Major Cold Shock Protein CspA Research Articles

A genome-wide survey of highly expressed non-coding RNAs and biological validation of selected candidates in Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a plant pathogen that has the natural ability of delivering and integrating a piece of its own DNA into plant genome. Although bacterial non-coding RNAs (ncRNAs) have been shown to regulate various biological processes including virulence, we have limited knowledge of how Agrobacterium ncRNAs regulate this unique inter-Kingdom gene transfer. Using whole transcriptome sequencing and an ncRNA search algorithm developed for this work, we identified 475 highly expressed candidate ncRNAs from A. tumefaciens C58, including 101 trans-encoded small RNAs (sRNAs), 354 antisense RNAs (asRNAs), 20 5′ untranslated region (UTR) leaders including a RNA thermosensor and 6 riboswitches. Moreover, transcription start site (TSS) mapping analysis revealed that about 51% of the mapped mRNAs have 5′ UTRs longer than 60 nt, suggesting that numerous cis-acting regulatory elements might be encoded in the A. tumefaciens genome. Eighteen asRNAs were found on the complementary strands of virA, virB, virC, virD, and virE operons. Fifteen ncRNAs were induced and 7 were suppressed by the Agrobacterium virulence (vir) gene inducer acetosyringone (AS), a phenolic compound secreted by the plants. Interestingly, fourteen of the AS-induced ncRNAs have putative vir box sequences in the upstream regions. We experimentally validated expression of 36 ncRNAs using Northern blot and Rapid Amplification of cDNA Ends analyses. We show functional relevance of two 5′ UTR elements: a RNA thermonsensor (C1_109596F) that may regulate translation of the major cold shock protein cspA, and a thi-box riboswitch (C1_2541934R) that may transcriptionally regulate a thiamine biosynthesis operon, thiCOGG. Further studies on ncRNAs functions in this bacterium may provide insights and strategies that can be used to better manage pathogenic bacteria for plants and to improve Agrobacterum-mediated plant transformation.

Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA

Homocysteine (Hcy) is a thiol-containing amino acid that is considered to be medically important because it is linked to the development of several life-threatening diseases in humans, including cardiovascular disease and stroke. It inhibits the growth of Escherichia coli when supplied in the growth medium. Growth inhibition is believed to arise as a result of partial starvation for isoleucine, which occurs because Hcy perturbs the biosynthesis of this amino acid. This study attempted to further elucidate the inhibitory mode of action of Hcy by examining the impact of exogenously supplied Hcy on the transcriptome. Using gene macroarrays the transcript levels corresponding to 68 genes were found to be reproducibly altered in the presence of 0.5 mM Hcy. Of these genes, the biggest functional groups affected were those involved in translation (25 genes) and in amino acid metabolism (19 genes). Genes involved in protection against oxidative stress were repressed in Hcy-treated cells and this correlated with a decrease in catalase activity. The gene showing the strongest induction by Hcy was cspA, which encodes the major cold-shock protein CspA. RT-PCR and reporter fusion experiments confirmed that cspA was induced by Hcy. Induction of cspA by Hcy was not caused by nutritional upshift, a stimulus known to induce CspA expression, nor was it dependent on the presence of a functional CspA protein. The induction of cspA by Hcy was suppressed when isoleucine was included in the growth medium. These data suggest that the induction of CspA expression in the presence of Hcy occurs because of a limitation for isoleucine. The possibility that Hcy-induced cspA expression is triggered by translational stalling that occurs when the cells are limited for isoleucine is discussed.

Extended −10 Motif Is Critical for Activity of thecspAPromoter but Does Not Contribute to Low-Temperature Transcription

Bacterial promoters belonging to the extended -10 class contain a conserved TGn motif upstream of the -10 promoter consensus element. Open promoter complexes can be formed on some extended -10 Escherichia coli promoters at temperatures as low as 6 degrees C, when complexes on most promoters are closed. The promoter of cspA, a gene that codes for the major cold shock protein CspA of E. coli, contains an extended -10 motif. CspA is dramatically induced upon temperature downshift from 37 to 15 degrees C, and its cold shock induction has been attributed to transcription, translation, and mRNA stabilization effects. Here, we show that though the extended -10 motif is critical for high-level expression of cspA, it does not contribute to low-temperature expression. In fact, transcription from the wild-type cspA promoter is cold sensitive in vitro and in vivo. Thus, transcription appears to play little or no role in low-temperature induction of cspA expression.

Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureus.

The identification of target structures is a prerequisite for the development of new treatment options, like antibody based therapy, against methicillin resistant Staphylococcus aureus (MRSA). In this study we identified immunodominant structures which were expressed in vivo during sepsis caused by MRSA. Using human sera we compared the immune response of humans with MRSA sepsis with the immune response of normal individuals and asymptomatically colonized individuals. We identified and characterized four staphylococcal specific antigenic structures. One target is a staphylococcal protein of 29 kDa that exhibited 29% identity to secreted protein SceA precursor of Staphylococcus carnosus. The putative function of this protein, which was designated IsaA (immunodominant staphylococcal antigen), is unknown. The second target is an immunodominant protein of 17 kDa that showed no homology to any currently known protein. This immunodominant protein was designated IsaB. The third and fourth antigens are both immunodominant proteins of 10 kDa. One of these proteins showed 100% identity to major cold shock protein CspA of S. aureus and the other protein was identified as the phosphocarrier protein Hpr of S. aureus. The identified immunodominant proteins may serve as potential targets for the development of antibody based therapy against MRSA.

Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureus

The identification of target structures is a prerequisite for the development of new treatment options, like antibody based therapy, against methicillin resistant Staphylococcus aureus (MRSA). In this study we identified immunodominant structures which were expressed in vivo during sepsis caused by MRSA. Using human sera we compared the immune response of humans with MRSA sepsis with the immune response of normal individuals and asymptomatically colonized individuals. We identified and characterized four staphylococcal specific antigenic structures. One target is a staphylococcal protein of 29 kDa that exhibited 29% identity to secreted protein SceA precursor of Staphylococcus carnosus. The putative function of this protein, which was designated IsaA (immunodominant staphylococcal antigen), is unknown. The second target is an immunodominant protein of 17 kDa that showed no homology to any currently known protein. This immunodominant protein was designated IsaB. The third and fourth antigens are both immunodominant proteins of 10 kDa. One of these proteins showed 100% identity to major cold shock protein CspA of S. aureus and the other protein was identified as the phosphocarrier protein Hpr of S. aureus. The identified immunodominant proteins may serve as potential targets for the development of antibody based therapy against MRSA.

Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA.

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.

Characterization of cspB, a cold-shock-inducible gene from Lactococcus lactis, and evidence for a family of genes homologous to the Escherichia coli cspA major cold shock gene.

Upon temperature downshift, the major cold shock protein CspA is highly induced in Escherichia coli. This protein being conserved in other bacteria, we used a PCR-based approach with a pair of degenerate primers derived from highly conserved regions of the CspA-related proteins to evidence the presence of at least three related genes in Lactococcus lactis. One of them, cspB, was cloned and sequenced. It encodes a 66-residue protein which possesses 60% sequence identity with E. coli CspA. Following a cold shock from 30 to 15 degrees C, the level of the cspB mRNA transcript increased, as shown by Northern blot hybridization. In addition, induction of cspB-directed beta-galactosidase activity was observed. These results indicate that the L. lactis cspB gene is cold shock inducible.

Post-transcriptional regulation of CspA expression in Escherichia coli.

The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible lambda PL promoter. After induction of transcription by thermal inactivation of the lambda ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10 degrees C, but poor expression was obtained if the cells were incubated at 37 degrees C or 30 degrees C. The reason for this differential temperature-dependent expression was investigated and it was found that: (i) the CspA content of the cells decreased more rapidly at 37 degrees C compared to 10 degrees C, regardless of whether transcription was turned off by addition of rifampicin; (ii) both the chemical and functional half-lives of the cspA transcript were substantially longer at 10 degrees C compared to 37 degrees C; (iii) S30 extracts as well as 70S ribosomes prepared from cold-shocked cells translated CspA mRNA (but not phage MS2 RNA) more efficiently than equivalent extracts or ribosomes obtained from control cells grown at 37 degrees C; and (iv) purified CspA stimulated CspA mRNA translation. Overall, these results indicate that a selective modification of the cold-shocked translational apparatus favouring translation of CspA mRNA, and an increased stability of this mRNA at low temperature, may play an important role in the induction of cspA expression during cold shock.