Bacteria are ubiquitous in all ecosystems and are often challenged by multiple stresses such as extreme temperatures, high salt concentrations, nutrient limitation, pH variations, radiation, predation and the presence of antibiotics/toxins. The most challenging among them is predation pressure which is one of the major causes of their mortality in different niches. Bacteria have evolved different adaptive measures to counter predation. Some of them include change in shape, size, motility, and unpalatable aggregate formation. Aromatic β-glucosides such as salicin, produced by plants as secondary metabolites, play a significant role in protecting them from herbivores. Members of the family Enterobaceriaceae primarily present in soil, e.g. Erwinia chrysanthemi (a phytopathogen) and Klebsiella aerogenes, can utilize the aromatic β-glucosides salicin and arbutin (likely to be present in soil derived from decomposing plant materials) as a carbon source unlike their fellow members such as Escherichia coli, Shigella sonnei, and Salmonella present in the gut environment. Bacteria can obtain energy by metabolizing β-glucosides in the form of glucose. Whether they can also use these molecules as defense tools in a manner similar to plants is an intriguing possibility. In such an event, Bgl+ bacteria could derive a dual advantage in terms of energy generation and protection from predation. The current study was initiated to investigate a possible link between β-glucoside metabolism and self-defense in Enterobacteriaceae. Different members of Enterobacteriaceae comprising of both laboratory strains and natural isolates were considered as prey. Predators included were laboratory strains and soil isolates of bacteriovorous nematodes of the Rhabditidae family, the amoeba Dictyostelium discoidium and a bacteriovorous Streptomyces sp. The predator-prey interaction was analyzed by performing viability and behavioral assays in the context of β-glucoside metabolism Results presented in Chapter 2 show that active catabolism of aromatic β¬glucosides like salicin, arbutin and esculin by Bgl+ bacteria decreases the viability of their predators. The aglycone products released during β-glucosides metabolism, e.g. saligenin in the case of salicin, are the causative agents of the mortality of the predators. The lethality is reversible up to a specific threshold of exposure. Saligenin acts as a chemo-attractant that lures and kills Caenorhabditis elegans N2. In the case of nematodes that succumb, bacteria can derive nutrition from the dead predators indicating a conversion of prey to predator. Experiments with mutant strains of Caenorhabditis elegans suggest that the dopaminergic receptor dop-1 is involved in mediating saligenin toxicity. Studies mentioned in Chapter 3 revolve around the relevance of the predator-prey interaction discussed in Chapter 2 in the natural environment. Members of Enterobacteriaceae and their predator amoebae (cellular slime molds) and nematodes were isolated from soil. They show coexistence in most of the soil samples analyzed. All the predators isolated from soil and other natural isolates of Caenorhabditis succumb to saligenin as their laboratory counterparts with higher sensitivity in some of the strains. Soil nematodes belonging to genera Oscheius and Mesorhabditis avoid saligenin unlike the members of Caenorhabditis genus which are attracted towards saligenin. This indicates that the soil nematodes are often exposed to saligenin or saligenin-like compounds, resulting in the evolution of a genetic machinery to avoid these toxic compounds. Studies with quasi-natural environments like soil and fruit indicate that β-glucoside metabolism have similar effects on predator prey interaction in these environments, reinforcing the relevance of these observations to the natural ecology of the organisms. The studies reported in Chapter 2 and 3 shed light on a novel defense strategy of otherwise non-pathogenic members of Enterobacteriaceae which comes with a dual advantage. These results have also brought into focus issues such as the benefit derived by bacterial populations that are genetically heterogeneous, consisting of both Bgl+ and Bgl-strains. The broad implications and future directions of the work are discussed in Chapter 4. Work presented in Appendix deals with the investigation of the pattern of cellobiose utilization in Shigella sonnei. As mentioned in Chapter 1, it is known that members of Enterobacteriaceae exhibit diversity in their pattern of β-glucoside utilization. Wild type strains of both E. coli and Shigella sonnei are unable to utilize Arbutin, Salicin and Cellobiose. While E. coli can acquire cellobiose utilizing ability directly from the wild type state (Arb-Sal-Cel-), Shigella sonnei strains, though closely related to E. coli, have to undergo a series of mutations in a specific sequence to become capable of utilizing these sugars. Characterization of a few Shigella sonnei Cel+ mutants showed a different mode of activation of the chb operon (known to be involved in cellobiose utilization in E. coli). Considering the ecological significance of the ability to hydrolyze aromatic β-glucosides, a detailed understanding of the metabolic capability of different strains and the molecular mechanism involved becomes significant.

