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.

FIG. 1.

(a) RT-PCR analysis of styE mRNA expression in P. putida CA-3 grown on various carbon sources. Lanes: 1, succinate; 2, styrene; 3, phenylacetic acid; 4, P. putida CA-3 genomic DNA as positive control; M, molecular weight markers. (b) RT-PCR analysis of citrate synthase mRNA expression in P. putida CA-3. Lanes: 1, succinate; 2, styrene; 3, phenylacetic acid as a sole carbon source; 4, P. putida CA-3 genomic DNA as a positive control; M, molecular weight markers. (c) RT-PCR analysis of total RNA from a styrene-grown culture of P. putida CA-3, assessing operonic expression of styE. Lanes: 1, 1,508-bp product obtained with styDF/styDR; 2, black arrow indicating 1,730-bp product obtained with styDF(1074)/styER; 3, 540-bp product obtained with CitF/CitR; M, molecular weight markers.

FIG. 2.

(a). Screening of pKGmE1 recombinant colonies for altered indole-to-indigo conversion phenotypes on minimal salts glucose with 3 mM indole. White arrows indicate colonies A6 and D9. (b) PCR screening of genomic DNA for the intact 1,296-bp styE gene. Lanes: 1, Hyperladder I (Promega); 2, negative control; 3, P. putida CA-3 genomic DNA; 4, A6; 5, D9.

FIG. 3.

SDS-PAGE analysis of outer membrane protein fraction of Pseudomonas putida CA-3 cells harboring pStyEF (lane 1) and Pseudomonas putida CA-3 wild-type cells (lane 2). M, protein markers (New England Biolabs); 62 kDa, glutamic dehydrogenase (bovine liver); 47.5 kDa, aldolase (rabbit muscle); 32.5 kDa, triosephosphate isomerase (E. coli). StyE (∼46 kDa) is indicated by a black arrow.

FIG. 4.

Determination of the MIC of vanadate on P. putida CA-3 wild-type and StyE-overexpressing strains. (a) Effects of vanadate on cells growing on minimal salts medium with styrene alone. (b) Effects of vanadate when the cultures were fed citrate as the sole carbon source. ⧫, styE-overexpressing strain; ▪, CA-3 wild-type strain.