Academic literature on the topic 'CcdA antitoxin'

The ccd system of the F plasmid encodes CcdB, a protein toxic to DNA-gyrase, and CcdA, its antitoxin. The function attributed to this system is to contribute to plasmid stability by killing bacteria that lose the plasmid during cell division. However, the function of ccd in resting bacteria is not clear. Results presented show that ccd transcription increases as bacteria enter stationary phase and that the amount of the Ccd proteins is higher in bacteria under nutritional stress than in growing bacteria. Moreover, an increase in the frequency of Lac+ "adaptive" mutations was observed in stationary-phase bacteria that over-express the Ccd proteins.Key words: ccd system, nutritional stress, adaptive mutation.

We report the nucleotide sequence of two novel cryptic plasmids (4357 and 14 662 base pairs) carried by aYersinia enterocoliticabiotype 1A strain isolated from pork. As distinguished from most biotype 1A strains, this isolate, designated 07-04449, exhibited adherence to eukaryotic cells. The smaller plasmid pYe4449-1 carries five attributable open reading frames (ORFs) encoding the first CcdA/CcdB-like antitoxin/toxin system described for aYersiniaplasmid, a RepA-like replication initiation protein, and mobilizing factors MobA and MobC. The deduced amino acid sequences showed highest similarity to proteins described inSalmonella(CcdA/B),Klebsiella(RepA), andPlesiomonas(MobA/C) indicating genomic fluidity among members of theEnterobacteriaceae. One additional ORF with unknown function, termed ORF5, was identified with an ancestry distinct from the rest of the plasmid. While the C+G content of ORF5 is 38.3%, the rest of pYe4449-1 shows a C+G content of 55.7%. The C+G content of the larger plasmid pYe4449-2 (54.9%) was similar to that of pYe4449-1 (53.7%) and differed from that of theY. enterocoliticagenome (47.3%). Of the 14 ORFs identified on pYe4449-2, only six ORFs showed significant similarity to database entries. For three of these ORFs likely functions could be ascribed: a TnpR-like resolvase and a phage replication protein, localized each on a low C+G island, and DNA primase TraC. Two ORFs of pYe4449-2, ORF3 and ORF7, seem to encode secretable proteins. Epitope-tagging of ORF3 revealed protein expression at4°Cbut not at or above27°Csuggesting adaptation to a habitat outside swine. The hypothetical protein encoded by ORF7 is the member of a novel repeat protein family sharing theDxxGN(x)nDxxGNmotif. Our findings illustrate the exceptional gene pool diversity within the speciesY. enterocoliticadriven by horizontal gene transfer events.

ABSTRACTPlasmid toxins CcdB and ParE are part of addiction systems promoting plasmid maintenance. Both target host DNA gyrase, as do quinolones and plasmid-determined Qnr proteins that protect gyrase from quinolone inhibition. We clonedqnrB4,qnrS1,ccdB,parE, and the antitoxin-encoding genesccdAandparDon compatible plasmids and tested them in combination. CcdB and ParE had no specific effect on quinolone susceptibility or Qnr protection, and Qnr did not act as a CcdB or ParE antitoxin.

Salmonella enterica serovar Javiana is the fourth most reported serovar of laboratory-confirmed human Salmonella infections in the U.S. and in Tennessee (TN). Although Salmonella ser. Javiana is a common cause of human infection, the majority of cases are sporadic in nature rather than outbreak-associated. To better understand Salmonella ser. Javiana microbial population structure in TN, we completed a phylogenetic analysis of 111 Salmonella ser. Javiana clinical isolates from TN collected from Jan. 2017 to Oct. 2018. We identified mobile genetic elements and genes known to confer antibiotic resistance present in the isolates, and performed a pan-genome-wide association study (pan-GWAS) to compare gene content between clades identified in this study. The population structure of TN Salmonella ser. Javiana clinical isolates consisted of three genetic clades: TN clade I (n = 54), TN clade II (n = 4), and TN clade III (n = 48). Using a 5, 10, and 25 hqSNP distance threshold for cluster identification, nine, 12, and 10 potential epidemiologically-relevant clusters were identified, respectively. The majority of genes that were found to be over-represented in specific clades were located in mobile genetic element (MGE) regions, including genes encoding integrases and phage structures (91.5%). Additionally, a large portion of the over-represented genes from TN clade II (44.9%) were located on an 87.5 kb plasmid containing genes encoding a toxin/antitoxin system (ccdAB). Additionally, we completed phylogenetic analyses of global Salmonella ser. Javiana datasets to gain a broader insight into the population structure of this serovar. We found that the global phylogeny consisted of three major clades (one of which all of the TN isolates belonged to) and two cgMLST eBurstGroups (ceBGs) and that the branch length between the two Salmonella ser. Javiana ceBGs (1,423 allelic differences) was comparable to those from other serovars that have been reported as polyphyletic (929–2,850 allelic differences). This study demonstrates the population structure of TN and global Salmonella ser. Javiana isolates, a clinically important Salmonella serovar and can provide guidance for phylogenetic cluster analyses for public health surveillance and response.