The chb operon constitutes the genes essential for utilization of chitooligosaccharides in Escherichia coli and related species. The six genes of the operon code for a transcriptional regulator (ChbR) of the operon, a permease (ChbBCA), a monodeacetylase (ChbG), and a phospho-beta-glucosidase (ChbF). In the absence of the substrate, the operon is maintained in a transcriptionally repressed state, while presence of the substrate leads to transcriptional activation. Regulation of the chb operon is brought about by the concerted action of three proteins, the negative regulator NagC coded by the nag operon, the dual function regulator ChbR coded by the chb operon and the universal regulatory protein CRP. Mutations that lead to alterations in the regulation of the operon can facilitate utilization of cellobiose, in addition to chitooligosaccharides by E. coli. The studies presented in Chapter II were aimed at understanding the evolution of cellobiose utilization in Shigella sonnei, which is phylogenetically very close to E. coli. Cel+ mutants were isolated from a Cel- wild type S. sonnei strain. Interestingly, Cel+ mutants arose relatively faster on MacConkey cellobiose agar from the S. sonnei wild type strain compared to E. coli. Similar to E. coli, the Cel+ phenotype in S. sonnei mutants was linked to the chb operon. Deletion of the phospho-β-glucosidase gene, chbF also resulted in loss of the Cel+ phenotype, indicating that ChbF is responsible for hydrolysis of cellobiose in these mutants. Previous work from the lab has shown that acquisition of two classes of mutations is necessary and sufficient to give rise to Cel+ mutants in E. coli. The first class of mutations either within the nagC locus or at the NagC binding site within the chb promoter, lead to NagC derepression. The second class consisting of gain-of-function mutations in chbR enable the recognition of cellobiose as an inducer by ChbR and subsequent activation of the operon. However, in S. sonnei a single mutational event of an IS element insertion resulted in acquisition of this phenotype. Depending on the type and location of the insertion, the mutants were grouped as Type I, and Type II. In Type I mutants an 1S600 insertion between the inherent -10 and -35 elements within the chb promoter leads to ChbR-independent constitutive activation of the operon, while in Type II mutants, an IS2/600 insertion at -113/-114, leads to ChbR-dependent, cellobiose-inducible expression of the operon. The results presented also indicate that in addition to relieving NagC mediated repression, the insertion in Type II mutants also leads to increase in basal transcription from the chb promoter. Constitutive expression of the chb operon also results in utilization of the aromatic β-glucosides salicin and arbutin, in addition to cellobiose in Type I mutants, which indicates the promiscuous nature of permease and hydrolysis enzyme of the chb operon. This part of the thesis essentially demonstrates the different trajectories taken for the evolution of new metabolic function under conditions of nutrient stress by two closely related species. It emphasizes the significance of the strain background, namely the diversity of transposable elements in the acquisition of the novel function. The second part of this research investigation, detailed in Chapter III deals with experiments to characterize the eukaryotic orthologs of the last gene of the chb operon. The chbG gene of E. coli codes for a monodeacetylase of chitooligosaccharides like chitobiose and chitotriose. The protein belongs to a highly conserved, but less explored family of proteins called YdjC, whose orthologs are present in many prokaryotes and eukaryotes including mammals. The human YDJC locus located on chromosome 22 is linked to a variety of inflammatory diseases and the transcript levels are relatively high in stem cells and a few cancer cells. In silico analysis suggested that the mammalian YdjC orthologs possess sequence and structural similarity with the prokaryotic counterpart. The full length mouse YdjC ortholog, which is 85% identical to the human ortholog was cloned into a bacterial vector and expressed in a chbG deletion strain of E. coli. The mouse YdjC ortholog could neither promote growth of the strain on chitobiose nor induce transcription from the chb promoter. The purified mouse YdjC ortholog could not deacetylate chitobiose in vitro as well, suggesting that the mouse ortholog failed to complement the function of the E. coli counterpart, ChbG under the conditions tested in this study. In order to characterize the mammalian YdjC orthologs more elaborately, further experimentation was performed in mammalian cell lines. The results indicate that YdjC is expressed in mammalian cell lines of different tissue origin and the expression was seen throughout the cell. Overexpression of mouse Ydjc in a few mammalian cells also resulted in increased proliferation and migration, indicating a direct or indirect role of this protein in cell growth/proliferation. The mammalian orthologs of ChbG therefore appear to have related but distinct activities and substrates compared to the bacterial counterpart that need to be elucidated further.

