Flagella-independent surface motility in Salmonella enterica serovar Typhimurium

The PhoP/PhoQ System Promotes Surface Motility on Low Mg²⁺ Agarose Media.

To examine the migratory behavior of wild-type S. enterica serovar Typhimurium strain 14028s under PhoP/PhoQ-inducing conditions, we grew bacteria in N-minimal liquid media with 10 μM Mg²⁺ for 4.5 h and then spotted the bacteria on the same media with different agarose concentrations (0.2–0.6%). The distance migrated was inversely correlated with the agarose concentration, and at 0.6% agarose, no movement was detected (Fig. S1).

In contrast to the wild-type strain, a phoP mutant displayed no motility on 0.3% agarose N-minimal media with 10 μM Mg²⁺ (Fig. 1A). Moreover, neither wild-type Salmonella nor the phoP mutant were motile on 0.3% agarose with 10 mM Mg²⁺ N-minimal media (Fig. 1A), presumably because the PhoP/PhoQ system is not active in high Mg²⁺ (11). The identified motility does not appear to be swarming motility because the PhoP/PhoQ system represses flagella-mediated motility (9, 10), and one would expect a phoP mutant to move more than the wild-type strain. In addition, swarming motility requires glucose, and the media had glycerol as the carbon source.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

The PhoP/PhoQ system promotes surface motility on low Mg²⁺ agarose media. (A) Surface motility of wild-type S. enterica serovar Typhimurium (14028s), phoP (MS7953s), mgtA (EG16735), mgtC (EL4), and pagM (SP244) strains grown as described in Materials and Methods and spotted onto 0.3% agarose low (10 µM) Mg²⁺ (HL) and high (10 mM) Mg²⁺ minimal media. (B) Surface behavior of wild-type S. enterica serovar Typhimurium (14028s) and mutants defective in the motA (MP7), fliC (SP255), flgE (SP370), or flhDC (EG11308) genes. Bacteria were grown as described in A and spotted onto 0.3% (Top) and 0.1% (Bottom) agarose. (C) Surface behavior of wild-type S. enterica serovar Typhimurium (14028s) and mutants defective in the fimA (SP347), pefA (SP371), stfA (SP372), bcfD (SP374), stbB (SP375), lpfB (SP376), csgB (MP362), stdA (SP379), stiA (SP380), stcA (SP381), sthA (SP382), safA (SP373), or stjC (SP378) fimbriae genes on 0.3% agarose HL. Motility was examined as described in A. Photo image was captured after bacteria were grown at 37 °C for 20 h. Shown are the representatives of three independent experiments.

We ruled out the participation of flagella in the identified motility because mutants defective in flagella rotation (motA), hook protein (flgE), flagellin subunit (fliC), or the master flagella regulator (flhDC) moved similarly to the parental strain on 0.3% agarose N-minimal media with 10 µM Mg²⁺ (Fig. 1B). By contrast, all four mutants were not motile or less motile than the wild-type strain if the agarose concentration was lowered to 0.1% (Fig. 1B), as expected for a process dependent on flagella. The fliC single mutant exhibited intermediate swimming behavior possibly because Salmonella harbors two flagellin genes, fliC and fliB (6), and only one was inactivated.

Fimbriae or pili are surface appendages required for twitching and gliding motility in certain bacterial species (22, 23). The S. enterica serovar Typhimurium genome has 13 fimbriae operons (24). Individual mutations in each of the 13 fimbriae operons (fimA, pefA, stfA, bcfD, stbB, lpfB, csgB, stdA, stiA, stcA, sthA, safA, and stjC) had no effect on bacterial migration on 0.3% agarose low Mg²⁺ media (Fig. 1C). The results suggest that the Salmonella PhoP/PhoQ system controls a previously unidentified form of surface motility. (Note that the participation of the fimbriae cannot be entirely ruled out until a strain deleted for all 13 fimbriae operons is constructed and evaluated.)

The PhoP-Activated mgtA, mgtC, and pagM Genes Are Required for Surface Motility.

