Salmonella typhimurium encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid

Abstract

Quorum sensing is a phenomenon in which bacteria sense and respond to their own population density by releasing and sensing pheromones. In gram-negative bacteria, quorum sensing is often performed by the LuxR family of transcriptional regulators, which affect phenotypes as diverse as conjugation, bioluminescence, and virulence gene expression. The gene encoding one LuxR family member, named sdiA (suppressor of cell division inhibition), is present in the Escherichia coli genome. In this report, we have cloned the Salmonella typhimurium homolog of SdiA and performed a systematic screen for sdiA-regulated genes. A 4.4-kb fragment encoding the S. typhimurium sdiA gene was sequenced and found to encode the 3' end of YecC (homologous to amino acid transporters of the ABC family), all of SdiA and SirA (Salmonella invasion regulator), and the 5' end of UvrC. This gene organization is conserved between E. coli and S. typhimurium. We determined that the S. typhimurium sdiA gene was able to weakly complement the E. coli sdiA gene for activation of ftsQAZ at promoter 2 and for suppression of filamentation caused by an ftsZ(Ts) allele. To better understand the function of sdiA in S. typhimurium, we screened 10,000 random lacZY transcriptional fusions (MudJ transposon mutations) for regulation by sdiA. Ten positively regulated fusions were isolated. Seven of the fusions were within an apparent operon containing ORF8, ORF9, rck (resistance to complement killing), and ORF11 of the S. typhimurium virulence plasmid. The three ORFs have now been named srgA, srgB, and srgC (for sdiA-regulated gene), respectively. The DNA sequence adjacent to the remaining three fusions shared no similarity with previously described genes.

FIG. 1

Restriction, ORF, and plasmid map of the sdiA region of S. typhimurium. Black boxes, ORFs; arrows, orientation. The percentages of amino acid identity between the S. typhimurium and E. coli homologs are indicated below each ORF. Plasmids discussed in the text are diagrammed above the ORFs, with black lines indicating the DNA region carried by each plasmid. The orientation of the insert with respect to the vector P_lac promoter is indicated.

FIG. 2

Sequence alignment of S. typhimurium SdiA, E. coli SdiA, V. fischeri LuxR, and P. aeruginosa LasR. Putative functional domains have been reviewed elsewhere (13). They include an autoinducer binding domain from residues 79 to 127, a helix-turn-helix DNA binding motif from residues 180 to 230, and a transcriptional activation domain from residues 211 to 250. Similar residues are boxed, while identical residues are boxed and shaded. Alignment was generated by using MacVector 6.0 and the ClustalW algorithm. Salmo, S. typhimurium.

FIG. 3

Activation of E. coli ftsQAZ promoter 2 by S. typhimurium sdiA. E. coli sdiA mutant carrying either pCX39 or pCX40 (encoding ftsQAZ promoter 2 or promoter 1 lacZ fusions, respectively) was transformed with either pJVR2 (S. typhimurium sdiA under arabinose control) or the vector alone (pBAD33). These strains were grown overnight in 5 ml of buffered LB containing the appropriate antibiotics (LB plus 0.1 M Tris-maleate [pH 8.0] in tubes [18 by 150 mm] with shaking at 37°C). On the following morning, the optical densities at 600 nm of the cultures were equalized to the least dense culture, followed by subculturing (at a dilution of 1:2) into buffered LB containing various arabinose (ara) or glucose (glu) concentrations. After 1 h of agitation at 37°C, the β-galactosidase activity of each culture was determined.

FIG. 4

The sdiA gene from S. typhimurium (Sty) can partially replace the native E. coli (Eco) sdiA gene for suppression of filamentation of an E. coli ftsZ(Ts) strain grown at 42°C. AW40 cells carrying the plasmids indicated above each panel were grown overnight in 5 ml of LB at the permissive temperature of 30°C (in tubes [18 by 150 mm] with shaking). The strains were subcultured at a dilution of 1:100 into LB containing the appropriate antibiotics and shaken at 42°C for 3 h. The cells were fixed in a final concentration of 2% paraformaldehyde and were examined by phase-contrast microscopy. Magnification, ×600.

FIG. 5

β-Galactosidase activities of sdiA-regulated lacZ fusions after growth in the presence of glucose (dark shaded bars) or arabinose (light shaded bars). Strains were grown overnight in LB (96-well plates at 37°C with no shaking) followed by subculture at a dilution of 1:2 into either LB plus 0.4% glucose or LB plus 0.4% arabinose (resulting in a final sugar concentration of 0.2% [also in 96-well plates]). β-Galactosidase activities were measured after 1 h of growth (37°C, no shaking). These data demonstrate that the fusions are not regulated by arabinose alone (BA612/pBAD33 background) (A) but are regulated by the arabinose-inducible sdiA gene present in BA612/pJVR2 (B). Each strain was assayed at least three times independently. The results shown are from a single representative experiment performed with duplicate cultures (error bars indicate standard deviations).

FIG. 6

ORF and mutation map of the 3′ end of the pef region (12). Black bars, ORFs (orientation is left to right for all genes); arrows, sdiA-regulated MudJ insertions.