Agric. Biol. Chem., 44 (11), 2689•`2693, 1980 2689 Supersensitivity of Escherichia coli Cells to Several -Lactam Antibiotics Caused by Rod- MorphologyƒÀ Mutations at the mrd Gene Cluster Shigeo TAMAKI, Hiroshi MATSUZAWA,* Sadayo NAKAJIMA-IIJIMA and Michio MATSUHASHI Institute of Applied Microbiology, and *Department of Agricultural Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received June 13, 1980 Two kinds of spherical mutants, mrdA and mrdB mutants, have been isolated from Escherichia coli strain K12. The mrdA mutants have thermosensitive penicillin-binding protein 2, while the mrdB mutants have normal penicillin-binding proteins. Both kinds of mutants form spherical cells at 42•Ž and are resistant to the amidinopenicillin, mecillinam, at the same temperature. The two mutations have been mapped very close to lip at 14.2 min (revised chromosome linkage map, 1980) on the E. coli chromosome. Both mutations cause supersensitivities of cell growth to various ƒÀ-lactam antibiotics, such as ampicillin, cephalexin, cefoxitin and nocardicin A at 42•Ž. There are several reports that mutations 14.4•`14.5 min on the new E. coli chromosome in penicillin-binding proteins (PBPs) in map.8) Mutation in PBP-2 is known to cause Escherichia coli can cause supersensitivity of formation of spherical cells.3,7) The gene which the cells to ƒÀ-lactam antibiotics. Mutations in is simultaneously responsible for the for PBP-l Bs caused supersensitivity to almost all mation of PBP-2 and rod shaped cells has been penicillins and cephalosporins and other types located at 14.5 min on the new E. coli of ƒÀ-lactam antibiotics1); mutations in PBP-5 chromosome map4,7,8) and has been named caused supersensitivity to most penicillins and mrd(murein cluster d)A.7) In addition we found cephalosporins, but not to the amidinopeni that another Rod - mutation, the mrdB mu cillin, mecillinam, or to cephamycins, which tation (probably identical to the previous rodA are 7ƒ¿-methoxy-substituted cephalosporins.2t mutation9)) at 14.4 min7,8) also causes similar Mutations in PBP-2 and PBP-3 have been supersensitivities to several ƒÀ-lactam anti found to be associated with resistance of the biotics to which the mrdA mutants are cells to the ƒÀ-lactam antibiotics that specifi supersensitive. cally bind to these respective PBPs, such as mecillinam for PBP-2 and cephalexin for PBP- MATERIALS AND METHODS 3.3) Mutations in PBP-1A1,4,5) and 46) caused Strains used. The two kinds of Rod- mutant strains, the no detectable change in sensitivity of the cells mrdA and mrdB strains, derived from E. coli K12 strain to any antibiotics tested. AT1325 lip910) (supplied by Dr. B. J. Bachmann) were as This report briefly describes the super described previously.7) Two mrdA (Rod- PBP-2-) mutant sensitivity to several ƒÀ-lactam antibiotics of strains, TMM1 and TMM3, were rod-shaped and Rod - morphological mutants of E. coli caused mecillinam sensitive at 30•Ž, while two mrdB (Rod - PBP- 2+) mutant strains, TMM4 and TMM6, were oval and by mutations in the mrd gene cluster7) at mecillinam-supersensitive at this temperature.7) The mrdA Abbreviations: ABPC, ampicillin; CEX, cephalexin; and mrdB mutant strains were both spherical and CFX, cefoxitin; NCA, nocardicin A; MPC, mecillinam; mecillinam-resistant at 42•Ž.7) The rodA mutant strain PBP, penicillin-binding protein; Rod-, symbol for spheri AOS151, isolated from strain JE1011 by mutagenization cal phenotype. with N-methyl-N'-nitro-N-nitrosoguanidine,9) was spheri 2690 S. TAMAKI, H. MATSUZAWA, S. NAKAJIMA-IUIMA and M. MATSUHASHI cal and had PBP-2 at 30•Ž as well as at 42•Ž. The rodA culture streaks. After incubation for 1 day at 30•Ž or transductant S19) was rod-shaped at 30•Ž, but spherical at 42•Ž the lengths of the zones of growth inhibition were 42•Ž. The other strains used are listed in Table I. measured. Assay of minimum inhibitory concentrations. The mi RESULTS AND DISCUSSION nimum inhibitory concentration (MIC) of compounds was assayed on freshly prepared plates of modified Lennox The results in Table II on the minimum broth consisting of lO g of polypeptone (Daigo Eiyo inhibitory concentration for growth show that Chemical Co., Osaka, Japan), 5 g of yeast extract (Ebios Pharmaceutical Co., Tokyo), 5 g of NaCl, 1 g of glucose, all the mrdA and mrdB mutant strains and 20 mg of thymine if required by the strains, per liter. (TMM1, TMM3, TMM4 and TMM6) showed The medium was adjusted to pH 7.0 with NaOH and higher sensitivities to ampicillin (ABPC), mixed with 1.5% agar (Wako Pure Chemical Industries, cephalexin (CEX), cefoxitin (CFX) and nocar Osaka, Japan), and appropriate antibiotics. Freshly dicin A (NCA) at 42•Ž than their parent prepared early stationary cultures (1 x 109 cells