This thesis attempts to broaden what is known about bacterial symbionts within amoebae by the use of a number of different molecular methods. Initially a number of different amoeba strains were screened for bacterial symbionts by 16S rRNA gene PCR, then the symbionts were identified by comparative sequence analysis and phylogenetic analysis. The amoeba strains containing bacterial symbionts were characterised by cell morphology, 18S rRNA gene sequencing, internal transcribed spacer sequencing and allozyme electrophoresis. Amoebae belonging to the genera Acanthamoeba, Naegleria, Ripidomyxa and Saccamoeba were identified as containing symbionts that belonged to a wide range of different bacterial genera. / Relationships between bacterial symbionts and their host amoebae were analysed by the use of transmission electron microscopy and fluorescent in situ hybridisation using symbiont specific probes. Also described are attempts that were made to isolate and grow the bacterial symbionts outside of their host amoebae as well as experiments to try to transfer bacterial symbionts from one amoeba strain to another. Lastly the results from this study are discussed as a whole to put into perspective how they contribute to the body of knowledge of symbionts within protozoa. / Thesis (PhDAppliedScience)--University of South Australia, 2006.

Clostridium scatologenes is an anaerobic bacterium that demonstrates some unusual metabolic traits such as the production of 3-methyl indole. The availability of genome level sequencing has lent itself to the exploration and elucidation of unique metabolic pathways in other organisms such as Clostridium botulinum. The Clostridium scatologenes genome, with an estimated length 4.2 million bp, was sequenced by the Applied Biosystems Solid method and the Roche 454 pyrosequencing method. The resulting DNA sequences were combined and assembled into 8267 contigs with an average length of 1250 bp with the Newbler Assembler program. Comparision of published subunits of csd gene and assembled contigs identified that one contig contained all three subunits. In addition a gene with similarity to clostridium carboxidivorans butyrate kinase was found lined next to csd gene. An alignment of the contig and csdgene sequences identified three deletions in the contig within the 4066 bases of the alignment. This implies that there is about 0.07% error rate in the sequencing itself requiring more finishing. Even without finishing the genome assembly into single contig, contigs were annotated in RAST pipeline predicting 2521 protein encoding genes (PEGs). The PEGs were classified by their metabolic function and compared to classified PEGs found in the closely related clostridium species, Clostridium carboxidivorans and Clostridium. ljungdahlii, which have similarly sized genomes. According to the RAST analysis, Clostridium scatologenes had 35% subsystem coverage of all known metabolic processes with its 2521 PEGs. This compares to 41% for Clostridium carboxidivorans with 4174 PEGs (29) and 42% for Clostridium ljungdahlii with 4184 PEGs (30), indicating that Clostridium scatologenesmay still have more genes to be identified. Comparison of the percent genes found in the metabolic subsystems was similar except in motility and chemotaxis. The contigs, on which the csd gene and tryptophan metabolizing genes lay, were examined to see if additional genes might support these metabolic pathways. Butyrate kinase was associated with the csd genes but no other associations were found for the two tryptophan metabolizing genes. The tryptophan biosynthesis operon genes were all found on one contig (contig 6771) and were syntenic with other bacterial species.

Thesis (M.S.)--University of Texas at Dallas, 2008. / Includes vita. Includes bibliographical references (leaves 39-43)

