Analysis of the genome and proteome composition of Bdellovibrio bacteriovorus: Indication for recent prey-derived horizontal gene transfer

AbstractThe genome/proteome composition of Bdellovibrio bacteriovorus, the predatory microorganism that preys on other Gram-negative bacteria, has been analyzed. The study elucidates that translational selection plays a major role in genome compositional variation with higher intensity compared to other deltaproteobacteria. Other sources of variations having relatively minor contributions are local GC-bias, horizontal gene transfer and strand-specific mutational bias. The study identifies a group of AT-rich genes with distinct codon composition that is presumably acquired by Bdellovibrio recently from Gram-negative prey-bacteria other than deltaproteobacteria. The proteome composition of this species is influenced by various physico-chemical factors, viz, alcoholicity, residue-charge, aromaticity and hydropathy. Cell-wall-surface-anchor-family (CSAPs) and transporter proteins with distinct amino acid composition and specific secondary-structure also contribute notably to proteome compositional variation. CSAPs, which are low molecular-weight, outer-membrane proteins with highly disordered secondary-structure, have preference toward polar-uncharged residues and cysteine that presumably help in prey-predator interaction by providing particular bonds of attachment

Variation in the strength of selected codon usage bias among bacteria

Among bacteria, many species have synonymous codon usage patterns that have been influenced by natural selection for those codons that are translated more accurately and/or efficiently. However, in other species selection appears to have been ineffective. Here, we introduce a population genetics-based model for quantifying the extent to which selection has been effective. The approach is applied to 80 phylogenetically diverse bacterial species for which whole genome sequences are available. The strength of selected codon usage bias, S, is found to vary substantially among species; in 30% of the genomes examined, there was no significant evidence that selection had been effective. Values of S are highly positively correlated with both the number of rRNA operons and the number of tRNA genes. These results are consistent with the hypothesis that species exposed to selection for rapid growth have more rRNA operons, more tRNA genes and more strongly selected codon usage bias. For example, Clostridium perfringens, the species with the highest value of S, can have a generation time as short as 7 min

A conserved gram-positive signalling gene cluster: Phylogeny and function.

All sequenced actinobacteria to date contain a conserved gene cluster found close to the origin of replication of the chromosome. This gene cluster contains a number of genes devoted to both signalling on serine and threonine residues a serine/threonine protein kinase (STPK), two ‘forkhead associated’(FHA) domains, and a ser/thr phosphatase as well as genes devoted to cell wall synthesis. Bioinformatic analysis reveals variations of the cluster conserved in firmicute genomes, implying a conservation of function. The key gene in this cluster is the STPK, as it is a transmembrane protein whose extracellular portion consists of four ‘PASTA’ domains which seem likely to be sensing peptidoglycan monomers associated with cell elongation. Mutation of the genes associated with signalling in Streptomyces coelicolor causes the early onset of secondary metabolism and aerial development, implying a link between the state of the cell wall and the control of metabolic flux. The supplementation of growth media with metabolic intermediates reveals a more direct link between FHA domains and metabolic flux, with excess citrate causing a particular increase in secondary metabolism. Further proteomic analysis confirms the involvement of proteins dedicated to metabolic functions, including the exclusive presence of phosphorylated citrate synthase in the FHA domain mutants. The emerging picture is one of a pleiotropic network of proteins, using serine / threonine phosphorylation as a signalling device, controlling metabolic flux in response to a variety of environmental signals

