Downloaded from www.microbiologyresearch.org by IP: 54.81.141.78 On: Fri, 22 Jan 2016 13:11:41 Phenotypic differentiation and seeding dispersal in non-mucoid and mucoid Pseudomonas aeruginosa biofilms B. Purevdorj-Gage, 1 W. J. Costerton 1 and P. Stoodley 1,2 Correspondence B. Purevdorj-Gage laura_p@erc.montana.edu 1 Center for Biofilm Engineering, 366 EPS Building – PO Box 173980, Montana State University-Bozeman, MT 59717, USA 2 Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, USA Received 2 August 2004 Revised 9 December 2004 Accepted 3 February 2005 There is growing evidence that Pseudomonas aeruginosa biofilms exhibit a multicellular developmental life cycle analogous to that of the myxobacteria. In non-mucoid PAO1 biofilms cultured in glass flow cells the phenotypic differentiation of microcolonies into a motile phenotype in the interior of the microcolony and a non-motile surrounding ‘wall phenotype’ are described. After differentiation the interior cells coordinately evacuated the microcolony from local break out points and spread over the wall of the flow cell, suggesting that the specialized microcolonies were analogous to crude fruiting bodies. A microcolony diameter of approximately 80 mm was required for differentiation, suggesting that regulation was related to cell density and mass transfer conditions. This phenomenon was termed ‘seeding dispersal’ to differentiate it from ‘erosion’ which is the passive removal of single cells by fluid shear. Using the flow cell culturing method, in which reproducible seeding phenotype in PAO1 wild-type was demonstrated, the effects of quorum sensing (QS) and rhamnolipid production (factors previously identified as important in determining biofilm structure) on seeding dispersal using knockout mutants isogenic with PAO1 was investigated. Rhamnolipid (rhlA) was not required for seeding dispersal but las/rhl QS (PAO1-JP2) was, in our system. To assess the clinical relevance of these data, mucoid P. aeruginosa cystic fibrosis isolate FRD1 was also investigated and was seeding-dispersal-negative. INTRODUCTION The proliferation and persistence of bacterial biofilms on various surfaces have been well documented in both in vitro and in vivo settings, and modern microscopic as well as molecular techniques have revealed that biofilm formation is a complex multifactorial process regulated by both genetic and environmental factors (Stoodley et al., 2002). Although much is known about the initial stages of biofilm develop- ment, very little is known about the mechanisms governing the detachment process (Stewart 1993; Stewart et al., 2000). Detachment has been shown to play an important role in shaping the morphological characteristics and structure of mature biofilms (Picioreanu et al., 2001; Stewart 1993; Van Loosdrecht et al., 1995, 1997) which further extends the implication of this process in biofilm function and beha- viour in general. Other studies have characterized two main types of detachment processes: erosion (the continual detachment of single cells and small portions of the biofilm) and sloughing (the rapid, massive loss of biofilm) (Bryers, 1988; Characklis, 1990; Stoodley et al., 2001). However, these types of detachment were generally thought of in terms of passive, shear-dependent processes. Only recently has detachment been considered as an active dispersal mechan- ism. A number of laboratories have observed the ‘hollowing’ out of microcolonies by cells actively leaving the interiors (Sauer et al., 2002) and the remaining ‘hollow mounds’ have been noted in Pseudomonas putida biofilms (Tolker-Nielsen et al., 2000). Based on previous reports, the suggested mechanisms for this particular hollowing process in the biofilm clusters differ from one biofilm species to another. For example, Kaplan et al. (2003b) have shown that the ‘non-motile’ Gram-negative bacterium Actinobacillus acti- nomycetemcomitans, grown statically on agar, released individual cells from within the biofilm colony via enzymic activity of b-hexosaminidase (Kaplan et al., 2003a). On the other hand transmission electron micrographs of Staphy- lococcus epidermidis cultured on agar plates suggested that the hollowing process may have occurred through the process of cell lysis (P. Stewart, personal communication). Also, Webb et al. (2003) have demonstrated that the cells in the interior of P. aeruginosa clusters lysed via phage Abbreviations: CF, cystic fibrosis; QS, quorum sensing. The online version of this paper at http://mic.sgmjournals.org has supplementary movie files. 0002-7536 G 2005 SGM Printed in Great Britain 1569 Microbiology (2005), 151, 1569–1576 DOI 10.1099/mic.0.27536-0