The extradiol dioxygenase diversity of a site highly contaminated with aliphatic and aromatic hydrocarbons under air-sparging treatment was assessed by functional screening of a fosmid library in Escherichia coli with catechol as substrate. The 235 positive clones from inserts of DNA extracted from contaminated soil were equivalent to one extradiol dioxygenase-encoding gene per 3.6 Mb of DNA screened, indicating a strong selection for genes encoding this function. Three subfamilies were identified as being predominant, with 72, 55 and 43 fosmid inserts carrying genes, related to those encoding TbuE of Ralstonia pickettii PK01 (EXDO-D), IpbC of Pseudomonas sp. JR1 (EXDO-K2) or DbtC of Burkholderia sp. DBT1 (EXDO-Dbt), respectively, whereas genes encoding enzymes related to XylE of Pseudomonas putida mt-2 were not observed. Genes encoding oxygenases related to isopropylbenzene dioxygenases were usually colocalized with genes encoding EXDO-K2 dioxygenases. Functional analysis of representative proteins indicated a subcluster of EXDO-D proteins to show exceptional high affinity towards different catecholic substrates. Based on Vmax/Km specificity constants, a task-sharing between different extradiol dioxygenases in the community of the contaminated site can be supposed, attaining a complementary and community-balanced catalytic power against diverse catecholic derivatives, as necessary for effective degradation of mixtures of aromatics.

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An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCRamplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA-and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.

A recombinant strain of bioluminescent Pseudomonas fluorescens 2–79 RLD containing a catabolic pathway for degradation of 2,5‐dichlorobenzoate (2,5‐DCB) was monitored in soil microcosms to examine the influence of plants on its growth and activity in a contaminated soil. Recombinant P. fluorescens 2–79 RLD was generated by mating a versatile chlorobenzoate utilizer, P. putida P 111, containing plasmid pPB111, with a bioluminescent strain of P. fluorescens that had been transformed previously with a Tn7‐luxCDABE marker. Plasmid pPB111 contains genes encoding for a chlorobenzoate‐1,2‐dioxygenase that converts ortho‐chlorobenzoates to their corresponding catechols. DNA hybridization experiments and cell‐free extract assays with parental and recombinant P. fluorescens 2–79 RLD suggested that the reaction product of the plasmid pPB111 encoded chlorobenzoate dioxygenase was degraded by an endogenous catechol dioxygenase in P. fluorescens 2–79. After introduction of P. fluorescens 2–79 RLD into soil containing 10 mg kg−1 2,5‐DCB, normally recalcitrant 2,5‐DCB was degraded rapidly over a period of 2 to 4 days in soil with plants. In contrast, 2,5‐DCB disappearance in nonplanted soil was significantly slower, requiring 7 days in one experiment, and more than 2 weeks in a second experiment. Population numbers of the degrader were similar in planted and nonplanted soil for the first 7 days, but declined in nonplanted soils by day 14. Physiological status, measured using an assay based on lag‐phase duration, was similar in planted and nonplanted soils at day 2, but rapidly declined in nonplanted soil by day 7. At day 14, plasmid stability in P. fluorescens 2–79 RLD was significantly greater in rhizosphere soil; only 10% of P. fluorescens 2–79 RLD cells in rhizosphere soil had lost the ability to degrade 2,5‐DCB, versus 94% of the cells in nonplanted soil. The plasmid also was transferred to indigenous bacteria in both planted and nonplanted soils, as determined by the appearance of novel degraders. The results demonstrate that the presence of plants promoted rapid degradation of DCB and provided a niche that enhanced maintenance of plasmid pPB111 in the degrader bacterium.