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The occurrence of NADP + -dependent malic enzymes (NADP + -MEs) in several Rhodococcus strains was analysed. The NADP + -ME number in Rhodococcus genomes seemed to be a strain-dependent property. Total NADP + -ME activity increased by 1.8- and 2.6-fold in the oleaginous Rhodococcus jostii RHA1 and Rhodococcus opacus PD630 strains during cultivation under nitrogen-limiting conditions. Total NADP + -ME activity inhibition by sesamol resulted in a significant decrease of the cellular biomass and lipid production in oleaginous rhodococci. A non-redundant ME coded by the RHA1_RS44255 gene located in a megaplasmid (pRHL3) of R. jostii RHA1 was characterized and its heterologous expression in Escherichia coli resulted in a twofold increase in ME activity in an NADP + -dependent manner. The overexpression of RHA1_RS44255 in RHA1 and PD630 strains grown on glucose promoted an increase in total NADP + -ME activity and an up to 1.9-foldincrease in total fatty acid production without sacrificing cellular biomass. On the other hand, its expression in Rhodococcus fascians F7 grown on glycerol resulted in a 1.3-1.4-foldincrease in total fatty acid content. The results of this study confirmed the contribution of NADP + -MEs to TAG accumulation in oleaginous rhodococci and the utility of these enzymes as an alternative approach to increase bacterial oil production from different carbon sources.

A previously identified sterol catabolic gene cluster is widely dispersed among actinobacteria, enabling them to degrade and grow on naturally occurring sterols. We investigated the physiological roles of various genes by targeted inactivation in mutant RG32 of Rhodococcus rhodochrous, which selectively degrades sterol side-chains. The ltp3 and ltp4 deletion mutants were each completely blocked in side-chain degradation of β-sitosterol and campesterol, but not of cholesterol. These results indicated a role for ltp3 and ltp4 in the removal of C24 branches specifically. Bioinformatic analysis of the encoded Ltp3 and Ltp4 proteins revealed relatively high similarity to thiolase enzymes, typically involved in β-oxidation, but the catalytic residues characteristic for thiolase enzymes are not conserved in their amino acid sequences. Removal of the C24-branched side-chain carbons of β-sitosterol was previously shown to proceed via aldolytic cleavage rather than by β-oxidation. Our results therefore suggest that ltp3 and ltp4 probably encode aldol-lyases rather than thiolases. This is the first report, to our knowledge, on the molecular characterization of genes with specific and essential roles in carbon-carbon bond cleavage of C24-branched chain sterols in Rhodococcus strains, most likely acting as aldol-lyases. The results are a clear contribution to our understanding of sterol degradation in actinobacteria.

The Rhodococcus erythropolis strain W2 has been shown previously to degrade the N-acylhomoserine lactone (AHL) quorum-sensing signal molecule N-hexanoyl-L-homoserine lactone, produced by other bacteria. Data presented here indicate that this Gram-positive bacterium is also capable of using various AHLs as the sole carbon and energy source. The enzymic activities responsible for AHL inactivation were investigated in R. erythropolis cell extracts and in whole cells. R. erythropolis cells rapidly degraded AHLs with 3-oxo substituents but exhibited relatively poor activity against the corresponding unsubstituted AHLs. Investigation of the mechanism(s) by which R. erythropolis cells degraded AHLs revealed that 3-oxo compounds with N-acyl side chains ranging from C8 to C14 were initially converted to their corresponding 3-hydroxy derivatives. This oxidoreductase activity was not specific to 3-oxo-AHLs but also allowed the reduction of compounds such as N-(3-oxo-6-phenylhexanoyl)homoserine lactone (which contains an aromatic acyl chain substituent) and 3-oxododecanamide (which lacks the homoserine lactone ring). It also reduced both the D- and L-isomers of n-(3-oxododecanoyl)-L-homoserine lactone. A second AHL-degrading activity was observed when R. erythropolis cell extracts were incubated with N-(3-oxodecanoyl)-L-homoserine lactone (3O,C10-HSL). This activity was both temperature- and pH-dependent and was characterized as an amidolytic activity by HPLC analysis of the reaction mixture treated with dansyl chloride. This revealed the accumulation of dansylated homoserine lactone, indicating that the 3O,C10-HSL amide had been cleaved to yield homoserine lactone. R. erythropolis is therefore capable of modifying and degrading AHL signal molecules through both oxidoreductase and amidolytic activities.