Bacterial species and communities play foundational roles in human health and therapeutics, in vital ecological and environmental processes, and in industrial applications for the biosynthesis of valuable compounds and materials. However, existing genetic engineering methods and technologies available for bacterial functional genetics or large-scale genomic integration are inefficient, unable to translate between different target species, or lacking precise targeting or reprogramming capabilities. In this work, we describe a novel class of CRISPR- associated transposons (CRISPR-Tn) that facilitate programmable RNA-guided DNA insertions. In particular, the Tn6677 CRISPR-Tn system from Vibrio cholerae comprises a Tn7-like transposase machinery that has co-opted a nuclease-deficient Type I-F3 CRISPR-Cas system to guide its target selection. We show that, similar to canonical CRISPR-Cas systems, this CRISPR- Tn system can be easily programmed using the CRISPR RNA (crRNA) spacer sequence, and directs highly target-specific DNA integration into the Escherichia coli genome. After defining their core biological and mechanistic principles, we developed these CRISPR-Tn systems into a genome engineering platform, which we named INTEGRATE (Insertions of Transposable Elements by Guide RNA-Assisted Targeting). Particularly, optimization of V. cholerae Tn6677 (Vch INTEGRATE, or VchINT) produced a system capable of programmable, broad-bacterial- host, and multiplexed integration of DNA payloads up to 10 kilobases in length, with genomic editing efficiencies reaching 100%. Our single-plasmid expression of system components enabled, for the first time, genome engineering of specific target strains within a complex fecal bacterial community. In addition, we performed extensive deep sequencing within transposition experiments to characterize and examine non-conventional transposition products, including cointegrates formed through replicative transposition, and long-range integration events resulting from on-target DNA binding. Finally, by individually inserting transposon ends into the E. coli genome, we demonstrated successful transposition-mediated mobilization of a genomic fragment 100 kilobases (kb) in length, demonstrating engineering at the genome-scale using VchINT. Altogether, this work highlights the potential of VchINT and other CRISPR-Tn systems as next- generation genome engineering technologies in bacteria and beyond.

Bacteria live under constant assault by bacteriophages and have evolved a diverse array of defense strategies. CRISPR-Cas systems are prokaryotic adaptive immune systems that rely on RNA-guided binding for the recognition and degradation of invading nucleic acids. Intriguingly, some bacteria also encode divergent CRISPR-Cas systems that can bind to — but cannot degrade — target nucleic acids. In this dissertation, I describe the study of nuclease-deficient CRISPR-Cas systems alongside the evolutionary pressures that led to their persistence in bacterial genomes. I present experimental data for the existence of CRISPR-associated transposons (CASTs) that utilize the RNA-guided DNA binding ability of Type I-F CRISPR-Cas systems to direct transposition to new target sites in a heterologous Escherichia coli host. This RNA-guided DNA integration pathway can tolerate large cargos of up to 10 kilo-base pairs in size, and is highly specific for the programmed target site, as determined by deep sequencing experiments. We further reveal the physical link between the CRISPR-Cas and transposition machineries through biochemical experiments and by determining cryo-EM structures of the transposition protein TniQ in complex with the CRISPR-Cas effector. After bioinformatic analyses and experimental validation we established an array of twenty diverse CAST systems for which a subset works completely orthogonally. This dataset revealed the modular nature of CASTs by showcasing the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Further analysis of the transposon-encoded cargo genes uncovered the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Finally, we exploit high-throughput screening assays to determine the specific sequence and spacing requirements of the transposon ends, and use this knowledge to develop a CAST-mediated endogenous gene-tagging approach. Intriguingly, our experiments uncover the involvement of a previously unknown cellular protein, integration host factor (IHF), which is critical for transposition of VchCAST, but not other homologous systems. Collectively, the work presented in this dissertation describes the discovery of RNA-guided DNA integration employed by CASTs, substantially advances our biological understanding of these systems, and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.

Genome evolution is not always shaped by a Darwinian-fashion of vertical inheritance from ancestral lineages. The historical gene content of a species contains many atypical gene sequences showing high similarity to those of distantly related taxa. This evolutionary phenomenon is referred to as lateral gene transfer (LGT). Lateral gene transfer permits the exchange of genetic material across lineages, completely ignoring any concept of taxonomic boundary. The rapid acquisition of foreign genes into bacterial genomes has greatly obscured the historical phylogeny of prokaryotes. In this thesis we calculate the rate of LGT on a Bacillaceae phylogeny, to determine the extent to which it controls species evolution. First, we examined the evolution of the phylogeny according to a simple model of maximum likelihood. We assume equal rates of gene insertion and deletion on the phylogeny and show high rates of evolution in the genomes of B. anthracis, B. cereus, and B. thuringiensis (Bc group), representative of adaptive evolution. We then improved the model to account for differential rates of gene insertion and deletion, thus offering a more realistic model of gene evolution. Again, we demonstrate that members of the Bc group are rapidly evolving, with the rate of gene insertion being significantly higher than the rated of gene deletion. Finally, we evaluate the sole effect of LGT on the phylogeny in a simple birth-death analysis with immigration. We show that LGT is the main vehicle of gene acquisition when the number of gene families substantially increases from external taxa to members of the Bc group. Collectively, our findings suggest that the Bacillaceae genome is rapidly expanding, and that laterally transferred genes may facilitate adaptive evolution and subsistence in a new niche. / Thesis / Master of Science (MSc)

