The genome of Bacillus tequilensis EA-CB0015 sheds light into its epiphytic lifestyle and potential as a biocontrol agent

Different Bacillus species have successfully been used as biopesticides against a broad range of plant pathogens. Among these, Bacillus tequilensis EA-CB0015 has shown to efficiently control Black sigatoka disease in banana plants, presumably by mechanisms of adaptation that involve modifying the phyllosphere environment. Here, we report the complete genome of strain EA-CB0015, its precise taxonomic identity, and determined key genetic features that may contribute to its effective biocontrol of plant pathogens. We found that B. tequilensis EA-CB0015 harbors a singular 4 Mb circular chromosome, with 3,951 protein-coding sequences. Multi-locus sequence analysis (MLSA) and average nucleotide identity (ANI) analysis classified strain EA-CB0015 as B. tequilensis. Encoded within its genome are biosynthetic gene clusters (BGCs) for surfactin, iturin, plipastatin, bacillibactin, bacilysin, subtilosin A, sporulation killing factor, and other natural products that may facilitate inter-microbial warfare. Genes for indole-acetic acid (IAA) synthesis, the use of diverse carbon sources, and a multicellular lifestyle involving motility, biofilm formation, quorum sensing, competence, and sporulation suggest EA-CB0015 is adept at colonizing plant surfaces. Defensive mechanisms to survive invading viral infections and preserve genome integrity include putative type I and type II restriction modification (RM) and toxin/antitoxin (TA) systems. The presence of bacteriophage sequences, genomic islands, transposable elements, virulence factors, and antibiotic resistance genes indicate prior occurrences of genetic exchange. Altogether, the genome of EA-CB0015 supports its function as a biocontrol agent against phytopathogens and suggest it has adapted to thrive within phyllosphere environments

Evolutionary Dynamics of Vibrio cholerae O1 following a Single-Source Introduction to Haiti

ABSTRACT Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics

mBio

Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics.|Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain

