Noritaka Iwai

Novel S-benzylisothiourea compound that induces spherical cells in Escherichia coli probably by acting on a rod-shape-determining protein(s) other than penicillin-binding protein 2.

Random screening for inhibitors of chromosome partitioning in Escherichia coli was done by the anucleate cell blue assay. A novel S-benzylisothiourea derivative, S-(3,4-dichlorobenzyl)isothiourea, tentatively named A22, was found to induce spherical cells and spherical anucleate cells in E. coli. Mecillinam, a specific inhibitor of penicillin-binding protein 2, which induces spherical cells in E. coli, also caused anucleate cell production. Spherical cells induced by treatment with either A22 or mecillinam varied in size, and anucleate cells seemed to be more frequent among the smaller cells. These results suggest that loss of the rod shape in E. coli leads to asymmetric cell division that results in production of anucleate cells. No competition was observed even in the presence of a 10-fold excess A22 in an in vitro assay of 14C-penicillin G binding, but mecillinam specifically inhibited binding of 14C-penicillin G to penicillin-binding protein 2. Simultaneous treatment with mecillinam and cephalexin, a specific inhibitor of penicillin-binding protein 3, induced lysis of E. coli cells, but a combination of A22 and cephalexin did not. These results suggest that the target molecule(s) of A22 was not penicillin-binding protein 2. A22 may act on a rod-shape-determining protein(s) other than penicillin-binding protein 2, such as RodA or MreB.

FtsZ-dependent localization of GroEL protein at possible division sites

When Escherichia coli is treated with penicillin, the envelopes bulge at the centre of the cells and the cells then lyse. The bulges expand into vesicle‐like structures termed penicillin‐induced vesicles. We have developed a method to isolate these structures and have shown that they contain mainly membrane proteins plus a high concentration of a 60 kDa protein. The N‐terminal amino acid sequence of the protein is identical to that of GroEL protein. Western blotting analysis using anti‐GroEL antibody showed that GroEL is indeed concentrated in the vesicles. Indirect immuno‐fluorescence microscopy showed that GroEL protein is localized at the centre of the cells at the site of formation of FtsZ‐rings. Localization of GroEL is dependent on FtsZ but not other Fts proteins. GroEL mutants formed elongated cells having no or asymmetrically localized FtsZ‐rings at the restrictive temperature. These findings suggest a possible role of the GroEL protein in cell division.

Structure-Activity Relationship of S-Benzylisothiourea Derivatives to Induce Spherical Cells in Escherichia coli

We have previously reported that a novel S-benzylisothiourea derivative, S-(3,4-dichlorobenzyl)isothiourea, tentatively named A22, induced spherical cells in Escherichia coli. To elucidate the structural element(s) required for inducing these spherical cells, the biological activity of S-benzylisothiourea derivatives and related compounds toward E. coli cells was investigated. S-(4-Chlorobenzyl)isothiourea revealed spherical cell-inducing activity, although being slightly less potent than A22, and S-benzylisothiourea itself showed much less activity. S-Cyclohexylmethylisothiourea did not show antibacterial activity and had little effect on the cell shape. S-Heptylisothiourea showed antibacterial activity and induced elongated cells rather than spherical cells. Benzylisothiocyanate inhibited cell growth but did not induce spherical cells. S-Ethylisothiourea, benzylthiocyanate, benzylisocyanate, and N-phenylthiourea did not show any activity under the present experimental conditions. These results indicate that the S-benzylisothiourea structure was necessary and sufficient for inducing spherical cells and that 3- and/or 4-chloro-substitution of the S-benzyl group enhanced this activity.

Antibacterial activities of imidazolium, pyrrolidinium and piperidinium salts

The antibacterial activity of various types of imidazolium, pyrrolidinium and piperidinium salts with both propargyl group and alkyl and/or silylalkyl chains of different lengths, are described. Especially, the MIC (μg/ml) of prepared each compound for Escherichia coli and other several bacteria was determined.

Irregular nuclear localization and anucleate cell production in Escherichia coli induced by a Ca2+ chelator, EGTA

A screening system for specific inhibitors of chromosome partitioning in Escherichia coli was constructed using the blue assay method developed for detection of anucleate cell production. Effects of known chemical compounds and antibiotics were examined in the system. It was found that a calcium-chelating reagent, EGTA, induced blue zones around the paper disks containing EGTA at concentrations which did not induce growth inhibition zones. Induction of anucleate cell production by EGTA was confirmed by fluorescence microscopy after DAPI staining. Even in the nucleated cells, irregular intracellular localization of nucleoids was frequently observed. The effect of EGTA was reversed by addition of Ca(2+). These results suggest the possible role of calcium ion in the process of chromosome partitioning in E. coli.

