DNA gyrase is an essential enzyme in DNA replication in Escherichia coli. It mediates the introduction of negative supercoils near oriC, removal of positive supercoils ahead of the growing DNA fork, and separation of the two daughter duplexes. In the course of purifying DNA gyrase from E. coli KL16, we found an 18-kDa protein that inhibited the supercoiling activity of DNA gyrase, and we coined it DNA gyrase inhibitory protein (GyrI). Its NH 2 -terminal amino acid sequence of 16 residues was determined to be identical to that of a putative gene product (a polypeptide of 157 amino acids) encoded by yeeB (EMBL accession no. U00009) and sbmC (Baquero, M. R., Bouzon, M., Varea, J., and Moreno, F. (1995) Mol. Microbiol. 18, 301-311) of E. coli. Assuming the identity of the gene (gyrI) encoding GyrI with the previously reported genes yeeB and sbmC, we cloned the gene after amplification by polymerase chain reaction and purified the 18-kDa protein from an E. coli strain overexpressing it. The purified 18-kDa protein was confirmed to inhibit the supercoiling activity of DNA gyrase in vitro. In vivo, both overexpression and antisense expression of the gyrI gene induced filamentous growth of cells and suppressed cell proliferation. GyrI protein is the first identified chromosomally nucleoid-encoded regulatory factor of DNA gyrase in E. coli.DNA gyrase, a type II topoisomerase in Escherichia coli, has the ability to cut a double-stranded DNA, pass an uncut portion of the duplex between the cut ends, and reseal the cut. It can introduce negative supercoils into covalently closed circular DNA and cause catenation and decatenation of two different DNA duplexes, in vitro (1). It has been established that the enzyme is essential for chromosomal replication in vivo (2). Moreover, there have been reports on the involvement of DNA gyrase in transcription from certain operons, DNA repair, and recombination in E. coli (2).DNA gyrase is composed of two subunits, A (GyrA) and B (GyrB), which are assembled in A 2 B 2 complexes, the active form (3-5). The active complex has been purified from E. coli (6) and reconstituted from the purified GyrA and GyrB (7-9). GyrA has an active center for the reactions of introducing and resealing the cuts of double-stranded DNA, whereas GyrB powers the reaction by catalyzing ATP hydrolysis.DNA gyrase is a target of two distinct classes of inhibitors, coumarins (10, 11) and quinolones (10, 12). Coumarins bind to GyrB and are competitive inhibitors with respect to ATP (11). In contrast, quinolones bind DNA gyrase when the enzyme is complexed with DNA and trap the enzyme in an abortive ternary complex, which, upon treatment with a denaturant, releases cleaved DNA with GyrA covalently attached to the 5Ј-phosphoryl ends generated at the cut site.There have been several reports on regulating DNA gyrase activity in E. coli. LetD (13) encoded on F factor inhibits DNA gyrase activity via the induction of synthesis of heat shock proteins (14). Another regulatory factor, cyclic AMP (cAMP) receptor (15), particip...

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Aspoxicillin, a newly developed acylureido-penicillin with a long half-life in mouse serum of 55 min, induced postantibiotic effects (PAEs) against Staphylococcus aureus Smith of 1.7 h in vitro and 5.2 h in vivo in a thigh infection model in neutropenic mice. The long serum half-life meant that in order to evaluate the in-vivo PAE, it was necessary to examine the contribution of the drug at a sub-minimal inhibitory concentration (sub-MIC). Growth suppression by sub-MICs of aspoxicillin was examined in vitro using either previously unexposed bacterial cells or cells which had been pre-exposed to twice the MIC of aspoxicillin for 2 h. At each sub-MIC tested, the duration of growth suppression for pre-exposed cells was longer than that for unexposed cells. In an attempt to eliminate the sub-MIC effect in vivo, penicillinase was injected into mice at the time after administration when the aspoxicillin serum concentration approached the MIC. The in-vivo PAE decreased to 2.7 h when penicillinase was injected. It was concluded that aspoxicillin induced a PAE in vivo which was additional to the effect of sub-inhibitory residual drug, but that sub-MIC levels of the drug were simultaneously involved in suppressing bacterial regrowth after the drug concentration decreased below the MIC. Similar postantibiotic sub-MIC effects may also occur with other long half-life antibiotics.

The bifunctional autolysin gene (all) of Staphylococcus aureus was transcribed into a 4.1-kb transcript. The transcription initiation site was located at an adenine residue 33-nt upstream from the putative atl start codon. Analysis using a promoter-reporter plasmid showed that promoter activity increased during the exponential growth phase. The Tn551 insertion site of the autolysis-deficient mutant S. aureus RUSAL2 was located in the putative catalytic region of the glucosaminidase domain.

The autolytic cell wall hydrolase of Staphylococcus aureus, Atl, contains three highly cationic repeats in the central region of the amino acid sequence, and the repeats are presumed to have the role of binding the enzyme to some components on the cell surface. To explain the possible function of the repeats, we synthesized a number of 10-to 30-mer oligopeptides based on the Atl amino acid sequence (Thr432-Lys610) containing repeat 1, and examined their effects on the autolysis of S. aureus cells. When the peptides were added to a cell suspension of S. aureus under low ionic strength conditions, five peptides, A10, All, A14, A16 and B9, showed immediate increases in optical density (OD) of the cell suspension accompanied by decreases in viable cell counts. After the immediate increases, the ODs for A10 and A14 changed little in the first 2 hr. In contrast, the ODs for All and Al6 decreased rapidly. When peptide A10 was added to suspensions of heat-killed whole cells, crude cell walls and a crude peptidoglycan preparation, their ODs were increased approximately 2-fold. In contrast, the OD was not increased when the peptide was added to a suspension of pure peptidoglycan from which anionic polymers had been removed. Light microscopic and transmission electron microscopic study showed that A10 and Al4 inhibited autolysis and that All and A16 induced autolysis earlier than the control. These results suggest strongly that the peptides adsorb to and precipitate on the anionic cell surface polymers such as teichoic acid and lipoteichoic acid via ionic interaction. The effects of peptides on the autolysis may be the results of the modification of S. aureus autolysin activities. These peptides, especially the 10-mer peptide B9 (PGTKLYTVPW) that represents the C-terminal half of A10 and N-terminal half of All, may be important segments for Atl to bind to the cell surface.