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:Transcription profile of Escherichia coli cells in mono-species pure biofilms was compared to that of E. coli cells in E. coli-Stenotrophomonas maltophilia dual-species biofilms. E. coli cells were separated from dual-species biofilms before total RNA extraction to eliminate possible cross hybridization from S. maltophilia transcripts. The separation method was developed by combining the use of reagent RNAlater and immuno-magnetic separation. Pure E. coli biofilms were processed with the same separation protocol before RNA extraction.
Project description:Transcription profile of Escherichia coli cells in mono-species pure biofilms was compared to that of E. coli cells in E. coli-Stenotrophomonas maltophilia dual-species biofilms. E. coli cells were separated from dual-species biofilms before total RNA extraction to eliminate possible cross hybridization from S. maltophilia transcripts. The separation method was developed by combining the use of reagent RNAlater and immuno-magnetic separation. Pure E. coli biofilms were processed with the same separation protocol before RNA extraction. Two condition experiments: E. coli mono-species biofilm vs E. coli in mixed-species biofilm. Two biological replicates with independently grown and harvested biofilms. Each biological replicate has two or three technical replicates of hybridization on microarray slides. Each slide has three built-in replicates for each probe.
Project description:In this study, we used an original system to produce C. difficile anaerobic biofilms consisting in continuous-flow microfermentors, that is compared to planktonic growth culture.
Project description:Pseudomonas aeruginosa is one of the most frequent pathogen dominant in complicated urinary tract infections (UTI). To unravel the adaptation strategies of P. aeruginosa to the conditions in the urinary tract and to define the underlying regulatory network an artificial growth system mimicking the conditions in the urinary tract was established. Transcriptome analyses were used to investigate the physiological status of P. aeruginosa under this conditions. We performed comparisons to identify genes induced under artificial urinary tract conditions to unravel the adaptive strategies and the underlying regulatory network used by Pseudomonas aeruginosa during urinary tract infections using Affimetrix GeneChips. Pseudomonas aeruginosa wild type strain PAO1 was grown in an artificial in vitro growth system mimicking the conditions in the urinary tract. Therefore, biofilms were grown on the surface of membrane filters placed on agar plates at 37 °C up to the late logarithmic state under aerobic and anaerobic conditions (incubated in an anaerobic beanch). An artificial urine medium (AUM) simulating the averaged urine of an human adult was used as nutrient souce. 10-fold diluted Luria Bertani (LB)-medium was used as reference medium. For growth under oxygen depletion the media were supplemented with 50 mM KNO3 to sustain anaerobic respiration. The biofilms were harveted at this time points and resuspsended in 0.9% (w/v) NaCl. The OD578 of biofilm suspension was 0.8 for all tested conditions. First comparison: Identification of genes induced or repressed under aerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown aerobically for 18 h to the late logarithmic phase in biofilms on AUM with the transcriptome profile of the PAO1 strain, which was grown aerobically for 18 h to the late logarithmic phase in biofilms on 10-fold diluted LB. Second comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown anaerobically for 2 days up to the late logarithmic phase in biofilms on AUM supplemented with 50 mM nitrate with the transcriptome profile of the PAO1 strain, which was grown anaerobically for 2 days up to the late logarithmic phase in biofilms on 10-fold diluted LB supplemented with 50 mM nitrate.
Project description:Pseudomonas aeruginosa undergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO3-) as an alternative electron acceptor. Understanding the mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments against P. aeruginosa biofilm infection. Anaerobic growth of PAO1 reached higher cell density in the presence of vitamin B12, an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape. These results suggest that vitamin B12, the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth of P. aeruginosa with normal cell division. We used microarray to elucidate the global gene expression profiles underlying vitamin B12-induced changes in bacterial cell shape and growth-associated properties.