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Media type:
E-Article
Title:
Cloning and Functional Characterization of the styE Gene, Involved in Styrene Transport in Pseudomonas putida CA-3
Contributor:
Mooney, Aisling;
O'Leary, Niall D.;
Dobson, Alan D. W.
imprint:
American Society for Microbiology, 2006
Published in:Applied and Environmental Microbiology
Language:
English
DOI:
10.1128/aem.72.2.1302-1309.2006
ISSN:
0099-2240;
1098-5336
Origination:
Footnote:
Description:
<jats:title>ABSTRACT</jats:title>
<jats:p>
A 1.5-kb region immediately downstream of the
<jats:italic>styABCD</jats:italic>
operon involved in styrene degradation in
<jats:italic>Pseudomonas putida</jats:italic>
CA-3 has been cloned. Sequence analysis revealed a 1,296-bp open reading frame, designated
<jats:italic>styE</jats:italic>
, and BLAST P database comparisons of the deduced StyE amino acid sequence revealed 33 to 98% identity with several membrane-associated ATPase-dependent kinase proteins involved in the active transport of aromatic hydrocarbons across bacterial membranes and also with FadL, an outer membrane protein necessary for the uptake of long-chain fatty acids in
<jats:italic>Escherichia coli</jats:italic>
. Transcription of
<jats:italic>styE</jats:italic>
is styrene dependent, and the gene is cotranscribed with the
<jats:italic>styABCD</jats:italic>
structural genes. StyE appears to be membrane associated, with a corresponding 45.9-kDa band being identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane preparations from styrene-grown cells.
<jats:italic>P. putida</jats:italic>
CA-3 cells in which the
<jats:italic>styE</jats:italic>
gene had been interrupted were no longer capable of growth on styrene. In contrast, overexpression of
<jats:italic>styE</jats:italic>
in
<jats:italic>P. putida</jats:italic>
CA-3 resulted in a 4.2-fold increase in styrene monooxygenase activity compared with wild-type cells grown on styrene, with a concomitant 8-fold increase in
<jats:italic>styA</jats:italic>
mRNA transcript levels. Experiments with the classic, ATPase inhibitor vanadate revealed that growth of wild-type cells on styrene was inhibited at a concentration of 1 mM, while 1.75 mM was required to achieve a similar effect in the StyE overexpression strain. Growth of either strain on citrate was not inhibited in the presence of up to 7 mM vanadate. These findings suggest a role for StyE in the active transport of styrene in
<jats:italic>Pseudomonas putida</jats:italic>
CA-3 and identify styrene transport as a potentially limiting factor with respect to mRNA transcript levels and associated enzymatic activity of the styrene degradative pathway.
</jats:p>