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The complete genome sequencing of a γ-hexachlorocyclohexane-degrading strain, Sphingobium japonicum UT26, revealed that the genome consists of two circular chromosomes [with sizes of 3.5 Mb (Chr1) and 682kb (Chr2)], a 191-kb large plasmid (pCHQ1), and two small plasmids with sizes of 32 and 5kb. The lin genes are dispersed on Chr1, Chr2, and pCHQ1. Comparison of the UT26 genome with those of other sphingomonad strains demonstrated that the "specific"lin genes for conversion of γ-HCH to β-ketoadipate (linA, linB, linC, linRED, and linF) are located on the DNA regions unique to the UT26 genome, suggesting the acquisition of these lin genes by horizontal transfer events. On the other hand, linGHIJ and linKLMN are located on the regions conserved in the genomes of sphingomonads, suggesting that the linGHIJ-encoded β-ketoadipate pathway and the LinKLMN-type ABC transporter system are involved in core functions of sphingomonads. Based on these results, we propose a hypothesis that UT26 was created by recruiting the specific lin genes into a strain having core functions of sphingomonads. Most of the specific lin genes in UT26 are associated with IS6100. Our analysis of spontaneous linA-, linC-, and linRED-deletion mutants of UT26 revealed the involvement of IS6100 in their deduced genome rearrangements. These facts strongly suggest that IS6100 plays important roles both in the dissemination of the specific lin genes and in the genome rearrangements., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A γ-hexachlorocyclohexane (HCH)-degrading bacterium, Sphingomonas sp. MM-1, was isolated from soil contaminated with HCH isomers. Cultivation of MM-1 in the presence of γ-HCH led to the detection of five γ-HCH metabolites, γ-pentachlorocyclohexene, 2,5-dichloro-2,5-cyclohexadiene-1,4-diol, 2,5-dichlorohydroquinone, 1,2,4-trichlorobenzene, and 2,5-dichlorophenol, strongly suggesting that MM-1 has the lin genes for γ-HCH degradation originally identified in the well-studied γ-HCH-degrading strain Sphingobium japonicum UT26. Southern blot, PCR amplification, and sequencing analyses indicated that MM-1 has seven lin genes for the conversion of γ-HCH to β-ketoadipate (six structural genes, linA to linF, and one regulatory gene, linR). MM-1 carried four plasmids, of 200, 50, 40, and 30 kb. Southern blot analysis revealed that all seven lin genes were dispersed across three of the four plasmids, and that IS6100, often found close to the lin genes, was present on all four plasmids.

Sphingobium japonicum strain UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a man-made chlorinated pesticide that causes serious environmental problems due to its toxicity and long persistence, as a sole source of carbon and energy. Here, we report the complete genome sequence of UT26, which consists of two chromosomes and three plasmids. The 15 lin genes involved in γ-HCH degradation are dispersed on the two chromosomes and one of the three plasmids.

gamma-Hexachlorocyclohexane (gamma-HCH, also called gamma-BHC and lindane) is a halogenated organic insecticide that causes serious environmental problems. The aerobic degradation pathway of gamma-HCH was extensively revealed in bacterial strain Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26. gamma-HCH is transformed to 2,5-dichlorohydroquinone through sequential reactions catalyzed by LinA, LinB, and LinC, and then 2,5-dichlorohydroquinone is further metabolized by LinD, LinE, LinF, LinGH, and LinJ to succinyl-CoA and acetyl-CoA, which are metabolized in the citrate/tricarboxylic acid cycle. In addition to these catalytic enzymes, a putative ABC-type transporter system encoded by linKLMN is also essential for the gamma-HCH utilization in UT26. Preliminary examination of the complete genome sequence of UT26 clearly demonstrated that lin genes for the gamma-HCH utilization are dispersed on three large circular replicons with sizes of 3.5 Mb, 682 kb, and 191 kb. Nearly identical lin genes were also found in other HCH-degrading bacterial strains, and it has been suggested that the distribution of lin genes is mainly mediated by insertion sequence IS6100 and plasmids. Recently, it was revealed that two dehalogenases, LinA and LinB, have variants with small number of amino acid differences, and they showed dramatic functional differences for the degradation of HCH isomers, indicating these enzymes are still evolving at high speed.

Sphingobium japonicum UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH) as its sole source of carbon and energy. In our previous studies, we cloned and characterized genes encoding enzymes for the conversion of gamma-HCH to beta-ketoadipate in UT26. In this study, we analyzed a mutant obtained by transposon mutagenesis and identified and characterized new genes encoding a putative ABC-type transporter essential for the utilization of gamma-HCH in strain UT26. This putative ABC transporter consists of four components, permease, ATPase, periplasmic protein, and lipoprotein, encoded by linK, linL, linM, and linN, respectively. Mutation and complementation analyses indicated that all the linKLMN genes are required, probably as a set, for gamma-HCH utilization in UT26. Furthermore, the mutant cells deficient in this putative ABC transporter showed (i) higher gamma-HCH degradation activity and greater accumulation of the toxic dead-end product 2,5-dichlorophenol (2,5-DCP), (ii) higher sensitivity to 2,5-DCP itself, and (iii) higher permeability of hydrophobic compounds than the wild-type cells. These results strongly suggested that LinKLMN are involved in gamma-HCH utilization by controlling membrane hydrophobicity. This study clearly demonstrated that a cellular factor besides catabolic enzymes and transcriptional regulators is essential for utilization of xenobiotic compounds in bacterial cells.

The growth of a gamma-hexachlorocyclohexane (gamma-HCH)-degrading bacterium Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 in rich medium was inhibited by gamma-HCH. This growth inhibition was not observed in a mutant that lacked the initial or second step enzymatic activity for gamma-HCH degradation, suggesting that metabolites of gamma-HCH are toxic to UT26. Two metabolites of gamma-HCH, 2,5-dichlorophenol (2,5-DCP) and 2,5-dichlorohydroquinone (2,5-DCHQ), showed a direct toxic effect on UT26 and other sphingomonad strains. Because only 2,5-DCP accumulated during gamma-HCH degradation, 2,5-DCP is thought to be a main compound for growth inhibition.

Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 utilizes the important insecticide gamma-hexachlorocyclohexane as a sole source of carbon and energy. In previous studies, we isolated and characterized six structural genes (linA to linF) and one regulatory gene (linR) of UT26 for the degradation of gamma-hexachlorocyclohexane to beta-ketoadipate. Our analysis in this study indicated that the UT26 genome consists of three large circular replicons of 3.6 Mb, 670 kb, and 185 kb. The 3.6 Mb and the 670 kb replicons had one and two copies, respectively, of the 16S ribosomal RNA gene, and these replicons were designated as chromosomes (Chr) I and II, respectively. Chr I was indicated to be a main chromosome carrying the dnaA gene. The first three lin genes, linA to linC, for conversion of gamma-hexachlorocyclohexane to 2,5-dichlorohydroquinone, were dispersed on Chr I. The 185 kb plasmid, pCHQ1, carried the linRED operon for the conversion of 2,5-dichlorohydroquinone to maleylacetate and was conjugatively transferred to another sphingomonad strain. The linF gene encoding maleylacetate reductase was located on Chr II. These results indicated that the genes for the complete gamma-hexachlorocyclohexane degradation are dispersed on the three large replicons of UT26.

Sphingomonas paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of gamma-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of gamma-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for gamma-HCH utilization.