Biosynthesis of Peptidoglycan in Gaffkya homari : Reactivation of Membranes by Freeze-Thawing in the Presence and Absence of Walls

Abstract

The reactivation of membranes from Gaffkya homari for the synthesis of sodium dodecyl sulfate-insoluble peptidoglycan (SDS-insoluble PG) was achieved by successive cycles of freeze-thawing (− 196 versus 25°C). The presence of G. homari walls during this process affected the synthesis of both SDS-soluble (nascent) and SDS-insoluble PG. At two cycles the synthesis of SDS-soluble PG decreased by 70%, whereas that of SDS-insoluble PG increased sevenfold when compared with membranes reactivated in the absence of walls but assayed in the presence of walls. Moreover, at six cycles the lag time for the synthesis of SDS-insoluble PG decreased from 15 min to 5 to 7 min. Walls from G. homari could not be replaced with walls from Bacillus megaterium or cellulose. In addition to these effects, the presence of walls from G. homari or B. megaterium or of cellulose during the incubation of membranes freeze-thawed in the absence of walls increased twofold the amount of SDS-insoluble PG. Reactivated membranes showed greater sensitivities to penicillin (an inhibitor of dd -carboxypeptidase) and d -methionine (an inhibitor of ld -carboxypeptidase) than did isolated membrane-walls. The percentage of cross-linking of the SDS-insoluble PG synthesized by the reactivated system was 34%, a value similar to that observed for the polymer synthesized by isolated membrane-walls. Freeze-thawing membranes and walls together gave a complex with a density different from that of either membranes or walls. Thus, the assembly system for the synthesis and processing of PG was reconstituted in a complex of membranes and walls prepared from the isolated components. Whether this complex has the exact interrelationship between membrane and wall found in the organism has not been established.