ORF 2 from the Bacillus cereus linear plasmid pBClin15 encodes a DNA binding protein.

AIMS: To isolate and identify DNA-binding protein(s) with affinity for the mobile chromosomal repeat element bcr1 in Bacillus cereus group bacteria. METHODS AND RESULTS: A biotinylated bcr1 element was immobilized to streptavidin-coated magnetic beads and used to pull out a 20 kDa DNA-binding protein from a whole cell protein extract of B. cereus ATCC 14579. The protein was identified as the product of ORF 2 encoded by the bacteriophage-related autonomously replicating linear genetic element pBClin15 carried by the strain. DNA binding was not bcr1-specific. By Northern blotting ORF 2 was co-transcribed with ORF 1, and also in certain instances with ORF 3 by transcriptional readthrough of the terminator located between ORF 2 and ORF 3. CONCLUSIONS: ORF 2 from pBClin15 encodes a DNA-binding protein. ORF 2 is co-transcribed with its upstream gene ORF 1, and in a subset of the transcripts also with the downstream gene ORF 3 through alternative transcription termination. SIGNIFICANCE AND IMPACT OF THE STUDY: The B. cereus group contains bacterial species of medical and economic importance. Bacteriophages or phage-encoded proteins from these bacteria have been suggested as potential therapeutic agents. Understanding the biology of bacteriophage-related genetic elements through functional characterization of their genes is of high relevance.

The Bacillus cereus group: novel aspects of population structure and genome dynamics.

AIMS: To provide new insights into the population and genomic structure of the Bacillus cereus group of bacteria. METHODS AND RESULTS: The genetic relatedness among B. cereus group strains was assessed by multilocus sequence typing (MLST) using an optimized scheme based on seven chromosomal housekeeping genes. A set of 48 strains from different clinical sources was included, and six clonal complexes containing several genetically similar isolates from unrelated patients were identified. Interestingly, several clonal groups contained strains that were isolated from similar human sources. Furthermore, comparative whole genome sequence analysis of 16 strains led to the discovery of novel ubiquitous genome features of the B. cereus group, such as atypical group II introns, IStrons, and hitherto uncharacterized repeated elements. CONCLUSIONS: The B. cereus group constitutes a coherent population unified by the presence of ubiquitous and specific genetic elements which do not show any pattern, either in their sequences or genomic locations, which allows to differentiate between the member species of the group. Nevertheless, the population is very dynamic, as particular lineages of clinical origin can evolve to form clonal complexes. At the genome level, the dynamic behaviour is indicated by the presence of numerous mobile and repeated elements. SIGNIFICANCE AND IMPACT OF THE STUDY: The B. cereus group of bacteria comprises species that are of medical and economic importance. The MLST data, along with the primers and protocols used, will be available in a public, web-accessible database (http://mlstoslo.uio.no).

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.

PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis.

Members of the Bacillus cereus group (B. anthracis, B. cereus, B. mycoides and B. thuringiensis) are well-known pathogens of mammals (B. anthracis and B. cereus) and insects (B. thuringiensis). The specific diseases they cause depend on their capacity to produce specific virulence factors, such as the lethal toxin of B. anthracis and the Cry toxins of B. thuringiensis. However, these Bacillus spp. also produce a variety of proteins, such as phospholipases C, which are known to act as virulence factors in various pathogenic bacteria. Few genes encoding these virulence factors have been characterized in pathogenic Bacillus spp. and little is known about the regulation of their expression. We had previously reported that in B. thuringiensis expression of the phosphatidylinositol-specific phospholipase C gene is regulated by the transcriptional activator PlcR. Here we report the identification of several extracellular virulence factor genes by the virtue of their PlcR-regulated expression. These PlcR-regulated genes encode degradative enzymes, cell-surface proteins and enterotoxins. The PlcR-regulated genes are widely dispersed on the chromosome and therefore do not constitute a pathogenic island. Analysis of the promoter region of the PlcR-regulated genes revealed the presence of a highly conserved palindromic region (TATGNAN4TNCATA), which is presumably the specific recognition target for PlcR activation. We found that the plcR gene is also present in and probably restricted to all the members of the B. cereus group. However, although the polypeptide encoded by the B. cereus PlcR gene is functionally equivalent to the B. thuringiensis regulator, the polypeptide encoded by the B. anthracis gene is truncated and not active as a transcriptional activator. PlcR is the first example described of a pleiotropic regulator involved in the control of extracellular virulence factor expression in pathogenic Bacillus spp. These results have implications for the taxonomic relationships among members of the B. cereus group, the virulence properties of these bacteria and the safety of B. thuringiensis-based biopesticides.

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 pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 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 pAMbeta1 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.

Insertional inactivation of a Tet(K)/Tet(L) like transporter does not eliminate tetracycline resistance in Bacillus cereus.

Bacillus cereus ATCC 10987 and ATCC 14579 can be induced to high levels of resistance to tetracycline. The chromosomal B. cereus gene bctl encodes a transmembrane protein with homology to Gram-positive tetracycline efflux proteins and relation to other members of the major facilitator superfamily of transport proteins. A mutant strain containing an insertionally inactivated bctl gene did not show impaired tetracycline resistance. No additional altered phenotype was observed in the mutant. Accumulation studies suggested that the resistance mechanism involves a reduced sensitivity to intracellular tetracycline.