Degradation of alkanes and highly chlorinated benzenes, and production of biosurfactants, by a psychrophilic Rhodococcus sp. and genetic characterization of its chlorobenzene dioxygenase

Rhodococcus sp. strain MS11 was isolated from a mixed culture. It displays a diverse range of metabolic capabilities. During growth on 1,2,4-trichlorobenzene, 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB) and 3-chlorobenzoate stoichiometric amounts of chloride were released. It also utilized all three isomeric dichlorobenzenes and 1,2,3-trichlorobenzene as the sole carbon and energy source. Furthermore, the bacterium grew well on a great number of n-alkanes ranging from n-heptane to n-triacontane and on the branched alkane 2,6,10,14-tetramethylpentadecane (pristane) and slowly on n-hexane and n-pentatriacontane. It was able to grow at temperatures from 5 to 30 °C, with optimal growth at 20 °C, and could tolerate 6 % NaCl in mineral salts medium. Genes encoding the initial chlorobenzene dioxygenase were detected by using a primer pair that was designed against the α-subunit (TecA1) of the chlorobenzene dioxygenase of Ralstonia (formerly Burkholderia) sp. strain PS12. The amino acid sequence of the amplified part of the α-subunit of the chlorobenzene dioxygenase of Rhodococcus sp. strain MS11 showed >99 % identity to the α-subunit of the chlorobenzene dioxygenase from Ralstonia sp. strain PS12 and the parts of both α-subunits responsible for substrate specificity were identical. The subsequent enzymes dihydrodiol dehydrogenase and chlorocatechol 1,2-dioxygenase were induced in cells grown on 1,2,4,5-TeCB. During cultivation on medium-chain-length n-alkanes ranging from n-decane to n-heptadecane, including 1-hexadecene, and on the branched alkane pristane, strain MS11 produced biosurfactants lowering the surface tension of the cultures from 72 to ⩽29 mN m−1. Glycolipids were extracted from the supernatant of a culture grown on n-hexadecane and characterized by 1H- and 13C-NMR-spectroscopy and mass spectrometry. The two major components consisted of α,α-trehalose esterified at C-2 or C-4 with a succinic acid and at C-2′ with a decanoic acid. They differed from one another in that one 2,3,4,2′-trehalosetetraester, found in higher concentration, was esterified at C-2, C-3 or C-4 with one octanoic and one decanoic acid and the other one, of lower concentration, with two octanoic acids. The results demonstrate that Rhodococcus sp. strain MS11 may be well suited for bioremediation of soils and sediments contaminated for a long time with di-, tri- and tetrachlorobenzenes as well as alkanes.

Biotransformation of Various Substituted Aromatic Compounds to Chiral Dihydrodihydroxy Derivatives

ABSTRACT The biotransformation of four different classes of aromatic compounds by the Escherichia coli strain DH5α(pTCB 144), which contained the chlorobenzene dioxygenase (CDO) fromPseudomonas sp. strain P51, was examined. CDO oxidized biphenyl as well as monochlorobiphenyls to the correspondingcis-2,3-dihydro-2,3-dihydroxy derivatives, whereby oxidation occurred on the unsubstituted ring. No higher substituted biphenyls were oxidized. The absolute configurations of several monosubstituted cis-benzene dihydrodiols formed by CDO were determined. All had an S configuration at the carbon atom in meta position to the substituent on the benzene nucleus. With one exception, the enantiomeric excess of several 1,4-disubstituted cis-benzene dihydrodiols formed by CDO was higher than that of the products formed by two toluene dioxygenases. Naphthalene was oxidized to enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. All absolute configurations were identical to those of the products formed by toluene dioxygenases of Pseudomonas putida UV4 and P. putida F39/D. The formation rate of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene was significantly higher (about 45 to 200%) than those of several monosubstituted cis-benzene dihydrodiols and more than four times higher than the formation rate of cis-benzene dihydrodiol. A new gas chromatographic method was developed to determine the enantiomeric excess of the oxidation products.

Genetic and Biochemical Analyses of thetec Operon Suggest a Route for Evolution of Chlorobenzene Degradation Genes

ABSTRACT The TecA broad-spectrum chlorobenzene dioxygenase ofBurkholderia sp. strain PS12 catalyzes the first step in the mineralization of 1,2,4,5-tetrachlorobenzene. The catabolic genes were localized on a small plasmid that belongs to the IncPβ incompatibility group. PCR analysis of the genetic environment of thetec genes indicated high similarity to the transposon-organized catabolic tcb chlorobenzene degradation genes of Pseudomonas sp. strain P51. Sequence analysis of the regions flanking the tecA genes revealed an upstream open reading frame (ORF) with high similarity to thetodF 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase gene ofPseudomonas putida F1 and a discontinuous downstream ORF showing high similarity to the todE catechol 2,3-dioxygenase gene of strain F1. Both homologues in strain P51 exist only as deletion remnants. We suggest that different genetic events thus led to inactivation of the perturbing meta-cleavage enzymes in strains P51 and PS12 during the evolution of efficient chlorobenzene degradation pathways. Biochemical characterization of TodF-like protein TlpF and a genetically refunctionalized TodE-like protein, TlpE, produced in Escherichia coli provided data consistent with the proposed relationships.

Identification of Chlorobenzene Dioxygenase Sequence Elements Involved in Dechlorination of 1,2,4,5-Tetrachlorobenzene

ABSTRACT The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities. Escherichia coli cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large α-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of specific todC1α-subunit subsequences by equivalent sequences of thetecA1 α-subunit. Substrate transformation specificities and rates by E. coli resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography. This allowed the identification of both a single amino acid and potentially interacting regions required for dechlorination of tetrachlorobenzene. Hybrids with extended substrate ranges were generated that exhibited activity toward both benzene and tetrachlorobenzene. The regions determining substrate specificity in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.