Grunddokument 2

Regulatory roles of sRNAs in pathogenesis of Vibrio cholerae

Dharmesh Sabharwal

Department of Molecular Biology The Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå University, Sweden Umeå 2015

To my parents….

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TABLE OF CONTENTS

ABSTRACT vi PAPERS INCLUDED IN THIS THESIS viii INTRODUCTION 1 1. Vibrio cholerae 1 1.1 Virulence factors of V. cholerae 3 1.2 Outer membrane proteins and their regulation in V. cholerae 4 1.3 Biofilm and its associated matrix proteins 5 1.4 Roles of ribosome binding proteins in stationary phase survival of bacteria 8 2. Bacterial sRNA 10 2.1 Small RNAs in Vibrio cholerae 11 2.2 RNA chaperone protein Hfq 16 2.3 Ribonucleases and RNA decay 18 3. VrrA and its roles in V. cholerae pathogenesis 19 3.1 Role of VrrA in release of outer membrane vesicles (OMVs) in V. cholerae 20 3.2. Roles of VrrA in V. cholerae virulence 22 4. Riboswitches as novel regulators in bacteria 23 4.1 Types of Riboswitches 24 4.2 Metabolite-sensing riboswitches in V. cholerae 26 4.3 Virulence factors controlled by a themosensor in V. cholerae 27 AIMS OF THE THESIS 29 RESULTS AND DISCUSSION 30

Paper 1 30 Paper II 31 Paper III 33 Paper IV 37 CONCLUSIONS 40 ACKNOWLEDGEMENTS 41 REFERENCES 43 Paper I-IV 57

ABSTRACT

The Gram-negative pathogen Vibrio cholerae uses variety of regulatory molecules to modulate expression of virulence factors. One important regulatory element of microorganisms is small non-coding RNAs (sRNAs), which control various cell functions such as expression of cell membrane proteins, mRNA decay and riboswitches. In this thesis studies, we demonstrated the roles of the sRNAs VrrA in regulation of outer membrane protein expression, biofilm formation and expression of ribosome binding proteins. In addition, we showed that VrrB, a newly discovered sRNA, played a role in amino acid dependent starvation survival of V. cholerae and might functioned as a riboswitch.

VrrA, a 140-nt sRNAs in V. cholerae, was controlled by the alternative sigma factor σE. The outer membrane protein, OmpT is known to be regulated by environmental signals such as pH and temperature via the ToxR regulon and carbon source signals via the cAMP–CRP complex. Our studies provide new insight into the regulation of OmpT by signals received via the σE regulon through VrrA. We demonstrated that VrrA down-regulate ompT translation by base-pairing with the 5′ region of the ompT mRNA in a Hfq (RNA chaperone protein) dependent manner.

V. cholerae biofilms contain three matrix proteins—RbmA, RbmC and Bap1—and exopolysaccharide. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. In our studies, we demonstrated that VrrA negatively regulated rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation was not stringently dependent on Hfq.

In V. cholerae, VC0706 (Vrp) and VC2530 proteins are homologous to ribosome-associated inhibitor A (RaiA) and hibernation promoting factor (HPF) of Escherichia coli, respectively. HPF facilitates stationary phase survival through ribosome hibernation. We showed that VrrA repressed Vrp protein expression by base-pairing to the 5´ region of vrp mRNA and that this regulation required Hfq. We also showed that Vrp was highly expressed during stationary phase growth and associated with the ribosomes of V. cholerae. We further demonstrated that Vrp and VC2530 were important for V. cholerae starvation survival under nutrient-deficient conditions. While VC2530 was down-regulated in bacterial cells lacking vrrA, mutation of vrp resulted in increased expression of VC2530.

Riboswitches are an important class of regulators in bacteria, which are most often located in the 5' untranslated region (5´ UTR) of bacterial mRNA. In this study, we discovered the novel non-coding sRNA, VrrB located at the 5´ UTR of a downstream gene encoding Vibrio auxotropic factor A (VafA) for phenylalanine. In V. cholerae, reduced production of VafA was observed in the presence of phenylalanine and phenylpyruvate in the culture media. Some analogs of phenylalanine and phenylpyruvate could also modulate the expression of VafA. Furthermore, bacterial cells lacking the vrrB gene exhibited high production of VafA, suggesting that VrrB might function as a riboswitch that controls VafA expression.

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PAPERS INCLUDED IN THIS THESIS

Paper I

Song T, Sabharwal D, Wai SN. 2010. VrrA mediates Hfq-dependent regulation of OmpT synthesis in Vibrio cholerae. J. Mol. Biol. 23;400(4):682-8.

Paper II

Song T*, Sabharwal D*, Gurung JM, Cheng AT, Sjöström AE, Yildiz FH, Uhlin BE, Wai SN. 2014. Vibrio cholerae utilizes direct sRNA regulation in expression of a biofilm matrix protein. PLoS One. 23;9(7):e101280. (*Shared first author).

Paper III Sabharwal D, Song T, Papenfort K, Wai SN. 2015. The VrrA sRNA controls stationary phase survival factors of Vibrio cholerae. (RNA Biology –in press)

Paper IV Sabharwal D, Johansson J, Wai SN. 2015. A new sRNA, VrrB, acts as a regulator for vafA, a gene involved in amino acid starvation survival of Vibrio cholerae. (Manuscript)

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INTRODUCTION

1. Vibrio cholerae

Vibrio cholerae is a Gram-negative bacterium that is transmitted to its host though contaminated food or water, causing the diarrheal disease called cholera. Historically cholera pandemics started in 1817, and O’Shaughnessy was the first to recognize the characteristics of the disease when he first demonstrated that rice water stools of patients contained salt and alkali, i.e., high in electrolyte content [1].

Further, an Italian anatomist, Filippa Pacini in 1854 found in the intestine of cholera victims a large number of curved bacteria, which he named Vibrio cholerae [2]. Later research led to classification of V. cholerae strains into 208 serogroups, based on the O-antigen in the LPS structure. Out of these, only serogroups O1 and O139 have caused pandemic cholera [3, 4]. The O1 serogroup can be further subdivided into two biotypes, classical and El Tor, based on their independent evolutionary lineage and different genotypic and phenotypic characteristics such as Voges-Proskauer reaction, hemolysis, hemagglutination and sensitivity to polymyxin B and phage infection [5, 6]. The V. cholerae O1 classical biotype was responsible for the first six cholera pandemics, while the seventh pandemic, which started in 1961, was caused by the El Tor biotype of V. cholerae O1. Later, a new serogroup, O139, caused an epidemic outbreak of cholera in India and Bangladesh. V. cholerae O139 is closely related to the strain that

caused the seventh pandemic, the El Tor biotype of V. cholerae O1, and is known to be originated by acquisition of novel DNA and subsequent replacement with part of the O-antigen gene cluster of the recipient strain [7]. The 22-kb sequence of the O1 El Tor rfb region was replaced by a ~ 35-kb DNA fragment encoding the O139-specific capsular polysaccharide resulting in creation of a new emerging O139 strain [8].

Serogroups O2 through O138 are referred to as non-O1/non-O139 (NOVC) strains and most of the NOVC strains lack the V. cholerae major virulence factors cholera toxin (CT) and toxin co-regulated pilus (TCP). Most NOVC strains are also not associated with epidemic diarrhea [9]. These strains are occasionally isolated from cases of mild diarrhea usually associated with consumption of shellfish and with extra-intestinal infections, including wounds, ear infections, lung infections, urinary tract infections, and central nervous system infections [10-12]. The presence of diarrhea might be associated with other toxins whose pathogenic mechanisms are still unknown.

