All bacterial strains are derivatives of ATCC 14028s and are listed in Table 1. Unless stated otherwise, bacteria were grown in Luria-Bertani (LB) broth or on LB agar supplemented with the following antibiotics as appropriate: kanamycin (50 mg/L), nalidixic acid (50 mg/L), carbenicillin (100 mg/L), streptomycin (100 mg/L), or chloramphenicol (20 mg/L). Mutants were constructed by a modification of the λ–red recombination technique and antibiotic cassettes were removed as described (Datsenko and Wanner, 2000; Santiviago et al., 2009). Chromosomal transcriptional fusions to lacZY were constructed as described previously (Ellermeier et al., 2002). All mutations were moved to a clean genetic background by bacteriophage P22-mediated transduction (Sternberg and Maurer, 1991).

The Texas A&M University and University of California Davis Institutional Animal Care and Use Committees approved all animal experiments (approval numbers TAMU 2012-084 and 2011-167; UCD 19001). Mouse experiments were performed using 10–12 week old female C57BL/6J or CBA/J mice as indicated (Jackson Laboratories) as previously described (Barthel et al., 2003).

In the acute murine colitis model, C57BL/6J mice were administered 20 mg streptomycin in 75 μL sterile water by gavage. Twenty-four hours after treatment, mice were infected with ~10⁸ CFU of an equivalent mixture of WT and mutant bacteria in 100 μL volume by gavage. Feces were collected 24 h after infection. Mice were euthanized 96 h post-infection and organs harvested, homogenized, serially diluted, and plated on LB agar with appropriate antibiotics for enumeration of CFU.

In the chronic murine colitis model, CBA/J mice were administered 20 mg streptomycin in 75 μL water or 75 μL sterile water by gavage. Forty-eight hours after treatment, mice were infected as above. Feces were collected on the indicated days and mice euthanized 14 days post-infection. Competitive index was determined by comparing the ratio of WT to mutant bacteria in the tissue to that of the inoculum.

Germ-free Swiss Webster mice were bred and housed under germ-free conditions inside gnotobiotic isolators (Park Bioservices, LLC). Weekly cultures were grown to monitor the germ-free status of the mice. For experiments, male and female 6–8-week-old mice were transferred to a biosafety cabinet and maintained in sterile cages for the duration of the experiment. Mice were infected with ~10⁸ CFU of an equivalent mixture of WT and mutant bacteria in 100 μL volume by gavage. Mice were euthanized 24 h post-infection, cecal and colon contents were homogenized, serially diluted, and plated on LB agar with appropriate antibiotics for enumeration of CFU. The competitive index was determined by normalizing the ratio of WT to mutant bacteria in intestinal contents to that of the inoculum.

Cell lines were purchased from American Type Culture Collection (ATCC) and used within 15 passages. HeLa cells (human cervical adenocarcinoma epithelial, ATCC CCL-2) were grown as recommended by ATCC. HeLa cells were seeded in 24-well plates at 5 × 10⁴ cells/well ~24 h prior to infection.

Invasion assays were performed as previously described (Ibarra et al., 2010). Late-log phase cultures were prepared by inoculating 10 ml LB-Miller broth with 0.3 ml overnight culture. Flasks were grown at 37°C with aeration for 3 h. Bacteria were collected by centrifugation at 8,000 × g for 90 s, resuspended in an equal volume of Hanks' buffered saline solution (HBSS, Mediatech) and added directly to mammalian cells seeded in 24-well plates for 10 min. The multiplicity of infection was ~50:1 (bacteria:eukaryotic cell). Non-internalized bacteria were removed by aspiration, monolayers washed three times in HBSS and then incubated in growth media until 30 min post-infection. Gentamicin was added at 50 μg/ml from 30 to 90 min post-infection to kill extracellular bacteria and the media was replaced with media containing 10 μg/ml gentamicin from 90 min post infection. For enumeration of intracellular bacteria, monolayers were washed once in phosphate-buffered saline, solubilized in 0.2% (w/v) sodium deoxycholate and serial dilutions were plated on LB agar.

For induction of SPI-1 expression, bacterial cells bearing plasmid constructs were grown overnight in LB with appropriate antibiotics. Overnight cultures were diluted 1:100 and incubated at 37°C with agitation for 3 h. SPI-2 inducing media was used as described (5 mM KCl, 7.5 mM (NH₄)₂SO₄, 0.5 mM K₂SO₄, 8 μM MgCl₂, 337 μM KH₂PO₄, 80 mM MES, 0.3% (v/v) glycerol, 0.1% (v/v) casamino acids, pH 5.8) (Coombes et al., 2004). Cells were grown overnight in SPI-2 inducing media, diluted 1:50, and incubated for an additional 24 h to evaluate SPI-2 expression. To evaluate the expression of phnA in the presence of PA, overnight cultures were diluted 1:100 in LB with 40 mM MOPS at pH 6.8 with the indicated phosphorus source. Cultures were incubated at 37°C with agitation for 3 h.

