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Based on structural, biochemical, and genetic data, the soluble diiron monooxygenases can be divided into four groups: the soluble methane monooxygenases, the Amo alkene monooxygenase of Rhodococcus corallinus B-276, the phenol hydroxylases, and the fourcomponent alkene/aromatic monooxygenases. The limited phylogenetic distribution of these enzymes among bacteria, together with available genetic evidence, indicates that they have been spread largely through horizontal gene transfer. Phylogenetic analyses reveal that the K-and L-oxygenase subunits are paralogous proteins and were derived from an ancient gene duplication of a carboxylate-bridged diiron protein, with subsequent divergence yielding a catalytic K-oxygenase subunit and a structural L-oxygenase subunit. The oxidoreductase and ferredoxin components of these enzymes are likely to have been acquired by horizontal transfer from ancestors common to unrelated diiron and Rieske center oxygenases and other enzymes. The cumulative results of phylogenetic reconstructions suggest that the alkene/aromatic monooxygenases diverged first from the last common ancestor for these enzymes, followed by the phenol hydroxylases, Amo alkene monooxygenase, and methane monooxygenases.

The ecology of hydrocarbon degradation by microbial populations in the natural environment is reviewed, emphasizing the physical, chemical, and biological factors that contribute to the biodegradation of petroleum and individual hydrocarbons. Rates of biodegradation depend greatly on the composition, state, and concentration of the oil or hydrocarbons, with dispersion and emulsification enhancing rates in aquatic systems and absorption by soil particulates being the key feature of terrestrial ecosystems. Temperature and oxygen and nutrient concentrations are important variables in both types of environments. Salinity and pressure may also affect biodegradation rates in some aquatic environments, and moisture and pH may limit biodegradation in soils. Hydrocarbons are degraded primarily by bacteria and fungi. Adaptation by prior exposure of microbial communities to hydrocarbons increases hydrocarbon degradation rates. Adaptation is brought about by selective enrichment of hydrocarbon-utilizing microorganisms and amplification of the pool of hydrocarbon-catabolizing genes. The latter phenomenon can now be monitored through the use of DNA probes. Increases in plasmid frequency may also be associated with genetic adaptation. Seeding to accelerate rates of biodegradation has been shown to be effective in some cases, particularly when used under controlled conditions, such as in fermentors or chemostats.

We have studied the appearance of whole-cell oxidizing activity for n-alkanes and their oxidation products in strains of Pseudomonas putida carrying the OCT plasmid. Our results indicate that the OCT plasmid codes for inducible alkane-hydroxylating and primary alcohol-dehydrogenating activities and that the chromosome codes for constitutive oxidizing activities for primary alcohols, aliphatic aldehydes, and fatty acids. Mutant isolation confirms the presence of an alcohol dehydrogenase locus on the OCT plasmid and indicated the presence of multiple alcohol and aldehyde dehydrogenase loci on the P. putida chromosome. Induction tests with various compounds indicate that inducer recognition has specificity for chain length and can be affected by the degree of oxidation of the carbon chain. Some inducers are neither growth nor respiration substrates. Growth tests with and without a gratuitous inducer indicate that undecane is not a growth substrate because it does not induce alkane hydroxylase activity. Using a growth test for determining induction of the plasmid alcohol dehydrogenase it is possible to show that heptane induces this activity in hydroxylase-negative mutants. This suggests that unoxidized alkane molecules are the physiological inducers of both plasmid activities.

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