In the past 200 years, several cholera pandemics have occurred, making V. cholerae organism a topic of interest for research. The recent cholera outbreak in Ghana in 2010 has resulted in thousands deaths [13]. Studies performed by Thompson, et al. showed that distinct V. cholerae O1 were the determinants of cholera outbreaks in Ghana [13].

1.1 Virulence factors of V. cholerae

In V. cholerae, the two main virulence factors responsible for cholera disease are cholera toxin (CT) and toxin co-regulated pilus (TCP). A filamentous bacteriophage (designated CTXΦ) contains genes encoding cholera toxin, [14], and a 41-kb pathogenicity island on the chromosome encodes the toxin co-regulated pilus (TCP) [15]. Cholera toxin, encoded by the ctxAB genes of lysogenic CTXΦ is a potent enterotoxin of the A-B toxin type. It consists of one enzymatic A subunit and five receptor binding B-subunits and is secreted by bacterial type II secretion system. After secretion, the CT-B subunit binds to ganglioside GM1 receptors in lipid rafts on the plasma membrane of the target cells [16]. The entire toxin complex is endocytosed by the cell and moved through the Golgi apparatus to the endoplasmic reticulum, where the A subunit dissociates from the toxin complex. CT-A is transported to the cytoplasm of the target cell where it ADP-ribosylates a GTP-binding protein that activates adenyl cyclase. This event leads to an increase in the intracellular cAMP level, resulting in an imbalance in electrolyte movement in the epithelial cell. This in turn leads to increased secretion of NaCl, which further enhances osmotic movement of water into the intestinal lumen, resulting in the watery diarrheal disease cholera that is a hallmark of cholera.

1.2 Outer membrane proteins and their regulation in V. cholerae

Outer membrane proteins (OMPs) are one of the components that bacteria regulate as a survival strategy in response to different environmental conditions. The exchanges across the outer membrane of the cell and the environment are ensured by OMPs involved in several functions, such as nutrient uptake, transport of signaling molecules, and secretion of various proteins, polysaccharides and drugs [17].

In V. cholerae, eight different outer membrane proteins have been reported. The most abundantly produced porins are OmpU (38 kDa) and OmpT (40 kDa), which are regulated in response to environmental signals such as bile and osmolarity. In response to environmental signals, a transcriptional regulator, ToxR activates expression of ompU and represses transcription of ompT [18-20]. ToxR interacts directly with long stretches of AT-rich sequences at the ompU and ompT promoters and functions as a versatile transcription factor.

Another regulator, cAMP receptor protein (CRP) is also known to interact at the ompT promoter and is required for ompT transcription activation. It was suggested that DNA looping is part of the mechanism involved in ToxR binding to the ompT promoter, which antagonizes CRP-dependent transcription activation by preventing the formation of a transcription complex [21].

The outer membrane protein OmpS (45 kDa) is a maltoporin whose synthesis is enhanced by maltose in the media. In contrast to OmpS, the

production of OmpT and OmpU is reduced by the presence of maltose [22]. OmpA (34 kDa) is important in maintaining the integrity of the cell envelope, thus it is involved in maintaining the release of outer membrane vesicles (OMVs) in V. cholerae. Deletion of ompA results in attenuated colonization of the intestines in an animal model [23]. Another osmoregulated outer membrane protein is OmpX (27 kDa), which unlike OmpU and OmpT, does not have pore-forming ability, and hence is not classified as a porin [24].

OmpV (25 kDa) is a highly immunogenic protein associated with peptidoglycan [25]. OmpW (22 kDa) is also immunogenic and seems to be present in all known heterologous V. cholerae Ogawa and O139 strains. V. cholerae cells preincubated with OmpW antisera elicited reduced fluid secretion in a rabbit intestinal infection model, therefore attracting interest for vaccine development [26]. Expression of OmpW is influenced by varied NaCl concentrations resulting in change in osmotic stress in several Vibrio species [27]. In addition, gene vc0972, which encodes an uncharacterized outer membrane protein, is negatively regulated by the small non-coding RNA MicX [28].

1.3 Biofilm and its associated matrix proteins

Vibrios are known to form biofilms on different environmental niches such as zooplankton, insects and intestines [29-31]. Biofilm formation in V. cholerae make three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters [32]. Bacteria within biofilms are more resistant to stress conditions such as osmotic,

oxidative, and acid stresses. It was also shown that bacterial biofilms could resist against several types of antibiotics [32]. Biofilm formation in V. cholerae was controlled by production of exopolysaccharide (VPS) and the synthesis of biofilm matrix proteins RbmA, RbmC and Bap1 [32-34].

Exopolysaccharide production in V. cholerae are controlled by three known signalling pathways: the quorum-sensing pathway, wherein the HapR mutant (transcriptional regulator) of V. cholerae strain lead to enhanced exopolysaccharide synthesis and biofilm formation, a flagellum-dependent pathway, wherein the cell lacking flagella induce the expression of exopolysaccharide synthesis and biofilm formation, and a phase variation pathway that result in two different morphological variants of V. cholerae strain termed as smooth and rugose (wrinkled). High exopolysaccharide production and biofilm formation is associated with rugose variants of bacterial strains [35].

The regulation of exopolysaccharide synthesis in V. cholerae is dependent on expression of the vps genes. Different environmental signal such as quorum sensing based autoinducers [36], polyamines [37, 38], nucleosides [39, 40], indole [41] and nutrient scarcity [42] can control transcription of the vps genes, thus biofilm formation. In the previous studies, glucose-specific enzyme IIA is known to regulate biofilm formation by interaction with a carbon storage regulator homolog MshH, demonstrating a phosphoenolpyruvate phosphotransferase system link to biofilm formation [43, 44].

The role of matrix proteins in biofilm formation appears to be different such as RbmA is responsible for formation of flexible linkages between bacterial cells and the extracellular matrix which result in the initial cell-cell adhesion step [32, 45], while Bap1 facilitates adherence of the developing biofilm to surfaces. The heterogeneous mixtures of exopolysaccharide, RbmC and Bap1 appear to form an envelope-like covering to encase the cell clusters [32] (Fig. 1). In the absence of RbmC, exopolysaccharide incorporation to maintain mature biofilm is significantly reduced. Regulation of the rbm gene was shown to be modulated by two factors: the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and a transcriptional regulator VpsR. Transcription of the rbm genes is activated by VpsR, while cAMP-CRP negatively regulate the rbm expression [46].

Fig. 1. Model of biofilm development in V. cholerae. (A) Single planktonic cell encounters a surface (B) RbmA (yellow) accumulates on the cell surface and promotes initial attachment (C) RbmA continues to accumulate and Bap1 (red) appears at the cell-surface interface at the initial cell division site, to ensure the new daughter cell adheres to the surface. (D) As the bacterial cell

continues to divide, a flexible envelope (blue) containing RbmC, Bap1, and VPS is generated and the cluster envelops formation is accompanied by more accumulation of RbmA (on individual cell surfaces) and Bap1 (on the cell- surface interface). (E) Individual cell clusters expand and contact other clusters to form mature biofilm [32].

