Faithful chromosome segregation is an essential component of cell division in all organisms. The eukaryotic mitotic machinery uses the cytoskeleton to move specific chromosomal regions. To investigate the potential role of the actin-like MreB protein in bacterial chromosome segregation, we first demonstrate that MreB is the direct target of the small molecule A22. We then demonstrate that A22 completely blocks the movement of newly replicated loci near the origin of replication but has no qualitative or quantitative effect on the segregation of other loci if added after origin segregation. MreB selectively interacts, directly or indirectly, with origin-proximal regions of the chromosome, arguing that the origin-proximal region segregates via an MreB-dependent mechanism not used by the rest of the chromosome.

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.

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