Analysis of biofilm structure and gene expression using fluorescence dual labeling.

The use of biofilms for the degradation of recalcitrant environmental contaminants or for the production of secondary metabolites necessitates understanding and controlling gene expression. In this work, dual labeling with green fluorescent protein (GFP) variants was used to investigate inducible gene expression in a biofilm. Colocalization of GFP emissions was used to determine regions of attached cells and to correlate structure and activity within the biofilm. The labeling strategy reported here is unique in that the two GFP signals were distinguished by differential excitation rather than differential emission.

Commensal interactions in a dual-species biofilm exposed to mixed organic compounds.

There is limited knowledge of interspecies interactions in biofilm communities. In this study, Pseudomonas sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and Pseudomonas putida DMP1, a p-cresol-degrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various p-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of p-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as the p-cresol concentrations increased, and biofilm formation was severely inhibited at high p-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all p-cresol concentrations, although at high p-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 and Escherichia coli ATCC 33456, an adhesive strain that does not mineralize p-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.

Dual labeling with green fluorescent proteins for confocal microscopy.

We report a dual labeling technique involving two green fluorescent protein (GFP) variants that is compatible with confocal microscopy. Two lasers were used to obtain images of (i) mixed cultures of cells, where one species contained GFPuv and another species contained GFPmut2 or GFPmut3, and (ii) a single species containing both GFPuv and GFPmut2 in the same cell. This method shows promise for monitoring gene expression and as a nondestructive and in situ technique for confocal microscopy of multispecies biofilms.

Ultrasonic flexural-plate-wave sensor for detecting the concentration of settling E. coli W3110 cells.

The flexural-plate-wave (FPW) sensor, a type of ultrasonic sensor, can detect changes in E. coli W3110 concentration in solution as the cells settle onto the sensor under the influence of gravity. A model of the sensor's response to cell settling has been developed and is in good agreement with the experimental data. The FPW technique improves on conventional methods for determining cell concentrations; this technique allows for on-line data collection, is nondestructive, and requires only small sample volumes. The FPW sensor has applications as a device to measure cell concentrations and growth rates in industrial fermentors, biofilms, and wastewater treatment facilities.