To identify the PhoP-regulated gene(s) responsible for motility on 0.3% agarose low Mg²⁺ media, we tested the behavior of strains mutated in each of 19 different PhoP-activated genes (mgtA, mgtB, mgtC, mig-14, pagC, pagK, pagM, pagN, pagO, pagP, pcgL, pgtE, phoN, pmrD, rstA, slyA, ugtL, virK, and yobG). Mutants lacking the mgtA, mgtC, or pagM genes were as defective as the phoP null mutant, whereas a slyA mutant was partially defective, displaying a motility intermediate between that of the wild-type strain and the phoP mutant (Fig. S2). The other mutants displayed a wild-type behavior (Fig. S2).

How do the mgtA, mgtC, and pagM genes promote surface motility in low Mg²⁺ conditions? Given that the Mg²⁺ transporter MgtA is required for full pagM transcription (16) (Fig. 2A), we reasoned that MgtA’s role in motility might be to promote pagM expression. In agreement with this notion, expression of the pagM gene from a heterologous promoter partially restored motility to the mgtA mutant, whereas the plasmid vector did not (Fig. 2B).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

The PhoP-activated mgtA, mgtC, and pagM genes are required for motility on 0.3% agarose low Mg²⁺ media. (A) pagM mRNA levels produced by wild-type (14028s), mgtA (EG16735), and mgtC (EL4) strains grown in low (10 µM) Mg²⁺ media at 37 °C for 4.5 h. Expression levels were determined as described in Materials and Methods. Shown are the mean and SD from three independent experiments. (B) Surface motility of pagM (SP244), mgtA (EG16735), and mgtC (EL4) strains harboring a plasmid expressing the wild-type pagM gene (pUH-pagM). Bacteria were grown in HL media for 4.5 h in the presence of ampicillin (50 µg/mL) and IPTG (0.1 mM) at 37 °C. The vector pUHE 21–2lacI^q was used as a control. Motility was evaluated as described in the Fig. 1A legend. (C) Surface motility of a wild-type S. enterica serovar Typhimurium (14028s) strain harboring a plasmid expressing the pagM gene (pUH-pagM). The vector pUHE 21–2lacI^q was used as a control. Motility was examined as described in B. (D) Surface motility of the pagM motA strain (SP247) harboring a plasmid expressing the pagM gene (pUH-pagM) or the vector pUHE 21–2lacI^q as a control. Bacteria were grown as described in B, and then 3 μL of a bacterial suspension (OD₆₀₀ ∼1) were spotted onto the 0.3% agarose low (10 µM) and high (10 mM) Mg²⁺. Photo image was captured after bacteria were grown at 37 °C for 20 h. Shown are the representatives of three independent experiments.

The mgtC gene specifies an inhibitor of the F₁F_o ATPase (25) and may also promote motility by allowing pagM expression given that MgtC controls ATP levels and cytosolic pH (25). In agreement with this notion, the mRNA levels of the pagM gene were fivefold higher in the wild-type strain than in the isogenic mgtC mutant following growth in low Mg²⁺ media for 4.5 h (Fig. 2A). Moreover, the pagM-expressing plasmid, but not the vector control, restored complete motility to the mgtC mutant (Fig. 2B). The pagM-expressing plasmid enhanced motility of the wild-type strain (Fig. 2C), and of course, it rescued motility in the pagM mutant (Fig. 2B).

That the mgtA and mgtC genes act upstream of pagM in the identified motility was reinforced by two independent sets of experiments. First, wild-type and pagM mutant strains produced similar amounts of mgtA mRNA and the same was true for the mgtC mRNA (Fig. S3A). And second, plasmids expressing the mgtA or mgtC gene from a heterologous promoter failed to restore motility to the pagM mutant and behaved like the vector control (Fig. S3B). By contrast, the pagM-expressing plasmid rescued motility in the pagM mutant (Fig. S3B).

To test the hypothesis that PagM-mediated motility requires low Mg²⁺ solely to activate the PhoP/PhoQ system, we investigated whether the pagM-expressing plasmid could restore motility to a pagM motA double mutant in high Mg²⁺ media. By conducting the experiment in a motA mutant background, we could rule out a role for flagella in any observed motility. As hypothesized, the pagM-expressing plasmid conferred motility upon the pagM motA strain in high Mg²⁺, whereas the vector control did not (Fig. 2D). The pagM motA strain harboring the pagM-expressing plasmid displayed more surface motility in high than in low Mg²⁺ (Fig. 2D). As Salmonella grows faster in high than in low Mg²⁺ (26), our findings suggest there is a correlation between PagM-mediated motility and bacterial growth.

