Biofilm formation at the air–liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose

Abstract

SummaryThe wrinkly spreader (WS) genotype of Pseudomonas fluorescens SBW25 colonizes the air-liquid interface of spatially structured microcosms resulting in formation of a thick biofilm. Its ability to colonize this niche is largely due to overproduction of a cellulosic polymer, the product of the wss operon. Chemical analysis of the biofilm matrix shows that the cellulosic polymer is partially acetylated cellulose, which is consistent with predictions of gene function based on in silico analysis of wss . Both polar and non-polar mutations in the sixth gene of the wss operon ( wssF ) or adjacent downstream genes ( wssGHIJ ) generated mutants that overproduce non-acetylated cellulose, thus implicating WssFGHIJ in acetylation of cellulose. WssGHI are homologues of AlgFIJ from P. aeruginosa , which together are necessary and sufficient to acetylate alginate polymer. WssF belongs to a newly established Pfam family and is predicted to provide acyl groups to WssGHI. The role of WssJ is unclear, but its similarity to MinD-like proteins suggests a role in polar localization of the acetylation complex. Fluorescent microscopy of Calcofluor-stained biofilms revealed a matrix structure composed of networks of cellulose fibres, sheets and clumped material. Quantitative analyses of biofilm structure showed that acetylation of cellulose is important for effective colonization of the air-liquid interface: mutants identical to WS, but defective in enzymes required for acetylation produced biofilms with altered physical properties. In addition, mutants producing non-acetylated cellulose were unable to spread rapidly across solid surfaces. Inclusion in these assays of a WS mutant with a defect in the GGDEF regulator (WspR) confirmed the requirement for this protein in expression of both acetylated cellulose polymer and bacterial attachment. These results suggest a model in which WspR regulation of cellulose expression and attachment plays a role in the co-ordination of surface colonization.

Comparative Genetics of the rdar Morphotype inSalmonella

“…The air-liquid interface represents a favorable niche that provides bacterial cells with increased access to oxygen. In other bacterial species (e.g., Pseudomonas fluorescens), reversion to pellicle formation, or adaptive divergence, can occur through mutations that increase the production of extracellular polymers (58,59). Therefore, we investigated whether smooth SARC16 isolates would form pellicles if grown in standing liquid culture for long time periods.…”

Evolution of exploitative interactions during diversification in Bacillus subtilis biofilms

“…Analogous interplay takes place in P. fluorescens where production of cellulose by a surface colonizer, the 'Wrinkly', creates an opportunity for exploitative invasion by the 'Smooth' which alone cannot colonize the surface (Hammerschmidt et al 2014). Invasion and, in consequence, premature pellicle collapse happens because the 'Wrinkly' variant secretes a costly extracellular product, an acetylated cellulose (Spiers et al 2003), that becomes vulnerable to exploitation by the nonproducer. The two morphotypes coexist since each has an advantage when present at low frequency (Rainey and Travisano 1998).…”

The structure–function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain

“…The effects of GGDEF proteins on exopolysaccharide levels have been probed via genetic studies in numerous species, including Pseudomonas putida (Gjermansen et al, 2005;Ude et al, 2006), Pseudomonas fluorescens SBW25 (Spiers et al, 2002(Spiers et al, , 2003Goymer et al, 2006;Ude et al, 2006), Salmonella typhimurium (Simm et al, 2004;Garcia et al, 2004;Simm et al, 2005), Thermotoga maritima (Johnson et al, 2005) and Vibrio cholerae (Bomchil et al, 2003;Rashid et al, 2003;Kovacikova et al, 2005).…”

“…A wspR : : mini-Tn5 WS mutant displays a smooth colony morphology (Spiers et al, 2002), and does not produce cellulose or attach to surfaces (Spiers et al, 2003). Expression of wspR in trans stimulates attachment and exopolysaccharide synthesis in various species (D'Argenio et al, 2002;Aldridge et al, 2003;Goymer et al, 2006;Ude et al, 2006).…”

“…fluorescens SBW25 WspR is a 333 residue protein comprising an N-terminal response-regulator receiver domain and a C-terminal GGDEF domain, separated by a linker region (Spiers et al, 2002(Spiers et al, , 2003. Recently, a combination of random and site-specific mutagenesis has been used to outline basic physical characteristics of WspR (Goymer et al, 2006).…”