Virulence of Salmonella typhimurium 1,4,[5],12:i:- : the new emergent strain

Tese de Doutoramento em Ciências Veterinárias na Especialidade Sanidade AnimalSalmonella serovar 1,4,[5],12:i:- is presently considered one of the major serovars responsible for human salmonellosis worldwide. A multidisciplinary approach, including the fields of epidemiology, spatial statistics, clinical and applied microbiology was used to perform an extensive characterization of Salmonella 1,4,[5],12:i:- isolates obtained by the National Health Institute Dr. Ricardo Jorge, which was lacking due to the recent emergence. It was observed that cases are reported in most districts, being more frequent in the Portuguese coastland. Spatial statistical analysis showed a significant geographic clustering, pointing out for the importance of evaluating these areas to identify risk factors, in order to establish adequate prevention programs. The most relevant antimicrobial profile in this serovar is the tetra-resistance pattern (R-type ASSuT), displaying resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. A high occurrence of R-type ASSuT isolates was observed in the isolates under study, with the majority harboring the resistance genes frequently associated with the European clone, namely blaTEM, sul2, straA-straB, tetB. Additionally, resistance to quinolones and 3rd generation cephalosporin was also detected. In Portugal, the rapid spread of Salmonella 1,4,[5],12:i:- R-type ASSuT might be related with the diversity of pulsotypes and also the presence of a core of virulence factors, including biofilm production. Biofilm-forming ability varied between sample locations and collection year, and can be one of the virulence features related with the rise of this serovar. Furthermore, biofilm formation was evaluated in vitro using a simulated human intestinal environment. In such conditions was observed an impairment of biofilm production, revealing that conditions mimicking the human intestinal tract can influence the biofilm-forming ability of the isolates under study. This research highlight the critical importance of close surveillance of Salmonella 1,4,[5],12:i:- in Portugal, including R-type ASSuT isolates. Information gathered may unravel Salmonella 1,4,[5],12:i:- features, prevent the dissemination to other regions and also benefit the medical community in order to rationalize salmonellosis antimicrobial therapeutics.RESUMO - Virulência de Salmonella Typhimurium 1,4,[5],12:i:-, a nova estirpe pandémica* - Salmonella é uma bactéria Gram-negativa pertencente à família Enterobacteriaceae, sendo uma das principais responsáveis pela morbilidade e mortalidade associadas a toxinfecções alimentares. Pode manifestar-se num espectro de sintomatologia variado, incluindo a gastroenterite, a bacteriémia e a infecção focal. Este género incluí mais de 2600 serovares descritos, distribuídos por apenas duas espécies: Salmonella enterica que inclui todos os serovares patogénicos para os humanos e Salmonella bongori. Actualmente, um dos principais serovares responsáveis pela salmonelose humana em todo o mundo é o 1,4,[5],12:i:-. Este serovar é uma variante monofásica de Salmonella Typhimurium, muito semelhante a nível molecular, sendo caracterizado pela ausência da expressão do gene fljB. Devido à sua recente emergência, estudos que avaliem este serovar são escassos, particularmente em Portugal, o que definiu o âmbito desta investigação, que teve como objectivo a caracterização epidemiológica e microbiológica, tanto do ponto de vista fenotípico e genotípico, de isolados de Salmonella 1,4,[5],12:i:- obtidos em Portugal a partir de diferentes origens, incluindo amostras humanas, animais e ambientais. Numa primeira fase foi realizada uma caracterização demográfica, epidemiológica e espacial de todos os casos de Salmonelose 1,4,[5],12:i:- humana notificados em Portugal pelo Instituto Nacional de Saúde Dr. Ricardo Jorge (INSA), durante um período de 10 anos, desde 2001 a 2011. Foram recolhidos dados sobre a origem, ano e mês de amostragem, género, idade, distrito e município de residência dos pacientes. Foi realizada a análise estatística descritiva, bem como, a análise estatística espacial através do software SaTScan™, combinada com análise através de software de georeferenciação, o QGIS™, de forma a caracterizar a epidemiologia e identificar agrupamentos espaciais de risco superior de infecção por Salmonella 1,4,[5],12:i:- em Portugal. Globalmente, observou-se que em Portugal, a maioria dos distritos tem casos notificados de infecção por Salmonella 1,4,[5],12:i:-. Verificou-se também um aumento da incidência durante o intervalo de 2004 a 2011, com um maior número de casos na região litoral do país, incluindo distritos como Porto, Lisboa e Aveiro, o que pode ser explicado pela maior densidade populacional nestas áreas. A maioria das infecções ocorreu durante Maio e Outubro, e o menor número em Fevereiro, afectando principalmente indivíduos jovens.[...]*O autor escreve segundo o antigo Acordo OrtográficoThis work was supported by National Health Institute Doutor Ricardo Jorge (INSA) and funded by Centre for Interdisciplinary Research in Animal Health (CIISA)N/

Doctor of Philosophy

dissertationThere are many bacteria that associate with insects in a mutualistic manner and offer their hosts distinct fitness advantages, and thus have likely played an important role in shaping the ecology and evolution of insects. Therefore, there is much interest in understanding how these relationships are initiated and maintained and the molecular mechanisms involved in this process, as well as interest in developing symbionts as platforms for paratransgenesis to combat disease transmission by insect hosts. However, this research has been hampered by having only a limited number of systems to work with, due to the difficulties in isolating and modifying bacterial symbionts in the lab. In this dissertation, I present my work in developing a recently described insect-bacterial symbiosis, that of the louse fly, Pseudolynchia canariensis, and its bacterial symbiont, Candidatus Arsenophonus arthropodicus, into a new model system with which to investigate the mechanisms and evolution of symbiosis. This included generating and analyzing the complete genome sequence of Ca. A. arthropodicus, which provided some evidence that Ca. A. arthropodicus has become recently associated with insects and may have evolved from an ancestor that was an insect pathogen. Additionally, I describe the development of methods for genetic modification of this bacterial symbiont and for introducing recombinant symbionts into louse fly hosts, as well as a new microinjection technique that enables the complete replacement of native symbionts with recombinant symbionts. With the generation of the symbiont genome sequence along with strategies for engineering recombinant symbionts and establishing them in an insect host, this work provides an interesting new system with which to investigate the function of specific genes in symbiosis as well as a promising new avenue of research involving paratransgenesis

