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Research Article

Flagella-independent surface motility in Salmonella enterica serovar Typhimurium

Sun-Yang Park, Mauricio H. Pontes, and Eduardo A. Groisman
PNAS February 10, 2015 112 (6) 1850-1855; first published January 26, 2015; https://doi.org/10.1073/pnas.1422938112
Sun-Yang Park
Howard Hughes Medical Institute and Department of Microbial Pathogenesis, Yale School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06536-0812; and Yale Microbial Sciences Institute, West Haven, CT 06516
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Mauricio H. Pontes
Howard Hughes Medical Institute and Department of Microbial Pathogenesis, Yale School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06536-0812; and Yale Microbial Sciences Institute, West Haven, CT 06516
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Eduardo A. Groisman
Howard Hughes Medical Institute and Department of Microbial Pathogenesis, Yale School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06536-0812; and Yale Microbial Sciences Institute, West Haven, CT 06516
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  • For correspondence: eduardo.groisman@yale.edu
  1. Edited by Howard C. Berg, Harvard University, Cambridge, MA, and approved December 31, 2014 (received for review December 1, 2014)

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Significance

We identified a form of surface motility in the bacterium Salmonella enterica serovar Typhimurium that is activated in low Mg2+ by PhoP/PhoQ, a regulatory system that hinders flagella expression and activity. PhoP furthers motility by promoting expression of the pagM gene, which specifies a small protein of unknown function, and also of two other genes that create the cytosolic conditions necessary for full pagM expression. Low Mg2+-promoted motility is a group behavior exclusive to a subset of S. enterica serovars harboring a particular allele of pagM. The pagM gene is present in nonfunctional allelic forms in certain S. enterica serovars and rarely found outside the genus Salmonella. Not required for virulence, PagM-mediated motility helps survival outside animal hosts.

Abstract

Flagella are multiprotein complexes necessary for swimming and swarming motility. In Salmonella enterica serovar Typhimurium, flagella-mediated motility is repressed by the PhoP/PhoQ regulatory system. We now report that Salmonella can move on 0.3% agarose media in a flagella-independent manner when experiencing the PhoP/PhoQ-inducing signal low Mg2+. This motility requires the PhoP-activated mgtA, mgtC, and pagM genes, which specify a Mg2+ transporter, an inhibitor of Salmonella’s own F1Fo ATPase, and a small protein of unknown function, respectively. The MgtA and MgtC proteins are necessary for pagM expression because pagM mRNA levels were lower in mgtA and mgtC mutants than in wild-type Salmonella, and also because pagM expression from a heterologous promoter rescued motility in mgtA and mgtC mutants. PagM promotes group motility by a surface protein(s), as a pagM-expressing strain conferred motility upon a pagM null mutant, and proteinase K treatment eliminated motility. The pagM gene is rarely found outside subspecies I of S. enterica and often present in nonfunctional allelic forms in organisms lacking the identified motility. Deletion of the pagM gene reduced bacterial replication on 0.3% agarose low Mg2+ media but not in low Mg2+ liquid media. Our findings define a form of motility that allows Salmonella to scavenge nutrients and to escape toxic compounds in low Mg2+ semisolid environments.

  • magnesium
  • PagM
  • PhoP/PhoQ
  • MgtA
  • MgtC

Cells have the ability to move on a variety of surfaces (1). This ability enables cells to search for nutrients, to avoid toxic compounds, to colonize new niches, and to form complex structures such as biofilms (2, 3). Bacterial motility has been classified into distinct types based on the energetic requirements and the structural elements involved (4, 5). For example, rotation of the multiprotein complex known as flagella can propel bacteria forward in a process that requires the proton motive force. Twitching motility is a surface movement mediated by the extension and retraction of surface appendages known as type IV pili, and gliding motility is an active surface movement that occurs along the long axis of the cell via focal adhesion complexes. By contrast, sliding motility is a passive surface translocation powered by growth and facilitated by a surfactant.

Salmonella enterica serovar Typhimurium exhibits two forms of flagella-mediated motility on semisolid agar media: swarming and swimming (6). Swarming motility entails morphological differentiation into swarmer cells, takes place on 0.4–0.7% agar surfaces, and requires an energy-rich carbon source such as glucose even in nutrient-rich media (6). Whereas swarming constitutes a multicellular group behavior on a surface, swimming is an individual bacterial movement experienced in liquid and low (0.1–0.3%) agar (2). Flagella-mediated motility is under the control of positive and negative regulators that respond to different environmental and cellular signals. For example, the nature of the carbon source is critical because the cAMP-receptor protein promotes transcription of the flagellar master regulator flhDC operon (7), and the RNA binding regulator CsrA furthers stability of the flhDC mRNA (8). By contrast, the PhoP/PhoQ regulatory system down-regulates flagella-mediated motility by repressing transcription of flagellar genes (9) and by decreasing the membrane potential (10).

