Identification of Cold Shock Gene Loci in Sinorhizobium meliloti by Using a luxAB Reporter Transposon

Using a luxAB reporter transposon, seven mutants of Sinorhizobium meliloti were identified as containing insertions in four cold shock loci. LuxAB activity was strongly induced (25- to 160-fold) after a temperature shift from 30 to 15°C. The transposon and flanking host DNA from each mutant was cloned, and the nucleic acid sequence of the insertion site was determined. Unexpectedly, five of the seven luxAB mutants contained transposon insertions in the 16S and 23S rRNA genes of two of the three rrn operons of S. meliloti. Directed insertion of luxAB genes into each of the three rrn operons revealed that all three operons were similarly affected by cold shock. Two other insertions were found to be located downstream of a homolog of the major Escherichia coli cold shock gene, cspA. Although the cold shock loci were highly induced in response to a shift to low temperature, none of the insertions resulted in a statistically significant decrease in growth rate at 15°C.

本研究利用luxAB报告转座子（luxAB reporter transposon），从苜蓿中华根瘤菌（Sinorhizobium meliloti）中筛选得到7株突变体，上述突变体的4个冷激位点（cold shock loci）均存在转座子插入突变。当培养温度由30℃转换至15℃后，各突变体的LuxAB活性均被显著诱导，诱导幅度达25至160倍。随后，我们克隆了每株突变体中的转座子及其侧翼宿主DNA，并测定了其插入位点的核酸序列。令人意外的是，7株luxAB突变体中有5株的转座子插入在了苜蓿中华根瘤菌3个rrn操纵子（rrn operons）中2个的16S及23S核糖体RNA（rRNA）基因内部。进一步将luxAB基因定向插入至3个rrn操纵子的各自位点后发现，所有3个操纵子均会受到冷激的相似调控。另外2处插入位点则位于大肠杆菌（Escherichia coli）主要冷激基因cspA的同源基因下游区域。尽管上述冷激位点在低温温度转换后均被高度诱导，但所有插入突变均未使菌株在15℃下的生长速率出现统计学显著的下降。