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A homolog of the major eubacterial cold shock genecspA was identified inSinorhizobium meliloti RM1021 byluxAB reporter transposon mutagenesis. Here we further characterize the organization and regulation of this locus. DNA sequence analysis indicated that the locus includes three open reading frames (ORFs) encoding homologs corresponding to CspA, a novel 10.6-kDa polypeptide designated ORF2, and a homolog of theEscherichia coli ribosomal protein S21. Transcription analysis indicated that this locus produced two different-sizedcspA -hybridizing transcripts upon cold shock, a 400-nucleotide (nt) RNA encodingcspA alone and a 1,000-nt transcript encodingcspA-ORF2-rpsU . The sizes of the transcripts agreed with the location of the transcription start site determined by primer extension and the locations of two putative transcriptional terminators. The promoter of thecspA-ORF2-rpsU locus had −10 and −35 elements similar to theE. coli ς70 consensus promoter and, like thecspA locus ofE. coli , included an AT-rich region upstream of the −35 hexamer. The promoter of theS. meliloti cspA locus was found to impart cold shock-induced mRNA accumulation. In addition, the 5′-untranslated region (5′ UTR) was found to increase the fold induction ofcspA transcripts after cold shock and depressed the level ofluxAB mRNA prior to cold shock, another feature similar tocspA regulation inE. coli . No “cold box” was identified upstream of theS. meliloti cspA gene, however, and there was no other obvious sequence identity between theS. meliloti 5′ UTR and that ofE. coli . DNA hybridization analysis indicated that outside thecspA-ORF2-rpsU cold shock locus there are several additionalcspA -like genes and a secondrpsU homolog.

applied and environmental microbiology

Transcriptional Organization and Regulation of a Polycistronic Cold Shock Operon in Sinorhizobium meliloti RM1021 Encoding Homologs of the Escherichia coli Major Cold Shock Gene cspA and Ribosomal Protein Gene rpsU