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.     

Global analysis of proteins synthesized by Mycobacterium smegmatis provides direct evidence for physiological heterogeneity in stationary-phase cultures

We have characterized the induction kinetics of approximately 1,700 proteins during entry into and survival in carbon-starved stationary phase by Mycobacterium smegmatis. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in > or =128-day stationary-phase cultures (Spr(128) proteins). A number of Spr(128) proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu). The identification of EF-Tu as an Spr(128) protein is significant, as ribosomal components are known to be expressed in a growth rate-dependent way. We interpreted these data in terms of a model whereby stationary-phase mycobacteria comprise populations of cells that differ in both their growth status and gene expression patterns. To investigate this further, we constructed gene fusions between the rpsL gene promoter (which heads the Mycobacterium smegmatis operon encoding the tuf gene encoding EF-Tu) or the rrnA promoter gene and an unstable variant of green fluorescent protein. While the majority of cells in old stationary-phase cultures had low levels of fluorescence and so rpsL expression, a small but consistently observed population of approximately 1 in 1,000 cells was highly fluorescent. This indicates that a small fraction of the cells was expressing rpsL at high levels, and we argue that this represents the growing subpopulation of cells in stationary-phase cultures.     

Family of the major cold-shock protein, CspA (CS7.4), of Escherichia coli, whose members show a high sequence similarity with the eukaryotic Y-box binding proteins

The cspA is a gene of Escherichia coli, whose expression is specifically induced at low temperatures to a level of 13% of total protein synthesis. The CspA protein consisting of 70 amino add residues has high sequence similarity with eukaryotic Y-box DNA-binding proteins. We found two independent clones from the Kohara miniset phage collection, which hybridized with a DNA fragment containing cspA. DNA sequencing of these clones confirmed that the two genes are highly homologous to cspA. One designated cspB is mapped at 35 min on the E. coli chromosome and encodes a 71-residue protein with 79% identity to CspA, while the other, cspC, is mapped at 40 min and encodes a 69-residue protein with 70% identity. In addition, a DNA sequence upstream of the clpA gene at 19 mm published elsewhere contains an open reading frame for a 74-residue protein with 45% identity to CspA. All csp genes were fused in the coding regions with the lacZ gene, and the expression of β-galactosidase was examined for these hybrid genes upon cold shock. A similar cold-shock induction to cspA was observed for cspB but not cspC and cspD. These results Indicate that E. coli has a family of the cspA gene, some of which are induced by cold shock.     

Structure and function of a cold shock domain fold protein, CspD, in Janthinobacterium sp. Ant5-2 from East Antarctica

A cold shock domain (CSD)-containing protein, CspD, of molecular mass ~7.28 kDa in a psychrotolerant Antarctic Janthinobacterium sp. Ant5-2 (ATCC BAA-2154) exhibited constitutive expression at 37, 22, 15, 4 and -1°C. The cspD gene encoding the CspD protein of Ant5-2 was cloned, sequenced and analyzed. The deduced protein sequence was highly similar to the conserved domains of the cold shock proteins (Csps) from bacteria belonging to the class Betaproteobacteria. Its expression was both time- and growth phase-dependent and increased when exposed to 37°C and UV radiation (UVC, dose: 1.8 and 2.8 mJ cm(-2)). The results from the electrophoretic mobility shift and subcellular localization study confirmed its single-stranded DNA-binding property. In silico analysis of the deduced tertiary structure of CspD from Ant5-2 showed a highly stable domain-swapped dimer, forming two similar monomeric Csp folds. This study established an overall framework of the structure, function and phylogenetic analysis of CspD from an Antarctic Janthinobacterium sp. Ant5-2, which may facilitate and stimulate the study of CSD fold proteins in the class Betaproteobacteria.     

OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium

Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria. Salmonella enterica serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422-1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene ompR. The ompR mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR, ompC and ompF, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of ompC. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an ack pta mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of ompR message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.     

Compensatory role of PspA, a member of the phage shock protein operon, in rpoE mutant Salmonella enterica serovar Typhimurium

Sigma(E) is an alternative sigma factor that responds to and ameliorates extracytoplasmic stress. In Salmonella enterica serovar Typhimurium (S. Typhimurium), sigma(E) is required for oxidative stress resistance, stationary-phase survival and virulence in mice. Microarray analysis of stationary-phase gene expression in rpoE mutant bacteria revealed a dramatic increase in expression of pspA, a member of the phage shock protein (psp) operon. The psp operon can be induced by filamentous bacteriophages or by perturbations of protein secretion, and is believed to facilitate the maintenance of proton motive force (PMF). We hypothesized that increased pspA expression may represent a compensatory response to the loss of sigma(E) function. Increased pspA expression was confirmed in rpoE mutant Salmonella and also observed in a mutant lacking the F(1)F(0) ATPase. Alternatively, expression of pspA could be induced by exposure to CCCP, a protonophore that disrupts PMF. An rpoE pspA double mutant strain was found to have a stationary-phase survival defect more pronounced than that of isogenic strains harbouring single mutations. The double mutant strains were also more susceptible to killing by CCCP or by a bactericidal/permeability-increasing protein (BPI)-derived anti-microbial peptide. Using fluorescence ratio imaging, differences were observed in the Deltapsi of wild-type and rpoE or pspA mutant bacteria. These findings suggest that pspA expression in S. Typhimurium is induced by alterations in PMF and a functional sigma(E) regulon is essential for the maintenance of PMF.     