Although increased genome sequencing e orts have increased our understanding of genomic variability within many bacterial species, there has been limited application of this knowledge towards assessing current molecular typing methods and developing novel molecular typing methods. This thesis reports a novel in silico comparative genomic framework where the performance of typing methods is assessed on the basis of the discriminatory power of the method as well as the concordance of the method with a whole-genome phylogeny. Using this framework, we designed a comparative genomic ngerprinting (CGF) assay for Listeria monocytogenes through optimized molecular marker selection. In silico validation and assessment of the CGF assay against two other molecular typing methods for L. monocytogenes (multilocus sequence typing (MLST) and multiple virulence locus sequence typing (MVLST)) revealed that the CGF assay had better performance than these typing methods. Hence, optimized molecular marker selection can be used to produce highly discriminatory assays with high concordance to whole-genome phylogenies. The framework described in this thesis can be used to assess current molecular typing methods against whole-genome phylogenies and design the next generation of high-performance molecular typing methods from whole-genome sequence data. / xiii, 100 leaves : ill. ; 29 cm

Les systèmes toxine-antitoxine sont divisés en trois classes suivant la nature et le mode d’action de l’antitoxine. Ils sont fortement représentés au sein du règne bactérien et se trouvent sur des éléments génétiques mobiles qu’ils stabilisent dans la population bactérienne, mais aussi sur les chromosomes bactériens où leur fonction n’a pas encore été établie avec certitude. Au cours de ce travail, nous avons étudié les systèmes toxine-antitoxine bactériens de classe II, qui sont généralement composés de deux gènes organisés en opéron. Le premier gène code pour une antitoxine qui antagonise l’activité de la toxine, le produit du second gène. L’antitoxine, en complexe avec la toxine, est également capable de réguler l’expression de l’opéron en se fixant au promoteur de l’opéron. Lors du commencement de cette étude, les systèmes toxine-antitoxine étaient divisés en 10 familles sur base des similarités de séquences partagées par les toxines. A chaque famille de toxine était associée une famille d’antitoxine. <p>\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to induce its expression and/or the operon might be carrying out function(s) apart from the utilization of aromatic β-glucosides. The experiments described in this thesis were carried out to test these possibilities. In cultures exposed to prolonged stationary phase, majority of the bacterial population dies and a few mutants that have the ability to scavenge the nutrients released by the dying cell mass survive. Bgl+ mutants were found to be enriched in twenty-eight-day-old Luria Broth grown cultures of E. coli that are wild type for bgl but carry the rpoS819 allele. Out of the five Bgl+ mutants that were isolated, four carried a mutation in the hns locus while one of them, ZK819-97, had an activating mutation linked to the bgl operon. Further analysis of ZK819-97 by DNA sequencing revealed the existence of a single C to T transition at the CAP binding site in the regulatory region. ZK819-97 was chosen for further analysis. Competition assays were carried out in which Bgl+ strain, ZK819-97 (Strr), and the parental Bgl- strain, ZK820 (Nalr), were grown independently for twenty-four hours in Luria Broth and then mixed in 1:1,000 (v/v) ratio reciprocally, without addition of fresh nutrients. ZK819-97, when present in minority, was found to increase in number and take over the parental strain, ZK820, i.e. ZK819-97 showed a Growth Advantage in Stationary Phase phenotype. To determine whether the GASP phenotype of ZK819-97 is associated with the bgl locus, the bgl allele from this strain was transferred by P1 transduction to its parental strain, ZK819. The resulting strain, ZK819-97T (Bgl+, Tetr), when competed with the parental strain, ZK819 Tn5 (Bgl-, Kanr), also showed a GASP phenotype when present in minority in the mixed cultures. To reconfirm this further, the bgl locus was deleted from ZK819-97T. The resulting strain, ZK819-97Δbgl, showed a loss of the GASP phenotype. When the bglB locus was disrupted in ZK819-97T, the resulting