Signaling-mediated cross-talk modulates swarming and biofilm formation in a coral pathogen Serratia marcescens

Abstract

Interactions within microbial communities associated with marine holobionts contribute importantly to the health of these symbiotic organisms formed by invertebrates, dinoflagellates and bacteria. However, mechanisms that control invertebrate-associated microbiota are not yet fully understood. Hydrophobic compounds that were isolated from surfaces of asymptomatic corals inhibited biofilm formation by the white pox pathogen Serratia marcescens PDL100, indicating that signals capable of affecting the associated microbiota are produced in situ. However, neither the origin nor structures of these signals are currently known. A functional survey of bacteria recovered from coral mucus and from cultures of the dinoflagellate Symbiodinium spp. revealed that they could alter swarming and biofilm formation in S. marcescens. As swarming and biofilm formation are inversely regulated, the ability of some native α-proteobacteria to affect both behaviors suggests that the α-proteobacterial signal(s) target a global regulatory switch controlling the behaviors in the pathogen. Isolates of Marinobacter sp. inhibited both biofilm formation and swarming in S. marcescens PDL100, without affecting growth of the coral pathogen, indicative of the production of multiple inhibitors, likely targeting lower level regulatory genes or functions. A multi-species cocktail containing these strains inhibited progression of a disease caused by S. marcescens in a model polyp Aiptasia pallida. An α-proteobacterial isolate 44B9 had a similar effect. Even though ∼4% of native holobiont-associated bacteria produced compounds capable of triggering responses in well-characterized N-acyl homoserine lactone (AHL) biosensors, there was no strong correlation between the production of AHL-like signals and disruption of biofilms or swarming in S. marcescens.

Figure 1

Inhibition of biofilm formation by Serratia marcescens PDL100 in the presence of compounds recovered from coral mucus surface layer. Left: Cellulose dialysis pouches containing reverse-phase C₁₈ Si or HP20SS resin were deployed on surfaces of the boulder coral Montastraea faveolata and the compounds were eluted with a bed volume of chloroform, isopropanol, 100% methanol, 75% methanol and 50% aqueous methanol, evaporated, and reconstituted in 200 μl methanol. A total of 30 μl of serial 10-fold dilutions (left to right) were added to the wells of polystyrene microtiter plates where the pathogen was allowed to settle in colonization factor antigen media. The highest amount corresponds approximately to 37.5 cm² of M. faveolata coral surface. Activities eluted from the reverse-phase C₁₈ Si with 75% methanol:25% water exhibited noticeable inhibitory properties, shown here. Middle: As a control, signals present on the sand bottom 10–15 m from the nearest living coral were similarly tested. Right: Biofilm formation by S. marcescens PDL100 in the absence of extracts in colonization factor antigen media. Error bars are standard errors of four technical replications.

Figure 2

Production of QS signals by bacteria isolated from marine holobionts. Bacterial cultures were extracted twice with half volumes of ethyl acetate acidified with glacial acetic acid (4 ml l⁻¹), and concentrated by rotary evaporation. Samples were spotted on reverse-phase C₁₈ Si TLC plates, developed with 60:40 methanol:water and overlaid with a suspension of the Agrobacterium tumefaciens NTL1 pZLR4 AHL reporter in soft agar with X-Gal. Synthetic AHL standards (all from Sigma) were similarly subjected to the bioassay-coupled thin-layer chromatography.

Figure 3

Swarming of Serratia marcescens in the presence of marine bacteria. Swarming of the model opportunistic pathogen S. marcescens MG1 (top row), coral white pox pathogen S. marcescens PDL100 (middle row) and the SwrI AHL synthase mutant S. marcescens MG44 (bottom row) was tested. Controls (Serratia spotted next to a glass fiber disk without a marine bacterium) are in the far left column. Identities of the tested marine bacteria are listed above the figure.

Figure 4

Biofilm formation by the white pox pathogen S. marcescens PDL100 on coral mucus in the presence of marine bacteria. Dual-species consortia consisting of the coral pathogen and a marine isolate were set up in microtiter plates, surfaces of which were coated with mucus of Acropora palmata. S. marcescens PDL100 was inoculated into each well, marine bacteria present within each dual-species consortium are listed at the bottom of the figure. The relative numbers of S. marcescens PDL100 and marine isolates were enumerated by dilution plating. S. marcescens PDL100 was distinguished from the other marine bacteria based on colony morphology: S. marcescens PDL100 forms small cream-color circular convex colonies with an entire edge on Marine Agar (1.5% agar). The black bars indicate the percentage of the coral pathogen and the gray bar is the percentage of the marine isolate in each sample. In each data set, the left bar indicates un-attached bacteria, and the right bar is the biofilm. Inhibition of biofilm formation by the coral pathogen was scored as the decrease of the relative percentage of S. marcescens PDL100 within biofilms, compared with the suspension. Data from the dual-species biofilms with Vibrio spp 52B8 are not shown because mixed swarms containing the two bacteria formed on agar plates, thus making enumeration of bacteria nearly impossible. Each panel represents data from three independent experiments using mucus harvested from three different colonies of A. palmata in April of 2010. Error bars represent standard errors of four biological replications (independent dual-species consortia).

Figure 5

Inhibition of serattiosis in Aiptasia pallida by antagonistic holobiont-associated bacteria. Clonal lines of Aiptasia pallida were maintained in aquaria. For the experiments, individual polyps were acclimated in six-well plates with 10 ml of sterile artificial seawater for 2 days. Antagonistic marine strains (columns C and E were added as a cocktail containing Marinobacter spp. 47E6, 47G8 and 46E2 as well as α-proteobacteria 46H6 and 45A11); polyps shown in columns D and F were treated with α-proteobacterium 44B9 at 10⁶ cfu ml⁻¹. Wells in column A contain polyps that were not exposed to S. marcescens or marine isolates; polyps in columns B, C and D were infected with S. marcescens PDL100 at 5 × 10⁷ cfu ml⁻¹ on day 1 of the experiment. Signs of the disease progression (darkened polyp and retracted tentacles) were documented daily for a week. Brightness and color balance of the images were adjusted in Adobe Photoshop CS4 using default settings. Experiments were performed with three biological and six technical replications.