Translational attenuation of the Bacillus subtilis spo0B cistron by an RNA structure encompassing the initiation region

Abstract

The spo0B gene, which exists as an operon with the obg gene, is required to initiate sporulation (stage 0) of Bacillus subtilis. This gene encodes a phosphotransferase in the multicomponent phosphorelay system. We here report the novel finding that a spo0B 5'-terminal SLR (stem-loop structure sequestering ribosome binding sequence; ACUCCUAA-X16-UUGGGAGU, ΔG = -8.71 kcal/mol) attenuated spo0B translation. The spo0B gene was efficiently transcribed but Spo0B protein was not overproduced in Escherichia coli when spo0B was induced using expression vectors carrying the SLR-spo0B region under control of the tac promoter. Deletion of the SLR from the vectors resulted in overexpression of spo0B. Therefore, to characterize expression of spo0B with a SLR in B. subtilis we constructed transcriptional and translational lacZ fusions combined with the spo0B 5'-terminal region with a deleted or mutagenized SLR These constructs were subsequently introduced into B. subtilis as multiple and single copies, then β-galactosidase activities were measured. The possible SLR also functioned as a negative cis element in B. subtilis. Furthermore, B. subtilis strain 1S16 (spo0B136) lysogenized φCD0B-S and -W, harboring spo0B with mutagenized SLRs that were more (ΔG = -14.0 kcal/mol) and less-stable (ΔG = -1.31 kcal/mol) compared with the wild-type, exhibited null and wild-type sporulation respectively. These results indicate that the spo0B 5'-SLR affects spo0B gene expression for sporulation but that low expression of spo0B through the wild-type SLR was sufficient to initiate sporulation in B. subtilis.