A chromosomal region of Escherichia coli contiguous to the fabE gene at 71 min on the chromosomal map contains multiple genes that are responsible for determination of the rod shape and sensitivity to the amidinopenicillin mecillinam. The so-called mre region was cloned and analyzed by complementation of two closely related but distinct E. coli mutants characterized, respectively, by the mutations mre-129 and mre-678, that showed a rounded to irregular cell shape and altered sensitivities to mecillinam; the mre-129 mutant was supersensitive to mecilinam at 30°C, but the mre-678 mutant was resistant. The mre-678 mutation also caused simultaneous overproduction of penicillin-binding proteins lBs and 3. A chromosomal region of the wild-type DNA containing the total mre region and the fabE gene was first cloned on a lambda phage; a 7-kilobase (kb) fragment containing the whole mre region, but not the fabE gene, was then recloned on a mini F plasmid, pLG339; and finally, a 2.8-kb fragment complementing only mre-129 was also cloned on this low-copy-number plasmid. The whole 7-kb fragment was required for complementing the mre-678 mutant phenotypes. Fragments containing fabE but not the mre-129 region could be cloned on a high-copy-number plasmid. Southern blot hybridization indicated that the mre-678 mutant had a large deletion of 5.25 kb in its DNA, covering at least part of the mre-129 gene.One of the most fundamental questions in cell biology is how the characteristic shape of a cell is determined. A clue to the mechanism of determining the rod shape of bacteria has been sought by isolating mutants that show different cell shapes and then analyzing them genetically and biochemically. Many mutants of Escherichia coli K-12 that could not form rod-shaped cells have been isolated. Among them, mutants with an osmotically stable, spherical shape were thought to have a defect in one of the steps involved in determination or formation of the rod shape of the cell. These rod mutations could be classified into three main groups on the basis of their positions in the gene map. In the first group, the mutations were located at 14.5 min (referred to as the mrd region) on the E. coli chromosome map. This group included the rodA (9), rodX (4), pbpA (16), and mrdAB (19) mutations. In the second group, the mutations were located at 71 min (referred to as the mre region), and this group included the rodY (4), envB (11), and mreBC (8) mutations. The third group consisted of mutations affecting formation of cyclic AMP (5) and the mechanism in which cyclic AMP is involved (5, 21), and their gene map positions were diverse. Most of these mutations showed altered sensitivities to an amidinopenicillin, mecillinam (MPC) (8,16,19,21,23). These results indicated that formation of the rod shape of the cell in E. coli involves a step that is sensitive to amidinopenicillin, and the mutation that causes a defect in this step results in a spherical shape of cell with altered sensitivity to MPC. Thus, this antibiotic was used for * Corresponding a...

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.

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Penicillin G, novobiocin, bacitracin, and enduracidin were used for selection of supersensitive mutants. This paper mainly reports results on novobiocin-supersensitive (NS) mutants. Most of these mutants were found to have defective lipopolysaccharides (LPS). The role of LPS in the adsorption of phages on E. coli was also studied. MATERIALS AND METHODS Bacteria and isolation of antibiotic-supersensitive mutants. Mutants were isolated from E. coli K-12 strain JEIOI I (F-thrleutrp-his-thythiara-lacgalxyl-mtlstr). (Genetic symbols are defined in Fig. 1.) Bacteria were usually cultured in nutrient broth at 37 C with shaking. Nutrient broth contained (per liter of deionized water): polypeptone (Daigo Eiyo Kagaku Co., Osaka, Japan), 10 g; Ehrlich meat extract (Kyokuto Seiyaku Co., Tokyo, Japan), 10 g; and NaCl, 2 g. It was adjusted to pH 7.4 by addition of I ml of 10 N NaOH per liter. For nutrient agar, nutrient broth was solidified by addition of 1.5% agar (Wako Pure Chemical Co., Osaka, Japan). Cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) essentially by the method of Adelberg et al. (1). Cells in the logarithmic phase of growth (5 ml of culture) were collected by centrifugation, 968

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