Molecular characterization of an inducible gentisate 1,2-dioxygenase gene, xlnE, from Pseudomonas alcaligenes NCIMB 9867.
Pseudomonas alcaligenes NCIMB 9867 (strain P25X) produces isofunctional enzymes of the gentisate pathway that enables the degradation of xylenols and cresols via gentisate. Previous reports had indicated that one set of enzymes is constitutively expressed whereas the other set is strictly inducible by aromatic hydrocarbon substrates. The gene encoding gentisate 1,2-dioxygenase (GDO), the enzyme that catalyzes the cleavage of the gentisate aromatic ring, was cloned from strain P25X. The GDO gene, designated xlnE, is 1,044 bp, and is part of a 5.4 kb operon which consists of six genes, xlnEFGHID. Transcription of this operon was driven by a sigma 70-type promoter, PxlnE, located 123 bp upstream of the xlnE start codon. Primer extension analysis showed that the xlnE transcription start point is located at the -87 adenine residue. In a P25X xlnE knockout mutant, GDO activity could only be detected when cells were grown in the presence of aromatic substrates, suggesting that xlnE encodes for the constitutive copy of GDO. This was verified by constructing a P25X strain with xlnE transcriptionally fused to a promoterless catechol 2,3-dioxygenase gene. In this strain, catechol 2,3-dioxygenase activity was detected in cells that were grown in the absence of aromatic inducers. However, catechol 2,3-dioxygenase activity increased up to four fold when these cells were grown in the presence of aromatic substrates, in particular 3-hydroxybenzoate. Thus, xlnE is in fact, inducible and the constitutive activity observed under non-inducing conditions was due to its relatively high basal levels of expression.
Yeo
Chew Chieng
CC
Programme in Environmental Microbiology, Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore.
Wong
Mark Vee-Meng
MV
Feng
Yongmei
Y
Song
Keang Peng
KP
Poh
Chit Laa
CL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D049308:Dioxygenases; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D023061:Gene Order; D005841:Gentisates / Q000378:metabolism / Q000494:pharmacology; D062385:Hydroxybenzoates / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D014992:Xylenes / Q000378:metabolism / Q000494:pharmacology
D004269:DNA, Bacterial; D005841:Gentisates; D062385:Hydroxybenzoates; D014992:Xylenes; C032948:3-hydroxybenzoic acid; D010105:Oxygenases; D049308:Dioxygenases; C026247:gentisate 1,2-dioxygenase; C048893:2,5-xylenol