title: 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.affiliation: Microbiology Department and Environmental Research Institute, University College Cork, National University of Ireland, Cork, Ireland.
authors.forename: Aisling
authors.initials: A
authors.lastname: Mooney
authors.forename: Niall D
authors.initials: ND
authors.lastname: O'Leary
authors.forename: Alan D W
authors.initials: AD
authors.lastname: Dobson
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001483:Base Sequence; D001673:Biodegradation, Environmental; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D009876:Operon; D016958:Pseudomonas putida / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic; D020058:Styrene / Q000378:metabolism*; D014158:Transcription, Genetic; D014638:Vanadates / Q000494:pharmacology
chemical_list: D004269:DNA, Bacterial; D014638:Vanadates; D020058:Styrene; D000251:Adenosine Triphosphatases