Regulation of spvR gene expression of Salmonella virulence plasmid pKDSC50 in Salmonella choleraesuis serovar Choleraesuis

The expression regulation of spvR, a regulatory gene on the virulence plasmid (pKDSC50) of Salmonella choleraesuis serovar Choleraesuis, was investigated by spvR–lacZ translational fusion. The spvR gene was found to be positively regulated by its own product, the SpvR protein, and this unusual positive auto-regulation was repressed by the products of spvA and spvB, virulence-associated genes present downstream from the spvR gene. Amino acid sequence analysis revealed that the amino-terminal region of SpvB had homology with the CatM repressor protein of Acineto-bacter calcoaceticus, which belongs to the MetR/LysR protein family. On the other hand, the sigma factor RpoS was required for expression of the spvR gene in the stationary phase of bacterial growth. The SpvR protein was also necessary for self-activation, suggesting that an RNA polymerase holoenzyme containing RpoS requires SpvR protein in order to recognize the spvR promoter.     

Regulation of spvR, the positive regulatory gene of Salmonella plasmid virulence genes

Abstract The regulation of the spvR promoter from the Salmonella dublin virulence plasmid was monitored using proter-reporter gene fusion constructs. Activity was dependent upon the presence of the spv region and was affected by the number of copies of the spv region present with the cell. Activity remained constant throughout exponential growth, and increased rapidly with the onset of stationary phase, under both aerobic and anaerobic conditions. Additionally, the level of spvR expression was controlled by the availability of iron, activity being greatest under low iron conditions in stationary phase. The spvA gene product negatively regulated spvR expression in a dose-dependent manner, indicating that SpvA provides a negative feedback mechanism for this operon.     

Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium

Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1.5 kbp from the TnA insertion in M51 and 6.5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes.     

In vitro binding of the Salmonella dublin virulence plasmid regulatory protein SpvR to the promoter regions of spvA and spvR.

The spv regulon of Salmonella dublin is essential for virulence in mice. SpvR, a LysR-type regulator, induces the expression of the spvABCD operon and its own expression in the stationary phase of bacterial growth and in macrophages. We constructed fusion proteins to the maltose-binding protein (MBP) and a His tag peptide (His) to overcome the insolubility and to facilitate purification of SpvR. We demonstrated that both fusion proteins, MBP-SpvR and His-SpvR, were able to induce spvA expression in vivo. MBP-SpvR was produced as soluble protein, whereas His-SpvR was only marginally present in the soluble cell fraction. Affinity chromatography resulted in at least 95% pure MBP-SpvR protein and in an enrichment of His-SpvR. Gel mobility shift assay revealed that the SpvR fusion proteins were able to bind to 125-and 147-bp DNA fragments of the spvA and spvR promoter regions, respectively. DNase I footprint experiments showed that the fusion proteins protected DNA regions of 54 and 50 bp within the spvA and spvR promoter regions, respectively.     

Unravelling the mysteries of virulence gene regulation in Salmonella typhimurium

Salmonella typhimurium, which causes gastroenteritis in calves and humans as well as a typhoid-like disease in mice, uses numerous virulence factors to infect its hosts. Genes encoding these factors are regulated by many environmental conditions and regulatory pathways in vitro. Many virulence genes are specifically induced at particular sites during infection or in cultured host cells. The complex regulation of virulence genes observed in vitro may be necessary to restrict their expression to specific locations within the host. In vitro and in vivo studies provide clues about how virulence genes might be regulated in vivo. Future studies must assess the actual environmental signals and regulators that modulate each virulence gene in vivo and determine how multiple regulatory pathways are integrated to co-ordinate the appropriate expression of virulence factors at specific sites in vivo.     

