SummaryWe report the identification of an ATP-binding cassette (ABC) transporter and an associated large cellsurface protein that are required for biofilm formation by Pseudomonas fluorescens WCS365. The genes coding for these proteins are designated lap for l arge a dhesion p rotein. The LapA protein, with a predicted molecular weight of ~ 900 kDa, is found to be loosely associated with the cell surface and present in the culture supernatant. The LapB, LapC and LapE proteins are predicted to be the cytoplasmic membranelocalized ATPase, membrane fusion protein and outer membrane protein component, respectively, of an ABC transporter. Consistent with this prediction, LapE, like other members of this family, is localized to the outer membrane. We propose that the lapEBCencoded ABC transporter participates in the secretion of LapA, as strains with mutations in the lapEBC genes do not have detectable LapA associated with the cell surface or in the supernatant. The lap genes are conserved among environmental pseudomonads such as P. putida KT2440, P. fluorescens PfO1 and P. fluorescens WCS365, but are absent from pathogenic pseudomonads such as P. aeruginosa and P. syringae . The wild-type strain of P. fluorescens WCS365 and its lap mutant derivatives were assessed for their biofilm forming ability in static and flow systems. The lap mutant strains are impaired in an early step in biofilm formation and are unable to develop the mature biofilm structure seen for the wild-type bacterium. Time-lapse microscopy studies determined that the lap mutants are unable to progress from reversible (or transient) attachment to the irreversible attachment stage of biofilm development. The lap mutants were also found to be defective in attachment to quartz sand, an abiotic surface these organisms likely encounter in the environment.

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.Cloning using Saccharomyces cerevisiae homologous recombination is a powerful technique in which PCR primers with stretches of homologous DNA are used to target recombination of an amplicon with a vector (9,21,25,27). Cloning of an amplicon with yeast recombination into an intact vector can be done without restriction enzymes, but this requires the use of a selectable or counterselectable marker. The formation of a gap or double-stranded break in the vector by restriction enzyme digestion allows selective cloning of unmarked amplicons (29). An amplicon can seamlessly replace any part of the DNA in a vector with no need for enzyme sites at the junctions of the recombined pieces of DNA, as long as the yeast replication machinery is not replaced and the ends of the amplicons (or other DNA with homologous ends) to be added to a vector are on either side of the digest site (gap) in the vector. Therefore, multiple unmarked pieces of DNA can efficiently be assembled together in a single step using this method. In addition to fusions, directed mutations, restriction sites, and other changes can be designed into the primers. This technique is particularly useful for building vectors and complex constructs.The basic concept of cloning with gap repair is as follows. A vector able to replicate in yeast with a selective marker, such as URA3, is digested (gapped) with a restriction enzyme. Primers that amplify the region of DNA that one wants to insert into the vector are designed, containing sequences of homology to the yeast-replicating plasmid (30 to 40 bp in length). One or more targeted amplicons and the gapped vector are simultaneously introduced into yeast cells. A subset of these cells recombines the amplicon(s) and vector via Saccharomyces cerevisiae native recombination enzymes, generating stable circularized plasmids. Linearized or gapped vectors are not stable in yeast, while vectors that circularize...

A role for the outer-membrane-associated LapA protein in early biofilm formation by Pseudomonas fluorescens WCS365 has previously been shown. This paper reports that lapD, a gene located adjacent to the lapA gene, also plays a role in biofilm formation. A mutation in lapD results in a conditional biofilm defect in a static assay -this biofilm phenotype is exacerbated when biofilm formation is assayed in a flow-cell system. Furthermore, a lapD mutation shows a partial defect in the transition from reversible to irreversible attachment, consistent with an early role for the lapD gene product in biofilm formation. LapD is shown to be localized to the inner membrane of P. fluorescens. The data show decreased LapA associated with the cell surface, but no apparent change in cytoplasmic levels of this protein or lapA transcription, in a lapD mutant. A model is proposed wherein the role of LapD in biofilm formation is modulating the secretion of the LapA adhesin.

No abstract