Genetic characterization of the resorcinol catabolic pathway in Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum grew on resorcinol as a sole source of carbon and energy. By genome-wide data mining, two gene clusters, designated NCgl1110-NCgl1113 and NCgl2950-NCgl2953, were proposed to encode putative proteins involved in resorcinol catabolism. Deletion of the NCgl2950-NCgl2953 gene cluster did not result in any observable phenotype changes. Disruption and complementation of each gene at NCgl1110-NCgl1113, NCgl2951, and NCgl2952 indicated that these genes were involved in resorcinol degradation. Expression of NCgl1112, NCgl1113, and NCgl2951 in Escherichia coli revealed that NCgl1113 and NCgl2951 both coded for hydroxyquinol 1,2-dioxygenases and NCgl1112 coded for maleylacetate reductases. NCgl1111 encoded a putative monooxygenase, but this putative hydroxylase was very different from previously functionally identified hydroxylases. Cloning and expression of NCgl1111 in E. coli revealed that NCgl1111 encoded a resorcinol hydroxylase that needs NADPH as a cofactor. E. coli cells containing Ncgl1111 and Ncgl1113 sequentially converted resorcinol into maleylacetate. NCgl1110 and NCgl2950 both encoded putative TetR family repressors, but only NCgl1110 was transcribed and functional. NCgl2953 encoded a putative transporter, but disruption of this gene did not affect resorcinol degradation by C. glutamicum. The function of NCgl2953 remains unclear.

FIG. 1.

Physical map and genetic organization (A) of the resorcinol pathway (C) in C. glutamicum and construction of plasmids for gene disruption and complementation (B). Restriction sites are abbreviated as follows: X, XbaI; H, HindIII; E, EcoRI; S, SacI; B, BanI. Right and left triangles with points touching represent deletion by restriction enzyme digestion; wide diamonds represent deletion by gene splicing by overlap extension.

FIG. 2.

(A through E) Growth curves of various C. glutamicum mutants cultivated with resorcinol; (F) transcriptional analysis by RT-PCR of gene expression during growth in MM broth with resorcinol or in LB broth. According to genome data analysis, NCgl1110 and NCgl2950 encode putative TetR family regulators; NCgl1111 encodes a putative hydroxylase; NCgl1112 and NCgl2952 encode putative maleylacetate reductases; NCgl1113 and NCgl2951 encode putative hydroxyquinol 1,2-dioxygenases; and NCgl2953 encodes a putative transporter. Symbols: •, RES167; (A) ▪, RES167ΔNCgl(2950-2953); (B) ▪, RES167ΔNCgl1111; ▴, RES167ΔNCgl1111/pXMJ19-NCgl1111; (C) ▪, RES167ΔNCgl1112; □, RES167ΔNCgl(2950-2953)/ΔNCgl1112; ▴, RES167ΔNCgl(2950-2953)/ΔNCgl1112/pXMJ19-NCgl1112; ▵, RES167ΔNCgl(2950-2953)/ΔNCgl1112/pXMJ19-NCgl2952; (D) ▪, RES167ΔNCgl1113; □, RES167ΔNCgl(2950-2953)/ΔNCgl1113; ▴, RES167ΔNCgl(2950-2953)/ΔNCgl1113/pXMJ19-NCgl1113; ▵, RES167ΔNCgl(2950-2953)/ΔNCgl1113/pXMJ19-NCgl2951; (E) ▪, RES167ΔNCgl1110; ▴, RES167ΔNCgl1110/pXMJ19-NCgl1110. (F) Strain RES167 was grown in MM with resorcinol (lanes 1 to 7) or in LB broth (lanes 8 to 11). Total RNAs extracted were used as templates for RT-PCR to detect the transcription of each gene as follows: lane 1, NCgl1110 (420 bp); lane 2, NCgl1111 (464 bp); lane 3, NCgl1112 (550 bp); lane 4, NCgl1113 (410 bp); lane 5, NCgl2950 (no corresponding fragment); lane 6, NCgl2951 (430 bp); lane 7, NCgl2952 (464 bp); lane 8, NCgl1110; lane 9, NCgl1112; lane 10, NCgl2950; lane 11, NCgl2952.

FIG. 3.

(A) Resorcinol hydroxylase activity in recombinant E. coli/pET28a-NCgl1111. (B) E. coli/pET28a was used as a negative control. The absorbances of the reaction mixture at wavelengths of 270 to 400 nm were recorded at time zero (top line) and at 60, 120, and 180 s (bottom line), Hydroxylase activity was also determined with 3-hydroxybenzoate, 3-aminophenol, 3,5-dihydroxytoluene, and 2,4-dihydroxybenzoate, but no activity was detected.