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The antibiotic alaremycin has a structure that resembles that of 5-aminolevulinic acid (ALA), a universal precursor of porphyrins, and inhibits porphyrin biosynthesis. Genome sequencing of the alaremycin-producing bacterial strain and enzymatic analysis revealed that the first step of alaremcyin biosynthesis is catalysed by the enzyme, AlmA, which exhibits a high degree of similarity to 5-aminolevulinate synthase (ALAS) expressed by animals, protozoa, fungi, and α-proteobacteria. Site-directed mutagenesis of AlmA revealed that the substitution of two amino acids residues around the substrate binding pocket transformed its substrate specificity from that of alaremycin precursor synthesis to ALA synthesis. To estimate the evolutionary trajectory of AlmA and ALAS, we performed an ancestral sequence reconstitution analysis based on a phylogenetic tree of AlmA and ALAS. The reconstructed common ancestral enzyme of AlmA and ALAS exhibited alaremycin precursor synthetic activity, rather than ALA synthetic activity. These results suggest that ALAS evolved from an AlmA-like enzyme. We propose a new evolutionary hypothesis in which a non-essential secondary metabolic enzyme acts as an ‘evolutionary seed’ to generate an essential primary metabolic enzyme.

We previously reported the extracellular production of antibody fragment Fab by Corynebacterium glutamicum. In the course of searching for genes which improve the secretion efficiency of Fab, we coincidentally found that the final growth increased significantly when the NCgl2986 gene encoding an amidase-like protein was overexpressed. This effect was observed when cells were grown on the production medium MMTG, which contains high concentrations of glucose and neutralizing agent CaCO3, but not on MMTG without CaCO3 or Lennox medium. Not only turbidity but also dry cell weight was increased by NCgl2986 overexpression, although the growth rate was not affected. It was recently reported that the Mycobacterium tuberculosis homolog Rv3915 functions as an activator of MurA protein, which catalyzes the initial step of peptidoglycan synthesis. Growth promotion was also observed when the MurA protein was overproduced. His-tagged NCgl2986 protein was purified, but its peptidoglycan hydrolyzing activity could not be detected. These results suggest that NCgl2986 promotes cell growth by activating the peptidoglycan synthetic pathway.

Corynebacterium glutamicum is used for the industrial production of various metabolites, including L-glutamic acid and L-lysine. With the aim of understanding the post-transcriptional regulation of amino acid biosynthesis in this bacterium, we investigated the role of RNase E/G in the degradation of mRNAs encoding metabolic enzymes. In this study, we found that the cobalamin-independent methionine synthase MetE was overexpressed in ΔrneG mutant cells grown on various carbon sources. The level of metE mRNA was also approximately 6- to 10-fold higher in the ΔrneG mutant strain than in the wild-type strain. A rifampicin chase experiment showed that the half-life of metE mRNA was approximately 4.2 times longer in the ΔrneG mutant than in the wild-type strain. These results showed that RNase E/G is involved in the degradation of metE mRNA in C. glutamicum.

Acid-resistance systems are essential for pathogenic Escherichia coli to survive in the strongly acidic environment of the human stomach (pH tolC mutant lacking the TolC outer membrane channel was defective in GAD induction. Here, we show that indole, a substrate of TolC-dependent efflux pumps and produced by the tryptophanase encoded by the tnaA gene, negatively regulates GAD expression. GAD expression was restored by deleting tnaA in the tolC mutant; in wild-type E. coli, it was suppressed by adding indole to the growth medium. RNA-sequencing revealed that tnaA mRNA levels drastically decreased upon exposure to moderately acidic conditions (pH 5.5). This decrease was suppressed by RNase E deficiency. Collectively, our results demonstrate that the RNase E-dependent degradation of tnaA mRNA is accelerated upon acid exposure, which decreases intracellular indole concentrations and triggers GAD induction.

We previously reported the extracellular production of antibody fragment Fab by Corynebacterium glutamicum. In the course of searching for genes which improve the secretion efficiency of Fab, we coincidentally found that the final growth increased significantly when the NCgl2986 gene encoding an amidase-like protein was overexpressed. This effect was observed when cells were grown on the production medium MMTG, which contains high concentrations of glucose and neutralizing agent CaCO

The ltsA gene of Corynebacterium glutamicum encodes a purF-type glutamine-dependent amidotransferase, and mutations in this gene result in increased susceptibility to lysozyme. Recently, it was shown that the LtsA protein catalyzes the amidation of diaminopimelate residues in the lipid intermediates of peptidoglycan biosynthesis. In this study, intracellular localization of wild-type and mutant LtsA proteins fused with green fluorescent protein (GFP) was investigated. The GFP-fused wild-type LtsA protein showed a peripheral localization pattern characteristic of membrane-associated proteins. The GFP-fusions with a mutation in the N-terminal domain of LtsA, which is necessary for the glutamine amido transfer reaction, exhibited a similar localization to the wild type, whereas those with a mutation or a truncation in the C-terminal domain, which is not conserved among the purF-type glutamine-dependent amidotransferases, did not. These results suggest that the C-terminal domain is required for peripheral localization. Differential staining of cell wall structures with fluorescent dyes revealed that formation of the mycolic acid-containing layer at the cell division planes was affected in the ltsA mutant cells. This was also confirmed by observation that bulge formation was induced at the cell division planes in the ltsA mutant cells upon lysozyme treatment. These results suggest that the LtsA protein function is required for the formation of a mycolic acid-containing layer at the cell division planes and that this impairment results in increased susceptibility to lysozyme.

