SummaryPseudomonas aeruginosa produces extracellular DNA which functions as a cell-to-cell interconnecting matrix component in biofilms. Comparison of extracellular DNA and chromosomal DNA by the use of polymerase chain reaction and Southern analysis suggested that the extracellular DNA is similar to whole-genome DNA. Evidence that the extracellular DNA in P. aeruginosa biofilms and cultures is generated via lysis of a subpopulation of the bacteria was obtained through experiments where extracellular β β β β -galactosidase released from lacZ -containing P. aeruginosa strains was assessed. Experiments with the wild type and lasIrhlI , pqsA , pqsL and fliMpilA mutants indicated that the extracellular DNA is generated via a mechanism which is dependent on acyl homoserine lactone and Pseudomonas quinolone signalling, as well as on flagella and type IV pili. Microscopic investigation of flow chamber-grown wild-type P. aeruginosa biofilms stained with different DNA stains suggested that the extracellular DNA is located primarily in the stalks of mushroom-shaped multicellular structures, with a high concentration especially in the outer part of the stalks forming a border between the stalk-forming bacteria and the cap-forming bacteria. Biofilms formed by lasIrhlI , pqsA and fliMpilA mutants contained less extracellular DNA than biofilms formed by the wild type, and the mutant biofilms were more susceptible to treatment with sodium dodecyl sulphate than the wild-type biofilm.

When grown as a biofilm in laboratory flow chambers Pseudomonas aeruginosa can develop mushroom-shaped multicellular structures consisting of distinct subpopulations in the cap and stalk portions. We have previously presented evidence that formation of the cap portion of the mushroom-shaped structures in P. aeruginosa biofilms occurs via bacterial migration and depends on type IV pili (Mol Microbiol 50: 61-68). In the present study we examine additional factors involved in the formation of this multicellular substructure. While pilA mutants, lacking type IV pili, are deficient in mushroom cap formation, pilH and chpA mutants, which are inactivated in the type IV pili-linked chemosensory system, showed only minor defects in cap formation. On the contrary, fliM mutants, which are non-flagellated, and cheY mutants, which are inactivated in the flagellum-linked chemotaxis system, were largely deficient in cap formation. Experiments involving DNase treatment of developing biofilms provided evidence that extracellular DNA plays a role in cap formation. Moreover, mutants that are deficient in quorum sensing-controlled DNA release formed microcolonies upon which wild-type bacteria could not form caps. These results constitute evidence that type IV pili, flagellum-mediated motility and quorum sensing-controlled DNA release are involved in the formation of mature multicellular structures in P. aeruginosa biofilms.

No abstract

BackgroundKlebsiella pneumoniae is an important gram-negative opportunistic pathogen causing primarily urinary tract infections, respiratory infections, and bacteraemia. The ability of bacteria to form biofilms on medical devices, e.g. catheters, has a major role in development of many nosocomial infections. Most clinical K. pneumoniae isolates express two types of fimbrial adhesins, type 1 fimbriae and type 3 fimbriae. In this study, we characterized the role of type 1 and type 3 fimbriae in K. pneumoniae biofilm formation.ResultsIsogenic fimbriae mutants of the clinical K. pneumoniae isolate C3091 were constructed, and their ability to form biofilm was investigated in a flow cell system by confocal scanning laser microscopy. The wild type strain was found to form characteristic biofilm and development of K. pneumoniae biofilm occurred primarily by clonal growth, not by recruitment of planktonic cells. Type 1 fimbriae did not influence biofilm formation and the expression of type 1 fimbriae was found to be down-regulated in biofilm forming cells. In contrast, expression of type 3 fimbriae was found to strongly promote biofilm formation.ConclusionBy use of well defined isogenic mutants we found that type 3 fimbriae, but not type 1 fimbriae, strongly promote biofilm formation in K. pneumoniae C3091. As the vast majority of clinical K. pneumoniae isolates express type 3 fimbriae, this fimbrial adhesin may play a significant role in development of catheter associated K. pneumoniae infections.

A flow cell-grown model consortium consisting of two organisms, Burkholderia sp. LB400 and Pseudomonas sp. B13(FR1), was studied. These bacteria have the potential to interact metabolically because Pseudomonas sp. B13(FR1) can metabolize chlorobenzoate produced by Burkholderia sp. LB400 when grown on chlorobiphenyl. The expected metabolic interactions in the consortium were demonstrated by high performance liquid chromatography (HPLC) analysis. The spatial structure of the consortium was studied by fluorescent in situ rRNA hybridization and scanning confocal laser microscopy. When the consortium was fed with medium containing a low concentration of chlorobiphenyl, microcolonies consisting of associated Burkholderia sp. LB400 and Pseudomonas sp. B13(FR1) bacteria were formed, and separate Pseudomonas sp. B13(FR1) microcolonies were evidently not formed. When the consortium was fed citrate, which can be metabolized by both species, the two species formed separate microcolonies. The structure development In the consortium was studied online using a gfp-tagged Pseudomonas sp. B13(FR1) derivative. After a shift In carbon source from citrate to a low concentration of chlorobiphenyl, movement of the Pseudomonas sp. B13(FR1) bacteria led to a change in the spatial structure of the consortium from the unassociated form towards the associated form within a few days. Experiments Involving a gfp-based Pseudomonas sp. B13(FR1) growth activity reporter strain Indicated that chlorobenzoate supporting growth of Pseudomonas sp. B13(FR1) is located close to the Burkholderia sp. LB400 microcolonies in chlorobiphenyl-grown consortia.