1.4 Roles of ribosome binding proteins in stationary phase survival of bacteria

In Bacteria, ribosome and ribosomal proteins play an important role in the adaptation to environmental stresses as a checkpoint for sensing shifts in temperature or nutrient levels. In all organism, ribosomes are composed of a small and a large subunit (30S and 50S, respectively, in bacteria) that cycle through stages of association and dissociation during protein synthesis. Bacteria can reduce the level of protein synthesis by inducing a state of hibernation during stationary phase or in nutrient starvation by switching actively functioning ribosomes into translationally inactive 100S dimmers [47] or 70S monomers [48], thereby inhibiting the growth of bacteria.

In E. coli, ribosome binding proteins involved in ribosome hibernation are RMF (ribosome modulation factor), HPF (hibernation promoting factor), RaiA (ribosome-associated inhibitor A) and SRA (stationary- phase-induced ribosome associated protein) [49]. RMF and HPF play a role in formation of 100S dimer ribosome particles. The binding of RMF causes dimerization of 70S ribosomes into 90S particles, which are further stabilized as 100S dimmers upon HPF binding thus leads to “ribosome hibernation” [49]. RaiA has a role in formation of

translationally inactive monomeric 70S ribosomes which consequently prevents the recycling of ribosomes for translation initiation [50]. Although RaiA and HPF share a 40% amino acid sequence similarity, HPF converts 90S into 100S particles, whereas RaiA prevents RMF- dependent 90S formation [51]. Either monomeric 70S ribosomes subunits formation promoted by RaiA or 100S dimmers particles promoted by HPF, both forms are translationally inactive [50, 52] leading longer cell survival under nutrient limited conditions. The ∆raiA and the ∆hpf single mutants E. coli survived better than that of wild-type while the survival efficiency of the ∆raiA∆hpf double mutant was similar to that of the wild-type strain [49].

The crystal structure of 70S ribosome in Thermus thermophilus demonstrated that RaiA and HPF share a common binding site in the ribosome. In addition, the same binding site overlaps with those of all tRNA and the initiation factors IF1 and IF3, suggesting that RaiA and HPF can interfere with protein synthesis [52]. Recent studies in Lactococcus lactis which lacks orthologues of rmf and hpf genes, showed that RaiA (also known as YfiA) is essential for ribosome dimerization and found in 100S ribosome particles [53].

Another ribosome binding protein YqjD of E. coli is regulated by stress response sigma factor RpoS. In many bacteria, sigma factor RpoS is essential for bacterial cell survival during nutrient deprivation condition [54]. The N-terminal region of YqjD protein was shown to be

associated with 70S and 100S ribosomes. The over-expression of YqjD can inhibit the growth of the E. coli cells during stationary phase [55].

2. Bacterial sRNA

Bacteria possess a wide range of regulatory molecules allowing them to perform various tasks such as modulating the expression of virulence factors, biofilm related genes and other survival factors in a hostile environment. In the last decade, small non-coding RNAs (sRNAs) have emerged as important regulatory molecules that contribute to a vast cascade of changes in gene expression and protein activity. sRNAs in bacteria can be subdivided into cis-encoded and trans-encoded RNAs.

Cis-encoded sRNAs mostly exhibit complete base pairing with their target mRNAs and are located on the DNA strand complementary to or at the 5´-UTR of the mRNAs they regulate, while trans-encoded sRNAs, transcribed from intergenic regions, can base pair partially to distally located target mRNAs (mostly at 5´ UTR mRNAs) and might have multiple targets. Trans-encoded sRNAs often require the RNA chaperone protein Hfq to facilitate efficient base pairing with their target mRNA [56]. In Mycobacterium tuberculosis, two of the cis- encoded sRNAs, AsDes and AsPks are responsible for targeting genes encoding enzymes involved in lipid metabolism, desA1 and pks12, respectively [57]. The trans-encoded sRNAs such as RybB of Salmonella typhimurium and Escherichia coli down-regulate multiple major omp mRNAs (outer membrane protein) in a Hfq-dependent manner [58-60].

2.1 Small RNAs in Vibrio cholerae

Bioinformatics and next-generation sequencing approaches have resulted in identification of hundreds of potential sRNAs in V. cholerae [61-64].

Livny et al. have created a sRNAPredict program for identification of putative intergenic sRNAs. Algorithms used in the program were based on sequence conservation and predicted Rho-independent terminators [63]. In V. cholerae, the sRNAPredict program has identified 9 of the 10 known or putative sRNAs. In addition, 32 novel candidates for sRNAs were identified, of which, 6 of 9 randomly chosen candidate sRNAs were confirmed by Northern-blot analysis.

In other studies, Liu et al. [62] performed direct cloning and parallel sequencing experiments followed by 5S/tRNA depletion in V. cholerae. Using this technique, 500 new, putative intergenic sRNAs (IGR-sRNA) and 127 putative antisense sRNAs (AS-sRNA) were identified. Some of these candidates were tested for Northern blot analysis to confirm the accuracy of this technique. Six of seven randomly chosen IGR- sRNA candidates, four of nine randomly chosen AS-sRNA and five of seven candidate sRNAs transcribed from within an annotated ORF (ORF-sRNA) were verified to transcripts of the expected sizes. Although many of sRNA are identified in V. cholerae, only some of them have been functionally analyzed (Table 1). One of the candidate sRNAs, IGR7 (later recognized as MtlS sRNA) was preliminary characterized for functional analysis and shown to be involved in

modulating carbon metabolism. IGR7 sRNA is transcribed antisense to the 5' UTR of mtlA (encodes a transporter for mannitol, a naturally occurring six-carbon sugar alcohol) [65].

In earlier studies, Mandlik et al. [61], performed parallel cDNA sequencing of V. cholerae transcripts, derived from V. cholerae infected rabbits and mice and compared these to transcripts derived from laboratory media-grown bacteria to identify in-vivio expressed virulence genes. The RNA sequencing approach led to detection of almost all previously known sRNAs, including four Qrr sRNAs [66] that have roles in quorum sensing and were expressed under all growth conditions tested. An iron-repressed sRNA, RyhB was less abundant in the rabbit cecum than in the mouse intestine, providing further evidence that iron is more limiting in the mouse intestine than in the cecum. Other previously described sRNA candidates such as TarA [67] and CsrC [68] were expressed at higher amounts in rabbits compared to LB and M9 media.

Further studies by Bradley et al. [64] identified a ToxT dependent virulence-related sRNA in V. cholerae. In their study, ToxT (the major transcriptional regulator within the virulence gene regulon) was over- expressed to indentify the sRNA promoters with ToxT binding sites. Analysis by pulldown of ToxT binding RNA followed by deep sequencing of transcripts revealed 16 putative new sRNAs, and two other sRNAs, TarA and TarB, within the Vibrio pathogenicity island. TarA (ToxT activated RNA A) has previously identified as an sRNA

and studied [67]. Like TarA, functional analysis of the sRNA TarB, also showed a role in a variable colonization phenotype in the infant mouse model of V. cholerae colonization. Target identification of TarB revealed an interaction with the tcpF mRNA (encodes for the secreted colonization factor) and showed that TarB negatively regulates expression of the secreted colonization factor TcpF.

The sRNA, RyhB, originally discovered in E. coli, is conserved in V. cholerae. As in E. coli, RyhB and Hfq interact in V. cholerae, and expression of RyhB is reduced in cells lacking Hfq, suggesting that Hfq may stabilize RyhB. Although the sequence length of RyhB in V. cholerae is significantly larger than in E. coli, in both bacterial species RyhB is regulated by the iron-dependent repressor Fur and involved in iron utilization [69].

