sites for diphosphate. That’s the reason OprP shows specificity towards phosphate and OprO shows towards diphosphate (24). OprP and OprO share very high sequence identity. Nevertheless, the major difference between the two channels in terms of phosphate transport is the presence of bulkier groups in the lumen of the pore.

The outcomes in this study provided a molecular basis to understand the substrate specificity of two structurally highly similar OM channels, as well as to engineer the substrate specificity properties of these channels. These results can be exploited to change or to fine tune the specificity of different channels and porins. Furthermore, understanding the permeation properties of OM channels is of utmost importance for antibiotics research because the improvement of translocation of these drugs into the bacteria is one of the challenging problems in the field (25,26).

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2.6 REFERENCES

1. Nikaido. H. (2003). Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev, 67.p.593–656. 2. Benz, R. (2006). Bacterial and Eukaryotic Porins: Structure, Function, Mechanism. Ed. John Wiley & Sons, Hoboken, NJ. 3. Aksimentiev, A. (2010). Deciphering ionic current signatures of DNA transport through a nanopore. Nanoscale, 2.p.468–483. 4. Howorka, S., and Z. Siwy. (2009). Nanopore analytics: sensing of single molecules. Chem. Soc. Rev, 38. p. 2360–2384. 5. Modi, N, M. Winterhalter, and U. Kleinekathöfer. (2012). Computational modeling of ion transport through nanopores. Nanoscale, 4 (20). p.6166–6180. 6. Nestorovich, E. M., C. Danelon, S.M. Bezrukov (2002). Designed to penetrate: time- resolved interaction of single antibiotic molecules with bacterial pores. Proc. Natl. Acad. Sci. USA, 99 (15). p.9789–9794. 7. Pagès. J. M., C. E. James, and M. Winterhalter (2008). The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria. Nat. Rev. Microbiol. 6, p.893–903. 8. Hancock, R. E.W., K. Poole, and R. Benz (1982). Outer membrane protein P of Pseudomonas aeruginosa: regulation by phosphate deficiency and formation of small anion-specific channels in lipid bilayer membranes. J. Bacteriol, 150 (2). p.730–738. 9. Moraes, T. F., M. Bains, Hancock, R. E.W, N. C. Strynadka (2007). An arginine ladder in OprP mediates phosphate-specific transfer across the outer membrane. Nat. Struct. Mol. Biol, 14 (1). p. 85–87. 10. Benz, R., and R. E. W. Hancock (1987). Mechanism of ion transport through the anion-selective channel of the Pseudomonas aeruginosa outer membrane. J. Gen. Physiol, 89.p. 275–295. 11. Benz, R., C. Egli, and R. E.W. Hancock (1993). Anion transport through the phosphate-specific OprP-channel of the Pseudomonas aeruginosa outer membrane: effects of phosphate, di- and tribasic anions and of negatively-charged lipids. Biochim. Biophys. Acta, 1149 (2). p. 224–230. 12. Hancock, R. E. W., and R. Benz (1986). Demonstration and chemical modification of a specific phosphate binding site in the phosphate starvation-inducible outer

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membrane porin protein P of Pseudomonas aeruginosa. Biochim. Biophys. Acta, 860 (3).p. 699–707. 13. Modi, N., I. Barcena-Uribarri, R. E.W. Hancock, U. Kleinekathöfer (2015). Tuning the affinity of anion binding sites in porin channels with negatively charged residues: molecular details for OprP. ACS Chem. Biol, 10 (2). p. 441–451. 14. Hancock, R. E. W., C. Egli, R. J. Siehnel (1992). Overexpression in Escherichia coli and functional analysis of a novel PPi-selective porin, OprO, from Pseudomonas aeruginosa. J. Bacteriol, 174 (2). p.471–476. 15. Siehnel, R. J., C. Egli, and R. E. W. Hancock (1992). Polyphosphate selective porin OprO of Pseudomonas aeruginosa: expression, purification and sequence. Mol. Microbiol, 6 (16). p. 2319–2326. 16. Van der Ley.P, Amesz.H, Tommassen.J and Lugtenberg.B (1985). Monoclonal antibodies directed against the cell-surface-exposed part of PhoE pore protein of the Escherichia coli K-12 outer membrane. Eur. J. Biochem. 147,401 –407. 17. Sukhan, A., and Hancock, R. E. W. (1995). Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP. J. Bacteriol, 177 (17).p. 4914–4920. 18. Chromatography, Theories, FPLC and beyond. http://web.mnstate.edu/biotech/chrom_fplc.pdf. 19. Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, p. 680-685. 20. Gross HJ, Baier H and Blum H (1987). Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8 (2), p. 93-99. 21. Winterhalter.M (2000). Black lipid membranes. Current Opinion in Colloid & Interface Science, 5 (3-4). p. 250-255. 22. Benz R, Janko K, Boos W, Läuger P (1978). Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli. Biochim Biophys Acta 511, p. 305–319. 23. Benz, R., Schmid, A., Nakae, T. and Vos Scheperkeuter, G. (1986). Pore formation by LamB of Escherichia coli in lipid bilayer membranes. J. Bacteriol, 165 (3). p. 978– 986. 24. Modi N, Ganguly S, Bárcena-Uribarri I, Benz R, van den Berg B, Kleinekathöfer U (2015) Structure, Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa. J. Biophysical Journal, 109.p.1429–1438. 55

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25. Ceccarelli, M., and P. Ruggerone (2008). Physical insights into permeation of and resistance to antibiotics in bacteria. Curr. Drug Targets.9, p.779–788. 26. Singh PR, Ceccarelli M, Lovelle M, Winterhalter M, Mahendran KR. (2012). Antibiotic permeation across the OmpF channel: modulation of the afﬁnity site in the presence of magnesium. J. Phys. Chem. B.116 (5). p.4433–4438.

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Note:- Modi N, Ganguly S, Bárcena-Uribarri I, Benz R, van den Berg B, Kleinekathöfer U (2015) Structure, Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa. Biophysical Journal; Vol. 109; p.1429–1438.

Hyperlink for the paper-Mutants of OprP

CHAPTER 3

MUTANTS OF DIPHOSPHATE SPECIFIC PORIN OprO OF PSEUDOMONAS AERUGINOSA

(NOTE: - Data in this chapter is taken from unpublished manuscript, which is attached at the end of the chapter.

Ganguly. S, Barcena-Uribarri. I, Kesireddy. A, Kleinekathöfer. U, Benz. R. Conversion of OprO - into OprP- porin of Pseudomonas aeruginosa by exchanging key amino acids at the channel constriction.)

3.1 Summary

Under phosphate constraining conditions, OprP and OprO specific porins are induced and expressed in the outer membrane of Pseudomonas aeruginosa (P. aeruginosa). Porins like OprO (pyrophosphate specific) and OprP (orthophosphate specific) show despite large homology but structural differences in their binding sites situated in the pore constriction. In previous studies about OprP, it was shown that the mutation of two amino acids, Y62F and Y114D, in OprP leads to an exchange in substrate specificity similar to OprO. In order to support the role of these key amino acids in the substrate sorting of the specific porins the reverse OprO (F62Y, D114Y single and F62YD114Y double) mutants were created. The phosphate and diphosphate binding of the generated porins were studied in planar lipid bilayers. Our findings in this study clears that just a few amino acids are mainly responsible for the substrate specificity in OprP as well as in OprO. Mutations of these amino acids in the porins allow interchanging of their characteristic properties and substrate specificity preference for orthophosphate.

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3.2 Introduction

The outer membrane of Gram-negative bacteria has a crucial role in providing an extra protection without affecting the exchange of materials required for existence of living. At the molecular level, the outer membrane of Gram-negative bacteria is organized such that it can have various protein channels involved in transport, uptake or efflux, of a large variety of compounds, nutrients or toxic molecules (sugars, drugs, small peptides, chemicals). Gram- negative bacteria form the major proportion of pathogenic bacteria showing antibiotic resistance due to the presence of outer membranes (1). One of the major pathogens among Gram-negative bacteria which show high resistance towards antibiotics is P. aeruginosa. One of the reasons for the antibiotic resistance of P. aeruginosa is the predominance of specific porins such as OprP/OprO, OprB and other porins of the OprD family, which provide a restricted substrate transport and the additional presence of efflux pumps like OprM (1). It is also one of the major cause of opportunistic nosocomial infections and the dominant cause of chronic lung infections in cystic fibrosis patients (2).

Like OprP, OprO is also regulated by Pho genes. For OprO to be regulated the cells should be in stationary phase along with being under phosphate- limiting stage. Crystal structures of OprO show trimeric confirmation, where each monomer has 16 antiparallel ß-strands connected by long extracellular loops and short periplasmic turns. In this porin, loops L3 and L5 bend into the cavity of the pore and state the shape and size of their constriction region (Figure 3.1). A closer consider the crystal structure of OprO porin explains, that diphoshpahte molecule are attracted from the external side into the periplasm of the cell through electropositive arginine ladder and by periplasmic lysine ladders (3).

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Note:- This figure is hidden due to copyright issues

Figure 3.1:- X-ray crystal structure of Pseudomonas aeruginosa OprO expressed in E. coli. (A) Overview of the trimer viewed from the extracellular side (B) Side view of the OprO monomer showing the basic ladder residues. Different positions of loops and turns are shown in the figure (4).

It was observed that several amino acids situated in the constriction zone seem to play a major role for the difference in single channel conductance and specificity of these porins in binding to phosphates and diphosphates. The previously reported exchange of amino acids Y62F and Y114D in OprP lead to a conversion of its pore properties into the ones displayed by OprO wild type. In this study OprP double mutant (DM) (Y62FY114D) resulted in an increase of single channel conductance and its binding affinity to pyrophosphate reaching values similar to those found in OprO (4). In order to find out if the reverse of OprO, will also display property of OprP, we created single and double mutants of OprO. The DM of OprO did not showed similar single channel conductance (SCC) but showed equivalent substrate values equal to OprP wild type.

3.3 Materials and methods

3.3.1 Bacterial strains and plasmids

Bacterial cells E. coli CE1248 (4) and XL-1 blue containing OprO and OprO mutant genes incorporated in pTZ19R plasmid are grown in 1000 ml baffled Erlenmeyer flasks containing 250ml of LB medium and DYT medium at 37°C with shaking at 220rpm. Agar plates and liquid media are supplemented with 100 µg/ml ampicillin antibiotic.