per ml; diluted 1 : 50 for assay of the MIC of mecillinam) at 30•Ž strain, AT1325 lip9, but were much more or 42•Ž were streaked on plates containing the antibiotic resistant to mecillinam (MPC) than the in 2- to 3-fold serially increasing concentrations and parent. These mutants were obtained spon growth of the cells was examined after incubation for 16 hr taneously and scarcely seemed to involve at 30•Ž or 42•Ž. double or multiple mutations. The results of Paper-strip plate inhibition test. The sensitivities of the transduction and mating experiments de strains to antibiotics were also examined by the paper scribed below were consistent with the con strip inhibition test. Freshly prepared early stationary clusion that the phenotypical differences be cultures, as described above, were streaked on a plate of tween the parent and the mutant cells were due modified Lennox broth (see above) containing no anti to single mutations, referred to as mrdA and biotics. A strip (2 mm x 8 cm) of Whatman No. 3 MM mrdB, respectively.7) At 30•Ž, the spontaneous filter paper wetted with a solution of an appropriate antibiotic was placed on the plate at right angles to the mrdA mutant strains were about as sensitive as TABLE 1. LIST OF STRAINS USED Supersensitivity of Escherichia coli Cells 2691 TABLE II. MINIMUM INHIBITORY CONCENTRATIONS OF SEVERAL ƒÀ-LACTAM ANTIBIOTICS IN WILD TYPE AND mrdA AND mrdB MUTANT TYPE STRAINS OF E. colica a Values are expressed as ƒÊg of antibiotic per ml of culture medium. the parent strain to all the antibiotics tested, ductants were almost equally sensitive to but the mrdB mutant strain TMM4 was ABPC, CEX, CFX, NCA and MPC, but the supersensitive to all the antibiotics tested and mrdB-type transductant involving the mrdB4 strain TMM6 was supersensitive to NCA and mutation was slightly more sensitive to CFX, MPC. NCA and MPC than the wild-type transduc To determine the exact correlation between tants. Essentially similar results were obtained the mutations and the changes in sensitivities when the sensitivities to ƒÀ-lactam antibiotics of the cell to ƒÀ-lactam antibiotics, we tested the were measured by the paper strip plate test. sensitivities of isogenic strains involving the These results are shown in Table III mrdA+ mrdB+, mrdA mrdB+ or mrdA+ mrdB (Experiment 1). The supersensitivities of the genes, isolated previously by joint transduc mrdB4 strain at 30•Ž were not clear in this tion (donor: mrdA mrdB+ lip+, recipient: simplified test. mrdA+ mrdB lip; or donor: mrdA+ mrdB lip+, The supersensitivities of the rodA strain recipient: mrdA mrdB+ lip; selection: Lip+ in isolated previously after mutagenization with each case)7) (Table II). Strains TMM341 and N-methyl-N'-vitro-N-nitrosoguanidine9) and TMM431 were wild-type transductants. the isogenic transductants involving the rodA+ Strains TMM342 and TMM432 were mrdA- and rodA genes were also examined by the type transductants and strain TMM433 was a paper strip plate test. From the results, shown mrdB-type transductant. The absence of dou as Experiment 2 in Table III, the super ble mutations, mrdA mrdB, in these two mrdA- sensitivities of the rodA strains to ABPC, type transductants was proved by formation of CEX, CFX and NCA and resistance to MPC wild-type recombinants in transductions to a at 42•Ž are clearly seen. At 30•Ž, there was recipient strain (mrdA+ mrdB4 lip) by Lip+ again no clear difference in the sensitivities of selection, and its absence in the mrdB-type rodA+ and rodA transductants to the anti transductant was proved by the presence of biotics. The rodA mutation seems to be a PBP-2 at both temperatures, 30•Ž and 42•Ž. mutation of the same gene as that of the mrdB At 42•Ž, all the mrdA and mrdB transduc mutations,7) but the identity of the two has not tants tested were more sensitive than the wild yet been established. type transductants to ABPC, CEX, CFX and The results presented in this paper suggest NCA, and were very resistant to MPC. At that mutations of the genes that seem to be 30•Ž, the wild-type and the mrdA-type trans responsible for formation of the normal, rod 2692 S. TAMAKI, H. MATSUZAWA, S. NAKAJIMA-IUIMA and M . MATSUHASHI TABLE III. PAPER STRIP PLATE INHIBITION TEST SHOWING THE CHANGE IN SENSITIVITIES TO -LACTAM ANTIBIOTICS CAUSED BY mrdA ƒÀ , mrdB AND rodA MUTATIONSa a Values are expressed as lengths of inhibition zones in mm. Solutions of ABPC (0.5 and 2 mg per ml, respectively for incubation at 30•Ž and 42•Ž), CEX (2 and 4), CFX (0.5 and 2), NCA (20 and 20) and MPC (0.2 and 2) were used to wet the paper strips. Data are averages for duplicate experiments, shaped cell morphology cause supersensitiv more fragile than a rod-shaped, normal pep ities to several ƒÀ-lactam antibiotics.