The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110 (pGEc47) affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subfraction.
The alkane hydroxylase system of Pseudomonas oleovorans, which catalyses the initial oxidation of aliphatic substrates, is encoded by three genes. One of the gene products, the alkane hydroxylase AlkB, is an integral cytoplasmic membrane protein. Induction leads to the synthesis of 1.5-2% AlkB relative to the total cell protein, both in P. oleovorans and in recombinant Escherichia coli DH1. We present a study on the induction and localization of the alkane hydroxylase in E. coli W3110, which appears to be an interesting host strain because it permits expression levels of AlkB of up to 10-15% of the total cell protein. This expression level had negative effects on cell growth. The phospholipid content of such cells was about threefold higher than that of wild-type W3110. Freeze-fracture electron microscopy showed that induction of the alk genes led to the appearance of membrane vesicles in the cytoplasm; these occurred much more frequently in cells expressing alkB than in the negative control, which contained all of the alk genes except for alkB. Isolation and separation of the membranes of cells expressing alkB by density gradient centrifugation showed the customary cytoplasmic and outer membranes, as well as a low-density membrane fraction. This additional fraction was highly enriched in AlkB, as shown both by SDS-PAGE and enzyme activity measurements. A typical cytoplasmic membrane protein, NADH oxidase, was absent from the low-density membrane fraction. alkB expression in W3110 changed the composition of the phospholipid headgroup in the membrane, as well as the fatty acid composition of the membrane. The major changes occurred in the unsaturated fatty acids: C16:1 and C18:1 increased at the expense of C17:0cyc and C19:0cyc.
Nieboer
M
M
Institute for Biotechnology, ETH Hönggerberg (HPT), Zürich, Switzerland.
Kingma
J
J
Witholt
B
B
D000473:Alkanes / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D003521:Cyclopropanes / Q000494:pharmacology; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D005614:Freeze Fracturing; D005798:Genes, Bacterial; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000378:metabolism; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014169:Transformation, Bacterial
D000473:Alkanes; D001426:Bacterial Proteins; D003521:Cyclopropanes; D008565:Membrane Proteins; D010743:Phospholipids; C010710:dicyclopropylketone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A