The Gram-positive bacteria Rhodococcus opacus ISO-5 and Rhodococcus sp. RHA1 utilized taurine (2-aminoethanesulfonate) as the sole source of carbon or of nitrogen or of sulfur for growth. Different gene clusters and enzymes were active under these different metabolic situations. Under carbon- or nitrogen-limited conditions three enzymes were induced, though to different levels: taurine-pyruvate aminotransferase (Tpa), alanine dehydrogenase (Ald) and sulfoacetaldehyde acetyltransferase (Xsc). The specific activities of these enzymes in R. opacus ISO-5 were sufficient to explain the growth rates under the different conditions. These three enzymes were purified and characterized, and the nature of each reaction was confirmed. Analyses of the genome of Rhodococcus sp. RHA1 revealed a gene cluster, tauR-ald-tpa, putatively encoding regulation and oxidation of taurine, located 20 kbp from the xsc gene and separate from two candidate phosphotransacetylase (pta) genes, as well as many candidate ABC transporters (tauBC). PCR primers allowed the amplification and sequencing of the tauR-ald-tpa gene cluster and the xsc gene in R. opacus ISO-5. The N-terminal sequences of the three tested proteins matched the derived amino acid sequences of the corresponding genes. The sequences of the four genes found in each Rhodococcus strain shared high degrees of identity (>95 % identical positions). RT-PCR studies proved transcription of the xsc gene when taurine was the source of carbon or of nitrogen. Under sulfur-limited conditions no xsc mRNA was generated and no Xsc was detected. Taurine dioxygenase (TauD), the enzyme catalysing the anticipated desulfonative reaction when taurine sulfur is assimilated, was presumed to be present because oxygen-dependent taurine disappearance was demonstrated with taurine-grown cells only. A putative tauD gene (with three other candidates) was detected in strain ISO-5. Regulation of the different forms of metabolism of taurine remains to be elucidated.

Aromatic-ring-hydroxylating dioxygenases (ARHDOs) are key enzymes in the aerobic bacterial metabolism of aromatic compounds. They are of biotechnological importance as they function as biocatalysts in the stereospecific synthesis of chiral synthons and the degradation of aromatic pollutants. This report describes the development and validation of a system for the rapid isolation and characterization of specific ARHDO activities. The system is based on the identification of ARHDO gene segments that encode the enzymes' major functional determinants, on consensus primers for the direct amplification of such partial genes and on a 'recipient' ARHDO gene cluster for the insertion of the amplified segments. Previously, it has been shown that neither the N- nor the C-terminal portions but only the core region of the large or alpha-subunit of a class II ARHDO significantly influence substrate and product spectra. On the basis of these observations, consensus primers were designed for the amplification of the gene segment encoding the catalytic core of the large subunit. These primers were tested on 11 bacterial isolates known to metabolize aromatic compounds. In 10 cases, a gene fragment of expected length was amplified. DNA sequencing confirmed similarity to ARHDO alpha-subunit gene cores. The heterologously well-expressible bphA gene cluster of Burkholderia sp. strain LB400 was modified to facilitate the in-frame insertion of amplified segments. It was used successfully to express the resulting hybrid gene clusters and to form catalytically active chimaeric ARHDOs. The metabolic properties of these enzymes differed significantly from each other and from the parental ARHDO of strain LB400. These results indicate that the system described here can be used to rapidly isolate and functionally characterize ARHDO activities, starting from isolated strains, mixtures of organisms or samples of nucleic acids. Applications of the system range from the recruitment of novel ARHDO activities to an improved characterization of natural ARHDO diversity.

