Cloning, mapping and nucleotide sequencing of a gene encoding a universal stress protein in Eschericha coli

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Abstract
The response of non-differentiating bacteria to nutrient starvation is complex and includes the sequential synthesis of starvation-inducible proteins. Although starvation for different individual nutrients generally provokes unique and individual patterns of protein expression, some starvation stimulons share member proteins. Two-dimensional polyacrylamide gel electrophoresis revealed that the synthesis of a small (13.5 kDa) cytoplasmic protein in Escherichia coli was greatly increased during growth inhibition caused by the exhaustion of any of a variety of nutrients (carbon, nitrogen, phosphate, sulphate, required amino acid) or by the presence of a variety of toxic agents including heavy metals, oxidants, acids and antibiotics. To determine further the mode of regulation of the protein designated UspA (üniversal stress pUrotein A) we cloned the gene encoding the protein by the technique of reverse genetics. We isolated the protein from a preparative two-dimensional polyacrylamide gel, determined its N-terminal amino acid sequence, and used this sequence to construct a degenerate oligonucleotide probe. Two phages of the Kohara library were found to contain the gene which then was subcloned from the DNA in the overlapping region of these two clones. The amino acid sequence, deduced from the nucleotide sequence of the uspA gene, shows no significant homology with any other known protein. The uspA gene maps at 77 min on the E. coli W3110 chromosome, and is transcribed in a clockwise direction. The increase in the level of UspA during growth arrest was found to be primarily a result of transcriptional activation of the corresponding gene. The induction was independent of the RelA/SpoT, RpoH, KatF, OmpR, AppY, Lrp, PhoB and H-NS proteins during stress conditions that are known to induce or activate these global regulators. The -10 and -35 regions upstream of the transcriptional start site of the uspA gene are characteristic of a σ70-dependent promoter.