Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G؉C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1's 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds (30) or steroids (4). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plantderived compounds in an O2-rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.biodegradation ͉ actinomycete ͉ linear chromosome ͉ aromatic pathways ͉ oxygenase

Cleavage of the ␤-aryl ether linkage is the most important process in lignin degradation. Here we characterize the three tandemly located glutathione S-transferase (GST) genes, ligF, ligE, and ligG, from lowmolecular-weight lignin-degrading Sphingomonas paucimobilis SYK-6, and we describe the actual roles of these genes in the ␤-aryl ether cleavage. Based on the identification of the reaction product by electrospray ionization-mass spectrometry, a model compound of ␤-aryl ether, ␣-(2-methoxyphenoxy)-␤-hydroxypropiovanillone (MPHPV), was transformed by LigF or LigE to guaiacol and ␣-glutathionyl-␤-hydroxypropiovanillone (GS-HPV). This result suggested that LigF and LigE catalyze the nucleophilic attack of glutathione on the carbon atom at the ␤ position of MPHPV. High-pressure liquid chromatography-circular dichroism analysis indicated that LigF and LigE each attacked a different enantiomer of the racemic MPHPV preparation. The ligG gene product specifically catalyzed the elimination of glutathione from GS-HPV generated by the action of LigF. This reaction then produces an achiral compound, ␤-hydroxypropiovanillone, which is further degraded by this strain. Disruption of the ligF, ligE, and ligG genes in SYK-6 showed that ligF is essential to the degradation of one of the MPHPV enantiomers, and the alternative activities which metabolize the substrates of LigE and LigG are present in this strain.

gamma-Hexachlorocyclohexane (gamma-HCH; also called BHC or lindane) is one of the highly chlorinated pesticides which can cause serious environmental problems. Sphingomonas paucimobilis UT26 degrades gamma-HCH under aerobic conditions. The unique degradation pathway of gamma-HCH in UT26 is revealed. In the upstream pathway, gamma-HCH is transformed to 2,5-dichlorohydroquinone (2,5-DCHQ) by two different dehalogenases (LinA and LinB) and one dehydrogenase (LinC) which are expressed constitutively. In the downstream pathway, 2,5-DCHQ is reductively dehalogenated, and then ring-cleaved by enzymes (LinD and LinE, respectively) whose expressions are regulated. We have cloned and sequenced five structural genes (linA, linB, linC, linD, and linE) directly involved in this degradation pathway. The linD and linE genes form an operon, and its expression is positively regulated by the LysR-type transcriptional regulator (LinR). The genes linA, linB, and linC are constitutively expressed, and are present separately from each other in the UT26 genome. Cell fractionation analysis, Western blotting, and immuno electron microscopy revealed that LinA and LinB are localized in the periplasmic space of UT26.

No abstract

The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of 2,4-Dichlorophenoxyacetic acid (2,4-D) is a manufactured herbicide that has been widely used for the control of broadleaf weeds since its introduction in the 1940s. Many 2,4-Ddegrading microorganisms have been isolated from agricultural, urban, and industrial soils and sediments (2,3,9,22,30,50), and the catabolic pathway of 2,4-D mineralization in Ralstonia eutropha JMP134 has been extensively characterized (8-10, 14, 19, 25, 26, 32-35, 38, 41-43, 48). In JMP134, 2,4-D is transformed to 2,4-dichlorophenol (2,4-DCP) by ␣-ketoglutarate-dependent 2,4-D dioxygenase encoded by tfdA, and 2,4-DCP is subsequently hydroxylated by 2,4-DCP hydroxylase encoded by tfdB to form 3,5-dichlorocatechol (3,5-DCC). 3,5-DCC is further metabolized through an intradiol ring cleavage pathway encoded by tfdCDEF (Fig. 1). These genes are located on plasmid pJP4.Most 2,4-D-degrading bacteria isolated from human-disturbed sites contain tfdA gene homologs. They include various copiotrophic, fast-growing genera in the ␤ and ␥ subdivisions of the Proteobacteria and have been classified as class I 2,4-D degraders (24). Ka et al. reported another group of 2,4-D degraders (class II) that were also isolated from disturbed sites but have neither tfdA gene homologs nor ␣-ketoglutarate-dependent 2,4-D dioxygenase activity (21-23). This group is composed of copiotrophic, fast-growing strains in the ␣ subdivision of the Proteobacteria, mostly belonging to the genus Sphingomonas. Fulthorpe et al. (17) and Kamagata et al. (24) isolated 2,4-D degraders from noncontaminated, pristine soils, degraders which have neither tfdA gene homologs nor ␣-ketoglutarate-dependent 2,4-D dioxygenase activity and, in contrast to those of the other two classes, grow slowly. This group of 2,4-D degraders (class III) is affiliated with the Bradyrhizobium-Agromyces-Nitrobacter-Afipia cluster (A. Saitou, H. Mitsui, and T. Hattori, Abstr. 11th Meet. Jpn. Soc. Microb. Ecol., p. 26, 1995) of oligotrophic bacteria in the ␣ subdivision of the Proteobacteria. The existence of three distinct ecological and genetic classes of 2,4-D degraders indicates a diversity of 2,4-D degradation genes and perhaps of pathways among 2,4-D degraders. However, the 2,4-D ...