Dioxygenases induced during benzoate degradation by the actinobacterium Rhodococcus wratislaviensis G10 strain degrading haloaromatic compounds were studied. Rhodococcus wratislaviensis G10 completely degraded 2 g/liter benzoate during 30 h and 10 g/liter during 200 h. Washed cells grown on benzoate retained respiration activity for more than 90 days, and a high activity of benzoate dioxygenase was recorded for 10 days. Compared to the enzyme activities with benzoate, the activity of benzoate dioxygenases was 10-30% with 13 of 35 substituted benzoate analogs. Two dioxygenases capable of cleaving the aromatic ring were isolated and characterized: protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase. Catechol inhibited the activity of protocatechuate 3,4-dioxygenase. Protocatechuate did not affect the activity of catechol 1,2-dioxygenase. A high degree of identity was shown by MALDI-TOF mass spectrometry for protein peaks of the R. wratislaviensis G10 and Rhodococcus opacus 1CP cells grown on benzoate or LB. DNA from the R. wratislaviensis G10 strain was specifically amplified using specific primers to variable regions of genes coding �\- and �]-subunits of protocatechuate 3,4-dioxygenase and to two genes of the R. opacus 1CP coding catechol 1,2-dioxygenase. The products were 99% identical with the corresponding regions of the R. opacus 1CP genes. This high identity (99%) between the genes coding degradation of aromatic compounds in the R. wratislaviensis G10 and R. opacus 1CP strains isolated from sites of remote location (1400 km) and at different time (20-year difference) indicates a common origin of biodegradation genes of these strains and a wide distribution of these genes among rhodococci.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants. Biphenyl 2,3-dioxygenase (BDO) is a key enzyme that determines the range of PCBs oxidized by a bacterial strain. BDO subunit �\ (BphA1) plays an essential role in substrate recognition and binding. The genes for dioxygenases that hydroxylate aromatic rings were screened and analyzed phylogenetically. Genes found in biphenyl-oxidizing Rhodococcus erythropolis strains G12a, B7b, and B106a proved to be similar to the published nucleotide sequences of the Rhodococcus sp. HA99 and R04 and Novosphingobium aromaticivorans F199 bphA1 genes, which code for the �\-subunits that do not belong to the biphenyl/toluene dioxygenase (B/TDO) family. PCB-destructing R. ruber P25 was found to possess a unique bphA1 gene, which clusters together with the phenylpropionate dioxygenase (PPDO) �\-subunits of Mycobacterium vanbaalenii PYR-1 and Frankia sp. EuI1c. The deduced amino acid sequences of the genes were analyzed. The amino acids of the BDO active site in R. wratislaviensis P1, P12, P13, and P20 (bphA1 genes of the B/TDO family) were identical to those of the active PCB degrader R. jostii RHA1. The Rhodococcus strains in question were shown to be active toward both orthoand parachlorinated ring of 2,4'-dichlorobiphenyl. The �\-subunit amino acids responsible for the substrate specificity of the enzyme in Pseudomonas sp. S9, S13, S210, S211, and S212 (B/TDO family) were the same as in P. pseudoalcaligenes KF707. The Pseudomonas strains were active toward the para-chlorinated ring of 2,4'-dichlorobiphenyl. The results of screening bacterial strains for bphA1 can be used to identify the biotechnologically promising PCB destructors.

Naturally occurring and anthropogenic petroleum hydrocarbons are potential carbon sources for many bacteria. The AlkB-related alkane hydroxylases, which are integral membrane non-heme iron enzymes, play a key role in the microbial degradation of many of these hydrocarbons. Several members of the genus Rhodococcus are well-known alkane degraders and are known to harbor multiple alkB genes encoding for different alkane 1-monooxygenases. In the present study, 48 Rhodococcus strains, representing 35 species of the genus, were investigated to find out whether there was a dominant type of alkB gene widespread among species of the genus that could be used as a phylogenetic marker. Phylogenetic analysis of rhodococcal alkB gene sequences indicated that a certain type of alkB gene was present in almost every member of the genus Rhodococcus. These alkB genes were common in a unique nucleotide sequence stretch absent from other types of rhodococcal alkB genes that encoded a conserved amino acid motif: WLG(I/V/L)D(G/D)GL. The sequence identity of the targeted alkB gene in Rhodococcus ranged from 78.5 to 99.2% and showed higher nucleotide sequence variation at the inter-species level compared to the 16S rRNA gene (93.9-99.8%). The results indicated that the alkB gene type investigated might be applicable for: (i) differentiating closely related Rhodococcus species, (ii) properly assigning environmental isolates to existing Rhodococcus species, and finally (iii) assessing whether a new Rhodococcus isolate represents a novel species of the genus.