Affiliation:
1. Marine Biotechnology Institute, Kamaishi Laboratories, Kamaishi, Iwate 026-0001, Japan
Abstract
ABSTRACT
Nocardioides
sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate,
o
-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to
o
-phthalate (
phd
) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight
phd
genes was cloned and sequenced. The
phdA
,
phdB
,
phdC
, and
phdD
genes, which encode the α and β subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster,
phdAB
, was located 8.3 kb downstream of the previously characterized
phdK
gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The
phdCD
gene cluster was located 2.9 kb downstream of the
phdB
gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the α and β subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The
phdABCD
genes were expressed poorly in
Escherichia coli
, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the
phdABCD
genes improved their expression in
E. coli. E. coli
cells carrying
phdBCD
or
phdACD
exhibited no phenanthrene-degrading activity, and those carrying
phdABD
or
phdABC
exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the
phdABCD
genes. It was thus concluded that all of the
phdABCD
genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the
phd
genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in
Nocardioides
sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
156 articles.
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