Identification and characterization of genes encoding a putative ABC-type transporter essential for utilization of gamma-hexachlorocyclohexane in Sphingobium japonicum UT26

Abstract

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.

FIG. 1.

Proposed degradation pathways of γ-HCH in S. japonicum UT26. Compounds: 1, γ-hexachlorocyclohexane (γ-HCH); 2, pentachlorocyclohexene (γ-PCCH); 3, 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN); 4, 1,2,4-trichlorobenzene (1,2,4-TCB); 5, 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (2,4,5-DNOL); 6, 2,5-dichlorophenol (2,5-DCP); 7, 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL); 8, 2,5-dichlorohydroquinone (2,5-DCHQ); 9, chlorohydroquinone (CHQ); 10, hydroquinone (HQ); 11, acylchloride; 12, γ-hydroxymuconic semialdehyde; 13, maleylacetate; 14, β-ketoadipate. GSH, glutathione (reduced form); GS-SG, glutathione (oxidized form). Square brackets show unstable materials that have yet to be detected.

FIG. 2.

Organization of the linKLMN gene cluster and constructs of linKLMN mutants and plasmids for complementation. Pentagons indicate the size and orientation of the ORFs. In S. japonicum UT953, TnMod-OKm was inserted into a position shown by the small black flag. The rightward direction of the flag represents the orientation of the Km^r gene on TnMod-OKm. S. japonicum RE1, RE2, RE3 and RE4 are linKLMN deletion mutants (Table 1), and gaps between the white bars indicate regions that were deleted by homologous recombination. Plasmids pKSR1000 to pKSR1009 were used for complementation analysis (Table 1), and white bars show the insertion regions of plasmids. Rightward black arrows indicate constitutive promoter Pu (36). Mutants and plasmids were constructed as described in Materials and Methods.

FIG. 3.

Degradation of γ-HCH and accumulation of 2,5-DCP in S. japonicum UT26 and its mutants. γ-HCH (30 μM) was incubated with intact cells (A) or crude extracts (B) of each strain in W medium at 30°C. The concentrations of γ-HCH and its metabolites in reaction medium were measured by GC with an ECD.

FIG. 4.

Assay for sensitivity of S. japonicum UT26 and its mutants to γ-HCH or 2,5-DCP. The cells at log phase cultured in 1/3LB medium were collected, washed in fresh 1/3LB medium, and diluted in 1/3LB medium, and cell suspensions (approximately 5 μl) containing 10¹ to 10³ cells were spotted on 1/3LB agar plate with either 2 mM γ-HCH or 25 μM 2,5-DCP or with DMSO (negative control). These plates were incubated at 30°C for 5 days.

FIG. 5.

Growth curves of S. japonicum UT26 (A) and RE1 (B) in 1/3LB medium in the presence of γ-HCH or 2,5-DCP. UT26 and its mutant cells at log phase cultured in 1/3LB were collected, washed in fresh 1/3LB medium, and diluted to an OD₆₆₀ of 0.05 in fresh 1/3LB with γ-HCH or 2,5-DCP. Each sample in the presence of 20 μM γ-HCH (open triangle), 6 μM 2,5-DCP (open diamond), or DMSO (negative control; filled circle), was grown at 30°C, and turbidity (OD₆₆₀) was recorded by using a TVS062CA biophotorecorder (Advantec, Toyko, Japan).

FIG. 6.

Change in the fluorescence of a hydrophobic probe, NPN (A) or BCECF (B), in the presence of intact cells of S. japonicum UT26 and RE1. a.u. arbitrary units.