Here we show that SadB, originally identified as required for early biofilm formation, is also a negative effector of swarming motility, a result consistent with our previous findings (3
). We also showed previously that RpoN and FleR, known regulators of flagellum and rhamnolipid production in P. aeruginosa
), also regulate SadB levels (2
), suggesting that SadB is coregulated with other functions required for swarming and biofilm formation. How does SadB contribute to both biofilm formation and swarming behaviors? A model summarizing the findings from this study is shown in Fig. . While we have yet to identify the biochemical function of the SadB protein, our results implicate this protein in two pathways that impact swarming motility and biofilm formation.
FIG. 5. Model for inverse control of biofilm formation and swarming motility. Planktonic bacteria (top) initially interact with the surface, likely via reversible polar attachment, although which end of the cell interacts with the abiotic surface is unclear. (more ...)
First, SadB is involved in mediating flagellar reversals, but only under high-viscosity conditions likely similar to those encountered during either biofilm formation or swarming, but not swimming. A role for the chemotaxis cluster in E. coli in controlling flagellar reversal rates prompted us to investigate the potential involvement of the five chemosensory-like clusters of P. aeruginosa as a mechanism for linking SadB to flagellar function. An in-frame deletion of pilJ, an MCP homolog, rendered the strain biofilm defective and a hyperswarmer and resulted in increased flagellar reversals. The biofilm, swarming and flagellar reversal phenotypes of the ΔpilJ mutant are identical to those of a sadB mutant strain. Epistasis analysis indicates that SadB is genetically upstream of pilJ, consistent with a model wherein sadB exerts its effects on flagellar rotation via the CheIV chemosensory system.
Based on the E. coli
), loss of the PilJ MCP should block signaling via this chemosensory system. In contrast, mutating the demethylase gene homolog, chpB
, should result in a hypermethylated MCP with higher basal receptor activity. Consistent with these hypotheses, the ΔchpB
mutant is defective for swarming but forms a more robust biofilm than the WT, phenotypes opposite those observed for the ΔpilJ
While mutations in sadB and pilJ resulted in increased flagellar reversals, to our surprise the ΔchpB mutant did not have the predicted decrease in reversals and in fact showed no discernible effect on this behavior. Perhaps the repression of flagellar reversals is accomplished via another pathway. Alternatively, while the ΔchpB mutation alters the basal activity of the MCP, perhaps a second input signal is still required to observe decreased reversals in this mutant background. We do show here that mutations in chpB impact another known biofilm-related factor, namely, the production of the putative pel-encoded matrix. A ΔchpB mutant has a CR-hyperbinding phenotype that is pelA dependent and results in increased matrix production as judged by SEM, but the ΔchpB mutation does not alter pel gene expression, suggesting that this mutation increased production of the Pel polysaccharide by a nontranscriptional mechanism. In contrast to the ΔchpB mutant, the sadB mutant showed decreased CR binding and matrix production, suggesting that SadB positively impacts production of the Pel polysaccharide. Despite the decrease in apparent production of the Pel polysaccharide by the sadB mutant, expression of the pelA and pelG mRNAs is slightly up-regulated in the sadB mutant versus the WT. These data indicate that the reduction of Pel polysaccharide production in a sadB mutant occurs via a nontranscriptional mechanism.
At this point, we do not understand how SadB or components of the CheIV cluster impact EPS production. Given the lack of change in pel
gene expression in the sadB
mutants, one obvious explanation for the changes in matrix production in strains mutated in these functions is that Pel production is controlled by a mechanism other than regulation of pel
operon gene expression. To date, the only known means of nontranscriptional regulation of EPS production in pseudomonads is thought to be via the nucleotide signaling molecule c-di-GMP (10
). However, there are no proteins with known c-di-GMP-related motifs in the CheIV cluster or in the CheI, CheII, or CheV cluster. The WspR protein, a component of the wsp
chemosensory system (CheIII cluster) of P. aeruginosa
, which plays a role in biofilm formation and EPS production, contains a GGDEF domain, an amino acid motif associated with the synthesis of c-di-GMP from GTP, and has been shown in vitro to catalyze c-di-GMP synthesis (12
), but mutations in wspR
do not yield SadB-like phenotypes. Furthermore, SadB lacks the EAL, GGDEF, and HD-GYP domains (39
) associated with c-di-GMP metabolism, and we have no biochemical evidence that SadB is involved in c-di-GMP metabolism (data not shown), nor does it appear to alter cellular pools of c-di-GMP (J. Hickman, J. Merritt, C. Harwood, and G. O'Toole, unpublished data). Therefore, the mechanism by which SadB and ChpB modulate Pel polysaccharide production remains to be elucidated.
