OVERPRODUCTION OF A FOREIGN MEMBRANE-PROTEIN IN ESCHERICHIA-COLI STIMULATES AND DEPENDS ON PHOSPHOLIPID-SYNTHESIS

When the Pseudomonas oleovorans alk system, consisting of the alkBFGHJ KL and alkST genes, is expressed in Escherichia coli W3110, significan t changes in phospholipid metabolism of the host are observed. A major role seems to be played by the cytoplasmic membrane protein alkane hy droxylase (AlkB), which is synthesized as up to 10-15% of the total pr otein in this strain [Nieboer, M., Kingma, J. & Witholt, B. (1993) The alkane oxidation system of Pseudomonas oleovorans: induction of the a lk genes in Escherichia coli W3110[pGEc47] affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cyt oplasmic membrane subfraction, Mol. Microbiol. 8, 1039-1051]. In the p resent paper, we have studied the link between synthesis of the membra ne protein and the synthesis of phospholipids and fatty acids by exami ning the kinetics of these processes. Using [S-35]methionine labeling, it was shown that induction of AlkB was maximal within 30-60 min afte r addition of inducer, when up to 15% of all newly synthesized protein is AlkB. Phospholipid synthesis was followed by measuring the incorpo ration of C-14-labeled acetate and P-33-labeled phosphoric acid into p hospholipids. Despite a negative effect of the inducer on the growth r ate of W3110[pGEc47], net phospholipid synthesis was significantly enh anced as a result of the expression of alkB. Synthesis of all three ma jor phospholipids were stimulated to comparable extents by the inducti on of alkB. Induction did not increase P-33 incorporation into lipids in the control recombinant alk(+) strain which lacked alkB. Simultaneo us with AlkB synthesis, the conversion of unsaturated 9-hexadecenoic a cid (C-16:1) into 9,10-methylene hexadecanoic acid (C-17:0cyc) was red uced in the alk(+) recombinant. Overall, these data show that the prod uction of a foreign membrane protein in E. coli can engender a respons e of the phospholipid-synthesizing system of the host. In the absence of such a response, induction of the alk system would be much more tox ic to the cells. Apparently, the increased phospholipid synthesis play s an important role in enabling the AlkB overproducing strain to grow.