Overproduction of a foreign membrane protein in Escherichia coli stimulates and depends on phospholipid synthesis.
When the Pseudomonas oleovorans alk system, consisting of the alkBFGHJKL and alkST genes, is expressed in Escherichia coli W3110, significant changes in phospholipid metabolism of the host are observed. A major role seems to be played by the cytoplasmic membrane protein alkane hydroxylase (AlkB), which is synthesized as up to 10-15% of the total protein in this strain [Nieboer, M., Kingma, J. & Witholt, B. (1993) 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, Mol. Microbiol. 8, 1039-1051]. In the present paper, we have studied the link between synthesis of the membrane protein and the synthesis of phospholipids and fatty acids by examining the kinetics of these processes. Using [35S]methionine labeling, it was shown that induction of AlkB was maximal within 30-60 min after addition of inducer, when up to 15% of all newly synthesized protein is AlkB. Phospholipid synthesis was followed by measuring the incorporation of 14C-labeled acetate and 33P-labeled phosphoric acid into phospholipids. Despite a negative effect of the inducer on the growth rate of W3110[pGEc47], net phospholipid synthesis was significantly enhanced as a result of the expression of alkB. Synthesis of all three major phospholipids were stimulated to comparable extents by the induction of alkB. Induction did not increase 33P incorporation into lipids in the control recombinant alk+ strain which lacked alkB. Simultaneous with AlkB synthesis, the conversion of unsaturated 9-hexadecenoic acid (C16:1) into 9,10-methylene hexadecanoic acid (C17:ocyc) was reduced in the alk+ recombinant. Overall, these data show that the production of a foreign membrane protein in E. coli can engender a response of the phospholipid-synthesizing system of the host. In the absence of such a response, induction of the alk system would be much more toxic to the cells. Apparently, the increased phospholipid synthesis plays an important role in enabling the AlkB overproducing strain to grow.
Nieboer
M
M
Institute of Biotechnology, ETH Hönggerberg (HPT), Zürich, Switzerland.
Vis
A J
AJ
Witholt
B
B
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics; D008954:Models, Biological; D010743:Phospholipids / Q000096:biosynthesis*; D011549:Pseudomonas / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D005227:Fatty Acids; D008565:Membrane Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A