Bacillus anthracis genome organization in light of whole transcriptome sequencing

Emerging knowledge of whole prokaryotic transcriptomes could validate a number of theoretical concepts introduced in the early days of genomics. What are the rules connecting gene expression levels with sequence determinants such as quantitative scores of promoters and terminators? Are translation efficiency measures, e.g. codon adaptation index and RBS score related to gene expression? We used the whole transcriptome shotgun sequencing of a bacterial pathogen Bacillus anthracis to assess correlation of gene expression level with promoter, terminator and RBS scores, codon adaptation index, as well as with a new measure of gene translational efficiency, average translation speed. We compared computational predictions of operon topologies with the transcript borders inferred from RNA-Seq reads. Transcriptome mapping may also improve existing gene annotation. Upon assessment of accuracy of current annotation of protein-coding genes in the B. anthracis genome we have shown that the transcriptome data indicate existence of more than a hundred genes missing in the annotation though predicted by an ab initio gene finder. Interestingly, we observed that many pseudogenes possess not only a sequence with detectable coding potential but also promoters that maintain transcriptional activity

The study of sigma factors o5816 and c7615 in Bacillus cereus

The Gram-positive and ubiquitous bacterium Bacillus cereus is an emerging human and animal pathogen, and is capable of adapting to numerous extracellular stresses such as the low pH environment of the gastrointestinal tract. One mechanism B. cereus might utilize to regulate the acid-tolerance response system is through alternative sigma factors: proteins that bind and direct RNA polymerase to promoter- specific genes. A previous study found that alternative sigma factors c5816 and o7615 were induced dramatically at low pH, but it was unclear whether these sigma factors mediated an acid-tolerance response. Here, molecular tools were developed to further study these a factors, including a regulatable expression system and sigma-specific polyclonal antibodies. We were unable to identify a growth-deficient or growth- enhanced phenotype in strains that had these sigma factors deleted or could over-express these sigma factors, respectively. Through transcriptome analysis and qRT-PCR, we attempted to define the a5816 regulon. The proteolytic subunit of a Cip protease, ClpA, was found to potentially be part of the a5816 regulon. Understanding how B. cereus uses sigma factors to adapt to environmental stresses may give insight on how this emerging pathogen interacts and survives in its host

Biological Consequences of Atypical Phage Conversion in Gram-Positive Pathogens

Temperate bacteriophage have a complex, dynamic relationship with bacteria: parasitizing in the lytic cycle, but often increasing bacteria’s fitness as lysogens. The phage-bacteria relationship is vast and has evolved over more than an estimated three billion years, and there are likely many uncharacterized, intricate events between host and phage with important impacts on bacterial pathogenesis. This Thesis explores some of these lesser-studied phage-bacteria interactions, describing atypical mechanisms (“conversion events”) by which phage shape the populations of Bacillus anthracis and Staphylococcus aureus, driving their increased diversity and likely impacting their natural behaviors. In B. anthracis, phage contributions to virulence are largely unknown. The first part of this Thesis describes how an induced phage from a highly virulent, B. anthracis-like isolate affects the well-characterized strain Sterne and selects for a phage-resistant variant with a markedly altered phenotype, but with no apparent difference in virulence potential. In this work, we characterize this variant strain by a variety of techniques, including whole-genome DNA and RNA-sequencing. In addition, we connect the Sterne variant phenotype to that of the phage’s parent strain, B. cereus Biovar anthracis CA, uncovering lytic phage-bacteria interactions (i.e., selection by lysis) that may act to promote phenotypic diversity and shape populations of B. anthracis and B. anthracis-like pathogenic species in the wild. Unlike B. anthracis, S. aureus has well-characterized bacteriophage contributions to its virulence potential, with known lysogens carrying virulence factors stably integrated into the host chromosome. The second part of this Thesis describes an extra-chromosomal DNA sequencing screening that uncovers the presence of episomal prophages in a number of S. aureus clinical isolates. QPCR characterization of one of these strains, MSSA476, reveals that the episomal nature of one of its prophages, ɸSa4ms, would have been missed if sequencing whole genomic and not specifically extra-chromosomal DNA. In addition, we find that ɸSa4ms excision into the cytoplasm is a temporal event, and that the prophage does not appear to undergo lytic cycle replication after excision—suggesting that its excision is part of a lysogenic switch. Follow-up experiments show that ɸSa4ms excision can alter expression of htrA2 and promote increased heat-stress tolerance. This work suggests that for S. aureus, in addition to carrying important virulence determinants, phage may also play a rather widespread role as DNA-level switches to control virulence factor expression and/or generate distinct subpopulations. While this Thesis discusses atypical phage conversion events, it also illustrates perhaps the most important, universal role of phage in bacterial pathogens: tools to create diversity and allow for bacteria’s increased infection and success under different evolutionary selections and environmental conditions

