A thermosensitive mutant of Escherichia coli K-12 was isolated in which the membrane fractions were deficient both in penicillin-binding protein-1Bs, the major components of protein 1 [Spratt, B.G. & Pardee, A.B. (1975) Nature 254, 516-517] and in activity for in vitro peptidoglycan synthesis. The mutant was also supersensitive to many kinds of beta-lactam antibiotics. All these phenotypic changes were found to be caused by a single mutation (mrc). Genetic mapping studies show that the mrc mutation was located at about 3.3 min on the E. coli chromosome linkage map [Bachmann, B.J., Low, K.B. & Taylor, A.L. (1976) Bacteriol. Rev. 40, 116-167]. Penicillin-binding protein-1Bs seemed to be identical to one of the essential enzymes involved in crosslinking of peptidoglycan and the target of cell-lytic action of penicillins. Possible functions of some other penicillin-binding proteins in compensating for lack of protein-1Bs were also proposed.

A composite plasmid (pAT2010) has been constructed in vitro from RSF2124 and Bacillus subtilis IFO3022 plasmid (pAT1060) by covalent joining of the two DNA molecules by means of Escherichia coli DNA ligase through the cohesive ends generated by restriction endonuclease RI (EcoRI) cleavage. The composite plasmid was selected by transformation of E. coliC600r-m- with the ligated mixture after enrichment for composite plasmid by preparative agarose gel electrophoresis, and plating of the transformants on a medium containing ampicillin and colicin E1. Treatment of the composite plasmid with EcoRI yielded two fragments corresponding to the linear forms of the parental plasmids. The composite plasmids replicated as biologically functionally units in E. coli, and expressed genetic information carried by RSF2124. In the presence of chloramphenicol, the composite plasmids continued to replicate and the copy number gradually increased. Such nature of replication in the presence of chloramphenicol is characteristic to RSF2124 derived from colicin E1 factor, and so it is suggested that the replicator of RSF2124 is functional in the composite plasmid. The composite plasmid was found to synthesize mRNA of B. subtilis plasmid in cell-free extracts of E. Coli, by hybridization of the mRNA to the original plasmid DNA of pAT1060.