Genes are able to replicate horizontally and vertically- a given gene may be more successful on horizontally mobile elements than others. This includes genes that exhibit a post-segregational killing (PSK) phenotype. PSK is generated by expression of a toxin and antitoxin from a mobile element, such that if a bacterium loses the element the toxin becomes active in the cell and the cell dies. All PSKs described to date involve a toxin and an antitoxin function, though within a given group of toxin and antitoxin gene pairs only some are likely to exhibit this phenotype. Here, I investigate what differentiates genes that induce PSK from biochemically similar genes that do not. One group of genes of which some are known to induce PSK is toxinantitoxin (TA) systems, composed of a stable toxin and an unstable antitoxin. I analyzed computational data on the distribution of type I TA systems (RNA antitoxin), which appear to be less mobile than type II TA systems (protein toxin). Data on validated TAs suggests a correlation between distribution, mobility and the PSK phenotype. Differences in phylogeny could be due to differences in tendency to exhibit PSK in different environments. This connection between distribution and PSK was explored by experimentally testing a computationally described operon, plasmid_Toxin-ptaRNA1, that exhibited structural and distributional similarities to a mobile type I TA system. Despite this, expression of the predicted toxin ORFs did not reduce growth (as measured by saturation density) in E. coli, and the operon did not induce PSK. The conditions of PSK were further tested with the toxin (barnase) and antitoxin (barstar), which are not known to have the phenotype. A number of heterologous expression systems were developed with these genes in E. coli to test their ability to exhibit PSK in a manner akin to both type II TA systems, with a cytoplasmic toxin, and bacteriocins,which have a secreted toxin. I used equations of logarithmic decay to model the necessary expression of the proteins in the cell and their rate of decay after plasmid loss to enable PSK. My results suggest there is likely to be an evolutionary trend toward TA systems with high expression levels of very unstable antitoxins. Secreted barnase was also tested experimentally for its ability to induce PSK similar to bacteriocins, which exhibit a PSK-like phenotype in monoculture by driving maintenance of the immunity encoding plasmid. Barnase did not induce PSK, possibly due to its inability to cause antibiosis in our test system. Structural similarities and biochemical similarities are not sufficient to determine whether a given system will act as a PSK because numerous contextual factors have an effect on whether the genes are addictive. A given set of genes may have the phenotype in one species but not another, under one set of environmental conditions but not another, or on one replicon but not another. This is consistent with the competition hypothesis, which states that genes will be selected for on mobile elements due to their ability to increase horizontal reproductive success, depending on the environmental conditions.

Les systèmes toxine-antitoxine (STA) sont généralement constitués de deux petites protéines cytoplasmiques : une toxine stable et une antitoxine instable capable de neutraliser la toxine et de réprimer l’expression de l’opéron toxine-antitoxine. Une étude menée au laboratoire avait mis en évidence que la perte du gène hicB3 (ypo3369) de Y. pestis, codant une antitoxine solitaire putative, entraine un retard de la croissance bactérienne in vitro et une atténuation de la virulence dans un modèle murin de peste bubonique (Pradel et al., 2014). Par analyse in silico, nous avons détecté, en amont de hicB3, un petit gène non annoté candidat pour coder la toxine HicA3. La surproduction de HicA3 provoque la bactériostase chez Escherichia coli et Y. pestis et la production subséquente de HicB3 restaure la croissance. HicA3 et HicB3 constituent donc un