CHAPTER 4 pTZ19R vector (Fisher Thermo scientific, Darmstadt Germany) is used for the cloning and overexpression of oprO gene. It is a phagemid vector with 2862 base pair in length. It is derived by pUC19 vector by inserting the DNA of phage intergenic region (IG) and the T7 promoter sequence near the multiple cloning sites (MCS) of pUC19 (Figure 3.2). pTZ19R plasmid contains pMB1 replicon rep (responsible for the replication of phagemid),a bla gene (resistance to ampicillin), f1 intergenic region (for initiation and termination of phage f1

DNA synthesis), a T7 promoter, lac containing a CAP protein binding site, promoter Plac, lac repressor binding site and the 5’-terminal part of lacZ gene encoding the N-terminal fragment of beta-galactosidase (5).

Figure 3.2:- Vector map of pTZ19R. It is derived by pUC19 vector by inserting the DNA of phage intergenic region and the T7 promoter sequence near the MCS of pUC19. pTZ19R plasmid contains pMB1 replicon rep, a bla gene, f1 intergenic region, a T7 promoter, lac containing a CAP protein binding site, promoter Plac, lac repressor binding site and the 5’-terminal part of lacZ gene encoding the N- terminal fragment of beta-galactosidase.

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3.3.2 Development of OprO wild type gene in E. coli

The genomic DNA from P. aeruginosa was isolated with Genomic DNA isolation kit (Fisher Thermo scientific, Darmstadt Germany) according to the company protocol. oprO gene of P. aeruginosa PAO1 was PCR amplified from genomic DNA by using the primers as given in Table 3. 1. The primers were designed such that it incorporates sites for restriction enzyme on both sides of the oprO gene and later on these restriction enzymes were used for digestion of the gene and to ligate it with the pTZ19R vector. oprO gene was amplified in a 50 µl PCR reaction tube containing 10X pfu buffer, 0.2mM dNTPs,1 U pfu DNA polymerase, oprO_fwd BamH1 forward primer (FP), oprO_rev SacI reverse primer (RP). PCR conditions were: Initial denaturation for 1 minute for 95°C, 30 cycles for denaturation at 95°C for 30 seconds, annealing at 59°C for 30 seconds, extension at 72°C for 3 minutes, and final extension for 7 minutes at 72°C.

The PCR amplified product and pTZ19R vector are loaded on to the agarose gel. A band corresponding to the gene is observed in the agarose gel at approximately 1317 base pair (b.p) (Figure 3.3 (b-f and h)), which is the size of the oprO gene and a band at 2862 b.p represents the linearized vector (Figure 3.3 (g)).

Figure 3.3:- Gel picture of amplified oprO gene and pTZ19R vector. a) Ladder; b-f and h) oprO amplified gene and g) pTZ19R linearized vector.

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Both gene and vector are purified from agarose gel and digested by incubating both at 37°C for 2 hours in separate tubes with BamH1/Sac1 restriction enzymes; the BamH1 recognizes and digests at 5´GGATCC 3´ and SacI digests at 5´GAGCTC 3´ site creating sticky ends in both. Ligation mixture is prepared by taking 1:3 concentration of vector to insert ratio, adding 2 µl of T4 ligation buffer and T4 ligase. This mixture is incubated at 4°C for overnight.

oprO_fwd CATGGATCCACATGATCCGTAAGCAC BamHI oprO_rev TCCGAGCTCTTAGAACACGTACTGC SACI

Table 3. 1:- Primers designed to clone oprO from genomic DNA of P. aeruginosa PAO1.

3.3.3 Transformation of pTZ19R plasmid with oprO gene into E.coli cells

The ligation product of OprO wild type gene into pTZ19R is transformed into XL-1 gold ultra-competent cells by standard electro-transformation protocol, plated on to the agar plates containing ampicillin and X-gal. After 16 hours of incubation, plates are screened for the colonies. The pTZ19R plasmid has the ability for the appearance of blue-white colonies due to the presence of lac Z gene when plated on agar plates containing X-gal. Colonies appearing white in color were chosen and sub-cultured. Blue color colonies represent absence of any insert in our case.

3.3.4 Site- directed mutagenesis in oprO gene using mismatch nucleotides

It is a process where a mutagenic primer binds to the single stranded template. The primer is then extended with DNA polymerase of E. coli using dNTPS (Deoxynucleotide triphosphate) in the presence of T4 ligase. oprO wild type gene in the plasmid pTZ19R was subjected to mutation using QuickChange Lighting site directed mutagenesis kit (Thermoscientific, Germany) where phenylalanine present in oprO gene is replaced by tyrosine at 62 amino acid position and an aspartic acid is mutated to tyrosine at 114 amino acid position. Primers were constructed using companies’ guidelines and the formula,

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Tm = 81.5 + 0.41(%GC) − (675/N) − % mismatch

Where,

N is the primer length in bases

Values for %GC and % mismatch are whole numbers

Such that it has Tm below 65°C and the desired mutation is in the middle of the primer. The primers used for mutagenesis are listed in Table 3. 2. 50 µl of mutagenesis PCR reaction comprised of 10X QuikChange Lightning reaction buffer, 0.2mM dNTPs, 1 U QuikChange Lightning DNA polymerase Enzyme, 1 U QuikSolution reagent, oligonucleotide forward primer (FP) (Table 3. 2) and oligonucleotide reverse primer (RP). Cycling Parameters for the Quik Change Lightning Site-Directed Mutagenesis were initial denaturation at 95°C 2 minutes, 18 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 10 seconds, elongation at 68°C for 2 minutes and final elongation at 68°C for 5 minutes.

Porins Codon Primer 5´-3´

F62 OprO TTC Non-mutated

Y114 OprO GAC Non-mutated

F62Y OprO TAC CTTCCGCCGCGCCTaCATCGAACTCGGCG

D114Y OprO TAC GTTCGGTCGCTTCtACCCCGACTTCGGCC

Table 3. 2:- Primers OprO to OprP to change the specificity towards diphosphate. Mutants are indicated by the residue and its position followed by the mutated residue, using the one letter amino acid code. The codons of OprO wild type and its mutants are indicated by the forward primers used for mutation. Letter in lower case shows the place of mutagenesis.

Add 2 µl of the Dpn I restriction enzyme to the PCR mixture directly and then immediately incubate at 37°C for 5 minutes to digest the parental. Transform the PCR mixture into XL10- Gold Ultracompetent Cells by heat-shock method and add 2 µl of the β-ME mix to make the

CHAPTER 4 cells more competent. Plate the cells on LB agar plate comprising of antibiotic. On the adjacent day, screen the colonies, subculture them and extract the plasmid and send for sequencing.

3.3.5 Expression and purification of OprO and OprO mutant recombinant proteins

Expression and purification steps of OprO and its mutant proteins are similar to OprP and its mutant proteins. The overexpressing plasmid pTZ19R with oprO wild type and mutant genes was transferred into E. coli CE1248 cells. These cells were sub-cultured on LB medium supplemented with 100 µg/ml ampicillin and 0.4 % glucose for suppressing the expression of

LamB porin and were grown until they reached an OD600 0.5-0.8. Protein expression was induced with 0.8mM to 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) (Applichem, Germany) and left for overnight growth at 37°C at 220 rpm shaking. The cells were harvested at 5,000 × g for 30 minutes. Cell pellet was washed with 10mM Tris-HCl, pH 8.0 and stored at -20°C.

The frozen cells were thawed and suspended again in 10mM Tris-HCl pH 8.0 including 50 µg/ml of pancreatic DNase I (Applichem, Germany). The cells were sonicated for 10 minutes at 50 % duty cycle or passed through French press. The cell debris was removed by centrifugation at 5,000 × g for 30 minutes at 4°C. The supernatant was taken out carefully and placed in ultracentrifuge tubes for 70.1Ti Beckman ultracentrifuge (Beckman, Germany) rotor. The tubes were ultracentrifuged at 146,000 × g (40,000 rpm) for 1 hour at 4°C. The pellet represents the membrane fraction. Supernatants are aparted from the pellet and stored for analysis with SDS-PAGE. With increases concentration of detergent, the pellet is solubilized with 8mL of 0.15% octyl-POE (N-octylpolyoxyethylene) in 10mM Tris-HCl pH 8.0 and the solution is ultracentrifuged again at 146,000 × g for 1hour at 4°C. Supernatants are again separated from pellets for further analysis. Protein pellets is solubilized with 8mL of 3% octyl-POE, 10mM Tris-HCl pH 8.0 and was incubated at 37°C for 1 hour and centrifuged again at 146,000 × g for 1hour at 20°C. This above-mentioned step is repeated and the supernatant is stored at 4°C. Samples from all washing steps and extractions steps are taken and are analyzed by SDS-PAGE (6). OprO single and double mutant proteins are also extracted by this method.

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Samples from wild type and mutants which shows the desired protein band on SDS-PAGE at 47 kDa were collected and concentrated. These proteins samples are then passed through a MonoQ column (Ion-exchange chromatography and the proteins were eluted gradually with increasing NaCl concentration from 0 to 1 M. The OprO wild type and its mutant proteins were eluted at a salt concentration of 300-400mM NaCl.

SDS-PAGE gels containing 10% polyacrylamide were used to study expression and purification of recombinant and mutant OprO proteins.10µg of samples are taken from whole cell lysates and different purification steps of wild type and mutant proteins from E. coli CE1248 and are loaded on to the gels. The samples are heated for 95°C for 2 × 10 minutes. The gels were stained with silver nitrate stain. For OprO wild type and OprO mutants, monomeric specific OprP antibody is used for blotting (7) and anti-OprP rabbit serum (Sigma-Aldrich, Germany). The bands were developed with BCIP® pills (Sigma Germany). E. coli CE1248 containing pTZ19R plasmid is taken as positive control.

3.3.6 Lipid bilayer experiments

The lipid bilayer experiments were performed in the similar way as it is explained in Chapter 2. Single channel conductance of OprO, OprP and the OprO mutants was measured in 0.1M and 1M KCl, 10mM MES (2-(N-morpholino)ethanesulfonic acid), pH 6 at +50 mV at RT. Addition of small amounts of protein samples on both sides of the cuvette to the salt solution produced a progressive increase in the membrane conductance in a step-like manner (Figure 3.4).

Substrates binding constant experiments were carried out to measure the inhibition of chloride conductance of OprO WT and its mutants by phosphate and diphosphates. For these experiments concentrated solutions of mono- and di-phosphates were used. The aqueous solution contained 0.1M KCl or 1M KCl, 10mM MES-KOH at pH 6, at applied voltage of +50mV at RT in these experiments. This method is explained in detail in Chapter 2, methods, and materials.