PagM-Mediated Motility Requires a Surface Protein(s) but Not Certain Wetting Agents.

The pagM gene encodes a 60 amino acid-long peptide that includes a 28 amino acid-long predicted signal sequence and a mature 32 amino acid-long product (Fig. S4). The amino acid composition of the mature PagM is unusual, as it has seven glycines and three cysteines (Fig. S4). Protein localization predictions suggest PagM is an extracytoplasmic peptide, and this prediction is supported by the alkaline phosphatase activity displayed by Salmonella harboring a translational fusion between pagM and a truncated phoA gene from Escherichia coli (27). (The subcellular location of the PagM protein could not be investigated because antibodies against PagM are not available, and introduction of epitope tags rendered PagM nonfunctional.)

We reasoned that if PagM and/or a surface protein required for motility in low Mg²⁺ were to localize to the outer leaflet of the outer membrane, protease treatment might abolish motility. In agreement with this notion, a disk impregnated with proteinase K abolished motility, whereas a disk with a proteinase K that had been previously heat- inactivated did not impact motility (Fig. 3A).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

PagM-mediated motility requires a surface protein(s) but not certain wetting agents. (A) Proteinase K treatment inhibits PagM-mediated motility. The pagM strain (SP244) harboring a plasmid expressing the pagM gene (pUH-pagM) or the vector pUHE 21–2lacI^q as a control was grown in N-minimal media containing 10 µM Mg²⁺ (HL) in the presence of ampicillin (50 µg/mL) and IPTG (0.1 mM) at 37 °C for 4.5 h. Three microliters of a bacterial suspension (OD₆₀₀ ∼1) were spotted onto the same media with 0.3% agarose and paper disk containing 5 µL proteinase K (20 µg/µl) or heat-inactivated proteinase K. (B) Surface behavior of wild-type S. enterica serovar Typhimurium (14028s) and mutants defective in the production of cellulose (bcsA; MP133), O-antigen (rfaL; MP70), capsule (yhiS; MP105), colanic acid (ugd; EG9524), curli (csgB; MP362), ECA (wecB; MP107), or LPS (waaB; YS10) on 0.3% agarose HL. Photo image was captured after bacteria were grown at 37 °C for 20 h. Shown are the representatives of three independent experiments.

Wetting agents often promote motility on cell surfaces by reducing the friction between a cell and its substrate (28, 29). We tested the potential role of various wetting agents by investigating the motility of mutants defective in the production of particular surface molecules. Strains defective in the production of capsule (yhiS), colanic acid (ugd), curli (csgB), or lipopolysaccharide (LPS) (waaB) retained a wild-type behavior (Fig. 3B). Mutants that cannot make cellulose (bcsA) or O-antigen (rfaL) displayed slightly reduced surface migration (Fig. 3B). A wecB mutant unable to synthesize enterobacterial common antigen (ECA) was less motile than wild-type Salmonella (Fig. 3B), and this could be due to poor growth.

Coculture with a PagM-Expressing Strain Confers Motility upon a pagM Mutant.

Bacterial motility can be an individual activity whereby a bacterium swims alone. Alternatively, motility may result from the collective behavior of groups of bacteria moving in a coordinated fashion (30, 31). We reasoned that if PagM-mediated motility is a group behavior, it might be possible to promote motility of a pagM mutant by coincubating it with a pagM-expressing strain. Thus, we used a mixture of two strains to inoculate 0.3% agarose low Mg²⁺ N-minimal media: a pagM mutant harboring a plasmid expressing the green fluorescence protein (GFP) constitutively and a pagM mutant harboring the plasmid expressing the pagM gene from a heterologous promoter.