Two-component signal transduction in Bacillus cereus and closely related bacteria

Bacillus cereus is a Gram-positive pathogen that is recognised as an important cause of food-borne disease worldwide. Within the genus Bacillus, B. cereus and its closest relatives form a homogeneous subdivision that has been termed the B. cereus group. This group includes B. anthracis, a pathogen that can cause anthrax in mammals, and B. thuringiensis, an insect pathogen that is used as an insecticide worldwide. Members of the B. cereus group can adapt to a wide range of environmental challenges. In bacteria, these challenges are generally monitored by two-component systems (TCS), which consist of a histidine kinase (HK) and a partner response regulator (RR). Upon sensing a specific environmental stimulus, the HK activates its cognate RR, which in turn controls the expression of genes that are involved in the appropriate response. This thesis describes the functional analysis of TCSs in the B. cereus group. By using in silico techniques, 50-58 HKs and 48-52 RRs were identified in eight different B. cereus group genomes. Biological functions, including the involvement in sporulation, biofilm formation and host-microbe interactions were predicted for these TCS proteins. A phylogenetic footprinting approach was developed and used to identify specific binding sites and target genes for over 50% of the B. cereus group DNA-binding RRs. These predictions allowed relating several RRs to a minimal regulon and thereby to a characteristic transcriptional response. To further support these predictions, the transcriptomes of two B. cereus TCS deletion mutants (ΔyvrHG and ΔyufLM) were analysed and compared with the transcriptome of wild-type B. cereus. This revealed that the minimal regulon predictions were correct for the two respective TCSs. Furthermore, the predicted biological roles for these TCSs, including roles in antibiotic resistance (YvrHG) and fumarate metabolism (YufLM), were supported by phenotypic tests. Besides the many “classical” HKs and RRs detected in the B. cereus group, several a-typical TCS proteins were found. These included five RRs without a DNA-binding output domain and two hybrid HKs (HK-RR fusions). Genome analyses revealed that one of the hybrid HK-encoding genes (BC1008) is located in a conserved gene cluster that also encodes the a-typical RR RsbY. In B. cereus, RsbY is known to activate the key stress-responsive sigma factor σB. As a partner HK for RsbY was still “missing”, the role of BC1008 in the σB-mediated stress response was tested. Indeed, a bc1008 deletion strain appeared incapable of inducing σB and its associated regulon upon stress conditions and appeared impaired in its heat adaptive response. In addition, truncation of the BC1008 fused RR receiver domain indicated that this domain plays a role in fine-tuning BC1008 activity. A comparative genome analysis further indicated that BC1008-type hybrid HKs control σB-like sigma factors in at least several other Gram-positive bacteria, including Geobacillus, Paenibacillus and actinobacteria. In summary, the research described in this thesis contributes to our understanding of B. cereus adaptive responses through TCSs. This knowledge may be applied for the development of novel intervention strategies for an improved control of B. cereus in food production environments. <br/

Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group

<p>Abstract</p> <p>Background</p> <p>The <it>Bacillus cereus </it><it>sensu lato </it>group consists of six species (<it>B. anthracis</it>, <it>B. cereus</it>, <it>B. mycoides</it>, <it>B. pseudomycoides</it>, <it>B. thuringiensis</it>, and <it>B. weihenstephanensis</it>). While classical microbial taxonomy proposed these organisms as distinct species, newer molecular phylogenies and comparative genome sequencing suggests that these organisms should be classified as a single species (thus, we will refer to these organisms collectively as the <it>Bc </it>species-group). How do we account for the underlying similarity of these phenotypically diverse microbes? It has been established for some time that the most rapidly evolving and evolutionarily flexible portions of the bacterial genome are regulatory sequences and transcriptional networks. Other studies have suggested that the sigma factor gene family of these organisms has diverged and expanded significantly relative to their ancestors; sigma factors are those portions of the bacterial transcriptional apparatus that control RNA polymerase recognition for promoter selection. Thus, examining sigma factor divergence in these organisms would concurrently examine both regulatory sequences and transcriptional networks important for divergence. We began this examination by comparison to the sigma factor gene set of <it>B. subtilis</it>.</p> <p>Results</p> <p>Phylogenetic analysis of the <it>Bc </it>species-group utilizing 157 single-copy genes of the family <it>Bacillaceae </it>suggests that several taxonomic revisions of the genus <it>Bacillus </it>should be considered. Within the <it>Bc </it>species-group there is little indication that the currently recognized species form related sub-groupings, suggesting that they are members of the same species. The sigma factor gene family encoded by the <it>Bc </it>species-group appears to be the result of a dynamic gene-duplication and gene-loss process that in previous analyses underestimated the true heterogeneity of the sigma factor content in the <it>Bc </it>species-group.</p> <p>Conclusions</p> <p>Expansion of the sigma factor gene family appears to have preferentially occurred within the extracytoplasmic function (ECF) sigma factor genes, while the primary alternative (PA) sigma factor genes are, in general, highly conserved with those found in <it>B. subtilis</it>. Divergence of the sigma-controlled transcriptional regulons among various members of the <it>Bc </it>species-group likely has a major role in explaining the diversity of phenotypic characteristics seen in members of the <it>Bc </it>species-group.</p

Comparative Transcriptional Profiling of Bacillus cereus Sensu Lato Strains during Growth in CO2-Bicarbonate and Aerobic Atmospheres

Bacillus species are spore-forming bacteria that are ubiquitous in the environment and display a range of virulent and avirulent phenotypes. This range is particularly evident in the Bacillus cereus sensu lato group; where closely related strains cause anthrax, food-borne illnesses, and pneumonia, but can also be non-pathogenic. Although much of this phenotypic range can be attributed to the presence or absence of a few key virulence factors, there are other virulence-associated loci that are conserved throughout the B. cereus group, and we hypothesized that these genes may be regulated differently in pathogenic and non-pathogenic strains.Here we report transcriptional profiles of three closely related but phenotypically unique members of the Bacillus cereus group--a pneumonia-causing B. cereus strain (G9241), an attenuated strain of B. anthracis (Sterne 34F(2)), and an avirulent B. cereus strain (10987)--during exponential growth in two distinct atmospheric environments: 14% CO(2)/bicarbonate and ambient air. We show that the disease-causing Bacillus strains undergo more distinctive transcriptional changes between the two environments, and that the expression of plasmid-encoded virulence genes was increased exclusively in the CO(2) environment. We observed a core of conserved metabolic genes that were differentially expressed in all three strains in both conditions. Additionally, the expression profiles of putative virulence genes in G9241 suggest that this strain, unlike Bacillus anthracis, may regulate gene expression with both PlcR and AtxA transcriptional regulators, each acting in a different environment.We have shown that homologous and even identical genes within the genomes of three closely related members of the B. cereus sensu lato group are in some instances regulated very differently, and that these differences can have important implications for virulence. This study provides insights into the evolution of the B. cereus group, and highlights the importance of looking beyond differences in gene content in comparative genomics studies