Previously, Rhodococcus erythropolis SQ1 kstD, encoding ketosteroid Delta(1)-dehydrogenase (KSTD1) was characterized. Surprisingly, a kstD gene deletion mutant (strain RG1) grew normally on steroids. UV mutagenesis of strain RG1 allowed isolation of strains (e.g. strain RG1-UV29) unable to perform the Delta(1)-dehydrogenation of 4-androstene-3,17-dione (AD) and 9alpha-hydroxy-4-androstene-3,17-dione (9OHAD). Functional complementation of strain RG1-UV29 with total genomic DNA of strain RG1 resulted in identification of a 1698 nt ORF (kstD2) showing clear similarity (35% identity at amino acid sequence level) with KSTD1. Expression of kstD2 in Escherichia coli resulted in high KSTD2 activity levels. Single gene deletion mutants of either kstD (strain RG1) or kstD2 (strain RG7) appeared unaffected in growth on the steroid substrates AD, 1,4-androstadiene-3,17-dione and 9OHAD. Strain RG7, but not strain RG1, showed temporary accumulation of 9OHAD during AD conversion. A kstD kstD2 double deletion mutant (strain RG8) was constructed. Strain RG8 was unable to grow on steroid substrates, had undetectable steroid Delta(1)-dehydrogenation activity and efficiently converted AD into 9OHAD. Strain SQ1 thus employs two KSTD isoenzymes in steroid catabolism. Analysis of two null mutants in KSTD2 showed that the semi-conserved Ser325 and the highly conserved Thr503 play a role in KSTD enzyme activity.

Protein engineering by segment exchange was used to distinguish between regions of major and minor influence on the structure of the substrate-binding pocket of a biphenyl dioxygenase (BDO). Eight chimaeric enzyme systems were generated that each consisted of a hybrid hydroxylase alpha subunit (BphA1) containing segments from Burkholderia sp. strain LB400 and Rhodococcus globerulus P6, and of a hydroxylase beta subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400. All hybrid bphA1 genes were expressed at high levels. Seven of the resulting fusion subunits functionally interacted with the other polypeptides of the dioxygenase system to yield catalytically active enzymes. Changes in the regiospecificity of substrate attack, monitored by the formation of seventeen different dioxygenation products obtained from seven chlorobiphenyls, were used to monitor effects of segment exchanges on the structure of the BDO substrate-binding site. Exchanges of neither the beta subunit nor the N- and C-terminal regions of the alpha subunit exerted significant influences. All BDO regions that showed major effects on the substrate-binding pocket were located between approximately positions 165 and 395 of the alpha subunit. Within this part of the enzyme, in addition to segments identified previously, a subregion which is involved in ligation of the mononuclear iron significantly influenced the regiospecificity of substrate dioxygenation. Moreover, the results indicate that the construction of appropriate hybrid genes may be used as a general strategy to overcome problems in obtaining heterologous BDO activities in Escherichia coli or other host organisms.

Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol (picric acid) or 2,4-dinitrophenol (2,4-DNP) as sole nitrogen source. A gene cluster involved in picric acid degradation was recently identified. The functional assignment of three of its genes, npdC, npdG and npdI, and the tentative functional assignment of a fourth one, npdH, is reported. The genes were expressed in Escherichia coli as His-tag fusion proteins that were purified by Ni-affinity chromatography. The enzyme activity of each protein was determined by spectrophotometry and HPLC analyses. NpdI, a hydride transferase, catalyses a hydride transfer from reduced F420 to the aromatic ring of picric acid, generating the hydride sigma-complex (hydride Meisenheimer complex) of picric acid (H(-)-PA). Similarly, NpdI also transformed 2,4-DNP to the hydride sigma-complex of 2,4-DNP. A second hydride transferase, NpdC catalysed a subsequent hydride transfer to H(-)-PA, to produce a dihydride sigma-complex of picric acid (2H(-)-PA). All three reactions required the activity of NpdG, an NADPH-dependent F420 reductase, for shuttling the hydride ions from NADPH to F420. NpdH converted 2H(-)-PA to a hitherto unknown product, X. The results show that npdC, npdG and npdI play a key role in the initial steps of picric acid degradation, and that npdH may prove to be important in the later stages.

