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bcr1 is a chromosomal ~155 bp repeated element found uniquely and ubiquitously in the Bacillus cereus group of Gram-positive bacteria; it exhibits several features characteristic of mobile elements, including a variable distribution pattern between strains. Here, highly similar bcr1 elements in non-conserved genomic loci are identified in a set of closely related B. cereus and Bacillus thuringiensis strains near the Bacillus anthracis phylogenetic cluster. It is also shown that bcr1 may be present on small RNA transcripts in the 100-400 bp size range. In silico folding of bcr1 at the RNA level indicated that transcripts may form a double-hairpin-like structure predicted to have high structural stability. A functional role of bcr1 at the RNA level is supported by multiple cases of G--U base-pairing, and compensatory mutations maintaining structural stability of the RNA fold. In silico folding at the DNA level produced similar predicted structures, with the potential to form a cruciform structure at open DNA complexes. The predicted structural stability was greater for bcr1 elements showing high sequence identities to bcr1 elements in non-conserved chromosomal loci in other strains, relative to other bcr1 copies, bcr1 mobility could thus be dependent on the formation of a stable DNA or RNA intermediate. Furthermore, bcr1 elements potentially encoding structurally stable and less stable transcripts were phylogenetically intermixed, indicating that loss of bcr1 mobility may have occurred multiple times during evolution. Repeated elements with similar features in other bacteria have been shown to provide functions such as mRNA stabilization, transcription termination and/or promoter function. Similarly, bcr1 may constitute a mobile element which occasionally gains a function when it enters an appropriate chromosomal locus.

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 eDNA to a 70-mer oligonueleotide 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.

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical ~272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one ~270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXOl-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Piasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.

A combination of sequence and structure analysis and reverse transcriptase PCR experiments was used to characterize the group II introns in the complete genomes of two strains of the pathogen Bacillus cereus. While B. cereus ATCC 14579 harbors a single intron element in the chromosome, B. cereus ATCC 10987 contains three introns in the chromosome and four in its 208-kb pBc10987 plasmid. The most striking finding is the presence in B. cereus ATCC 10987 of an intron [B.c.I2(a)] located on the reverse strand of a gene encoding a putative cell surface protein which appears to be correlated to strains of clinical origin. Because of the opposite orientation of B.c.I2(a), the gene is disrupted. Even more striking is that B.c.I2(a) splices out of an RNA transcript corresponding to the opposite DNA strand. All other intragenic introns studied here are inserted in the same orientation as their host genes and splice out of the mRNA in vivo, setting the flanking exons in frame. Noticeably, B.c.I3 in B. cereus ATCC 10987 represents the first example of a group II intron entirely included within a conserved replication gene, namely, the [alpha] subunit of DNA polymerase III. Another striking finding is that the observed 3' splice site of B.c.I4 occurs 56 bp after the predicted end of the intron. This apparently unusual splicing mechanism may be related to structural irregularities in the 3' terminus. Finally, we also show that the intergenic introns of B. cereus ATCC 10987 are transcribed with their upstream genes and do splice in vivo.

Bacillus cereus strains ATCC 10987 and ATCC 14579 harbor a ~155-hp repeated element, bcr1, which is conserved in B. cereus, B. anthracis, B. thuringiensis, and B. mycoides but not in B. subtilis and B. licheniformis. In this study, we show by Southern blot hybridizations that bcr1 is present in all 54 B. cereus group strains tested but absent in 11 Bacillus strains outside the group, suggesting that bcr1 may be specific and ubiquitous to the B. cereus group. By comparative analysis of the complete genome sequences of B. cereus ATCC 10987, B. cereus ATCC 14579, and B. anthracis Ames, we show that bcr1 is exclusively present in the chromosome but absent from large plasmids carried by these strains and that the numbers of full-length bcrl repeats for these strains are 79, 54, and 12, respectively. Numerous copies of partial bcr1 elements are also present in the three genomes (91, 128, and 53, respectively). Furthermore, the genomic localization of bcr1 is not conserved between strains with respect to chromosomal position or organization of gene neighbors, as only six full-length bcr1 loci are common to at least two of the three strains. However, the intergenic sequence surrounding a specific bcr1 repeat in one of the three strains is generally strongly conserved in the other two, even in loci where bcr1 is found exclusively in one strain. This finding indicates that bcr1 either has evolved by differential deletion from a very high number of repeats in a common ancestor to the B. cereus group or is moving around the chromosome. The identification of bcr1 repeats interrupting genes in B. cereus ATCC 10987 and ATCC 14579 and the presence of a flanking TITAT motif in each end show that bcr1 exhibits features characteristic of a mobile element.

Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis are three closely related species of the B.cereus group with each of them being economically important and their DNA isolates were analyzed using fluorescent amplified fragment length polymorphism (AFLP). Phytogenetic analysis of AFLP data showed diversity with B.thuringinesis and B.cereus compared to the monomorphic nature of B.anthracis.