Genome Mining Actinobacteria: Eliciting the Production of Natural Products

Antimicrobial resistance is an imminent threat that is expected to kill 10 million people per year by 2050. Natural products have been a major source of antimicrobial compounds and encompass a chemical space far greater than synthetic chemistry can provide and have evolved over the ages to have biological activities. The natural products produced by Streptomyces have provided us with two-thirds of the antibiotics currently used, as well as chemotherapeutics, antifungals, and immunosuppressants. In recent years, the drug discovery pipeline from Streptomyces has run dry, largely because laboratory culture conditions lack their natural stimuli, resulting in the rediscovery of the same natural products. However, with the advent of modern genomics, we now realize that the genomes of Streptomyces have the have a greater capacity for natural product production than what we have observed, which providing us with the hope of new drug leads. My doctoral research focused on two aims. First is the concept of eliciting Streptomyces to produce novel natural products; in other words, what triggers the production of natural products. Additional perspectives from ecology, evolution, and regulation evoked the idea that microbe-microbe interactions could be the key. Microscopic observations of the interactions between Streptomyces and yeast suggested that physical contact is essential for elicitation. Genomics, transcriptomics, and proteomics showed that 31% of the silent biosynthetic gene clusters are activated, notably an antifungal polyene cluster, as well as a suite of enzymes capable of digesting the cell wall of yeast. Arguably, Streptomyces can prey on yeast. The differential regulation of a homologous polyene gene clusters further suggested that natural product production is triggered by different ecological needs. Second, genome mining provides insight into the genetic potential of Actinobacteria to produce natural products via the identification of their gene clusters and is a proven method that aids in drug discovery. Here, I sequenced the genome of a rare Streptomonospora isolate and identified the novel persiamycin gene cluster and its associated product. Moreover, genome mining was applied to publicly available Streptomyces genomes, which resulted in the identification of two new gene clusters that produce the antibiotic komodoquinone B. KEYWORDS: Streptomyces, Actinobacteria, secondary metabolism, natural products, genome miningMikrobilääkeresistenssi on välitön uhka, joka uhkaa tappaa vuosittain 10 miljoonaa ihmistä vuoteen 2050 mennessä. Luonnontuotteet ovat olleet merkittävä mikrobilääkkeiden lähde. Lisäksi luonnontuotteet kattavat paljon laajemman kemiallisen alueen kuin synteettinen kemia voi tarjota, ja ne ovat aikojen kuluessa kehittyneet niin, että niillä on biologisia vaikutuksia. Streptomykeetti-organismin tuottamat luonnontuotteet ovat tuottaneet kaksi kolmasosaa käytössä olevista antibiooteista sekä kemoterapeuttisia aineita, sienilääkkeitä, immunosuppressantteja ja matolääkkeitä. Viime vuosina Streptomykeetti-bakteerin lääkkeiden kehittämiskanava on kuitenkin kuivunut. Nykyaikaisen genomiikan myötä olemme kuitenkin nyt ymmärtäneet, että Streptomykeeteillä on potentiaalia tuottaa vielä löytämättömiä luonnontuotteita, jotka antavat meille toivoa uusista lääkkeistä, erityisesti antibiooteista. Väitöstutkimuksellani oli kaksi tavoitetta. Ensimmäinen tarkoitus oli saada Streptomykeetit tuottamaan uusia luonnontuotteita; toisin sanoen tutkia sitä, mikä saa Streptomykeetin tuottamaan luonnontuotteita. Ekologian, evoluution ja säätelyn lisänäkökulmat herättivät ajatuksen, että mikrobien väliset vuorovaikutus voisivat olla avainasemassa. Mikroskooppiset havainnot Streptomykeetin ja hiivan välisestä vuorovaikutuksesta osoittivat, että fyysinen kontakti oli välttämätön yhdisteiden tuoton aktivoinnissa. Genomiikka, transkriptomiikka ja proteomiikka osoittivat, että jopa 31 prosenttia hiljaisista biosynteettisistä geeniryhmistä aktivoitui, samoin kuin joukko entsyymejä, jotka kykenevät pilkkomaan hiivan soluseinää. Tämä osoitti, että Streptomykeetit pystyvät saalistamaan hiivasoluja. Homologisten geeniryhmien erilainen säätely viittaa lisäksi siihen, että luonnontuotteiden tuotanto perustuu erilaisiin ekologisiin tarpeisiin. Toisena tavoitteena oli käyttää genomien louhintaa selvittääksemme Streptomykeetin geneettisestä potentiaalista tuottaa luonnontuotteita. Tässä työssä selvitimme harvinaisen Streptomonaspora bakteerin genomin ja tunnistin uuden persiamysiini-yhdisteen biosynteesireitin. Tämän lisäksi löysimme genomin louhinnan avulla kaksi uutta biosynteettistä geeniryhmää julkisista tietokannoista, joiden osoitimme olevan vastuussa komodokinoni B antibiootin tuotannosta. ASIASANAT: Streptomykeetit, Aktinobakteerit, sekundaarinen aineenvaihdunta, luonnonyhdisteet, genomin louhint