STA fonctionnel. Cependant, la perte du STA HicA3B3 n’affecte pas la virulence de Y. pestis dans un modèle murin de peste bubonique. Nous avons ensuite purifié et caractérisé les protéines HicA3 et HicB3. La toxine HicA3 est une ribonucléase monomérique de 66 aa qui comporte un résidu histidine catalytique essentiel pour son activité. L’antitoxine HicB3 a une double fonction : elle interagit avec HicA3 pour la neutraliser et elle réprime le promoteur de l’opéron hicA3B3. Des expériences de retard sur gel et de fusions transcriptionnelles avec un gène rapporteur ont révélé que l’antitoxine HicB3 et le complexe HicA3-HicB3 se fixent sur deux opérateurs chevauchant les boîtes -10 et -35 du promoteur PhicA3. Nous avons également résolu la structure cristalline de l’antitoxine HicB3 et celle du complexe HicA3-HicB3. HicB3 est un tétramère qui comporte deux domaines de fixation à l’ADN du type ruban-hélice-hélice et deux domaines de neutralisation de la toxine. / Toxin-antitoxin systems (TAS) are generally constituted by two small cytoplasmic proteins: a stable toxin and an unstable antitoxin which neutralizes the toxin and represses the expression of the toxin-antitoxin operon. In previous research, our lab found that Yersinia pestis lacking the hicB3 (ypo3369) gene, encoding a putative orphan antitoxin, has a growth defect in vitro and is attenuated for virulence in a murine model of bubonic plague (Pradel et al., 2014). In silico analysis revealed a small gene upstream of hicB3, encoding a putative toxin that we called HicA3. HicA3 overproduction generates bacteriostasis of Escherichia coli and Y. pestis, and the subsequent production of HicB3 restores cell growth. HicA3 and HicB3 thus constitute a functional TAS. However, the lack of the HicA3B3 TAS does not affect Y. pestis virulence in a murine model of bubonic plague. We then purified and characterized the HicA3 and HicB3 proteins. The HicA3 toxin is a monomeric 66-aa ribonuclease with a catalytic histidine residue required for its activity. The HicB3 antitoxin has two functions: it binds and neutralizes HicA3 and it represses the hicA3B3 operon promoter. Gel-shift assays and transcriptional reporter fusion experiments showed that both HicB3 and the HicA3-HicB3 complex bind to two operators overlapping the -10 and -35 boxes of the PhicA3 promoter. We also solved the crystal structures of the HicB3 antitoxin and the HicA3-HicB3 complex. HicB3 is a tetramer with two DNA binding domains of the ribbon-helix-helix type and two toxin neutralization domains.

The aim of this study was to identify and characterise type II toxin-antitoxin (TA) systems in Burkholderia pseudomallei, the causative agent of the human disease melioidosis. 8 putative TA systems were identified within the genome of B. pseudomallei K96243. 5 of these were located witihn genome islands. Of the candidate toxins, BPSL0175 (RelE1) or BPSS1060 (RelE2) caused growth to cease when expressed in Escherichia coli, whereas expression of BPSS0390 (HicA) or BPSS1584 (HipA) (in an E. coli ΔhipBA background) caused a reduction in the number of culturable bacteria. HicA also caused growth arrest in B. pseudomallei K96243 ΔhicAB. These toxin induced phenotypes were enhanced by an <3kDa extracellular factor that accumulated in the spent medium during growth. Expression of the cognate antitoxins could restore growth and culturability of cells. Expression of hicA in E. coli gave an increased number of persister cells in response to ciprofloxacin or ceftazidime. Site directed mutagenesis studies identified two key residues within the HicA toxin that were essential for both the reduced culturability and increased persistence phenotypes. Deletion of hicAB from B. pseudomallei K96243 did not affect persister cell or survival frequencies compared to the wild type following treatment with a variety of stress conditions. Deletion of the ΔhipBA locus from B. pseudomallei K96243 also had no affect on bacterial persistence or survival under the conditions tested.

