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  • 1
    ISSN: 1617-4623
    Keywords: Salmonella typhimurium ; rpoS ; spv ; Virulence plasmid ; Regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The SpvR protein is a DNA-binding protein of the LysR family, required for the transcription of thespvABCD virulence operon ofSalmonella typhimurium. An alternative sigma factor, σS (RpoS), in conjunction with SpvR, controls the transcription of thespvR gene. In this study, we used a combination of primer extension experiments and deletion/fusion analyses of thespvR gene to identify sequences involved inspvR transcription inS. typhimurium. When induced in the stationary phase of growth in rich medium or during carbon starvation, transcription ofspvR inS. typhimurium is driven by a single promoter (spvRp1) and initiates 17 nucleotides upstream of thespvR start codon. The level ofspvR transcription originating atspvRp1 was 20-fold higher in the wild-type strain than in therpoS mutant. In both strains, however, transcription atspvRp1 requires the SpvR protein. 5′ Deletions up to position −86, relative to thespvR start codon, did not inhibit inducibility by σS and/or SpvR. In contrast, 5′ deletion up to −75 abolished the activation ofspvRp1 by SpvR in both the wild-type strain andrpoS mutant. Within the 11-bp sequence lying between position −86 and position −75, a 10-bp consensus motif TNTNTGCANA, present in both thespvR andspvA promoter regions, was identified and may contain the DNA recognition site for SpvR. In addition, we detected initiation of transcription within thespvR coding region. This finding may have implications for comparative studies of regulation withspvR gene fusions.
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  • 2
    ISSN: 1617-4623
    Keywords: Key wordsSalmonella typhimurium ; rpoS ; spv ; H-NS ; DNA curvature
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The σS-regulated gene spvR of Salmonella typhimurium encodes an autoregulatory protein required for transcriptional activation of the virulence operon spvABCD. A mutation in the histone-like protein H-NS, which negatively controls the σS level, has been reported to increase spv gene expression in S. typhimurium strain LT2. In agreement with this, we found that transcription of spvR and spvABCD was derepressed in hns strains of Escherichia coli and S. typhimurium. Moreover, levels of spv gene expression in hns rpoS double mutants were higher than expression levels in mutants deficient in rpoS alone, and were close to those measured in wild-type strains. This demonstrates that H-NS contributes to spv gene regulation independently of its function in controlling the σS level. Since the same start site was used for spvR gene transcription in wild-type as in hns and hns rpoS mutant strains, it is likely that the spvR promoter, spvRp1, can be recognized efficiently by an RNA polymerase containing σ70. The spvR promoter region shows an intrinsic DNA curvature that might be a determinant in H-NS- and/or σS-mediated control. A single amino acid substitution, Leu to Pro at position 265, abolished the regulatory function of SpvR in E. coli and Salmonella, implicating the C-terminal domain of SpvR in its structure and/or regulatory function. The spvR 265 allele is not transcribed at detectable levels in hns or hns rpoS strains, suggesting that activation of spvRp1 in these strains remains dependent on SpvR. Thus, we propose a model for spvR gene regulation in which SpvR acts as a co-regulator of an RNA polymerase containing either σ70 (in the absence of H-NS) or σS, to induce transcriptional initiation at spvRp1. Moreover, growth-phase regulation of spv gene expression was maintained in hns and hns rpoS strains, indicating that an additional element, besides σS, is involved in the growth-phase regulation in rich medium.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1617-4623
    Keywords: Key wordsSalmonella ; rpoS ; spv ; Downstream box ; Regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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 β-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.
    Type of Medium: Electronic Resource
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