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Biofilms

Bioaggregation of unicellular organisms is a remarkably common motif in microbiology. It appears that high density living is advantageous. But this is not without its challenges. Thin layers of biomass can generate steep concentration gradients resulting in limitations to diffusion of compounds inside the aggregate out (eg. quorum sensing signals) and compounds outside the aggregate in (eg. electron acceptors). This portfolio brings together diverse research interests including bacterial quorum sensing, phenazine activity and biodegradation, Pseudomonas aeruginosa biofilms and activated sludge flocculate formation in wastewater treatment plants.

Bacterial adhesion and biofilm formation are both dependent on the production of extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA). eDNA promotes biofilm establishment in a wide range of bacterial species. In Pseudomonas aeruginosa eDNA is major component of biofilms and is essential for biofilm formation and stability. In this study we report that production of pyocyanin in P. aeruginosa PAO1 and PA14 batch cultures is responsible for promotion of eDNA release. A phzSH mutant of P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H(2)O(2)) generation, cell lysis, and eDNA release in comparison to its wildtype strain. A ΔphzA-G mutant of P. aeruginosa PA14 deficient in pyocyanin production generated negligible amounts of H(2)O(2) and released less eDNA in comparison to its wildtype counterpart. Exogenous addition of pyocyanin or incubation with H(2)O(2) was also shown to promote eDNA release in low pyocyanin producing (PAO1) and pyocynain deficient (PA14) strains. Based on these data and recent findings in the biofilm literature, we propose that the impact of pyocyanin on biofilm formation in P. aeruginosa occurs via eDNA release through H(2)O(2) mediated cell lysis.

Activated sludge used for wastewater treatment globally is composed of a high-density microbial community of great biotechnological significance. In this study the presence and purpose of quorum sensing via N-acylated-l-homoserine lactones (AHLs) in activated sludge was explored. The presence of N-heptanoyl-l-homoserine lactone in organic extracts of sludge was demonstrated along with activation of a LuxR-based AHL monitor strain deployed in sludge, indicating AHL-mediated gene expression is active in sludge flocculates but not in the bulk aqueous phase. Bacterial isolates from activated sludge were screened for AHL production and expression of phenotypes commonly but not exclusively regulated by AHL-mediated gene transcription. N-acylated-l-homoserine lactone and exoenzyme production were frequently observed among the isolates. N-acylated-l-homoserine lactone addition to sludge upregulated chitinase activity and an AHL- and chitinase-producing isolate closely related to Aeromonas hydrophila was shown to respond to AHL addition with upregulation of chitinase activity. N-acylated-l-homoserine lactones produced by this strain were identified and genes ahyI/R and chiA, encoding AHL production and response and chitinase activity respectively, were sequenced. These experiments provide insight into the relationship between AHL-mediated gene expression and exoenzyme activity in activated sludge and may ultimately create opportunities to improve sludge performance.

Phenazines are a large class of nitrogen-containing aromatic heterocyclic compounds produced and secreted by bacteria from phylogenetically diverse taxa. Due to their roles in ecological fitness of their producers and biocontrol of plant pathogens, it is of interest to understand the fate of phenazines. Compared to phenazine biosynthesis, little is known about phenazine-degrading microorganisms. In this study, a phenazine-degrading bacterial strain Rhodanobactersp. PCA2 was isolated and characterized to degrade phenazine-1-carboxylic acid (PCA), phenazine, pyocyanin and 1-hydroxyphenazine as the sole carbon, nitrogen and energy source. Sequencing analysis (genomic and specific PCR products) revealed that genes (ubiDand the homolog of the MFORT_16269 gene) involved in PCA degradation were plasmid borne in strain PCA2. Gene abundance (qPCR) and transcriptional activity (RT-qPCR) of the MFORT_16269 gene homolog significantly increased during incubation with PCA, supporting its involvement in PCA degradation by strain PCA2. Furthermore, results from LC-MS analysis together with proteomics indicated that strain PCA2 degraded PCA via decarboxylation and cleavage of aromatic and nitrogen-containing rings, which was potentially catalysed by UbiD, UbiX, phenylpropionate dioxygenase, biphenyl-2,3-diol 1,2-dioxygenase, amidohydrolase and nitroreductase and produced phenazine, (4Z)-2-hydroxy-5-{[(1Z)-6(hydroxyamino)cyclohexa -2,4-dien-1-ylidene]carbamoyl} penta-2,4-dienoic acid (HCCPD) and phenylhydroxylamine as the intermediates.