PMC

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

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.

Sequence alignment of S. typhimurium SdiA, E. coli SdiA, V. fischeri LuxR, and P. aeruginosa LasR. Putative functional domains have been reviewed elsewhere (). 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.

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.

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.

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).