Thesis (DSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Plasmids belonging to the IncQ family have an exceptionally broad host-range and are highly mobilizable in the presence of the self-transmissible IncP plasmids. All IncQ plasmids identified to date have certain features in common. The feature that distinguishes them most from all other plasmids is that they have a unique mechanism of replication. Their replicons consist of repA, repB and repC genes encoding a replicase, primase and DNA-binding proteins respectively. All IncQ plasmids contain at least three 22-bp iterons (or 20-bp iterons with 2-bp spacers) that are identical in sequence and to which the RepC DNA-binding protein binds. They replicate by means of a unique strand-displacement mechanism that is considered to place a limit on their size. Replication proceeds by a partially single-stranded intermediate that is believed to result in an increased likelihood of structural instability with an increase in plasmid size. The most compact backbone of IncQ plasmids is approximately 5.9-kb and the largest natural IncQ plasmid reported is 14.2-kb. Although the mobilization regions of IncQ plasmids are not as unique as the replicons, they are all characterized by the primase of the replicon being fused to the relaxase of the mobilization genes. The remainder of the mobilization genes may vary substantially in number and sequence between plasmids and have been subdivided into at least four distinct lineages. This dissertation consists of twenty one manuscripts published during the period 1984 to 2012. The focus is almost entirely on the IncQ plasmid subfamily known as IncQ2. Most of the earlier work was on determining the nature and extent of the replicons, mobilization genes and the toxin-antitoxin plasmid stability system. A strong theme in the latter work focussed on using the natural variation among the IncQ2 plasmids as a means to understand IncQ plasmid evolution. The collection of articles comprises a combination of original research and reviews. / AFRIKAANSE OPSOMMING: Plasmiede wat aan die IncQ familie behoort kom ‘n uitsonderlike wye gasheerselreeks voor en is hoogs mobiliseerbaar deur middel van die selfoordraagbaar IncP plasmiede. Alle IncQ plasmiedes wat tot datum identifiseer is het sekere gemeenskaplike eienskappe. Die eienskap wat hulle van alle ander plasmiedes onderskei is hul unieke dupliseringsmeganisme. Hul dupliseringsmeganisme bestaan uit repA, repB en repC gene wat onderskeidlik ‘n helikase, ‘n ‘primase’ en ‘n DNSbindingsproteïen enkodeer. Die IncQ plasmiede het ten minste drie 22-bp iterone (of 20-bp iterone met 2-bp skeidingsnukleotiede) met ‘n identiese nukleotiedvolgorde en waaraan die RepCbindingsproteïen bind. Hulle dupliseer deur middel van ‘n unieke DNA-string-vervangingsmeganisme wat ‘n beperking op hul grootte plaas. Tydens replikasie word ‘n intermediêre struktuur gevorm wat gedeeltelik enkelstring is en dit is blykbaar die rede vir ‘n verhoging in strukturële onstabilitiet as die plasmied groter word. Die kleinste ruggraat onder die IncQ plasmiede is min of meer 5.9-kb en die grootste natuurlike IncQ plasmied wat gerapporteer is, is 14.2-kb. Alhoewel die mobiliseringsgebied van die IncQ plasmiede nie so duidelik uitkenbaar as die replikons nie, hierdie gebied is gekenmerk deur ‘n ‘primase’ wat aan ‘n ‘relaxase’ in die mobiliseringsgene gekoppel is. Die oorblywende mobiliseringsgene verskil in beide getal en nukleotiedvolgorde tussen plasmiede en is gebruik om die plasmiede in vier duidelike oorsponggroepe in te deel. Hierdie proefskrif bestaan uit een-en-twintig artikels wat tussen 1984 en 2012 gepubliseer is. Die fokus is hoofsaaklik op die IncQ plasmiedsubfamilie wat as IncQ2 bekend is. Baie van die vroeër werk het oor die aard en omvang van die duplisering en mobiliseringsgene asook die toksienteentoksien plasmiedstabiliseringsmeganisme hanteer. ‘n Sterk tema in die latere werk was om die natuurlike variasie onder die IncQ2 plasmiede te bestudeer ten einde IncQ plasmiedevolusie te verstaan. Die publikasie versameling bestaan uit ‘n kombinasie van oorspronklike navorsing en oorsigartikels.

