Journal of Bacteriology, February 2003, p . 1357-1366, Vol . 185, No . 4

Detection of Other Microbial Species by Salmonella: Expression of the SdiA Regulon

Jenée N . Smith and Brian M . M . Ahmer^*

Department of Microbiology, The Ohio State University, Columbus Ohio 43210

Received 4 September 2002/ Accepted 22 November 2002

The final step in AHL synthesis is catalyzed by enzymes of the LuxI family (13, 39) . Each LuxI family member typically generates a single AHL, along with smaller amounts of related molecules . The primary difference between the AHLs synthesized by different LuxI enzymes is the length of the acyl chain, which usually varies between 4 and 14 carbons . AHLs can also vary at the 3-carbon position by the presence of a carbonyl or hydroxyl group . The AHLs discussed in this report are abbreviated C₆, C₈, etc . to denote chain length, and oxoC₆, oxoC₈, etc . to denote a carbonyl modification . Many bacteria encode more than one LuxI-type enzyme and synthesize several AHLs . The AHL production and response profile of different species can overlap, providing the potential for cross talk between species (27, 30, 31, 40) .

The genera Escherichia, Salmonella, and Klebsiella provide an unusual case . The existing DNA sequence databases for these organisms show that each species carries a single luxR homolog named sdiA but does not carry a luxI family member . Other types of AHL synthase enzymes have been identified in Vibrio and Pseudomonas (luxLM and hdtS [5, 14, 20]), but Escherichia, Salmonella, and Klebsiella do not encode homologs of these enzymes either . Consistent with the genome sequence data, representatives of these genera fail to produce detectable levels of AHLs in standard bioassays under laboratory growth conditions (22, 36) . Since these organisms contain an AHL receptor but do not synthesize AHLs, they cannot be using SdiA to determine the population density of their own species . Instead, they may use SdiA to detect AHLs produced by other microbial species (22) .

Before the signals for SdiA had been identified plasmid-based expression of sdiA was used to identify responsive transcriptional fusions in Salmonella enterica serovar Typhimurium (1) . The fusions were created with the MudJ transposable element, which creates lacZY fusions on insertion into a coding sequence (8, 17) . Seven fusions were isolated in the rck operon (for "resistance to complement killing") on the serovar Typhimurium virulence plasmid . Three additional fusions were obtained, but the DNA sequence flanking the MudJ insertions did not match the existing database entries . Here we report the locations of these three fusions now that the serovar Typhimurium genome sequence has been completed . These 10 fusions were isolated based on their response to plasmid-based expression of sdiA, but they failed to show any sdiA-dependent expression when placed into isogenic wild-type and sdiA mutant backgrounds (1) . It was hypothesized that there were no AHLs in the culture and that overexpression of sdiA had bypassed the AHL requirement (1) . Evidence supporting this hypothesis was obtained when a plasmid-based luciferase fusion to the rck operon promoter was found to be activated in an sdiA-dependent manner in response to synthetic oxoC₆ and oxoC₈ at concentrations of 1 to 10 nM . The fusion also responded to C₆ and C₈ when the concentrations were approximately 10-fold higher (22) . However, responses to AHL were not observed when the chromosomal MudJ fusions were used (we refer to all of the MudJ fusions, including those on the virulence plasmid, as chromosomal to distinguish them from the multicopy plasmid-based luciferase fusions [22]) . The MudJ fusions failed to respond in cross-streak assays on 1.5% agar plates, in filter disk assays performed with 0.7% top agar, and in early-exponential-phase liquid cultures (22) . In this report, we show that growth in motility agar or to the late exponential phase in liquid culture allows the sdiA-dependent activation of chromosomal fusions in response to AHL or to bacterial species producing AHL . This is a definitive demonstration that SdiA detects and responds to AHL production by other bacterial species . We also reveal the locations of three previously uncharacterized MudJ fusions that are regulated by SdiA .

 
Plasmid constructions. To isolate the promoter of srgE, a plasmid-based transcriptional fusion was constructed (see Fig . 1) . The putative promoter region was amplified from S . enterica serovar Typhimurium strain 14028 using Pfu Turbo DNA polymerase (Stratagene) . Each primer contained a BglII restriction site at the 5' end . The PCR product spans nucleotides 16638 to 17114 of Genbank accession number AE008767 . The resulting PCR product was cloned using pCR-Blunt II Topo (Invitrogen) . The insert was removed from pCR-Blunt II Topo by using EcoRI and cloned into pSB401 from which the luxR-containing EcoRI fragment had been removed . Clones were screened for insert and orientation using PCR . One isolate forming a srgE-luxCDABE fusion was named pJNS25, while a clone in the opposite orientation was named pJNS35 . These plasmids were electroporated into strains 14028 and BA612 using a Bio-Rad Gene Pulser II .

