Cloning and functional characterization of the styE gene, involved in styrene transport in Pseudomonas putida CA-3.

Abstract

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.

• Authors: Aisling Mooney, Niall D O'Leary, Alan D W Dobson
• Journal: Applied and environmental microbiology (Appl Environ Microbiol) Vol. 72 Issue 2 Pg. 1302-9 (Feb 2006) ISSN: 0099-2240 [Print] United States
• PMID: 16461680 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA, Bacterial
• Vanadates
• Styrene
• Adenosine Triphosphatases

Topics

• Adenosine Triphosphatases (metabolism)
• Base Sequence
• Biodegradation, Environmental
• Biological Transport, Active
• Cloning, Molecular
• DNA, Bacterial (genetics)
• Gene Expression
• Genes, Bacterial
• Operon
• Pseudomonas putida (drug effects, genetics, metabolism)
• Recombination, Genetic
• Styrene (metabolism)
• Transcription, Genetic
• Vanadates (pharmacology)