A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms

Pseudomonas 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 ?-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 biofil

The development of a FACS-based strategy for the isolation of Shigella flexneri mutants that are deficient in intercellular spread.

In the disease course of bacillary dysentery, pathogenic Shigella flexneri invade colonic epithelial cells and spread both within and between host cells. The ability to spread intercellularly allows the organism to infect an entire epithelial layer without significant contact with the extracellular milieu. Using fluorescence activated cell sorter (FACS)-based technology, we developed a rapid and powerful selection strategy for the isolation of S. flexneri mutants that are unable to spread from cell to cell. The majority of mutants identified using this strategy harbour mutations that affect the structure of their lipopolysaccharide or the ability of the bacteria to move intracellularly via actin-based motility; both factors have previously been shown to be essential for cell-to-cell spread. However, using a modified strategy that eliminated both of these types of mutants, we identified several mutants that provide us with evidence that bacterial proteins of the type III secretion system, which are essential for bacterial entry into host cells, also play a role in cell-to-cell spread.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Putative mechanisms and biological role of coccoid form formation in Campylobacter jejuni.

In certain conditions Campylobacter jejuni cells are capable of changing their cell shape from a typically spiral to a coccoid form (CF). By similarity to other bacteria, the latter was initially considered to be a viable but non-culturable form capable of survival in unfavourable conditions. However, subsequent studies with C. jejuni and closely related bacteria Helicobacter pylori suggested that CF represents a non-viable, degenerative form. Until now, the issue on whether the CF of C. jejuni is viable and infective is highly controversial. Despite some preliminary experiments on characterization of CF cells, neither biochemical mechanisms nor genetic determinants involved in C. jejuni cell shape changes have been characterized. In this review, we highlight known molecular mechanisms and genes involved in CF formation in other bacteria. Since orthologous genes are also present in C. jejuni, we suggest that CF formation in these bacteria is also a regulated and genetically determined process. A possible significance of CF in the lifestyle of this important bacterial pathogen is discussed