The pagM-proficient strain rescued motility of the pagM mutant when inoculated at a ratio of 1:4 (Fig. 4A). This rescue is independent of flagella because motility was restored by the motA mutant harboring the plasmid vector as a helper strain (Fig. 4B). By contrast, motility requires a live pagM-expressing strain because there was no rescue if the bacteria were previously killed by heating at 95 °C for 10 min (Fig. 4C).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Coculture with a PagM-expressing strain confers motility upon a pagM mutant. (A) Surface motility of a pagM mutant (SP244) harboring a plasmid expressing the gfp gene in coculture with the pagM mutant harboring a plasmid expressing the pagM gene. Bacteria were grown in N-minimal media containing 10 µM Mg²⁺ in the presence of ampicillin (50 µg/mL) and IPTG (0.2 mM) at 37 °C for 4.5 h. Two bacterial suspensions (OD₆₀₀ ∼1) were mixed at the indicated ratios, and 3 μL of the mixture were spotted on 0.3% agarose HL containing ampicillin and IPTG. (B) Surface motility of coculture of the motA strain (MP7) with the plasmid vector pFPV25 carrying a promoterless gfp gene and the pagM strain with a plasmid (pFPV25.1) expressing the gfp gene from a constitutive promoter. Motility was examined as described in A. (C) Surface motility of coculture of the pagM strain (SP244) with a plasmid expressing the pagM gene, which had been previously killed by heat treatment at 95 °C for 10 min, and the pagM strain (SP244) with a plasmid expressing the gfp gene. Motility was examined as described in A. Photo images (A–C) were captured after 20 h of growth at 37 °C under a transilluminator for GFP detection. (D) Phase-contrast (Left) and fluorescent micrograph (Right) images of edge of coculture at a ratio of 1:4 as described in A.

Microscopic examination of the cocultures on 0.3% agarose low Mg²⁺ media revealed that the GFP-expressing pagM mutant reached the edge of the growth line together with the pagM-expressing strain (Fig. 4D). The intermixing of GFP-expressing and pagM-expressing bacteria is reminiscent of swarming in Serracia liquefaciens (32). Cumulatively, these results indicate that PagM-mediated motility is a group behavior rather than an individual one.

The PagM Allele Determines Surface Motility.

We conducted a Tblastn search of the complete Salmonella genomes using the deduced amino acid sequence of the pagM gene from S. enterica serovar Typhimurium strain 14028s. We identified sequences exhibiting 100% identity to the 14028s PagM in certain isolates from subspecies I, one of the six subspecies that comprise S. enterica (33). These isolates belong to the serovars Anatum, Bovimobificans, Dublin, Heidelberg, Javiana, Paratyphi B, Schwarzegrund, Thompson, and Typhimurium. We found that the PagM proteins differ in length and sequence even among independent isolates of a given serovar (Fig. 5A and Fig. S4). For example, there is a single nucleotide difference between the pagM genes from virulent strain 14028s and certain Typhimurium strains such as strain LT2. This difference results in a stop codon early in the pagM sequence (Fig. 5A and Fig. S4) that renders LT2 nonmotile on 0.3% agarose low Mg²⁺ N-minimal media (Fig. 5B).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 5.

The PagM allele determines surface motility. (A) Alignment of the deduced amino acid sequence of pagM homologs from S. enterica serovar Typhimurium 14828s (Typhimurium 14828s), serovar Dublin (Dublin), serovar Pullorum (Pullorum), serovar Typhimurium LT2 (Typhimurium LT2), and serovar Enteritidis (Enteritidis). (B) Surface motility of Typhimurium 14028s, Dublin, Enteritidis, Typhimurium LT2, and Pullorum. Bacteria were grown in N-minimal media with 10 µM Mg²⁺ and 0.3% casamino acids at 37 °C for 5.5 h. Three microliters of a bacterial suspension (OD₆₀₀ ∼1) were spotted onto the same media containing 0.3% agarose. (C) Surface motility of the S. enterica serovar Typhimurium 14828s pagM mutant (SP244) harboring a plasmid expressing the wild-type pagM (pUH-pagM) or pagM variants (pUH-pagM-A39K and pUH-pagM-K43E). The vector pUHE 21–2lacI^q was used as a control. (D) Surface motility of the nonmotile serovar Enteritidis harboring a plasmid expressing the 14028s pagM gene or the vector control. Bacteria were grown in N-minimal media containing 10 µM Mg²⁺ in the presence of ampicillin (50 µg/mL) and IPTG (0.1 mM) at 37 °C for 4.5 h. Motility was examined as described in B. Photo image was captured after bacteria were grown at 37 °C for 20 h. Shown are the representatives of three independent experiments.