The sRNAs (Qrr1, Qrr2, Qrr3 and Qrr4), together with the sRNA binding protein Hfq, are involved in quorum sensing repression by destabilizing the V. cholerae hapR mRNA, specifically at low cell density [66]. Other studies demonstrated that three sRNAs CsrB, CsrC and CsrD were involved in controlling the activity of the global regulatory protein, CsrA. Expression of CsrB, CsrC and CsrD was activated by the VarS/VarA two-component sensory system [68]. The VarS/VarA-CsrA/BCD system provides an additional input control for regulating quorum sensing by modulating expression of the Qrr sRNAs [66], thus also regulating hapR expression [68].

The ToxT-dependent sRNA, TarA was shown to modulate infant mouse colonization of V. cholerae. In addition, TarA regulates PtsG, a glucose transporter involved in regulation of glucose uptake. The RNAhybrid program revealed strong base-pairing between the TarA and the ptsG mRNA leader with a favorable free energy of -54.4 kcal mol-1. The TarA sequence also consists of a putative Hfq binding site and further analysis by Northern blot showed reduced expression of TarA in the hfq mutant compared to V. cholerae wild-type strain O395 [67].

The MicX sRNA (previously known as A10) was identified by the sRNAPredict program [63], and shown to directly regulate the gene vc0972 (encoding an uncharacterized OMP) and vc0620 (encoding the periplasmic component of a peptide ABC transporter) [28]. The regulation of vc0972 and vc0620 was Hfq independent, although RNase E and Hfq were required to process the primary transcript for MicX, which overlaps with the 3′ end of vca0943, to make a shorter and more stable form [28].

Previous studies showed that chitin disaccharide (GlcNAc)2 was needed to activate the transcription and translation of tfoX, which is involved in induction of natural competence for DNA uptake from the environment [70]. Later studies identified the presence of a 102- nucleotide sRNA known as TfoR which is located inside the intergenic region between vc2078-vc2079 and is transcriptionally activated in the presence of (GlcNAc)2 [71]. In addition, TfoR positively regulated the

translation of tfoX mRNA involved in natural competence induction [71].

Table 1. List of regulatory sRNAs and their functions in V. cholerae sRNAs Target Functional role Reference Genes

IGR7 mtlA Carbon metabolism [65] (MtlS)

RyhB sodB Iron homeostasis, biofilm [72] (vc2045) formation

TarA ptsG Glucose uptake [67]

TarB tcpF Colonization of the mouse [64] small intestine

CsrB, CsrC, Titrating CsrA protein [68] CsrD involved in quorum sensing

Qrr1-4 hapR Quorum sensing [66]

MicX (A10) vc0972, Regulates uncharacterized [28, 63] vc0620 OMP and periplasmic component of a peptide ABC transporter

VrrA ompA Outer membrane vesicle [23] synthesis and colonization of mouse intestine

TfoR tfoX Natural competence [71] induction

2.2 RNA chaperone protein Hfq

The Hfq protein was originally identified in E. coli as a host factor required for replication of the bacteriophage Qβ [73]. Characterization of Hfq revealed similarity with the family of Sm and Sm-like (Lsm) proteins found in eukaryotes, which bind RNA and have roles in RNA decapping, mRNA splicing and stabilization [74].

The crystal structures of Hfq proteins from Staphylococcus aureus [75], E. coli [76], Pseudomonas aeruginosa [77], Bacillus subtilis [78] and Herbaspirillum seropedicae [79] revealed that Hfq has conserved Sm like protein topology. In eukaryotes, Sm-like proteins form heteroheptameric rings [74], whereas in bacteria, Hfq forms homohexameric rings [80].

The Hfq protein consists of two major RNA binding faces, one termed a proximal face where Hfq binds to the hepta-oligoribonucleotide AU5, as shown in S. aureus [75], and the other termed a distal face where Hfq binds to poly(A) tails [80]. In E. coli, Hfq binds preferentially to two or more sites including U-rich and/or the poly(A) tail of mRNA. However, in Bacillus subtilis, the distal site of Hfq showed additional binding to AG(3)A sites in mRNA, unlike in E. coli [78] .

The role of Hfq in sRNA-mediated regulation was first observed in OxyS sRNA which is known to repress rpoS expression in an Hfq dependent manner [81]. The binding interaction between OxyS and Hfq

was revealed by co-immunoprecipitation and gel mobility shift experiments [81]. Later studies illustrated that Hfq interacts with most of the regulatory sRNAs (Sittka et al., 2008; Zhang et al., 2003), facilitates annealing of two complementary transcripts [82] and is often required for stability of the sRNA molecules [56].

Geissmann and Touati showed that Hfq can act as chaperones during formation of RNA-duplexes [83]. Hfq protein strongly binds to sodB mRNA, which results in alteration of its mRNA structure by partially opening the loop, thus making it accessible for interaction with RyhB sRNA [83]. Hfq has also been described as having ATPase activity, limited to the intrinsic activity of Hfq rather than association with any unknown protein [84]. In another studies, the ATP-binding site in Hfq was recognized using biochemical and genetic techniques [85]. In addition, RNA foot-printing and binding analyses showed that ATP binding to the Hfq–RNA complex results in significant destabilization [85].

In V. cholerae, Hfq is required for efficient expression of the stationary phase sigma factor RpoS, which is also true for E. coli and Salmonella typhimurium [86]. Strains lacking the hfq gene in V. cholerae fail to colonize suckling mouse intestine, thus showing the importance of Hfq in V. cholerae virulence [86]. Recent studies by Vincent et al. [87] revealed the structure of V. cholerae Hfq in complex with Qrr1 sRNA. During complex formation, Hfq does not alter its own shape but instead alters the conformation of the RNA, thus adding further insight into the

chaperone role of Hfq in its interaction with Qrr sRNA [87]. In the regulatory pathway of some sRNAs in V. cholerae, Hfq is not necessity for interaction with its target mRNAs, for example MicX sRNA does not required Hfq for interactions with its target mRNA [28]. However, the Qrr sRNAs do require Hfq to interact with hapR mRNA [66].

2.3 Ribonucleases and RNA decay

Ribonuclease plays important roles in sRNA-mediated gene regulation in bacteria. It has been shown that Hfq, the key molecule involved in modulation of mRNA stability, binds at the ribonuclease E (RNase E) cleavage site on the target mRNA, thus Hfq can protect the transcript against RNase E cleavage [88].

The RNA degradosome is a multiprotein complex involved in processing ribosomal RNA and degrading messenger RNA. It is mainly composed of a DEAD-box RNA helicase (RhlB), a glycolytic enzyme (enolase), a polynucleotide phosphorylase (3´-exoribonuclease- PNPase), RNase E and other proteins [89]. Alternative forms of the RNA degradosome have been described under different growth conditions or in the presence of other factors. These alternative forms showed different RNase E activity during mRNA degradation, thus they might contribute to bacterial adaptation to different environmental niches [90]. RNase E and PNPase of the RNA degradosome complex are ribonucleases involved in the degradation of mRNA and the maturation of stable RNA [91]. RNase E cleaves single-stranded regions with preference for 5’ monophosphate termini and AU-rich

sequences of RNAs [92]. Another component of RNA degradosome complex, enolase, is a glycolytic enzyme although its role in the degradosome is still a mystery [93]. RhlB is a DEAD-box RNA helicase that enhances the degradation of transcripts by RNase E and PNPase [94-96]. Apart from RNase E, other RNases such as RNase III can directly process and degrade mRNA transcripts and specifically cleave double-stranded RNA [97].

