The ability to recognize and predict non-
σ54 promoters in the alphaproteobacteria is not well developed. In this study, 25 experimentally verified Sinorhizobium melilotipromoter sequences were compiled and used to predict the location of other related promoters in the S. melilotigenome. Fourteen candidate predictions were targeted for verification and of these at least 12 proved to be genuine promoters. As a result, the experimental identification of 12 novel promoters linked to genes rpoD, topA, rpmJ, trpS, ropB1, metC, rpsT, secE, trkHand three tRNA genes is reported. In all, 99 predicted and verified promoters are reported, including those linked with 13 tRNA genes, eight ribosomal protein genes and a number of other physiologically important or essential genes. On the basis of sequence conservation and a mutational analysis of promoter activity, the −35 and −10 consensus for these promoters is 5-CTTGAC-N17-CTATAT. This promoter structure, which seems to be widely conserved amongst several other genera in the alphaproteobacteria, shares significant similarity with, but is skewed by a 1 nt step from, the canonical Escherichia coli σ70 promoter. Perhaps this difference is responsible for the observation that S. melilotipromoters are often poorly expressed in E. coli. In this regard, expression data from plasmid-borne gfp-reporter fusions to eight of the S. melilotipromoters verified in this work revealed that while these promoters were very active in S. melilotiand Agrobacterium tumefaciensonly very low, near-background activity was detected in E. coli.