 
Measurement of luciferase and ß-galactosidase activity. Liquid cultures were grown in 5-ml volumes in 18- by 150-mm tubes placed near horizontal in a drum roller to maintain agitation . The optical density (OD) of the cultures was measured with a Spectronic 20D+ instrument at 550 nm . The light production of 10-µl samples was integrated for 10 s with a Turner Designs TD-20/20 luminometer and expressed as Light/OD . Expression of luciferase by bacteria on plates or in motility agar was imaged and quantitated using a C2400-32 intensified charge-coupled device camera with an Argus 20 image processor (Hamamatsu Photonics) (9) .

Expression of lacZY reporters was measured using ß-galactosidase assays as described by Miller with the following modifications (23) . The liquid cultures were grown as described above for luciferase assays, except that at each time point a 300-µl sample was taken and placed on ice until all samples had been collected . A 20-µl portion of each sample was placed into a polystyrene flat-bottom 96-well plate containing 180 µl of LB for reading of the OD at 570 nm with a Dynatech Instruments MR700 plate reader . The readings using this instrument were multiplied by 3.47 to account for path length differences between the MR700 and the Spectronic 20D+ used for the luciferase assays . A 100-µl volume of sample was placed into a polypropylene round-bottom 96-well plate containing 100 µl of Z-buffer and solubilized with 4 µl of chloroform and 4 µl of 0.1% sodium dodecyl sulfate . A 40-µl volume of 4-mg/ml o-nitrophenyl-ß-D-galactopyranoside (ONPG) was added to start the reaction, and 100 µl of 1M NaCO₃ was added to stop it . The plate was then centrifuged at 5,000 x g for 10 min to pellet the chloroform and bacteria . Supernatant (100 µl) was transferred to a polystyrene flat-bottom 96-well plate for determination of A₄₁₀ with the Dynatech MR700 plate reader . Units of activity were calculated as (1,000 x A₄₁₀)/[reaction time (minutes) x sample volume (milliliters) x OD₅₇₀] .

Comparisons of lacZY fusions in motility agar were qualitative, with each strain being stabbed, via a pipette tip, into agar containing 40 µg of X-Gal per ml . This was followed by an overnight incubation, during which the bacteria spread across the plate . However, in some experiments the agar concentration was increased to a point at which the bacteria could no longer swim through it . For those experiments, the plates were seeded with bacteria when the plates were poured . Each reporter strain (100 µl of an overnight culture) was added to 25 ml of molten LB agar (cooled to 50°C) containing X-Gal and AHL, poured into a petri plate, allowed to solidify at room temperature, and then incubated overnight at 37°C .

Of the three MudJ fusions located outside the rck operon, the first is srg-5::MudJ (Fig . 1A) . According to the nomenclature of the recently completed serovar Typhimurium genome (21), this insertion is within an open reading frame (ORF) named STM1554 at 33.6 centisomes (between nucleotides 15335 and 15336 of accession number AE008767) . The insertion is at the extreme 3' end of the gene so that only the last 5 codons are disrupted and the lacZ fusion is in the sense orientation . Because this gene is strongly regulated by sdiA and because the promoter region has been isolated and confirmed to be sdiA dependent (see below), we propose the name srgE for STM1554 (for "sdiA-regulated gene E") and the allele number srgE5::MudJ for the insertion . srgE appears to be within a 39-kb "island" because eight ORFs upstream of srgE (STM1555 to STM1562) and 26 ORFs downstream of srgE (STM1553 to STM1528) are predominantly Salmonella specific . The boundaries of the island are the osmC gene in the clockwise direction and the yne genes in the counterclockwise direction . However, the G + C content varies widely among the ORFs, suggesting that the island is a mosaic of acquisition and/or deletion events . The organization of the region suggests that srgE is expressed from a monocistronic transcript . Three ORFs are divergently transcribed from the srgE promoter region (STM1555 to STM1557), but these are not regulated by sdiA (see below) . The G + C content of Salmonella is typically 53%, while the G + C content of srgE is much lower at 36% (21) . The G + C content abruptly increases on both sides of srgE, suggesting that srgE may have been a single-gene acquisition .

