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PMID: 7574644 Published · ppublish English Comparative Study Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Nucleotide sequence analysis of genes encoding a toluene/benzene-2-monooxygenase from Pseudomonas sp. strain JS150.

Applied and environmental microbiology ·Vol. 61 ·No. 9 ·1995-09-00 ·Pages 3336-46

Johnson GR, Olsen RH

Abstract

It was previously shown by others that Pseudomonas sp. strain JS150 metabolizes benzene and alkyl- and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, we found that strain JS150 also carries genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen (18O2) incorporation experiments using Pseudomonas aeruginosa strains that carried the cloned genes confirmed that toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Regions encoding the toluene-2-monooxygenase and regulatory gene product were localized in two regions of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined. Analysis of this sequence revealed six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. In addition to these gene products, all the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomonas putida CF600, P35X, and BH than with those from the toluene monooxygenases of Pseudomonas mendocina KR1 and Burkholderia (Pseudomonas) pickettii PKO1. The relationship found between the phenol hydroxylases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested that DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest.

MeSH Terms
Amino Acid Sequence Base Sequence Cloning, Molecular DNA, Bacterial/genetics Genes, Bacterial Mixed Function Oxygenases/genetics Molecular Sequence Data Multigene Family Open Reading Frames Pseudomonas/enzymology,genetics Restriction Mapping Sequence Homology, Amino Acid
Chemicals
DNA, Bacterial Mixed Function Oxygenases toluene ortho-monooxygenase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Johnson G R
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109-0620, USA.
Olsen R H
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Article Info
Journal
Applied and environmental microbiology
Abbr.
Appl Environ Microbiol
ISSN
0099-2240
DOI
10.1128/aem.61.9.3336-3346.1995
Published
1995-09-00
Pages
3336-46
Language
English
Region
United States
NLM ID
7605801
PMCID
PMC167614
Subset
IM
Grants
NIEHS NIH HHS · ES-04911 · United States
NCRR NIH HHS · MO1RR00042 · United States
Databases
GENBANK
L40033
External Links
PubMed source DOI: 10.1128/aem.61.9.3336-3346.1995 PMC Full Text View journal details
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