Characterization of cspB, a Bacillus subtilis inducible cold shock gene affecting cell viability at low temperatures

Abstract

A new class of cold shock-induced proteins that may be involved in an adaptive process required for cell viability at low temperatures or may function as antifreeze proteins in Escherichia coli and Saccharomyces cerevisiae has been identified. We purified a small Bacillus subtilis cold shock protein (CspB) and determined its amino-terminal sequence. By using mixed degenerate oligonucleotides, the corresponding gene (cspB) was cloned on two overlapping fragments of 5 and 6 kb. The gene encodes an acidic 67-amino-acid protein (pI 4.31) with a predicted molecular mass of 7,365 Da. Nucleotide and deduced amino acid sequence comparisons revealed 61% identity to the major cold shock protein of E. coli and 43% identity to a family of eukaryotic DNA binding proteins. Northern RNA blot and primer extension studies indicated the presence of one cspB transcript that was initiated 119 bp upstream of the initiation codon and was found to be induced severalfold when exponentially growing B. subtilis cell cultures were transferred from 37 degrees C to 10 degrees C. Consistent with this cold shock induction of cspB mRNA, a six- to eightfold induction of a cspB-directed beta-galactosidase synthesis was observed upon downshift in temperature. To investigate the function of CspB, we inactivated the cold shock protein by replacing the cspB gene in the B. subtilis chromosome with a cat-interrupted copy (cspB::cat) by marker replacement recombination. The viability of cells of this mutant strain, GW1, at freezing temperatures was strongly affected. However, the effect of having no CspB in GW1 could be slightly compensated for when cells were preincubated at 10 degrees C before freezing. These results indicate that CspB belongs to a new type of stress-inducible proteins that might be able to protect B. subtilis cells from damage caused by ice crystal formation during freezing.

A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures

“…Fifteen hours after reaching the stationary phase, about one-third of the cells from the wild type had lysed, as judged from the optical density. Thereafter, the (Willimsky et al, 1992) was chosen as a reference. Sequence of cspD from Capuano et al (1996).…”

“…A Zap II library (Stratagene) containing JH642 chromosomal DNA was screened with a radiolabelled cspC probe from pcspC as described in Willimsky et al (1992). Six independent clones were isolated and inserts were analysed by restriction mapping and Southern blotting.…”

“…Transcription starts of cspC and cspD were obtained by primer extension using CspCextP (complementary to sequence from ¹32 to ¹15) for cspC and using CspDextP (¹12 to þ9) for cspD as described in Willimsky et al (1992).…”

“…In addition, a recent report suggests that CIPs can be translated under conditions in which general protein synthesis is blocked as a result of transient malfunction of ribosomes , indicating that mRNA of CIPs (including CSPs) may be more translatable after cold shock relative to mRNA coding for other proteins. Ensuing release of CSP induction was shown to target, in part, the unusually long untranslated 5Ј mRNA leader region (UTR) of cspA (160 bases) in E. coli , which is also present in cspB and in cspG of E. coli (approximately 150 b) as well as in cspB of B. subtilis (119 b) (Willimsky et al, 1992). Overproduction of mRNA containing the first 25 bases of the cspA UTR resulted in prolonged induction of CSPs after cold shock, which was normalized by simultaneous overproduction of CspA .…”

Identification and freedom to operate analysis of potential genes for drought tolerance in maize

“…The third selected gene is CspB, which codes for a coldshock induced protein involved in cellular adaptation to low temperature in Escherichia coli and Saccharomyces cerevisiae and has also been identified in Bacillus subtilis (Willimsky et al, 1992). Cold-shock proteins are nucleic acid-binding chaperones that are induced to stabilize mRNA after a significant drop in temperature (Mazzon et al, 2012).…”