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Figure 3.4:- Pore forming events of OprP wild type, OprO wild type, OprO double mutant, OprO D114Y and OprO F62Y. Single-channel conductance observed for OprO, OprP WT and OprO mutants in a diphytanoyl phosphatidylcholine/n-decane (DiPhPC) membrane bathed in 0.1 M, 10 mM MES-KOH, pH 6, at +50mV.

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3.4 Results

3.4.1 Confirmation of presence of OprO wild type and tyrosine mutant genes in pTZ19R vector plasmid

One of the methods for confirmation of presence of OprO WT and OprO mutant gene insert is blue-white screening of the colonies. But sometimes they can give false positive results, so to confirm the presence of right insert, colony PCR is also used. It is done in 20µl reaction mixture comprising of 10X DreamTaq Green Buffer, 0.2 mM dNTP Mix, FP and RP, 25 mM

MgCl2, 1 U Taq DNA Polymerase. PCR cycles were: Initial denaturation at 95°C for 3 minutes, 35 cycles for denaturation at 95°C for 30 seconds, annealing at 72°C for 3 minute and final extension at 72°C for 7 min. Colonies showing the respected amplification are further subcultured, plasmids are extracted from them and send to GATC biotech sequencing center (Köln, Germany) to check whether the plasmid has any other mutation except the desired mutation for mutants and whether the gene has been inserted and in proper orientation or not for the wild type OprO gene.

3.4.2 Cell wall extraction and purification of OprO and its mutants

Samples from different steps of expression, extraction, and purification of OprO wild type and OprO mutants are taken and loaded on to 10% SDS gels (Figure 3.5). An overexpression protein band corresponding to OprO or OprO mutant proteins at 47 KDa is seen (8). For further confirmation of presence of OprO or OprO mutant proteins in the extracted samples, dot blot test is performed with extraction steps. Dot blot test (Figure 3.6) was performed with anti-OprP protein as reported by Hancock etal that OprP antibody also shows antigenic property towards OprO (8).

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Figure 3.5:- Gel picture of different steps of expression and purification of OprO wild type in E. coli. Samples are taken at different point of expression and purification are taken and loaded onto 10% SDS gels. Each sample is heated at 95°C for 2*10 minutes. The gel is stained with silver staining. An overexpressing band is seen between 55 and 40 kDa giving an indication of the presence of OprO protein.

After the confirmation of the presence of OprO or OprO mutant proteins in the solubilizing samples, they are concentrated by amicon filters and loaded onto the anion exchange chromatography for further purification of OprO wild type or mutant proteins. OprO protein is eluted out at 300 to 400 mM NaCl (fraction 36) salt concentration (Figure 3.7).

Figure 3.6:- Dot blot test for the confirmation of OprO or OprO mutant proteins.

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Figure 3.7:- Purification of OprO wild type protein with anion exchange chromatography. Salt gradient of 0 to 1 M NaCl is applied to the column. OprO wild type or OprO mutants are eluted out at 36th fraction.

3.4.3 Single channel conductance of OprO double mutant does not show similar conductance as of OprP wild type.

The SCC of OprO, OprP WT and OprO mutants was measured in 0.1M and 1M KCl, pH 6 10mM MES at +50 mV applied voltage at RT. The single channel recordings show that they form defined channels. Each channel was facing upwards demonstrating that the channels are in open position. A typical single channel recording in a step-like manner caused by insertion of OprO WT, OprO mutants and OprP WT is shown in Table 3.4. Conductance was measured in solutions having 2 different KCl salt concentrations in order to determine whether the substitution of amino acids F62 and D114 has any detrimental effect on OprO pore or not. Comparison between the values of SCC of 0.1 M and 1 M KCl showed no clear effect on the anion binding site of any mutants, as the values did not show linear concentration-conductance relationship. Based on previous studies the single channel conductance of OprO is clearly higher than the one obtained for OprP (240 vs. 160 pS in 0.1 M KCl) (9). At the single amino acid level, the exchange of phenylalanine by tyrosine in OprO F62Y produced a decrease in the overall conductance of the channel (200 pS in 0.1M KCl). The slight decrease in conductance is probably caused by the introduction of an extra - OH group at 62nd position. On the other hand, the replacement of aspartic acid with bulky tyrosine in OprO D114Y did not showed any effect on the conductance (240pS in 0.1M KCl).

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When both the mutation is introduced together, it surprisingly increases the conductance of the OprO channel (400 pS in 0.1M KCl) (Table 3.3).

Proteins Average single channel conductance

0.1M KCl 1M KCl

OprO WT 240 440

OprO (F62Y) 200 400

OprO (D114Y) 240 440

OprO DM 400 600

OprP WT 160 260

Table 3.3:- Single-channel conductance of OprO WT, three OprO mutant and OprP WT channels in 0.1 M and 1 M KCl at pH 6 solutions. The experiments were carried out in DiPhPC membranes with an applied voltage of +50 mV at RT. At least 100 independent events were used to calculate the conductance.

These SSC results are in agreement with the recent theoretical studies performed by Modi et al. (4), where even in computational modeling, the conversion of OprO to OprP by OprO DM does not yield similar conductance as OprP WT. Whereas in the same study, conversion of OprP to OprO with DM of OprP experimentally and theoretically yielded similar conductance as of OprO WT.

Figure 3.8:- Top-view of cartoon representation of a) OprO WT and b) OprO DM monomers. D94, OprO WT and mutated residues are shown in blue color. Astrid symbol represents the conducting pathway. The oxygen molecule is shown in red.

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The increase in approximately 2-fold KCl conductance of OprO DM compared to OprO WT might have resulted from the faster permeation of chloride ions through the channel. This could be a result of effect put together by of replacement of phenylalanine with a less hydrophobic tyrosine and on the other hand, replacing the negatively charged aspartic acid with a neutralizing tyrosine. Along with these effects, the two-mutated tyrosine also partially masks the D94 (as shown in Figure 3.8b) (only negative amino acid present in the channel), allowing free flow of Cl- ions through the channel. From our studies, we also expected that the radius of the channel should decrease due to the introduction of bulkier groups. But as it can be observed that the radius of the OprO DM did not showed any significant difference compared to WT OprO when studied in silico using HOLE software in computational modeling. (10) (Figure 3.9).

Figure 3.9:- Figure showing average radius of OprO wild type and OprO double mutant channels along with the corresponding standard deviation derived from unbiased MD simulations. The pore radii have been determined using the HOLE program (10).

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3.4.4 Substrate specificity studies of the proteins

In order to understand whether the single and double mutation of OprO had any effect on the interaction of phosphate and diphosphate, so the ability of these ions to inhibit the SCC was measured in BLM. The data of the binding kinetics experiments were plotted and analyzed using Lineweaver-Burk plot or Langmuir isotherms to calculate the stability constants, K, for diphosphate or monophosphate binding.

Figure 3.10:- Lineweaver -Burk plots for OprO WT and its mutants for the inhibition of membrane conductance. The straight lines correspond to stability constants K given by the equation 1/ (Fraction of closed channels) = Kc/ (Kc +1). a) Lineweaver -Burk plots for diphosphate - OprO WT shows highest stability constant for diphosphate K =1450 1/M. Followed by intermediate stability constant OprO D114Y with K = 630 1/M and OprO F62Y with stability constant K = 490 1/M; OprP WT having the least stability constant K = 310 1/M for diphosphate and OprO DM showing similar stability constant as of OprP WT with K = 343 1/M

b) Lineweaver -Burk plots for phosphate - OprP WT shows highest stability constant K = 770 1/M; Followed by OprO DM stability constant with K = 720 1/M. OprO WT showing the least stability constant K = 220 1/M and intermediate stability constants are represented by D114Y with stability constant K = 500 1/M and OprO F62Y with K = 690 1/M

Examples for the first type of analysis are shown in Figure 3.10 and yield half saturation concentration constants, K for diphosphate or monophosphate binding respectively.

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Diphosphate Phosphate Proteins 1/K (mM) K (1/M) 1/K (mM) K(1/M) OprO WT 0.6 1450± 120 4.5 220 ± 50 OprO (F62Y) 2 490±110 1.4 690 ± 140 OprO(D114Y) 1.5 630±90 2 500 ± 70 OprO DM 2.9 343±20 1.4 720 ± 90 OprP WT 3.1 310 ± 35 1.3 770 ± 150

Table 3.4:- Phosphate and diphosphate mediated inhibition of chloride conductance of OprO WT, OprO mutants and OprP WT in 0.1M KCl, 10mM MES, pH 6, with applied voltage +50 mV at RT. The half stability constants for inhibition of Cl- conductance by diphosphate or phosphate were obtained from titration experiments using either the Lineweaver Burk plot or the Langmuir equation. Mean values (± SD) of at least three individual titration experiments are shown.

The results of chloride inhibition experiments of OprO WT and mutant proteins with monophosphate and diphosphate are summarized in Table 3.4. The stability constants for binding of monophosphate and diphosphate to OprO WT and to the OprO DM showed considerably differences. As shown in Figure 3.10(a), OprO WT shows a high stability constant for diphosphate with K =1,450 1/M as known (represented by the green straight line). In contrast OprO DM (red line), showed a significantly reduced affinity for diphosphate; K=343 1/M. This value is very close to the stability constant for binding to OprP WT (black line). Intermediate binding affinity was found for OprO F62Y (blue line) and OprOY114D (yellow line); K = 490 1/M and K = 630 1/M respectively. In contrast to this, phosphate binding to the OprO DM is increased and has similar stability constant as shown by OprP WT Figure 3.10(b); K =720 1/M and K =770 1/M respectively. As known previously, OprO WT has a relatively low stability constant with K = 220 1/M for phosphate (4).

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3.5 Discussion

3.5.1 OprO mutants shows similar specificity as of OprP wild type and future aspects

In the present studies, we could successfully exhibit that by exchanging the two amino acid residues F62Y and D114Y at the lumen of the constriction region of OprO, the substrate specificity of OprO WT can be changed to that of the properties similar to OprP WT. As known, OprO is a homologue of OprP and previous studies on this pore helps us in emphasizing the importance of specific amino acids present at the constriction region for binding of phosphate molecules (11, 12, 13). And any change in these amino acids can alter the functionality of the complete pore. Similarly, from our studies we can conclude that these two residues F62 and D114 are of major importance in exhibiting diphosphate specificity in OprO and any change in these amino acids can modify the whole characteristic properties of this pore. This could be seen via OprO DM where OprO resembles OprP in substrate binding. This study together with other techniques can be useful for understanding the principles of ion and molecule specificity of different pores. And also gives an important insight in the transport of antibiotics, such as fosfomycin and fosmidomycin and related molecules.

