CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone, which is thought to facilitate translation at low temperature by destabilizing mRNA structures. Here we demonstrate that CspA, as well as homologous RNA chaperones CspE and CspC, are transcription antiterminators. In vitro, the addition of physiological concentrations of recombinant CspA, CspE, or CspC decreased transcription termination at several intrinsic terminators and also decreased transcription pausing. In vivo, overexpression of cloned CspC and CspE at 37°C was sufficient to induce transcription of the metY-rpsO operon genes nusA, infB, rbfA, and pnp located downstream of multiple transcription terminators. Similar induction of downstream metY-rpsO operon genes was observed at cold shock, a condition to which the cell responds by massive overproduction of CspA. The products of nusA, infB, rbfA, and pnp-NusA, IF2, RbfA, and PNP-are known to be induced at cold shock. We propose that the cold-shock induction of nusA, infB, rbfA, and pnp occurs through transcription antitermination, which is mediated by CspA and other cold shock-induced Csp proteins.CspA proteins ͉ cold shock ͉ transcription antitermination

SummaryThe gene for CspA, the major cold-shock protein of Escherichia coli is known to be dramatically induced upon temperature downshift. Here, we report that three-base substitutions around the Shine-Dalgarno sequence in the 159-base 5Ј-untranslated region of the cspA mRNA stabilizes the mRNA 150-fold, resulting in constitutive expression of cspA at 37ЊC. This stabilization was found to be at least partially due to resistance against RNase E degradation. The coldshock induction of cspA was also achieved by exchanging its promoter with the non-cold-shock lpp promoter. The results presented indicate that the cspA gene is efficiently transcribed even at 37ЊC. However, the translation of the cspA mRNA is blocked because of its extreme instability at 37ЊC. The presented results also demonstrate that the cspA gene is constitutively transcribed at all temperatures; however, its expression at 37ЊC is prevented by destabilizing its mRNA.

When the gene for CspA, the major cold shock protein of Escherichia coli, was disrupted by a novel positive/negative selection method, the ⌬cspA cells did not show any discernible growth defect at either 37 or 15°C. By two-dimensional gel electrophoresis, total protein synthesis was analyzed after temperature downshift in the ⌬cspA strain. The production of the CspA homologs CspB and CspG increased, and the duration of their expression was prolonged, suggesting that both CspB and CspG compensate for the function of CspA in the absence of CspA during cold shock adaptation. Interestingly, the production of the 159-base 5-untranslated region (5-UTR) of cspA from the chromosomal cspA::cat gene, detected by primer extension, failed to be repressed after cold shock. When an independent system to produce CspA was added to the ⌬cspA strain, the 5-UTR production for the cspA::cat gene was significantly reduced compared to that of the ⌬cspA strain. By examining the expression of translationally fused cspA and cspB genes to lacZ in the ⌬cspA strain, it was found that cspA is more strongly regulated by CspA than cspB is. We showed that the increased expression of the 5-UTR of the cspA mRNA in the ⌬cspA strain occurred mainly at the level of transcription and, to a certain extent, at the level of mRNA stabilization. The mRNA stabilization in the ⌬cspA strain was observed for other mRNAs, supporting the notion that CspA functions as an mRNA chaperone to destabilize secondary structures in mRNAs.

Escherichia coli contains nine members of the CspA protein family from CspA to CspI. To elucidate the cellular function of CspE, we constructed a ΔcspE strain. CspE is highly produced at 37°C. The synthesis level of CspE transiently increased during the growth lag period after dilution of stationary‐phase cells into the fresh medium at 37°C. This is consistent with the ΔcspE phenotype of the longer growth lag period after dilution. The protein synthesis patterns of the ΔcspE strain and the wild‐type strain were compared using two‐dimensional gel electrophoresis. In the ΔcspE strain, the synthesis of a number of proteins at 37°C was found to be altered and cspA was derepressed. The derepression of cspA in the ΔcspE strain was at the level of transcription in a promoter‐independent fashion but was not caused by stabilization of the cspA mRNA, which was shown to be a major cause of CspA induction after cold shock. In vitro transcription assays demonstrated that both CspE and CspA enhanced transcription pause at the region immediately downstream of the cold box, a putative repressor binding site on the cspA mRNA. In a cell‐free protein synthesis system using S‐30 cell extracts, CspA production was specifically inhibited by the addition of CspE. These results indicate that CspE functions as a negative regulator for cspA expression at 37°C, probably by interacting with the transcription elongation complex at the cspA cold box region.

The yycFG two-component signal transduction system (TCSTS) has been shown to be essential to the viability of several gram-positive bacteria. However, the function of the gene pair remains unknown. Interestingly, while both components are essential to Staphylococcus aureus and Bacillus subtilis, only the response regulator (yycF) is essential to Streptococcus pneumoniae. To study this essential TCSTS further, the S. pneumoniae and S. aureus truncated YycG histidine kinase and full-length YycF response regulator proteins were characterized at a biochemical level. The recombinant proteins from both organisms were expressed in Escherichia coli and purified. The YycG autophosphorylation activities were activated by ammonium. The apparent K_m (ATP) of S. aureus YycG autophosphorylation was 130 µM and S. pneumoniae was 3.0 µM. Each had similar k_cat values of 0.036 and 0.024 min^–1, respectively. Cognate phosphotransfer was also investigated indicating different levels of the phosphorylated YycG intermediates during the reaction. The S. pneumoniae YycG phosphorylated intermediate was not detectable in the presence of its cognate YycF, while phosphorylated S. aureus YycG and YycF were detected concurrently. In addition, noncognate phosphotransfer was demonstrated between the two species. These studies thoroughly compare the essential YycFG TCSTS from the two species at the biochemical level and also establish methods for assaying the activities of these antibacterial targets.

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