Downloaded from www.microbiologyresearch.org by IP: 54.237.57.119 On: Sun, 08 May 2016 00:58:29 Glucosyltransferase A (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 contribute to cell aggregation, in vitro biofilm formation, and colonization of the mouse gastrointestinal tract Jens Walter, 1,3 Clarissa Schwab, 2 Diane M. Loach, 1 Michael G. Ga ¨ nzle 2 and Gerald W. Tannock 1 Correspondence Jens Walter jwalter2@unl.edu 1 Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand 2 Department of Agricultural Food and Nutritional Science, University of Alberta, Edmonton, Canada 3 Department of Food Science and Technology, University of Nebraska, Lincoln, NE 68583-0919, USA Received 14 June 2007 Revised 14 September 2007 Accepted 16 October 2007 Members of the genus Lactobacillus are common inhabitants of the proximal gastrointestinal tract of animals such as mice, rats, chickens and pigs, where they form epithelial biofilms. Little is known about the traits that facilitate biofilm formation and gut colonization. This study investigated the ecological role of a glucosyltransferase (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 and a fructosyltransferase (FtfA) of L. reuteri LTH5448. In vitro experiments using isogenic mutants revealed that GtfA was essential for sucrose-dependent autoaggregation of L. reuteri TMW1.106 cells under acidic conditions, while inactivation of Inu slowed the formation of cell aggregates. Experiments using an in vitro biofilm assay showed that GtfA and Inu contributed to biofilm formation of L. reuteri TMW1.106. Experiments using ex-Lactobacillus-free mice revealed that the ecological performance of the inu mutant, but not of the gtfA or ftfA mutant, was reduced in the gastrointestinal tract when in competition with the parental strain. In the absence of competition, the gtfA mutant showed delayed colonization of the murine gut relative to the wild-type. In addition, the gtfA mutant showed reduced ecological performance in competition experiments with Lactobacillus johnsonii # 21. From the evidence provided in this study we conclude that GtfA and Inu confer important ecological attributes of L. reuteri TMW1.106 and contribute to colonization of the mouse gastrointestinal tract. INTRODUCTION The gastrointestinal tract of mammals is colonized by a complex collection of micro-organisms (the gut micro- biota) that influences biochemical, physiological, immuno- logical and non-specific disease-resistance characteristics of the host (Gordon & Pesti, 1971). Bacteria of the genus Lactobacillus are commonly detected as inhabitants of gut ecosystems and predominate in the proximal (gastric) regions of animals such as pigs, chickens, mice and rats (Tannock, 1992; Walter, 2005). The high population levels of lactobacilli in the gut of these animals are facilitated through bacterial adherence to the non-secretory, strati- fied, squamous epithelia present in the forestomach, pars oesophagea and crop of mice and rats, pigs and chickens, respectively. The epithelial associations formed by lacto- bacilli show characteristics of bacterial biofilms (Donlan & Costerton, 2002) because the bacteria are firmly attached to a surface (epithelium) and are embedded in a matrix of extracellular polymeric substances (Fuller & Brooker, 1974; Savage et al., 1968). The mechanisms by which lactobacilli form these epithelial associations are not well understood, but preliminary in vitro investigations have shown that carbohydrate molecules are likely to be involved, while a large surface protein (Lsp) appears to initiate adherence in vivo (Tannock, 1997; Walter et al., 2005). Extracellular polysaccharides (exopolysaccharides, EPS) are synthesized by a wide variety of bacteria including lactic acid bacteria, and they have been shown to contribute to dental biofilm formation and cell aggregation of strep- tococci (Burne et al., 1996; Munro et al., 1991; Yamashita et al., 1993). Many strains of lactobacilli produce homo- polysaccharides (HoPS) and oligosaccharides (OS) consisting of either glucose residues (glucans and gluco- oligosaccharides, GOS) or fructose residues (fructans and fructo-oligosaccharides, FOS) (Ga ¨nzle & Schwab, 2005; Korakli & Vogel, 2006; van Hijum et al., 2006). These Abbreviations: EPS, extracellular polysaccharides; FOS, fructo-oligo- saccharides; GOS, gluco-oligosaccharides; HoPS, homopolysaccharides; OS, oligosaccharides; TEM, transmission electron microscopy. Microbiology (2008), 154, 72–80 DOI 10.1099/mic.0.2007/010637-0 72 2007/010637 G 2008 SGM Printed in Great Britain