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

A homolog of the major eubacterial cold shock gene cspA was identified in S inorhizobium meliloti RM1021 by luxAB 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 fra mes (ORFs) encoding homologs corresponding to CspA a novel 10.6-kDa polypep tide designated ORF2, and a homolog of the Escherichia coli ribosomal prote in 821, Transcription analysis indicated that this locus produced two diffe rent-sized cspA-hybridizing transcripts upon cold shock, a 400-nucleotide ( nt) RNA encoding cspA alone and a 1,000-nt transcript encoding cspA-ORF2-rp sU. The sizes of the transcripts agreed with the location of the transcript ion start site determined by primer extension and the locations of two puta tive transcriptional terminators. The promoter of the csp,4-ORF2-rpsU locus had -10 and -35 elements similar to the E. coli sigma(70) consensus promot er and, like the cspA locus of E. coli, included an AT-rich region upstream of the -35 hexamer. The promoter of the S. meliloti cspA locus was found t o impart cold shock-induced mRNA accumulation, In addition, the 5'-untransl ated region (5' UTR) was found to increase the fold induction of cspA trans cripts after cold shock and depressed the level of lux LB mRNA prior to col d shock, another feature similar to cspA regulation in E, coli, No "cold bo x" was identified upstream of the S. meliloti cspA gene, however, and there was no other obvious sequence identity between the S. meliloti 5' UTR and that of E, coli, DNA hybridization analysis indicated that outside the cspA -ORF2-rpsU cold shock locus there are several additional cspA-like genes an d a second rpsU homolog.