Accumulation of the lipid A precursor UDP-2,3-diacylglucosamine in an Escherichia coli mutant lacking the lpxH gene.
The lpxH gene encodes the UDP-2,3-diacylglucosamine-specific pyrophosphatase that catalyzes the fourth step of lipid A biosynthesis in Escherichia coli. To confirm the function of lpxH, we constructed KB21/pKJB5. This strain contains a kanamycin insertion element in the chromosomal copy of lpxH, complemented by plasmid pKJB5, which is temperature-sensitive for replication and harbors lpxH(+). KB21/pKJB5 grows at 30 degrees C but loses viability at 44 degrees C, demonstrating that lpxH is essential. CDP-diglyceride hydrolase (Cdh) catalyzes the same reaction as LpxH in vitro but is non-essential and cannot compensate for the absence of LpxH. The presence of Cdh in cell extracts interferes with the LpxH assay. We therefore constructed KB25/pKJB5, which contains both an in-frame deletion of cdh and a kanamycin insertion mutation in lpxH, covered by pKJB5. When KB25/pKJB5 cells are grown at 44 degrees C, viability is lost, and all in vitro LpxH activity is eliminated. A lipid migrating with synthetic UDP-2,3-diacylglucosamine accumulates in KB25/pKJB5 following loss of the covering plasmid at 44 degrees C. This material was converted to the expected products, 2,3-diacylglucosamine 1-phosphate and UMP, by LpxH. Pseudomonas aeruginosa contains two proteins with sequence similarity to E. coli LpxH. The more homologous protein catalyzes UDP-2,3-diacylglucosamine hydrolysis in vitro. The corresponding gene complements KB25/pKJB5 at 44 degrees C, but the less homologous gene does not. The accumulation of UDP-2,3-diacylglucosamine in our lpxH mutant is consistent with the observation that the lipid A disaccharide synthase LpxB, the next enzyme in the pathway, cannot condense two UDP-2,3-diacylglucosamine molecules, but instead utilizes UDP-2,3-diacylglucosamine as its donor and 2,3-diacylglucosamine 1-phosphate as its acceptor.
Babinski
Kristen J
KJ
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Kanjilal
Suparna J
SJ
Raetz
Christian R H
CR
D000595:Amino Acid Sequence; D004075:Diglycerides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007612:Kanamycin; D008050:Lipid A / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D010957:Plasmids; D011550:Pseudomonas aeruginosa; D011755:Pyrophosphatases / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D014530:Uridine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism
D004075:Diglycerides; D008050:Lipid A; D014530:Uridine Diphosphate; D007612:Kanamycin; D017351:N-Acetylglucosaminyltransferases; C050305:lipid A disaccharide synthase; D011755:Pyrophosphatases; C464799:UDP-2,3-diacylglucosamine pyrophosphatase