Downloaded from www.microbiologyresearch.org by IP: 23.20.70.183 On: Mon, 12 Sep 2016 17:45:08 Burkholderia cenocepacia requires RpoE for growth under stress conditions and delay of phagolysosomal fusion in macrophages Ronald S. Flannagan 1 and Miguel A. Valvano 1,2 Correspondence Miguel A. Valvano mvalvano@uwo.ca 1 Infectious Disease Research Group, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada 2 Medicine, Siebens–Drake Research Institute, University of Western Ontario, London, Ontario, Canada Received 27 September 2007 Revised 28 October 2007 Accepted 5 November 2007 Burkholderia cenocepacia is an opportunistic pathogen causing serious infections in patients with cystic fibrosis. The widespread distribution of this bacterium in the environment suggests that it must adapt to stress to be able to survive. We identified in B. cenocepacia K56-2 a gene predicted to encode RpoE, the extra-cytoplasmic stress response regulator. The rpoE gene is the first gene of a predicted operon encoding proteins homologous to RseA, RseB, MucD and a protein of unknown function. The genomic organization and the co-transcription of these genes were confirmed by PCR and RT-PCR. The mucD and rpoE genes were mutated, giving rise to B. cenocepacia RSF24 and RSF25, respectively. While mutant RSF24 did not demonstrate any growth defects under the conditions tested, RSF25 was compromised for growth under temperature (44 6C) and osmotic stress (426 mM NaCl). Expression of RpoE in trans could complement the osmotic growth defect but exacerbated temperature sensitivity in both RSF25 and wild-type K56-2. Inactivation of rpoE altered the bacterial cell surface, as indicated by increased binding of the fluorescent dye calcofluor white and by an altered outer-membrane protein profile. These cell surface changes were restored by complementation with a plasmid encoding rpoE. Macrophage infections in which bacterial colocalization with fluorescent dextran was examined demonstrated that the rpoE mutant could not delay the fusion of B. cenocepacia-containing vacuoles with lysosomes, in contrast to the parental strain K56-2. These data show that B. cenocepacia rpoE is required for bacterial growth under certain stress conditions and for the ability of intracellular bacteria to delay phagolysosomal fusion in macrophages. INTRODUCTION The Burkholderia cepacia complex (Bcc) comprises a group of nine closely related bacterial species that are phenoty- pically very similar but genetically distinct (Coenye et al., 2003; Mahenthiralingam et al., 2000). These bacteria are opportunistic pathogens that cause infections in immuno- compromised individuals and often infect cystic fibrosis (CF) patients (Mahenthiralingam & Vandamme, 2005). Burkholderia cenocepacia, a member of the Bcc, is one of the species most often recovered from CF patients world- wide and is also associated with the most severe infections (Brisse et al., 2004; LiPuma, 2005; Manno et al., 2004; Reik et al., 2005; Speert et al., 2002). Furthermore, B. cenocepacia infections in CF patients are associated with an accelerated decline in lung function as compared to other CF-related pathogens such as Pseudomonas aerugi- nosa (Courtney et al., 2004; Jones et al., 2004). The treatment of infection is difficult because this bacterium is multi-drug resistant (Zhou et al., 2007) and can be transmitted from person to person (Govan et al., 1993; LiPuma et al., 1990; Smith et al., 1993). Infections are further complicated by the ‘cepacia syndrome’, a poten- tially fatal necrotizing pneumonia that occurs in a subset of patients (Isles et al., 1984; Thomassen et al., 1985). Although B. cenocepacia can be transmitted between patients, it is also acquired from environmental sources (Coenye & Vandamme, 2003) as these bacteria occupy many different niches, including the rhizosphere, plants and humans. Therefore, it is likely that B. cenocepacia can readily adapt to many different stresses. One way by which bacteria adapt to stress is through the activity of the alternative sigma factor RpoE, a key regulator of the extra- cytoplasmic stress response that has been extensively characterized in the enteric bacterium Escherichia coli (De Abbreviations: Bcc, Burkholderia cepacia complex; BcCV, B. cenocepa- cia-containing vacuole; CF, cystic fibrosis; OM, outer membrane; Tp, trimethoprim. Microbiology (2008), 154, 643–653 DOI 10.1099/mic.0.2007/013714-0 2007/013714 G 2008 SGM Printed in Great Britain 643