Environmental Microbiology (2006) 8(11), 1997–2011 doi:10.1111/j.1462-2920.2006.01080.x © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd Blackwell Publishing LtdOxford, UKEMIEnvironmental Microbiology1462-2912© 2006 The Authors; Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd 200681119972011Original Article Pseudomonas biofilms and cellulose expressionS. Ude et al. Received 21 February, 2006; accepted 9 May, 2006. *For correspondence. E-mail: andrew.spiers@plants.ox.ac.uk; Tel. (+44) 1865 275000; Fax (+44) 1865 275074. † Present address: AgResearch Ltd, Grasslands Research Centre, Palmerston North, New Zealand. Biofilm formation and cellulose expression among diverse environmental Pseudomonas isolates Susanne Ude, 1 Dawn L. Arnold, 2 Christina D. Moon, 1† Tracey Timms-Wilson 3 and Andrew J. Spiers 1 * 1 Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK. 2 Centre for Research in Plant Science, Faculty of Applied Sciences, University of the West of England, Bristol, UK. 3 NERC Centre for Ecology and Hydrology, Oxford, UK. Summary The ability to form biofilms is seen as an increasingly important colonization strategy among both patho- genic and environmental bacteria. A survey of 185 plant-associated, phytopathogenic, soil and river Pseudomonas isolates resulted in 76% producing bio- films at the air–liquid (A–L) interface after selection in static microcosms. Considerable variation in biofilm phenotype was observed, including waxy aggrega- tions, viscous and floccular masses, and physi- cally cohesive biofilms with continuously varying strengths over 1500-fold. Calcofluor epifluorescent microscopy identified cellulose as the matrix compo- nent in biofilms produced by Pseudomonas asplenii, Pseudomonas corrugata , Pseudomonas fluorescens , Pseudomonas marginalis , Pseudomonas putida, Pseudomonas savastanoi and Pseudomonas syrin- gae isolates. Cellulose expression and biofilm forma- tion could be induced by the constitutively active WspR19 mutant of the cyclic-di-GMP-associated, GGDEF domain-containing response regulator involved in the P. fluorescens SBW25 wrinkly spreader phenotype and cellular aggregation in Pseudomonas aeruginosa PA01. WspR19 could also induce P. putida KT2440, which otherwise did not pro- duce a biofilm or express cellulose, as well as Escher- ichia coli K12 and Salmonella typhimurium LT2, both of which express cellulose yet lack WspR homo- logues. Statistical analysis of biofilm parameters sug- gest that biofilm development is a more complex process than that simply described by the production of attachment and matrix components and bacterial growth. This complexity was also seen in multivariate analysis as a species-ecological habitat effect, under- scoring the fact that in vitro biofilms are abstractions of those surface and volume colonization processes used by bacteria in their natural environments. Introduction Biofilms are part of the range of aggregations employed by bacteria in the colonization of surfaces and volumes, both in human infections and in other environments, such as the surfaces and tissues of plants, the rhizosphere and in water. Despite the lack of clear distinctions between different types of aggregations (e.g. between microcolo- nies, biofilms, flocs and slimes), these small bacterial pop- ulations face the same difficulties such as the need to be firmly attached in an optimal location, to resist environ- mental stresses and predation, and to be successfully competitive et cetera . Bacteria probably employ similar attachment and matrix factors [e.g. fimbrae, flagella, extra- cellular polysaccharide (EPS)] to overcome these prob- lems, although perhaps in different combinations and as a result of different environmental, physiological and pop- ulation cues. (For a selection of biofilm reviews, see Cos- terton et al ., 1995; Davey and O’Toole, 2000; Morris and Monier, 2003; Hall-Stoodley et al ., 2004; Ramey et al ., 2004; Branda et al ., 2005.) Recent molecular analysis of the wrinkly spreader (WS) mutant of Pseudomonas fluorescens SBW25 has identi- fied partially acetylated cellulose, fimbrae-like attachment factor and lipopolysaccharide (LPS) as being important to the strength and integrity of the WS biofilm (Spiers et al ., 2002, 2003; Spiers and Rainey, 2005) (Fig. 1). This robust structure, unlike the archetypal submerged surface–liquid interface biofilms of Pseudomonas aeruginosa PA01, is located at the air–liquid (A–L) interface and is maintained by bacterial attachment to the vial walls at the meniscus region and a general hydrophobic nature. The selective advantage of the A–L interface biofilm to the WS mutant is greater access to oxygen, allowing a more rapid rate of growth compared with the ancestral non-biofilm-forming P. fluorescens SBW25 which grows in the liquid column. Unlike many biofilms, the WS biofilm is not the result of a quorum decision (Juhas et al ., 2005), but due to a single point-mutation in a chemosensory-like regulatory appara- tus ( wsp ) which controls both attachment and cellulose