Rhodococcus erythropolis DCL14 assimilates all stereoisomers of carveol and dihydrocarveol as sole source of carbon and energy. Induction experiments with carveol- or dihydrocarveol-grown cells showed high oxygen consumption rates with these two compounds and with carvone and dihydrocarvone. (Dihydro)carveol-grown cells of R. erythropolis DCL14 contained the following enzymic activities involved in the carveol and dihydrocarveol degradation pathways of this micro-organism: (dihydro)carveol dehydrogenase (both NAD+- and dichlorophenolindophenol-dependent activities), an unknown cofactor-dependent carvone reductase, (iso-)dihydrocarvone isomerase activity, NADPH-dependent dihydrocarvone monooxygenase (Baeyer-Villiger monooxygenase), epsilon-lactone hydrolase and an NAD+-dependent 6-hydroxy-3-isopropenylheptanoate dehydrogenase. Product accumulation studies identified (4R)-carvone, (1R,4R)-dihydrocarvone, (4R,7R)-4-isopropenyl-7-methyl-2-oxo-oxepanone, (3R)-6-hydroxy-3-isopropenylheptanoate, (3R)-3-isopropenyl-6-oxoheptanoate, (3S,6R)-6-isopropenyl-3-methyl-2-oxooxepanone and (5R)-6-hydroxy-5-isopropenyl-2-methylhexanoate as intermediates in the (4R)-carveol degradation pathway. The opposite stereoisomers of these compounds were identified in the (4S)-carveol degradation pathway. With dihydrocarveol, the same intermediates are involved except that carvone was absent. These results show that R. erythropolis DCL14 metabolizes all four diastereomers of carveol via oxidation to carvone, which is subsequently stereospecifically reduced to (1R)-(iso-) dihydrocarvone. At this point also dihydrocarveol enters the pathway, and this compound is directly oxidized to (iso-)dihydrocarvone. Cell extracts contained both (1R)-(iso-)dihydrocarvone 1,2-monooxygenase and (1S)-(iso)-dihydrocarvone 2,3-monooxygenase activity, resulting in a branch point of the degradation pathway; (1R)-(iso-)dihydrocarvone was converted to 4-isopropenyl-7-methyl-2-oxo-oxepanone, while (1S)-(iso)-dihydrocarvone, which in vivo is isomerized to (1R)-(iso-)dihydrocarvone, was converted to 6-isopropenyl-3-methyl-2-oxo-oxepanone. 4-Isopropenyl-7-methyl-2-oxooxepanone is hydrolysed to 6-hydroxy-3-isopropenylheptanoate, which is subsequently oxidized to 3-isopropenyl-6-oxoheptanoate, thereby linking the (dihydro)carveol degradation pathways to the limonene degradation pathway of this micro-organism. 6-Isopropenyl-3-methyl-2-oxo-oxepanone is, in vitro, hydrolysed to 6-hydroxy-5-isopropenyl-2-methylhexanoate, which is thought to be a dead-end metabolite.

Extracts from benzyl-alcohol-grown Rhodococcus erythropolis DSM 1069 showed NAD(P)-independent, N,N-dimethyl-4-nitrosoaniline (NDMA)-dependent alcohol dehydrogenase activity. The enzyme exhibiting this activity was purified to homogeneity and characterized. It appears to be a typical nicotinoprotein as it contains tightly bound NADH acting as cofactor instead of coenzyme. Other characteristics indicate that it is highly similar to the known nicotinoprotein alcohol dehydrogenase (np-ADH) from Amycolatopsis methanolica: it is a homotetramer of 150 kDa; N-terminal amino acid sequencing (22 residues) showed that 77% of these amino acids are identical in the two enzymes; it has optimal activity at pH 7.0; it lacks NAD(P)H-dependent aldehyde reductase activity; it catalyses the oxidation of a broad range of (preferably) primary and secondary alcohols, either aliphatic or aromatic, and formaldehyde, with the concomitant reduction of the artificial electron acceptor NDMA. NDMA could be replaced by an aldehyde, but not formaldehyde, the substrate specificity of the enzyme for the aldehydes reflecting that for the corresponding alcohols. The latter also applied to the low aldehyde dismutase activity displayed by the enzyme. From this, together with the results of the induction studies, it is concluded that np-ADH functions as the main alcohol-oxidizing enzyme in the dissimilation of many, but not all, alcohols by R. erythropolis and may also catalyse coenzyme-independent interconversion of alcohols and aldehydes under certain circumstances. It is anticipated that the enzyme may be of even wider significance since structural data indicate that np-ADH is also present in other (nocardioform) actinomycetes.