Abstract Deep mutational scanning studies suggest that synonymous mutations are typically silent and that most exposed, non active-site residues are tolerant to mutations. Here we show that the ccdA antitoxin component of the E.coli ccdAB toxin-antitoxin system is unusually sensitive to mutations when studied in the operonic context. A large fraction (∼80%) of single-codon mutations, including many synonymous mutations in the ccdA gene shows inactive phenotype, but they retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure/stability, consistent with a large region of CcdA being intrinsically disordered. E. coli codon preference and strength of ribosome-binding associated with translation of downstream ccdB gene are found to be major contributors of the observed mutant phenotypes. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that the ccdA mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by studying single-site synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their operonic context, genes are likely to be more sensitive to both synonymous and non-synonymous point mutations than inferred previously.

Les systèmes toxine-antitoxine (TA) sont très répandus au sein des génomes bactériens. Ces opérons bicistroniques de petite taille ont été découverts sur des plasmides à bas nombre de copies. Dans ce contexte génétique, les systèmes TA confèrent un avantage sélectif à leurs molécules-hôtes en tuant les bactéries-filles qui ne les ont pas héritées par le mécanisme de tuerie post-ségrégationnelle (PSK, post-segregational killing). Ces systèmes génétiques sont également appelés modules d’addiction étant donné qu’ils rendent la descendance des bactéries qui les contiennent dépendantes de leur présence. Alors que leur rôle dans les molécules d’ADN épisomiques est relativement bien établi, le sens biologique de la présence d’homologues à ces systèmes épisomiques au sein des chromosomes bactériens est sujet à d’intenses débats. L’idée que les systèmes TA chromosomiques confèrent un avantage sélectif a été mise en évidence dans plusieurs modèles. Selon ces modèles, les systèmes TA permettent aux bactéries de mieux faire face à des conditions environnementales stressantes. <p>Entre-temps, la compréhension de l’évolution des génomes bactériens a connu des avancées significatives. L’impressionnante capacité d’adaptation des bactéries est aujourd’hui majoritairement attribuée au transfert horizontal de gènes (THG) provoqué par les éléments génétiques mobiles (phages, plasmides, transposons…). Dans le débat du rôle des systèmes TA chromosomiques, très peu d’attention a été accordée aux relations phylogénétiques et interactions entre systèmes plasmidiques et chromosomiques co-existant au sein d’un même hôte ainsi qu’à l’impact du THG sur leur évolution. Notre travail de thèse vise à mieux comprendre la biologie des systèmes TA en tenant compte de ces paramètres. Nous nous sommes intéressés à des systèmes homologues au système plasmidique ccdF. Nous avons étudié expérimentalement les 4 systèmes ccd (ccd1, ccd2, ccd3 et ccd4) qui co-habitent au sein du chromosome d’Erwinia chrysanthemi 3937 (une bactérie phytopathogène), leurs interactions intragénomiques et les interactions de ces systèmes avec le système plasmidique ccdF. Ce cadre expérimental a mené à la construction du modèle d’anti-addiction. Ce modèle propose que certains systèmes chromosomiques puissent conférer un avantage sélectif à leurs hôtes bactériens en interférant avec le PSK médié par leurs homologues plasmidiques. Cet avantage sélectif pourrait permettre la fixation de systèmes TA latéralement acquis au sein des populations bactériennes. Nous avons également recherché de nouveaux systèmes ccd au sein des génomes bactériens afin d’avoir un aperçu de leur distribution, des contextes génétiques dans lesquels ils existent et de l’implication du THG dans leur dispersion. Les réflexions qui ont accompagné notre recherche nous ont mené à proposer une synthèse sur le rôle des systèmes TA (plasmidiques et chromosomiques). Celle-ci se nourrit des avancées qui ont été effectuées, ces dernières années, dans la compréhension de l’évolution des génomes bactériens, de la théorie hiérarchique de la sélection naturelle et des processus non-adaptatifs et contingents qui pourraient expliquer la présence et la propagation des systèmes TA au sein des génomes bactériens sans que ceux-ci en soient les agents causaux. <p><p><br>Doctorat en sciences, Spécialisation biologie moléculaire<br>info:eu-repo/semantics/nonPublished

