Control of formation and cellular detachment from Shewanella oneidensis MR-1 biofilms by cyclic di-GMP

Abstract

Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral-reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3',5')guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a DeltamxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.

FIG. 1.

Organization of the mxdABCD genes in S. oneidensis MR-1. Horizontal arrows indicate the positions of primers used in transcription analyses, and vertical arrows mark the positions of transposon insertions that led to the identification of the gene cluster. Hatched areas indicate the in-frame deletions generated (see Materials and Methods). The mxdABCD genes correspond to the SO gene annotations designated SO4180-4177 (The Institute for Genomic Research).

FIG.2.

Involvement of the mxdABCD genes in cell attachment and three-dimensional biofilm architecture. (A) Biofilm phenotypes of (from top to bottom) AS93 (wild-type control), ΔmxdA, ΔmxdB, and ΔmxdC in hydrodynamic flow chambers. Images display shadow projections of biofilms formed after 48 h. x-z and y-z cross-sectional images at selected positions in the biofilm are shown at the bottom and right side, respectively. Scale bar, 70 μm. (B) Quantification of biomass of the ΔmxdABCD mutants. CLSM images of the experiments shown in panel A were quantified by COMSTAT (20). (C) Complementation of ΔmxdA and ΔmxdB by GGDEF-encoding VCA0956. Biofilm formation in 96-well microtiter plates was measured by crystal violet staining of cells attached to the walls, as described in Materials and Methods.

FIG. 3.

Intracellular content of c-di-GMP. c-di-GMP content in planktonically grown cells were determined as described in Materials and Methods and is expressed per milligram (wet weight) of cells. The strains assayed included AS145 (AS93 plus pARA:yhjH), AS146 (AS93 plus pARA:VCA0956), AS147 (AS93 plus empty vector), AS152 (AS140 plus pLacTac:mxdA), and AS160 (AS140 plus empty vector). Error bars represent one standard deviation.

FIG. 4.

Truncation analysis of MxdA. 5′ and 3′ deletions were introduced in mxdA, and strains expressing these alleles in a ΔmxdA genetic background were tested for biofilm formation by rescuing the ΔmxdA phenotype. Rectangles on the left indicate the size and region of the expressed protein relative to the wild type; bars on the right indicate biofilm biomass formed in a 96-well plate assay. The MxdA NVDEF region spans amino acids at positions 358 to 362.

FIG. 5.

Effects of VCA0956 and yhjH expression on S. oneidensis biofilm architecture. Images display shadow projections of biofilms formed after 24 h of AS147 (A), AS146 constitutively overexpressing VCA0956 (B), and AS145 constitutively overexpressing yhjH (C). The inocula were grown in LB containing 25 μg/ml kanamycin, diluted to an optical density at 600 nm of 0.01, and injected into the flow chamber. After 40 min, the flow of LM containing 25 μg/ml kanamycin and 0.2% arabinose as inducer was initiated. Scale bar, 200 μm.

FIG. 6.

Effects of VCA0956 and yhjH expression on developed S. oneidensis wild-type biofilms. Displayed are the amounts of biomass detached relative to the detached biomass of wild-type AS147 (set to 100%). Biofilms of strains AS147, AS145, and AS146 were grown in flow chambers for 20 h prior to induction with 0.2% (wt/vol) l-arabinose. After 90 min of induction, detachment was induced by a stop of flow (gray bars). The white bar indicates biomass detached from AS145 after 120 min without a stop of flow.

FIG. 7.

Model for control of attachment and detachment by c-di-GMP in S. oneidensis. An environmental cue is sensed by a sensor protein(s), which modulates the enzymatic activity of a c-di-GMP-forming diguanylate cyclase(s), such as GGDEF domain-containing proteins and MxdA, and/or of c-di-GMP-hydrolyzing phosphodiesterase(s), such as EAL domain-containing proteins. An altered general or localized c-di-GMP pool allosterically affects the activity of proteins or enzymes involved in attachment and/or detachment, such as MxdB. MxdA is postulated to be a key c-di-GMP-forming enzyme and to function in (structural) context with the putative glycosyl transferase MxdB. The c-di-GMP level (of a general intracellular or a localized pool) can be controlled in several ways: via activation and/or inhibition of a diguanylate cyclase(s) or by inhibition and/or activation of phosphodiesterases. Upon activation, MxdA catalyzes the formation of c-di-GMP, which stimulates the polysaccharide synthesis activity of MxdB, resulting in increased attachment. Downregulation of MxdA or activation of a phosphodiesterase(s) and subsequent decreased glycosyl transferase activity could result in decreased attachment (i.e., detachment). At this point, it is unclear whether detachment is due to the activation of a detachase or the inhibition of an attachment activity such as MxdB by low c-di-GMP levels.