Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases: minimal lipopolysaccharide structure and induction of envelope stress response.
To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Delta(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 degrees C, besides showing accumulation of free lipid IV(A) precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo. In contrast, LPS of Delta(waaC lpxL lpxM lpxP) strains showed primarily Kdo(2)-lipid IV(A), indicating that these minimal LPS structures are sufficient to support growth of E. coli under slow-growth conditions at 21/23 degrees C. These lipid IV(A) derivatives could be modified biosynthetically by phosphoethanolamine, but not by 4-amino-4-deoxy-l-arabinose, indicating export defects of such minimal LPS. DeltawaaA and Delta(waaC lpxL lpxM lpxP) exhibited cell-division defects with a decrease in the levels of FtsZ and OMP-folding factor PpiD. These mutations led to strong constitutive additive induction of envelope responsive CpxR/A and sigma(E) signal transduction pathways. Delta(lpxL lpxM lpxP) mutant, with intact waaC, synthesized tetraacylated lipid A and constitutively incorporated a third Kdo in growth medium inducing synthesis of P-EtN and l-Ara4N. Overexpression of msbA restored growth of Delta(lpxL lpxM lpxP) under fast-growing conditions, but only partially that of the Delta(waaC lpxL lpxM lpxP) mutant. This suppression could be alleviated by overexpression of certain mutant msbA alleles or the single-copy chromosomal MsbA-498V variant in the vicinity of Walker-box II.
Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany.
Gracjana
G
Klein
Buko
B
Lindner
Werner
W
Brabetz
Helmut
H
Brade
Satish
S
Raina
D000217:Acyltransferases / Q000172:deficiency*; D002872:Chromosome Deletion; D002876:Chromosomes, Bacterial / Q000235:genetics; D048168:Escherichia coli K12 / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D016695:Glycosyltransferases / Q000172:deficiency*; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry*; D009154:Mutation; D010957:Plasmids; D013489:Suppression, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D008070:Lipopolysaccharides; D000217:Acyltransferases; D016695:Glycosyltransferases; C086735:heptosyltransferase; D001616:beta-Galactosidase