Les systèmes toxine-antitoxine (TA) bactériens ont été découverts il y a une vingtaine d’année sur les plasmides à bas nombre de copie. Ils sont composés de deux gènes organisés en opéron, l’un codant pour une toxine stable et l’autre pour une antitoxine instable capable de neutraliser l’effet de la toxine. Les systèmes TA sont fortement représentés au sein de l’ensemble des génomes bactériens. Ils se localisent aussi bien sur des éléments génétiques mobiles (plasmides, phages, transposons,…) que dans les chromosomes, ce qui suggère que le transfert horizontal de gènes participe à leur dissémination. Le système TA ccd du plasmide F d’Escherichia coli (ccdF) est composé de l’antitoxine CcdA et de la toxine CcdB. Le système ccdF contribue à la stabilité du plasmide F en tuant les bactéries-filles n’ayant pas reçu de copies plasmidiques lors de la division bactérienne (tuerie post-ségrégationelle).<p>Au cours de ce travail, nous avons caractérisé un homologue du système toxine-antitoxine ccd du plasmide F (ccdF) qui se situe dans le chromosome de la souche pathogène E. coli O157:H7 EDL933 entre les gènes folA et apaH (ccdO157). Les systèmes ccdF et ccdO157 coexistent naturellement dans les souches d’E. coli O157:H7, le système ccdF se trouvant sur le plasmide pO157 qui dérive du plasmide F. Nos résultats montrent que l’antitoxine plasmidique CcdAF neutralise l’effet de la toxine chromosomique CcdBO157, tandis que l’antitoxine chromosomique CcdAO157 ne contrecarre pas la toxicité de la toxine plasmidique CcdBF. Nous avons également montré que le système ccdF cause une tuerie post-ségrégationelle, lorsqu’il est cloné dans un plasmide instable, dans une souche possédant le système chromosomique ccdO157. Le système ccdF est donc fonctionnel en présence de son homologue chromosomique. <p>Le système ccdO157 est absent du chromosome de la souche de laboratoire E. coli K-12 MG1655, où une région intergénique de 77 pb sépare les gènes folA et apaH. Celle-ci contient une séquence cible pour la transposition. Nous avons étudié la distribution du système ccdO157 au sein de 523 souches d’E. coli représentatives de l’ensemble des sérogroupes décrits. Nos résultats montrent que le système ccdO157 est présent au sein de souches appartenant à 47 sérogroupes différents. Nos résultats mettent en évidence la diversité de la région intergénique folA-apaH d’E. coli. Celle-ci peut contenir gènes codant pour des protéines présentant de l’homologie avec des protéines d’espèce bactériennes éloignées d’E. coli ou d’organismes eucaryotes, ainsi qu’un élément génétique mobile, l’IS621, ce qui montre que le système ccdO157 a intégré le chromosome d’E. coli via le transfert horizontal de gènes.<p><br>Doctorat en Sciences<br>info:eu-repo/semantics/nonPublished

