SadC reciprocally influences biofilm formation and swarming motility via modulation of exopolysaccharide production and flagellar function

Abstract

Pseudomonas aeruginosa has served as an important organism in the study of biofilm formation; however, we still lack an understanding of the mechanisms by which this microbe transitions to a surface lifestyle. A recent study of the early stages of biofilm formation implicated the control of flagellar reversals and production of an exopolysaccharide (EPS) as factors in the establishment of a stable association with the substratum and swarming motility. Here we present evidence that SadC (PA4332), an inner membrane-localized diguanylate cyclase, plays a role in controlling these cellular functions. Deletion of the sadC gene results in a strain that is defective in biofilm formation and a hyperswarmer, while multicopy expression of this gene promotes sessility. A DeltasadC mutant was additionally found to be deficient in EPS production and display altered reversal behavior while swimming in high-viscosity medium, two behaviors proposed to influence biofilm formation and swarming motility. Epistasis analysis suggests that the sadC gene is part of a genetic pathway that allows for the concomitant regulation of these aspects of P. aeruginosa surface behavior. We propose that SadC and the phosphodiesterase BifA (S. L. Kuchma et al., J. Bacteriol. 189:8165-8178, 2007), via modulating levels of the signaling molecule cyclic-di-GMP, coregulate swarming motility and biofilm formation as P. aeruginosa transitions from a planktonic to a surface-associated lifestyle.

FIG. 1.

The sadC gene (PA4332) is involved in the transition to irreversible attachment during biofilm formation. (A) Biofilm formation of the WT and ΔPA4332 mutant in a microtiter plate assay. Image of CV-stained wells (top) and quantification of staining by OD₅₅₀ readings of the ethanol-solubilized dye (bottom). The graph depicts averages of four replicates with the standard deviation indicated by error bars. (B) Biofilm assay as described above comparing the addition of a vector control (pMQ72) or a sadC-containing plasmid (psadC) to either the WT or ΔsadC mutant strains. (C) Static attachment assay in minimal medium containing glucose and Casamino Acids. Surface-attached bacteria were quantified from 10 fields of view using phase-contrast microscopy. Average numbers of attached cells per field of view are shown. (D) Reversible attachment under static conditions. Bacteria associated with the substratum were observed and scored as to whether they were reversibly or irreversibly attached defined on the basis of whether movement was observed. The average percentage of reversibly attached cells is represented comparing the WT to the ΔsadC mutant (n = 10 fields of view; *, P = 0.00069).

FIG. 2.

The ΔsadC mutant displays hypermotility phenotypes. (A) Swarming motility assays on 0.5% agar plates. Overnight cultures of the WT or ΔsadC strains carrying a vector control (pMQ72) or a sadC-containing plasmid (psadC) were spotted onto swarm agar and incubated at 37°C for 16 h. (B) Average percentage of the plate surface occupied by the respective swarms. Replicate swarm plates (n = 5) for each test strain were grown under identical conditions and then photographed for calculation of the surface area coverage. Averages for the WT (WT/pMQ72) and ΔsadC (ΔsadC/pMQ72) strains carrying vector controls differ significantly (P = 0.001). In both cases, addition of a multicopy sadC-containing plasmid (WT/psadC, ΔsadC/psadC) results in a significant decrease in respective swarm coverage at this time point (P = 0.0088, P = 0.0004). (C) Swim reversals in 15% Ficoll-containing medium quantified from six separate fields of view. The ΔsadC mutant cells on average are observed to reverse more frequently than the parental strain (*, P = 0.01).

FIG. 3.

sadC influences production of the Pel EPS. (A) CR binding assay for EPS production. Aliquots of WT, ΔsadC, and ΔpelA strains carrying a vector control (pMQ72) or a plasmid expressing the sadC gene (psadC) were spotted onto CR plates. Plates were incubated for 24 h at 37°C, followed by 24 h at room temperature. (B) CR binding in biofilms. Representative wells showing attached cells grown in the presence of CR (left) or post-stained with crystal violet (right) after a 24-h incubation at 37°C. (C) qRT-PCR analysis of gene expression from agar-grown strains. The picograms of input cDNA is plotted on the y axis. There is no statistically significant difference comparing expression between the strains for the pelA (P > 0.09) or pelG gene (P > 0.3).