The molecular mass of SrgE is predicted to be 55.7 kDa, with a pI of 6.8 . The annotation of the serovar Typhimurium genome predicts that SrgE is an inner membrane protein based on identification of a transmembrane domain between residues 418 and 435, using the program PSORT (25) . However, another transmembrane helix prediction program, TMHMM 2.0, suggests that there are no transmembrane helices (19), leaving the predicted subcellular localization of SrgE uncertain . The genome annotation suggests that SrgE has a coiled-coil domain (21) . The PairCoil program also suggests that SrgE has a coiled-coil domain, located between residues 344 and 374 (6) . The function and significance of this domain are unknown . The only similar protein in the current databases, using BLAST programs, is an apparent ortholog of srgE in the S . enterica serovar Typhi genome, ORF STY1509 (3, 4, 26) . STY1509 is 98% identical to SrgE throughout the majority of the protein . However, STY1509 lacks 15 N-terminal residues (possibly due to a start codon prediction discrepancy) and 82 C-terminal residues compared to SrgE . The PairCoil program predicts a coiled-coil domain in the serovar Typhi protein between residues 329 and 359 .

The second fusion, srg-6::MudJ, is located within STM1026 (gtgA) in the antisense orientation (nucleotide 29215 of accession number AE008743) . This gene lies within the gifsy-2 prophage at 24 centisomes (12) . GtgA has 97% amino acid identity to GogA of the gifsy-1 phage and 71% identity to PipA of SPI5 (12, 41) . The function of this gene and the significance of the antisense regulation by sdiA are unknown .

The third fusion, srg-7::MudJ, resides on the virulence plasmid of serovar Typhimurium . The fusion is in the antisense orientation within ORF PSLT61 (nucleotide 50951 of accession number AE006471) (21) . PSLT61 has a high G + C content (61%) and is predicted to encode an inner membrane protein of 28.5 kDa with a pI of 5.2 . This protein is at least 38% identical to the products of numerous plasmid-located genes of unknown function including ycfA from plasmid ColIb-P9, protein L7076 from enterohemorrhagic Escherichia coli plasmid pO157, and ORF248 of the E . coli F plasmid . As with srg-6, the function of this gene and the significance of the antisense regulation by sdiA are unknown .

Cloning of the srgE promoter region. The intergenic region between srgE and the divergently transcribed gene was amplified by PCR and cloned into pSB401 to place the putative promoter region upstream of the promoterless luxCDABE operon . A clone with the srgE promoter in the appropriate orientation was named pJNS25, while a clone with the fragment in the opposite orientation was named pJNS35 . These two reporter constructs were electroporated into the wild-type and sdiA mutant serovar Typhimurium strains 14028 and BA612, respectively . Cross-streak assays were performed with these strains to detect sdiA-dependent responses to AHL . A 20-µl volume of 10 µM C₆ was streaked across one end of an LB plate, and the reporter strains were streaked perpendicular to the AHL . Increases in light production of at least 12-fold were observed from the wild-type strain carrying pJNS25 near the C₆, while the sdiA mutant strain did not respond (Fig . 1B) . This confirms that the region cloned within pJNS25 does contain an sdiA-dependent promoter . The strains carrying pJNS35 did not respond to AHL (data not shown), demonstrating that the sdiA-dependent activation of the srgE promoter is unidirectional . Similar attempts to clone the srg-6 and srg-7 promoters were unsuccessful .

Conditions that allow chromosomal fusions to respond to SdiA and AHL. Previously we determined that SdiA expressed from the chromosome could activate a plasmid-based Prck-luxCDABE reporter in the presence of particular AHLs (22) . This activation occurred under a variety of conditions including cross-streak assays on solid LB agar plates (1.2 or 1.5% agar), filter disk assays in 0.7% top agar, and liquid culture . However, the MudJ insertions in the rck operon and the other three srg loci did not respond to chromosomally encoded SdiA and AHL (they responded only to plasmid-encoded SdiA) . Since this observation cast doubt on the ability of SdiA to truly detect and respond to AHL production by other species, our recent focus has been on identifying growth conditions that allow the activation of chromosomal fusions by chromosomal sdiA . It was recently discovered that the regulator adjacent to sdiA, named sirA, is much more active during growth in motility agar (LB broth containing 0.25% agar) than in liquid culture (15) . Therefore, we tested the ability of SdiA to activate chromosomal fusions in motility agar . All plates contained either 1 µM C₆ or 0.1% acidified EA as a solvent control . MudJ fusions to all four sdiA-regulated loci were tested in each type of medium at four different incubation temperatures . While this is a qualitative assay, the results clearly demonstrated that three of the four loci can be activated under particular growth conditions . srgA1::MudJ (representing the rck operon), srgE5::MudJ, and srg-7::MudJ responded to AHL in an sdiA-dependent manner, while srg-6::MudJ was not regulated by sdiA under any condition (Fig . 2) . Activation of the three fusions was primarily sdiA and AHL dependent, although both sdiA- and AHL-independent expression was observed .

