Hydrocarbon-degrading microorganisms play an important role in the natural attenuation of spilled petroleum in a variety of anoxic environments. The role of benzylsuccinate synthase (BSS) in aromatic hydrocarbon degradation and its use as a biomarker for field investigations are well documented. The recent discovery of alkylsuccinate synthase (ASS) allows the opportunity to test whether its encoding gene, assA, can serve as a comparable biomarker of anaerobic alkane degradation. Degenerate assA- and bssA-targeted PCR primers were designed in order to survey the diversity of genes associated with aromatic and aliphatic hydrocarbon biodegradation in petroleum-impacted environments and enrichment cultures. DNA was extracted from an anaerobic alkane-degrading isolate (Desulfoglaeba alkenexedens ALDC), hydrocarbon-contaminated river and aquifer sediments, a paraffin-degrading enrichment, and a propane-utilizing mixed culture. Partial assA and bssA genes were PCR amplified, cloned, and sequenced, yielding several novel clades of assA genes. These data expand the range of alkane-degrading conditions for which relevant gene sequences are available and indicate that considerable diversity of assA genes can be found in hydrocarbon-impacted environments. The detection of genes associated with anaerobic alkane degradation in conjunction with the in situ detection of alkylsuccinate metabolites was also demonstrated. Comparable molecular signals of assA/bssA were not found when environmental metagenome databases of uncontaminated sites were searched. These data confirm that the assA gene is a useful biomarker for anaerobic alkane metabolism.

Plasmid pRO1957, which contains a 26.5-kb fragment from the chromosome of Pseudomonas pickettii PKO1, allows P. aeruginosa PAO1 to grow on toluene or benzene as a sole carbon and energy source. A subclone of pRO1957, designated pRO1966, when present in P. aeruginosa PAO1 grown in lactate-toluene medium, accumulates m-cresol in the medium, indicating that m-cresol is an intermediate of toluene catabolism. Moreover, incubation of such cells in the presence of 1802 followed by gas chromatography-mass spectrometry analysis of m-cresol extracts showed that the oxygen in m-cresol was derived from molecular oxygen.Accordingly, this suggests that toluene-3-monooxygenation is the first step in the degradative pathway. Toluene-3-monooxygenase activity is positively regulated from a locus designated tbuT. Induction of the toluene-3-monooxygenase is mediated by either toluene, benzene, ethylbenzene, or m-cresol. Moreover, toluene-3-monooxygenase activity induced by these eflectors also metabolizes benzene and ethylbenzene to phenol and 3-ethylphenol, respectively, and also after induction, o-xylene, m-xylene, and p-xylene are metabolized to 3,4-dimethylphenol, 2,4-dimethylphenol, and 2,5-dimethylphenol, respectively, although the xylene substrates are not effectors. Styrene and phenylacetylene are transformed into more polar products.The degradation of toluene by bacteria incorporating molecular oxygen into toluene has been studied by several laboratories, resulting in the characterization of four pathways in pseudomonads. The best characterized of these pathways is the TOL plasmid pathway of Pseudomonas putida mt-2 (25). The TOL plasmid pathway converts toluene to benzyl alcohol, benzaldehyde, benzoate, and finally to catechol, which undergoes meta cleavage. The structural and regulatory genes of this pathway have been mapped and consist of two regulons. The upper pathway operon, xylCMAB, encodes enzymes for the metabolism of toluene to benzoate and is positively regulated byxylR (7,11,24) together with the sigma factor NtrA (5). The lower pathway enzymes for the metabolism of catechol are encoded by the xylXYZLTEGFJQKIH operon (9) and regulated by xylS (6,10,19,24). Transcription of xylS is also mediated by xylR and the NtrA sigma factor, which results in overproduction of xylS and subsequent transcription of the meta-cleavage pathway operon in the absence of meta-cleavage intermediates (19). The enzymes of the upper pathway also exhibit broad substrate specificity, transforming not only toluene and xylene but also ethyl-, methyl-and chloro-substituted toluene (1).P. putida Fl also metabolizes toluene; however, the first step in the pathway is the transformation of toluene to cis-toluene dihydrodiol, followed by conversion to 3-methylcatechol, which undergoes meta cleavage. The genetic organization (29, 30) and biochemistry (8,20,27) of the enzymes responsible for the catabolism of toluene to 3-methylcatechol have been studied extensively. The toluene 2,3-dioxygenase of P. putida Fl metabolizes a wide range of hydrocarbon...

Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.

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