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

Andrew J. Spiers, Paul B. Rainey

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Abstract

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.
Original languageEnglish
Pages (from-to)2829-2839
Number of pages11
JournalMicrobiology
Volume151
Issue number9
DOIs
StatePublished - 1 Sep 2005

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Pseudomonas fluorescens
Biofilms
Cellulose
Lipopolysaccharides
Operon
Air
Propidium
Gram-Negative Bacteria
Hydrophobic and Hydrophilic Interactions
Mutagenesis
Sucrose
Coloring Agents
Phenotype
Membranes

Cite this

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title = "The Pseudomonas fluorescens SBW25 wrinkly spreader biofilm requires attachment factor, cellulose fibre and LPS interactions to maintain strength and integrity",
abstract = "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.",
author = "Spiers, {Andrew J.} and Rainey, {Paul B.}",
year = "2005",
month = "9",
doi = "10.1099/mic.0.27984-0",
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journal = "Microbiology",
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N2 - 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.

AB - 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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