Identification of bacterial pathogenic gene classes subject to diversifying selection

Philosophiae Doctor - PhD (Biotechnology)Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host’s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.South Afric

Induction of antibiotic tolerance in bacteria by self-produced and inter-species signaling

Thesis (Ph.D.)--Boston UniversityThough most bacteria within a population are killed by high concentrations of antibiotics, tolerant bacteria survive and can re-grow once antibiotics are removed. Bacterial persisters are dormant cells within an isogenic bacterial population that are tolerant to antibiotic treatment and have been implicated in chronic and recurrent infections. Tolerant and persistent bacteria are generated heterogeneously within populations, and a complete understanding of the processes by which these cells are formed remains elusive. However, there is increasing evidence that bacterial communication by chemical signaling plays a role in establishing population heterogeneity. Here I show that bacterial communication induces persistence in Escherichia coli using the self-produced signaling molecule indole. Indole-induced persister formation was monitored using microfluidics, and oxidative stress and phage-shock pathways were determined to play a role in this phenomenon. I propose a model in which indole signaling "inoculates" a bacterial sub-population against antibiotics by activating stress responses, leading to persister formation. Having demonstrated that communication using the signaling molecule indole controls persistence in the intestinal bacterium E. coli, I sought to determine whether indole could be used as an interspecies signal to control antibiotic tolerance in mixed microbial communities. The common bacterial pathogen Salmonella typhimurium was chosen for these experiments because this species, though closely related to E. coli, does not produce indole. The results demonstrated that indole signaling by E. coli induces tolerance to antibiotics in S. typhimurium. Further, the data suggest that indole-induced tolerance in S. typhimurium is mediated at least in part by the phage shock and oxidative stress response pathways, which were previously implicated in control of indole-induced persistence in E. coli. I used C. elegans as a simple in vivo model for gastrointestinal infection with S. typhimurium, demonstrating that indole signaling increased Salmonella tolerance and altered heterogeneity of infection in this system. These results suggest that antibiotic tolerance in pathogens may be induced by interception of bacterial signals in the host environment

The Application of Genetic, In Silico and In Vitro Tools to Elucidate the Biology of a Functional Amyloid Fiber.

Curli are thin aggregative fimbriae produced by many Enterobacteriaceae as a structural component of biofilms. Curli share many biochemical and biophysical properties with amyloid fibers which are often associated with human neurological diseases including Alzheimer’s, mad cow, and Parkinson’s. However, curli are the product of a dedicated assembly system that consists of a complex gene regulatory network featuring CsgD; a secretion system including CsgG, CsgE, and CsgF; and the major and minor fiber subunits CsgA and CsgB. As a model system, many aspects of curli formation have been explored including subunit secretion, regulation, biological function, and amyloidogenesis. My work focused on the genetics of curli formation in Escherichia coli, the economic constraints on the evolution of CsgA and other extracellular proteins, and the in vitro amyloidogenesis of CsgA-His. I screened the Keio collection of single gene deletions to discover new genes that affect curli production. More than 300 genes modulate curli production including the sodium antiporter nhaA, a regulator of the glycine cleavage system gcvA, multiple LPS biosynthesis genes, and genes involved in many fundamental cellular processes. This analysis suggests that curli production is part of a highly regulated and complex developmental pathway. The regulation of glyA by CsgD and the curli phenotype of gcvA focused my attention on the amino acid composition of CsgA. CsgA is incredibly rich in glycine and serine. As simple amino acids, both are inexpensive to synthesize. Consequently, CsgA is relatively cheap to produce on a per unit basis. Strikingly, other extracellular proteins including those in Escherichia coli, Pseudomonas syringae, Mycobacterium tuberculosis, Saccharomyces cerevisiae, and other microbes are also inexpensive relative to intracellular proteins. Since extracellular proteins are often lost to the environment, evolution has in turn selected them for increased economy to counteract lost resources. Finally, we studied the in vitro amyloid formation of CsgA-His. Like disease-associated amyloids, CsgA-His bound Thioflavin T upon polymerizing into fibers, reacted with an amyloid specific antibody, self seeded, and displayed other aspects of amyloid formation. Collectively, this work sheds new light on the biology of the functional amyloid fiber curli and hopefully will beget novel directions of inquiry.Ph.D.Molecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/78834/1/dnlsmith_1.pd

