Cloning of the rpoD analog from Rhizobium etli: sigA of R. etli is growth phase regulated

Abstract

Rhizobium bacteria fix atmospheric nitrogen during symbiosis with legume plants only after bacterial division is arrested. The role of the major vegetative sigma factor, SigA, utilized by Rhizobium bacteria during symbiosis is unknown. By using PCR technology, a portion of the sigA gene corresponding to domain II was directly amplified from Rhizobium etli total DNA by using two primers designed in accordance with the published sequence of sigA from Agrobacterium tumefaciens. The amplified fragment was cloned and used as a hybridization probe for cloning of the R. etli sigA gene. Sequencing data revealed an open reading frame of 2,055 bp showing extensive similarity to various vegetative sigma factors. The 5' end of the sigA transcript was determined and revealed a long, seemingly untranslated region of 170 nucleotides. Quantitative analysis of the sigA transcript by RNase protection and by primer extension assays indicated its down-regulation during entry into the stationary phase. On the basis of the structures of various vegetative sigma factors and considering previous information on heterologous expression, we speculate on the function of domain I of vegetative sigma factors.