Core genome multilocus sequence typing scheme for Bacillus cereus group bacteria.

Core genome multilocus sequence typing (cgMLST) employs a strategy where the set of orthologous genes common to all members of a group of organisms are used for phylogenetic analysis of the group members. The Bacillus cereus group consists of species with pathogenicity towards insect species as well as warm-blooded animals including humans. While B. cereus is an opportunistic pathogen linked to a range of human disease conditions, including emesis and diarrhoea, Bacillus thuringiensis is an entomopathogenic species with toxicity toward insect larvae, and therefore used as a biological pesticide worldwide. Bacillus anthracis is a classical obligate pathogen causing anthrax, an acute lethal condition in herbivores as well as humans, and which is endemic in many parts of the world. The group also includes a range of additional species, and B. cereus group bacteria have been subject to analysis with a wide variety of phylogenetic typing systems. Here we present, based on analyses of 173 complete genomes from B. cereus group species available in public databases, the identification of a set of 1568 core genes which were used to create a core genome multilocus typing scheme for the group which is implemented in the PubMLST system as an open online database freely available to the community. The new cgMLST system provides unprecedented resolution over existing phylogenetic analysis schemes covering the B. cereus group.

CalY is a major virulence factor and a biofilm matrix protein.

The extracellular biofilm matrix often contains a network of amyloid fibers which, in the human opportunistic pathogen Bacillus cereus, includes the two homologous proteins TasA and CalY. We show here, in the closely related entomopathogenic species Bacillus thuringiensis, that CalY also displays a second function. In the early stationary phase of planktonic cultures, CalY was located at the bacterial cell-surface, as shown by immunodetection. Deletion of calY revealed that this protein plays a major role in adhesion to HeLa epithelial cells, to the insect Galleria mellonella hemocytes and in the bacterial virulence against larvae of this insect, suggesting that CalY is a cell-surface adhesin. In mid-stationary phase and in biofilms, the location of CalY shifted from the cell surface to the extracellular medium, where it was found as fibers. The transcription study and the deletion of sipW suggested that CalY change of location is due to a delayed activity of the SipW signal peptidase. Using purified CalY, we found that the protein polymerization occurred only in the presence of cell-surface components. CalY is, therefore, a bifunctional protein, which switches from a cell-surface adhesin activity in early stationary phase, to the production of fibers in mid-stationary phase and in biofilms.

Spores and soil from six sides: interdisciplinarity and the environmental biology of anthrax (Bacillus anthracis).

Environmentally transmitted diseases are comparatively poorly understood and managed, and their ecology is particularly understudied. Here we identify challenges of studying environmental transmission and persistence with a six-sided interdisciplinary review of the biology of anthrax (Bacillus anthracis). Anthrax is a zoonotic disease capable of maintaining infectious spore banks in soil for decades (or even potentially centuries), and the mechanisms of its environmental persistence have been the topic of significant research and controversy. Where anthrax is endemic, it plays an important ecological role, shaping the dynamics of entire herbivore communities. The complex eco-epidemiology of anthrax, and the mysterious biology of Bacillus anthracis during its environmental stage, have necessitated an interdisciplinary approach to pathogen research. Here, we illustrate different disciplinary perspectives through key advances made by researchers working in Etosha National Park, a long-term ecological research site in Namibia that has exemplified the complexities of the enzootic process of anthrax over decades of surveillance. In Etosha, the role of scavengers and alternative routes (waterborne transmission and flies) has proved unimportant relative to the long-term persistence of anthrax spores in soil and their infection of herbivore hosts. Carcass deposition facilitates green-ups of vegetation to attract herbivores, potentially facilitated by the role of anthrax spores in the rhizosphere. The underlying seasonal pattern of vegetation, and herbivores' immune and behavioural responses to anthrax risk, interact to produce regular 'anthrax seasons' that appear to be a stable feature of the Etosha ecosystem. Through the lens of microbiologists, geneticists, immunologists, ecologists, epidemiologists, and clinicians, we discuss how anthrax dynamics are shaped at the smallest scale by population genetics and interactions within the bacterial communities up to the broadest scales of ecosystem structure. We illustrate the benefits and challenges of this interdisciplinary approach to disease ecology, and suggest ways anthrax might offer insights into the biology of other important pathogens. Bacillus anthracis, and the more recently emerged Bacillus cereus biovar anthracis, share key features with other environmentally transmitted pathogens, including several zoonoses and panzootics of special interest for global health and conservation efforts. Understanding the dynamics of anthrax, and developing interdisciplinary research programs that explore environmental persistence, is a critical step forward for understanding these emerging threats.

The putative drug efflux systems of the Bacillus cereus group.

The Bacillus cereus group of bacteria includes seven closely related species, three of which, B. anthracis, B. cereus and B. thuringiensis, are pathogens of humans, animals and/or insects. Preliminary investigations into the transport capabilities of different bacterial lineages suggested that genes encoding putative efflux systems were unusually abundant in the B. cereus group compared to other bacteria. To explore the drug efflux potential of the B. cereus group all putative efflux systems were identified in the genomes of prototypical strains of B. cereus, B. anthracis and B. thuringiensis using our Transporter Automated Annotation Pipeline. More than 90 putative drug efflux systems were found within each of these strains, accounting for up to 2.7% of their protein coding potential. Comparative analyses demonstrated that the efflux systems are highly conserved between these species; 70-80% of the putative efflux pumps were shared between all three strains studied. Furthermore, 82% of the putative efflux system proteins encoded by the prototypical B. cereus strain ATCC 14579 (type strain) were found to be conserved in at least 80% of 169 B. cereus group strains that have high quality genome sequences available. However, only a handful of these efflux pumps have been functionally characterized. Deletion of individual efflux pump genes from B. cereus typically had little impact to drug resistance phenotypes or the general fitness of the strains, possibly because of the large numbers of alternative efflux systems that may have overlapping substrate specificities. Therefore, to gain insight into the possible transport functions of efflux systems in B. cereus, we undertook large-scale qRT-PCR analyses of efflux pump gene expression following drug shocks and other stress treatments. Clustering of gene expression changes identified several groups of similarly regulated systems that may have overlapping drug resistance functions. In this article we review current knowledge of the small molecule efflux pumps encoded by the B. cereus group and suggest the likely functions of numerous uncharacterised pumps.

Correction: Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis.

[This corrects the article DOI: 10.1371/journal.ppat.1002629.].

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 ΔacrAB. 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.

An ace up their sleeve: a transcriptomic approach exposes the AceI efflux protein of Acinetobacter baumannii and reveals the drug efflux potential hidden in many microbial pathogens.

The era of antibiotics as a cure-all for bacterial infections appears to be coming to an end. The emergence of multidrug resistance in many hospital-associated pathogens has resulted in "superbugs" that are effectively untreatable. Multidrug efflux pumps are well known mediators of bacterial drug resistance. Genome sequencing efforts have highlighted an abundance of putative efflux pump genes in bacteria. However, it is not clear how many of these pumps play a role in antimicrobial resistance. Efflux pump genes that participate in drug resistance can be under tight regulatory control and expressed only in response to substrates. Consequently, changes in gene expression following antimicrobial shock may be used to identify efflux pumps that mediate antimicrobial resistance. Using this approach we have characterized several novel efflux pumps in bacteria. In one example we recently identified the Acinetobacterchlorhexidine efflux protein (AceI) efflux pump in Acinetobacter. AceI is a prototype for a novel family of multidrug efflux pumps conserved in many proteobacterial lineages. The discovery of this family raises the possibility that additional undiscovered intrinsic resistance proteins may be encoded in the core genomes of pathogenic bacteria.

Survey of chimeric IStron elements in bacterial genomes: multiple molecular symbioses between group I intron ribozymes and DNA transposons.

IStrons are chimeric genetic elements composed of a group I intron associated with an insertion sequence (IS). The group I intron is a catalytic RNA providing the IStron with self-splicing ability, which renders IStron insertions harmless to the host genome. The IS element is a DNA transposon conferring mobility, and thus allowing the IStron to spread in genomes. IStrons are therefore a striking example of a molecular symbiosis between unrelated genetic elements endowed with different functions. In this study, we have conducted the first comprehensive survey of IStrons in sequenced genomes that provides insights into the distribution, diversity, origin and evolution of IStrons. We show that IStrons have a restricted phylogenetic distribution limited to two bacterial phyla, the Firmicutes and the Fusobacteria. Nevertheless, diverse IStrons representing two major groups targeting different insertion site motifs were identified. This taken with the finding that while the intron components of all IStrons belong to the same structural class, they are fused to different IS families, indicates that multiple intron-IS symbioses have occurred during evolution. In addition, introns and IS elements related to those that were at the origin of IStrons were also identified.

SecDF as part of the Sec-translocase facilitates efficient secretion of Bacillus cereus toxins and cell wall-associated proteins.

The aim of this study was to explore the role of SecDF in protein secretion in Bacillus cereus ATCC 14579 by in-depth characterization of a markerless secDF knock out mutant. Deletion of secDF resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the ΔsecDF mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of secDF indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen B. cereus, and further supports the notion that B. cereus enterotoxins are secreted by the Sec-system.

SinR controls enterotoxin expression in Bacillus thuringiensis biofilms.

The entomopathogen Bacillus thuringiensis produces dense biofilms under various conditions. Here, we report that the transition phase regulators Spo0A, AbrB and SinR control biofilm formation and swimming motility in B. thuringiensis, just as they control biofilm formation and swarming motility in the closely related saprophyte species B. subtilis. However, microarray analysis indicated that in B. thuringiensis, in contrast to B. subtilis, SinR does not control an eps operon involved in exopolysaccharides production, but regulates genes involved in the biosynthesis of the lipopeptide kurstakin. This lipopeptide is required for biofilm formation and was previously shown to be important for survival in the host cadaver (necrotrophism). Microarray analysis also revealed that the SinR regulon contains genes coding for the Hbl enterotoxin. Transcriptional fusion assays, Western blots and hemolysis assays confirmed that SinR controls Hbl expression, together with PlcR, the main virulence regulator in B. thuringiensis. We show that Hbl is expressed in a sustained way in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses Hbl. The gene coding for SinI, an antagonist of SinR, is expressed in the same biofilm subpopulation as hbl, suggesting that hbl transcription heterogeneity is SinI-dependent. B. thuringiensis and B. cereus are enteric bacteria which possibly form biofilms lining the host intestinal epithelium. Toxins produced in biofilms could therefore be delivered directly to the target tissue.

Different bacterial communities in ectomycorrhizae and surrounding soil.

Several eukaryotic symbioses have shown to host a rich diversity of prokaryotes that interact with their hosts. Here, we study bacterial communities associated with ectomycorrhizal root systems of Bistorta vivipara compared to bacterial communities in bulk soil using pyrosequencing of 16S rRNA amplicons. A high richness of Operational Taxonomic Units (OTUs) was found in plant roots (3,571 OTUs) and surrounding soil (3,476 OTUs). The community composition differed markedly between these two environments. Actinobacteria, Armatimonadetes, Chloroflexi and OTUs unclassified at phylum level were significantly more abundant in plant roots than in soil. A large proportion of the OTUs, especially those in plant roots, presented low similarity to Sanger 16S rRNA reference sequences, suggesting novel bacterial diversity in ectomycorrhizae. Furthermore, the bacterial communities of the plant roots were spatially structured up to a distance of 60 cm, which may be explained by bacteria using fungal hyphae as a transport vector. The analyzed ectomycorrhizae presents a distinct microbiome, which likely influence the functioning of the plant-fungus symbiosis.

Gene transcription from the linear plasmid pBClin15 leads to cell lysis and extracellular DNA-dependent aggregation of Bacillus cereus ATCC 14579 in response to quinolone-induced stress.

The Bacillus cereus type strain ATCC 14579 harbours pBClin15, a linear plasmid with similar genome organization to tectiviruses. Since phage morphogenesis is not known to occur it has been suggested that pBClin15 may be a defect relic of a tectiviral prophage without relevance for the bacterial physiology. However, in this paper, we demonstrate that a pBClin15-cured strain is more tolerant to antibiotics interfering with DNA integrity than the WT strain. Growth in the presence of crystal violet or the quinolones nalidixic acid, norfloxacin or ciprofloxacin resulted in aggregation and lysis of the WT strain, whereas the pBClin15-cured strain was unaffected. Microarray analysis comparing the gene expression in the WT and pBClin15-cured strains showed that pBClin15 gene expression was strongly upregulated in response to norfloxacin stress, and coincided with lysis and aggregation of the WT strain. The aggregating bacteria experienced a significant survival benefit compared with the planktonic counterparts in the presence of norfloxacin. There was no difference between the WT and pBClin15-cured strains during growth in the absence of norfloxacin, the pBClin15 genes were moderately expressed, and no effect was observed on chromosomal gene expression. These data demonstrate for the first time that although pBClin15 may be a remnant of a temperate phage, it negatively affects the DNA stress tolerance of B. cereus ATCC 14579. Furthermore, our results warrant a recommendation to always verify the presence of pBClin15 following genetic manipulation of B. cereus ATCC 14579.

Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and pairwise genome comparisons of the species of the group by means of ANI calculations.

Strain BCT-7112(T) was isolated in 1966 in Japan from a survey designed to obtain naturally occurring microorganisms as pure cultures in the laboratory for use as probiotics in animal nutrition. This strain, which was primarily identified as Bacillus cereus var toyoi, has been in use for more than 30 years as the active ingredient of the preparation TOYOCERIN(®), an additive for use in animal nutrition (e.g. swine, poultry, cattle, rabbits and aquaculture). Despite the fact that the strain was initially classified as B. cereus, it showed significant genomic differences from the type strains of the B. cereus group that were large enough (ANI values below 92%) to allow it to be considered as a different species within the group. The polyphasic taxonomic study presented here provides sufficient discriminative parameters to classify BCT-7112(T) as a new species for which the name Bacillus toyonensis sp. nov. is proposed, with BCT-7112(T) (=CECT 876(T); =NCIMB 14858(T)) being designated as the type strain. In addition, a pairwise comparison between the available genomes of the whole B. cereus group by means of average nucleotide identity (ANI) calculations indicated that besides the eight classified species (including B. toyonensis), additional genomospecies could be detected, and most of them also had ANI values below 94%. ANI values were on the borderline of a species definition only in the cases of representatives of B. cereus versus B. thuringiensis, and B. mycoides and B. weihenstephanensis.

A new family of proteins related to the HEAT-like repeat DNA glycosylases with affinity for branched DNA structures.

The recently discovered HEAT-like repeat (HLR) DNA glycosylase superfamily is widely distributed in all domains of life. The present bioinformatics and phylogenetic analysis shows that HLR DNA glycosylase superfamily members in the genus Bacillus form three subfamilies: AlkC, AlkD and AlkF/AlkG. The crystal structure of AlkF shows structural similarity with the DNA glycosylases AlkC and AlkD, however neither AlkF nor AlkG display any DNA glycosylase activity. Instead, both proteins have affinity to branched DNA structures such as three-way and Holliday junctions. A unique ß-hairpin in the AlkF/AlkG subfamily is most likely inserted into the DNA major groove, and could be a structural determinant regulating DNA substrate affinity. We conclude that AlkF and AlkG represent a new family of HLR proteins with affinity for branched DNA structures.

BC4707 is a major facilitator superfamily multidrug resistance transport protein from Bacillus cereus implicated in fluoroquinolone tolerance.

Transcriptional profiling highlighted a subset of genes encoding putative multidrug transporters in the pathogen Bacillus cereus that were up-regulated during stress produced by bile salts. One of these multidrug transporters (BC4707) was selected for investigation. Functional characterization of the BC4707 protein in Escherichia coli revealed a role in the energized efflux of xenobiotics. Phenotypic analyses after inactivation of the gene bc4707 in Bacillus cereus ATCC14579 suggested a more specific, but modest role in the efflux of norfloxacin. In addition to this, transcriptional analyses showed that BC4707 is also expressed during growth of B. cereus under non-stressful conditions where it may have a role in the normal physiology of the bacteria. Altogether, the results indicate that bc4707, which is part of the core genome of the B. cereus group of bacteria, encodes a multidrug resistance efflux protein that is likely involved in maintaining intracellular homeostasis during growth of the bacteria.

Global mRNA decay analysis at single nucleotide resolution reveals segmental and positional degradation patterns in a Gram-positive bacterium.

BACKGROUND: Recent years have shown a marked increase in the use of next-generation sequencing technologies for quantification of gene expression (RNA sequencing, RNA-Seq). The expression level of a gene is a function of both its rate of transcription and RNA decay, and the influence of mRNA decay rates on gene expression in genome-wide studies of Gram-positive bacteria is under-investigated. RESULTS: In this work, we employed RNA-Seq in a genome-wide determination of mRNA half-lives in the Gram-positive bacterium Bacillus cereus. By utilizing a newly developed normalization protocol, RNA-Seq was used successfully to determine global mRNA decay rates at the single nucleotide level. The analysis revealed positional degradation patterns, with mRNAs being degraded from both ends of the molecule, indicating that both 5' to 3' and 3' to 5' directions of RNA decay are present in B. cereus. Other operons showed segmental degradation patterns where specific ORFs within polycistrons were degraded at variable rates, underlining the importance of RNA processing in gene regulation. We determined the half-lives for more than 2,700 ORFs in B. cereus ATCC 10987, ranging from less than one minute to more than fifteen minutes, and showed that mRNA decay rate correlates globally with mRNA expression level, GC content, and functional class of the ORF. CONCLUSIONS: To our knowledge, this study presents the first global analysis of mRNA decay in a bacterium at single nucleotide resolution. We provide a proof of principle for using RNA-Seq in bacterial mRNA decay analysis, revealing RNA processing patterns at the single nucleotide level.

Necrotrophism is a quorum-sensing-regulated lifestyle in Bacillus thuringiensis.

How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading.

Diversity, mobility, and structural and functional evolution of group II introns carrying an unusual 3' extension.

BACKGROUND: Group II introns are widespread genetic elements endowed with a dual functionality. They are catalytic RNAs (ribozymes) that are able of self-splicing and they are also mobile retroelements that can invade genomic DNA. The group II intron RNA secondary structure is typically made up of six domains. However, a number of unusual group II introns carrying a unique extension of 53-56 nucleotides at the 3' end have been identified previously in bacteria of the Bacillus cereus group. METHODS: In the present study, we conducted combined sequence comparisons and phylogenetic analyses of introns, host gene, plasmid and chromosome of host strains in order to gain insights into mobility, dispersal, and evolution of the unusual introns and their extension. We also performed in vitro mutational and kinetic experiments to investigate possible functional features related to the extension. RESULTS: We report the identification of novel copies of group II introns carrying a 3' extension including the first two copies in bacteria not belonging to the B. cereus group, Bacillus pseudofirmus OF4 and Bacillus sp. 2_A_57_CT2, an uncharacterized species phylogenetically close to B. firmus. Interestingly, the B. pseudofirmus intron has a longer extension of 70 bases. From sequence comparisons and phylogenetic analyses, several possible separate events of mobility involving the atypical introns could be identified, including both retrohoming and retrotransposition events. In addition, identical extensions were found in introns that otherwise exhibit little sequence conservation in the rest of their structures, with the exception of the conserved and catalytically critical domains V and VI, suggesting either separate acquisition of the extra segment by different group II introns or a strong selection pressure acting on the extension. Furthermore, we show by in vitro splicing experiments that the 3' extension affects the splicing properties differently in introns belonging to separate evolutionary branches. CONCLUSIONS: Altogether this study provides additional insights into the structural and functional evolution of unusual introns harboring a 3' extension and lends further evidence that these introns are mobile with their extension.

Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4.

Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions.

Evolutionary history and functional characterization of three large genes involved in sporulation in Bacillus cereus group bacteria.

The Bacillus cereus group of bacteria is a group of closely related species that are of medical and economic relevance, including B. anthracis, B. cereus, and B. thuringiensis. Bacteria from the Bacillus cereus group encode three large, highly conserved genes of unknown function (named crdA, crdB, and crdC) that are composed of 16 to 35 copies of a repeated domain of 132 amino acids at the protein level. Bioinformatic analysis revealed that there is a phylogenetic bias in the genomic distribution of these genes and that strains harboring all three large genes mainly belong to cluster III of the B. cereus group phylogenetic tree. The evolutionary history of the three large genes implicates gain, loss, duplication, internal deletion, and lateral transfer. Furthermore, we show that the transcription of previously identified antisense open reading frames in crdB is simultaneously regulated with its host gene throughout the life cycle in vitro, with the highest expression being at the onset of sporulation. In B. anthracis, different combinations of double- and triple-knockout mutants of the three large genes displayed slower and less efficient sporulation processes than the parental strain. Altogether, the functional studies suggest an involvement of these three large genes in the sporulation process.