Chromosome Architecture and Evolution in Bacteria

Inferences of organismal molecular evolution have been dominated by comparisons of their constituent genes. Yet the evolutionary histories of genes within Bacterial genomes are not necessarily congruent. Here, Horizontal Gene Transfer (HGT) of sequences across species boundaries can confound these analyses. There does appear to be phylogenetic cohesion, where members of higher taxonomic groups share genotypic similarity despite gene transfer. Herein I examine the rules for governing HGT to determine the impact this process has played in the evolution of Bacteria and Archaea. Bacterial chromosomes are more than simple lists of genes. Genomes must maintain information beyond component genes to direct efficient replication and segregation of their chromosomes. I propose that this structure constrains the process of HGT so that transfer among certain pairs of donors and recipients is favored. I present methods to detect this structure and new theories of bacterial cell biology and evolution based on what this structure reveals. I present evidence that bacterial chromosomes are structured by repetitive sequences termed Architecture IMparting Sequences (AIMS). AIMS are found primarily on leading strands and increase in abundance towards the replication terminus. Bacteria with robustly-identified replication origins and termini all have AIMS, and related AIMS are conserved amongst families of bacteria. We propose that AIMS are under selection to provide DNA binding proteins with polarity information, facilitating identification of the location of the replication terminus. Although AIMS evolved to direct the biology of cell division and replication, the conservation of AIMS among related taxa leads to a secondary effect. Because AIMS are counterselected when in nonpermissive orientations, AIMS constrain both intragenomic and intergenomic rearrangements. Thus HGT frequency will depend on AIMS compatibility between different species. We predict that HGT is most common between bacterial genomes which are more closely related and will impede transfer between species which have dissimilar genome architecture. The additional level of selection reflected by AIMS has resulted in cohesive bacterial groups that reflect common gene pools as a result of biased rates of gene transfer

Development and application of -omics and bioinformatics approaches for a deeper understanding of infectious diseases systems

