Incorporation of LL-diaminopimelic acid into peptidoglycan of Escherichia coli mutants lacking diaminopimelate epimerase encoded by dapF.
Recently a dapF mutant of Escherichia coli lacking the diaminopimelate epimerase was found to have an unusual large LL-diaminopimelic acid (LL-DAP) pool as compared with that of meso-DAP (C. Richaud, W. Higgins, D. Mengin-Lecreulx, and P. Stragier, J. Bacteriol. 169:1454-1459, 1987). In this report, the consequences of high cellular LL-DAP/meso-DAP ratios on the structure and metabolism of peptidoglycan were investigated. For this purpose new efficient high-pressure liquid chromatography techniques for the separation of the DAP isomers were developed. Sacculi from dapF mutants contained a high proportion of LL-DAP that varied greatly with growth conditions. The same was observed with the two DAP-containing precursors, UDP-N-acetylmuramyl-tripeptide and UDP-N-acetylmuramyl-pentapeptide. The limiting steps for the incorporation of LL-DAP into peptidoglycan were found to be its addition to UDP-N-acetylmuramyl-L-alanyl-D-glutamate and the formation of the D-alanyl-DAP cross-bridges. The Km value of the DAP-adding enzyme for LL-DAP was 3.6 x 10(-2) M as compared with 1.1 x 10(-5) M for meso-DAP. When isolated sacculi were treated with Chalaropsis N-acetylmuramidase and the resulting soluble products were analyzed by high-pressure liquid chromatography, the proportion of the main peptidoglycan dimer was lower in the dapF mutant than in the parental strain. Moreover, the proportion of LL-DAP was higher in the main monomer than in the main dimer, where it was almost exclusively located in the donor unit. There are thus very few D-alanyl-LL-DAP cross-bridges, if any. We also observed that large amounts of LL-DAP and N-succinyl-LL-DAP were excreted in the growth medium by the dapF mutant.
Mengin-Lecreulx
D
D
Unité Associée du Centre National de la Recherche Scientifique, Orsay, France.
Michaud
C
C
Richaud
C
C
Blanot
D
D
van Heijenoort
J
J
D000591:Amino Acid Isomerases; D000596:Amino Acids / Q000032:analysis; D000599:Amino Acids, Diamino / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D003960:Diaminopimelic Acid / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D007535:Isomerases / Q000235:genetics*; D009154:Mutation; D010457:Peptidoglycan / Q000032:analysis / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D019998:Racemases and Epimerases / Q000235:genetics*
D000596:Amino Acids; D000599:Amino Acids, Diamino; D003470:Culture Media; D010457:Peptidoglycan; D011498:Protein Precursors; D003960:Diaminopimelic Acid; D007535:Isomerases; D019998:Racemases and Epimerases; D000591:Amino Acid Isomerases; C042032:diaminopimelate epimerase