strain, ZK819-97ΔB, also failed to show a GASP phenotype, indicating that the phospho-β-glucosidase B activity is essential for this phenotype. The strain, ZK819-IS1, carrying an activating IS1 insertion within the bgl regulatory region also showed a GASP phenotype, confirming that this phenotype of the Bgl+ strain is independent of the nature of the activating mutation. All the above mentioned strains used in the competition assays carry a mutant allele of rpoS, rpoS819. Introduction of the wild type rpoS allele in these strains resulted in the loss of the GASP phenotype of the Bgl+ strain, suggesting that the two mutations work in a concerted manner. The Bgl+ strain was found to show the GASP phenotype only when present in minority of 1:1,000 or 1:10,000 in the mixed cultures and showed a slight disadvantage at higher ratios, indicating that the GASP phenotype of the Bgl+ strain is a frequency dependent phenomenon. In competition assays carried out between 24-hour-old cultures of Bgl+ and Bgl- strains resuspended in five-day-old spent medium prepared from a wild type E. coli strain, Bgl+ strain did not show any extra or early GASP phenotype. In addition, a reporter strain, which has a lacZ transcriptional fusion with the activated bgl promoter, was resuspended in spent medium prepared from a five-day-old culture of wild type strain of E. coli and bgl promoter activity was measured by β-galactosidase assay. The bgl promoter did not show any induction in this medium. These experiments suggest the absence of any β-glucoside like molecules in the spent medium within the sensitivity of these assays. A reporter strain that has a lacZ transcriptional fusion to the wild type bgl promoter was used to measure the expression level of this promoter during exponential and stationary phase of growth in LB. Expression of the wild type as well as various activated promoters of bgl was found to be enhanced in stationary phase. To investigate a possible role of the rpoS encoded stationary phase specific sigma factor, RpoS (σs), and another stationary phase factor, Crl, known to be important for the regulation of many genes of the σs regulon, the bgl promoter activity measurements were carried out in the presence or the absence of RpoS and/or Crl. RpoS along with Crl was found to negatively regulate the expression of wild type as well as activated promoters of bgl, both in exponential and stationary phase. In the absence of the negative regulation by RpoS and Crl, the increase in the bgl promoter activity was more pronounced as compared to that in its presence. rpoS and crl mutations are common in nature and it has been suggested that crl deletion gives a growth advantage to the strain in stationary phase. To test this possibility crl deletion was created in wild type as well as in attenuated rpoS allele background. The strain carrying the crl deletion was found to have a growth advantage in stationary phase over the wild type strain in the presence of wild type rpoS allele, while it shows a slight disadvantage in combination with mutant rpoS. Over expression of LeuO or BglJ is known to activate the bgl operon. To study a possible role of these factors in the regulation of the bgl expression in stationary phase, the bgl promoter activity was measured in strains that were deleted for leuO and/or bglJ, in the absence or presence of crl. These studies indicated that BglJ had a moderate effect on the bgl promoter activity in stationary phase in the absence of Crl but not in its presence. LeuO did not have a significant effect on the bgl promoter activity in either condition. Thus under the conditions tested, the physiological increase in the levels of LeuO and BglJ in stationary phase was insufficient to regulate the bgl expression. Preliminary results show that the bgl operon might be involved in the regulation of oppA, an oligopeptide transporter subunit, in stationary phase. Implications of these findings are discussed. The studies reported in this thesis highlight the involvement of the bgl operon of E. coli in stationary phase. This could be mediated by genetic as well as physiological mechanisms. This study also underscores the importance of observing organisms closer to their natural context and the need to reconsider the concept of ‘cryptic genes’.

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> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished