HyperCAT: an extension of the SuperCAT database for global multi-scheme and multi-datatype phylogenetic analysis of the Bacillus cereus group population

The Bacillus cereus group of bacteria includes species that are of significant medical and economic importance. We previously developed the SuperCAT database, which integrates data from all five multilocus sequence typing (MLST) schemes available to infer the genetic relatedness within this group. Since large numbers of isolates have been typed by other techniques, these should be incorporated in order to provide the most comprehensive and truly global view of the B. cereus group population. The SuperCAT system has been extended into a new database, HyperCAT, with two main additions. First, an extended supertree approach was applied to combine the phylogenetic information available from MLST, amplified fragment length polymorphism and multilocus enzyme electrophoresis. Secondly, a tree-independent clustering algorithm was designed to build superclusters of genetically closely related isolates sharing identical genotyping data. The superclusters were then mapped onto the supertree to generate an integrative genetic and phylogenetic snapshot of the B. cereus group population currently incorporating 2143 isolates. HyperCAT is freely accessible at the University of Oslo’s typing website, which has also been upgraded with TNT software, allowing improved and ultra-fast supertree reconstructions. In addition, novel and advanced tools have been included for interactive viewing and navigation of trees, clusters and networks

The PlcR Virulence Regulon of Bacillus cereus

PlcR is a Bacillus cereus transcriptional regulator, which activates gene expression by binding to a nucleotidic sequence called the ‘PlcR box’. To build a list of all genes included in the PlcR regulon, a consensus sequence was identified by directed mutagenesis. The reference strain ATCC14579 sequenced genome was searched for occurrences of this consensus sequence to produce a virtual regulon. PlcR control of these genes was confirmed by comparing gene expression in the reference strain and its isogenic Δ-plcR strain using DNA microarrays, lacZ fusions and proteomics methods. The resulting list included 45 genes controlled by 28 PlcR boxes. Forty of the PlcR controlled proteins were exported, of which 22 were secreted in the extracellular medium and 18 were bound or attached to cell wall structures (membrane or peptidoglycan layer). The functions of these proteins were related to food supply (phospholipases, proteases, toxins), cell protection (bacteriocins, toxins, transporters, cell wall biogenesis) and environment-sensing (two-component sensors, chemotaxis proteins, GGDEF family regulators). Four genes coded for cytoplasmic regulators. The PlcR regulon appears to integrate a large range of environmental signals, including food deprivation and self cell-density, and regulate the transcription of genes designed to overcome obstacles that hinder B. cereus growth within the host: food supply, host barriers, host immune defenses, and competition with other bacterial species. PlcR appears to be a key component in the efficient adaptation of B. cereus to its host environment

Complete Genome Sequence of Pseudomonas aeruginosa K34-7, a Carbapenem-Resistant Isolate of the High-Risk Sequence Type 233

Carbapenem-resistant Pseudomonas aeruginosa is defined as a “critical” priority pathogen for the development of new antibiotics. Here we report the complete genome sequence of an extensively drug-resistant, Verona integron-encoded metallo-- lactamase-expressing isolate belonging to the high-risk sequence type 233

Complete Genome Sequence of Pseudomonas aeruginosa K34-7, a Carbapenem-Resistant Isolate of the High-Risk Sequence Type 233

Carbapenem-resistant Pseudomonas aeruginosa is defined as a “critical” priority pathogen for the development of new antibiotics. Here we report the complete genome sequence of an extensively drug-resistant, Verona integron-encoded metallo-β-lactamase-expressing isolate belonging to the high-risk sequence type 233

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

Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMβ1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMβ1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMβ1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti

Low Concentrations of Bile Salts Induce Stress Responses and Reduce Motility in Bacillus cereus ATCC 14570▿ †

Tolerance to bile salts was investigated in forty Bacillus cereus strains, including 17 environmental isolates, 11 dairy isolates, 3 isolates from food poisoning outbreaks, and 9 other clinical isolates. Growth of all strains was observed at low bile salt concentrations, but no growth was observed on LB agar plates containing more than 0.005% bile salts. Preincubation of the B. cereus type strain, ATCC 14579, in low levels of bile salts did not increase tolerance levels. B. cereus ATCC 14579 was grown to mid-exponential growth phase and shifted to medium containing bile salts (0.005%). Global expression patterns were determined by hybridization of total cDNA to a 70-mer oligonucleotide microarray. A general stress response and a specific response to bile salts were observed. The general response was similar to that observed in cultures grown in the absence of bile salts but at a higher (twofold) cell density. Up-regulation of several putative multidrug exporters and transcriptional regulators and down-regulation of most motility genes were observed as part of the specific response. Motility experiments in soft agar showed that motility decreased following bile salts exposure, in accordance with the transcriptional data. Genes encoding putative virulence factors were either unaffected or down-regulated

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

Bacillus thuringiensis CbpA is a collagen binding cell surface protein under c-di-GMP control

Cyclic diguanylate (c-di-GMP) signalling affects several cellular processes in Bacillus cereus group bacteria including biofilm formation and motility, and CdgF was previously identified as a diguanylate cyclase promoting biofilm formation in B. thuringiensis. C-di-GMP can exert its function as a second messenger via riboswitch binding, and a functional c-di-GMP-responsive riboswitch has been found upstream of cbpA in various B. cereus group strains. Protein signature recognition predicted CbpA to be a cell wall-anchored surface protein with a fibrinogen or collagen binding domain. The aim of this study was to identify the binding ligand of CbpA and the function of CbpA in cellular processes that are part of the B. cereus group c-di-GMP regulatory network. By global gene expression profiling cbpA was found to be down-regulated in a cdgF deletion mutant, and cbpA exhibited maximum expression in early exponential growth. Contrary to the wild type, a ΔcbpA deletion mutant showed no binding to collagen in a cell adhesion assay, while a CbpA overexpression strain exhibited slightly increased collagen binding compared to the control. For both fibrinogen and fibronectin there was however no change in binding activity compared to controls, and CbpA did not appear to contribute to binding to abiotic surfaces (polystyrene, glass, steel). Also, the CbpA overexpression strain appeared to be less motile and showed a decrease in biofilm formation compared to the control. This study provides the first experimental proof that the binding ligand of the c-di-GMP regulated adhesin CbpA is collagen