Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.Surface adhesion of bacteria and subsequent cell binary fission and exopolymer production lead to the formation of bacterial biofilms (see reference 6 for a review). Application of confocal scanning laser microscopy (CSLM) has led to the suggestion that microcolonies are the basic structural units in sessile communities (4). It was found that biofilms are highly hydrated open structures containing a high fraction of exopolymers and large void spaces between the microcolonies (18). Mushroom-shaped microcolonies, separated by channels and voids, were observed in Pseudomonas fluorescens biofilms (15, 16). Investigations of multispecies biofilm communities on granular activated carbon in fluidized-bed reactors revealed that growth occurred as discrete microcolony structures separated by channel boundaries (19). Microbial biofilms in river water were shown to have a ridged structure, with microcolonies forming ridges parallel to the direction of flow (23).Evidence is now emerging that motility may play a role in structure formation in biofilms. After the initial attachment to the substratum, Pseudomonas aeruginosa evidently moves on the substratum by means of twitching motility, and it has been suggested that the initial microcolonies are formed by aggregation of bacteria (25). Furthermore, it has been suggested that the initial microcolonies in Vibrio cholerae El Tor biofilms are formed via flagellar motility of the cells along the substratum (32). In addition, Wolfaardt et al. (34) and Nielsen et al. (24) observed structural changes in biofilms in response to changing environments, suggesting that established biofilms in some cases may display dynamic behavior. Since it appears that structures in both young and mature biofil...

Cystic fibrosis (CF) patients have increased susceptibility to chronic lung infections by Pseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate the in vivo growth physiology of P. aeruginosa within lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescence in situ hybridization (PNA-FISH)-based method was used to estimate the in vivo growth rates of P. aeruginosa directly in lung tissue samples from CF patients and the growth rates of P. aeruginosa in infected lungs in a mouse model. The growth rate of P. aeruginosa within CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect on P. aeruginosa by PMNs was also observed in vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding that P. aeruginosa growth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O 2 consumption, which slows the growth of P. aeruginosa in infected CF lungs. In support of this, the growth of P. aeruginosa was significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronic P. aeruginosa infection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration.

The phenylurea herbicides are an important group of pesticides used extensively for pre- or post-emergence weed control in cotton, fruit and cereal crops worldwide. The detection of phenylurea herbicides and their metabolites in surface and ground waters has raised the awareness of the important role played by agricultural soils in determining water quality. The degradation of phenylurea herbicides following application to agricultural fields is predominantly microbial. However, evidence suggests a slow degradation of the phenyl ring, and substantial spatial heterogeneity in the distribution of active degradative populations, which is a key factor determining patterns of leaching losses from agricultural fields. This review summarises current knowledge on the microbial metabolism of isoproturon and related phenylurea herbicides in and below agricultural soils. It addresses topics such as microbial degradation of phenylurea herbicides in soil and subsurface environments, characteristics of known phenylurea-degrading soil micro-organisms, and similarities between metabolic pathways for different phenylurea herbicides. Finally, recent studies in which molecular and microbiological techniques have been used to provide insight into the in situ microbial metabolism of isoproturon within an agricultural field will be discussed.

No abstract

Expression of the functional gene tfdA involved in degradation of phenoxyacetic acids such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) was investigated during degradation scenarios in natural unseeded soil samples. The results illustrate how messenger RNA (mRNA)-based analysis is well suited to quantitatively study the activity of specific microbial populations in soil using phenoxyacetic acid biodegradation as a model system. Via quantitative real-time PCR, a clear response to the presence of phenoxy acids was shown during degradation in soil amended with 20 mg 2,4-D or MCPA per kg soil. Further, we found a relatively high degree of correlation between expression of the functional gene and the rates of mineralization. Melting curve analyses of real-time PCR products, supported by tfdA-denaturing gradient gel electrophoresis analysis showed that, although only class I tfdA genes were apparent in the indigenous microbial population, class III tfdA genes became predominant during incubation, and were the only genes expressed during degradation of MCPA in soil. In contrast, both classes were expressed during degradation of the structurally similar compound 2,4-D. The ability to quantify microbial transcripts directly in environmental samples will have a profound impact on our understanding of microbial processes in the environment in future studies.