The Dawning Era of Comprehensive Transcriptome Analysis in Cellular Microbiology

Bacteria rapidly change their transcriptional patterns during infection in order to adapt to the host environment. To investigate host–bacteria interactions, various strategies including the use of animal infection models, in vitro assay systems and microscopic observations have been used. However, these studies primarily focused on a few specific genes and molecules in bacteria. High-density tiling arrays and massively parallel sequencing analyses are rapidly improving our understanding of the complex host–bacterial interactions through identification and characterization of bacterial transcriptomes. Information resulting from these high-throughput techniques will continue to provide novel information on the complexity, plasticity, and regulation of bacterial transcriptomes as well as their adaptive responses relative to pathogenecity. Here we summarize recent studies using these new technologies and discuss the utility of transcriptome analysis

Bacillus cereus efflux protein BC3310 – a multidrug transporter of the unknown major facilitator family, UMF-2

Phylogenetic classification divides the major facilitator superfamily (MFS) into 82 families, including 25 families that are comprised of transporters with no characterized functions. This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a member of the “unknown major facilitator family-2” (UMF-2). BC3310 was shown to be a multidrug efflux pump conferring resistance to ethidium bromide, SDS and silver nitrate when heterologously expressed in Escherichia coli DH5α acrAB. A conserved aspartate residue (D105) in putative transmembrane helix 4 was identified, which was essential for the energy dependent ethidium bromide efflux by BC3310. Transport proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF- 2 proteins revealed that they carry a variant of the MFS motif A, which may be used as a marker to distinguish easily between this family and other MFS proteins. Genes orthologous to bc3310 are highly conserved within the B. cereus group of organisms and thus belong to the core genome, suggesting an important conserved functional role in the normal physiology of these bacteria

Temperature-dependent regulation in the Bacillus cereus-Bacillus anthracis crossover strain, Bacillus cereus G9241

Bacillus cereus G9241 was originally isolated from a Louisiana welder suffering from an anthrax-like infection. A member of the Bacillus cereus sensu lato complex, strain G9241 is closely related to the mammalian pathogen Bacillus anthracis. It contains two plasmids pBCX01 and pBC218, which are homologous and analogous to pX01 and pX02 respectively, from Bacillus anthracis. In addition, it contains a phagemid, pBFH_1 encoding a putative prophage. The gene for PlcR, which is the pleiotropic quorum sensing regulator of secreted proteins, is truncated in all B. anthracis isolates. The current dogma suggests this truncation evolved to accommodate the acquisition of the anthrax toxin regulator, AtxA encoded on the pX01 plasmid. B. cereus G9241 appears to break this dogma as it encodes intact copies of both plcR and atxA. Work prior to this study showed when cultured at 25 °C, cell free B. cereus G9241 culture supernatant is cytotoxic to human macrophages, PMNs and T2 lymphocytes in addition to insect haemocytes from Manduca sexta. However, the cytotoxic activity of the culture supernatant is lost at 37 °C. B. cereus G9241 is also motile at 25 °C but immotile at 37 °C. This study proposes that BcG9241 is able to switch between B. cereus and B. anthracis –like phenotypes in a temperature-dependent manner. A combination of RNAseq, whole cell and secretome proteomics suggests that differential regulation of PlcR at a post transcriptional level is responsible for the temperature-dependent cytotoxic activity of the culture supernatant and temperature-dependent motility. Furthermore, expression from the extrachromosomal elements increases at 37 °C, particularly from the phagemid pBFH_1. This study shows that pBFH_1 encoded phage particles are expressed at 37 °C and this may be a link to a rapid sporulation phenotype also seen at this temperature