SYSTEMATIC INVESTIGATION OF QUORUM SENSING IN Escherichia coli

High throughput techniques and advanced mathematical tools have enabled systematic investigations of biological systems with unparalleled precision. Not only molecular interactions between components but mechanisms and the dynamic behaviors associated with these systems are revealed, suggesting that comprehensive systems biology can be realized in the near future. Quorum sensing, especially the auto-inducer2 (AI-2) system, has been extensively studied due to its commonality among bacteria and connections to pathogenic phenotypes. In this study, the E. coli quorum sensing AI-2 system was studied combing system-based mathematical modeling and high throughput genomic profiling. First, a Stochastic Petri Network (SPN) model was constructed based on available regulatory information. Simulations together with experimental data demonstrated that the apparent stimulation of AI-2 in the presence of glucose is not from the increased transcriptional or translational expression of AI-2 synthases luxS and pfs, nor from the increased metabolic flux associated with LuxS-related pathways but from an alternative AI-2 synthesis pathway. The conversion of adenosine with cellular extracts from both luxS and pfs mutants validated our prediction about the existence of an alternative non-LuxS related AI-2 synthesis pathway. Second, AI-2 uptake regulatory network was investigated in detail: lsrR-lacZ, lsrK-lacZ fusion reporters were constructed and the analysis found that lsrR is subject to its own repression and is induced by both lsrK and luxS. Further transcriptome analysis demonstrated that lsrR and lsrK, together with quorum signal AI-2, coregulate lsrRK regulon, which influences phenotypes (biofilm, small RNAs). Importantly, this regulation is in a distinctly different manner than that mediating the lsr operon. We hypothesize that lsrR acts together with AI-2 to mediate cellular processes and that the phosphorylation of AI-2 molecule through lsrK triggers different response pathways. These investigations demonstrated that lsrR, lsrK are indispensable for AI-2 uptake. These newly elucidated regulatory mechanisms and associations undoubtedly broaden the scope of the AI-2 quorum sensing system, and provide a solid foundation for further mathematical modeling of the dynamics and system behaviors in E. coli . Finally, a tight coupling of experimental manipulation with mathematical analysis, as demonstrated in this study, provides a good example for systematically investigating biological systems

Local Diversification of Methicillin- Resistant Staphylococcus aureus ST239 in South America After Its Rapid Worldwide Dissemination

The global spread of specific clones of methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem, and understanding the dynamics of geographical spread requires worldwide surveillance. Over the past 20 years, the ST239 lineage of MRSA has been recognized as an emerging clone across the globe, with detailed studies focusing on isolates from Europe and Asia. Less is known about this lineage in South America, and, particularly, Brazil where it was the predominant lineage of MRSA in the early 1990s to 2000s. To gain a better understanding about the introduction and spread of ST239 MRSA in Brazil we undertook a comparative phylogenomic analysis of ST239 genomes, adding seven completed, closed Brazilian genomes. Brazilian ST239 isolates grouped in a subtree with those from South American, and Western, romance-language-speaking, European countries, here designated the South American clade. After an initial worldwide radiation in the 1960s and 1970s, we estimate that ST239 began to spread in South America and Brazil in approximately 1988. This clone demonstrates specific genomic changes that are suggestive of local divergence and adaptational change including agrC single-nucleotide polymorphisms variants, and a distinct pattern of virulence-associated genes (mainly the presence of the chp and the absence of sea and sasX). A survey of a geographically and chronologically diverse set of 100 Brazilian ST239 isolates identified this virulence genotype as the predominant pattern in Brazil, and uncovered an unexpectedly high prevalence of agr-dysfunction (30%). ST239 isolates from Brazil also appear to have undergone transposon (IS256) insertions in or near global regulatory genes (agr and mgr) that likely led to rapid reprogramming of bacterial traits. In general, the overall pattern observed in phylogenomic analyses of ST239 is of a rapid initial global radiation, with subsequent local spread and adaptation in multiple different geographic locations. Most ST239 isolates harbor the ardA gene, which we show here to have in vivo anti-restriction activity. We hypothesize that this gene may have improved the ability of this lineage to acquire multiple resistance genes and distinct virulence-associated genes in each local context. The allopatric divergence pattern of ST239 also may suggest strong selective pressures for specific traits in different geographical locations

