Surfing Motility: a Conserved yet Diverse Adaptation among Motile Bacteria

Abstract

Bacterial rapid surfing motility is a novel surface adaptation of Pseudomonas aeruginosa in the presence of the glycoprotein mucin. Here, we show that other Gram-negative motile bacterial species, including Escherichia coli, Salmonella enterica, Vibrio harveyi, Enterobacter cloacae, and Proteus mirabilis, also exhibit the physical characteristics of surfing on the surface of agar plates containing 0.4% mucin, where surfing motility was generally more rapid and less dependent on medium viscosity than was swimming motility. As previously observed in Pseudomonas aeruginosa, all surfing species exhibited some level of broad-spectrum adaptive resistance, although the antibiotics to which they demonstrated surfing-mediated resistance differed. Surfing motility in P. aeruginosa was found to be dependent on the quorum-sensing systems of this organism; however, this aspect was not conserved in other tested bacterial species, including V. harveyi and S. enterica, as demonstrated by assaying specific quorum-sensing mutants. Thus, rapid surfing motility is a complex surface growth adaptation that is conserved in several motile bacteria, involves flagella, and leads to diverse broad-spectrum antibiotic resistance, but it is distinct in terms of dependence on quorum sensing.IMPORTANCE This study showed for the first time that surfing motility, a novel form of surface motility first discovered in Pseudomonas aeruginosa under artificial cystic fibrosis conditions, including the presence of high mucin content, is conserved in other motile bacterial species known to be mucosa-associated, including Escherichia coli, Salmonella enterica, and Proteus mirabilis Here, we demonstrated that key characteristics of surfing, including the ability to adapt to various viscous environments and multidrug adaptive resistance, are also conserved. Using mutagenesis assays, we also identified the importance of all three known quorum-sensing systems, Las, Rhl, and Pqs, in P. aeruginosa in regulating surfing motility, and we also observed a conserved dependence of surfing on flagella in certain species.

Keywords: antibiotic resistance; bacterial motility; mucin; quorum sensing; surface motility; surfing.

FIG 1

Mucin triggered rapid surface motility in a range of bacterial species. Bacterial strains were grown under swimming conditions (0.3% agar), surfing conditions (0.3% agar in the presence of 0.4% mucin), and solid medium conditions (1.5% agar) in SCFM. The rate of motility zone growth, depicted on the graphs, was assessed as the diameter of the motility zone over 10 h of incubation at 37°C; surfing is represented by the continuous lines, and swimming is represented by the dashed lines (n = 3).

FIG 2

Effect of medium viscosity on surfing motility. Bacterial strains were point inoculated onto SCFM at various agar concentrations with and without mucin and grown for 18 h at 37°C to test the effects on surfing (Surf) and swimming (Swim) motility. Percent plate coverage as a function of agar concentration was measured using ImageJ (n = 3). Corresponding images are depicted in Fig. S2.

FIG 3

Effects of alternative wetting agents on surfing motility. Mucin was replaced with carboxymethyl cellulose (CMC) at 1% (wt/vol) or Tween 20 at 0.01% added into 0.3% agar LB.

FIG 4

Surfing motility was dependent on flagella but not on pili/fimbriae. Flagellum-deficient mutants in P. aeruginosa (ΔfliC), S. enterica (ΔfliC), P. mirabilis (ΔflaD), and E. coli (ΔflhDC) demonstrated complete inhibition of surfing motility in 0.3% agar SCFM supplemented with 0.4% agar after 13 to 15 h of incubation. Pilus- or fimbria-deficient mutants of P. aeruginosa (ΔpilC), P. mirabilis (ΔmrpA), and E. coli (Δfim) still exhibited surfing motility under the same conditions.

FIG 5

P. aeruginosa surfing was dependent on quorum sensing. (A) Quorum-sensing PA14 mutants (ΔpqsA, ΔpqsB, ΔpqsC, ΔpqsD, ΔpqsE, ΔpqsR, ΔlasI, ΔrhlI, and ΔrhlR) exhibited surfing deficiency, as shown by the negative control (ΔfliC), or conversion to swarming. Surface coverage was determined by analyzing the percent surface coverage relative to that of wild-type PA14 using ImageJ. (B) Complements of quorum-sensing mutants (rhlI⁺ and lasI⁺) exhibited complete or partial surfing restoration. Addition of exogenous autoinducer molecules restored surfing, with a slight increase in motility zone compared to that of the wild type. Significance levels between the plate coverage area of the mutants relative to that of the wild type were calculated using 2-way analysis of variance (ANOVA) where all the mutants had a P value of <0.0001 (****).

FIG 6

Surfing dependence on quorum sensing did not extend to bacterial species other than P. aeruginosa. Motility assays were performed on SCFM containing 0.3% agar and 0.4% mucin (surfing) or 0.3% agar (swimming). Swimming for the three test species, P. aeruginosa, S. enterica, and V. harveyi, showed no dependence on quorum sensing, since their respective quorum sensing mutants continued to exhibit wild-type swimming. Although the P. aeruginosa lasI mutant was surfing deficient, quorum-sensing mutants from S. enterica (ΔluxS) and V. harveyi (ΔluxR) continued to show surfing.