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-cresoldegrading 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.Biofilms of environmental and medical significance frequently consist of diverse populations of microorganisms (4, 12). A range of metabolic interactions have been observed among microorganisms in biofilms, including mutualistic and commensal relationships (16,26). Moreover, metabolic interactions within biofilms may be facilitated by the spatial arrangement of interacting cells (11,19,20,27). In biofilms that detoxify mixed organic wastes, the metabolic interactions among bacteria could potentially influence biofilm structure and development, since the metabolism of complex organic pollutants often involves multispecies bacterial consortia (10, 23). For example, fluctuating toxicant concentrations could provide selective pressure that alters the species distribution in a biofilm, ultimately influencing biofilm activity.Another feature of biofilms that may respond to changing toxicant concentrations is the exopolysaccharide (EPS) matrix. EPS is an integral structural and functional component of biofilm systems (6, 24), can account for up to 90% of the organic matter in a biofilm (25), and helps protect organisms in the biofilm community from environmental stresses (1; T. R. Neu, G. Packroff, and J. R. Lawrence, Abstr. 97th Gen. Meet. Am. Soc. Microbiol. 1997, p. 396, 1997. Recent research has characterized the composition and quantity of EPS produced (17, 18), as well as its presence in natural biofilms (8). Few studies have invest...
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.Over the past decade, green fluorescent protein (GFP) from the jellyfish Aequorea victoria has emerged as a versatile reporter gene and in situ cell marker. Advantages such as species independence and the lack of a requirement for substrates and cofactors make GFP unique as a reporter gene (3). GFP has become an especially valuable marker for nondestructively visualizing cells, particularly in biofilms. The use of GFP in combination with confocal laser scanning microscopy (CLSM) has led to new insights into biofilm processes (6). Several GFP variants with excitation and emission properties different from those of the wild-type protein have been developed (1). One such protein, GFPuv (5) (Clontech, Palo Alto, Calif.), emits bright green light (maximum at 509 nm) when exposed to UV or blue light (395 or 470 nm). Mutant proteins GFPmut2 and GFPmut3 (4) have emission maxima of 507 and 511 nm when excited by blue light (481 and 501 nm, respectively). Unlike GFPuv, GFPmut2 and GFPmut3 are not excited by UV light, a difference that allows differential imaging of these proteins in the same sample.A dual-labeling technique based on two GFPs has been used for epifluorescent microscopy (11). However, dual labeling with fluorescent proteins for confocal microscopy has not been developed, because of the limited selection of excitation wavelengths available with Kr/Ar lasers, which are frequently features of commercially available confocal microscopes. In this study, we used a confocal microscope equipped with Kr/Ar and UV lasers to expand the range of fluorescent proteins that could be imaged. The two-laser system was used to image (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.Strains, plasmids, and media. The strains and plasmids used are listed in Table 1. All strains were grown in minimal salts medium (MSM) (7) with 0.1% glycerol, 0.1% Casamino Acids, and 100 g of ampicillin ml Ϫ1 . Escherichia coli and Pseudomonas strains were cultured at 37 and 30°C, respectively. Cultures containing pCSAK50 were induced with 0.02% arabinose.Bench-scale flow cell. Biofilms were prepared in bench-scale parallel plate flow cells (reactor volume of 0.35 ml) (Fig. 1). A cover glass was glued to the plastic frame with silicone rubber adhesive sealant RTV 102 (GE Silicones, Waterford, N.Y.). Flow cells were operated in recirculating (start-up) or continuous modes at a constant flow rate (0.8...
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.
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.
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