Proteins interact with other proteins to maintain functional homeostasis of the cell by tightly regulating cellular processes. Hence, it becomes important to understand not only the downstream effects of protein-protein interactions (PPIs), but its impact on interacting partners too. Much of the work embodied in this thesis pertains to sequence, structure, dynamics and functional analysis of protein-protein complexes. Firstly, the impact of PPIs on structure and dynamics of individual interacting partners was studied using a dataset of 58 protein-protein complexes of known 3-D structure. It was observed that binding elicits change in structure and dynamics of many interacting proteins, not only at the interface, but also at the regions away from the interfaces. Analyses of various examples showed that such changes could a) contribute towards the stability of the complex by adding to the positive gain in entropy, b) regulate a downstream function such as binding of another protein and c) alter the functional capacity of an enzyme. Next, this learning was extended to a protein-protein complex between DNA gyrase and CcdB toxin from E. coli to understand the dissociation of the former due to binding of another protein, the CcdA antitoxin. It was observed that binding of CcdA to the CcdB-gyrase complex elicits a series of response in the CcdB toxin. This response could be recorded in terms of change in residue flexibility/conformation at strategic locations, that facilitates release of gyrase from the assembly. The toxin-antitoxin (TA) systems are small, genetic elements composed of a toxin gene (encoding toxin protein) and a neighbouring antitoxin gene (encoding antitoxin protein) regulated under the same operon. These specific protein pairs from M. tuberculosis (Mtb) were analysed to understand the basis of their unique expansion. Sequence analysis of VapBC and MazEF TA systems of Mtb suggested similarities in their binding modes and functional residues. Structure of known TA complexes were utilised to detect interface as well as substrate-binding residues, which were further validated by laboratory experiments performed by the collaborators. To check the uniqueness of Mtb TA systems, their homologues from other micro-organisms were identified. Interestingly, while most orthologues were found well-conserved in the members of the Mtb complex, the soil-inhabiting, free-living Actinobacteria also harboured as many as 12 toxin-antitoxin pairs. A detailed analysis of Mtb TA systems helped in identifying sequence features of these systems. Further, these features along with TA features from other organisms led to the identification of four novel TA systems, namely, Rv2515c-Rv2514c, Rv3642c-Rv3641c, Rv0367c-Rv0366c, and Rv0023-Rv0024. Detailed computational analysis supported by laboratory experiments (performed by collaborators) suggested novel VapBC system for Rv2515c-Rv2514c and non-canonical PezAT for Rv0367c-Rv0366c. Lastly, because there exist many TA systems in Mtb, attempts were made to explore the potential of these TA systems to cross-react with one another. To this end, extensive analyses of experimental and modelled structures and promoter sequences of TA systems were performed. The results obtained helped in identification of specificity conferring residues in VapB15 and MazEFs and enabled proposition of cross-talks. Taken together, results presented in this thesis shed light on the characteristics of specific PPIs and how they evolve, function and impact their partners to control cellular processes.

A major challenge in biology is to understand and predict the effect of mutations on protein structure, stability and function. Chapter 1 provides a general introduction on protein sequence-structure relationships and use of the CcdAB toxin-antitoxin system as a model to study molecular determinants of mutant phenotypes. In Chapter 2, we describe the use of saturation mutagenesis combined with deep sequencing to determine phenotypes for 1664 single-site mutants of the E. coli cytotoxin, CcdB. We examined multiple expression levels, effects of multiple chaperones and proteases and employed extensive in vitro characterization to understand how mutations affect these phenotypes. While general substitution preferences are known, eg polar residues preferred at exposed positions and non-polar ones at buried positions, we show that depth from the surface is important and that there are distinctly different energetic penalties for each specific polar, charged and aromatic amino acid introduced at buried positions. We also show that over-expression of ATP independent chaperones can rescue mutant phenotypes. Other studies have primarily looked at effects of ATP dependent chaperone expression on phenotype, where it is not possible to say whether mutational effects on folding kinetics or thermodynamic stability are the primary determinant of altered phenotypes, since there is energy input with these chaperones. The data suggest that mutational effects on folding rather than stability determine the in vivo phenotype of CcdB mutants. This has important implications for efforts to predict phenotypic effects of mutations and in protein design. While looking at the mutational landscape of a given gene from an evolutionary perspective, it is important to establish the genotype-phenotype relationships under physiologically relevant conditions. At the molecular level, the relationship between gene sequence and fitness has implications for understanding both evolutionary processes and functional constraints on the encoded proteins. Chapter 3 describes a methodology to test the fitness of individual CcdB mutants in E.coli over several generations by monitoring the rate of plasmid loss. We also propose a methodology for high throughput analysis of a pool of CcdB mutants using deep sequencing to quantitate the relative population of each mutant in a population of E.coli cells, grown for several generations and build the fitness landscape. While the F-plasmid based CcdAB system is known to be involved in plasmid maintenance through post-segregational killing, recent identification of ccdAB homologs on the chromosome, including in pathogenic strains of E.coli and other bacteria, has led to speculations on their functional role on the chromosome. In Chapter 4, we show that both the native ccd operon of the E.coli O157 strain as well as the ccd operon from the F- plasmid when inserted on the E.coli chromosome lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters. Both the ccdF and ccdO157 operons may share common mechanisms for activation under stress conditions and also display weak cross activation. The chromosomal toxin shows weaker activity as compared to the plasmidic counterpart and is therefore less efficient in causing cell death. This has important implications in generation of potential therapeutics that target these TA systems. Chapter 5 describes the use of site-saturation mutagenesis coupled with deep sequencing to infer mutational sensitivity for the intrinsically disordered antitoxin, CcdA. The data allows us to make comparisons between overall as well as residue specific mutational sensitivity patterns with that of globular proteins, like CcdB (described in Chapter 2) and study toxin- antitoxin interaction and regulation through saturation suppressor mutagenesis. Interestingly, we found several examples of synonymous point mutations in CcdA that lead to loss of its activity. In Chapter 6 we attempt to explore the molecular bases for some of these synonymous mutations. In most cases the mutated codon has a similar overall codon preference to the WT one. Initial findings suggest a change in mRNA structure leading to change in CcdB: CcdA ratio, thereby causing cell death. These observations have important implications, because TA systems are ubiquitous, highly regulated and are known to be involved in multiple functions including drug tolerance. However a role for RNA structure in their regulation has not been shown previously. Appendix–I lists the mutational sensitivity scores for the CcdB mutants. Phenotypes for CcdA mutants obtained through deep sequencing have been tabulated in Appendix-II. Overall, we provide extensive datasets for mutational sensitivities of a globular (CcdB) and an intrinsically disordered protein (CcdA). Exploration of the molecular determinants of these mutant phenotypes not only provides interesting insights into CcdAB operon function but is also useful in understanding various aspects of protein stability, folding and activity as well as regulation of gene expression in bacteria.