3. VrrA and its roles in V. cholerae pathogenesis

VrrA (Vibrio regulatory RNA of ompA) was discovered during the analysis of a mini-Tn5 transposon mutant (SNW6) from a library of V. cholerae El Tor O1 strain A1552 [98], which was found to carry a mini-Tn5 insertion in the intergenic region between the vc1741 and vc1743 genes. Further inspection of the sequence in this region followed by Northern blot analysis led to identification of a ∼140 nt sRNA expressed in samples of the wild type strain but not in the SNW6 mutant strain. 5’ RACE analysis identified the transcription start site (+1) of VrrA which is located approximately 140 bp upstream of a putative Rho-independent terminator. The +1 site analysis of the vrrA downstream gene vc1743 showed that both VrrA and vc1743 shared a common transcriptional start site [23]. The VrrA promoter region of V. cholerae, contains a previously reported consensus binding site for sigma factor, σE [99, 100]. The VrrA homologues were identified using a BLASTN search and multiple alignment in other Vibrio species such as V. splendidus (VS), V. alginolyticus (VA), V. parahaemolyticus

(VP), V. harveyi (VH), V. vulnificus (VV), V. shilonii (AK1), Vibrionales bacterium SWAT-3 (SWAT-3), Vibrio sp. MED222 (MED222) and Vibrio sp. Ex25 (EX25). All the VrrA homologue genes have the conserved σE binding sites at the vrrA promoter. Furthermore, Northern blot analysis showed that expression of VrrA was completely abolished in the ΔrpoE mutant strain [23].

Previous studies in E. coli and V. cholerae indicated involvement of σE in cell envelope integrity [101]. In addition, mutation of the σE gene led to reduce survival ability of V. cholerae within the host intestinal environment [102].

VrrA, which is controlled by sigma factor σE, thus provides additional signal input for modulating the expression of outer membrane proteins and stationary phase survival factors in V. cholerae [23, 103-106]. The secondary structure of VrrA predicted by RNA fold software (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) revealed an open-loop sequence from nucleotides 70 to 106. This open loop single stranded sequence might be important for the interaction of VrrA with its target mRNA.

3.1 Role of VrrA in release of outer membrane vesicles (OMVs) in V. cholerae

Gram-negative bacteria are known to produce outer membrane vesicles (OMVs) in the 20 - 200 nm diameter size range [107, 108]. OMVs are composed of outer membrane proteins, lipopolyaccharide (LPS),

phospholipids and periplasmic constituents. OMVs perform different roles in bacterial pathogenesis such as transfer of bacterial DNA, delivery of virulence factors to bacterial or eukaryotic cells, and bacterial communication through OMV-associated signal molecules [108-112]. OMVs have been considered an important factor for biotechnological applications such as the delivery of antibiotics to target cell or as potential vaccine candidates [113]. Different environmental conditions that can lead to changes in the envelope structure of bacteria could modulate the release of OMVs. Disturbance of the intracellular proteins that link the outer membrane to the peptidoglycan layer or are involved in a structural network between the inner and outer membranes and the peptidoglycan layer lead to shedding of large amounts of OMVs (Fig. 2) [114].

In V. cholerae, the bioinformatics based RNAhybrid program [115] predicted the interaction between VrrA and ompA mRNA and revealed that a region of VrrA was partially complementary to nucleotides encompassing the ribosome binding site and part of the coding region of ompA mRNA (Song et al. 2008). Furthermore, this interaction was validated experimentally by a Toe-printing assay [116] which suggested that VrrA specifically and directly pairs with the ompA coding region in vitro thereby inhibiting ribosome binding (Song et al. 2008). The RNA chaperone Hfq is not stringently required for VrrA to regulate ompA. In Gram-negative bacteria, changes in outer membrane protein composition can result in altered formation and release of

OMVs [117]. In V. cholerae, VrrA is involved in regulating OmpA protein production, resulting in modulation of OMV release (Song et al. 2008).

Fig. 2. Proposed model showing OMVs originate from regions of the cell without peptidoglycan-associated lipoproteins [112].

3.2. Roles of VrrA in V. cholerae virulence

In E. coli, the OmpA protein is involved in promoting adhesion to HeLa cells and Caco-2 colonic epithelial cells [118]. OmpA of E. coli has 47.8% similarity to V. cholerae OmpA, and analysis of V. cholerae colonization showed that inactivation of ompA resulted in a ~10-fold attenuation in colonization of the infant mouse small intestine. The vrrA mutant strain, which had higher OmpA protein production, displayed an approximately five-fold increase in colonization compared with the wild-type strain. Taken together with our earlier studies, we

conclude that VrrA has a role in V. cholerae virulence by modulating the expression of OmpA [23].

Two other factors critical to V. cholerae virulence are cholera toxin (CT) and an intestinal colonization factor known as the toxin co- regulated pilus (TCP). Previous studies showed that TCP, a type IV pilus, is required for intestinal colonization [119]. In V. cholerae, VrrA can regulate the level of TCP expression, indicating that VrrA is either a direct or indirect regulator of TCP. However, bioinformatics analysis by the RNAhybrid program suggested that VrrA regulates TCP directly by interacting at the region that includes the translation start and the Shine–Dalgarno region of tcpA mRNA. In addition to OmpA, the increased colonization of the vrrA mutant in V. cholerae could partially be caused due to increased production of TCP.

4. Riboswitches as novel regulators in bacteria

Riboswitches, an important class of regulators in bacteria, which are most often located in the 5' UTR of bacterial mRNA. Riboswitches mainly consist of genetic switch elements located on mRNAs that contain a conserved ligand-binding (aptamer) domain and a variable sequence, called an expression platform. In particular, bacteria use structural variations in expression platforms upon ligand binding at the aptamer domain of the RNA sequences, which directly affects the expression of genes encoded in the full transcript. Upon ligand binding, bacteria typically turn off gene expression either by using an

intrinsic transcription terminator or by occluding the ribosome binding site and start codon to inhibit translation initiation [120].

4.1 Types of Riboswitches

The first riboswitch class was discovered a decade ago in 2002, and it was experimental proven showed that mRNAs carry a sensing domain enabling vitamin derivatives to control gene expression [121-123]. The 5'-UTR of the btuB mRNA of E. coli contains a metabolite-sensing genetic switch that binds coenzyme B12 independent of any protein. This binding influences the synthesis of the cobalamin transport protein BtuB by modulating btuB mRNA [121]. In another study in E. coli, mRNAs encoding enzymes involved in thiamine (vitamin B1) biosynthesis shown to bound thiamine or its pyrophosphate derivative. The mRNA–metabolite complex brings structural changes that sequester the ribosome-binding site and leads to a reduction in gene expression [122]. Similar studies in Bacillus subtilis showed that small molecules like flavin mononucleotide (FMN) or thiamine pyrophosphate (TPP) can interact with the conserved leader region of the mRNA, resulting in creation of a termination hairpin that controls transcription elongation of riboflavin and thiamine operons [123]. In Listeria monocytogenes, the 5´ UTR preceding the prfA gene forms a secondary structure at temperature 30 ºC that masks the ribosome binding site, thus limiting translation. However, the secondary structure tends to disappear with increased production of PrfA at 37 ºC [124].

