We will continue to study aspects of the biosynthesis, assembly and degradation of the bacteria cell wall, especially in relationship to cell growth and cell division. Emphasis will be placed on studies of Streptococcus faecium ATCC 9790 as a """"""""model system,"""""""" particularly because of its relatively simple shape and mode of division. Studies during the next 5-year grant period will be directed towards studies of the possible roles of two separate and distinct muramidases of SF in surface assembly and division. Thus we propose to: (i) Study several of the properties of the two very unusual autolytic murasidases of SF. Included will be studies of the molecular mechanism of hydrolysis of PGs by muramidase-2 (E- 2). The possibility that E-2 is a transglycosidase is of particular interest, as is the possibility that E-2 is a """"""""two- headed"""""""" type of transglycosidase-transpeptidase, similar to at least two of the penicillin-binding proteins (PBPs) of Escherichia coli. Additionally we will study and compare the binding of and catalytic properties of both E-l and E-2 on a number of different soluble and insoluble substrates. The ultimate goal of these studies is to obtain an insight into the action of and regulation of these two activities in growing and dividing bacteria. (ii) Study the possible in vivo functions of these two, redundant, PG hydrolases in cell wall assembly, surface enlargement and/or cell division. Towards this goal we will obtain and characterize isogenic pairs, one of which will contain a mutation in the structural gene for E-1 or E-2. The genes for E-l and E-2 will be cloned in E. coli and the two genes will be sequenced. (iii) Additional studies will include investigations of the relationship of E-2 to PBPs, and studies of cell wall organization using monoclonal antibodies to epitopes present in the cell wall PG.