Author(s): Timothy D. Read (corresponding author) [1, 2]; Scott N. Peterson [1, 3]; Nicolas Tourasse [4, 7]; Les W. Baillie [1, 2, 5]; Ian T. Paulsen [1, 6]; Karen E. [...]

Researchers used fluorescent amplified fragment length polymorphism to analyze 154 Bacillus cereus and Bacillus thuringiensis isolates taken from five different locations and compare them to reference strains. This method can be automated, allowing scientists to process very large samples.

Research has been conducted on the members of Bacillus cereus group of bacteria. Results indicate that B. anthracis is closely related to B. cereus and B. thuringiensis strains.

Research was conducted to investigate the effect of inactivation on the toxicity of haemolysin BL in a mutant of Bacillus cereus. Results show that inactivation of the first gene of the hbl operon reduces the enterotoxigeneic and haemolytic activity against sheep erythrocytes.

Research was conducted to characterize the PlcR-regulated three virulence gene loci employing cloning and sequencing techniques. The data from analyzing a 37.1 kb DNA sequence surrounding hbl, plcA and plcR genes indicated they were not involved in the virulence process.

DNA sequence analysis was done on the cloned 1.9 kb Bacillus cereus gene encoding a protein of 635 amino acids (aa) that is homologous to the SNF2 family of putative helicases which are involved in transcriptional control, DNA repair or maintenance of chromosome stability. There were two open-reading frames identified, orf-1 composed of 981 nucleotides (nt) and the 3207-nt Bcsnf. The latter predicted a protein of 1064 aa where the conserved SNF2 domain was found at the carboxy terminus while its 633 amino-terminal aa exhibited no homology with any protein in the databases.

The physical and genetic mapping of Bacillus (B) thuringiensis subsp. berliner 1715 chromosome reveals 24 restriction enzyme sites and 28 probes. The genes are nonrandomly located on the 5.7 Mb chromosome. A comparison of the genetic maps of B. thuringiensis and B. cereus shows plasmid-encoded genes on the more variable half of the chromosome and rRNA genes and origin of replication on the other conserved half. The cryIA or Tn4430 probes' hybridization indicates large extrachromosomal DNA elements which show genome size to be at least 6.9 Mb.

The chromosome of the green sulfur bacterium Chlorobium tepidum has been mapped and contains fifteen genes and operons, most of which express proteins that play a role in photosynthesis. Researchers used restriction enzyme digestion with XbaI and PacI, pulsed-gel electrophoresis, and hybridization to fragments to construct a map of the chromosome. The size of the chromosome is approximately 2,150 kb.

Pulsed-field gel electrophoresis and multilocus enzyme electrophoresis analyses of 24 Bacillus thuringiensis and 12 B. cereus heterogenesis strains indicate that both belong to the same species. Seven B. cereus strains encode the DNA homologous to the Tn4430 transposon probe of B. thuringiensis, with all the strains containing the NotI restriction enzyme that affects digestion.

A physical map of a Bacillus thuringiensis strain was presented. The size of the chromosome was 5.4 Mb. Like Bacillus cereus strains, B. thuringiensis strains show different NotI fingerprint patterns. It was also demonstrated that several of the B. thuringiensis strains produce enterotoxins.It was concluded that the taxonomy of B. cereus and B. thuringiensis should be reevaluated.

Physical maps of Bacillus cereus strains ATCC 10876, ATCC 11778 and ATCC 14579 (type strain) were constructed using pulsed-field gel electrophoresis. Restriction fragment analysis revealed 10 to 14 NotI fragments whose sizes ranged from 15 to 1,300 kb. The three genomes ranged in size from 5.4 to 6.3 megabases. Comparisons with a previously mapped strain ATCC 10987 showed that all four strains exhibit divergent NotI restriction patterns. Additional differentiation was achieved using DNA probes obtained from ATCC 10987 sequences.

Results reveal that the population structure of the Bacillus cereus group is weakly clonal as determined by a multilocus sequence typing. Data indicae that the five isolates of B. anthracis display the same sequence types.

Eighty kb of genome sequence sampled from various regions of the Bacillus cereus ATCC 10987 chromosome was investigated. Examination of the sequence and comparison of the localization of the putative genes with that of B. subtilis orthologues revealed that gene organization is not conserved between B. cereus and B. subtilis, several putative genes are more closely related to genes from other bacteria and archaea than to B. subtilis, and B. cereus has a 155 bp repetitive sequence that is not found in B. subtilis.

Presents a physical map of the chromosome of Chlorobium tepidum. Estimation of chromosome size; Hybridization of csmDE, csmAC, mdh, petBC and rRNA genes; Analysis of results from partial digestion with the restriction endonuclease XbaI.