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3.6 REFERENCES

1. S, Falanga A, Cantisani M, Tarallo R, Pepa M E D, D’Oriano V,and Galdiero M (2012). Microbe-Host Interactions: Structure and Role of Gram-Negative Bacterial Porins. Curr Protein Pept Sci, 13(8). p. 843–854. 2. Hancock R.E.W and Brinkman F. S. L (2002). Function of Pseudomonas Porins in Uptake and Efflux. Annu. Rev. Microbiol, 56, p.17–38. 3. Høiby. N; Ciofu. O; Bjarnsholt. T (2010). Pseudomonas aeruginosa Biofilms in Cystic Fibrosis. Future Microbiol, 5(11). P.1663-74. 4. Modi N, Ganguly S, Bárcena-Uribarri I, Benz R, van den Berg B, Kleinekathöfer U (2015) Structure, Dynamics, and Substrate Specificity of the OprO Porin

from Pseudomonas aeruginosa. Biophys J. ,109 (7), p.1429–38. 5. Van der ley.P, Amesz.H, Tommassen.J and Lugtenberg.B (1985). Monoclonal antibodies directed against the cell-surface-exposed part of PhoE pore protein of the Escherichia coli K-12 outer membrane. Eur. J. Biochem. 147 (2), p. 401 –407. 6. Hoheisel.J. D (1989). A cassette with seven unique restriction sites, including octanucleotide sequences: extension of multiple-cloning-site plasmids. Elsevier Gene, 80.p. 151-4. 7. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, p. 680-685. 8. Sukhan, A., and Hancock, R. E. W. (1995) Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP. J. Bacteriol. 177 (17), p. 4914–4920. 9. Hancock, R. E. W., C. Egli, R. J. Siehnel (1992). Overexpression in Escherichia coli and functional analysis of a novel PPi-selective porin, oprO, from Pseudomonas aeruginosa. J. Bacteriol. 174, p. 471–476. 10. Smart.O.S., Goodfellow. J.M and Wallace B.A (1993).The Pore Dimensions of Gramicidin A. Biophys J., 65 (6), p. 2455-60. 11. Modi N, Bárcena-Uribarri I, Bains M, Benz R, Hancock RE, Kleinekathöfer U (2014). Tuning the affinity of anion binding sites in porin channels with negatively charged residues: molecular details for OprP. ACS Chem Biol.; 10(2), p. 441-51. 12. Sukhan .A and Hancock R.E.W (1996). The Role of Specific Lysine Residues in the Passage of Anions through the Pseudomonas aeruginosa Porin OprP J Biol Chem., 271 (35),p. 21239-42.

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13. Modi N, Bárcena-Uribarri I, Bains M, Benz R, Hancock RE, Kleinekathöfer U (2013). Role of the central arginine R133 toward the ion selectivity of the phosphate specific channel OprP: effects of charge and solvation. Biochemistry: 52(33):p.5522- 32.

CHAPTER 4

DIETZIA MARIS GENOME ANNOTATION

(NOTE: - This chapter is derived from accepted manuscript; under Genome Announc. 4(3). pii: e00542-16.)

Sonalli Ganguly, Guadalupe Jimenez-Galisteo, Daniel Pletzer, Mathias Winterhalter, Roland Benz and Miguel Viñas. Draft genome sequences of Dietzia maris DSM 43672 a Gram- positive bacterium of mycolata group.)

4.1 Summary

Dietzia maris is a Gram-positive, non-spore forming soil bacterium. Dietzia species has become an emerging opportunistic pathogen in recent years. They are also good source of enzyme used in bioremediation and biodegradation. Previously, a channel forming protein was observed in Dietzia maris. So, to know the gene sequence of this porin, the genome project was undertaken. In this chapter, we report the draft genome sequence of Dietzia maris, earlier known as Rhodococcus maris. The genome contains 3,505,372 base pairs in size with 73% of G+C content. The draft genome sequence will improve our understanding of Dietzia maris related to other mycolata species as well as constitutes a basic tool to explore the cell wall proteins.

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4.2 Introduction

Formerly known as Rhodococcus maris, Dietzia maris was initially isolated from soil, skins, and intestinal tracts of carp (Cyprinus carpio). It is a Gram-positive cocci growing sometimes as short rods, aerobic and non- spore forming (1). Its colony appearance is circular, raised or convex, glistening, and has a deep orange color (2). Dietzia maris holds potential source of enzymes which are used in oil degradation and biosurfactant production (3), industrial fermentation and to produce the antioxidant canthaxanthin (4). Recently, Dietzia species are emerging as a new group of opportunistic pathogens. Cases have been reported to cause bacteremia and septic shocks including prosthetic hip infection in patients having lower immune system (5, 6, 7).

Phenotypically and taxonomically Dietzia species are closely related to actinomycetes group. Actinomycete comprises the genera Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, Tsukamurella, and Gordonia (2). The cell wall in these species contains mycolic acids of different length depending, which act as a permeability barrier for the passage of solutes (8). These mycolic acids act in a similar manner as do the outer membrane of Gram-negative bacteria. Previously, many reports dealing with channel forming proteins have been published about these species (9). These channels forming proteins are responsible for the uptake of hydrophilic compounds across the mycolic acid layer.

From the cell wall extracts of Dietzia maris a channel-forming protein was isolated and studied in our laboratory (9). To have an insight of structure and function of this channel forming protein and to understand its relation with other channel forming proteins in mycolata, we have sequenced and annotated the complete genome of Dietzia maris.

Genome annotation is a process for the proper understanding of genes and the function to which they are associated. Genome sequences are strings of nucleotide, which has only limited application in research and molecular biology. Various analysis procedures are required to assign biological interpretation to a genome sequence. Goal of genome annotation is to describe the function of every single nucleotide, in any cell or cell compartment to know how these genes functions together to direct the growth, development and maintenance of an organism (10). Bioinformatics tools are used for the annotating the genome sequence. But along with bioinformatics, other approaches are used such as molecular biology, information

CHAPTER 4 technology, mathematics and statics. Biologists deliver the raw data and biological context for the annotation of a sequence. Often this results in new hypotheses that lead to more experiments by both biologists and bioinformaticians and ultimately contribute to the advancement of biological understanding.

Genome annotation starts with identification of protein coding domains that has codon usage and translation start sites and then follows the functional annotation which predicts the biological function for each of those elements and the biological process in which it takes part. A genome is divided into parts- the protein and RNA-encoding genes and the other, non-coding DNA (11, 12). These protein coding genes are selected and compared to known genes found in other organisms and the coding domain is adjusted, if necessary. If the gene matches the gene sequence of other organism whose function is known, then the function of the protein coding genes can be concluded. Many genome projects are being undertaken, so proper assembly and high throughput annotation tools are required. Sometimes these tools are not upto the mark and can lead to misannotation. Therefore, these automated annotations need to undergo human review for each gene before the final annotation is released.

For annotation of Dietzia maris genome, we used automatic annotation tools such as Rapid Annotations using Subsystems Technology (RAST) server, Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) via NCBI and for manual annotation NCBI Basic Local Alignment Search Tool (BLAST) was used.

4.3 Methods and material

4.3.1 Isolation of Dietzia maris genomic DNA and sequencing

For the extraction of genomic DNA, 100 ml of bacterial cells were grown in brain-heart infusion medium at 28°C in aerobic condition for 2-3 days. The cells were centrifuged, pelleted down, and were extracted with commercially available bacterial genomic DNA isolation kit (GenElute bacterial genomic DNA kit, NA2110; Sigma-Aldrich Co., Germany). DNA was sequenced by LGC Genomic (Berlin, Germany). Approximately 12µg of genomic DNA was used for sequencing. Shortgun libraries were generated and both the ends of DNA fragments were attached with adaptors. Illumina MiSeq sequencer was used to sequence

CHAPTER 4 these libraries. Reads with final length < 20 bases were discarded and read having more than one “N” were removed. After trimming and error correction with Musket version 1.0.6 of the sequences, they were assembled and scaffolded using Ray v 2.3.1 de Novo assembler. It produced 56 scaffolds in “dna” and FASTA formats.

4.3.2 RAST (rapid annotation using subsystem technology)

RAST is a fully automated tool used for annotation for bacterial and archaeal genomes. It was built in 2008 for annotation of complete or nearly complete genomes (13). It identifies protein coding genes, tRNAs and rRNAs. It was built up on the infrastructure of SEED system (14).

Note: - This figure is hidden due to copyright issues

Figure 4. 1:- Flow chart annotation in the SEED system. Annotation of the genome in the SEED system works on the curation of subsystem of genes. From these curative subsystems, we extract the group of protein families. Then these new annotated sybsyytems will become new FIGfams (http://www.slideshare.net/ramykaram/rka-evergeen2015-phantomefinalweb).

Annotation of the genome in the SEED system works on the curation of subsystem of genes that are similar in functional role, such as a metabolic pathway. From these curative subsystems, we extract the group of protein families (FIGfams) (Figure 4. 1). FIGfams are a set of protein sequences which have over 70% sequence similarity and are considered to

CHAPTER 4 follow out the same functional role. Based on these systems, RAST attempts to achieve accuracy, consistency, and completeness in its annotation. RAST server not only allots initial gene function for the particular gene and a metabolic pathway, but also serves as a collection point for these new subsystems such that, when subsequently curated, will become new FIGfams. It also allows comparing it to the hundreds of genomes maintained within the SEED integration (15).

The Basic Steps in Annotating a Genome Using RAST

RAST services are freely available online tools for the annotation of prokaryotic genomes. The genomes may be in the form of "complete" or they may be in hundreds of contigs (which does impact the quality of the derived annotations). A new user has to register in the server initially by acquiring a password, such that the user can have access to those genomes that they have submitted. It also allows the server also to contact the user once the automatic annotation has finished or in case user intervention is required (13). Once the account has been created, import the prokaryote genome in the form of a set of contigs or whole genome in FASTA format. After login, the user can monitor his/her submitted job/jobs on the Job Overview page (Figure 4.2).

Figure 4.2:- RAST Job detail page.

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After uploading of genome is finished, they are checked and any duplicate sequences are deleted and then a unique internal id is generated for each of the sequences. Then these sequences are screened for protein coding genes via BlastX (16). After the annotation is finished the results are available to view and download (GenBank, FASTA, GFF3, Excel)) or the genome can be browse in the SEED-Viewer without having the data actually installed in the SEED. The output files can be saved on a personal computer for further review.

