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Soils contaminated with mineral oil hydrocarbons are often cleaned in off-site bioremediation systems. In order to find out which bacteria are active during the degradation phase in such systems, the diversity of the active microflora in a degrading soil remediation system was investigated by small-subunit (SSU) rRNA analysis. Two sequential RNA extracts from one soil sample were generated by a procedure incorporating bead beating. Both extracts were analysed separately by generating individual SSU rDNA clone libraries from cDNA of the two extracts. The sequencing results showed moderate diversity. The two clone libraries were dominated by Gammaproteobacteria, especially Pseudomonas spp. Alphaproteobacteria and Betaproteobacteria were two other large groups in the clone libraries. Actinobacteria, Firmicutes, Bacteroidetes and Epsilonproteobacteria were detected in lower numbers. The obtained sequences were predominantly related to genera for which cultivated representatives have been described, but were often clustered together in the phylogenetic tree, and the sequences that were most similar were originally obtained from soils and not from pure cultures. Most of the dominant genera in the clone libraries, e.g. Pseudomonas, Acinetobacter, Sphingomonas, Acidovorax and Thiobacillus, had already been detected in (mineral oil hydrocarbon) contaminated environmental samples. The occurrence of the genera Zymomonas and Rhodoferax was novel in mineral oil hydrocarbon-contaminated soil.

A rapid oligonucleotide probe design strategy based on subtractive hybridization was devised to eliminate the need to sequence many similar clones of dominant members of a community. The method yields probes for 16S rRNA or rRNA genes of individual members of microbial communities specific within the context of those communities. Results revealed that only those clones with 100% sequence identity with the probe fragment were detected. Probes also were inclined to correspond to more variable regions of the 16S rRNA if the target sequences of other clones in the library.

The genes responsible for the degradation of 2,4-dichlorophenoxyacetate (2,4-D) by a-Proteobacteria have previously been difficult to detect by using gene probes or polymerase chain reaction (PCR) primers. PCR products of the chlorocatechol 1,2-dioxygenase gene,tfdC, now allowed cloning of two chlorocatechol gene clusters from theSphingomonassp. strain TFD44. Sequence characterization showed that the first cluster,tfdD,RFCE, comprises all the genes necessary for the conversion of 3,5-dichlorocatechol to 3-oxoadipate, including a presumed regulatory gene,tfdR, of the LysR-type family. The second gene cluster,tfdC2E2F2, is incomplete and appears to lack a chloromuconate cycloisomerase gene and a regulatory gene. Purification and N-terminal sequencing of selected enzymes suggests that at least representatives of both gene clusters (TfdD of cluster 1 and TfdC2 of cluster 2) are induced during the growth of strain TFD44 with 2,4-D. A mutant constructed to contain an insertion in the chloromuconate cycloisomerase genetfdDstill was able to grow with 2,4-D, but more slowly and with a longer lag phase. This, and the detection of additional activity peaks during protein purification suggest that strain TFD44 harbors at least another chloromuconate cycloisomerase gene. The sequence of thetfdCEregion was almost identical to that of a partially characterized chlorocatechol catabolic gene cluster ofSphingomonas herbicidovoransMH, whereas the sequence of thetfdC2E2F2cluster was different. The similarity of the predicted proteins of thetfdD,RFCEandtfdC2E2F2clusters to known sequences of other Proteobacteria in the database ranged from 42 to 61% identical positions for the first cluster and from 45.5 to 58% identical positions for the second cluster. Between both clusters, the similarities of their predicted proteins ranged from 44.5 to 64% identical positions. Thus, both clusters (together with those ofS. herbicidovoransMH) represent deep-branching lines in the respective dendrograms, and the sequence information will help future primer design for the detection of corresponding genes in the environment. [ABSTRACT FROM AUTHOR]