Background: Research in infectious diseases underwent a revolution with the uprising of Omics approaches, including, but not limited to, genomics, metagenomics and metatranscriptomics. In fact, there are several examples where Omics approaches showed their potential to tackle different challenges related to the versatile nature of infectious diseases by promoting “studies of one” to “system-wide studies”. In the frame of this PhD programme, we focused on the development and validation of Omics approaches and bioinformatics workflow aiming at tackling mainly diagnostics but also to some extents the treatment of infectious diseases. The four applications presented in this thesis had following specific objectives; (i) to develop and validate a bioinformatics approach aiming at selecting high quality markers among a large amount of complete genomic sequences; (ii) to characterise the viral metagenome of a plant to determine aetiology of a disease that could not be identified and/or fully characterised with other tools; (iii) to assess the potential of metagenomics in the field of personalised medicine and compare its diagnostics accuracy with validated diagnostics tools; and (iv) to make a system-wide survey of microbial populations and estimate its potential to cause harm to humans. Methods: Methodology was specific for each application but as a general rule, we only used published bioinformatics tools that have been used and validated in other studies. This includes, but is not limited to, the BLAST algorithm for the comparison of sequences to various databases and the MIRA assembler to assemble the metagenomics datasets obtained within the different projects. Results: For clarity, the results are summarised by project, corresponding to the different applications investigated during this PhD. Project (i): The developed bioinformatics workflow allowed the selection of highly conserved and specific molecular markers among various viral species with inputs of up to several hundred complete genomic sequences. The quality of the selected markers was successfully validated using several types of molecular assays including real-time PCR, LAMP and Sanger sequencing. Project (ii): We were able to find the aetiology of a grapevine plant presenting leafroll symptoms. A new virus, named Grapevine Leafroll-associated virus 4 Ob, with a thirteen kilobases genome was found in the viral metagenome. Other viruses that were co-identified in the virome were known to be asymptomatic viruses for grapevine, and with the help of additional serological experiences, we were able to confirm that this GLRaV-4 Ob was the causative agent of the Leafroll symptoms. Project (iii): The gut pathobiomes from four patients presenting persistent digestive disorders were fully characterised using a metagenomics approach. Comparison of validated diagnostics tools with this approach showed that the diagnostics rate was in favour of the latter for the detection of bacterial and helminths pathogens and in favour of the validated tools for the detection of viruses and protozoa. Using the same datasets, but compared to a different database, we were also able to screen the stool samples for antimicrobial resistance genes and retrieve potential resistance genes that might interfere with the treatment of these patients. Project (iv): In this project, a system-wide assessment of the microbial communities of the wastewater treatment system was done using a metagenomics approach. We were able to demonstrate how closely the genetic diversity of Escherichia coli and the overall genetic diversity were linked in this environment. We were also able to map the repartition of different pathogenic classes, including bacteria, helminths, intestinal protozoa and viruses as well as to show if and how human waterborne pathogens spread throughout this ecosystem. Conclusion: Omics offer new strategies of how challenges, mainly related to the vast diversity within the research area of infectious diseases, can be tackled. Meta-analyses, like metagenomics or metatranscriptomics are the applications that benefited most from the use of Next-Generation Sequencing technologies, and they now allow system-wide studies where previous studies were only focusing on one parameter (one microbe or one specific gene for instance). However, these Omics approaches have their limitations, mainly due to the bioinformatics challenges they give rise to. As a general conclusion, it is foreseeable that, because of the increased amount of results they generate, Omics approaches, once matured, will be more widely used and will replace standard approaches in the field of infectious diseases

