title: Burkholderia cenocepacia requires RpoE for growth under stress conditions and delay of phagolysosomal fusion in macrophages.
abstract: 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 degrees C) 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.
authors.affiliation: Infectious Disease Research Group, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Flannagan
authors.affiliation: Medicine, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario, Canada.|Infectious Disease Research Group, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D001557:Benzenesulfonates / Q000378:metabolism; D019117:Burkholderia / Q000166:cytology / Q000254:growth & development* / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008247:Lysosomes / Q000382:microbiology / Q000502:physiology*; D008264:Macrophages / Q000166:cytology / Q000382:microbiology*; D008561:Membrane Fusion; D016254:Mutagenesis, Insertional; D009997:Osmotic Pressure; D010587:Phagocytosis; D010588:Phagosomes / Q000382:microbiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D001557:Benzenesulfonates; C007061:C.I. Fluorescent Brightening Agent 28