4.3.3 Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP)

Another automatized annotation tool for prokaryotic genomes is Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) via NCBI. It makes use of statistical and similarity- based methods. Similarity based methods is used when sufficient quantities of comparative data are available and statistical based method is used when there is absence of supporting material for the gene in database. It also provides annotation services to GenBank submitters using this pipeline, which is also used to annotate NCBI reference sequence (RefSeq) in prokaryotic genomes (17).

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Note:- This figure is hidden due to copyright issues

Figure 4.3:- Flow chart of annotation Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) via NCBI. Each box represents the step how a genome is proceeding in this pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/process/).

GenBank submitters can submit their genomes in the form of complete genomes as well as whole genome sequences (WGS) consisting of multiple contigs. But the genome sequence should match the standards required for submission. The RefSeq are the collection of sequences that provides a comprehensive, integrated, well-annotated set of sequences, including genomic DNA, transcripts, and proteins. These sequences form the foundation of medical, functional, and diversity studies and provide a stable reference for genome annotation, gene identification and characterization, mutation and polymorphism analysis, expression studies, and comparative analyses.

NCBI’s PGAAP uses second-generation gene prediction approaches and was developed in 2001-2002. The second-generation approach uses a combination of hidden Markov model (HMM)-based gene prediction algorithms with protein homology methods. Genes are predicted using a combination of tools like GeneMark (18) and Glimmer (19). Ribosomal RNAs were predicted by sequence similarity searching using BLAST against an RNA

CHAPTER 4 sequence database and/or using Infernal and Rfam models. Transfer RNAs were predicted using tRNAscan-SE (20).

Submission of genome in NCBI pipeline initiate with creating each contig in FASTA format (Figure 4.3),

Example :- >contig02 [organism=Clostridium difficile] [strain=ABDC] [gcode=11] [plasmid- name=pABDC1] [topology=circular] [completeness=complete]

Then by creating a submission put all the files in a single directory and run tbl2asn. If any problem arises, fix it and proceed to next level of submission where you need to write a letter stating, registered BioProject and organism name in the message and the requested release date of the genome, including a request to annotate the genome through PGAAP. After successful completion of submission, an accession number is issued. Then it is released to the public database after requested date.

4.3.4 BLAST (Basic Local Alignment Search Tool)

It is a widely and mostly used bioinformatics tool for homology searches in databases. It was released in 1990. It is based on alignment algorithm for biological sequences and it allows a person to compare a query of nucleotide or a protein sequence against a nucleotide or protein database of sequences. It allows in identifying database sequences that resemble the query sequence above a certain threshold (21).

BLAST is a very good tool for initial identiﬁcation of possible functions for a new sequence, but it should be used with caution. Due to widespread usage of this tool, many of the genes in are annotated using homology-based function prediction. Sometimes his may lead to misannotations and other errors.

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Figure 4.4:- An example of BLAST query search

4.4 Results

4.4.1 Results from RAST

Genomic DNA from Dietzia maris was isolated and was send for sequencing to LGC Genomic (Berlin, Germany). Sequencing of the whole genome resulted in 3,505,372 base pairs divided into 56 scaffolds with a K-mer value of 57. Scaffold N50 was 121,122. It has 70% of G+C content.

These scaffolds were run through RAST automatic annotation systems. RAST gave us an overview containing basic information, on the genome such as taxonomy, size and the number of contigs, the number of coding sequences, RNAs and subsystems that were automatically determined to be present in the genome. Along with these features a bar graph and a pie chart represents the distribution of genes connected to the various subsystem groups (Figure 4.5).

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Figure 4.5:- Information obtained from RAST server. It gives information on taxonomy, size and the number of contigs, the number of coding sequences, RNAs and subsystems

Prediction based on DNA sequences The subsystem shows that whole genome of Dietzia maris consists of total 3311 features divided into following categories. The numbers before individual categories depicts the number of genes belonging to the each category in Dietzia maris genome.

 Cofactors, Vitamins, Prosthetic Groups, Pigments (264)  Cell Wall and Capsule (76)  Virulence, Disease and Defense (65)  Potassium metabolism (26)  Photosynthesis (0)  Miscellaneous (36)  Phages, Prophages, Transposable elements, Plasmids (0)

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 Membrane Transport (46)  Iron acquisition and metabolism (0)  RNA Metabolism (78)  Nucleosides and Nucleotides (102)  Protein Metabolism (230)  Cell Division and Cell Cycle (24)  Motility and Chemotaxis (2)  Regulation and Cell signaling (40)  Secondary Metabolism (0)  DNA Metabolism (78)  Fatty Acids, Lipids, and Isoprenoids (213)  Nitrogen Metabolism (38)  Dormancy and Sporulation (2)  Respiration (46)  Stress Response (92)  Metabolism of Aromatic Compounds (7)  Amino Acids and Derivatives (324)  Sulfur Metabolism (18)  Phosphorus Metabolism (36)  Carbohydrates (276)

Comparative genomics Dietzia maris genome is compared with other genome present in the SEED viewer. The genome which is mostly similar to Dietzia maris is noted first. Top 10 genomes have been quoted here.

Genome ID Score Genome Name

1077144.3 518 Dietzia alimentaria 72

596309.3 489 Rhodococcus erythropolis SK121

101510.15 478 Rhodococcus jostii RHA1

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685727.3 478 Rhodococcus equi 103S

101510.16 462 Rhodococcus jostii RHA1

632772.3 445 Rhodococcus opacus B4

234621.6 412 Rhodococcus erythropolis PR4

257309.1 358 Corynebacterium diphtheriae NCTC 13129

257309.4 348 Corynebacterium diphtheriae NCTC 13129

553207.3 326 Corynebacterium matruchotii ATCC 14266

1166015.3 304 Corynebacterium diphtheriae bv. intermedius str. NCTC 5011

Comparative genomics data produced by RAST server suggests that Dietzia maris is closely related to Dietzia alimentaria 72 genome and then with Rhodococcus erythropolis SK121 genome.

4.4.2 Automatic annotation by PGAAP

The various contigs of Dietzia maris obtained from sequencing center was uploaded onto PGAAP and after the annotation the sequences were submitted onto GenBank and NCBI. The following results were obtained. We identified a total of 3,199 protein-coding genes, 49 tRNAs, 10 rRNAs, and 3 non-coding RNAs.

DEFINITION Dietzia maris strain DSM 43672, whole genome shotgun sequence. ACCESSION VERSION DBLINK BioProject: PRJNA315745 BioSample: SAMN04543680 KEYWORDS WGS. SOURCE Dietzia maris

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ORGANISM Dietzia maris Bacteria; Actinobacteria; Corynebacteriales; Dietziaceae; Dietzia. REFERENCE 1 (bases 1 to 450) AUTHORS Ganguly,S., Jimenez-Galisteo,G., Vinas,M. and Benz,R. TITLE Direct Submission JOURNAL Submitted (21-MAR-2016) Life Sciences & Chemistry, Jacobs University, Campus Ring 1, Bremen 28759, Germany COMMENT Annotation was added by the NCBI Prokaryotic Genome Annotation Pipeline (released 2013). Information about the Pipeline can be found here: https://www.ncbi.nlm.nih.gov/genome/annotation_prok/

##Genome-Assembly-Data-START## Assembly Method :: Ray v. 2.3.1 Assembly Name :: scaffolds Genome Representation :: Full Expected Final Version :: No Genome Coverage :: 1.0x Sequencing Technology :: Illumina MiSeq ##Genome-Assembly-Data-END##

##Genome-Annotation-Data-START## Annotation Provider :: NCBI Annotation Date :: 03/21/2016 16:25:10 Annotation Pipeline :: NCBI Prokaryotic Genome Annotation Pipeline Annotation Method :: Best-placed reference protein set; GeneMarkS+ Annotation Software revision :: 3.1 Features Annotated :: Gene; CDS; rRNA; tRNA; ncRNA; repeat_region Genes (total) :: 3,261 CDS (total) :: 3,199 Genes (coding) :: 3,078 CDS (coding) :: 3,078 Genes (RNA) :: 62 rRNAs :: 3, 3, 4 (5S, 16S, 23S) complete rRNAs :: 3, 1 (5S, 16S) partial rRNAs :: 2, 4 (16S, 23S) tRNAs :: 49 ncRNAs :: 3 Pseudo Genes (total) :: 121 Pseudo Genes (ambiguous residues) :: 0 of 121 Pseudo Genes (frameshifted) :: 24 of 121 Pseudo Genes (incomplete) :: 92 of 121 Pseudo Genes (internal stop) :: 13 of 121 Pseudo Genes (multiple problems) :: 8 of 121 ##Genome-Annotation-Data-END##

Nucleotide sequence accession number: - The draft genome sequence of Dietzia maris DSM 43672 has been deposited in GenBank under the accession number. LVFF00000000.

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4.4.3 Manual annotation by BLAST

Automatic annotation is less precise than manual annotation, but manual annotation is very labor intensive. Contigs from 0 to 56 was carefully observed and it was run through RAST server. RAST created some gene id and gave an estimation of protein coding genes. These protein coding genes from each contigs were taken individually and blasted in NCBI- BLAST. The highest query that matched the particular sequence was taken into account. The genes which showed difference in annotation by automatic annotation were adjusted and annotated again. It was observed that automatic annotation failed to recognize few genes correctly including recognition of conserved domains. But with manual annotation, it was easy to find these.

4.5 Discussion

In this chapter, the main goal was to sort out the whole raw genome sequence of Dietzia maris, as only the nucleotide sequences cannot give all the required information regarding the structural features of it. This genome project can also help us in improving our understanding about its relation to other mycolata species as well as to gain knowledge about the cell wall proteins. An important step in understanding about Dietzia maris was to annotate the whole genome by both automatic and manual annotation. Both annotation processes helped in identifying the location of protein coding and non-protein coding genes, the functional identity of genes and the manual curation of these identities improved our output.

Genome annotation is not a routine, easy and everyday activity, but it is an exciting research, somewhat similar to detective’s work, which has the potential of chasing out the deep mysteries of life from genome sequences.