Molecular cloning and functional expression of the rfaE gene required for lipopolysaccharide biosynthesis in Salmonella typhimurium

The rfaE (WaaE) gene of Salmonella typhimurium is known to be located at 76min on the genetic map outside of the rfa gene cluster encoding core oligosaccharide biosynthesis of lipopolysaccharide(LPS). The rfaE mutant synthesizes heptose-deficient LPS; its LPS consists of only lipid A and 3-deoxy-D-manno-octulosonic acid (KDO), and the rfaE gene is believed to be involved in the formation of ADP-L-glycero-D-manno-heptose. Mutants, which make incomplete LPS, are known as rough mutants. Salmonella typhimurium deep-rough mutants affected in the heptose region of the inner core often show reduced growth rate, sensitivity to high temperature and hypersensitivity to hydrophobic antibiotics. We have cloned the rfaE gene of S. typhimurium. The chromosomal region carrying this gene was isolated by screening a genomic library of S. typhimurium using the complementation of S. typhimurium rfaE mutant. The 2.6-Kb insert in the plasmid pHEPs appears to carry a functional rfaE gene. SL1102 (rfaE543) makes heptose-deficient LPS and has a deep rough phenotype, but pHEPs complement the rfaE543 mutation to give the smooth phenotype. The sensitivity of SL1102 to bacteriophages (P22.c2, Felix-O, Br60) which use LPS as their receptor for adsorption is changed to that of wild-type strain. The permeability barrier of SL1102 to hydrophobic antibiotics (novobiocin) is restored to that of wild-type. LPS produced by SL1102 (rfaE543) carrying pHEPs makes LPS indistinguishable from that of smooth strains. The rfaE gene encoded a polypeptide of 477 amino acid residues highly homologous to the S. enterica rfaE protein (98% identity), E. coli (93% identity), Yersenia pestis (85% identity), Haemophilus influenzae (70% identity) and Helicobacter pyroli (41% identity) with a molecular weight 53 kDa.     

Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella

The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively. This system increases the expression of virulence genes and decreases the expression of motility genes. In this study, we examined the pathways by which SirA affects these genes. We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that hilA had no effect on flhDC. SirA was able to repress flhDC in a hilA mutant and activate hilA in an flhDC mutant. Therefore, although the flhDC and hilA regulatory cascades interact, sirA affects each of them independently. A form of BarA lacking the two N-terminal membrane-spanning domains, BarA198, autophosphorylates in the presence of ATP and transfers the phosphate to purified SirA. Phosphorylated SirA was found to directly bind the hilA and hilC promoters in gel mobility shift assays but not the flhD, fliA, hilD, and invF promoters. Given that the CsrA/csrB system is known to directly affect flagellar gene expression, we tested the hypothesis that SirA affects flagellar gene expression indirectly by regulating csrA or csrB. The sirA gene did not regulate csrA but did activate csrB expression. Consistent with these results, phosphorylated SirA was found to directly bind the csrB promoter but not the csrA promoter. We propose a model in which SirA directly activates virulence expression via hilA and hilC while repressing the flagellar regulon indirectly via csrB.     

Evidence for factors regulating transfer cell-specific expression in maize endosperm

In maize, a layer of basal endosperm cells adjacent to the pedicel is modified for a function in solute transfer. Three genes specifically expressed in this region, termed the basal endosperm transfer layer (BETL-2 to -4), were isolated by differential hybridization. BETL-2 to -4 are coordinately expressed in early and mid-term endosperm development, but are absent at later stages. BETL-2 to -4 coding sequences all predict small (<100 amino acids), secreted, cysteine-rich polypeptides which lack close relatives in current database accessions. BETL-3 and BETL-1 display some sequence similarities with each other and to plant defensins. BETL-2 to -4 promoter regions were isolated and compared, revealing the presence of a promoter-proximal microsatellite repeat as the most highly conserved sequence element in each sequence. Electrophoretic mobility shift assays (EMSA) showed that specific BETL-2 to -4 promoter fragments competed for binding to the same DNA-binding activity in nuclear extracts prepared from maize endosperm. Although BETL-2 to -4 are only expressed in basal endosperm cells, the DNA-binding activities detected were of two types: distal endosperm-specific, or present in both basal and distal endosperm extracts. On the basis of these findings, a model to account for the coordinate regulation of BETL genes in endosperm cells is proposed.