Cell–cell and cell–surface interactions mediated by cellulose and a novel exopolysaccharide contribute to Pseudomonas putida biofilm formation and fitness under water‐limiting conditions

Abstract

The composition of the exopolysaccharide matrix of Pseudomonas putida mt2 biofilms is relatively undefined as well as the contributions of each polymer to ecological fitness. Here, we describe the role of two putative exopolysaccharide gene clusters, putida exopolysaccharide A (pea) and bacterial cellulose (bcs) in biofilm formation and stability, rhizosphere colonization and matrix hydration under water-limiting conditions. Our findings suggest that pea is involved in the production of a novel glucose, galactose, and mannose-rich polymer that contributes to cell-cell interactions necessary for pellicle and biofilm formation and stability. In contrast, Bcs plays a minor role in biofilm formation and stability, although it does contribute to rhizosphere colonization based on a competition assay. We show that pea expression is highly induced transiently under water-limiting conditions but only slightly by high osmolarity, as determined by qRT-PCR. In contrast, both forms of water stress highly induced bcs expression. Cells deficient in making one or more exopolysaccharide experienced greater dehydration-mediated cell-envelope stress, leading to increased alginate promoter activity. However, this did not lead to increased exopolysaccharide production, except in bcs or pea mutants unable to produce alginate, indicating that P. putida compensates by producing, presumably more Pea or Bcs exopolysaccharides, to facilitate biofilm hydration. Collectively, the data suggest that Pea and Bcs contribute to biofilm formation and in turn their presence contributes to fitness under water-limiting conditions, but not to the extent of alginate.

Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads

“…A similar observation has been made for P. putida mt-2 (the progenitor of KT2440) where water stress was also found to increase cellulose expression [76]. Many bacteria respond to desiccation by producing exopolysaccharides, many of which are hygroscopic and retain water entropically [92], and amorphous cellulose is more hygroscopic and retains more liquid than crystalline cellulose [62].…”

“…In a test of 16 pseudomonads known to form biofilms and express cellulose, WspR19 was found to significantly increase biofilm attachment, strength, and cellulose expression in P. fluorescens 54/96, P. syringae DC3000, P. syringae T1615 and P. syringae 6034 [15] (WspR19 induction of cellulose production by SBW25 and DC3000 is shown in Figure 5). WspR19 also induced a WS-like phenotype in P. putida KT2440, despite the fact that biofilm-formation or cellulose expression in this pseudomonad had not been observed in the initial survey (cellulose expression was subsequently reported for both wild-type and WspR19-carrying strains under different experimental conditions by [76]). Similarly, nine of ten non-biofilmforming and non-cellulose expressing P. syringae isolates were found to produce biofilms when induced with WspR19, and two of these also expressed detectable levels of cellulose [15].…”

Adaptive Divergence in Experimental Populations ofPseudomonas fluorescens. V. Insight into the Niche Specialist Fuzzy Spreader Compels Revision of the ModelPseudomonasRadiation

“…The role of LPS as a major component of WS biofilm strength and integrity, as a consequence of interactions between LPS and the cellulose matrix of the biofilm, was revealed in an earlier study (Spiers and Rainey 2005) and is well supported (Lau et al 2009;Nielsen et al 2011;Nilsson et al 2011). Alteration of the bacterial cell surface, with consequent changes to cell hydrophobicity, adhesive properties, and motility, likely perturbs the cell-cell interactions that maintain biofilm structure.…”

“…Furthermore, mutants lacking O antigen produce biofilms with weaker viscoelastic properties. Recent studies with P. putida KT2440 EPS mutants support a model in which LPS mediates the necessary cell-cell interactions that maintain biofilm matrix stability following attachment (Nilsson et al 2011), with EPS in a stabilizing role and cellulose polymers providing additional structural support (Nielsen et al 2011;Nilsson et al 2011). …”

Roles of Cyclic Di-GMP and the Gac System in Transcriptional Control of the Genes Coding for the Pseudomonas putida Adhesins LapA and LapF

“…P. putida KT2440 is also able to develop biofilms on biotic and abiotic surfaces, and molecular determinants participating in these processes have been described. They include surface proteins, exopolysaccharides (EPS), and elements involved in the synthesis, regulation, and functionality of the flagellar system (8,9,(21)(22)(23)(24)(25). Two very large secreted proteins, LapA and LapF, have been recognized as significant players with different roles (8,22).…”