Subsequently new classes of riboswitches have been discovered that sense purines and their derivatives, amino acids, cofactors and their derivatives, phosphorylated sugar, and inorganic ligands including metal ions [125].

A study by Mandal et al. [126] showed that the xpt-pbuX operon in B. subtilis, is controlled by a riboswitch that exhibits high affinity and high selectivity for guanine. The direct binding of guanine by mRNAs serves a function for metabolic homeostasis in purine metabolism [126]. However, the C U mutation in the conserved core of the aptamer results in adenine preference for metabolite binding, suggesting a mechanism by which a single nucleotide determines the binding specificity between guanine and adenine [127]. In other studies, the amino acid-based riboswitch senses glycine to control expression of the gcvT operon, which codes for proteins that form a glycine cleavage system [128]. Another class of riboswitches utilizes S- adenosyl-methionine (SAM) as a cofactor for binding the conserved regulatory leader sequence (S-box) located in operons involved in methionine (Met) and cysteine (Cys) biosynthesis. SAM binding to S- box RNA resulted in formation of an intrinsic terminator, thus interrupting transcription prematurely [129].

The glmS riboswitch-ribozyme uses phosphorylated sugar (glucosamine-6-phosphate) as a metabolite which, upon interaction induces cleavage at glmS mRNA, thus repressing the expression of GlmS [130]. In Salmonella enterica serovar Typhimurium, expression

of the Mg2+ transporter MgtA is controlled by its 5´ UTR by sensing Mg2+. A cation-responsive riboswitch, in response to Mg2+ levels, leads to an altered stem-loop structure at the 5´ UTR of the mgtA gene, thus modulating expression of the target mRNA [131].

4.2 Metabolite-sensing riboswitches in V. cholerae

In previous studies, Sudarsan et al. showed that the highly conserved RNA domain called GEMM, which are mostly located upstream of the open reading frames (ORFs) for diguanylate cyclase (DGC) and phosphodiesterase (PDE), encode proteins in some organisms [132]. GEMM RNAs with similar architectures are further classified into type 1 and type 2 GEMM RNAs based on the presence of specific tetraloop and tetraloop receptor sequences. These GEMM RNA domains are responsive to cyclic di-GMP binding and involved in modulation of gene expression important for numerous fundamental cellular processes such as virulence gene expression, pilus formation, and flagellum biosynthesis [132, 133]. In V. cholerae, genomic analysis identified two sequences for the type 1 GEMM RNA domain located upstream of the gbpA gene and the vc1722 gene (homologous to tfoX) of V. cholerae. Biochemical and genetic analyses confirmed the interaction of these domains with cyclic di-GMP, which leads to spontaneous cleavage and altered structure of the transcript, thus modulating the expression of downstream mRNA [132]. The crystal structure of a c- di-GMP riboswitch aptamer from V. cholerae bound to c-di-GMP was solved at a resolution of 2.7 Å [134]. The structure analysis depicted

that the three-helix junction bound to the ligand involves extensive base-stacking and base-pairing [134].

In earlier studies, it was demonstrated that glycine-sensing RNAs from Bacillus subtilis operated as rare genetic switches for the gcvT operon. The aptamer domain consists of two forms of the gcvT RNA motif; type I and type II had been identified on the basis of differences in the sequences at 5´ and 3´ ends that flank a common central core [135] . Bioinformatics analysis revealed that both motif types are also present in V. cholerae, residing adjacent to each other in the region immediately upstream of the vc1422 gene (a putative sodium and alanine symporter). An in-line probing experiment showed that the metabolite-binding capabilities of V. cholerae vc1422 mRNA, where addition of glycine at 1 mM caused changes in the pattern of mRNA structures [128].

4.3 Virulence factors controlled by a themosensor in V. cholerae

During host intestine infection, V. cholerae uses two major virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP) that are required to cause diarrheal disease. The transcription factor protein, ToxT activates the expression of both ctx and tcp genes. Recent studies showed that the shift of bacteria from contaminated water at lower temperature into the human intestine at 37°C resulted in enhanced production of ToxT by inducing a structural alteration at the toxT 5´ UTR [136]. The studies showed that the four-U element residing in the 5´ UTR of toxT is an anti-Shine-Dalgarno (SD) element that base pairs

with the SD sequence to regulate ribosome access to the mRNA. However, at 37 °C, the four-U element allows access to the SD sequence for translation, unlike at 20 °C. The result reveals the characteristics of an RNA thermometer that activates virulence under environmental conditions suitable for infecting the human intestine in a temperature-dependent manner by regulating ToxT production [136].

AIMS OF THE THESIS

To analyze the role of V. cholerae sRNA (VrrA) in regulation of

1) Outer membrane protein expression

2) Biofilm formation

3) Ribosome binding proteins expression

To characterize a newly discovered sRNA, VrrB in V. cholerae

To investigate the function of VrrB and Vibrio auxotropic factor A (VafA) in V. cholerae

RESULTS AND DISCUSSION

A. Regulatory roles of sRNA, VrrA in V. cholerae

In the thesis studies, we demonstrated the roles of sRNAs, VrrA in regulation of outer membrane protein expression (Paper I), biofilm formation (Paper II) and expression of ribosomal binding proteins (Paper III). A summary of VrrA-mediated regulation of V. cholerae factors was illustrated in Fig. 3.

Paper 1

VrrA Mediates Hfq-Dependent Regulation of OmpT Synthesis in V. cholerae

V. cholerae encounters drastic environmental changes when it infects the host. In order to survive, bacteria regulate clusters of genes in response to environmental signals. One of such approach used by bacteria is to modify outer membrane proteins. In this study, we showed that vrrA gene, which requires the membrane stress sigma factor σE, regulates outer membrane protein OmpT expression. By Western blot analysis, we demonstrated that the expression of OmpT was increased in the vrrA mutant compared to the wild-type strain. The elevated level of OmpT in the vrrA mutant was restored to the wild type level in the complemented strain (Paper I, Fig. 1a, lanes 2 and 3). We also showed that in the absence of RNA chaperone protein Hfq,

OmpT levels appear to be unrelated to VrrA (Paper I, figures 1c to 1f), suggesting that Hfq is required for VrrA-mediated regulation of OmpT.

To understand the mechanism of VrrA-mediated regulation of OmpT, we further tested if the 5′ region of ompT mRNA is responsive to VrrA regulation. The RNAhybrid algorithm [115] was used to predict possible RNA duplexes formed by VrrA and the 5′ region of ompT mRNA. Based on the interacting sequences obtained by RNAhybrid algorithm prediction, nucleotide substitution mutants of VrrA were constructed. The VrrA mutant variants (VrrAM1 to VrrAM5) expressed from the plasmids pTS2-M1 to pTS2-M5 (Paper 1, Fig. 4e) were tested for the regulation of OmpT. The result showed that VrrA acts as an antisense RNA to repress ompT mRNA.

Previously it was shown that, two transcriptional regulators, CRP and ToxR, compete at the ompT promoter region. Transcription of ompT is activated by CRP in a loop-forming mechanism, while ToxR acts as an antiactivator by competing with CRP [21]. In addition to pH/temperature signals via the ToxR regulon and carbon source signals via the cAMP–CRP complex, our study provided further insight into the regulation of V. cholerae outer membrane proteins expression in response to signals received via the σE regulon through VrrA.