Toksino-antitoksino (TA) sistemos – tai poros viename operone esančių bakterijų ir archėjų genų, kurių vienas koduoja toksišką baltymą, o antras – jį neutralizuojantį baltymą-antitoksiną. Tol, kol ląstelėje gaminamas pakankamas abiejų baltymų kiekis, antitoksinas jungiasi su toksinu ir jį išaktyvina. Tačiau, esant nepalankioms aplinkos sąlygoms, labilesnis antitoksinas suardomas aktyvintų proteazių, o likęs laisvas stabilesnis toksinas slopina gyvybiškai svarbius ląstelinius procesus – baltymų arba DNR biosintezę, dėl ko stabdomas ląstelių augimas arba jos žūva. Escherichia coli chromosomoje aprašyta daugiau nei dešimt TA sistemų, kurių viena yra dinJ-yafQ, apie kurią žinoma labai nedaug. Anksčiau laboratorijoje atliktuose darbuose nustatyta, kad dinJ-yafQ koduoja transliaciją slopinantį toksiną YafQ, o DinJ ir YafQ baltymai sudaro stiprų baltymų kompleksą, slopinantį YafQ toksišką poveikį. Kol kas nieko nėra žinoma apie YafQ molekulės sritis, svarbias sąveikai su antitoksinu DinJ. Šiame darbe sekai atrankios mutagenezės metodu buvo tirtos YafQ baltymo sritys, svarbios sąveikai su „savuoju“ toksinu DinJ. TA sistemoms būdinga savo operono transkripcijos autoreguliacija. DNR sulėtinimo gelyje eksperimentais parodėme atrankią DNR ir antitoksino DinJ bei DinJ-YafQ baltymų komplekso sąveiką. Laisvas antitoksinas DinJ silpniau sąveikauja su DNR nei būdamas komplekse su YafQ, o sąveikai su DNR svarbi DinJ baltymo N galinė dalis. Iš dviejų dinJ-yafQ operono promotoriaus srityje... [toliau žr. visą tekstą] / Prokaryotic toxin antitoxin systems consist of two adjacent genes, where one encodes a stable toxin harmful to essential cellular processes (translation or DNA synthesis), and the other a labile antitoxin, capable of blocking the toxin's activity by binding into stable protein complex. TA systems are proposed to be involved in bacterial adaptation to stress conditions by modulating the level of essential biological processes. There are at least ten characterized chromosome-encoded TA loci in Escherichia coli. The dinJ-yafQ operon codes for YafQ toxin which is neutralized by its cognate antitoxin, DinJ. YafQ is known to inhibit translation in vivo and belongs to the RelE toxin family of toxin ribonucleases. By using site-specific mutagenesis of YafQ, we have investigated the protein regions important for its interaction with DinJ antitoxin. Transcriptional autoregulation has been reported for members of all known TA gene families and appears to be general characteristic of regulation of TA loci. In this work electrophoretic mobility shift assay was used to investigate the interaction between the antitoxin DinJ and DinJ-YafQ complex and dinJ-yafQ operon promoter DNA. Antitoxin DinJ in the complex with YafQ had an enhanced DNA-binding affinity compared to free DinJ. N-terminal domain of antitoxin is crucial for interaction with DNA. Bioinformatic analysis of dinJ-yafQ operon promoter region revealed several palindromic DNA islands and their importance for interaction with DinJ... [to full text]

<p>The <em>Sinorhizobium meliloti</em> genome consists of 6204 predicted protein-coding regions of which approximately 2000 are proteins of unknown function (PUFs). To identify functions of <em>S. meliloti</em> PUFs, we employed the FRT/Flp recombination system to delete large gene clusters and then screened for phenotypes. Large-scale deletions have been mainly used to define minimal gene sets that contain only those genes that are essential and sufficient to sustain a functioning cell. To adapt FRT/Flp for use in <em>S. meliloti</em>, we used an already constructed pTH1522-derived integration gene library of the <em>S. meliloti</em> genome (pTH1522 carries a single FRT site). A second FRT site was inserted at defined locations in the genome through integration of a second plasmid (pTH1937) that also carries a single FRT site. Here we outline how this Flp/FRT system was used to delete defined regions and hence generate multiple gene knock-out mutants. This system was used to delete 32 and 56 defined regions from the 1340 Kb pSymA and 1678 Kb pSymB megaplasmid, respectively. The structures of the resulting megaplasmid deletion mutants were confirmed by PCR analysis. Carbohydrate and nitrogen utilization phenotypes were associated with the deletion of specific regions. Deleting large, regions of the genome helped us to identify phenotypes such as inability to grow on minimal media with fucose, maltotriose, maltitol, trehalose, palatinose, lactulose and galactosamine as sole carbon source. For several FRT-flanked regions, few or no recombinants were recovered which suggested the presence of essential genes. Through this strategy, two essential genes <em>tRNA^arg </em>and<em> engA</em> located on the pSymB and three toxin/antitoxin-like systems, <em>sma0471</em>/<em>sma0473</em>, <em>sma2105</em> and <em>sma2230</em>/<em>sma2231</em> on pSymA megaplasmid were identified.</p> / Doctor of Philosophy (PhD)

Bacillus subtilis is a gram positive soil bacterium that is surrounded by many other microorganisms its environment. That is why it is necessary for the bacterium to be able to fight with these microorganisms for the nutrients and living space. B. subtilis contains the modules in its genetic make-up that improve its ability to compete. These modules are called the toxin-antitoxin systems. This Diploma Thesis is trying to identify yet undescribed extracellular toxins produced by the wild type BSB1 strain of B. subtilis. The related microorganism Bacillus megaterium was used as a competing bacterium. The contact-dependent or independent manner of killing the competing bacterium was demonstrated using this model. By deletion analysis and comparisons of the genomes of the various strains of B. subtilis, the SPβ prophage was first identified as a region containing an unknown toxin(s). Analysis of the extracellular proteome of B. subtilis subsequently revealed an unknown toxin (or toxin complex, respectively) of the molecular weight exceeding 100 kDa. Even more fascinating was the finding that such a large protein molecule is resistant to the pancreatic protease, trypsin. Subsequent non-enzymatic cyanogen bromide cleavage of the extracellular proteins and their analysis by mass spectrometry revealed...