ß-galactosidase activity was determined using standard methodology (Miller, 1972). Briefly, bacterial cells were pelleted by centrifugation and resuspended in Z-buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 mM ß-mercaptoethanol). Culture density was determined by OD₆₀₀. Bacteria were permeabilized with chloroform and 0.1% (w/v) SDS prior to addition of substrate (o-nitrophenyl-β-D-galactoside; ONPG 4 mg/mL). The reactions were performed at 28°C and were stopped with 1 M Na₂CO₃ for determination of OD₄₂₀ and OD₅₅₀. β-galactosidase activity (Miller units) was calculated using the following equation: 1000 × [OD₄₂₀–(1.75 × OD₅₅₀)] / [time × volume × OD₆₀₀].

Modifications were made to a phosphorus-limited minimal medium (Neidhardt et al., 1974) to assess the ability of different bacterial strains to utilize different phosphorus sources. The final media composition (MMMM) was as follows: 20 mM NH₄Cl, 2.5 mM Na₂SO₄, 80 mM NaCl, 0.35 mM CaCl₂, 20 mM KCl, 40 mM MOPS, 1 mM MgSO₄, 0.01 mM FeSO₄, 0.2% (w/v) glucose, pH 6.8. Phosphorus sources were Na₂HPO₄ (Pi, Sigma), 2-aminoethylphosphonate (2-AEP, Sigma), and phosphonoacetic acid (PA, Sigma) added at the concentrations indicated. Bacterial strains were grown overnight at 37°C with agitation in MMMM with Na₂HPO₄ at the indicated concentration. Overnight cultures were pelleted and pellets were resuspended in an equal volume MMMM supplemented with the indicated phosphorous sources. After re-centrifugation and resuspension in the starting volume or the original culture, bacteria were diluted 1:100 into fresh MMMM with indicated phosphorus source. Aliquots were removed, serially diluted, and plated to determine CFU.

All data were log transformed prior to analysis. Statistical significance was determined using Student's t-test or analysis of variance with significance set at P < 0.05.

STM3602 is necessary for STm to colonize the intestine of calves in ligated ileal loops (Elfenbein et al., 2013). We used the murine colitis model to further dissect the function of this gene during infection (Barthel et al., 2003). In this model, mice are treated with high doses of streptomycin prior to infection promoting STm colonization and development of a neutrophilic inflammatory response. We found that the ΔSTM3602 mutant is shed in lower numbers than the wild type organism in the feces by 24 h post-infection, and colonizes Peyer's patches and mesenteric lymph nodes at lower levels than the wild type organism at 4 days post-infection (Figure 2). This colonization defect was reversed by complementation in trans. Colonization of the cecum by the ΔSTM3602 mutant was highly variable in different mice making it difficult to determine with statistical confidence whether or not mutants lacking STM3602 have a colonization defect in the cecum. Regardless, our results are consistent with previous data suggesting a requirement of STM3602 in colonization of the intestine in the presence of a profound neutrophilic inflammatory response.

Salmonellae possess two type-3 secretion systems, encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). The TTSS-1 and its associated effectors are essential for invasion of non-phagocytic epithelial cells and penetration of the intestinal epithelium (Zhang et al., 2002; Raffatellu et al., 2005). The TTSS-2 is needed for maintenance of the Salmonella-containing vacuole and intracellular replication (Figueira and Holden, 2012). We hypothesized that the intestinal colonization defect of ΔSTM3602 mutants could be due to reduced expression of TTSS-1.

We used plasmids containing lacZY under the control of promoters of genes on SPI-1 or SPI-2 to determine whether deletion of STM3602 resulted in an effect on activation of these promoters (Zheng et al., 2013). Using a plasmid containing lacZY under the control of the prgH promoter (a TTSS-1 structural gene), we found that the prgH promoter is activated less efficiently in SPI-1 inducing conditions in bacteria lacking STM3602 than in the WT (Figure 3A). Consistent with this result, deletion of STM3602 also results in a similar reduction in invasion in cultured epithelial cells (Figure 3B). Conversely, the ΔSTM3602 mutant is capable of activation of TTSS-2 apparatus (ssaG) and effector promoters (sseJ; Figure 3C) as well as survival within cultured epithelial cells to the same level as the wild type (Figure 3D). These results suggest that STM3602 plays a role in the complex regulatory network of the TTSS-1.

