SdiA of Salmonella enterica is a LuxR homolog that detects mixed microbial communities

Abstract

Proteins of the LuxR family detect the presence of N-acylhomoserine lactones (AHLs) and regulate transcription accordingly. When AHLs are synthesized by the same species that detects them, the system allows a bacterium to measure the population density of its own species, a phenomenon known as quorum sensing. The sdiA genes of Escherichia coli and Salmonella enterica serovar Typhimurium are predicted to encode LuxR homologs. However, these species do not appear to synthesize AHLs or any other molecule detected by SdiA. It has previously been demonstrated that overexpression of sdiA results in the activation of the ftsQAZ locus in E. coli and four other loci in Salmonella serovar Typhimurium. Here we report that transcriptional fusions to these five loci fall into two classes. The first class requires overexpression of sdiA for activation. The second class responds to sdiA expressed from its natural position in the chromosome if the appropriate AHLs are added to the culture. The only member of the second class is a series of Prck-luxCDABE fusions in Salmonella serovar Typhimurium. SdiA responds with highest sensitivity to AHLs that have a keto modification at the third carbon and an acyl chain length of 6 or 8 (half-maximal response between 1 and 5 nM). Growth of Salmonella in proximity to species known to synthesize these AHLs results in sdiA-dependent activation of the Prck-luxCDABE fusions. SdiA appears to be the first AHL receptor discovered that detects signals emanating exclusively from other species.

FIG. 1

ORF maps of the sdiA (A) and rck (B) regions of the Salmonella serovar Typhimurium genome derived from GenBank accession numbers U88651 (A) and L08613 (B). Numbers along the bottom of each map represent nucleotide positions.

FIG. 2

Expression of the Prck-luxCDABE fusion, pBA428, in various strain backgrounds. Levels of expression are identical in the wild-type (14028/pBA428 [⧫]) and sdiA mutant (BA612/pBA428 [▪]) backgrounds. In a strain where sdiA is plasmid borne under the control of the arabinose-dependent araBAD promoter (BA612/pBA428/pBA321), Prck-luxCDABE expression is high in the presence of 0.2% arabinose (▴) and low in the presence of 0.2% glucose (53). Results shown are means of results from triplicate cultures. Error bars representing standard deviations were smaller than the symbols used and are therefore not shown.

FIG. 3

Testing of individual chemically synthesized AHLs for activation of a Prck-luxCDABE fusion in either a wild-type background (14028/pBA405) (A) or the isogenic sdiA mutant background (BA612/pBA405) (B) using a filter disk assay. A soft agar overlay containing the appropriate reporter strain was poured onto an LB agar plate and allowed to harden at room temperature for 2 h. Filter disks impregnated with 100 pmol of the indicated AHL were then placed onto the overlay, and the plate was incubated at 37°C for 8 h. Similar results were obtained using reporter plasmids pBA403 and pBA428 (data not shown).

FIG. 4

Quantitation of Salmonella serovar Typhimurium responses to chemically synthesized AHLs. The reporter strain 14028/pBA428 was incubated in LB broth with 0.3% agar containing varying amounts of each AHL. The responses of triplicate cultures were measured after 6 h of incubation at 37°C. Error bars are not shown for clarity, but standard deviations did not exceed 32% of any value. This experiment is representative of experiments performed on four separate occasions.

FIG. 5

Detection of Y. enterocolitica by Salmonella serovar Typhimurium. Y. enterocolitica strain NCTC 10460 is struck across the bottom of the plate (A and B). The wild-type Salmonella serovar Typhimurium reporter strain 14028/pBA428 is struck in duplicate perpendicular to the Y. enterocolitica on the left side of the plate (A through D). The isogenic sdiA mutant, BA612/pBA428, is struck in duplicate on the right side of the plate (A through D). Raw luminescence data are shown in panels B and D, while the pseudocolored version of the same data is shown in panels A and C. The luminescence of the wild-type reporter strain near the Y. enterocolitica strain is 13-fold greater than that of the sdiA mutant control (A and B). No response is obtained using an isogenic yenI::kan mutant of Y. enterocolitica (C and D). These data show that SdiA expressed from its natural position in the chromosome of Salmonella serovar Typhimurium can detect the physical proximity of other species that are capable of synthesizing AHLs. (E) MudJ insertions in the rck operon fail to respond to AHL. The wild-type Salmonella serovar Typhimurium reporter strain BA1103 (14028 srgA1::MudJ) is struck in duplicate on the left side of the LB kanamycin–X-Gal plate, while the isogenic sdiA mutant, BA1303, is struck in duplicate on the right side of the plate. Across the bottom is 20 μl of 10 μM oxoC8. All other Salmonella serovar Typhimurium MudJ insertions previously shown to respond to sdiA overexpression also failed to respond when present in the wild-type 14028 background in this assay (data not shown). (F) The E. coli P2ftsQ-lacZYA reporter fails to respond to AHL. The wild-type E. coli reporter strain UT481/pCX39 is struck in duplicate on the left side of the LB ampicillin–X-Gal plate, while the isogenic sdiA mutant, WX2/pCX39, is struck in duplicate on the right side of the plate. Across the bottom is 20 μl of 10 μM oxoC8. The E. coli P2ftsQ-lacZYA reporter and the Salmonella serovar Typhimurium MudJ insertions also failed to respond in cross-streak assays against Y. enterocolitica, in filter disk assays with synthetic AHL, and in liquid cultures supplemented with synthetic AHL (data not shown).

FIG. 6

Comparison of Prck-luxCDABE (pBA428) activation when sdiA is expressed from its natural position in the Salmonella serovar Typhimurium chromosome and when it is expressed from a pSC101-derived plasmid (pBM1). Strains were incubated for 6 h at 37°C in LB broth with 0.3% agar containing various concentrations of AHL before measurement of luminescence. Error bars represent standard deviations of triplicate samples.