Staphylococcus aureus (S. aureus) est un pathogène humain majeur dont l’impact sur la santé publique est majoré par les phénomènes de résistance et de tolérance aux antibiotiques. Au cours de cette thèse, nous avons identifié deux nouveaux systèmes toxine-antitoxine (STA) de type I appartenant au core génome et exprimés par S. aureus nommés sprG2/SprF2, sprG3/SprF3. Ces nouveaux STA sont homologues au STA sprG1/SprF1 localisé dans un îlot de pathogénie. Nous avons montré que des interactions croisées influençant le niveau des ARN sprG et SprF pouvaient avoir lieu entre les STA homologues sprG/SprF, mais que celles-ci n’avaient pas d’impact sur la neutralisation spécifique de chaque toxine SprG par son antitoxine SprF. Nous avons démontré que les peptides encodés par sprG2 et sprG3 sont bactériostatiques, contrairement aux peptides encodés par sprG1 qui sont bactéricides. Nous avons démontré que l’expression des ARN sprG et SprF pouvait varier en réponse à des stress environnementaux comme un stress hyperosmotique ou un stress oxydatif. Pour le STA sprG1/SprF1, nous avons démontré que l’antitoxine SprF1 pourrait participer à l’entrée en persistance de S. aureus, en atténuant la traduction globale via son association aux ribosomes. Ainsi, SprF1 est le premier exemple d’une antitoxine ARN avec une double fonction de neutralisation de la toxine sprG1 via son extrémité 3’ et de fixation aux ribosomes pour atténuer la traduction de S. aureus via son extrémité 5’. Ensemble, ces travaux de thèse suggèrent que les STA sprG/SprF seraient impliqués dans l’adaptation de S. aureus au stress antibiotique ou dans l’échappement au système immunitaire. / Staphylococcus aureus (S. aureus) is a human pathogen that causes nosocomial and community-associated infections. The antibiotic resistance and tolerance of S. aureus increase its impact on public health. During my PhD thesis, we identified two novel type I toxin-antitoxin systems (TAS) located in the core genome and expressed in S. aureus named sprG2/SprF2 and sprG3/SprF3. These TAS are homologues of the sprG1/SprF1 TAS, encoded in a pathogenicity island. We showed that cross-interactions affecting sprG and SprF RNA level can occur between sprG/SprF homologous TAS, but without any impact on the specific neutralization of a sprG toxin by its SprF antitoxin. We demonstrated that overexpression of sprG2- and sprG3-encoded peptide induce bacteriostasis, as opposed to the sprG1-encoded peptides that induced S. aureus death. We showed that sprG and SprF RNA levels can be modulated by environmental triggers such as hyperosmotic and oxidative stresses. Concerning sprG1/SprF1, we demonstrated in S. aureus strain N315 that the SprF1 antitoxin could be involved in persister cells formation, by a translation attenuation mechanism via its association with the ribosomes. SprF1 is the first example of an untranslated RNA antitoxin with a dual function that neutralize sprG1 toxin and that bind to ribosome to attenuate S. aureus translation. Altogether, these thesis experiments suggest an involvement of the sprG/SprF TAS in S. aureus adaptation to antibiotic stress or in the escape of the immune system.