Beijerinckia sp., bactéria de vida livre fixadora de nitrogênio, comumente encontrada em solos tropicais lateríticos. A cianoficina produzida por cianobactérias é a única reserva nitrogenada intracelular descrita até hoje. O presente trabalho teve como principal objetivo verificar o acúmulo de material nitrogenado intracelular associado à Fixação Biológica de Nitrogênio em cinco isolados de Beijerinckia da rizosfera de cana-de-açúcar (Saccharum sp.). Os resultados mostraram um aumento na concentração de proteína celular total concomitantemente a atividade da nitrogenase durante a fase estacionária de todos os isolados. A fixação de nitrogênio durante esta fase sugere que o destino do nitrogênio fixado seriam os grânulos de armazenamento. A análise química desta reserva confirmou a presença de arginina em teor muito elevado em relação aos demais aminoácidos sugerindo uma reserva nitrogenada diferente da cianoficina. Em recombinantes de Escherichia coli confirmou-se um possível gene envolvido no armazenamento de material nitrogenado em Beijerinckia sp. / Beijerinckia sp. bacteria free-living nitrogen-fixing, commonly found in tropical lateritic soils. The cyanophycin produced by cyanobacteria is the only intracellular nitrogen reserve described to date. This study aimed to verify the intracellular buildup of nitrogen associated with Biological Nitrogen Fixation in five Beijerinckia isolated from the rhizosphere of sugarcane (Saccharum sp.). The results showed an increase in total cellular protein concentration concomitantly nitrogenase activity during the stationary phase of all isolates. The nitrogen fixation during this phase suggests that the fate of fixed nitrogen would be the storage granules. Chemical analysis of the reserve confirmed the presence of very high content of arginine in relation to other amino acids suggesting a different reserve of cyanophycin. In recombinant Escherichia coli confirmed a possible gene involved in nitrogen storage material in Beijerinckia sp.

A diversidade microbiana em ambientes aquáticos e sua associação com plantas carnívoras ainda é pouco estudada. Assim, a comunidade bacteriana da planta carnívora Utricularia gibba e do seu meio aquático foi avaliada por meio do seqüenciamento em larga escala (454 Roche) de uma biblioteca do gene 16S rRNA. Os resultados indicaram que a comunidade bacteriana na água é significativamente diferente da comunidade dos utrículos. Além disso, a comunidade bacteriana da água é composta principalmente por membros dos filos Proteobacteria, Actinobacteria, Firmicutes e Verrucomicrobia, enquanto que a comunidade presente me U. gibba é composta por membros dos filos Proteobacteria, Firmicutes, Cyanobacteria e Acidobacteria. O gênero Polynucleobacter foi dominante nos dois ambientes aquáticos, mas não foi detectado no interior dos utrículos, onde Acidobacterium e Methylococcus foram os gêneros dominantes. Assim, uma comunidade bacteriana específica no interior dos utrículos deve ter sido selecionada a partir do ambiente, podendo esta atuar na degradação das presas. / The microbial diversity of aquatic environments and their association to carnivorous plants is still poor studied. Thus, the bacterial community associated to traps of Utricularia gibba and its aquatic environment was evaluated by large-scale sequencing (454 Roche) of 16S rRNA library from these environments. The results indicated the bacterial community in water is significantly different from the community of utricles. In addition, the bacterial community detected in water environment is mainly composed by Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while in utricules of U. gibba the community is composed by Proteobacteria, Firmicutes, Cyanobacteria and Acidobacteria. The genus Polynucleobacter was dominant in water, but was not detected in association with the plant. Inside the plant, the genus Acidobacterium and Methylococcus were dominant, but were not detected in water samples. Thus, a specific bacterial community within the utricles should have been selected from the environment, and could play a role in prey degradation.