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4.6 REFERENCES

1. Rainey FA, Klatte S, Kroppenstedt RM & Stackebrandt E (1995). Dietzia, a new genus including Dietzia maris comb. nov. formerly Rhodococcus maris. Int J Syst Bacteriol 45, p.32–36. 2. Koerner RJ, Goodfellow M & Jones AL (2009). The genus Dietzia: a new home for some known and emerging opportunist pathogens. FEMS Immunol Med Microbiol 55, p.296–305. 3. Wang.W, Cai. B, and Shao.Z (2014). Oil degradation and biosurfactant production by the deep sea bacterium Dietzia maris As-13-3. Front Microbiol 5, p.711. 4. Venugopalan V, Tripathi SK, Nahar P, Saradhi PP, Das RH, Gautam HK (2013).Characterization of canthaxanthin isomers isolated from a new soil Dietzia sp. and their antioxidant activities. J Microbiol Biotechnol. 23(2), p. 237-45. 5. Bemer-Melchior.P, Haloun. A, Riegel.P, and Drugeon1.H.B (1999). Bacteremia due to Dietzia maris in an immunocompromised patient. Clin Infect Dis. 29(5), p.1338-40. 6. Pidoux.O, Argenson.JN, Jacomo.V, and Drancourt.M (2001). Molecular Identification of a Dietzia maris Hip Prosthesis Infection Isolate.J Clin Microbiol. 39(7), p.2634–2636. 7. Reyes G, Navarro JL, Gamallo C, delas Cuevas MC (2006). Type A aortic dissection associated with Dietzia maris. Interact Cardiovasc Thorac Surg. 5(5), p.666-8. 8. Marrakchi. H, Lanéelle M-A, Daffé. M (2014). Mycolic Acids: Structures, Biosynthesis, and Beyond. Chem Biol., 21, p. 67–85. 9. Mafakheri S, Bárcena-Uribarri I, Abdali N, Jones AL, Sutcliffe IC, Benz R (2014). Discovery of a cell wall porin in the mycolic-acid-containing actinomycete Dietzia maris DSM 43672. FEBS J, 281(8), p.2030-41. 10. Fleischmann RD1, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM (1995). Whole-genome random sequencing and assembly of Haemophilus inﬂuenzae Rd. Science, 269(5223), p.496– 512. 11. Snyder.M and Gerstein.M (2003) Genomics. Deﬁning genes in the genomics era. Science, 300(5617), p.258–60. 12. Pearson.H (2006). Genetics: What is a gene? Nature, 441(7092), p.398–401.

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13. Aziz RK , Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL,Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008).The RAST Server: rapid annotations using subsystems technology. BMC Genomics, 9, p.75. 14. Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, Vonstein V, Wattam AR, Xia F, Stevens R (2014). The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res.; p.42(Database issue). 15. The SEED framework for comparative genomics. [http://www.theseed.org] 16. Markowitz, V. M., Mavromatis, K., Ivanova, N. N., Chen, I. A., Chu, K., & Kyrpides, N. C. (2009). Img er: a system for microbial genome annotation expert review and curation. Oxford Bioinformatics. 25, p.2271–2278. 17. Tatusova.T, DiCuccio.M, Badretdin.A, Chetvernin.V, Ciufo.S, and Li.W. (2013). Prokaryotic Genome Annotation Pipeline. Rd. The NCBI Handbook [Internet]. 2nd edition. 18. Lukashin AV, Borodovsky M (1998). GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res. 26(4), p.1107–15. 19. Salzberg SL, Delcher AL, Kasif S, White O (1998). Microbial gene identification using interpolated Markov models.Nucleic Acids Res. 26(2), p. 544–8. 20. Lowe T.M., Eddy S.R (1997). tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucl. Acids Res.; 25, p.955–964. 21. Altschul SF1, Gish W, Miller W, Myers EW, Lipman DJ (1990). Basic local alignment search tool. J Mol Biol. 215(3), p. 403-10.

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Note: - Sonalli Ganguly, Guadalupe Jimenez-Galisteo, Daniel Pletzer, Mathias Winterhalter, Roland Benz and Miguel Viñas. Draft genome sequences of Dietzia maris DSM 43672 a Gram-positive bacterium of mycolata group

Link for the paper: - Dietzia maris annotation

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CHAPTER 5

IDENTIFICATION OF MspA PROTEIN LIKE GENES IN RHODOCOCCUS SPECIES

5.1 Summary

Genus Rhodococcus has gained its importance in the field of environmental, pharmaceutical, chemical and energy sectors, as it consists of potential source of enzymes for degradation or synthesis of various compounds. They are also great threat to humans as emerging pathogens. This genus belongs to mycolata group, with distinct cell wall composition, except than that found in Gram-positive bacteria. Previous studies on cell wall composition of Rhodococcus, identified channel forming proteins. In this chapter, we have identified porins which are similar to MspA porin from Mycobacterium smegmatis in Rhodococcus species.

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5.2 Introduction

“Rhodococcus” genus name was given by Zopf 1891(1), later it was revived and redefined by Goodfellow & Alderson in 1977 (2). It belongs to mycolata group consisting of suborder Corynebacterineae, phylum Actinobacteria, family Norcardiae. Rhodococci are Gram- positive, aerobic, non-motile, high G+C content, mycolate containing actinomycetes (3) and have been isolated from soil, rocks, marine sediments, gut of insects, and from healthy and diseased animals and plants (2). In recent years, they have gained attentions of researchers due to its wide range of application. It inhabits a large source of compounds needed for degradation of a range of environmental pollutants and to transform or synthesize various compounds. Another reason for gaining importance is that these bacteria are also emerging as new pathogens. Few species of Rhodococcus has been reported to cause human infections including animals (4). One of the major diseases causing species is Rhodococcus equi which is found to effect immunosuppressed patients (3).

The cell wall of Rhodococci are made up of thick layer of mycolic acids (hence the name mycolata) which are in turn attached to cell wall. Cell wall consists of various components such as channel-forming pore proteins, free lipids, lipoglycans, lipoproteins and capsules or cell envelope polysaccharides. Porins are one of the major components of the cell wall as it allows transport of hydrophilic molecules inside the cell. Many pore forming channels has been reported previously in Rhodococcus, Mycobacterium and Nocardia (6). In our studies, we will be mainly focusing on the porins which are similar to MspA derived from Mycobacterium smegmatis (7).

MspA porin

MspA is the major porin of Mycobacterium smegmatis (Figure 5. 1) and the most studied porin from mycolata group. It mediates the exchange of hydrophilic solutes across the outer membrane (7). It is cation selective in nature and eight monomers of MspA constitute a single channel of 9.6 nm in length. MspA porins are being used for DNA sequencing technologies (8). Sequence homology to Mycobacterium smegmatis MspA can also be identified in the Rhodococcus jostii genome (9). Here we are trying to find other MspA homologous proteins present in Rhodococcus species, hypothesizing that these MspA homologues can also be used for DNA sequencing technologies.

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Note:- The figure is hidden due to copyright isuues

Figure 5. 1:- MspA porin from Mycobacterium smegmatis. This is the only known porin in this organism (10)

5.3 Materials and methods

5.3.1 Bacterial strains and culture

Rhodococcus ruber DSM 43338 strain was grown on heart and brain infusion (HBI) medium (Roth) at 28°C for 3-4 days shaking at 200 rpm. The cells were washed twice with sterile distilled water and lyophilized.

5.3.2 Amino acid alignment of Rhodococcus MspA like proteins

For our studies we chose two different species of Rhodococcus, Rhodococcus sp. AW25M09 and Rhodococcus ruber, when we blasted MspA porin of Mycobacterium smegmatis against the genome of these porins, we found few sequences showing homology to this porin.

10 20 30 40 50 60 | | | | | | AW25M09 MTEIQKSRRSRGLKSMSVAVAASASVVGLVLG-TGTASAAVDSSNSVVDADGNTITVTLS Rxxxxx2 ----MKMQTRRGVKAARNVTVAAAAVFGLVLGSTGTAGAVVDDQNRIV-SDGHEVIVTQE Mspaxx1 ------MKAISRVLIAMVAAIAALFTSTGTSHAGLDNELSLVDGQDRTLTVQQW Consensus k rg Ka s v A aav glvlgsTGTa A vD ns Vd #g t tVtq Prim.cons. MTEI2K2222RG3KA3S3V33A3AAV3GLVLGSTGTA3A3VD33NS3VD3DG3T3TVTQ3

70 80 90 100 110 120

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| | | | | | AW25M09 DTFINGVAPLDGSPLTREWFANGKASYDVQGPSADDFEG-TLKIGYQIGYPMSLGGGVTV Rxxxxx2 DTFIQGVPPIGGSPLSREWFHNGRGIANIVGPDAADFEGSTFQFGYQFAWSGSLDGSIGV Mspaxx1 DTFLNGVFPLDRNRLTREWFHSGRAKYIVAGPGADEFEG-TLELGYQIGFPWSLGVGINF Consensus DTFi#GV PldgspLtREWFhnGra y ! GP Ad#FEG Tl GYQig p SLggg! v Prim.cons. DTFINGV3PLDGSPLTREWFHNGRA3Y3V3GP3ADDFEGSTL33GYQIG3P3SLGGGI3V

130 140 150 160 170 180 | | | | | | AW25M09 SWASPTASLGIGSSNT---STVTGTTTAGELLSPGGLGAFVAGAAIEGAGASTSGLDVGV Rxxxxx2 AWSSPQAELEVAPGEVYEPATETKTVDGKETEVPVTAPVLNEDGSPILDADGKPVTEQLY Mspaxx1 SYTTPNILID------DGDITAPPFGL------Consensus sw sP a l t t t dg et P gl Prim.cons. SW3SP3A3L3222222YEP2T2T2T2DG3ET33P3GL22222222222222222222222

190 200 210 220 230 240 | | | | | | AW25M09 DLLPQATGTIALTNGPGVVQSESAYAVKNDSADITS---AEGDAAGTSGSIQVANLHGTA Rxxxxx2 RIKPDATATENTLTVGGLLPQLTAGIELTPAPGIEELVVAEGEFDGDFKSVQIANVHGAA Mspaxx1 ------NSVITPNLFPGVSISADLGNGPGIQEVATFSVDVSGAEGGVAVSNAHGTV Consensus p at t n pgl p sa a l pgI e aeg# G gs!q!aN HGta Prim.cons. 222P2AT2T2N3333PGL3P33SA3A3L333PGI3E222AEGD33G33GSVQVAN3HGTA

250 260 270 | | | AW25M09 TEILGNVTIRPYVSLTSATGDTAITYGVPVRLN Rxxxxx2 TGVIGAVSVRPFVRVITANGDNVTTYGKVWTL- Mspaxx1 TGAAGGVLLRPFARLIASTGDSVTTYGEPWNMN Consensus Tg G V RP%vrli atGD vtTYG pw $n Prim.cons. TG33G3V33RPFVRLI3ATGD3VTTYG3PW3LN

For my studies, I started focusing on Rhodococcus ruber, when MspA porin sequence was blasted against Rhodococcus ruber, we found 3 genes to be homologous to MspA porin. When these three porins of Rhodococcus ruber were aligned, only few amino acids difference was found between them. (The alignment was performed using https://npsa-prabi.ibcp.fr/cgi- bin/align_multalin.pl)

10 20 30 40 50 60 | | | | | | SEQ1xx0 --MKMQTRRGVKAARNVTVAAAAVFGLVLGSTGTAGAVVDDQNRIVS-DGHEVIVTQEDT seqxxx1 ------RGFKAARNAAVSGAAVLGLVLGATGSAQAAVDDQNRIVSTDGYEVVVTQEDT seqxxx2 MINTFSGNRFRQCARATALGAAAVLGMVVGS-GSAGAAVDNARTLPLGNNNAIEVLQADT Consensus Rg kaARn av aAAVlG$VlGstGsAgAaVD#qnrivs #g e! VtQeDT Prim.cons. MI222222RG3KAARN3AV3AAAVLGLVLGSTGSAGAAVDDQNRIVS2DG3EV3VTQEDT

70 80 90 100 110 120 | | | | | | SEQ1xx0 FIQGVPPIGGSPLSREWFHNGRGIANIVGPDAADFEGSTFQFGYQFAWSGSLDGSIGVAW seqxxx1 FIQGVPALGGSPFNREFFHNGRGTANLVGADAADAEGTTFQFGYQFAWAGSIDGAIGVTY seqxxx2 SIQSYPPLDSSPVSFEFFHDEVVSVNFTGPDADSFQDTKLTIGYQIGYPVALNGAV-VHI Consensus fIQgvPplggSP srEfFH#grg aN vGpDAadf#gttfqfGYQfaw gsl#Ga!gV Prim.cons. FIQGVPPLGGSP3SREFFHNGRG3AN3VGPDAADFEGTTFQFGYQFAW3GSLDGAIGV33

130 140 150 160 170 180 | | | | | | SEQ1xx0 SSPQAELEVAPGEVYEPATETKTVDGKETEVPVTAPVLNEDGSPILDADGKPVTEQLYRI

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CHAPTER 5 seqxxx1 STPGLELELSA------KDG------seqxxx2 NTPSLDWEVG------NEFGIGV------ELAPEF Consensus stP l#lEv nedG e Prim.cons. STP3LELEV32GEVYEPATETKTVDGKETEVPVTAPVLNEDG222LDADGKPVTE22222

190 200 210 220 230 240 | | | | | | SEQ1xx0 KPDATATENTLTVGGLLPQLTAGIELTPAPGIEELVVAEGEFDGDFKSVQIANVHGAATG seqxxx1 -PTATVTD------ILPQAYAELELTPAPGIEELVVAEGEFDGDFKSVQFSNVHGTASG seqxxx2 ALGLDASTGATIGGSIIPSQEIEVDLSPGGITDVAFVEDMEFDGRSATVRLAGVHGSVSG Consensus p atat g ilPq ae #LtPapgi#elvVa#gEFDGdfksVq anVHG asG Prim.cons. 2P3ATAT322222G2ILPQ33AE3ELTPAPGIEELVVAEGEFDGDFKSVQ3ANVHG3ASG

250 260 270 | | | SEQ1xx0 VIGAVSVRPFVRVITANGDNVTTYGKVWTL seqxxx1 VLGAVQVRPFVRAITANGDNVTTYGKPWTV seqxxx2 ALGPVTIRPYARAVTSNNDTVITYGVPHRL Consensus vlGaV !RP%vRa!TaNgDnVtTYGkpwtl Prim.cons. VLGAV3VRPFVRAITANGDNVTTYGKPWTL

Sequence 0001: SEQ1xx0 (267 residues) - MspA protein (WP_003937791) Sequence 0002: seqxxx1 (208 residues) - porin protein MspA (WP_003937792) Sequence 0003: seqxxx2 (231 residues) - porin MspA (WP_003935716)

When proteins with NCBI accession number WP_003937791 and WP_003937792 were aligned together, it was found that these proteins have common promoter sequence.

5.3.2 Purification of Rhodococcus ruber cell wall

For isolation of the cell wall proteins of Rhodococcus ruber, same method was used for extraction of the cell wall channel as of Nocardia farcinica (11). This method is used for isolation of the cell wall proteins from Rhodococcus ruber lyophilized cells. The lyophilized cells are resuspended with 10 mM Tris-HCl, pH 8 and spin down at 2,900xg for 20 minutes. The pellet in the next step is treated with 0.2% SDS and spun down at 11,000xg, 4 °C for 30 minutes. This step is repeated twice. In the next step, the pellet is treated with 10 mM EDTA and then with 1% Genapol X-80 and spun down for 11,000xg, 4 °C for 10 min. All the supernatants are stored for further analysis in SDS-PAGE and for bilayer. In the last step, the final pellet is homogenized in 1% Genapol and 10 mM Tris-HCl, pH 8, for 20 h at 50 °C under agitation.

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5.4 Results

5.4.1 MALDI- tof

Samples from washing step with 10mM EDTA and 1% genapol are taken and are loaded onto the preparative SDS-gel (Figure 5.2). Band of 120KDa and 48 KDa are excised and send for detection by MALDI-tof to Rudolf-Virchow-Zentrum der Universität Würzburg, Würzburg.

Figure 5.2:- Gel picture showing the sample for MALDI-tof from Rhodococcus ruber. The samples on the left hand side of ladder is from washing step with 10 mM EDTA and right hand side samples are taken from washing step with 1% genapol.

MALDI results also confirmed the presence of both proteins together with NCBI accession number WP_003937791 and WP_003937792.

5.4.2 Single channel analysis of Rhodococcus ruber

Single channel experiments are performed similarly as it is explained in chapter 2. Crude samples from the cell wall extracts of Rhodococcus ruber are taken and are tested in bilayer. Mainly sample from washing step with 10mM EDTA is used. It showed channel forming activity.

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Figure 5.3:- Histogram figure showing pore forming activity from cell wall extracts of Rhodococcus ruber for the probability P(G) of occurrence of a given conductivity unit observed with membranes formed of 1% PC dissolved in n-decane. It is an initial histogram with only 38 insertions. Among this histogram two frequent conductive units were observed for 1.2nS and 1nS. The aqueous phase contained 0.1 M KCl, pH 6 with the applied membrane potential of 20 mV, T = 20°C.

Single-channel measurements were done in PC/n-decane membrane. The aqueous phase contained 0.1 M KCl, pH 6 and crude cell wall extracts of Rhodococcus ruber. The applied membrane potential was 20 mV; T = 20°C. Figure 5.3 shows initial histogram of the probability P (G) for the occurrence of a given conductivity unit in the presence of 1% PC dissolved in n-decane. Highest probability of conductance was observed for 1.2 nS and 1nS.

5.5 Conclusion

Much information is could not be obtained from above experiements. Further extensive purification steps must be taken place for identification and characterization of these proteins. Charaterization can be done by BLM assays.

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5.5 REFERENCES

1. Zopf, W. (1891). Über Ausscheidung von Fettfarbstoffen (Lipochromen) seitens gewisser Spaltpilze. Ber Dtsch Bot Ges 9, p. 22–28 (in German). 2. Goodfellow, M. & Alderson, G. (1977). The actinomycete-genus Rhodococcus: a home for the ‘‘rhodochrous’’ complex. J Gen Microbiol 100, p. 99–122. 3. Bell. K.S, Philp. J.C, Aw. D.W.J and Christof. N (1998).The genus Rhodococcus, A review; J.of Applied Microbio., 85, p.195–210. 4. Prescott, J.F. (1991) Rhodococcus equi: an animal and human pathogen. Clinical Microbiology 4, p.20–34. 5. Sutcliffe. I. C, Brown. A. K. and Dover L. G (2010). The Rhodococcal Cell Envelope: Composition, Organisation and Biosynthesis. Microbiology Monographs 16,p. 29-71. 6. Nikaido H (2003). Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67, p.593–656. 7. Faller M, Niederweis M, Schulz GE (2004). The structure of a mycobacterial outer- membrane channel. Science 303, p.1189–1192. 8. Derringtona. I.M, Butlera. T. Z, Collinsa. M.D, Manraoa. E, Mikhail Pavlenokb, Niederweis M, and Gundlacha. J. H (2010). Nanopore DNA sequencing with MspA. PNAS 107 (37), p.16060–16065. 9. Somalinga.V, Mohn.W.W (2013). Rhodococcus jostii Porin A (RjpA) Functions in Cholate Uptake; Applied and Environmental Microbiology 79,p. 6191– 6193. 10. Mahfoud. M, Sukumaran. S, Hülsmann. P, Grieger. K and Niederweis. M (2005). Topology of the Porin MspA in the Outer Membrane of Mycobacterium smegmatis. J. of bio. chemistry 281(9), p. 5908 –5915. 11. Riess, F. G., Lichtinger, T., Cseh, R., Yassin, A. F., Schaal, K. P. & Benz, R. (1998). The cell wall porin of Nocardia farcinica: biochemical identification of the channel- forming protein and biophysical characterization of the channel properties. Mol Microbiol 29, p. 139-150.

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CONCLUSION

CHAPTER 6

6.1 Conclusion

In this thesis our work was mainly focused on the characterization of OprP and OprO porin mutants from Pseudomonas aeruginosa. According to recent report on P. aeruginosa in 2014, it is becoming a global threat due to its multi-drug resistance. One of the reasons for its antibiotic resistance is absence of general porins and presence of more specific porin. Porins are water filled channels which allows passive diffusion. Study of these wild type porins and also its mutants from P. aeruginosa can give an insight on its metabolism function. OprP and OprO are phosphate specific porins of P. aeruginosa and helps in uptake of phosphate at time phosphate starvation by the cells. OprP and OprO both porins have sequence homology but with few differences. In our studies we tried to mutate two important residues present in the constriction zone which are different in OprP and OprO. In OprP we chose two tyrosine present at 62 and 114 positions to mutate it to phenylalanine and aspartic acid respectively. We create single and double mutants, single mutants behaved similar to OprP wild type protein, but double mutant of OprP behaved similar to OprO wild type in its single channel conductance as well as in its substrate specificity. We can conclude that, we could successfully change the specificity of OprP porin to that of OprO.

In the next study, we tried to reverse the mutations i.e, we changed phenylalanine at 62 position to tyrosine and aspartic acid at 114 position to tyrosine to know if these mutations could change OprO wild type to behave as OprP wild type protein. So we created single and double mutants of OprO. Single mutants didn’t behaved as OprP wild type in single channel conductance as well as substrate specificity but double mutants indeed behaved as OprP wild type protein in its substrate specificity. But these double mutants showed two-fold higher conductance than OprP protein. It could be concluded that the two-fold increase in single channel conductance of OprP double mutant can be due to different artifacts.

