Specific labeling of diaminopimelate: a radioassay for the determination of the peptidoglycan cross-linking index.
A radioassay for the determination of the peptidoglycan cross-linking index (CLI) was devised. It is based on specific radioactive labeling of diaminopimelic acid (DAP) by diverting [14C]aspartate into the DAP pathway, while inhibiting incorporation of label into other cell wall components. Purified [14C]DAP-labeled cell walls were treated with fluorodinitrobenzene, hydrolyzed, and chromatographed by TLC. The radioactivity in well-separated mono dinitrophenyl-diaminopimelate (DNP-DAP) and DAP spots was counted and the CLI was determined from the ratio of DAP to the total of mono DNP-DAP and DAP counts. The method, suitable for bacteria such as Bacillus subtilis unable to incorporate exogenous DAP, can be applied to other systems. A CLI of 50.8 +/- 1.3% and 55.5 +/- 0.9% was obtained for B. subtilis 168 cells growing exponentially in rich and minimal medium, respectively. Comparison of these to results previously obtained on B. subtilis suggested the existence of a hitherto unreported peptidoglycan endopeptidase activity.
Roten
C A
CA
Institut de génétique et de biologie microbiennes, Lausanne, Switzerland.
Pagni
M
M
Margot
P
P
Touri
F
F
Karamata
D
D
D001412:Bacillus subtilis / Q000737:chemistry; D002250:Carbon Radioisotopes; D002473:Cell Wall / Q000737:chemistry; D002855:Chromatography, Thin Layer; D003432:Cross-Linking Reagents; D003960:Diaminopimelic Acid / Q000032:analysis*; D004139:Dinitrofluorobenzene; D006868:Hydrolysis; D010457:Peptidoglycan / Q000737:chemistry*
D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents; D010457:Peptidoglycan; D003960:Diaminopimelic Acid; D004139:Dinitrofluorobenzene