The PhoP/PhoQ system consists of the DNA binding transcriptional regulator PhoP (11) and the sensor PhoQ, which responds to low extracytoplasmic Mg2+ (12), mildly acidic pH (13), and certain antimicrobial peptides (14) by promoting the active (i.e., phosphorylated) form of PhoP. Transcription of ∼5% of the Salmonella genes is under PhoP control (15). The PhoP-activated mgtA gene specifies a Mg2+ transporter that enhances PhoP-P levels by transporting Mg2+ away from the periplasm, where it acts as an inhibitory signal for PhoQ (16). Because transcription elongation into the mgtA coding region is controlled by the Mg2+-responding mgtA leader RNA, the MgtA protein and the resulting higher levels of PhoP-P are produced only when cytoplasmic Mg2+ levels drop below a certain threshold (17, 18). This regulatory architecture defines a two-tier structure among PhoP-activated genes based on whether they require mgtA for maximal expression (16). This architecture allows Salmonella to delay transcription of a subset of the PhoP regulon until the cytosolic conditions triggering MgtA production are met.

The PhoP-activated genes most dependent on the MgtA protein specify proteins of unknown function and, like the mgtA gene, are not required for virulence in an animal (19⇓–21). Therefore, we hoped that the investigation of mgtA-dependent phenotypes might reveal a novel aspect(s) of Salmonella’s lifestyle. We now report that the MgtA-dependent pathway governs a form of surface migration that does not appear to involve flagella or fimbriae. We establish that this surface migration is dependent on the PhoP-activated MgtA-dependent pagM gene. The rare occurrence of pagM outside the species S. enterica suggests that the uncovered motility is a property exclusive to this species, allowing it to explore surroundings under conditions unfavorable to flagella-mediated motility.

Results

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.

Fig. 1.
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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).

Fig. 2.
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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 21–2lacIq 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 21–2lacIq 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 21–2lacIq 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 F1Fo 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).

Fig. 3.
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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 21–2lacIq 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).

Fig. 4.
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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).

Fig. 5.
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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 21–2lacIq 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.

Fig. 6.
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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 21–2lacIq) 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.

Discussion

We have now identified a form of motility that S. enterica serovar Typhimurium uses to move on surfaces when experiencing low Mg2+ semisolid conditions. This form of motility is independent of flagella (Fig. 1B) and still takes place in mutants defective in each of 13 fimbriae operons (Fig. 1C). By contrast, it requires the PhoP/PhoQ regulatory system to promote expression of the mgtA, mgtC, and pagM genes (Fig. 1A). The pagM gene appears to be directly involved in the uncovered motility because, first, pagM expression from a heterologous promoter conferred motility even when Salmonella experienced high Mg2+, which is a noninducing condition for the PhoP/PhoQ system (Fig. 2D). Second, pagM expression from a heterologous promoter restored motility to mgtA and mgtC mutants (Fig. 2B), but mgtA- and mgtC-expressing plasmids failed to rescue motility in a pagM mutant (Fig. S3B). Third, mgtA and mgtC mutants displayed low pagM mRNA levels (Fig. 2A), but inactivation of pagM had no effect on the mRNA levels of mgtA or mgtC (Fig. S3A). Fourth, nonmotile Salmonella isolates have pagM alleles different from that present in motile Typhimurium strain 14028s (Fig. 5 A and B). And fifth, motility was eliminated or conferred simply by altering the pagM allele (Fig. 5 C and D).

PagM-mediated motility appears to constitute a group behavior because a pagM-expressing strain rescued motility of a pagM mutant when incubated together with it (Fig. 4). PagM-dependent motility appears to involve a surface protein(s) because proteinase K and heat treatment abolished motility (Figs. 3A and 4C). The surface protein could be PagM itself, given that it has a predicted signal sequence (Fig. S4).

PagM may promote sliding motility by which bacterial movement results from expansive forces in a growing colony combined with surface properties that reduce the friction between the cell and its substrate (4). In support of this notion, Salmonella displayed faster surface migration in high than low Mg2+ when the pagM gene was expressed constitutively (Fig. 2D), which correlates with better bacterial replication at high than low Mg2+ (26). Sliding motility typically requires surfactants to lower surface friction—for example, serrawettin in Serratia marcescens (28, 34), acetylated glycopeptidolipids in Mycobacteria smegmatis (35, 36), and surfactin in Bacillus subtilis (37). In S. enterica, however, low Mg2+-promoted motility was not affected when the genes responsible for the production of cellulose, capsule, colanic acid, LPS, ECA, or O-antigen were inactivated (Fig. 3B), and the identity of a potential wetting agent reducing surface friction in Salmonella remains unknown.

Vibrio cholerae and E. coli can move on low-melting-temperature agarose minimal media in a flagella-independent manner (38). However, the motility of these two species appears to be unrelated to that promoted by PagM in Salmonella because V. cholerae requires LPS for motility (38), whereas Salmonella does not (Fig. 3B), and also because V. cholerae moves on a surface independently of growth (38), whereas pagM-mediated motility is stimulated by growth (Fig. 2D). In addition, V. cholerae and E. coli lack homologs of PagM, which exhibits a fairly limited phylogenetic distribution.