 
Expression of the srgE5::MudJ fusion was observed at 30, 37, and 42°C but not at 22°C . At each temperature the expression was completely sdiA dependent . Consistent with the sdiA dependence, the expression at 37°C was almost entirely AHL dependent . However, at 30°C, sdiA-dependent activation was observed in the absence of AHL .

The srgA1::MudJ fusion differs from the srgE5::MudJ in two respects . First, the rck operon is more tightly temperature dependent in that expression was observed only at 37 and 42°C . Second, the rck operon can be expressed in the absence of sdiA under certain conditions . For example, in Fig . 2 the sdiA mutant shows an increase in srgA1::MudJ expression at 37 and 42°C compared to 22 and 30°C .

The regulation of srg-7::MudJ by sdiA was very weak, but it had an expression pattern similar to that of srgA1::MudJ (Fig . 2) . This is interesting given that both fusions are present on the serovar Typhimurium virulence plasmid . Like srgA1, the srg-7 fusion was not expressed at 22°C or 30°C . Also like srgA1, there was expression in the absence of sdiA and AHL at higher temperatures .

The assays throughout this report used C₆ at a concentration of 1 µM . SdiA detects oxoC₈ at much lower concentrations than C₆, but oxoC₈ is not commercially available and is not easy to synthesize . To determine if the use of oxoC₈ would give different results from the use of C₆, the motility agar assays in this section were performed once with oxoC₈ at a final concentration of 10 nM . The results were essentially identical to those obtained using 1 µM C₆ (data not shown) .

Agar percentages that allow sdiA-dependent activation of chromosomal fusions. To examine the effect of agar concentration more closely, we seeded plates of various agar concentrations with each reporter strain and 1 µM C₆ and incubated them overnight at 37°C . SdiA-dependent activation of srgE was observed with 0.3, 0.4, and 0.5% agar but not with 0.7% agar (data not shown) . The response in 0.6% agar was very weak . The srgA1::MudJ and srg-7::MudJ fusions were both activated in an sdiA-dependent manner in 0.3 and 0.4% agar . Interestingly, both fusions were more active using 0.5, 0.6, and 0.7% agar, but this was not an sdiA-dependent activation, and in fact the presence of sdiA seemed to inhibit the increased response (the sdiA mutant was slightly darker blue than was the wild type [data not shown]) . The srg-6::MudJ fusion did not respond to AHL under any conditions . To summarize, all of the chromosomal MudJ fusions except srg-6 respond to AHL in an sdiA-dependent manner when the bacteria are grown in 0.25 to 0.4% motility agar but not in 0.7% top agar or on 1.2% agar plates .

Quantitation of sdiA-dependent responses in liquid media. Attempts to measure ß-galactosidase activity in motility agar were unsuccessful . Therefore, we evaluated the response of the chromosomal and plasmid-based fusions in liquid culture . Mild responses were observed for chromosomal fusions at late-exponential and stationary phases (maximum of 4.4-fold for srgE5::MudJ at 37°C [Fig . 3; Table 2]) . These responses allowed us to quantitate and compare the levels of AHL-dependent and AHL-independent SdiA activity . The pattern of response in liquid culture was similar to that obtained in motility agar in that srgE5::MudJ was the strongest responder (with regard to fold differences), followed by srgA1::MudJ and srg-7::MudJ (Fig . 3 and Table 2 for srgE5 and srgA1; data not shown for srg-7) . srg-6::MudJ failed to respond (data not shown) . One difference between liquid and motility agar is that the rck operon was activated weakly in liquid at 30°C (2.4-fold) while no expression was detected in motility agar at the same temperature . Another difference is that the AHL-independent SdiA activity observed at 30°C was much stronger in motility agar than in liquid (Fig . 3; Table 2) . Overall, fold induction values in the presence of AHL increased as the temperature was increased from 30 to 37°C (Fig . 3; Table 2) . However, as the temperature was reduced from 37 to 30°C, SdiA became active in the absence of AHL (Fig . 3; Table 2) .

