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.

BackgroundRecent 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.ResultsIn 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.ConclusionsTo 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.

Many short (<400 bp) interspersed sequence repeats exist in bacteria, yet little is known about their origins, mode of generation, or possible function. Here, we present a comprehensive analysis of 18 different previously identified repeated DNA elements, bcr1-bcr18 (Økstad OA, Hegna I, Lindback T, Rishovd AL, Kolstø AB. 1999. Genome organization is not conserved between Bacillus cereus and Bacillus subtilis. Microbiology. 145:621-631.; Tourasse NJ, Helgason E, Økstad OA, Hegna IK, Kolstø AB. 2006. The Bacillus cereus group: novel aspects of population structure and genome dynamics. J Appl Microbiol. 101:579-593.), in 36 sequenced genomes from the Bacillus cereus group of bacteria. This group consists of genetically closely related species with variable pathogenic specificity toward different hosts and includes among others B. anthracis, B. cereus, and B. thuringiensis. The B. cereus group repeat elements could be classified into three categories with different properties: Group A elements (bcr1-bcr3) exhibited highly variable copy numbers ranging from 4 to 116 copies per strain, showed a nonconserved chromosomal distribution pattern between strains, and displayed several features characteristic of mobile elements. Group B repeats (bcr4-bcr6) were present in 0-10 copies per strain and were associated with strain-specific genes and disruptions of genome synteny, implying a possible contribution to genome rearrangements and/or horizontal gene transfer events. bcr5, in particular, was associated with large gene clusters showing resemblance to integrons. In agreement with their potentially mobile nature or involvement in horizontal transfers, the sequences of the repeats from Groups A and B (bcr1-bcr6) followed a phylogeny different from that of the host strains. Conversely, repeats from Group C (bcr7-bcr18) had a conserved chromosomal location and orthologous gene neighbors in the investigated B. cereus group genomes, and their phylogeny matched that of the host chromosome. Several of the group C repeats exhibited a conserved secondary structure or had parts of the structure conserved, possibly indicating functional RNAs. Accordingly, five of the repeats in group C overlapped regions encoding previously characterized riboswitches. Similarly, other group C repeats could represent novel riboswitches, encode small RNAs, and/or constitute other types of regulatory elements with specific biological functions. The current analysis suggests that the multitude of repeat elements identified in the B. cereus group promote genome dynamics and plasticity and could contribute to the flexible and adaptive life style of these bacteria.

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