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SummaryPseudomonas aeruginosa forms diverse matrixenclosed surface-associated multicellular assemblages (biofilms) that aid in its survival in a variety of environments. One such biofilm is the pellicle that forms at the air-liquid interface in standing cultures. We screened for transposon insertion mutants of P. aeruginosa PA14 that were unable to form pellicles. Analysis of these mutants led to the identification of seven adjacent genes, named pel genes, the products of which appear to be involved in the formation of the pellicle's extracellular matrix. In addition to being required for pellicle formation, the pel genes are also required for the formation of solid surface-associated biofilms. Sequence analyses predicted that three pel genes encode transmembrane proteins and that five pel genes have functional homologues involved in carbohydrate processing. Microscopic and macroscopic observations revealed that wild-type P. aeruginosa PA14 produces a cellulase-sensitive extracellular matrix able to bind Congo red; no extracellular matrix was produced by the pel mutants. A comparison of the carbohydrates produced by the wild-type strain and pel mutants suggested that glucose was a principal component of the matrix material. Together, these results suggest that the pel genes are responsible for the production of a glucose-rich matrix material required for the formation of biofilms by P. aeruginosa PA14.

Pseudomonas aeruginosa forms biofilms, which are cellular aggregates encased in an extracellular matrix. Molecular genetics studies of three common autoaggregative phenotypes, namely wrinkled colonies, pellicles, and solid-surface-associated biofilms, led to the identification of two loci, pel and psl, that are involved in the production of carbohydrate-rich components of the biofilm matrix. The pel gene cluster is involved in the production of a glucose-rich matrix material in P. aeruginosa strain PA14 (L. Friedman and R. Kolter, Mol. Microbiol. 51:675-690, 2004). Here we investigate the role of the pel gene cluster in P. aeruginosa strain ZK2870 and identify a second genetic locus, termed psl, involved in the production of a mannose-rich matrix material. The 11 predicted protein products of the psl genes are homologous to proteins involved in carbohydrate processing. P. aeruginosa is thus able to produce two distinct carbohydrate-rich matrix materials. Either carbohydrate-rich matrix component appears to be sufficient for mature biofilm formation, and at least one of them is required for mature biofilm formation in P. aeruginosa strains PA14 and ZK2870.Pseudomonas aeruginosa has been extensively utilized as a model organism for the study of biofilm formation (16,20). A key feature of mature P. aeruginosa biofilms is the presence of an extracellular matrix that encases the constituent cells. This matrix has been reported to contain a mixture of polymeric substances, including nucleic acids, proteins, and polysaccharides (11,(33)(34)(35). Matrix production occurs at a late stage in biofilm development, when cells display a high degree of autoaggregation (6, 27). The ability to autoaggregate leads to several macroscopic phenotypes; among them are the production of pellicles at the air-liquid interface of standing liquid cultures and the production of highly structured colonies on agar plates. Different strains of P. aeruginosa display variability in the abilities to form pellicles under different culture conditions and to show different colony morphologies. This phenotypic diversity most likely results from genetic differences among isolates (6, 11). Thus, we consider it important to perform genetic analyses of multiple strains.We recently identified the pel locus of P. aeruginosa strain PA14 (11). This locus contains seven genes whose products are required for matrix formation and which are therefore critical for the autoaggregative properties of the strain; pel mutants do not form pellicles or mature solid-surface-associated (SSA) biofilms. In addition, while the parent strain gives rise to wrinkled colonies under some growth conditions, the P. aeruginosa PA14 pel mutant colonies are invariably flat and smooth. The predicted protein products of the pel genes share sequence similarity with proteins involved in carbohydrate processing, and pel mutants lack a glucose-rich component of their extracellular matrix. Whether the pel genes are involved in the synthesis of a matrix component in other strains of P. aerugi...

Daptomycin is a lipopeptide antibiotic with potent activity against gram-positive bacteria. Complete-genome comparisons of laboratory-derived Staphylococcus aureus with decreased susceptibility to daptomycin and their susceptible parent were used to identify genes that contribute to reduced susceptibility to daptomycin. Selective pressure of growth in sublethal concentrations of daptomycin resulted in the accumulation of mutations over time correlating with incremental decreases in susceptibility. Single point mutations resulting in amino acid substitutions occurred in three distinct proteins: MprF, a lysylphosphatidylglycerol synthetase; YycG, a histidine kinase; and RpoB and RpoC, the ␤ and ␤ subunits of RNA polymerase. Sequence analysis of mprF, yycF, yycG, rpoB, and rpoC in clinical isolates that showed treatment-emergent increases in daptomycin MICs revealed point mutations in mprF and a nucleotide insertion in yycG, suggesting a role for these genes in decreased susceptibility to daptomycin in the hospital setting.

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pseudomonas aeruginosa virulence

Sequencing of a highly virulent strain of Pseudomonas aeruginosa and comparison to a previously sequenced, less pathogenic, strain, together with experimental testing in a C. elegans model, suggests that Pseudomonas virulence is multifactorial and combinatorial.