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CONCLUSION

These studies on the mutants can be useful in understanding the evolutionary process of porin which has evolved by just mutating one or two amino acids keeping their motifs conserved. These studies can also be helpful for designing new drugs or antimicrobial metabolites for destruction of these pathogenic bacteria P. aeruginosa. But still a lot more has to be known about these porins.

Dietzia genus is classified as a member of mycolata group and they contain mycolic acids in their cell walls. In our lab, a pore forming channel was identified from the cell wall extracts of Dietzia maris. But the genome sequence of this pore was unknown. To find the sequence of this pore, whole genome of Dietzia maris was sequenced and annotated. Annotation was done manually as well as with the help of automatic annotation. BLAST, RAST and PGAAP were used. Various categories of genes were found during annotation. We are also trying to find the gene sequence of the pore with this annotation process. With this annotation process, we could know a lot more about the metabolic process of Dietzia maris. These informations can be useful in exploiting this microorganism at genome or amino acid level.

Rhodococcus species are another group of mycolic acid containing species. It is been reported earlier that these species have many pore forming proteins on their cell wall, which acts like in the similar to the porins of Gram-negative bacteria. Many MspA homologous proteins have been reported in these species. MspA is the major porin of Mycobacterium smegmatis. This porin is modified to be used for sequencing purposes. We also want to use the porins from Rhodococcus species for sequencing purposes similar to MspA porin. To reach this goal, more intensive work has to be done in detail.

115

ABBREVIATIONS

2-(N-morpholino)ethanesilfonate -MES

Base pairs- b.ps

Basic local Alignment search tool- BLAST

Coding sites -CDS

Computational molecular dynamics -MD

Aspartic acid- D

Diphytanoyl phosphatidylcholine- DiphPC

Deoxynucleotide triphosphate - dNTPS -

Double mutant -DM

Forward primer FP

Phenylalanine- F

Heart and brain infusion -HBI

Hidden Markov model -HMM

Human immunodeficiency virus -HIV

Intergenic region -IG

Isopropyl β-D-1-thiogalactopyranoside -IPTG

Lipopolyscaccharides -LPS

Lipoteichoic acids -LTAs

Luria-Bertani -LB

Free-energy molecular dynamics simulations- MD simulations

116

Multiple cloning sites -MCS

N-actylmuramic acid -NAM

NCBI reference sequence -RefSeq

N-octylpolyoxyethylene- octyl-POE

Polymerase chain reaction- PCR

Prokaryotic Genomes Automatic Annotation Pipeline -PGAAP

Rapid Annotations using Subsystems Technology-RAST

Reverse primer -RP

Ribosomal-binding sites -RBSs

Single channel conductance -SCC

Sodium dodecyl sulfate polyacrylamide gel electrophoresis -SDS-PAGE

ß 1, 4 linked N-actylglucosamin -NAG

Wall teichoic acids -WTAs

Western immunoblotting -WB

Tyrosine – Y

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List of figures

Figure 1.3:- Fluid-mosaic model. The plasma membrane is composed of phospholipid molecules, integral proteins, carbohydrates bound to proteins forming glycoproteins or lipids forming glycolipids. The lipids in the plasma membrane are arranged in such a way that their polar heads are facing outward while their hydrophobic side chains faces inward.( http://johnfredycastro.blogspot.de/2011_07_01_archive.html)...... 19

Figure 1.5:- Structure of outer membrane of Mycolata (Corynebacteriales) (22). In mycolata, the mycolic acids are attached via an arabinogalactan skeleton to the peptidoglycan...... 24

Figure 1.6:- Colonies of different Dietzia species on iso sensitest agar after 48 hours of incubation at 36°C (24)...... 25

118

domain is tried to find. Depending on the predicted domains, annotation is added to the gene. Later on any other information related to its tRNA etc are added to it.... 27

Figure 2.4:- An example of MonoQ column and FPLC (Biorad). It consists of various parts such as pumps, injection valve, column, monitor/recorder, fraction collector. For our purification purpose we used MonoQ column which is a strong anionic column.(https://aswebsaxs.synchrotron.org.au/saxswiki/index.php/SR13_ID01_Size _Exclusion_Chromatography) ...... 40

119

mutgenesis; Lane 5- Y62FY114D mutagenesis PCR product. PCR products show only a single amplified band, representing the presence of only amplified product without any contamination. B) Gel picture showing amplified bands of OprP gene in colony PCR. Colony PCR was performed with Forward and reverse primers of OprP gene wild type or OprP mutants to confirm the presence of insert. The size of OprP gene or OprP mutant genes is 1305 b.ps...... 45

Figure 2.8:- SDS-PAGE gel picture of different purification steps of OprP wild type. Lane 1- Extraction with 10mM Tris-HCl; Lane 2- Extraction with 0.15% octyl- POE; Lane 3 -1st extraction with Octyl-POE; Lane 4 -2nd extraction with 3%Octyl-POE; , Lane L- Protein Ladder. The protein band of OprP can be observed below the 55KDa in lane 3 and 4. But the band in lane 4 is more near to the molecular weight of OprP. Protein samples representing lane 4 were taken for further purification steps...... 47

Figure 2.9:- Purification protocol of OprP protein by FPLC. The chromatogram shows various peaks and each peak represent some protein that is eluted out. OprP protein in eluted out at 39th fraction...... 48

120

region, a T7 promoter, lac containing a CAP protein binding site, promoter Plac, lac repressor binding site and the 5’-terminal part of lacZ gene encoding the N- terminal fragment of beta-galactosidase...... 70

Figure 3.3:- Gel picture of amplified oprO gene and pTZ19R vector. a) Ladder; b-f and h) oprO amplified gene and g) pTZ19R linearized vector...... 71

Figure 3.4:- Pore forming events of OprP wild type, OprO wild type, OprO double mutant, OprO D114Y and OprO F62Y. Single-channel conductance observed for OprO, OprP WT and OprO mutants in a diphytanoyl phosphatidylcholine/n- decane (DiPhPC) membrane bathed in 0.1 M, 10 mM MES-KOH, pH 6, at +50mV...... 76

Figure 3.5:- Gel picture of different steps of expression and purification of OprO wild type in E.coli. Samples are taken at different point of expression and purification are taken and loaded onto 10% SDS gels. Each sample is heated at 95°C for 2*10 minutes. The gel is stained with silver staining. An overexpressing band is seen between 55 and 40 kDa giving an indication of the presence of OprO protein...... 78

Figure 3.6:- Dot blot test for the confirmation of OprO or OprO mutant proteins...... 78

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Figure 3.10:- Lineweaver -Burk plots for OprO WT and its mutants for the inhibition of membrane conductance. The straight lines corresponds to stability constants K given by the equation 1/ (Fraction of closed channels) = Kc/ (Kc +1). a) Lineweaver -Burk plots for diphosphate - OprO WT shows highest stability constant for diphosphate K =1450 1/M. Followed by intermediate stability constant OprO D114Y with K = 630 1/M and OprO F62Y with stability constant K = 490 1/M; OprP WT having the least stability constant K = 310 1/M for diphosphate and OprO DM showing similar stability constant as of OprP WT with K = 343 1/M .... 82

Figure 4. 1:- Flow chart annotation in the SEED system. Annotation of the genome in the SEED system works on the curation of subsystem of genes. From these curative subsystems we extract the group of protein families. Then these new annotated sybsyytems will become new FIGfams (http://www.slideshare.net/ramykaram/rka- evergeen2015-phantomefinalweb)...... 90

Figure 4.2:- RAST Job detail page...... 91

Figure 4.4:- An example of BLAST query search ...... 95

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Figure 4.5:- Information obtained from RAST server. It gives information on taxonomy, size and the number of contigs, the number of coding sequences, RNAs and subsystems ...... 96

Figure 5. 1:- MspA porin from Mycobacterium smegmatis. This is the only known porin in this organism (10) ...... 108

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List of Tables

Table 2.2:- Single-channel conductance of OprP, OprO, and three OprP mutants channels in 0.1 M and 1 M KCl solutions buffered with 10 mM MES, pH 6, T=20 °C. The applied voltage was 50 mV...... 51

Table 3. 1:- Primers designed to clone oprO from genomic DNA of P. aeruginosa PAO1. ………………………………………………………………………………………..72

Table 3. 2:- Primers OprO to OprP in order to change the specificity towards diphosphate. Mutants are indicated by the residue and its position followed by the mutated residue, using the one letter amino acid code. The codons of OprO wild type and its mutants are indicated by the forward primers used for mutation. Letter in lower case shows the place of mutagenesis...... 73

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Table 3.4:- Phosphate and diphosphate mediated inhibition of chloride conductance of OprO WT, OprO mutants and OprP WT in 0.1M KCl, 10mM MES, pH 6, with applied voltage +50 mV at RT. The half stability constants for inhibition of Cl- conductance by diphosphate or phosphate were obtained from titration experiments using either the Lineweaver burk plot or the Langmuir equation. Mean values (± SD) of at least three individual titration experiments are shown...... 83

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Appendix

Publications-

Ganguly.S, Bárcena-Uribarri I , Kesireddy.A, Kleinekathöfer U, Benz R (2016). Conversion of OprO - into OprP- porin of Pseudomonas aeruginosa by exchanging key amino acids at the channel constriction. (For submission).

Ganguly.S, Jimenez-Galisteo.M.G, Pletzer .D, Winterhalter.M, Benz R, Viñas.M. (2016). Draft genome sequences of Dietzia maris DSM 43672 a Gram positive bacterium of mycolata group. Genome Announc. 4(3). pii: e00542-16.

Seminars and Conferences

November 2013, Heidelberg - 15th Embl PhD symposium. Competitive in biology- The race for survival from molecules to system. Poster-OprP versus OprO of Pseudomonas aeruginosa outer membrane: structure, dynamics and ion-selectivity. (Published in abstract book)

March 2014, Hünfeld – Molecular membrane biophysics meeting Poster-Phosphate specific porins OprP versus OprO of Pseudomonas aeruginosa outer membrane: structure, dynamics and ion-selectivity. (Published in abstract book)

March 2015, Marburg- VAAM-International conference on microbiology 2015. Oral presentation- Phosphate specific porins of Pseudomonas aeruginosa outer membrane: structure, dynamics and ion-selectivity. (Published in abstract book)

January 2014 and 2015. Borstel- Biophysical meeting Poster-OprP versus OprO of Pseudomonas aeruginosa outer membrane: structure, dynamics, and ion-selectivity.

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