Complementation of cold shock proteins by translation initiation factor IF1 in vivo.

The cold shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins. This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans. For B. subtilis it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock. Analysis of the B. subtilis cspB cspC double deletion mutant revealed that removal of these csp genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores. We show here that heterologous expression of the translation initiation factor IF1 from E. coli in a B. subtilis cspB cspC double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s). Two of the possible explanation models are discussed.     

Expression of cspH, Encoding the Cold Shock Protein in Salmonella enterica Serovar Typhimurium UK-1

Both Salmonella enterica serovar Typhimurium and Escherichia coli contain the cspH gene encoding CspH, one of the cold shock proteins (CSPs). In this study, we investigated the expression of cspH in S. enterica serovar Typhimurium and found that it was induced in response to a temperature downshift during exponential phase. The cspH promoter was activated at 37 degrees C, and its mRNA was more stable than the other csp mRNAs at 37 degrees C. Moreover, lacZ expression of the translational cspH-lacZ fusion was induced at that temperature. Interestingly, the cspH mRNA had a much shorter 5'-untranslated region than those in the other cold-shock-inducible genes, and the promoter sequence, which was only 55 bp, was sufficient for cspH expression. The 14-base downstream box located 12 bases downstream of the initiation codon of cspH mRNA was essential for its cold shock activation.     

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

Like other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity > 70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15°C as well as at 37°C, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37°C and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B . subtilis . After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.     

Cold shock induction of the cspL gene in Lactobacillus plantarum involves transcriptional regulation

Fragments of the cspL promoter region were fused to the gusA reporter and reintroduced into Lactobacillus plantarum cells, either on multicopy plasmids or through single-copy chromosomal integration. beta-Glucuronidase activity and primer extension data demonstrate that the cspL promoter is induced in response to cold shock and that multicopy constructs quench the induction of the resident cspL gene.     

Complementation analysis of the cold-sensitive phenotype of the Escherichia coli csdA deletion strain

The cold shock response of Escherichia coli is elicited by downshift of temperature from 37 degrees C to 15 degrees C and is characterized by induction of several cold shock proteins, including CsdA, during the acclimation phase. CsdA, a DEAD-box protein, has been proposed to participate in a variety of processes, such as ribosome biogenesis, mRNA decay, translation initiation, and gene regulation. It is not clear which of the functions of CsdA play a role in its essential cold shock function or whether all do, and so far no protein has been shown to complement its function in vivo. Our screening of an E. coli genomic library for an in vivo counterpart of CsdA that can compensate for its absence at low temperature revealed only one protein, RhlE, another DEAD-box RNA helicase. We also observed that although not detected in our genetic screening, two cold shock-inducible proteins, namely, CspA, an RNA chaperone, and RNase R, an exonuclease, can also complement the cold shock function of CsdA. Interestingly, the absence of CsdA and RNase R leads to increased sensitivity of the cells to even moderate temperature downshifts. The correlation between the helicase activity of CsdA and the stability of mRNAs of cold-inducible genes was shown using cspA mRNA, which was significantly stabilized in the DeltacsdA cells, an effect counteracted by overexpression of wild-type CsdA or RNase R but not by that of the helicase-deficient mutant of CsdA. These results suggest that the primary role of CsdA in cold acclimation of cells is in mRNA decay and that its helicase activity is pivotal for promoting degradation of mRNAs stabilized at low temperature.     

Loss of expression of cspC, a cold shock family gene, confers a gain of fitness in Escherichia coli K-12 strains

The CspA family of cold shock genes in Escherichia coli K-12 includes nine paralogs, cspA to cspI. Some of them have been implicated in cold stress adaptation. Screening for mutations among common laboratory E. coli strains showed a high degree of genetic diversity in cspC but not in cspA and cspE. This diversity in cspC was due to a wide spectrum of variations including insertions of IS elements, deletion, and point mutation. Northern analysis of these mutants showed loss of cspC expression in all but one case. Further analysis of the loss-of-function cspC mutants showed that they have a fitness advantage in broth culture after 24 h over their isogenic wild-type derivatives. Conversely, introduction of mutated cspC alleles conferred a competitive fitness advantage to AB1157, a commonly used laboratory strain. This provides the evidence that loss of cspC expression is both necessary and sufficient to confer a gain of fitness as seen in broth culture over 24 h. Together, these results ascribe a novel role in cellular growth at 37 degrees C for CspC, a member of the cold shock domain-containing protein family.