Paper II

V. cholerae utilizes direct sRNA regulation in expression of a biofilm matrix protein

In bacteria, biofilms consists of three distinct stages of development: (i) planktonic stage (ii) a transient monolayer stage and (iii) mature biofilm [137]. The third phase is primarily involved in promoting resistance of bacteria against environmental stresses [35].

In V. cholerae, biofilms contain three matrix proteins RbmA, RbmC and Bap1 and exopolysaccharide [32]. In this study, we showed that sRNA, VrrA down-regulates the biofilm matrix protein RbmC by base- pairing with the 5´-UTR of rbmC mRNA. Using Western blot analysis, we observed that the RbmC level was elevated in the absence of VrrA in the wild-type strain and the elevated RmbC level was restored to the the wild-type level when the VrrA over-expressed from a plasmid. We also showed that the VrrA mediated regulation of RbmC expression occured in the absence of Hfq suggesting that Hfq is not essential for this regulation.

To determine the interaction region between VrrA and 5` UTR region of the rbmC mRNA, the RNAhybrid algorithm [115] was used. The duplex formation was observed at residues 91– 106 of VrrA interacting with the -8 to -25 region of the rbmC mRNA (numbering of rbmC is relative to the AUG start codon) (Paper II, Fig. 2A). This 13 bp duplex is interrupted by a bulge dividing the stretch into a 7-bp and a 6-bp duplex masking the ribosome binding site required for translation initiation. Since the predicted interaction regions of VrrA were different from the interaction sites described in the previous study [104], additional VrrA mutant variants (VrrAM7 to VrrAM10) expressed

from plasmids (pTS2-M7 to pTS2-M10) (Paper II, Fig. 2B) were constructed. To detect the levels of RbmC in these constructs, Western blot analyses were performed. It was showed that VrrAM9 and VrrAM10 (expressed from pTS2-M9 and pTS2-M10, respectively) completely lost their ability to repress RbmC expression (Paper II, Fig. 4A, lanes 5 and 6). Our studies provide additional information that the VrrA- mediated suppression of RbmC might be an additional mechanism of biofilm regulation in V. cholerae. Over-expression of VrrA decreased the biofilm formation in V. cholerae suggesting that RbmC is essential for maintaining the stability of mature biofilms. In previous studies, it was shown that many sRNA has been involved in biofilm formation in bacteria, such as OmrA/B [138], McaS [139, 140], RprA [141] and GcvB [139]. None of these sRNA directly targets the biofilm matrix components, however, instead these sRNA target biofilm master regulator, CsgD, which in turn regulates biofilm components. Our studies revealed VrrA is the first example of an sRNA molecule that directly targets expression of a biofilm matrix component, RbmC.

Paper III

The VrrA sRNA controls a stationary phase survival factor Vrp of V. cholerae

In E. coli, the ribosome binding proteins RMF (ribosome modulation factor), HPF (hibernation promoting factor), RaiA (ribosome-associated inhibitor A) and SRA (stationary-phase-induced ribosome associated protein) are expressed in stationary phase and

involved in ribosome hibernation [49]. In our study, we demonstrated that VrrA is the first sRNA that directly regulates Vrp (a homolog of RaiA) in an Hfq-dependent manner by base-pairing with the 5´ region of the vrp mRNA in V. cholerae.

In order to analyze how VrrA targets to the 5´ region of the vrp mRNA in V. cholerae, the pairing region between VrrA and vrp mRNA was examined using the RNA hybrid programme. The RNA hybrid programme [115] predicted residues 72-84 of VrrA forms an 13-bp duplex with the -3 to -15 region (numbers relative to AUG start codon) of the vrp mRNA with free energy -28.2 kcal/mol (Paper III, Fig. 1A). This interaction partially masks the ribosome binding site of vrp mRNA required for translation initiation.

To examine the function of Vrp, we performed ribosome profile of overnight grown wild-type V. cholerae O1 strain A1552 on a sucrose density gradient followed by Western blot analysis and found that Vrp is a ribosome binding protein in V. cholerae. We also analyzed Vrp expression during different bacterial growth phases and showed that Vrp was highly expressed in stationary phase. To investigate the role of VrrA in Vrp expression, we determined the Vrp levels by Western blot analysis and found that 2.2 fold increase of Vrp expression in the vrrA mutant and elevated Vrp expression could be restored to the wild-type level by expressing VrrA from a plasmid harboring the wild-type allele of vrrA. In addition, we showed that Vrp regulation by VrrA is Hfq- dependent.

We demonstrated the role of Vrp and VC2530 (homolog of HPF) in survival of V. cholerae under nutrient limited conditions. In V. cholerae, the vrp and vc2530 single-deletion mutants were able to survive to an extent similar to that of the wild-type strain, probably by maintaining stabilized 70S monomeric and 100S dimeric forms of ribosomes, respectively, during stationary phase. However, the starvation survival of the double deletion mutant ∆vrp∆vc2530 was reduced 3.3 fold compared to the wild-type (Paper III, Fig. 3E). The previous studies in E. coli showed that RaiA and HPF have the same binding site on the 30S subunit of the ribosome indicating that both proteins might have similar function in stabilizing ribosomes during stationary phase [52]. In our studies, we showed that the expression level of Vrp was higher in the vc2530 deletion mutant than in the wild- type. Increased level of Vrp may therefore be involved in stabilization of ribosomal subunits potentially allowing the vc2530 deletion mutant to live as long as the wild-type. The vrp mutant also showed the same survival ability as the wild-type under nutrient-limited conditions. In the vrp mutant, the level of VC2530 expression was also higher than that of the wild-type. Increased level of VC2530 protein may stabilize the ribosome, thereby protecting the ribosome dimers from degradation and allowing the same survival time as that of the wild type. Deletion of both vrp and vc2530 genes might result in a less stable ribosome which leads to a reduction in starvation survival ability.

Taken together, we suggest that Vrp, which is under the direct regulation of VrrA, and VC2530 which is indirectly regulated by VrrA, are important for V. cholerae starvation survival under nutrient-limited conditions.

Fig. 3. Proposed model of regulation circuit of VrrA in Vibrio cholerae.

B. Regulatory roles of sRNA, VrrB in Vibrio cholerae

We demonstrated a newly discovered sRNA, VrrB role(s) in amino acid starvation survival of Vibrio cholerae (Paper IV)

Paper IV

A new sRNA, VrrB, acts as a regulator for vafA, a gene involved in amino acid starvation survival of V. cholerae

The amino acids availability is vital to all living organisms. Bacteria can respond to changes in their environment by modulating their gene expression–they express different enzymes depending on the metabolite sources available to them. In our studies, we discovered a new sRNA by thorough analysis of the rho independent terminator at the intergenic region between the vrp and the Vibrio auxotropic factor A (vafA) genes. We named this sRNA as Vibrio regulatory RNA B (VrrB). We demonstrated that VafA is an autotrophic factor for pyruvic acid derivative “phenylpyruvate” a metabolite required to produce phenylalanine. In addition, we have unraveled the contribution of VafA in the amino acid starvation survival of V. cholerae and the regulation of its synthesis by a possible riboswitch, VrrB. We showed that 5’ UTR sequences of vafA mRNA contains 147bp non-coding sRNA gene, vrrB and that the deletion of vrrB leads to high production of VafA (Paper IV, Fig. 3A). We suggest that vrrB sequence consists of the transcription attenuator region and might have the metabolite- sensing domain, deletion of which therefore lead to the enhanced VafA production. We also observed that reduced production of VafA in wild- type V. cholerae strain when the bacteria were grown in the minimal

media in the presence of phenylalanine or phenylpyruvate. Some analogs of phenylalanine and phenylpyruvate could also modulate the expression of VafA.