Rhodococcus corallinus (formerly Nocardia corallina) B-276, isolated with propene as sole carbon and energy source, is able to oxidize trichloroethene (TCE). Glucose- or propene-grown R. corallinus B-276 cells exhibited no difference in TCE degradation efficiency. TCE degradation was found to be growth-phase-dependent and maximum rates were monitored with stationary-phase cells. K(m) and Vmax values for TCE degradation of R. corallinus B-276 grown in nutrient broth medium in the presence of glucose were 187 microM and 2.4 nmol min-1 (mg protein)-1, respectively. Escherichia coli recombinants harbouring and expressing the alkene monooxygenase genes of R. corallinus B-276 exhibited the ability to degrade TCE. This result provides clear evidence that the alkene monooxygenase of R. corallinus B-276 catalyses TCE oxidation. R. corallinus B-276 was shown to contain four linear plasmids, pNC10 (70 kb), pNC20 (85 kb), pNC30 (185 kb) and pNC40 (235 kb). The observation that pNC30-deficient strains had lost the ability to grow on propene suggested that the genes of the propene degradation pathway are encoded by the linear plasmid pNC30. Southern blot analysis with cloned alkene monooxygenase genes from R. corallinus B-276 revealed a positive hybridization signal with the linear plasmid pNC30. This result clearly shows that the alkene monooxygenase is encoded by the linear plasmid pNC30. Eleven short-chain-alkene-oxidizing strains were screened for the presence of linear plasmids. Among these, four propene-oxidizing Rhodococcus strains and one ethene-oxidizing Mycobacterium strain were found to contain linear megaplasmids. Southern blot analysis with the alkene monooxygenase revealed positive signals with linear plasmids of two propene-oxidizing Rhodococcus ruber strains. These results indicate that homologous alkene monooxygenases are encoded by linear plasmids in R. ruber strains.

Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined. A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis. Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases. The EdoA and EdoB dioxygenases were classified as belonging to the third family of type I oxygenases and represented two new subfamilies, whereas the EdoD dioxygenase was a type II enzyme. Analysis of six Rhodococcus strains revealed a wide distribution of the above dioxygenase genes. Rhodococcus sp. 11 was shown to harbour all four of the analysed dioxygenase genes. Nucleotide sequences homologous to the edoB gene were present in all of the strains, including R. erythropolis NCIMB 13065, which did not utilize any of the aromatic compounds analysed. The latter finding points to the existence of a silent pathway(s) for degradation of aromatic compounds in this Rhodococcus strain.

Rhodococcus sp. strain IGTS8 (ATCC 53968) is able to utilize dibenzothiophene (DBT) as a sole source of sulphur. The carbon skeleton of DBT is not metabolized and is conserved as 2-hydroxybiphenyl (HBP), which accumulates in the medium. This phenotype is due to the expression of the plasmid-encoded DBT-desulphurization (dsz) operon, which encodes three proteins, DszA, B and C. In this paper it is shown, using [35S]DBT radiolabelling studies, that sulphur is released in the form of inorganic sulphite. The pathway of DBT desulphurization is described in detail. In summary, DszC catalyses the stepwise S-oxidation of DBT, first to dibenzothiophene 5-oxide (DBTO) and then to dibenzothiophene 5,5-dioxide (DBTO2); DszA catalyses the conversion of DBTO2 to 2-(2'-hydroxyphenyl)benzene sulphinate (HBPSi-) and DszB catalyses the desulphination of HBPSi- to give HBP and sulphite. Studies with cell-free extracts show that DszA and DszC, but not DszB, require NADH for activity. 18O2-labelling studies show that each incorporated oxygen atom is derived directly from molecular oxygen. These results are consistent with the role of DszC as a mono-oxygenase, of DszA as an apparently unique enzyme which catalyses the reductive hydroxylation of DBTO2 leading to cleavage of the thiophene ring, and of DszB as an aromatic sulphinic acid hydrolase.

