A coding segment of the virulence regulatory gene spvR enhances expression of spvR-lacZ and spvR-gfp translational fusions in Salmonella typhimurium.

Abstract

The Salmonella gene spvR is regulated by RpoS, and encodes an autoregulatory DNA-binding protein of the LysR family, which is required for transcriptional activation of the virulence operon spvABCD. We found that the 12-bp sequence between codons 3 and 8 of spvR (12bp-box) increased the expression of spvR'-lacZ translational fusions by at least two orders of magnitude. The 12bp-box did not affect the level of expression of a transcriptional spvR'-lacZ fusion, as determined by measurement of beta-galactosidase activity and mRNA levels. This suggests that the 12bp-box does not play a major role at the transcriptional level. However, the amounts of both SpvR-LacZ hybrid protein and lacZ mRNA produced from a translational spvR'-lacZ fusion were significantly lower if the 12bp-box was removed. Thus, the 12bp-box directly or indirectly affects the amount of spvR'-lacZ mRNA produced from a translational fusion but not from a transcriptional fusion. The 12bp-box still appeared to be functional when the spvR'-lacZ mRNA was elongated at its 5' end. In that case, however, while the 12bp-box greatly reduced the level of downstream mRNA produced, it did not affect the level of upstream mRNA. The effect of the 12bp-box was not restricted to lacZ fusions because expression of a translational spvR'-gfp fusion was also decreased by removal of the 12bp-box. The sequence of the 12bp-box is complementary to a sequence near the decoding region of 16S rRNA thought to be involved in translation initiation. This box may thus function as a translational enhancer, in which case the effect of the 12bp-box on lacZ mRNA levels might result indirectly from the tight coupling of translation and mRNA degradation. However, a direct role of the 12bp-box in increasing mRNA stability or in reducing the incidence of premature termination of transcription cannot be excluded.