FIG. 4.

SadC is localized to the cytoplasmic membrane. Subcellular protein localization by Western blotting of fractions separated by differential centrifugation. Shown are the total cell lysate (Tot) and the cytoplasmic (Cyt), total-membrane (TM), and inner-membrane (IM) fractions.

FIG. 5.

The GGDEF domain of SadC has diguanylate cyclase activity. (A) Resolution of labeled extracts by two-dimensional TLC from WT carrying a vector control (WT/pMQ72) or the sadC gene on a multicopy plasmid (WT/psadC) grown in the presence of [³²P]orthophosphate. Shown is a representative image from an experiment performed with three independent replicates. c-di-GMP is indicated in the white oval. (B) The percent c-di-GMP (normalized to the total ³²P label) present in the WT/psadC strain is ∼10-fold greater than that of the WT/pMQ72 strain (*, P < 0.05). (C) Diagram of PleD/SadC chimeric proteins and respective phenotypes on CR plates and in biofilm assays. Both chimeric proteins are translational fusions comprised of the PleD* N terminus and the SadC C terminus, the net effect being to replace all or most of the GGDEF domain of PleD* with that of SadC. The D/C637 construct encodes the entire SadC GGDEF domain while the D/C700 construct lacks the first 21 amino acids of the cognate SadC GGDEF domain (left). Plasmids containing the pD/C637 or pD/C700 construct expressed in the ΔsadC background were spotted onto CR plates and assayed for biofilm formation (right). Arabinose to 0.2% was added to both assays. (D) In vitro diguanylate cyclase activity assay. Purified D/C700 or D/C637 proteins were incubated with [³²P]GTP under diguanylate cyclase reaction conditions. Reaction products at 2 and 20 h are shown resolved by TLC with concomitantly run no-protein and PleD* 1:100 dilution control reactions.

FIG. 6.

sadC is in a genetic pathway with other genes that influence biofilm formation, swarming motility, and EPS production. (A) Biofilm formation was assessed for the strains indicated after 4 h. Images of representative wells (top) are shown with averaged OD readings from dye solubilized from four replicate wells (bottom). (B) CR binding of the WT and sadB strains carrying a vector control or a sadC-containing multicopy plasmid. CR plates were incubated for 24 h at 37°C. (C) Biofilm formation by the indicated strains was assayed after 8 h at 37°C. (D) The indicated strains were assayed for CR binding (top) and swarming motility (bottom). CR plates were incubated for 24 h at 37°C, followed by 24 h at room temperature, while swarm plates were imaged after a 16-h incubation at 37°C.

FIG. 7.

Model for the role of SadC in modulating biofilm formation, swarming motility, and related phenotypes. A diagram of the putative roles of SadC and other factors in this pathway involved in the inverse regulation of biofilm formation and swarming motility. SadC, presumably upon receipt of the proper environmental signals (such as nutritional cues or contact with an appropriate surface), transmits this information to the cytoplasm by modulating the production of c-di-GMP. This signal can be further regulated by the activity of the BifA protein, a c-di-GMP phosphodiesterase. We hypothesize that the status of this c-di-GMP pool, which may also be impacted by the action of additional DGCs, acts as a signal to the downstream constituents of this pathway. SadB is predicted to play a role in the transmission of this signal to the Pel machinery and components of the CheIV chemotaxis-like cluster. We propose that when there are relatively high c-di-GMP levels, biofilm formation is promoted at the expense of swarming motility. This is consistent with the observed increase in EPS production and decrease in swarming motility. Based on its previously reported function as an allosteric effector of EPS production, we suggest a similar role for c-di-GMP-based regulation of Pel production. If the c-di-GMP pool is low, this state is associated with decreased biofilm formation and increased swarming motility. In the case of modulating flagellar function, PilJ or potentially other components of the Che IV chemotaxis cluster may be involved. How c-di-GMP communicates with the CheIV cluster is not known, but it may parallel regulation of the pel locus.