Analysis of bacterial community structure in sulfurous-oil-containing soils and detection of species carrying dibenzothiophene desulfurization (dsz) genes

The selective effects of sulfur-containing hydrocarbons, with respect to ch anges in bacterial community structure and selection of desulfurizing organ isms and genes, were studied in soil. Samples taken from a polluted field s oil (A) along a concentration gradient of sulfurous oil and from soil micro cosms treated,vith dibenzothiophene (DBT)-containing petroleum (FSL soil) w ere analyzed. Analyses included plate counts of total bacteria and of DBT u tilizers, molecular community profiling via soil DNA-based PCR-denaturing g radient gel electrophoresis (PCR-DGGE), and detection of genes that encode enzymes involved in the desulfurization of hydrocarbons, i.e., dszA, dszB, and dszC. Data obtained from the A soil showed no discriminating effects of oil levels on the culturable bacterial numbers on either medium used. Gene rally, counts of DBT degraders were 10- to 100-fold lower than the total cu lturable counts. However, PCR-DGGE showed that the numbers of bands detecte d in the molecular community profiles decreased with increasing oil content of the soil. Analysis of the sequences of three prominent bands of the pro files generated,vith the highly polluted soil samples suggested that the un derlying organisms were related to Actinomyces sp., Arthrobacter sp., and a bacterium of uncertain affiliation. dszA, dszB, and dszC genes were presen t in all A soil samples, whereas a range of unpolluted soils gave negative results in this analysis. Results from the study of FSL soil revealed minor effects of the petroleum-DBT treatment on culturable bacterial numbers and clear effects on the DBT-utilizing communities. The molecular community pr ofiles were largely stable over time in the untreated soil, whereas they sh owed a progressive change over time following treatment with DBT-containing petroleum. Direct PCR assessment revealed the presence of dszB-related sig nals in the untreated FSL soil and the apparent selection of dszA- and dszC -related sequences by the petroleum-DBT treatment. PCR-DGGE applied to sequ ential enrichment cultures in DBT-containing sulfur-free basal salts medium prepared from the A and treated FSL soils revealed the selection of up to LO distinct bands. Sequencing a subset of these bands provided evidence for the presence of organisms related to Pseudomonas putida, a Pseudomonas sp. , Stenotrophomonas maltophilia, and Rhadococcus erythropolis. Several of 52 colonies obtained from the A and FSL soils on agar plates with DBT as the sole sulfur source produced bands that matched the migration of bands selec ted in the enrichment cultures. Evidence for the presence of dszB in 12 str ains was obtained, whereas dszA and dszC genes were found in only 7 and 6 s trains, respectively. Most of the strains carrying dszA or dszC were classi fied as R. erythropolis related, and all revealed the capacity to desulfuri ze DBT. A comparison of 37 dszA sequences, obtained via PCR from the A and FSL soils, from enrichments of these soils, and from isolates, revealed the great similarity of all sequences to the canonical (R. erythropolis strain IGTS8) dszA sequence and a large degree of internal conservation. The 37 s equences recovered were grouped in three clusters. One group, consisting of 30 sequences, was minimally 98% related to the IGTS8 sequence, a second gr oup of 2 sequences was slightly different, and a third group of 5 sequences was 95% similar. The first two groups contained sequences obtained from bo th soil types and enrichment cultures (including isolates), but the last co nsisted of sequences obtained directly from the polluted ii soil.