 
SdiA can activate chromosomal fusions in response to other species of bacteria. A plasmid-based Prck-luxCDABE fusion was previously demonstrated to respond in an sdiA-dependent fashion to the presence of other bacterial species in a cross-streak assay on a standard 1.5% agar LB plate (22) . Two species that were potent activators of SdiA in this assay are Yersinia enterocolitica and Hafnia alvei (22) . However, the chromosomal MudJ fusions to sdiA-regulated loci did not respond in this assay (22), which cast doubt on the ability of SdiA to detect and respond to other species . To determine if the MudJ fusions would respond to Y . enterocolitica or H . alvei in motility agar, we stabbed one side of a motility agar plate with an S . enterica serovar Typhimurium reporter strain and the other side with Y . enterocolitica or H . alvei . The best results were obtained when the Yersinia and Hafnia strains were given a 1-day head start at either 22 or 30°C, respectively, before the Salmonella was added to the plate . Once the Salmonella was stabbed into the plates, the plates were incubated for a further 24 h at 37°C . During this time, the serovar Typhimurium cells swam outward and surrounded the Y . enterocolitica or H . alvei cells . As shown in Fig . 4, the chromosomal MudJ fusions were activated in an sdiA-dependent manner in the region immediately surrounding the AHL-producing species . The fusions did not respond to a yenI mutant of Y . enterocolitica that cannot synthesize AHLs (Fig . 4) . The sdiA-dependent response of the serovar Typhimurium chromosomal MudJ fusions in this assay conclusively demonstrates that Salmonella can detect and respond to other bacterial species that are synthesizing AHLs .

 
SdiA-dependent expression in the absence of exogenously added AHL is not due to AHL production by Salmonella. We have previously reported that the three genera known to encode SdiA (Escherichia, Klebsiella, and Salmonella) do not synthesize AHLs at concentrations that can be detected by biosensors (22) . However, those studies were performed at 37°C . The studies performed at 30°C in this report show SdiA activity in the absence of exogenously supplied AHL . It was possible that sufficient concentrations of AHL are generated by Salmonella at 30°C to partially activate SdiA .

To test this hypothesis, we grew two Salmonella strains at 30°C in LB motility agar containing X-Gal, adjacent to four different AHL biosensor strains (Fig . 5) . The Salmonella strain BA1101 (14028 srgE5::MudJ) and the isogenic sdiA mutant, BA1301, were used to demonstrate that there was indeed AHL-independent SdiA activity during the assay (BA1101 was light blue, while BA1301 was white) . The four reporters were CviR, LasR, LuxR, and AhyR . The CviR reporter strain was Chromobacterium violaceum strain CVOblu, while the other three reporters are E . coli based: DH5/pSB536 (AhyR), JM109/pSB401 (LuxR), and JM109/pSB1075 (LasR) . CVOblu synthesizes the purple pigment violacein when short-chain AHLs are detected (C₄ and C₆ [34]) . The pSB536 reporter encodes AhyR from Aeromonas hydrophila, which responds to C4 (34, 35) . The pSB401 plasmid encodes LuxR from Vibrio fischeri, which detects oxoC₆ with highest sensitivity but also detects oxoC₈, C₆, and C₈, the same AHLs detected by SdiA (40) . Plasmid pSB1075 encodes the Pseudomonas aeruginosa LasR protein, which detects oxoC₁₂ with highest sensitivity but also detects C₁₀, C₁₂, oxoC₁₀, and oxoC₁₄ (40) . We also attempted to use the TraR reporter Agrobacterium tumefaciens strain NT1/pDCI41E33 (31), but it failed to detect positive control strains under the conditions used, and so it was not used .

