Cold-Induced Putative DEAD Box RNA Helicases CshA and CshB Are Essential for Cold Adaptation and Interact with Cold Shock Protein B in Bacillus subtilis

Abstract

The nucleic acid binding cold shock proteins (CSPs) and the cold-induced DEAD box RNA helicases have been proposed separately to act as RNA chaperones, but no experimental evidence has been reported on a direct cooperation. To investigate the possible interaction of the putative RNA helicases CshA and CshB and the CSPs from Bacillus subtilis during cold shock, we performed genetic as well as fluorescence resonance energy transfer (FRET) experiments. Both cshA and cshB genes could be deleted only in the presence of a cshB copy in trans, showing that the presence of one csh gene is essential for viability. The combined gene deletion of cshB and cspD resulted in a cold-sensitive phenotype that was not observed for either helicase or csp single mutants. In addition to the colocalization of the putative helicases CshA and CshB with CspB and the ribosomes in areas surrounding the nucleoid, we detected a strong FRET interaction in vivo between CshB and CspB that depended on active transcription. In contrast, a FRET interaction was not observed for CshB and the ribosomal protein L1. Therefore, we propose a model in which the putative cold-induced helicases and the CSPs work in conjunction to rescue misfolded mRNA molecules and maintain proper initiation of translation at low temperatures in B. subtilis.A sudden decrease in temperature is a serious challenge for all living microorganisms. The cellular reaction required for efficient adaptation to low temperatures is termed cold shock response. The cell has to cope with changes at different physiological levels, such as reduced fluidity of the membrane and slowdown of protein synthesis and protein folding (39). Accordingly, a general decrease of protein synthesis was observed in Escherichia coli and Bacillus subtilis cells following a cold shock (9, 17). The stress arising from a decrease in temperature can in principle be traced back to a reduction in molecular dynamics. The initiation of translation was shown to be a limiting factor for bacterial growth at low temperatures (3, 6). In addition to the delicate interaction of the complex translation machinery, the formation of secondary structures of mRNA prevents efficient initiation of translation (12). The tendency of RNA to fold and become kinetically trapped was described as a general problem and interferes with proper biological function (14). The formation of unfavorable secondary structures is even more intricate at lower temperatures. In analogy to protein folding mechanisms, RNA chaperones have been proposed to resolve and prevent misfolding of RNA molecules by either destabilizing RNA duplexes or binding to singlestranded nucleic acids (21). In the cold shock response, DEAD box RNA helicases have already been identified to destabilize RNA duplexes and the major cold shock proteins (CSPs) to bind RNA. DEAD box RNA helicases belong to the large family of DExD/H-box proteins, which generally have an RNA-dependent ATPase activity in vitro. They are involved in various cellular processes, such as ribosome assembly, i...

Bacterial RNA Chaperones Confer Abiotic Stress Tolerance in Plants and Improved Grain Yield in Maize under Water-Limited Conditions

“…The chaperone function of CspA, as well as other CSP proteins, is thought to be important for stimulating growth following stress acclimation and during periods of high metabolic activity. A recently proposed model describes CSPs working in conjunction with a DEAD box helicase to rescue misfolded mRNA molecules and maintain proper initiation of translation (Hunger et al, 2006). This model is consistent with previously reported evidence for the colocalization of CSPs with ribosomes in transcriptionally active cells, where CSP proteins are proposed to be implicated in coupling transcription with translation (Mascarenhas et al, 2001;Weber et al, 2001;for review, see El-Sharoud and Graumann, 2007).…”

Mutational Analysis of the Escherichia coli DEAD Box Protein CsdA

“…These data are consistent with a physiological function of cold shock protein CsdA as an RNA helicase at low temperature. Bacillus subtilis, cyanobacteria, the Antarctic archaeon Methanococcoides burtonii, Hordeum vulgare, Pisum sativum, and Arabidopsis plants also increase the synthesis of a DEAD box protein upon a shift to low temperature (6,13,20,25,31,37). These observations suggest an evolutionarily conserved requirement for helicase function to optimize cellular growth at low temperatures.…”

Cyanobacterial RNA Helicase CrhR Localizes to the Thylakoid Membrane Region and Cosediments with Degradosome and Polysome Complexes in Synechocystis sp. Strain PCC 6803

“…Although RNA helicases do not contain canonical membrane-spanning domains, they are membrane associated in some bacteria (12,21,22) but not all (13). For example, RNA helicases associated with RNA degradosomes localize to the cytoplasmic membrane via RNase E in E. coli (4,12) and RNase Y in B. subtilis (5) whereas CshA and CshB colocalize with CspB and ribosomes in areas surrounding the B. subtilis nucleoid, the localization being dependent on active transcription (23). Localization of these RNA helicase-containing complexes to specific cellular sites therefore confines the associated processes to restricted cellular regions.…”