There is a correlation between the pagM allele and surface motility because, first, serovar Dublin encodes a PagM protein identical to the 14028s PagM (Fig. 5A and Fig. S4) and exhibits surface motility similar to strain 14028s (Fig. 5B). And second, serovar Enteritidis specifies a truncated form of PagM, and serovar Pullorum encodes a PagM protein that differs in four amino acid residues from the serovar Typhimurium 14028s PagM (Fig. 5A and Fig. S4), and neither Enteritidis nor Pullorum displays motility (Fig. 5B). (We verified the allelic differences by sequencing the pagM genes from the investigated strains.)

If the surface motility phenotypes are due solely to the pagM allele, it should be possible to convert a motile strain into a nonmotile one and vice versa simply by swapping pagM genes. As proposed, derivatives of serovar Typhimurium strain 14028s specifying a PagM protein with a lysine at position 39 or a glutamate at position 43 did not move on 0.3% agarose low Mg²⁺ N-minimal media (Fig. 5C). These amino acid differences distinguish the PagM proteins from those in Typhimurium 14028s and Pullorum. And the nonmotile serovar Enteritidis gained motility when harboring a plasmid with the 14028s pagM gene but not with the vector control (Fig. 5D).

The genus Salmonella consists of two species: S. enterica and Salmonella bongori. Sequences sharing 39–40% amino acid identity were found in the deduced amino acid sequences of three different S. bongori genomes (Fig. S5). A search for PagM homologs among other members of the family Enterobacteriaceae revealed the presence of related sequences in the genomes of Sodalis sp. HS1 (43% amino acid identity), Candidatus Sodalis pierantonius str. SOPE (43% amino acid identity), and Citrobacter rodentium ICC168 (36% amino acid identity) (Fig. S5). However, other strains of the latter species lack pagM-related sequences. This analysis suggests that S. enterica acquired pagM by horizontal gene transfer after it split from the common ancestor that gave rise to S. bongori.

PagM Aids Replication on Low Mg²⁺ Semisolid Media.

We reasoned that PagM-mediated motility might aid bacterial replication on 0.3% agarose low Mg²⁺ media by enabling exploration of sites away from the site of inoculation. Such sites would still have nutrients and/or lack potentially toxic metabolic products. To explore this possibility, we determined the bacterial numbers of pagM-expressing and pagM mutant strains following incubation in low Mg²⁺ liquid versus semisolid media. There were similar numbers of pagM-expressing and pagM mutants when bacteria were grown in low Mg²⁺ liquid media. By contrast, the number of pagM-expressing bacteria was 30-fold higher than that of the pagM mutant after a 20-h incubation on 0.3% agarose low Mg²⁺ media (Fig. 6). We conclude that the pagM gene provides a growth advantage to Salmonella experiencing low Mg²⁺ semisolid conditions.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 6.

PagM aids replication on low Mg²⁺ semisolid media. Fold change in colony forming units (cfu) of the pagM mutant (SP244) harboring either the vector control (pUHE 21–2lacI^q) or the pagM-expressing plasmid (pUH-pagM) following incubation on 0.3% agarose media with 10 µM Mg²⁺ at 37 °C for 20 h. Bacteria were grown in N-minimal media containing 10 µM Mg²⁺ (HL) in the presence of ampicillin (50 µg/mL) and IPTG (0.1 mM) at 37 °C for 4.5 h. Three microliters of a bacterial suspension (OD₆₀₀ ∼0.6) were spotted onto 0.3% HL with ampicillin and IPTG (0.5 mM) and grown at 37 °C for 20 h. To measure viable bacterial numbers, bacteria were collected, serially diluted in phosphate saline buffer, and then plated on Luria–Bertani agar plates containing ampicillin. The number of cfus was determined following incubation at 37 °C overnight.