 
The CviR reporter was able to detect the positive control species Serratia marcescens and Hafnia alvei but showed absolutely no response to the Salmonella strains (Fig . 5A) . The lux-based reporters all showed a basal level of light in the presence of Salmonella but responded strongly to control strains (Fig . 5B) . To be sure that the low levels of light emitted in the presence of Salmonella are indeed basal levels of reporter expression, indicative of no signal, negative control strains were used . For the LuxR reporter, the positive control species was Y . enterocolitica and the negative control was an isogenic yenI mutant which cannot synthesize AHLs . For the AhyR and LasR reporters, the positive control species was P . aeruginosa and the negative control was an isogenic lasI rhlI double mutant of P . aeruginosa that cannot synthesize AHLs . The response of these three reporter systems to Salmonella was less than or equal to the response generated by the negative control strains . Given that all of these reporters can detect AHLs at nanomolar concentrations, similar to SdiA (1 to 10 nM for SdiA [22], 1 nM for CviR [7], 1 to 10 nM for LuxR [40], and 0.1 to 1 nM for LasR [40]), we conclude that Salmonella is not producing AHL at the nanomolar concentrations required to activate SdiA . Because low levels of SdiA activity were observed during the assay (BA1101 was light blue, while BA1301 was white), we conclude that this is a basal level of SdiA activity that does not represent AHL detection .

We have demonstrated that chromosomal MudJ fusions to srgE, srg-7, and the rck operon respond to exogenous AHL in an sdiA-dependent fashion when sdiA is expressed from its natural position in the chromosome . This is the first study in which chromosomal fusions have been activated by chromosomal sdiA . The chromosomal fusions respond in both liquid medium and in motility agar (0.25%) but do not respond in top agar (0.7% agar) or on solid agar plates (1.2 or 1.5% agar) . The plasmid-based reporter systems for srgE and rck are not limited in this way and respond to SdiA and AHL under all these conditions . Therefore, the chromosomal fusions are the more relevant reporters, although as a simple AHL detection system the plasmid-based fusions are more useful since they can be used in a filter disk assay or in a cross-streak assay . There is a regulator downstream of sdiA that has requirements that seem inverted compared to sdiA . The regulator is named sirA in Salmonella, and it regulates motility and invasion functions (2, 15, 18) . SirA is active on solid agar and in motility agar but is only weakly active in liquid (15) . Therefore, motility agar containing AHL is currently the in vitro medium of choice to ensure that both regulators are active .

The significance of two of the srg loci with regard to the SdiA regulon is questionable at this point . The MudJ insertions for both are in the antisense orientation . One of these, srg-7, can be activated slightly by chromosomal sdiA in response to exogenous AHL . The srg-7::MudJ fusion is located within PSLT61 on the S . enterica serovar Typhimurium virulence plasmid which does not have a known function . There are homologs of PSLT61 on many plasmids including the F plasmid of E . coli and pO157 of enterohemorrhagic E . coli . The second antisense fusion is srg-6::MudJ . This fusion resides within the gtgA gene of the gifsy-2 prophage . The function of this gene is not known . This fusion is the weakest in response to plasmid-located sdiA (1) and is the only fusion for which we have not yet detected a response to chromosomal sdiA and exogenous AHL . Our preliminary attempts to isolate the sdiA-dependent promoters for both srg-6 and srg-7 were unsuccessful .

In contrast, the srg-5::MudJ is in the sense orientation within ORF STM1554 and is strongly activated by sdiA . The promoter region for this gene was successfully isolated and confirmed to be sdiA dependent (Fig . 1B) . Therefore, we propose the name srgE (for "sdiA-regulated gene") for ORF STM1554 and the allele name srgE5::MudJ for the insertion . In addition, we propose a name change within the rck operon . We recently reported that the sdiA-dependent promoter of the rck operon was further upstream than expected (22) . This added two genes to the operon, pef1 and ORF7 (Fig . 1a) . Because other genes within this operon are named srgA, srgB, and srgC, we propose that ORF7 be named srgD .

The rck operon and srg-7 are carried on the serovar Typhimurium virulence plasmid and share several traits that differ from srgE. Fusions to both the rck operon and srg-7 are tightly temperature dependent in motility agar in that they fail to respond to AHL at 30°C and instead respond only at 37°C and higher . In contrast, the srgE promoter responds strongly to AHL at 30°C and higher . However, this difference between the rck operon and srgE largely disappears in liquid culture, where both loci can be activated at 30°C . None of the fusions are active at room temperature, and the mechanism of temperature-dependence is unknown . However, the 37°C optimal temperature for the SdiA regulon suggests that the mammalian gastrointestinal tract may be the location where SdiA detects and responds to AHL . Consistent with this hypothesis is the recent detection in the bovine rumen of compounds that can activate AHL biosensors (11) . A second trait shared by the rck operon and srg-7 is that both can be expressed in the absence of sdiA, a characteristic that complicates studies of sdiA. Under the conditions examined to date, the srgE promoter is not expressed in the absence of sdiA and therefore provides the simplest and strongest reporter of SdiA activity .