The dissociation of the soluble NAD-reducing hydrogenase of Rhodococcus opacus MR11 into two dimeric proteins with different catalytic activities and cofactor composition is unique among the NAD-reducing hydrogenases studied so far. The genes of the soluble hydrogenase were localized on a 7.4 kbp Asnl fragment of the linear plasmid pHG201 via heterologous hybridization. Analysis of the nucleotide sequence of this fragment revealed the seven open reading frames ORF1, hoxF, -U, -Y, -H, -W and ORF7. The six latter ORFs belong to the gene cluster of the soluble hydrogenase. Their gene products are highly homologous to those of the NAD-reducing enzyme of Alcaligenes eutrophus H16. The genes hoxF, -U, -Y and -H encode the subunits alpha, gamma, delta and beta, respectively. The gene hoxW encodes a putative protease, which may be essential for C-terminal processing of the beta subunit. Finally, ORF7 encodes a protein which has similarities to cAMP- and cGMP-binding protein kinases, but its function is not known. ORF1, which lies upstream of the hydrogenase gene cluster, encodes a putative transposase found in IS elements of other bacteria. Northern hybridizations and primer extensions using total RNA of autotrophically and heterotrophically grown cells of R. opacus MR11 indicated that the hydrogenase genes are under control of a delta 70-like promoter located at the right end of ORF1 and are even transcribed under heterotrophic conditions at a low level. Furthermore, this promoter was shown to be active in the recombinant Escherichia coli strain LHY1 harbouring the 7.4 kbp Asnl fragment, resulting in overexpression of the hydrogenase genes. Although all four subunits of the soluble hydrogenase were shown via Western immunoblots to be synthesized in E. coli, no active enzyme was detectable.

The haloalkane dehalogenase (dhaA) gene from Rhodococcus rhodochrous NCIMB 13064 was cloned and sequenced. Its comparison with the previously studied dhlA gene from Xanthobacter autotrophicus GJ10 did not show homology. However, the amino acid sequences of the products of these genes showed approximately 30% identity and several of the catalytic amino acid residues were conserved in the NCIMB 13,064 dehalogenase. A high level of dhaA expression was demonstrated in Escherichia coli cells and this gene was shown to encode a dehalogenase with the activity against chloroalkanes of chain length C3-C10. Also, some dehalogenase activity against 1,2-dichloroethane encoded by the cloned dhaA gene was detected. The analysis of NCIMB 13,064 derivatives lacking dehalogenase activity showed that the dhaA gene was located on the 100 kbp pRTL1 plasmid. It was also found that reversible rearrangements of DNA in the dhaA region may be responsible for the control of expression of haloalkane dehalogenase in R. rhodochrous NCIMB 13064. A number of repeated and inverted sequences which may cause genetic instability at the locus were found in the haloalkane dehalogenase gene region.

The enzymes responsible for the degradation of isopropylbenzene (IPB) and co-oxidation of trichloroethene (TCE) by Rhodococcus erythropolis BD2 are encoded by the linear plasmid pBD2. Fragments containing IPB catabolic genes were cloned from pBD2 and the nucleotide sequence was determined. By means of database searches and expression of the cloned genes in recombinant strains, we identified five clustered genes, ipbA1A2A3A4C, which encode the three components of the IPB 2,3-dioxygenase system, reductaseIPB (ipbA4), ferredoxinIPB (ipbA3) and the two subunits of the terminal dioxygenase (ipbA1A2), as well as the 3-isopropylcatechol (IPC) 2,3-dioxygenase (ipbC). The protein sequences deduced from the ipbA1A2A3A4C gene cluster exhibited significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative and Gram-positive bacteria, but the gene order differed from most of them. IPB 2,3-dioxygenase and 3-IPC 2,3-dioxygenase could both be expressed in Escherichia coli, but the IPB 2,3-dioxygenase activities were too low to be detected by polarographic and TCE degradative means. However, inhibitor studies with the R. erythropolis BD2 wild-type are in accordance with the involvement of the IPB 2,3-dioxygenase in TCE oxidation.

In cell-free extracts of Escherichia coli clones harbouring the 3.5 kb Bg/II fragment of plasmid pTC1 from Rhodococcus rhodochrous CTM a catechol 2,3-dioxygenase (C23O) accepting both 3-methylcatechol and 2,3-dihydroxybiphenyl as substrates could be detected. The plasmid-encoded gene for C23O of R. rhodochrous CTM and its flanking regions were sequenced. In front of the gene a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The derived amino acid sequence of the C23O was compared to that of nine other enzymes, which all catalyse the extradiol cleavage of an aromatic ring. These nine sequences were from different Pseudomonas strains, in contrast to the sequence described here, from a Gram-positive bacterium. The role of four strongly conserved histidines was examined by chemical modification of the histidyl residues of the native enzyme by diethylpyrocarbonate. For that purpose the C23O was purified to homogeneity from E. coli harbouring pSC1701. However, the enzyme lost its activity during the purification. Activity could partially be restored by treatment with Fe2+ and reducing agents.