title: Genetic analysis of functions involved in the late stages of biofilm development in Burkholderia cepacia H111.
abstract: Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, we report the isolation of 13 random mini-Tn5 insertion mutants of B. cepacia H111 that are defective in biofilm formation on a polystyrene surface. We show that the screening procedure used in this study is biased towards mutants defective in the late stages of biofilm development. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains revealed that the isolated mutants are impaired in their abilities to develop a typical three-dimensional biofilm structure. Molecular investigations showed that the genes required for biofilm maturation fall into several classes: (i). genes encoding for surface proteins; (ii). genes involved in the biogenesis and maintenance of an integral outer membrane; and (iii). genes encoding regulatory factors. It is shown that three of the regulatory mutants produce greatly reduced amounts of N-octanoylhomoserine lactone (C8-HSL). This compound serves as the major signal molecule of the cep quorum-sensing system. As this density-dependent regulatory system is involved in the regulation of biofilm maturation, we investigated the interplay between the three regulatory genes and the quorum-sensing cascade. The results of these investigations show that the identified genes encode for regulatory elements that are positioned upstream of the cep system, indicating that the quorum-sensing system of B. cepacia is a major checkpoint for biofilm formation.
authors.affiliation: Lehrstuhl für Mikrobiologie, Technische Universität München, Freisburg, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Huber
authors.forename: Kathrin
authors.initials: K
authors.lastname: Riedel
authors.forename: Manuela
authors.initials: M
authors.lastname: Köthe
authors.forename: Michael
authors.initials: M
authors.lastname: Givskov
authors.forename: Søren
authors.initials: S
authors.lastname: Molin
authors.forename: Leo
authors.initials: L
authors.lastname: Eberl
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development*; D019121:Burkholderia Infections / Q000382:microbiology; D016956:Burkholderia cepacia / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D003550:Cystic Fibrosis / Q000382:microbiology; D004251:DNA Transposable Elements / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D016254:Mutagenesis, Insertional; D011137:Polystyrenes; D015398:Signal Transduction; D013499:Surface Properties
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D011137:Polystyrenes; C088386:homoserine lactone; D015107:4-Butyrolactone