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The Pseudomonas fluorescens SBW25 wrinkly spreader biofilm requires attachment factor, cellulose fibre and LPS interactions to maintain strength and integrity

¹ Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
² School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand

Correspondence
Andrew J. Spiers
andrew.spiers{at}plants.ox.ac.uk

The wrinkly spreader (WS) isolate of Pseudomonas fluorescens SBW25 forms a substantial biofilm at the air–liquid interface. The biofilm is composed of an extracellular partially acetylated cellulose-fibre matrix, and previous mutagenesis of WS with mini-Tn5 had identified both the regulatory and cellulose-biosynthetic operons. One uncharacterized WS mutant, WS-5, still expressed cellulose but produced very weak biofilms. In this work, the mini-Tn5 insertion site in WS-5 has been identified as being immediately upstream of the tol-pal operon. Like Tol-Pal mutants of other Gram-negative bacteria, WS-5 showed a ‘leaky-membrane’ phenotype, including the serendipitous ability to utilize sucrose, increased uptake of the hydrophilic dye propidium iodide, and the loss of lipopolysaccharide (LPS) expression. WS-5 cells were altered in relative hydrophobicity, and showed poorer recruitment and maintenance in the biofilm than WS. The WS-5 biofilm was also less sensitive to chemical interference during development. However, growth rate, cellulose expression and attachment were not significantly different between WS and WS-5. Finally, WS-5 biofilms could be partially complemented with WS-4, a biofilm- and attachment-deficient mutant that expressed LPS, resulting in a mixed biofilm with significantly increased strength. These findings show that a major component of the WS air–liquid biofilm strength results from the interactions between LPS and the cellulose matrix of the biofilm – and that in the WS biofilm, cellulose fibres, attachment factor and LPS are required for biofilm development, strength and integrity.

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