Genome-wide screening of Escherichia coli genes involved in execution and promotion of cell-to-cell transfer of non-conjugative plasmids: rodZ (yfgA) is essential for plasmid acceptance in recipient cells

The HSP30 gene of the budding yeast Saccharomyces cerevisiae encodes a seven-transmembrane heat shock protein expressed in response to various types of stress including heat shock. Although Hsp30p contains a potential N-glycosylation consensus sequence (Asn(2)-Asp(3)-Thr(4)), whether it is actually N-glycosylated has not been verified. Here we demonstrate that N-glycosylation is induced at Asn(2) of Hsp30p by severe heat shock, ethanol stress, and acetic acid stress. Mild heat shock and glucose depletion induced the expression but not N-glycosylation of Hsp30p, indicating the N-glycosylation to be dependent on temperature and environmental conditions. N-glycosylation did not affect on the intracellular localization of Hsp30p but its physiological role under severe heat shock conditions. Since limited information is available on stress-responsive or condition-induced N-glycosylation, our findings provide new insight into the regulation of cellular stress response in yeast.     

Dps protects cells against multiple stresses during stationary phase

Dps, the nonspecific DNA-binding protein from starved cells, is the most abundant protein in stationary-phase Escherichia coli. Dps homologs are found throughout the bacteria and in at least one archaeal species. Dps has been shown to protect cells from oxidative stress during exponential-phase growth. During stationary phase, Dps organizes the chromosome into a highly ordered, stable nucleoprotein complex called the biocrystal. We show here that Dps is required for long-term stationary-phase viability under competitive conditions and that dps mutants have altered lag phases compared to wild-type cells. We also show that during stationary phase Dps protects the cell not only from oxidative stress but also from UV and gamma irradiation, iron and copper toxicity, thermal stress, and acid and base shock. The protective roles of Dps are most likely achieved through a combination of functions associated with the protein-DNA binding and chromosome compaction, metal chelation, ferroxidase activity, and regulation of gene expression.     

The p53 family member p73 modulates the proproliferative role of IGFBP3 in short children born small for gestational age

The regulation of insulin-like growth factor–binding protein 3 (IGFBP3) gene expression is complex, because it can be induced by agents that both stimulate and inhibit the proliferation. The principal aim of this study was to investigate whether p73, a member of the p53 gene family, has a role in the regulation of the IGFBP3 expression and whether this regulation occurs in a context of cell survival or death. We demonstrate that IGFBP3 is a direct TAp73α (the p73 isoform that contains the trans-activation domain) target gene and activates the expression of IGFBP3 in actively proliferating cells. As IGFBP3 plays a key role in regulating the growth hormone/insulin-like growth factor type 1 (GH/IGF1) axis, whose alterations in gene expression appear to have a role in the growth failure of children born small for gestational age (SGA), we measured the mRNA expression levels of p73 and IGFBP3 in a group of SGA children. We found that mRNA expression levels of p73 and IGFBP3 are significantly lower in SGA children compared with controls and, in particular, p73 mRNA expression is significantly lower in SGA children with respect to height. Our results shed light on the intricate GH/IGF pathway, suggesting p73 as a good biomarker of the clinical risk for SGA children to remain short in adulthood.     

Selective mRNA degradation by polynucleotide phosphorylase in cold shock adaptation in Escherichia coli

Upon cold shock, Escherichia coli cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A pnp mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15 degrees C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.     

Proteomic features of potential tumor suppressor NESG1 in nasopharyngeal carcinoma

We previously defined the recently revised NESG1 gene as a potential tumor suppressor in nasopharyngeal carcinoma (NPC). Here, we further used proteomics technology to globally examine NESG1‐controlled proteins in NPC cells. Twenty‐six proteins were found to be deregulated by NESG1 using proteomics analysis while enolase 1 (alpha) (ENO1), heat shock protein 90 kDa beta (Grp94), member 1 (HSP90B1), and cathepsin D (CTSD) proteins were differentially expressed by Western blot. Interestingly, a‐enolase (ENO1), an overexpressed gene in NPC, was confirmed as a NESG1‐regulated protein in NPC cells. Overexpressed ENO1 not only restored cell proliferation and cell‐cycle progression, but also antagonized the regulation of NESG1 to cell‐cycle regulators p21 and CCNA1 expression as well as induced the expression of C‐Myc, pRB, and E2F1 in NESG1‐ovexpressed NPC cells. Real‐time PCR and immunohistochemistry analysis showed that NESG1 expression is negatively correlated with ENO1 expression in NPC tissues. Our observations suggest that ENO1 downregulation plays an important role in NESG1‐induced growth inhibition of NPC cancer cells.