Complex regulation of the synthesis of the compatible solute ectoine in the halophilic bacterium Chromohalobacter salexigens DSM 3043T

Abstract

The synthesis of the compatible solute ectoine, mediated by the ectABC gene products, is the main mechanism used by the halophilic bacterium Chromohalobacter salexigens to cope with osmotic stress. Evidence was found that this process is regulated at the transcriptional level. S1 protection analyses performed with RNA extracted from cells grown in minimal medium at low (0?75 M NaCl) or high (2?5 M NaCl) osmolarity suggested the existence of four promoters upstream of ectA. Two of these (PectA1 and PectA2) might be recognized by the main vegetative sigma factor s 70 , and one (PectA3) might be dependent on the general stress sigma factor s S .The S1 protection assays suggest that PectA1 and PectA3 may be osmoregulated promoters. In addition, an internal promoter showing sequences homologous to promoters dependent on the heat-shock sigma factor s 32 was found upstream of ectB. Transcription from PectA in C. salexigens followed a pattern typical of s S -dependent promoters, and was reduced by 50 % in an E. coli rpoS background. These data strongly suggest the involvement of the general stress sigma factor s S in ectABC transcription in C. salexigens. Expression of PectA-lacZ and PectB-lacZ trancriptional fusions was very high at low salinity, suggesting that ectABC may be a partially constitutive system. Both transcriptional fusions were induced during continuous growth at high temperature and their expression was reduced in cells grown in the presence of osmoprotectants (ectoine or glycine betaine) or the DNA gyrase inhibitor nalidixic acid. Moreover, PectA-lacZ expression was negatively modulated in cells grown with an excess of iron (FeCl 3 ). Measurement of ectoine levels in the presence of glycine betaine at different NaCl concentrations suggests that an additional post-transcriptional control may occur as well.

Genetic and Biochemical Aspects of Ectoine Biosynthesis in Moderately Halophilic and Halotolerant Methylotrophic Bacteria

“…It was demonstrated that in C. salexigens and Streptomyces griseus, intracellular levels of ectoine increase in response to high growth temperature (Calderon et al, 2004;Malin and Lapidot, 1996). These findings suggest a role of ectoine as a protectant against the detrimental effects of high temperatures.…”

“…Participation of different transcriptional sigma factors in expression of the ect-genes was not confirmed by an appropriate genetic study. Supplementation of the growth media with ectoine or glycine betaine decreased the transcription of PectA-lacZ and PectB-lacZ (Calderon et al, 2004). Altogether with constitutive ect-genes transcription from PectA and PectB at low osmolarity, this implies an involvement of other transcriptional factors.…”

“…Four putative promoters (РectA1-4) are located upstream of the ectA gene and one internal promoter PectB is upstream of the ectB gene (Calderon et al, 2004). The consensus sequences of the ect promoters closely resembled those of σ 70 -(РectA1 and PectA2), σ S -(PectA3) and σ 32 -(PectB) dependent promoters of E. coli.…”

“…In Bacillus pasteurii (Kuhlmann and Bremer, 2002), Marinococcus halophilus (Bestvater and Galinski, 2002), Halobacillus halophilus (Saum and Muller, 2008b) and S. salexigens (Bursy et al, 2007), transcription of the ectABC-ask or ectABC genes proceeded as polycistronic mRNAs and subjected to osmotic conditions. The osmoregulated expression of ectABC was also revealed in gramnegative Brevibacterium epidermis (Onraedt et al, 2004), Mm alcaliphilum (Reshetnikov et al, 2006;Mustakhimov et al, 2010) and Chromohalobacter salexigens (Calderon et al, 2004).…”

“…This was consistent with maximal ectoine accumulation in the stationary growth phase (Canovas et al, 1999). It could be reasonable to propose an involvement of the general stress factor σ S , however, no rpoS-like gene has been identified in C. salexigens (Calderon et al, 2004). Participation of different transcriptional sigma factors in expression of the ect-genes was not confirmed by an appropriate genetic study.…”