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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.

ABSTRACT: 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.

SUBMITTER: Chapot-Chartier MP

PROVIDER: S-EPMC179434 | biostudies-other | 1997 Sep

REPOSITORIES: biostudies-other

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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.

Chapot-Chartier M P MP Schouler C C Lepeuple A S AS Gripon J C JC Chopin M C MC

Journal of bacteriology 19970901 17

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. Followi ...[more]

PMID: 9287018

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Project description:Inverse PCR was used to amplify major cold shock protein (MCSP) gene families from a diverse range of bacteria, including the psychrotolerant Yersinia enterocolitica, which was found to have two almost identical MCSP coding regions (cspA1 and cspA2) located approximately 300 bp apart. This tandem gene duplication was also found in Y. pestis, Y. pseudotuberculosis, and Y. ruckeri but not in other bacteria. Analysis of the transcriptional regulation of this MCSP gene in Y. enterocolitica, performed by using both reverse transcriptase-PCR and Northern blot assays, showed there to be two cold-inducible mRNA templates arising from this locus: a monocistronic template of approximately 450 bp (cspA1) and a bicistronic template of approximately 900 bp (cspA1/A2). The former may be due to a secondary structure between cspA1 and cspA2 causing either 3' degradation protection of cspA1 or, more probably, partial termination after cspA1. Primer extension experiments identified a putative transcriptional start site (+1) which is flanked by a cold-box motif and promoter elements (-10 and -35) similar to those found in Escherichia coli cold-inducible MCSP genes. At 30 degrees C, the level of both mRNA molecules was negligible; however, upon a temperature downshift to 10 degrees C, transcription of the bicistronic mRNA was both substantial (300-fold increase) and immediate, with transcription of the monocistronic mRNA being approximately 10-fold less (30-fold increase) and significantly slower. The ratio of bicistronic to monocistronic mRNA changed with time after cold shock and was higher when cells were shocked to a lower temperature. High-resolution, two-dimensional protein gel electrophoresis showed that synthesis of the corresponding proteins, both CspA1 and CspA2, was apparent after only 10 min of cold shock from 30 degrees C to 10 degrees C. The data demonstrate an extraordinary capacity of the psychrotolerant Y. enterocolitica to produce major cold shock proteins upon cold shock.

Project description:Research involving the cold shock gene cspA of the medically important bacterium Staphylococcus aureus is steadily increasing as the relationships between the activity of this gene at 37 °C and a spectrum of virulence factors (e.g., biofilm formation, capsule production) as well as stress-related genes (e.g., alkaline shock protein, asp-23 and the alternative sigma factor, sigB) are distinguished. Fundamental to each of these discoveries is defining the regulation of cspA and the production of its protein product CspA.In this paper, primer extension analysis was used to identify a transcriptional start point at 112 bp upstream of the initiation codon of the cspA coding sequence from S. aureus Newman RNA collected at 37 °C. Based on the location of the putative -10 and -35 sites as well as putative cold shock protein binding sites, a 192 bp sequence containing an 80 bp promoter + a 112 bp 5' UTR was generated by polymerase chain reaction. The activity of this 192 bp sequence was confirmed in a pLL38 promoter::xylE reporter gene construct. In addition, Western blots were used to confirm the production of CspA at 37 °C and demonstrated that production of the protein was not constitutive but showed growth-dependent production with a significant increase at the 6 h time point.The results presented identify another regulatory region for the cold shock gene cspA of S. aureus and show growth-dependent activity of both this cspA regulatory sequence, presented as a 192 bp sequence of promoter + 5' UTR and the production of the CspA protein at 37 °C. The presence of two active transcription start points, a -112 bp sequence defined in this work and a second previously defined at -514 bp upstream of the cspA initiation codon, suggests the possibility of interactions between these two regions in the regulation of cspA. The growth-dependent production of the cold shock protein CspA supports the availability of this protein to be a modulator of virulence and stress factor genes at 37 °C.

Dataset Information

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.

Publications

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.

Similar Datasets

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