Organization of lin genes and IS6100 among different strains of hexachlorocyclohexane-degrading Sphingomonas paucimobilis: evidence for horizontal gene transfer

Abstract

The organization of lin genes and IS6100 was studied in three strains of Sphingomonas paucimobilis (B90A, Sp+, and UT26) which degraded hexachlorocyclohexane (HCH) isomers but which had been isolated at different geographical locations. DNA-DNA hybridization data revealed that most of the lin genes in these strains were associated with IS6100, an insertion sequence classified in the IS6 family and initially found in Mycobacterium fortuitum. Eleven, six, and five copies of IS6100 were detected in B90A, Sp+, and UT26, respectively. IS6100 elements in B90A were sequenced from five, one, and one regions of the genomes of B90A, Sp+, and UT26, respectively, and were found to be identical. DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA, respectively, compared to only one copy of these genes in strains Sp+ and UT26. Although the copy number and the sequence of the remaining genes of the HCH degradative pathway (linB, linC, linD, and linE) were nearly the same in all strains, there were striking differences in the organization of the linA genes as a result of replacement of portions of DNA sequences by IS6100, which gave them a strange mosaic configuration. Spontaneous deletion of linD and linE from B90A and of linA from Sp+ occurred and was associated either with deletion of a copy of IS6100 or changes in IS6100 profiles. The evidence gathered in this study, coupled with the observation that the G+C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis, strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by IS6100. The association of IS6100 with the rest of the lin genes further suggests that IS6100 played a role in shaping the current lin gene organization.

FIG. 1.

Pathway for degradation of HCH isomers in S. paucimobilis strains (data adapted from references and 20). γ-PCCH, gamma-pentachlorocyclohexene; 1,4-TCDN, 1,3,4,6-tetrachloro-1,4-cyclohexadiene; 1,2,4-TCB, 1,2,4 trichlorobenzene; 2,4,5-DNOL, 2,4,5-trichloro-2,5-cyclohexadiene-1-ol; DCP, 2,5-dichlorophenol; 2,5-DDOL, 2,5-dichloro-2,5-cyclohexadiene-1,4-diol; 2,5-DCHQ, 2,5-dichlorohydroquinone; 2-CHQ, 2-chlorohydroquinone; HQ, hydroquinone; γ-HMSA, gamma-hydroxymuconic semialdehyde. The asterisk indicates that in B90 the degradation of α-, γ-, and δ-HCH isomers stops at the level of 2,5-dichlorohydroquinone.

FIG. 2.

(A) Southern blot of total DNAs of S. paucimobilis strains digested with HindIII and hybridized with [α-³²P]dATP-labeled linX. Lane 1, S. paucimobilis UT26; lane 2, S. paucimobilis Sp+; lane 3, S. paucimobilis B90; lane 4, S. paucimobilis B90A; lane 5, Gene Ruler DNA ladder mixture (MBI Fermentas). (B) Southern blot of total DNAs of S. paucimobilis strains digested with BamHI and HindIII and hybridized with [α-³²P]dATP-labeled linA. Lane 1, Gene Ruler DNA ladder mixture; lane 2, BamHI-digested DNA of S. paucimobilis B90A; lane 3, HindIII-digested DNA of S. paucimobilis B90A; lane 4, BamHI-digested DNA of S. paucimobilis B90; lane 5, HindIII-digested DNA of B90; lane 6, BamHI-digested DNA of S. paucimobilis UT26; lane 7, HindIII-digested DNA of UT26; lane 8, BamHI-digested DNA of S. paucimobilis Sp+; lane 9, HindIII-digested DNA of Sp+.

FIG. 3.

(A) Partial physical and genetic map of pLINA57 containing the ∼41-kb fragment from S. paucimobilis B90A. The ORFs deduced from the complete 13-kb nucleotide sequence are indicated by arrows showing the direction of transcription. Details of the coding regions are summarized in Table 3. (B) Comparison of the nucleotide sequences adjacent to five different IS6100 copies, Nucleotide sequences in lowercase letters are common sequences at the terminal ends of each IS6100. IS6100A, IS6100B, and IS6100C are present in pLINA57, and IS6100D and IS6100E are present in pLINB23 and pLIND33, respectively. The sequence of one of the flanking regions of IS6100D could not be determined. (C) Physical and genetic map of the region of pLINB35 containing linB and IS6100. (D) Physical and genetic map of the region of pLIND33 containing linD, linE, linR, and IS6100.

FIG. 4.

(A) Southern blot hybridization of genomic DNAs of S. paucimobilis B90A, B90, Sp+, and UT26 digested with BamHI and hybridized with [α-³²P]dATP-labeled IS6100. Lane 1, Gene Ruler DNA ladder mixture; lane 2, B90A; lane 3, B90; lane 4, Sp+; lane 5, UT26. (B) Southern blot hybridization of PstI-digested genomic DNAs of S. paucimobilis B90A, B90, Sp+, and UT26 hybridized with [α-³²P]dATP-labeled linD (panel a) and linE (panel b) as probes. Lane 1, Gene Ruler DNA ladder mixture; lane 2, B90A; lane 3, B90; lane 4, Sp+; lane 5, UT26. (C) Southern blot hybridization of genomic DNAs of S. paucimobilis Sp+ mutants digested with HindIII and BamHI and hybridized with [α-³²P]dATP-labeled IS6100 (panel a) and linA (panel b). Lane1, Gene Ruler DNA ladder mixture; lanes 2 to 6, HindIII-digested genomic DNAs of Sp+ and mutants 1 to 4; lanes 7 to 11, BamHI-digested genomic DNAs of Sp+ and mutants 1 to 4.