Les systèmes toxine-antitoxine (TA) sont de petits modules génétiques largement présents dans les génomes bactériens. Ils codent pour une petite protéine toxique et une antitoxine. Ils sont classés en six types en fonction de la nature et du mode d'action de l'antitoxine. Ce travail a porté sur l'étude du type I, pour lequel l'antitoxine est un ARN antisens qui cible l'ARNm de la toxine afin de réprimer son expression. Au cours de cette thèse, nous avons étudié le système aapA3/IsoA3, codé sur le chromosome du pathogène gastrique humain Helicobacter pylori. À ce jour, la plupart des systèmes TA ont été étudiés à l'aide de systèmes d'expression artificiels, qui ne permettent pas de caractériser la régulation transcriptionnelle ou post-transcriptionnelle. En utilisant la létalité induite par l’expression chromosomique de la toxine obtenue en absence d’antitoxine, nous avons développé une sélection génétique de mutants suppresseurs révélés par séquençage haut-débit. Cette approche, appelée FASTBAC-Seq, nous a permis de cartographier une myriade de déterminants de toxicité localisés dans les régions codantes et non codantes du gène de la toxine AapA3. En particulier, certaines de ces mutations ont révélé l'existence de tige-boucles ARN transitoires qui agissent de manière co-transcriptionnelle pour empêcher l'initiation de la traduction pendant la synthèse de l'ARNm codant pour la toxine. Ces structures ARN métastables fonctionnelles sont nécessaires pour découpler les processus de transcription et de traduction et permettent la présence de ces gènes toxiques sur le chromosome bactérien. Bien que les ARNm non traduits deviennent rapidement instables, nos travaux ont également révélé l'existence de deux tige-boucles protectrices situées aux deux extrémités de l'ARNm. Ces structures secondaires empêchent des activités exonucléolytiques agissant en 5' et 3'. Dans l’ensemble, notre travail met en évidence les conséquences de la forte pression de sélection pour limiter l'expression des toxines sous laquelle évoluent les systèmes TA. Cela nous a permis de mieux comprendre l’influence du repliement secondaire des ARNm, non seulement lors de la régulation posttranscriptionnelle, mais aussi co-transcriptionnelle de l’expression de cette famille particulière de gènes. Ces caractéristiques de régulation basées sur l'ARN peuvent être exploitées à l'avenir pour des applications biotechnologiques (p. ex., production accrue de protéines par stabilisation d'ARNm) ou biomédicales (p.ex., développement de stratégies antimicrobiennes alternatives pour l'activation de la synthèse de toxines). / Toxin-antitoxin (TA) systems are small genetic modules widely present in bacterial genomes. They usually code for a small toxic protein and its cognate antitoxin and can be classified into six types depending on the nature and mode of action of the antitoxin. This work focuses on the study of type I, for which the antitoxin is an antisense RNA that targets the toxin mRNA to inhibit its expression. We characterized the aapA3/IsoA3 system, encoded on the chromosome of the human gastric pathogen Helicobacter pylori. To date, most TAs have been studied using artificial expression systems, which do not allow the characterization of transcriptional or post-transcriptional regulation. Taking advantage of the lethality induced by the toxin chromosomal expression in the absence of antitoxin, we developed a high-throughput genetic selection of suppressor mutations revealed by Next-Generation Sequencing. This approach, named FASTBAC-Seq, allowed us to map a myriad of toxicity determinants located in both, coding and noncoding regions, of the aapA3 toxic gene. More precisely, some suppressor mutations revealed the existence of transient RNA hairpins that act co-transcriptionally to prevent translation initiation while the toxinencoding mRNA is being made. Such functional RNA metastable structures are essential to uncouple the transcription and translation processes and allow the presence of these toxic genes on bacterial chromosomes. Although untranslated mRNAs become rapidly unstable, our work also revealed the presence of two protective stem-loops located at both mRNA ends that prevent from both, 5’ and 3’ exonucleolytic activity. Altogether, our work evidenced the consequences of the strong selection pressure to silence toxin expression under which the TAs evolve, and highlighted the key role of mRNA folding in the co- and post-transcriptional regulation of this family of genes. These RNA-based regulatory mechanisms may be exploited in the future for biotechnological (e.g., increased protein production through mRNA stabilization) or biomedical (e.g., development of alternative antimicrobial strategies aiming at the activation of toxin synthesis) applications.