Cold-induced transcripts have a long 5′ UTR containing cold-box elements described in E. coli, B. subtilis or archeabacteria (Jiang et al., 1996; Chamot et al., 1999; Hunger et al., 2006). These cis-elements modulate the stability of cold-induced mRNAs at low temperatures (Gualerzi et al., 2003). Analysis of BC0259 5′-UTR revealed the presence of such cold-box elements, with (1) one box possibly located downstream of the +1 of transcription (thus on BC0259 mRNA) and (2) two other conserved Erastin sequences located upstream from the +1 of transcription. The significance of these sequences upstream of the BC0259 promoter remains to be determined. However, the role of cold boxes in
the transcriptional regulation of cold genes has already been suggested elsewhere (Fang et al., 1997; Mitta et al., 1997). The cold phenotype of the 9H2 mutant is not due to a complete defect of RNA helicase encoding gene expression, as reported previously in other species with knockout mutants (Charollais et al., 2004; Ando & Nakamura, 2006). This study clearly shows that the expression level of the BC0259 gene and consequently the amount of transcripts in the cell has a huge impact on low-temperature adaptation of B. cereus. BC0259 was expressed at a higher level (about twofold) in WT cells grown at OD600 nm=0.2 when compared with cells grown at OD600 nm=1.0.
This suggests the importance of this gene during this stage of the kinetics of growth, Amobarbital and is in agreement with the growth defect observed with the 9H2 mutant during the find more lag phase. Four other RNA helicase-encoding genes are present in the B. cereus ATCC 14579 genome and may play a role in cold adaptation. Yet, they did not totally
counteract the effect of mutation in the BC0259 gene at 10 °C. The 9H2 mutant survived better than WT at a nonpermissive growth temperature, suggesting that the lower amount of BC0259 in the mutant had a positive effect on survival. Survival was improved in the presence of chloramphenicol for both the WT and the mutant, showing that a limited amount of protein synthesis was required for survival. Moreover, it has been shown that addition of chloramphenicol increases the level of cspA transcripts (Jiang et al., 1993), which is also dramatically induced in an E. coli RNA-helicase csdA mutant (Yamanaka & Inouye, 2001). This may suggest interactions between Csp and RNA helicases in B. cereus as described in B. subtilis (Hunger et al., 2006). Our transpositional approach revealed several genes that were clearly involved in low-temperature adaptation, with some also implicated in pH or salt stresses, suggesting possible cross responses in the adaptive potential of B. cereus ATCC 14579. This study also emphasizes the important role played by a DEAD-box RNA helicase in the cold-adaptive response of B. cereus, and further research is now needed to define the molecular function of this protein.