Genome evolution and systems biology in bacterial endosymbionts of insects

Gene loss is the most important event in the process of genome reduction that appears associated with bacterial endosymbionts of insects. These small genomes were derived features evolved from ancestral prokaryotes with larger genome sizes, consequence of a massive process of genome reduction due to drastic changes in the ecological conditions and evolutionary pressures acting on these prokaryotic lineages during their ecological transition to host-dependent lifestyle. In the present thesis, the process of genome reduction is studied from different perspectives. In the first chapter, genome rearrangements have been studied in a set of 31 complete γ-proteobacterial genomes that includes five genomes of bacterial endosymbionts of insects. This is carried out by comparing the order of a subset of 244 single-copy orthologous genes presents in all the genomes and calculating the number of inversions and breakpoints between each genome pair. This reveals that inversions were the main rearrangement event in γ-proteobacteria evolution, with a progressive increase in the number of rearrangements with increased evolutionary distance. However, significant heterogeneity in different γ-proteobacterial lineages was also detected, with a significant acceleration in the rates of genome rearrangements in bacterial endosymbionts of insects at initial stages of the association. In the second chapter, the structure and functional capabilities of Sodalis glossinidius has been studied. S. glossinidius is the secondary endosymbiont of tsetse flies, and it´s at very initial stages of genome reduction process. It´s genome is experiencing a massive process of gene inactivation, with 972 pseudogenes (inactivated genes) that were described but not annotated in the original annotation of the genome. In this chapter, a complete functional re-annotation of this genome was carried out, that includes the characterization of 1501 pseudogenes though analysis of S. glossinidius intergenic regions. A massive presence of CDSs related with mobile genetic elements and surface proteins were detected, being also the functional classes most affected by pseudogenization. The reconstruction of the metabolic map of S. glossinidius revealed a functional profile very similar to that of free-living enterics, with inactivation of L-arginine biosynthesis pathway, whereas the comparison with Wigglesworthia glossinidia (tsetse primary endosymbiont) reveals possible cases of metabolic complementation between both tsetse endosymbionts at thiamine, coenzyme A and tetrahydrofolate biosynthesis level. Finally, in the third chapter of the thesis, the complete reductive evolution process associated with S. glossinidius was studied from a systems biology perspective through the reconstruction of their genome-scale metabolic networks at different stages of this process and the prediction of their internal reaction fluxes under different external conditions through Flux Balance Analysis. This revealed the decisive role of the pseudogenization of genes involved in L-arginine and glycogen biosynthesis and specially the pseudogenization of the key anaplerotic enzyme phosphoenolpyruvate carboxylase in the ecological transition to a host-dependent lifestyle experienced by S. glossinidius. A progressive decrease in network robustness to gene deletion events and to changes in particular reaction fluxes were detected. Finally, reductive evolution simulations over the functional network of S. glossinidius under different external conditions revealed a higher plasticity in minimal networks evolved in a nutrient-rich environment, and allow defining different sets of essential and disposable genes based on their presence or absence in minimal metabolic networks. These essential genes had more optimized patterns of codon usage and more restricted patterns of sequence evolution than disposable genes that could be lost without affecting the functionality of the network. However, lineage-specific estimates of dN and dS in S. glossinidius and Escherichia coli revealed that common features of ancient bacterial endosymbionts like acceleration in the rates of sequence evolution and the loss of adaptative codon usage were starting to affect S. glossinidius evolution.En esta tesis doctoral, el proceso de reducción genómica característico de bacterias endosimbiontes de insectos ha sido estudiado utilizando diferentes aproximaciones computacionales basadas en la genómica comparada y la biología de sistemas. Por un lado, las dinámicas de reordenaciones genómicas han sido estudiadas en un subconjunto de 31 genomas completos de γ-proteobacterias que incluyen 5 genomas completos de endosimbiontes bacterianos de insectos, revelando una aceleración significativa de las tasas de reordenaciones en estos genomas en etapas iniciales del proceso de reducción. Posteriormente, el genoma de Sodalis glossinidius, el endosimbionte secundario de la mosca tsétsé, fue re-anotado con el objetivo de evaluar el impacto de los procesos de inactivación génica y proliferación de elementos genéticos móviles en etapas tempranas del proceso de reducción, asi como su impacto sobre las capacidades funcionales de la bacteria en el contexto ecológico de su coexistencia con el endosimbionte primario ancestral Wigglesworthia glossinidia. Finalmente, el proceso completo de reducción genómica en S. glossinidius ha sido estudiado a través de la reconstrucción de su red metabólica a diferentes etapas de este proceso y su análisis funcional mediante Análisis de Balance de Flujos, evaluando la robustez de las redes frente a sucesos de deleción asi como las dinámicas evolutivas de genes esenciales y no esenciales en base a su presencia en redes mínimas evolucionadas a partir de la red funcional. Este análisis permitió identificar sucesos de inactivación génica con efectos drásticos sobre las capacidades funcionales del sistema como los genes implicados en la biosíntesis de arginina y glicógeno, y especialmente la inactivación de la enzima fosfoenolpiruvato carboxilasa, asi como una disminución progresiva de la robustez de las redes frente a diferentes sucesos mutacionales asociada al proceso de pérdida génica. Finalmente, simulaciones de evolución reductiva sobre la red funcional bajo diferentes condiciones de entorno ha permitido definir conjuntos de genes esenciales y delecionables en base a su presencia o ausencia en las redes mínimas producto de las simulaciones, revelando una mayor conservación a nivel de secuencia y un uso de codones más optimizado en genes esenciales frente a genes cuya pérdida no afecta a la funcionalidad del sistema