Purification and Properties of p-Hydroxybenzoate Hydroxylases from Rhodococcus Strains.

Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon source. The PHBH enzymes were purified to apparent homogeneity and appeared to be homodimers of about 95 kD with each subunit containing a relatively weakly bound FAD. In contrast to their counterparts from gram-negative microorganisms, the Rhodococcus PHBH enzymes prefer NADH to NADPH as external electron donor. All purified enzymes were inhibited by Cl– and for five of six enzymes more pronounced substrate inhibition was observed in the presence of chloride ions

Fluorene transformation by bacteria of the genus Rhodococcus

Of the four investigated Rhodococcus strains (R. rhodochrous172, R. opacus 4a and 557, and R. rhodnii 135), the first three strains were found to be able to completely transform fluorene when it was present in the medium as the sole source of carbon at a concentration of 12¿25 mg/l. At a fluorene concentration of 50¿100 mg/l in the medium, the rhodococci transformed 50% of the substrate in 14 days. The addition of casamino acids and sucrose (1¿5 g/l) stimulated fluorene transformation, so that R. rhodochrous 172 could completely transform it in 2¿5 days. Nine intermediates of fluorene transformation were isolated, purified, and structurally characterized. It was found that R. rhodnii 135 and R. opacus strains 4a and 557 hydroxylated fluorene with the formation of 2-hydroxyfluorene and 2,7-dihydroxyfluorene. R. rhodochrous 172 transformed fluorene via two independent pathways to a greater degree than the other rhodococci studied

Reductive deamination as a new step in the anaerobic microbial degradation of halogenated anilines

In this paper we report the isolation and characterization of an anaerobic enrichment culture as well as of a Rhodococcus sp. strain 2 capable of degrading 3,4-dihaloanilines under nitrate reducing conditions. Using mass spectrometry several of the intermediates formed in the process of 3,4-dichloroaniline conversion were identified. Most interesting is the observation of reductive deamination and the formation of 1,2-dichlorobenzene as one of the intermediates. Using 19F NMR and fluorinated 3,4-dihaloaniline model substrates it was corroborated that reductive deamination of the anilines to give dihalobenzene intermediates represents a new initial step in the anaerobic microbial degradation of these halogenated anilines

Identification of fluorpyrogallols as new intermediates in the biotransformation of monofluorphenols in Rh. opacus 1cp

The transformation of monofluorophenols by whole cells of Rhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and 19F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The 19F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2,3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus 1cp