Project description:The physiological and transcriptional response of Nitrosomonas europaea biofilms to phenol and toluene was examined and compared to suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors under continuous flow conditions of growth medium containing ammonia as growth substrate. The responses of N. europaea biofilms to the aromatic hydrocarbons phenol and toluene were determined during short-term (3 h) additions of each compound to the biofilms. Ammonia oxidation in the biofilms was inhibited 50% by 60 uM phenol and 100 uM toluene. These concentrations were chosen for microarray analysis of phenol- and toluene-exposed N. europaea biofilms. Liquid batch cultures of exponentially growing N. europaea cells were harvested alongside the biofilms to determine differential gene expression between attached and suspended growth of N. europaea. Four sample groups of N. europaea cells were used in this study, with biological triplicates of each group. Groups were: Control (untreated) biofilms, phenol-exposed biofilms, toluene-exposed biofilms, and exponentially growing suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors containing 4 independent growth channels and subject to 2 hour inhibition tests. During each experiment, 2 biofilm channels served as control with no inhibitor present and the other 2 biofilm channels were exposed to either 60 uM phenol or 100 uM toluene. Nitrite production was monitored throughout the experiment, and the given concentrations of phenol and toluene resulted in 50% inhibition of ammonia oxidation by the biofilms. Suspended cells were grown in batch reactors. Three 4-plex NimbleGen microarray chips were used, and each chip contained one sample from each experimental group. QC of samples was determined by spectrophotometric methods and using Agilent bioanalyzer traces to determine purity and integrity of RNA and cDNA. A sample tracking report was used to verify the correct hybridization of each sample to the intended array.
Project description:Transcriptomic, metabolomic, physiological, and computational modeling approaches were integrated to gain insight into the mechanisms of antibiotic tolerance in an in vitro biofilm system. Pseudomonas aeruginosa biofilms were grown in drip-flow reactors on a medium composed to mimic the exudate from a chronic wound (CWE). After 72 hours, the biofilms were treated with CWE (control biofilms) or CWE containing ciprofloxacin (treated biofilms) for an additional 24 hours. Planktonic samples were cultivated to early logarithmic phase in CWE. The biofilm specific growth rate was estimated via elemental balances to be approximately 0.37 h-1, or one-third of the planktonic maximum specific growth rate. Global analysis of gene expression indicated decreased anabolic activity in biofilms compared to planktonic cells. A focused transcriptomic analysis revealed the induction of multiple stress responses in biofilm cells, including those associated with growth arrest, zinc limitation, hypoxia, and acyl-homoserine lactone quorum sensing.
Project description:Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here, we operated two laboratory-scale sequence batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal (EBPR). Reactors formed two distinct biofilms, a floccular biofilm, consisting of small, loose, microbial aggregates, and a granular biofilm, forming larger, dense, spherical aggregates. Using metaproteomic methods we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. Both biofilms contained proteins that were indicative of core EBPR metabolisms and cellular function. To understand the proteomic differences between floccular and granular biofilm communities, we compared protein abundances that were statistically enriched in both biofilm states (alpha level = 0.05). Floccular biofilms were enriched with pathogenic secretion systems suggesting a previously unrecognized, highly competitive, mixed microbial community. Comparatively, granular biofilms revealed a high stress environment with evidence of nutrient starvation, phage predation pressure, extracellular polymeric substance (EPS) synthesis, and increased cell lysis. Granular biofilms enriched outermembrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement core EBPR metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter–enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.
Project description:The physiological and transcriptional response of Nitrosomonas europaea biofilms to phenol and toluene was examined and compared to suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors under continuous flow conditions of growth medium containing ammonia as growth substrate. The responses of N. europaea biofilms to the aromatic hydrocarbons phenol and toluene were determined during short-term (3 h) additions of each compound to the biofilms. Ammonia oxidation in the biofilms was inhibited 50% by 60 uM phenol and 100 uM toluene. These concentrations were chosen for microarray analysis of phenol- and toluene-exposed N. europaea biofilms. Liquid batch cultures of exponentially growing N. europaea cells were harvested alongside the biofilms to determine differential gene expression between attached and suspended growth of N. europaea.
Project description:Low concentrations of pharmaceutical compounds were shown to induce transcriptional responses in isolated microorganisms, which could have consequences on ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two distinct rivers and supplemented with environmentally relevant doses of four pharmaceutical products (erythromycin-ER, gemfibrozil-GM, sulfamethazine-SN and sulfamethoxazole-SL). To follow the expression of functional genes, we constructed a 9,600 features anonymous DNA microarray platform onto which cDNA from the various biofilms was hybridized. The reactor design for biofilm development has been previously described (Lawrence et al., 2004; Lawrence et al., 2000). Two duplicate experiments were carried out, with reactors being inoculated with either water from the WC (nutrient rich) or the SSR (nutrient poor). Treatments consisted in the addition of various pharmaceutical compounds: 1 µg l-1 erythromycin (ER), 1 µg l-1 gemfibrozil (GM), 0.5 µg l-1 sulfamethazine (SN), 0.5 µg l-1 sulfamethoxazole (SL). Nothing was added to control reactors (CO). All treatments were replicated independently three times. A reference sample (composite sample from Wascana Creek reactors used to construct the microarray) was hybridized (Cy5) on each slide.
Project description:Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants.
Project description:Transcriptome analysis was applied to characterize the physiological activities of Psuedomonas aeruginosa cells grown for three days in drip flow biofilm reactors when compared to the activities of P. aeruginosa grown planktonically to exponential phase in the same media. Here, rather than examining the effect of an individual gene on biofilm antibiotic tolerance, we used a transcriptomics approach to identify regulons and groups of related genes that are induced during biofilm growth of Pseudomonas aeruginosa. We then tested for statistically significant overlap between the biofilm-induced genes and independently compiled gene lists corresponding to stress responses and other putative antibiotic protective mechanisms. This data was evaluated and used to select strains that carry transposon mutations in genes that might play a role in antibiotic tolerance of biofilms. The strains were evaluated for defects in biofilm tolerance.
Project description:Transcriptome analysis was applied to characterize the physiological activities of Psuedomonas aeruginosa cells grown for three days in drip flow biofilm reactors when compared to the activities of P. aeruginosa grown planktonically to exponential phase in the same media. Here, rather than examining the effect of an individual gene on biofilm antibiotic tolerance, we used a transcriptomics approach to identify regulons and groups of related genes that are induced during biofilm growth of Pseudomonas aeruginosa. We then tested for statistically significant overlap between the biofilm-induced genes and independently compiled gene lists corresponding to stress responses and other putative antibiotic protective mechanisms. This data was evaluated and used to select strains that carry transposon mutations in genes that might play a role in antibiotic tolerance of biofilms. The strains were evaluated for defects in biofilm tolerance. One planktonic condition with four biological replicates; One drip flow biofilm condition grown for 72 hours with three biological replicates; One drip flow biofilm condition grown for 84 hours with three biological replicates.