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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Corynebacterium glutamicum is a biotin auxotroph that secretes L-glutamic acid in response to biotin limitation; this process is employed in industrial L-glutamic acid production. Fatty acid ester surfactants and penicillin also induce L-glutamic acid secretion, even in the presence of biotin. However, the mechanism of L-glutamic acid secretion remains unclear. It was recently reported that disruption of odhA, encoding a subunit of the 2-oxoglutarate dehydrogenase complex, resulted in L-glutamic acid secretion without induction. In this study, we analyzed odhA disruptants and found that those which exhibited constitutive L-glutamic acid secretion carried additional mutations in the NCgl1221 gene, which encodes a mechanosensitive channel homolog. These NCgl1221 gene mutations lead to constitutive L-glutamic acid secretion even in the absence of odhA disruption and also render cells resistant to an L-glutamic acid analog, 4-fluoroglutamic acid. Disruption of the NCgl1221 gene essentially abolishes L-glutamic acid secretion, causing an increase in the intracellular L-glutamic acid pool under biotin-limiting conditions, while amplification of the wild-type NCgl1221 gene increased L-glutamate secretion, although only in response to induction. These results suggest that the NCgl1221 gene encodes an L-glutamic acid exporter. We propose that treatments that induce L-glutamic acid secretion alter membrane tension and trigger a structural transformation of the NCgl1221 protein, enabling it to export L-glutamic acid.L-Glutamate has a distinctive taste, known as "umami," that is neither sweet, sour, salty, nor bitter (24), and it is widely used as a flavor enhancer. About 1.8 million tons of monosodium glutamate are produced worldwide per year by fermentation using coryneform bacteria. These are rod-shaped, nonsporulating, gram-positive bacteria containing mycolic acids and are widely distributed in the natural world. A nonpathogenic species, Corynebacterium glutamicum, was originally isolated as an L-glutamate-producing bacterium (12, 34). Wild-type C. glutamicum, without breeding, releases more than 80 g/liter of L-glutamic acid under appropriate culture conditions (27).The mechanism of L-glutamate secretion by C. glutamicum is unique. The presence of biotin, which is required by C. glutamicum for growth, inhibits L-glutamate production in the culture medium, while production is induced under biotinlimiting conditions (26) and in response to fatty acid ester surfactants (31) and penicillin (22). It is also induced by ethambutol treatment, which inhibits formation of the mycolic acid layer of the cell wall (25). Since biotin limitation and the other inducing treatments cause damage to cell surface structures of this microorganism, it has long been assumed that L-glutamate leaks through the cell membrane (32).The dtsR1 gene, isolated as a multicopy suppressor of a mutant hypersensitive to fatty acid ester surfactants, encodes a protein showing strong homology to the ␤ subunit of acetylcoenzyme A (CoA) carboxylase (10). Sinc...

Although (-)-epigallocatechin gallate (EGCG) has been reported to induce apoptosis in a variety of tumor cells, detailed mechanisms remain to be explored. In the present study, we investigated the antitumor mechanism of EGCG by using human T-cell acute lymphoblastic leukemia Jurkat cells. We focused on the involvement of reactive oxygen species, as we found previously that EGCG caused apoptotic cell death in osteoclastic cells due mainly to promotion of the reduction of Fe(III) to Fe(II) to trigger Fenton reaction, which affords hydroxyl radical from hydrogen peroxide [H(2)O(2) + Fe(II) --> (*)OH + OH(-) + Fe(III)]. EGCG (12.5-50 micro M) decreased the viability of Jurkat cells and caused concomitant increase in cellular caspase-3 activity. Catalase and the Fe(II)-chelating reagent o-phenanthroline suppressed the EGCG effects, indicating involvements of both H(2)O(2) and Fe(II) in the mechanism. Unexpectedly, epicatechin gallate (ECG), which has Fe(III)-reducing potency comparable with EGCG, failed to decrease the viability of Jurkat cells, while epigallocatechin (EGC), which has low capacity to reduce Fe(III), showed cytotoxic effects similar to EGCG. These results suggest that, unlike in osteoclastic cells, a mechanism other than Fe(III) reduction plays a role in catechin-mediated Jurkat cell death. We found that EGCG causes an elevation of H(2)O(2) levels in Jurkat cell culture, in cell-free culture medium and sodium phosphate buffer. Catechins with a higher ability to produce H(2)O(2) were more cytotoxic to Jurkat cells. Hydrogen peroxide itself exerted Fe(II)-dependent cytotoxicity. Amongst tumor and normal cell lines tested, cells exhibiting lower H(2)O(2)-eliminating activity were more sensitive to EGCG. From these findings, we propose the mechanism that make catechins cytotoxic in certain tumor cells is due to their ability to produce H(2)O(2) and that the resulting increase in H(2)O(2) levels triggers Fe(II)-dependent formation of highly toxic hydroxyl radical, which in turn induces apoptotic cell death.

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