Components of the CheIV cluster, including pilJ
and the previously described chpA
), also play a role in twitching motility, indicating that this putative chemosensory system participates in coordinating all three known surface behaviors of this microbe. We also showed that mutations in pilJ
had no effect on swimming motility, further reinforcing a role for the CheIV cluster specifically in surface-associated behaviors of this microbe.
Our data suggest that SadB and PilJ modulate flagellar reversals under high-viscosity conditions but do not contribute to the control of flagellar reversals under the low-viscosity conditions that favor swimming. We hypothesize that SadB-dependent control of flagellar reversals upon polar, reversible attachment to a solid surface might decrease rotation about the cell pole and thus increase the time of interaction between the bacterium and its substratum, thereby promoting biofilm formation. In contrast, increased reversal rates appear to favor swarming motility. Wolfe and Berg postulated that for E. coli
, increasing flagellar reversals might in some circumstances facilitate the ability to move through a semisolid matrix (50
). Based on their microscopic observations, they concluded that “cells that do not tumble tend to get trapped in agar” and move less efficiently through this matrix (50
). While that study was performed in the context of swimming through 0.3% agar, our data suggest that this phenomenon might also be extended to swarming conditions. Also consistent with our data is the finding that E. coli
strains locked in the “tumbling” chemotaxis mode by mutation had a reduced ability to attach to an abiotic surface compared to the WT or mutants locked in the “running” mode (28
). In addition to controlling flagellar function, by coordinating the production of the Pel polysaccharide, SadB can modulate another facet of biofilm initiation and swarming. Work presented here and recent published studies (24
) show that a functional pel
locus contributes to early biofilm formation, and we show here that mutating the pel
locus promotes swarming motility (Fig. ). This inverse relationship between polysaccharide production and motility has been noted in several other studies of P. aeruginosa
Our studies may provide a mechanistic basis for a recent exciting report by Shrout and colleagues (43
). They proposed that early in biofilm formation, the extent of swarming motility helps dictate the final structure of the biofilm. That is, under conditions that promote swarming early in biofilm formation, the resulting mature biofilm is flat, while under conditions inhibitory to swarming motility, a biofilm with aggregates (distinct macrocolonies) will result. Based on their experimental work and accompanying mathematical simulations, they also postulated a role for a polysaccharide-containing matrix in the formation of the aggregates during biofilm development (43
). One interpretation of the study of Shrout et al. is that P. aeruginosa
must be able to integrate several important cell functions early in biofilm formation, namely, swarming motility and matrix production. The data presented in our report suggest that SadB and the CheIV cluster provide a molecular means for coregulating these functions.
We propose that P. aeruginosa
inversely regulates the surface-associated behaviors of biofilm formation and swarming by controlling both flagellar reversals and the production of the Pel polysaccharide. Flagellar reversal rates in E. coli
are largely regulated nontranscriptionally via the Che signal transduction pathway (25
), and given the high sequence similarity of the cheIV
cluster components to their E. coli
counterparts, this is likely also the case in P. aeruginosa
. CR binding and SEM data, together with the pelA
transcriptional analysis presented here, indicate that production of the pel
-encoded EPS may also be controlled by SadB and the CheIV cluster via a mechanism other than transcriptional control. Given that PilJ is also involved in twitching motility, the CheIV cluster may coordinate three different surface behaviors: swarming motility, twitching motility and biofilm formation. An appealing aspect of this regulatory strategy for coregulating surface behaviors is that in adapting to a surface-associated lifestyle, P. aeruginosa
may be able to seamlessly and rapidly transition among its surface behaviors as it encounters ever-changing substratum properties and environmental conditions.