The RNAfold web server [142] was used to predict the part of the 5’UTR and coding region of the vafA gene for the secondary structure formation. We observed the two stem loops (Paper IV, Fig. 6A) one of which contains vrrB terminator and other might be involved in occluding the ribosome binding site of the vafA mRNA.

We hypothesized that metabolites such as phenylpyruvate or phenylalanine and their anologs might bind to the 5’ UTR sequences of vafA mRNA and alter the structural conformation by creating either the stable terminator or anti-terminator resulting in eliminating or stabilizing the secondary stem loops near ribosome binding site of vafA mRNA which might modulate the production of VafA (Paper IV, Fig. 6B2). Another possibility of vafA regulation might be that the binding of ligands can induce the cleavage of the vafA transcript by RNase compromising its stability and production of VafA (Paper IV, Fig. 6B3).

Taken together, our results suggest that VrrB repress the production of VafA. The vafA mRNA (Paper IV, Fig. 4) which co-transcribe together with VrrB sRNA might function as a potential riboswitch to control production of VafA in response to amino acid signals such as tyrosine, 4-hydroxyphenylpyruvate, phenylalanine, phenyllactic acid or

phenylpyruvate (Fig. 3A & 3B). Further investigations are required for clarification of role(s) of VrrB as a riboswitch which modulate expression of VafA for the survival of V. cholerae in the amino acids deficient media.

Fig. 4. Proposed model of regulation circuit of VrrB in Vibrio cholerae. Metabolites that activates the VafA are marked by arrow in green color, while those repress the VafA are shown in red color.

CONCLUSIONS

 VrrA regulates the expression of outer membrane protein OmpT in Vibrio cholerae.  Vibrio cholerae utilizes VrrA to regulate the expression of biofilm matrix protein, RbmC.  Over expression of VrrA results in decreased biofilm formation.  Vrp, a ribosome binding protein in V. cholerae express highly during stationary phase.  VrrA down-regulates the expression of Vrp.  ∆vrp ∆vc2530 double deletion mutant in V. cholerae showed reduced starvation survival compared to wild-type and single deletion mutant ∆vrp and ∆vc2530.  VrrB is a new sRNA in V. cholerae located in the intergenic region between vrp and vafA.  vafA cannot grow in M9 media lacking either phenylpyruvate or phenylalanine.  vrrB modulates the production of VafA.  Differential expression of VafA is observed in M9 media in the presence of any of the metabolites such as tyrosine, 4- hydroxyphenylpyruvate, phenylalanine, phenyllactic acid, 2- nitro-phenylpyruvate or phenylpyruvate.

ACKNOWLEDGEMENTS

Finally, it’s time for my dissertation. I still remember the day I reached Umeå – 4th November 2008, after a long journey of 16 hours, first time ever outside India. Since then, I have spent more than six fruitful years in Umeå.

During my Master’s studies in Umeå, I attended a encouraging lecture about Vibrio cholerae by Prof. Sun Nyunt Wai, following which I got chance to pursue my Master’s thesis project in her laboratory (Thinking that it will be fun to work in lab., though now I admit that research is fun only when you get good results, which happen so rarely). I feel lucky to continue my Master’s project as PhD student in the same lab. First, foremost and earnestly I would like to thank to my supervisor Prof. Wai, for letting me do my PhD in her lab. She helped me during each and every stage of my research work. This PhD would not have been possible, without her great support and scientific insights.

I am also very thankful to my co- supervisors Prof. Brent Eric Uhlin and Prof. Jörgen Johansson, for their help and suggestions during my project updates and article presentations. I would like to acknowledge funding agencies: Swedish Research Council, the Faculty of Medicine; Umeå University and part of this work was performed within the Umeå Centre for Microbial Research (UCMR), the Laboratory for Molecular Infection Medicine Sweden (MIMS).

I express my deepest gratitude to my parents, sister, and brother, for always encouraging me during my PhD studies.

In random order, I would like to thank - Dr. Takahiko, who supervised me during my Master’s thesis, thanks for teaching me many techniques to handle Vibrio. Prof. Sven Bergström, for lots of signatures. Bhupender, my friend from neighbouring city in Haryana thanks for your company during Fatmomakke trip and always helping with research projects. Mitesh, for offering delicious food, helping with any knowledge I needed and fruitful discussions. Sridhar, for introducing me to new online electronic technologies. Pramod, for always helping me with research in the lab. and suggesting future career guidance. Annika, for helpful in the lab. and teaching me some Swedish. Svitlana, for nice talks. Tetsuya, for your nice company and kindness. Monika, for always helpful and for providing AFM images. Marylise, Kyaw, Constance, for all help and nice talks during lab. work. Sabine, Stefanie,

Christopher, Teresa, Sakura, Tianyan, Caroline and Karolis, for nice suggestion during JC clubs. Anne-Laure, Anjana and Nikola you all are amazing assistants which made teaching fun, easy and efficient. Thanks for all the cooperation and support. Mikael Wikström, for all the help related to credential update in Ladok system. Maria Westling, Ingela Sandström and Selma Grönlund for all the adminstrative work. Marek Wilczynski, for helping with computer installations. Johnny Stenman, for ordering all the stuff.

Satadal, Shashank, Kumar and Asif are my initial friends, whom I met in Umeå, I still miss our parties at Gluntens Väg- 9. Sanjay, that’s for you “Chak de phatte, nap de killi, raat nu Jalandar, sawere Dilli” and for long Skype scientific discussions. Sri Kanth, for Best cricket bowler I have faced in Umeå. Rajdeep, for idea about cover page design, always helpful and for offering Potato salad. Pardeep Singh, for being the best cricket player in Umeå and offering nice Punjabi food. Philip, Devendra, Naresh for polite talks. Deepak, Rohit Ruhal, for always helpful and nice discussions. Ganesh, for always asking how are you Dharmesh?. Patricia, for your politeness. Khanzeb khan, for always helping and for the cake during cricket world cup. It was very generous of you. Jyoti, for always happy in all environment conditions. Mridula, for helping with RNA related equipments. Steffi Jimmy, for nice talks. Damini, Radha, Geetanjali, for organising Indian festival and for always been helpful. Nabil, for always showing inspiration. Kemal, for nice time during course in Argentina. Bernard, for disclosing Nobel Prize winning research ideas. They might work one day. Jalal, for all business discussions on Viber calls, they too might work one day. Sandeep, for generous nature and butter chicken recipe and for frozen vodka. Tanmay, for fun talk. Shrikant, for orgainsing traditional Indian festivals. Prashant, for being Rahul Dravid of Umeå. Dilip, for good discussions.

Special thanks to my IKSU Badminton group: Syam, Tushar, Edvin, Jakob, Edward, Brijesh, Yogesh, Amol, Vishal, Bhupender it was fun and good memory with interesting matches we played. And now we are almost close to the level of being professionals. Kent, it was fun with Kayaking, can’t believe we took that risk. Stina, for teaching me cross country skiing. Aislinn, for all fika talk. My hometown friends from India, Sagar, Mohit, Golu, Chandan, Pawan, Jitesh, Rana, Ajay, Shitiz - it’s always nice to have funny conversation on phone.

Thanks to all !!!

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