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Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

This report describes a high-throughput assay to identify substances that reduce the frequency of conjugation in Gram-negative bacteria. Bacterial conjugation is largely responsible for the spread of multiple antibiotic resistances in human pathogens. Conjugation inhibitors may provide a means to control the spread of antibiotic resistance. An automated conjugation assay was developed that used plasmid R388 and a laboratory strain of Escherichia coli as a model system, and bioluminescence as a reporter for conjugation activity. Frequencies of conjugation could be measured continuously in real time by the amount of light produced, and thus the effects of inhibitory compounds could be determined quantitatively. A control assay, run in parallel, allowed elimination of compounds affecting cell growth, plasmid stability or gene expression. The automated conjugation assay was used to screen a database of more than 12 000 microbial extracts known to contain a wide variety of bioactive compounds (the NatChem library). The initial hit rate was 1.4%. From these, 48 extracts containing active compounds and representing a variety of organisms and extraction conditions were subjected to fractionation (24 fractions per extract). The 52 most active fractions were subjected to a secondary analysis to determine the range of plasmid inhibition. Plasmids R388, R1 and RP4 were used as representatives of a variety of plasmid transfer systems. Only one fraction (of complex composition) affected transfer of all three plasmids, while four other fractions were active against two of them. Two separate compounds were identified from these fractions: linoleic acid and dehydrocrepenynic acid. Downstream analysis showed that the chemical class of unsaturated fatty acids act as true inhibitors of conjugation.

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell-cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and [H.sub.2][O.sub.2], and are readily phagocytosed by PMNs, in contrast to bacteria with functional QS systems. In contrast to the wild-type, QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently, the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

Research has been conducted on Pseudomonas sp. DSS73 isolated from sugar beet seedling rhizoplane. The ability of DSS73 to contain root-pathogenic microfungi, arising from amphisin-dependent surface translocation and growth, has been investigated, and the results are reported.

Research has been conducted on Pseudomonas sp. DSS73 isolated from sugar beet seedling rhizoplane. The ability of DSS73 to contain root-pathogenic microfungi which arises from amphisin-dependent surface translocation has been investigated, and the details are reported.

The synthetic halogenated furanone compound interferes with the N-acyl homoserine lactone-mediated quorum sensing in Pseudomonas aeruginosa. Data indicate that furanone penetrates microcolonies in biofilms and blocks cell signalling and quorum sensing, leading to bacterial detachment and loss of bacterial biomass in biofilms.

Research has been conducted on the Pseudomonas aeruginosa and Burkholderia cepacia which form mixed biofilms in the lungs of the cystic fibrosis patients. The ability of these bacteria to communicate with each other via the N-acylhomoserine-lactone signal molecules has been investigated and the results indicate that in mixed biofilms intergeneric signalling occurs in one direction, from P. aeruginosa to B. cepacia.

Results indicate that cepR gene, involved in the biofilm formation, and the cepI N-acylhomoserine lactone synthase gene make up the cep quorum-sensing system in Burkholderia cepacia H111. Genetic analysis of the cepR mutant reveals that swarming motility and biofilm formation are independent.

Green fluorescent protein (GFP) fluorescence in Streptococcus gordonii was assessed under low-oxygen concentration and low-pH conditions. GFP, it was found, can be used in studies of oxygen-tolerant anaerobic bacteria. Densely packed, flow cell-grown biofilms of S. gordonii do not develop oxygen gradients that inhibit GFP fluorescence development. The transient nature of GFP fluorescence in acid-producing bacteria can often be overcome in flowcells.

Plasmid transfer in the animal intestine is discussed relative to other dynamic bacterial populations and the role of community structure/environment. Experiments have been carried out with the goal of clarifying whether plasmid transfer in the mouse gut shows kinetics expected of a mixed liquid-flow system. Transfer kinetics seen were very like those of a biofilm and not like those anticipated in a mixed, liquid culture.

A study was conducted to analyze the influence of activated polymorphonuclear leukocytes and their release of toxic oxygen by-products in the cystic fibrosis lung environment in the generation and/or selection of mucoid variants during inflammatory responses to Pseudomonas aeruginosa. Results indicated that polymorphonuclear leukocytes and their oxygen radicals can promote phenotypic and genotypic changes that are typical of the intractable form of P.aeruginosa in the cystic fibrosis lung.

Previous research has suggested poor survival rates among Pseudomonas putida strain KT2442 under a condition of sulphur or phosphate deprivation. To understand this process further, the physiological responses of P. putida strain KT2442 to phosphate deprivation are determined with respect to cell morphology, macromolecular content and rates of total protein synthesis. It is shown that phosphate-deprived cultures undergo population shifts that allow them to be taken over mutants that have adjusted to phosphate deprivation.

Research has been conducted on N-Acyl-L-homoserine-lactone-producing Serratia species. The role of quorum sensing in these species' food spoiling properties has been investigated, and the results demonstrate that quorum-sensing-controlled exoenzymic activities influence food quality.