A major challenge in biology is to understand and predict the effect of mutations on protein structure, stability and function. Chapter 1 provides a general introduction on protein sequence-structure relationships and use of the CcdAB toxin-antitoxin system as a model to study molecular determinants of mutant phenotypes. In Chapter 2, we describe the use of saturation mutagenesis combined with deep sequencing to determine phenotypes for 1664 single-site mutants of the E. coli cytotoxin, CcdB. We examined multiple expression levels, effects of multiple chaperones and proteases and employed extensive in vitro characterization to understand how mutations affect these phenotypes. While general substitution preferences are known, eg polar residues preferred at exposed positions and non-polar ones at buried positions, we show that depth from the surface is important and that there are distinctly different energetic penalties for each specific polar, charged and aromatic amino acid introduced at buried positions. We also show that over-expression of ATP independent chaperones can rescue mutant phenotypes. Other studies have primarily looked at effects of ATP dependent chaperone expression on phenotype, where it is not possible to say whether mutational effects on folding kinetics or thermodynamic stability are the primary determinant of altered phenotypes, since there is energy input with these chaperones. The data suggest that mutational effects on folding rather than stability determine the in vivo phenotype of CcdB mutants. This has important implications for efforts to predict phenotypic effects of mutations and in protein design. While looking at the mutational landscape of a given gene from an evolutionary perspective, it is important to establish the genotype-phenotype relationships under physiologically relevant conditions. At the molecular level, the relationship between gene sequence and fitness has implications for understanding both evolutionary processes and functional constraints on the encoded proteins. Chapter 3 describes a methodology to test the fitness of individual CcdB mutants in E.coli over several generations by monitoring the rate of plasmid loss. We also propose a methodology for high throughput analysis of a pool of CcdB mutants using deep sequencing to quantitate the relative population of each mutant in a population of E.coli cells, grown for several generations and build the fitness landscape. While the F-plasmid based CcdAB system is known to be involved in plasmid maintenance through post-segregational killing, recent identification of ccdAB homologs on the chromosome, including in pathogenic strains of E.coli and other bacteria, has led to speculations on their functional role on the chromosome. In Chapter 4, we show that both the native ccd operon of the E.coli O157 strain as well as the ccd operon from the F- plasmid when inserted on the E.coli chromosome lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters. Both the ccdF and ccdO157 operons may share common mechanisms for activation under stress conditions and also display weak cross activation. The chromosomal toxin shows weaker activity as compared to the plasmidic counterpart and is therefore less efficient in causing cell death. This has important implications in generation of potential therapeutics that target these TA systems. Chapter 5 describes the use of site-saturation mutagenesis coupled with deep sequencing to infer mutational sensitivity for the intrinsically disordered antitoxin, CcdA. The data allows us to make comparisons between overall as well as residue specific mutational sensitivity patterns with that of globular proteins, like CcdB (described in Chapter 2) and study toxin- antitoxin interaction and regulation through saturation suppressor mutagenesis. Interestingly, we found several examples of synonymous point mutations in CcdA that lead to loss of its activity. In Chapter 6 we attempt to explore the molecular bases for some of these synonymous mutations. In most cases the mutated codon has a similar overall codon preference to the WT one. Initial findings suggest a change in mRNA structure leading to change in CcdB: CcdA ratio, thereby causing cell death. These observations have important implications, because TA systems are ubiquitous, highly regulated and are known to be involved in multiple functions including drug tolerance. However a role for RNA structure in their regulation has not been shown previously. Appendix–I lists the mutational sensitivity scores for the CcdB mutants. Phenotypes for CcdA mutants obtained through deep sequencing have been tabulated in Appendix-II. Overall, we provide extensive datasets for mutational sensitivities of a globular (CcdB) and an intrinsically disordered protein (CcdA). Exploration of the molecular determinants of these mutant phenotypes not only provides interesting insights into CcdAB operon function but is also useful in understanding various aspects of protein stability, folding and activity as well as regulation of gene expression in bacteria.