The srgE promoter is the third sdiA-dependent promoter to be isolated . The other two promoters are the rck operon promoter in Salmonella and the ftsQ promoter in E . coli. The promoter for the rck operon resides between ORF6 and pef1 (22) and is shown in Fig . 1A . In E . coli, the ftsQP2 promoter cloned on a multicopy plasmid as a fusion to lacZY is activated 5- to 13-fold in response to plasmid-encoded sdiA but only 1.3-fold in response to chromosomal sdiA (38) . Addition of AHL to the culture increases expression by twofold but only when using plasmid-encoded sdiA (32) . Therefore, unlike the rck and srgE promoters, the E . coli ftsQP2 promoter has never been shown to respond to AHL using sdiA expressed from its natural position in the chromosome, and our preliminary results indicate that it does not (unpublished data) . However, biochemical experiments using purified SdiA and the ftsQP2 promoter (in the absence of AHL) revealed a putative SdiA binding site (42) . This sequence is not present within the promoter regions of either srgE or the rck operon, suggesting that this is either not a specific SdiA binding site or SdiA of E . coli has a different binding site than SdiA of Salmonella or that rck and srgE are only indirectly affected by SdiA . We feel that prediction of SdiA binding sites is premature at this point and should await the results of biochemical experiments with more promoters .

To date, SdiA is the only LuxR homolog that is hypothesized to detect only the AHL production of other species . For this to be true, it must be clear that Salmonella fails to synthesize AHLs and that SdiA is active only when AHLs are supplied exogenously . This has been demonstrated conclusively in this study at 37°C . In liquid culture we observed a 1.3-fold difference between the wild type and the sdiA mutant when measuring the regulation of the srgA1::MudJ and srgE5::MudJ fusions (Table 2) . However, there is no detectable AHL production by S . enterica serovar Typhimurium at this temperature (22), so we ascribe this 1.3-fold difference to basal levels of SdiA activity . Furthermore, as shown in Fig . 4, Salmonella can detect and respond to the presence of H . alvei or Y . enterocolitica. This detection event requires that Salmonella carry sdiA and that the other species produce AHL (the yenI mutant of Yersinia is not detected) . We feel that this combination of evidence conclusively demonstrates that at 37°C Salmonella fails to synthesize AHLs but detects and responds to AHLs produced by other species .

The conclusions to be drawn from results obtained at 30°C are not so clear . At this temperature there is substantial SdiA activity in the absence of AHL (Fig . 2 and 3; Table 2) . This activity is strongest in motility agar, where the expression of srgE in the presence and absence of AHL is nearly identical but still entirely sdiA dependent (this is seen only with srgE, since the other fusions are not expressed at 30°C [Fig . 2]) . In liquid culture, the AHL-independent SdiA activity is less intense but is seen with both srgE and the rck operon (Fig . 3; Table 2) . Since Salmonella is not producing AHL at this temperature (Fig . 5), there are two potential explanations for this activity . First, SdiA may be detecting some type of molecule synthesized by Salmonella at 30°C that is not an AHL . While we cannot eliminate this possibility, we feel that it is unlikely because the molecule would have to be detected by SdiA but not by the other four LuxR-type biosensors, it would have to be produced only at 30°C, and it would have to be produced by a currently unrecognized type of synthase . We favor the second possibility, in which the activity does not represent signal detection at all but simply represents high basal levels of SdiA activity in the absence of signal . This immediately raises the question of why the basal level of activity is so much higher at 30°C than at 37°C . We hypothesize that this could be due to either increased sdiA expression or increased oligomerization of SdiA at 30°C . Both hypotheses need to be tested, but we already know that expression of sdiA from a plasmid increases the expression of SdiA-regulated genes in the absence of signal (1, 22) . Regardless, this is the first demonstration of chromosomally encoded sdiA activating chromosomal fusions . At 37°C, this activation requires the addition of AHL from another source . We conclude that SdiA is used to detect AHLs that are synthesized by other species .

This publication was made possible by grant 1 R01 AI50002-01 from the National Institute of Allergy and Infectious Diseases .

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