STM3602 belongs to the GntR family of transcriptional regulators and is a part of the PhnR clade (Marchler-Bauer et al., 2015). This gene shares 29.2% sequence similarity and 45.1% amino acid identity with PhnR (STM0430; Sievers et al., 2011). We hypothesized that STM3602 might regulate one or both of the phosphonate utilization loci in STm. We characterized the survival and growth of the ΔSTM3602 mutant in media containing either 2-AEP or PA as the sole phosphorus source. When strains were grown in the presence of 5 mM PA, the ΔSTM3602 mutant replicated poorly (Figure 4A). However, this mutant grows normally in 5 mM 2-AEP or Pi (Figures 4B,C) as sole phosphorus sources. PA is degraded to acetate and inorganic phosphate (Figure 1). One possible explanation for the phenotype of the ΔSTM3602 mutant in PA-containing medium is that this mutant may be unable to utilize or excrete acetate. However, the ΔSTM3602 mutant exhibits similar growth kinetics to WT in the presence of both 5 mM acetate and 5 mM Pi (Figure 4D) suggesting a mechanism specific to use of PA. Overall, these data suggest a role for STM3602 in phosphonoacetate utilization.

Next, we determined the growth kinetics of the ΔSTM3602 mutant in media with 5 mM PA after phosphorus deprivation. When bacteria were grown in phosphorus-limiting conditions (0.5 mM Pi) and transferred to media containing 10 times more phosphorus in the form of PA, the ΔSTM3602 mutant lost viability over the course of the 6-h experiment (Figure 5). A mutant deleted for phnA, a gene that encodes the phosphonoacetate hydrolase, had growth kinetics similar to the WT in these conditions (Figure 5). These results suggest that STM3602 is needed for activation of pathways necessary for growth in the presence of PA and that there is a pathway for metabolism of this compound that operates independently of phnA.

The ΔSTM3602 mutant grows poorly in the presence of PA. We hypothesized that STM3602 regulates the phnABO operon because the annotation of phnA suggests that PhnA may degrade PA. In order to test this hypothesis, we generated a mutant strain bearing a lacZY fusion to the first 10 amino acids of phnA to monitor transcription from the native phnA promoter. Using this construct, we found that in rich media the expression of the phnA promoter was not affected by the addition of PA at varying concentrations (Figure 6). In addition, deletion of STM3602 did not affect the expression of phnA in these conditions during log phase growth (Figure 6), a condition where STM3602 is expressed (Kroger et al., 2013). These data suggest that STM3602 does not regulate phnA in rich medium in the presence or absence of PA.

The first phosphonate to be discovered, 2-AEP is present in both ruminal and duodenal contents of sheep (Ankrah et al., 1989). This compound is associated with ruminal protozoal and bacterial populations and is less abundant in defaunated animals compared with those with a normal microbial composition. Phosphonoacetic acid is one product of 2-AEP metabolism mediated by the enzyme PhnY, phosphonoacetaldehyde oxidase (Agarwal et al., 2014). Although salmonellae lack a gene with this annotated function, phyla found in the murine intestinal microflora including Cyanobacteria, Proteobacteria and Firmicutes encode genes to produce PA from 2-AEP (Stecher et al., 2007; Martinez et al., 2010; Villarreal-Chiu et al., 2012).

We hypothesized that the colonization defect of the ΔSTM3602 mutant might correlate with microbial colonization of the host intestine. We used the chronic carriage mouse model to determine the longer-term kinetics of the colonization defect of this mutant. In streptomycin pre-treated mice (altered microbiota) in competitive infection with the WT, the ΔSTM3602 mutant failed to colonize the intestine beginning very early at 1-day post-infection. In animals with an intact microbiota (no streptomycin pretreatment) the colonization defect of the ΔSTM3602 mutant took longer to develop becoming apparent at 5 days post infection (Figure 7A). Thus, the colonization defect of the ΔSTM3602 mutant occurs much earlier in animals with disrupted microbiota than with intact microbiota.

One possible explanation for the reduced ability of the ΔSTM3602 mutant to colonize the murine intestine is due to reduced expression of the TTSS-1 (Figures 3A,B). To rule out this possibility, we performed a competitive infection experiment in a ΔSPI-1 genetic background (ΔSPI-1 mutant vs. ΔSPI-1ΔSTM3602 double mutant). In these experiments, the double ΔSPI-1ΔSTM3602 mutant colonizes the intestine poorly on the first day post-infection in animals regardless of whether the intestinal microflora were intact or disrupted (Figure 7B). The phenotype of the ΔSTM3602 mutant was significantly stronger 10 days post-infection in the animals that began with disrupted microflora. These data suggest that the observed colonization defects are unlikely to be due solely to poor activity of the TTSS-1 but more likely a result of inability to compete in the intestine.

Finally, we evaluated the fitness of the ΔSTM3602 mutant using competitive infections in germ-free mice to eliminate any effects of microbiota on colonization efficiency of the ΔSTM3602 mutant. In the germ free murine model, the ΔSTM3602 mutant exhibited a small but significant colonization defect in the cecal contents in the WT and ΔSPI-1 genetic backgrounds (Figure 7C) but only a significant colonization defect in the colon contents in the ΔSPI-1 genetic background. Taken together, these data suggest that STM3602 is required for colonization of the murine intestine regardless of the host inflammatory response or the microbial composition of the intestine.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.