Affiliation:
1. Environmental Engineering and Science, Department of Civil and Environmental Engineering,1 and
2. Department of Biological Sciences,2 Stanford University, Stanford, California 94305-4020
Abstract
ABSTRACT
Biochemical studies in
Azoarcus
sp. strain T have demonstrated that anaerobic oxidation of both toluene and
m
-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from
Azoarcus
sp. strain T and examined the role of this enzyme in both anaerobic toluene and
m
-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase,
bssCAB
, together with two additional genes,
bssD
and
bssE
, were organized in an operon in the order
bssDCABE. bssD
is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase.
bssE
shows homology to
tutH
from
Thauera aromatica
strain T1, whose function is currently unknown. A second operon that is upstream of
bssDCABE
and divergently transcribed contains two genes,
tdiS
and
tdiR
. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null
bssA
mutant was constructed (the
bssA
gene encodes the α-subunit of benzylsuccinate synthase). This
bssA
null mutant strain was unable to grow under denitrifying conditions on either toluene or
m
-xylene, while growth on benzoate was unaffected. The growth phenotype of the Δ
bssA
mutant could be rescued by reintroducing
bssA
in
trans
. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and
m
-xylene.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
88 articles.
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