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Bacterial adaptation is characterized by a lag-phase during which cells do not multiply and modify their physiology to cope with the constraints of their environment. Our aim was to determine a sequence of events during the lag-phase of growth at low temperature and pH for three Bacillus cereus strains. The onsets of expression of two genes, essential for stress adaptation (cshA coding for a RNA helicase) or involved in the transition between lag and exponential phase (abrB coding for a transition regulator), were determined using fluorescent transcriptional reporter systems. Regardless of the stressing conditions and the tested strains, cshA promoter was active very early, while the biomass increased and always before the first cell division. At 12°C and pH 7.0, onset of cshA promoter activity occurred between 3 h and 7 h, while bacterial counts started between 12 h and 13 h. At pH 5.0 and at 20°C or 30°C, onset of cshA promoter activity occurred before 1 h and earlier than at pH 7.0. In contrast, the onset of abrB promoter activity depended on the strain and stressing conditions. In the ATCC 14579 strain, onset of abrB promoter activity always started between 30 min and 3 h, before biomass increased and cell division occurred. For the other strains, it took place along with the first cell division at 12°C, but much later during growth in the other tested conditions. [ABSTRACT FROM AUTHOR]

The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H2O2 and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation. [ABSTRACT FROM AUTHOR]

The different strains of Bacillus cereus can grow at temperatures covering a very diverse range. Some B. cereus strains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in lowtemperature B. cereus growth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature (Tmin). CasKR was also involved in optimal cold growth above Tmin and in cell survival below Tmin. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the casKR genes in a AcasKR mutant restored its ability to grow at Tmin. Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of the B. cereus group. We show that the role of CasKR in cold growth is similar in other B. cereus sensu lato strains with different growth temperature ranges, including psychrotolerant strains. [ABSTRACT FROM AUTHOR]

In this study, growth rates and lag times of the five RNA helicase-deleted mutants of Bacillus cereus ATCC 14579 were compared to those of the wild-type strain under thermal, oxidative, and pH stresses. Deletion of cshD and cshE had no impact under any of the tested conditions. Deletion of cshA, cshB, and cshC abolished growth at 12°C, confirming previous results. In addition, we found that each RNA helicase had a role in a specific temperature range: deletion of cshA reduced growth at all the tested temperatures up to 45°C, deletion of cshB had impact below 30°C and over 37°C, and deletion of cshC led mainly to a cold-sensitive phenotype. Under oxidative conditions, deletion of cshB and cshC reduced growth rate and increased lag time, while deletion of cshA increased lag time only with H2O2 and reduced growth rate at a high diamide concentration. Growth of the ΔcshA strain was affected at a basic pH independently of the temperature, while these conditions had a limited effect on ΔcshB and ΔcshC strain growth. The RNA helicases CshA, CshB, and CshC could participate in a general adaptation pathway to stressful conditions, with a stronger impact at low temperature and a wider role of CshA. [ABSTRACT FROM AUTHOR]

The mechanisms involved in the ability of Bacillus cereus to multiply at low temperatures were investigated. It was assumed that many genes involved in cold acclimation would be upregulated at low temperatures. Recombinase-based in vivo expression technology (IVET) was adapted to the detection of the transient activation of B. cereus promoters during growth at 10°C. Four independent screenings of a promoter library from type strain ATCC 14579 were performed, and 17 clones were isolated. They corresponded to 17 promoter regions that displayed reproducibly elevated expression at 10°C relative to expression at 30°C. This analysis revealed several genes that may be important for B. cereus to grow successfully under the restrictive conditions of cold habitats. Among them, a locus corresponding to open reading frames BC5402 to BC5398, harboring a lipase-encoding gene and a putative transcriptional regulator, was identified three times. While a mutation in the putative regulator-encoding gene did not cause any particular phenotype, a mutant deficient in the lipase-encoding gene showed reduced growth abilities at low temperatures compared with the parental strain. The mutant did not change its fatty acid profiles in the same way as the wild type when grown at 12°C instead of 37°C. This study demonstrates the feasibility of a promoter trap strategy for identifying cold-induced genes. It outlines a first picture of the different processes involved in B. cereus cold acclimation. [ABSTRACT FROM AUTHOR]