Escherichia coli enteropatogênica atípica (aEPEC) faz parte de um grupo de patógenos capazes de formar um tipo de lesão característica em cultura de tecidos epiteliais, denominada attaching and effacing (A/E). Os genes que são necessários para produção da lesão A/E estão localizados em uma ilha de patogenicidade denominada região LEE (locus of enterocyte effacement). A transcrição de genes da região LEE está sujeita a regulação por vários fatores, entre eles quorum sensing, termo utilizado para designar um mecanismo de regulação gênica dependente da concentração celular. Esse mecanismo é usado por bactérias Gram-positivas e Gram-negativas e em ambos os casos envolve a produção e detecção de moléculas sinalizadoras extracelulares, denominadas autoindutores. Até o momento, pelo menos quatro sistemas de quorum sensing foram descritos, entre eles o sistema de autoindutor AI-3 encontrado em bactérias Gram-positivas e Gram-negativas. Diversos mecanismos celulares, entre eles a expressão de fatores de virulência em amostras de EPEC e EHEC, são regulados por esse fenômeno. O principal objetivo deste estudo foi verificar se existe uma possível regulação por quorum sensing na interação in vitro de uma amostra de E. coli da microbiota intestinal com amostras de aEPEC. Após a confirmação da produção de AI-3 por amostras de E.coli da microbiota intestinal foram realizados ensaios de adesão e quantificação utilizando meio pré-condicionado com esta amostra, epinefrina e bloqueadores que confirmaram que os padrões de adesão de aEPEC obtidos em menor tempo são devidos a presença de AI-3 no meio pré-condicionado, indicando a participação de quorum sensing nessa interação. Além disso, foi observado um fenômeno citotóxico nas células que não é produzido pelo AI-3. / Atypical Enteropathogenic Escherichia coli (aEPEC) are part of a group of pathogens capable of forming a type of lesion characteristic of epithelial tissues in culture, called attaching and effacing (A/E). The genes that are required for production of A/E lesion are located in a pathogenicity island called LEE region (locus of enterocyte effacement). The transcription of LEE genes in the region is subject to regulation by various factors, including quorum sensing, a term used to describe a mechanism of gene regulation dependent on cell concentration. This mechanism is used by Gram-positive and Gram-negative and in both cases involves the production and detection of extracellular signaling molecules, called autoinducers. So far, four systems of quorum sensing have been described, including the system of autoinducers AI-3 found in Gram-positive and Gram-negative bacteria. Several cellular mechanisms, including expression of virulence factors in EPEC and EHEC are regulated by this phenomenon. The main objective of this study was to determine whether there is a possible regulation by quorum sensing in the in vitro interaction of a strains of E. coli of the intestinal microbiota with strains aEPEC. After confirming the production of AI-3 in E. coli of the intestinal microbiota were performed adhesion assays and quantification using means preconditioned with this strains, epinephrine, and blockers who confirmed that patterns of adherence of aEPEC obtained in less time are due to the presence of AI-3 in the preconditioned means, indicating the involvement of quorum sensing in this interaction. Furthermore, we observed a phenomenon that cytotoxic cells is not produced by AI-3.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

Approximately 10% of sequenced bacterial genomes are multipartite, consisting of two or more large chromosome-sized replicons. This genome organization can be found in many plant, animal, and human pathogens and symbionts. However, the advantage of harbouring multiple replicons remains unclear. One species with a multipartite genome is Sinorhizobium meliloti, a model rhizobium that enters into N2-fixing symbioses with various legume crops. In this work, S. meliloti derivatives lacking one or both of the secondary replicons (termed pSymA and pSymB) were constructed. Phenotypic characterization of these strains, including growth rate, metabolic capacity, and competitive fitness, provided some of the first experimental evidence that secondary replicons evolved to provide a niche specific advantage, improving fitness in a newly colonized environment. These results were further supported by characterizing the symbiotic phenotypes of 36 large-scale pSymA and pSymB deletion mutants. To further this analysis, an in silico S. meliloti genome-scale metabolic network reconstruction was developed and flux balance analysis used to examine the contribution of each replicon to fitness in three niches. These simulations were consistent with the hypothesis that metabolic pathways encoded by pSymB improve fitness specifically during growth in the plant-associated rhizosphere. Phylogenetic analysis of a pSymB region containing two essential genes provided a clean example of how a translocation from the primary chromosome to a secondary replicon can render the secondary replicon essential. Moreover, an experimental analysis of genetic redundancy indicated that 10-15% of chromosomal genes are functionally redundant with a pSymA or pSymB encoded gene, providing an alternative method for how secondary replicons can become essential and influence the evolution of the primary chromosome. Finally, the work presented here provides a novel framework for forward genetic analysis of N2-fixing symbiosis and the identification of the minimal N2-fixing symbiotic genome, which will help facilitate the development of synthetic symbioses. / Thesis / Doctor of Philosophy (PhD) / Many bacteria that enter into symbiotic or pathogenic relationships with plants, animals, and humans contain a genome that is divided into multiple chromosome-like molecules. One example is the N2-fixing legume symbiont Sinorhizobium meliloti, whose genome contains three chromosome-sized molecules. Here, the functions associated with each molecule in the S. meliloti genome were examined through a combination of experimental genetic analyses and computer based simulations. Results from these approaches suggested that adaptation to unique environments selected for the evolution of secondary chromosome-like molecules, with each predominately contributing to growth in a specific environment, including environments associated with an eukaryotic host. The genes on these replicons are therefore prime targets for manipulation of bacterium-host interactions, and represent reservoirs of valuable genes for use in synthetic biology applications. Additionally, the genome reduction approach employed in this study laid out a ground work for identification of the minimal N2-fixing symbiotic genome. This represents a crucial step towards successfully engineering improved nitrogen fixation, and the engineering of synthetic N2-fixing symbioses involving non-legumes and/or non-rhizobia.

No / Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting. / European Research Council grant (INTERRUPTB; no. 311725), European Research Council grant (TB-ACCELERATE; no. 638553), Foundation for Innovative New Diagnostics, German Center for Infection Research (DZIF), Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany’s Excellence Strategy (EXC 22167–390884018), FWO Odysseus G0F8316N, US National Institutes of Health BD2K K01 (MRF ES026835), Agence Nationale de la Recherche (ANR-16-CD35-0009)

As Hsp60 acham-se conservadas em todos os organismos, participando da estruturação de proteínas e em processos crônico-degenerativos. Foi avaliada a ação das Hsp65r WT e sua mutante K409A de M. leprae no envelhecimento. Análises do Tempo Médio de Sobrevida (TMS), titulação dos isótipos, ensaios de avidez e análises histopatológicas foram realizadas em camundongos das linhagens HIII e LIII inoculados intraperitonealmente aos 120 ou 270 dias de vida com 2.5µg/mL das Hsp65r. Verificou-se redução no TMS de fêmeas HIII, envelhecidas e adultas, inoculadas com WT. A inoculação da Hsp65r WT em fêmeas LIII envelhecidas resultou no aumento do TMS. A dosagem dos anticorpos não revelou alterações marcantes na produção dos isótipos, e a avidez de IgG foi menor nas fêmeas HIII envelhecidas inoculadas com WT. A análise histopatológica mostrou inflamação nos rins de fêmeas HIII velhas tratadas com Hsp65r WT. Os resultados indicaram a interferência da Hsp65r WT na imunidade durante o processo de senescência de fêmeas envelhecidas constitutivamente boas produtoras de anticorpos. / The Hsp60 are conserved in all organisms, involved in proteins folding and chronic-degenerative processes. We evaluated the action of M. leprae rHsp65 WT and its mutant K409A in aging. Analyses of mean survival time (MST), antibody titration, avidity assays and histopathological examinations were performed in mice from HIII and LIII lines intraperitoneally inoculated at 120 or 270 days of life with 2.5µg/mL of rHsp65. There was a decrease in MST in adult and aged HIII females mice inoculated with WT protein. WT rHsp65 treatment in aged LIII females resulted in the increase of MST. The antibodies titration showed no marked changes in the production of isotypes and IgG avidity was lower in aged HIII females inoculated with WT. Histopathology showed inflammation in the kidneys of old HIII females treated with WT rHsp65. The results indicated the interference of WT rHsp65 in immunity during the senescence of aged females from HIII line constitutively producing good antibodies.

O Fator estimulador de colônias de granulócitos humano recombinante (rhG-CSF) produzido em Escherichia coli é uma proteína não glicosilada com 175 aminoácidos, de grande importância clínica para o tratamento de neutropenias. O presente trabalho propõe a construção de dois sistemas de expressão em E. coli, um sistema para obtenção do rhG-CSF no citoplasma e outro para secreção da proteína recombinante no meio de cultura utilizando a sequência sinal da L-asparaginase II. Os dois sistemas de expressão foram testados e comparados. A partir desses dados, passou-se para as etapas de obtenção do rhG-CSF com o sistema de expressão sem a sequência sinal. As etapas de renaturação e purificação foram eficientes obtendo-se uma proteína com adequado grau de pureza, integridade estrutural e atividade biológica. Essa proteína também foi utilizada com sucesso para a produção de anticorpos policlonais em camundongos. Com os resultados obtidos, a proteína rhG-CSF mostrou-se viável para estudos posteriores em bioreatores e produção em escala-piloto. / The recombinant human granulocyte colony stimulating factor (rhG-CSF) is a non-glycosylated protein with 175 amino acids. This factor plays an important role in hematopoietic cell proliferation and has been widely used for treating neutropenia. The purpose of this work is to construct two expression systems in E. coli; a system for obtaining rhG-CSF in the cytoplasm and the other for secretion of recombinant protein in the culture medium using the signal sequence of L-asparaginase II. The two expression systems were tested and compared. From these data, the next steps for obtaining the rhG-CSF were done with the expression system without the signal sequence. The refolding and purification steps were efficient, resulting in a protein with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of polyclonal antibodies in mice. With these results, the protein rhG-CSF was feasible for further studies in bioreactors and pilot scale production.