The Complete Genome of \u3cem\u3eTeredinibacter turnerae\u3c/em\u3e T7901: An Intracellular Endosymbiont of Marine Wood-Boring Bivalves (Shipworms)

Here we report the complete genome sequence of Teredinibacter turnerae T7901. T. turnerae is a marine gamma proteobacterium that occurs as an intracellular endosymbiont in the gills of wood-boring marine bivalves of the family Teredinidae (shipworms). This species is the sole cultivated member of an endosymbiotic consortium thought to provide the host with enzymes, including cellulases and nitrogenase, critical for digestion of wood and supplementation of the host\u27s nitrogen-deficient diet. T. turnerae is closely related to the free-living marine polysaccharide degrading bacterium Saccharophagus degradans str. 2–40 and to as yet uncultivated endosymbionts with which it coexists in shipworm cells. Like S. degradans, the T. turnerae genome encodes a large number of enzymes predicted to be involved in complex polysaccharide degradation (\u3e100). However, unlike S. degradans, which degrades a broad spectrum (\u3e10 classes) of complex plant, fungal and algal polysaccharides, T. turnerae primarily encodes enzymes associated with deconstruction of terrestrial woody plant material. Also unlike S. degradans and many other eubacteria, T. turnerae dedicates a large proportion of its genome to genes predicted to function in secondary metabolism. Despite its intracellular niche, the T. turnerae genome lacks many features associated with obligate intracellular existence (e.g. reduced genome size, reduced %G+C, loss of genes of core metabolism) and displays evidence of adaptations common to free-living bacteria (e.g. defense against bacteriophage infection). These results suggest that T. turnerae is likely a facultative intracellular ensosymbiont whose niche presently includes, or recently included, free-living existence. As such, the T. turnerae genome provides insights into the range of genomic adaptations associated with intracellular endosymbiosis as well as enzymatic mechanisms relevant to the recycling of plant materials in marine environments and the production of cellulose-derived biofuels

From Customized Cellular Adhesion to Synthetic Ecology: Characterizing the Cyanobacterium Synechocystis PCC 6803 for Biofuel Production

abstract: ABSTRACT Sustainable global energy production is one of the grand challenges of the 21st century. Next-generation renewable energy sources include using photosynthetic microbes such as cyanobacteria for efficient production of sustainable fuels from sunlight. The cyanobacterium Synechocystis PCC 6803 (Synechocystis) is a genetically tractable model organism for plant-like photosynthesis that is used to develop microbial biofuel technologies. However, outside of photosynthetic processes, relatively little is known about the biology of microbial phototrophs such as Synechocystis, which impairs their development into market-ready technologies. My research objective was to characterize strategic aspects of Synechocystis biology related to its use in biofuel production; specifically, how the cell surface modulates the interactions between Synechocystis cells and the environment. First, I documented extensive biofouling, or unwanted biofilm formation, in a 4,000-liter roof-top photobioreactor (PBR) used to cultivate Synechocystis, and correlated this cell-binding phenotype with changes in nutrient status by developing a bench-scale assay for axenic phototrophic biofilm formation. Second, I created a library of mutants that lack cell surface structures, and used this biofilm assay to show that mutants lacking the structures pili or S-layer have a non-biofouling phenotype. Third, I analyzed the transcriptomes of cultures showing aggregation, another cell-binding phenotype, and demonstrated that the cells were undergoing stringent response, a type of conserved stress response. Finally, I used contaminant Consortia and statistical modeling to test whether Synechocystis mutants lacking cell surface structures could reduce contaminant growth in mixed cultures. In summary, I have identified genetic and environmental means of manipulating Synechocystis strains for customized adhesion phenotypes, for more economical biomass harvesting and non-biofouling methods. Additionally, I developed a modified biofilm assay and demonstrated its utility in closing a key gap in the field of microbiology related to axenic phototrophic biofilm formation assays. Also, I demonstrated that statistical modeling of contaminant Consortia predicts contaminant growth across diverse species. Collectively, these findings serve as the basis for immediately lowering the cost barrier of Synechocystis biofuels via a more economical biomass-dewatering step, and provide new research tools for improving Synechocystis strains and culture ecology management for improved biofuel production.Dissertation/ThesisDoctoral Dissertation Biological Design 201