碩士<br>長庚大學<br>醫學生物技術暨檢驗學系<br>98<br>Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its cognate antitoxin and are found on the chromosomes of many bacteria including pathogens. It has been demonstrated that chromosomal TA systems influence bacterial multicellular behaviors such as biofilm formation and fruiting body formation. However, the role of TA systems in swarming behavior, another type of bacterial multicellular behavior, remains elusive. Swarming is a complex multicellular behavior requires the integration of chemical and physical signals, which leads to the physiological and morphological differentiation of the bacteria from nonmotile vegetative cells into highly-motile swarmer cells. Here we demonstrated that approximately 90% of cells undergo cell death in vegetative cell population during swarming development in Serratia marcescens. A pair of chromosomal TA module named ccdAB(Sm) was identified. Deletion of ccdAB(Sm) caused dramatic reduction of dead cells during swarming development and showed precocious swarming motility. Interestingly, deletion of rssBA, a two-component system regulating swarming initiation, also reduced cell death during swarming. Furthermore, the transcription of ccdAB(Sm) and their upstream genes are negatively regulated by RssAB signaling which led to cell death in early swarming development. Taken together, these findings provide evidences that RssAB regulates cell death through a chromosomal TA module, CcdAB(Sm), in swarming development.

The primary amino acid sequence typically dictates the ultimate conformation of the protein. Mutations in the sequence demonstrate neutral, positive or negative effects on the structure-function relationship of the protein. Destabilising mutations often reduce the soluble levels of the protein in vivo, leading to complete or partial loss of its function. Global suppressors are diverse compensatory mutations that can alleviate the detrimental effects of multiple destabilised, inactive mutants, despite being physically distant from the site of the original inactive mutations. The molecular mechanisms responsible for suppression are still unknown, despite the characterisation of suppressors in several proteins since the late 1980s. Another modulator of a protein’s structure is the pH of the solution. It has long been known that proteins form molten globules at acidic pH conditions, which are compact denatured states with fluctuating tertiary structures. CcdB (Controller of Cell Division or Death B), a 101-residue homodimeric toxin which is a part of the CcdA:CcdB toxin:antitoxin module and poisons intracellular Gyrase, has been utilised as the model protein of choice in the course of the studies. Chapter 1 presents the importance of global suppressors, gives an overview of the role of pH on the protein’s structure and discusses various Gyrase inhibitors and outlines how the CcdB:Gyrase interaction can be utilised to design antibacterial peptides based on the toxin’s sequence. Chapter 2 provides a detailed thermodynamic and kinetic investigation of a global suppressor in CcdB. The studies show that the suppressor restores the stability and function of inactive mutants by marginally enhancing their apparent thermodynamic stabilities and lowering their unfolding rates. Chapter 3 shows the broad-spectrum antibacterial action of a novel CcdB-derived peptide on E.coli and pathogenic strains of S.aureus, S.Typhimurium, as well as a multi-drug resistant clinical isolate of A.baumannii. Chapter 4 reports a preliminary investigation of the low pH-induced molten globule-like state of CcdB using NMR spectroscopy.<br>MHRD, India