Bacillus cereus ATCC 14579 possesses five RNA helicase-encoding genes overexpressed under cold growth conditions. Out of the five corresponding mutants, only the ⌬cshA, ⌬cshB, and ⌬cshC strains were cold sensitive. Growth of the ⌬cshA strain was also reduced at 30°C but not at 37°C. The cold phenotype was restored with the cshA gene for the ⌬cshA strain and partially for the ⌬cshB strain but not for the ⌬cshC strain, suggesting different functions at low temperature.

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 H 2 O 2 and reduced growth rate at a high diamide concentration. Growth of the ⌬cshA strain was affected at a basic pH independently of the temperature, while these conditions had a limited effect on ⌬cshB and ⌬cshC 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.The DEAD-box RNA helicases are encoded by viral, archaeal, eukaryotic, and prokaryotic genomes (9) and play an important role in RNA processing, transport, and degradation and in many other processes involving RNA (4, 19, 26), such as translation or ribosome biogenesis (10,11,22). DEAD-box RNA helicases act as molecular motors that unwind doublestranded RNA, thereby affecting the rearrangement of RNA secondary structures (9, 21). RNA helicases could also be implicated in rearrangement of ribonucleoprotein (RNP) complexes by removing protein from RNA or by the combination of both RNA-unwinding and RNA-annealing activity to promote RNA strand exchange through a potential branch migration (5,13,17,24). Bacterial cells often encounter stressful conditions that tend to decrease the cellular fitness. Consequently, bacteria have to maintain RNA pathway functionalities and control their RNA turnover. Most of the synthesized mRNA is rapidly degraded to allow adaptation to environmental changes (14). RNA helicases could be involved in stress adaptation by maintaining and regulating RNA functions.Studies reporting the involvement of prokaryotic RNA helicases in the adaptation to abiotic stress mainly deal with response to cold, light, and salt conditions (17). The RNA helicase CrhC maintains the photosynthetic capacity of the cyanobacterium Synechocystis. Its expression is regulated by the changes on the redox potential of the electron transport chain caused by variations in light, temperature, and salt concentrations (12). CrhC catalyzes the unwinding of RNA secondary structures but also ensures rearrangements in RNA complexes (5,25). A Bacillus subtilis CshA homolog of Clostridium perfringens is involved in the adaptation to oxidative stress, with the corresponding null mutant strain showing better survival under oxidative stress cond...

The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates the interactions of spores with their environment, modulates spore adhesion and germination, and has been implicated in pathogenesis. In Bacillus cereus, the exosporium consists of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, surrounded by a hairy nap composed of glycoproteins. The morphogenetic protein CotE is necessary for the integrity of the B. cereus exosporium, but how CotE directs exosporium assembly remains unknown. Here, we used super-resolution fluorescence microscopy to follow the localization of SNAP-tagged CotE, CotY, and ExsY during B. cereus sporulation and evidenced the interdependencies among these proteins. Complexes of CotE, CotY, and ExsY are present at all sporulation stages, and the three proteins follow similar localization patterns during endospore formation that are reminiscent of the localization pattern of Bacillus subtilis CotE. We show that B. cereus CotE guides the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process, as previously proposed, but occurs through the formation of two caps, as observed during B. subtilis coat morphogenesis, suggesting that a general principle governs the assembly of the spore surface layers of Bacillaceae. IMPORTANCE Spores of Bacillaceae are enveloped in an outermost glycoprotein layer. In the B. cereus group, encompassing the Bacillus anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance and separation from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize the formation of the exosporium during the sporulation of B. cereus, and we studied the localization and interdependencies of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaffold before a complete exosporium or coat is detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore, and we present a model for the assembly of the exosporium.