The opposite regulation of flagella and pagM by the Salmonella PhoP/PhoQ system is reminiscent of the control exerted by proteins that repress flagella but promote expression of other surface molecules. For example, the fimZ and pefI genes specify activators of distinct fimbriae operons and repress flagella expression when transcribed from heterologous promoters (39, 40). However, PagM promotes motility (Figs. 1–5), whereas fimbriae further surface adhesion.

PagM may enable Salmonella to move and scavenge Mg2+, and perhaps other nutrients, when experiencing low Mg2+ for an extended period. This is because, first, low Mg2+ activates the PhoP/PhoQ system, which hinders flagella-mediated motility by repressing transcription of flagellin (9) and by decreasing membrane potential, which hinders flagellar rotation (10). And second, pagM transcription requires the Mg2+ transporter MgtA and the F1Fo ATPase inhibitor MgtC. This makes PagM-mediated motility contingent on the cytosolic conditions that promote transcription elongation into the mgtA and mgtC coding regions: a decrease in Mg2+ (18) and an increase in ATP (25), respectively. These cytosolic conditions may be indicative of a need to explore other locales. Indeed, PagM helps Salmonella replication (Fig. 6), possibly by facilitating access to nutrients and escape from toxic metabolic byproducts. Finally, unlike flagella-mediated motility, which has been implicated in virulence (41⇓–43), deletion of the pagM gene did not alter Salmonella pathogenicity (21), suggesting PagM-mediated motility aids Salmonella survival outside a mammalian host.

Materials and Methods

Bacterial Strains, Plasmids, and Growth Conditions.

Bacterial strains and plasmids used in this study are listed in Table S1. Details of strains and plasmid constructions are described in SI Materials and Methods. Primers are listed in Tables S2 and S3. Bacteria were grown at 37 °C in Luria–Bertani broth or in N-minimal media, pH 7.7 (44) supplemented with 0.1% casamino acids, 38 mM glycerol, and the indicated concentrations of MgCl2. For genetic manipulations, such as transformation and transduction, ampicillin was used at a final concentration of 50 µg/mL and chloramphenicol at 20 µg/mL. To induce the LacI-repressed lac promoter derivative in plasmid pUHE21-2 lacIq, we used isopropyl-thio-β-galactoside (IPTG) at 0.1–0.5 mM.

Determination of Transcript Levels.

Total RNA was extracted using RNeasy Mini Kit (Qiagen). cDNA was synthesized using High Capacity RNA to cDNA Master Mix (Applied Biosystems) following the manufacturer’s instructions. Quantification of transcripts was performed by real-time PCR using Fast SYBR Green Master Mix (Applied Biosystems) in an ABI 7500 Sequence Detection System (Applied Biosystems). Data were normalized to the levels of the 16S ribosomal RNA rrs gene. A list of primers used for quantitative RT-PCR is presented in Table S2.

Surface Motility Assay.

Bacteria were grown overnight in N-minimal media, pH 7.7, containing 10 mM MgCl2. They were then washed with N-minimal media, inoculated into 2 mL of N-minimal media containing 10 µM MgCl2 (1:50 dilution), and grown at 37 °C for 4.5–5 h with aeration. The harvested bacteria were adjusted to OD600 ∼1 with N-minimal media. Three microliters of cell suspension were spotted on the 0.3% agarose N-minimal media with 10 µM or 10 mM MgCl2 and grown at 37 °C for 20 h. To test the motility of strains with plasmid harboring the pagM gene, bacteria were grown in the presence of ampicillin (50 µg/mL) and IPTG (0.1 mM) and spotted on the same media with 0.3% agarose and IPTG (0.1–0.5 mM). To test swimming motility, bacteria were spotted onto 0.1% agarose N-minimal media with 10 µM MgCl2. The data are representative of three independent experiments, which gave similar results.

Acknowledgments

We thank Jennifer Aronson for editorial assistance on the manuscript and Sangjin Kim for help with fluorescence microscopy. This research was supported, in part, by Grant AI49561 from the National Institutes of Health (to E.A.G.). E.A.G. is an investigator of the Howard Hughes Medical Institute.

Footnotes

  • ↵1To whom correspondence should be addressed. Email: eduardo.groisman{at}yale.edu.
  • Author contributions: S.-Y.P. and E.A.G. designed research; S.-Y.P. performed research; M.H.P. contributed new reagents/analytic tools; S.-Y.P. and E.A.G. analyzed data; and S.-Y.P. and E.A.G. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1422938112/-/DCSupplemental.

View Abstract

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Flagella-independent motility in Salmonella
Sun-Yang Park, Mauricio H. Pontes, Eduardo A. Groisman
Proceedings of the National Academy of Sciences Feb 2015, 112 (6) 1850-1855; DOI: 10.1073/pnas.1422938112

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Flagella-independent motility in Salmonella
Sun-Yang Park, Mauricio H. Pontes, Eduardo A. Groisman
Proceedings of the National Academy of Sciences Feb 2015, 112 (6) 1850-1855; DOI: 10.1073/pnas.1422938112
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