Structure-Activity Relationship Study of the Bacterial Actin-Like Protein MreB Inhibitors: Effects of Substitution of Benzyl Group in S-Benzylisothiourea

We comprehensively investigated the effects of substitution of the benzyl group in S-benzylisothiourea derivatives on antibacterial activity, because we found previously that some substitutions enhanced it. A 2,4-Cl2-derivative was found to be the most effective compound, it was stronger than the original one in Gram-negative rod shaped-bacteria such as Escherichia coli and Salmonella typhimurium.

Transcriptional Analysis of the Escherichia coli mreBCD Genes Responsible for Morphogenesis and Chromosome Segregation

The Escherichia coli mreB gene encodes an actin-like cytoskeletal protein and is required for rod shape formation of cells and chromosome segregation. Just downstream of mreB, the mreC and mreD genes are located. They are also required for rod shape formation, though their role in chromosome segregation is unclear. lacZ fusion analysis and Northern hybridization showed that the mreB, mreC, and mreD genes formed an operon. Most of the transcripts were expressed as a monocistronic mreB mRNA, and only 1–2% of the transcripts were expressed as a polycistronic mreBCD mRNA. Introduction of a frame-shift mutation in the mreB gene resulted in a significant decrease in the amount of polycistronic mreBCD mRNA but not in that of monocistronic mreB mRNA, suggesting that an attenuation-like regulation was involved in this transcriptional control. Primer extension analysis identified three transcriptional initiation sites. Three possible σD-dependent promoter-like sequences were found just upstream of these transcriptional initiation sites. lacZ fusion analysis confirmed that these three promoters contributed to the expression of mreBCD. On the basis of these findings, the essentiality of the mreB gene was confirmed.

Anucleate Cell Blue Assay: a Useful Tool for Identifying Novel Type II Topoisomerase Inhibitors

ABSTRACT About 95,000 compounds were screened by the anucleate cell blue assay. Fifty-one of the hit compounds had various structures and showed inhibitory activity against DNA gyrase and/or topoisomerase IV. Moreover, the compounds exhibited antibacterial activity against a fluoroquinolone- and novobiocin-resistant strain of Staphylococcus aureus. The anucleate cell blue assay is therefore a useful tool for finding novel type II topoisomerase inhibitors.

Isolation of a New Antibiotic, Alaremycin, Structurally Related to 5-Aminolevulinic Acid from Streptomyces sp. A012304

A new antibiotic, which is structurally related to 5-aminolevulinic acid, a precursor of heme biosynthesis, and named alaremycin, was isolated from the culture broth of an actinomycete strain through a random screening with the blue assay to detect the formation of anucleate cells in Escherichia coli. The producing strain was identified as Streptomyces sp. by morphological, physiological, chemical and genetic criteria. Alaremycin was purified from the culture supernatant by HP-20 hydrophobic-interaction chromatography, sequential solvent/water extraction in the acidic or alkaline pH range, and QMA cation-exchange chromatography. The chemical structure of alaremycin was determined as 5-acetamido-4-oxo-5-hexenoic acid by analyses of mass and NMR spectra. The antibacterial activity of alaremycin was enhanced in the presence of 5-aminolevulinic acid.

Degradation of benzotrifluoride via the dioxygenase pathway in Rhodococcus sp. 065240

We previously isolated Rhodococcus sp. 065240, which catalyzes the defluorination of benzotrifluoride (BTF). In order to investigate the mechanism of this degradation of BTF, we performed proteomic analysis of cells grown with or without BTF. Three proteins, which resemble dioxygenase pathway enzymes responsible for isopropylbenzene degradation from Rhodococcus erythropolis BD2, were induced by BTF. Genomic PCR and DNA sequence analysis revealed that the Rhodococcus sp. 065240 carries the gene cluster, btf, which is highly homologous to the ipb gene cluster from R. erythropolis BD2. A mutant strain, which could not catalyze BTF defluorination, was isolated from 065240 strain by UV mutagenesis. The mutant strain had one mutation in the btfT gene, which encodes a response regulator of the two component system. The defluorinating ability of the mutant strain was recovered by complementation of btfT. These results suggest that the btf gene cluster is responsible for degradation of BTF. Rhodococcus sp. 065240, which catalyzes the defluorination of benzotrifluoride (BTF), carries the gene cluster, btf. The dioxygenase pathway enzymes encoded by btf genes are responsible for degradation of BTF.