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Applied and Environmental Microbiology, February 2006, p. 1302-1309, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1302-1309.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Cloning and Functional Characterization of the styE Gene, Involved in Styrene Transport in Pseudomonas putida CA-3

Aisling Mooney, Niall D. O'Leary, and Alan D. W. Dobson*

Microbiology Department and Environmental Research Institute, University College Cork, National University of Ireland, Cork, Ireland

Received 29 June 2005/ Accepted 18 November 2005

A 1.5-kb region immediately downstream of the styABCD operon involved in styrene degradation in Pseudomonas putida CA-3 has been cloned. Sequence analysis revealed a 1,296-bp open reading frame, designated styE, 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 Escherichia coli. Transcription of styE is styrene dependent, and the gene is cotranscribed with the styABCD 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. P. putida CA-3 cells in which the styE gene had been interrupted were no longer capable of growth on styrene. In contrast, overexpression of styE in P. putida 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 styA 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 Pseudomonas putida 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.

* Corresponding author. Mailing address: Microbiology Department, National University of Ireland, Cork, Ireland. Phone: 353 21 4902743. Fax: 353 21 4903101. E-mail: a.dobson{at}ucc.ie.

Applied and Environmental Microbiology, February 2006, p. 1302-1309, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1302-1309.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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