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 new /?-Iactam-inducible penicillin-binding protein (PBP) that has extremely low affinity to penicillin and most other D-lactam antibiotics has been widely found in highly /?-lactam(methicillin)-resistant Staphylococcus aureus (MRSA). The gene for this protein was sequenced and the nucleotide sequence in its promoter and close upstream area was found to show close similarity with that of staphylococcal penicillinase, while the amino acid sequence over a wide range of the molecule was found to be similar to those of two PBPs of Escherichia coli, the shape-determining protein (PBP 2) and septum-forming one (PBP 3). Probably the MRSA PBP (Mr 76462) evolved by recombination of two genes: an inducible type I penicillinase gene and a PBP gene of a bacterium, causing the formation of a /?-lactam-inducible MRSA PBP.

A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus.

The 6.5-kilobase mre region at 71 min in the Escherichia coli chromosome map, where genes involved in formation of a rod-shaped cell form a gene cluster, was analyzed by in vivo protein synthesis in a maxicell system and by base sequencing of DNA. An open reading frame that may code for a protein with an Mr of about 37,000 on sodium dodecyl sulfate-polyacrylamide gels was found and was correlated with the mreB gene. N-terminal amino acid sequencing of the hybrid mreB-lacZ protein confirmed the production by mreB of a protein of 347 amino acid residues with a molecular weight of 36,958. The amino acid sequence of this protein deduced from the DNA sequence showed close similarity with that of a protein of theftsA gene which is involved in cell division of E. coli. Three other contiguous genes that formed three proteins with Mrs of about 40,000, 22,000, and 51,000, respectively, were detected downstream of the mreB gene by in vivo protein synthesis. The mreB protein and some of these three proteins may function together in determination of cell shape.Two regions called mrd (15) and mre (11) on the Escherichia coli chromosome involve clusters of genes responsible for determination of the cell shape and the sensitivity of cell growth to an amidinopenicillin, mecillinam. The mrd region located at 15 min on the E. coli chromosome map involves two genes for formation of the rod shape of the cell, mrdA (= pbpA), which codes for penicillin-binding protein 2, a peptidoglycan synthetase, and mrdB (= rodA), which codes for the RodA protein which may also be necessary for functioning of the mrdA protein (5). The mre region located at 71 min on the chromosome map involves genes that probably function together in shape determination and mecillinam sensitivity of the cell (17). Previously, we reported preliminary results of gene analysis of the apparent 7-kilobase (kb) mre region neighboring the fabE gene (17). The gene we called mreB (11), which may be allelic to envB (18) and in which the mutation mre-129 causing a round cell and supersensitivity to mecillinam is located, was cloned in a 2.8-kb DNA fragment. However, we found that another mutation, mre-678, causing a round cell, resistance to mecillinam, and overproduction of penicillin-binding proteins lBs and 3 was due to deletion of a 5.2-kb DNA fragment extending from the mreB gene and could not be complemented by the 2.8-kb fragment (17).The present report describes the in vivo identification of the protein products of the genes located in the mre region (the size of the SaII fragment is corrected to 6.5 kb from that in the previous report) by using a maxicell system (13) MATERIALS AND METHODSBacterial strains. E. coli K-12 strain CSR603 (uvrA6 recAl phr-J thr leu pro his thi arg lac gal ara xyl mtl rpsL) used for protein synthesis in the maxicell system was obtained from B. J. Bachmann, Yale University School of Medicine. Strain JM109 (Alac-proAB recAl endAl gyrA96 thi hsdRJ7 supE44 relAl A-F' traD36 proAB lacIq AlacZMJ5) used for DNA sequencing experiments was obtained...

New shape-determining genes in the mre cluster at 71 min on the Escherichia coli chromosome map, named mreC and mreD, were identified by complementation experiments using Amre-678 mutant cells, which have a 5-kilobase-pair deletion encompassing the mre region, and by DNA sequencing. The Amre-678 mutant cells required three genes, the previously reported mreB gene and the two new genes, to restore the normal rod shape of the cells and normal sensitivity of growth to mecillinam. The mreC gene is preceded by the mreB gene and by a 65-base-pair spacing sequence containing a palindrome sequence and a possible Shine-Dalgarno sequence. The deduced amino acid sequence of the MreC protein consists of 367 amino acid residues with a molecular weight of 39,530. The initiation codon of the mreD gene overlaps the termination codon of the mreC gene by one nucleotide residue. The deduced amino acid sequence of the MreD protein consists of 162 amino acid residues with a molecular weight of 18,755. In vitro, the coding frames of mreC and mreD produced proteins with M,s of 40,000 and 15,000, respectively, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.