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

A 1.5-kb region immediately downstream of thestyABCD operon involved in styrene degradation inPseudomonas putida CA-3 has been cloned. Sequence analysis revealed a 1,296-bp open reading frame, designatedstyE , 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 inEscherichia coli . Transcription ofstyE is styrene dependent, and the gene is cotranscribed with thestyABCD 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 thestyE gene had been interrupted were no longer capable of growth on styrene. In contrast, overexpression ofstyE inP. 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 instyA 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 inPseudomonas 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.