Downloaded from www.microbiologyresearch.org by IP: 93.91.26.97 On: Fri, 06 Nov 2015 17:00:38 Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli Caroline Blumer, 1 3 Alexandra Kleefeld, 1 Daniela Lehnen, 1 Margit Heintz, 1 Ulrich Dobrindt, 2 Ga ´ bor Nagy, 3 Kai Michaelis, 2 Levente Emo ¨ dy, 3 Tino Polen, 4 Reinhard Rachel, 5 Volker F. Wendisch 4 and Gottfried Unden 1 Correspondence Gottfried Unden unden@uni-mainz.de 1 Institut fu ¨ r Mikrobiologie und Weinforschung, Johannes Gutenberg Universita ¨ t Mainz, Becherweg 15, 55099 Mainz, Germany 2 Institut fu ¨ r Molekulare Infektionsbiologie der Universita ¨t Wu ¨ rzburg, 97070 Wu ¨ rzburg, Germany 3 Institute of Medical Microbiology and Immunology, University of Pe ´ cs, 7624 Pe ´ cs, Hungary 4 Forschungszentrum Ju ¨ lich, Institut fu ¨ r Biotechnologie I, 52425 Ju ¨ lich, Germany 5 Institut fu ¨ r Mikrobiologie, Universita ¨ t Regensburg, 93040 Regensburg, Germany Received 7 April 2005 Revised 20 June 2005 Accepted 29 June 2005 Type 1 fimbriae of Escherichia coli facilitate attachment to the host mucosa and promote biofilm formation on abiotic surfaces. The transcriptional regulator LrhA, which is known as a repressor of flagellar, motility and chemotaxis genes, regulates biofilm formation and expression of type 1 fimbriae. Whole-genome expression profiling revealed that inactivation of lrhA results in an increased expression of structural components of type 1 fimbriae. In vitro, LrhA bound to the promoter regions of the two fim recombinases (FimB and FimE) that catalyse the inversion of the fimA promoter, and to the invertible element itself. Translational lacZ fusions with these genes and quantification of fimE transcript levels by real-time PCR showed that LrhA influences type 1 fimbrial phase variation, primarily via activation of FimE, which is required for the ON-to-OFF transition of the fim switch. Enhanced type 1 fimbrial expression as a result of lrhA disruption was confirmed by mannose-sensitive agglutination of yeast cells. Biofilm formation was stimulated by lrhA inactivation and completely suppressed upon LrhA overproduction. The effects of LrhA on biofilm formation were exerted via the changed levels of surface molecules, most probably both flagella and type 1 fimbriae. Together, the data show a role for LrhA as a repressor of type 1 fimbrial expression, and thus as a regulator of the initial stages of biofilm development and, presumably, bacterial adherence to epithelial host cells also. INTRODUCTION The lrhA gene of Escherichia coli encodes a transcriptional regulator of the LysR family (Bongaerts et al., 1995). Recently, the function of LrhA has been elucidated by a microarray-based comparison of the transcriptional profiles of wild-type E. coli and an isogenic lrhA mutant. LrhA has been identified as a regulator of genes involved in fla- gellation, motility and chemotaxis (Lehnen et al., 2002). The lrhA mutants showed an increased expression of flagellar, motility and chemotaxis genes (e.g. flhDC, fliA, fliC) and a corresponding increased motility and chemotactic response. LrhA acts as transcriptional repressor of the flhDC genes encoding the FlhD 2 C 2 master regulator, and thus of the flhDC regulon. The flhDC regulon contains nearly 50 genes required for flagellum biosynthesis and function, which are arranged in at least 13 operons and whose expression is under hierarchical control (Kalir et al., 2001; Macnab, 1996). LrhA also controls its own expression, but unlike most LysR- type regulators, LrhA is positively autoregulated (Lehnen et al., 2002). In addition, the LrhA protein also controls the stationary-phase sigma factor s S (RpoS), which regulates gene expression in response to general stress in enteric bacteria (Gibson & Silhavy, 1999; Mukherjee et al., 2000). The concentration of s S is regulated through degradation by the ClpXP protease. LrhA represses RpoS stability by affecting the function of ClpXP and the response regulator RssB (or SprE), which serves as a s S recognition factor. 3Present address: Miltenyi Biotec GmbH, 51429 Bergisch-Gladbach, Germany. Abbreviations: Ap, ampicillin; H-NS, histone-like nucleoid-structuring protein; IHF, integration host factor; IRL, left inverted repeat; Km, kanamycin; Lrp, leucine-responsive regulatory protein; Sm, spectino- mycin; UPEC, uropathogenic E. coli. 0002-8098 G 2005 SGM Printed in Great Britain 3287 Microbiology (2005), 151, 3287–3298 DOI 10.1099/mic.0.28098-0