Sangita Phadtare

Cold-shock response and cold-shock proteins.

Both prokaryotes and eukaryotes exhibit a cold-shock response upon an abrupt temperature downshift. Cold-shock proteins are synthesized to overcome the deleterious effects of cold shock. CspA, the major cold-shock protein of Escherichia coli, has recently been studied with respect to its structure, function and regulation at the level of transcription, translation and mRNA stability. Homologues of CspA are present in a number of bacteria. Widespread distribution, ancient origin, involvement in the protein translational machinery of the cell and the existence of multiple families in many organisms suggest that these proteins are indispensable for survival during cold-shock acclimation and that they are probably also important for growth under optimal conditions.

Cold-shock induced high-yield protein production in Escherichia coli

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.

Genome-Wide Transcriptional Analysis of the Cold Shock Response in Wild-Type and Cold-Sensitive, Quadruple-csp-Deletion Strains of Escherichia coli

A DNA microarray-based global transcript profiling of Escherichia coli in response to cold shock showed that in addition to the known cold shock-inducible genes, new genes such as the flagellar operon, those encoding proteins involved in sugar transport and metabolism, and remarkably, genes encoding certain heat shock proteins are induced by cold shock. In the light of strong reduction in metabolic activity of the cell after temperature downshift, the induction of sugar metabolism machinery is unexpected. The deletion of four csps (cspA, cspB, cspG, and cspE) affected cold shock induction of mostly those genes that are transiently induced in the acclimation phase, emphasizing that CspA homologues are essential in the acclimation phase. Relevance of these findings with respect to the known RNA chaperone function of CspA homologues is discussed.

Role of CspC and CspE in Regulation of Expression of RpoS and UspA, the Stress Response Proteins in Escherichia coli

Nine homologous proteins, CspA to CspI, constitute the CspA family of Escherichia coli. Recent studies are aimed at elucidating the individual cellular functions of these proteins. Two members of this family, CspC and CspE, are constitutively produced at 37 degrees C. In the present study, these two proteins were evaluated for their cellular role(s). The expression of three stress proteins, OsmY, Dps, and UspA, is significantly affected by the overexpression and deletion of CspC and CspE. RpoS is a regulatory element for osmY and dps. Further analysis showed a larger amount and greater stability of the rpoS mRNA as well as a higher level of RpoS itself with the overexpression of CspC and CspE. This suggests that CspC and CspE upregulate the expression of OsmY and Dps by regulating the expression of RpoS itself. Indeed, this upregulation is lost in the Delta rpoS strain. Other RpoS-controlled proteins such as ProP and KatG, are also upregulated by the overexpression of CspC. The present study suggests that CspC and CspE are the important elements involved in the regulation of the expression of RpoS, a global stress response regulator, and UspA, a protein responding to numerous stresses. In the light of these observations, it seems plausible that CspC and CspE function as regulatory elements for the expression of stress proteins in the complex stress response network of E. coli.

Sequence‐selective interactions with RNA by CspB, CspC and CspE, members of the CspA family of Escherichia coli

The CspA family of Escherichia coli comprises nine homologous proteins, CspA to CspI. CspA, the major cold shock protein, binds RNA with low sequence specificity and low binding affinity. This is considered to be important for its proposed function as an RNA chaperone to prevent the formation of secondary structures in RNA molecules, thus facilitating translation at low temperature. The cellular functions of other Csp proteins are yet to be fully elucidated, and their sequence specific binding capabilities have not been identified. As a step towards identification of the target genes of Csp proteins, we investigated the RNA binding specificities of CspB, CspC and CspE by an in vitro selection approach (SELEX). In the present study, we show that these proteins are able to bind preferentially to specific RNA/single‐stranded DNA sequences. The consensus sequences for CspB, CspC and CspE are U/T stretches, AGGGAGGGA and AU/AT‐rich regions, especially AAAUUU, respectively. CspE and CspB have Kd values in the range 0.23–0.9 × 10−6 M, while CspC has 10‐fold lower binding affinity. Consistent with our recent findings of transcriptional regulation of cspA by CspE, we have identified a motif identical to the CspE consensus. This motif is the putative CspE‐mediated transcription pause recognition site in a 5′‐untranslated region of the cspA mRNA.

The mRNA interferases, MazF-mt3 and MazF-mt7 from Mycobacterium tuberculosis target unique pentad sequences in single-stranded RNA

mRNA interferases are sequence‐specific endoribonucleases encoded by toxin‐antitoxin (TA) systems in bacterial genomes. Previously, we demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF‐mt1 to ‐mt7) and determined cleavage specificities for MazF‐mt1 and MazF‐mt6. Here we have developed a new general method for the determination of recognition sequences longer than three bases for mRNA interferases with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. Using this method, we determined that MazF‐mt3 cleaves RNA at UU˘CCU or CU˘CCU and MazF‐mt7 at U˘CGCU (˘indicates the cleavage site). As pentad sequence recognition is more specific than those of previously characterized mRNA interferases, bioinformatics analysis was carried out to identify M. tuberculosis mRNAs that may be resistant to MazF‐mt3 and MazF‐mt7 cleavage. The pentad sequence was found to be significantly underrepresented in several genes, including members of the PE and PPE families, large families of proteins that play a role in tuberculosis immunity and pathogenesis. These data suggest that MazF‐mt3 and MazF‐mt7 or other mRNA interferases that target longer RNA sequences may alter protein expression through differential mRNA degradation, a regulatory mechanism that may allow adaptation to environmental conditions, including those encountered by pathogens such as M. tuberculosis during infection.

RNA remodeling and gene regulation by cold shock proteins.

One of the many important consequences that temperature down-shift has on cells is stabilization of secondary structures of RNAs. This stabilization has wide-spread effects, such as inhibition of expression of several genes due to termination of their transcription and inefficient RNA degradation that adversely affect cell growth at low temperature. Several cold shock proteins are produced to counteract these effects and thus allow cold acclimatization of the cell. The main RNA modulating cold shock proteins of E. coli can be broadly divided into two categories, (1) the CspA family proteins, which mainly affect the transcription and possibly translation at low temperature through their RNA chaperoning function and (2) RNA helicases and exoribonucleases that stimulate RNA degradation at low temperature through their RNA unwinding activity.

CHARACTERIZATION OF ESCHERICHIA COLI CSPE, WHOSE PRODUCT NEGATIVELY REGULATES TRANSCRIPTION OF CSPA, THE GENE FOR THE MAJOR COLD SHOCK PROTEIN

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.

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.

Analysis of Escherichia coli Global Gene Expression Profiles in Response to Overexpression and Deletion of CspC and CspE

The Escherichia coli cold shock protein CspA family consists of nine proteins (CspA to CspI), of which two, CspE and CspC, are constitutively produced at 37 degrees C and are involved in regulation of expression of genes encoding stress response proteins but can also perform an essential function during cold acclimation. In this study, we analyzed global transcript profiles of cells lacking cspE and cspC as well as cells individually overexpressing these proteins or a CspE mutant that is unable to melt nucleic acids and is defective in cold acclimation. The analysis reveals sets of genes whose expression (i) is regulated by CspC and CspE at physiological temperature or cold shock conditions and (ii) depends on the nucleic acid melting function of CspE. Bioinformatic analysis of the latter group reveals that many of those genes contain promoter-proximal sequences that can block transcript elongation and may be targeted by the nucleic acid melting function of CspE.