Corynebacterium glutamicum is used for the industrial production of various metabolites, including L-glutamic acid and L-lysine. With the aim of understanding the post-transcriptional regulation of amino acid biosynthesis in this bacterium, we investigated the role of RNase E/G in the degradation of mRNAs encoding metabolic enzymes. In this study, we found that the cobalamin-independent methionine synthase MetE was overexpressed in ΔrneG mutant cells grown on various carbon sources. The level of metE mRNA was also approximately 6- to 10-fold higher in the ΔrneG mutant strain than in the wild-type strain. A rifampicin chase experiment showed that the half-life of metE mRNA was approximately 4.2 times longer in the ΔrneG mutant than in the wild-type strain. These results showed that RNase E/G is involved in the degradation of metE mRNA in C. glutamicum.

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.

Corynebacterium glutamicum is known to perform a unique form of cell division called post-fission snapping division. In order to investigate the mechanism of cell division of this bacterium, we isolated temperature-sensitive mutants from C. glutamicum wild-type strain ATCC 31831, and found that one of them, M45, produced high frequencies of mini-cells with no nucleoids. Cell pairs composed of an elongated cell, with one nucleoid, connected to a mini-cell, with no nucleoids, were occasionally observed. The temperature sensitivity and mini-cell formation of M45 was complemented by a 2-kb DraI-EcoRI fragment derived from the ATCC 31831 chromosomal DNA, which carried a dnaB homolog encoding a replicative DNA helicase. DNA sequence analysis revealed that M45 carried a missense mutation in the dnaB gene, which caused a substitution of Thr364 to Ile. Microscopic observation after 4',6-diamidino-2-phenylindole staining revealed that the DNA content of single cells was decreased by culturing at the restrictive temperature, suggesting that the mutation affects chromosomal replication. These results suggest that the C. glutamicum dnaB mutant performs an asymmetric cell division even after DNA replication is inhibited, which results in the production of mini-cells.

Rhodosporidium toruloides is a lipid-producing yeast, the growth of which is severely suppressed when hydrolysates of lignocellulosic biomass are used as carbon source. This is probably due to the toxic substances, such as organic acids, furans, and phenolic compounds produced during the preparation of the hydrolysates. In order to solve this problem, R. toruloides cultures were subjected to atmospheric room-temperature plasma mutagenesis, resulting in the isolation of mutants showing tolerance to sugarcane bagasse hydrolysate (SBH). Three mutant strains, M11, M13, and M18, were found to grow with producing lipids with SBH as carbon source. M11 in particular appeared to accumulate higher levels (up to 60% of dry cell weight) of intracellular lipids. Further, all three mutant strains showed tolerance of vanillin, furfural, and acetic acid, with different spectra, suggesting that different genetic determinants are involved in SBH tolerance.

Escherichia coli RNase G is involved in the degradation of several mRNAs, including adhE and eno, which encode alcohol dehydrogenase and enolase respectively. Previous research indicates that the 5' untranslated region (5'-UTR) of adhE mRNA gives RNase G-dependency to lacZ mRNA when tagged at the 5'-end, but it has not been elucidated yet how RNase G recognizes adhE mRNA. Primer extension analysis revealed that RNase G cleaved a phosphodiester bond between -19A and -18C in the 5'-UTR (the A of the start codon was defined as +1). Site-directed mutagenesis indicated that RNase G did not recognize the nucleotides at -19 and -18. Random deletion analysis indicated that the sequence from -145 to -125 was required for RNase G-dependent degradation. Secondary structure prediction and further site-directed deletion suggested that the stem-loop structure, with a bubble in the stem, is required for RNaseG-dependent degradation of adhE mRNA.

Biosensors of single-gene knockout mutants and physical methods using mesh and quartz glass are employed to discriminate plasma agents and assess their lethal effects generated in a Direct-Current atmospheric-pressure oxygen plasma jet. Radicals generated in plasma are determined by optical emission spectroscopy, along with the O3 density measurement by UV absorption spectroscopy. Besides, thermal effect is investigated by an infrared camera. The biosensors include three kinds of Escherichia coli (E. coli) K-12 substrains with their mutants, totalling 8 kinds of bacteria. Results show that oxidative stress plays a main role in the inactivation process. Rather than superoxide O2-, neutral reactive oxygen species such as O3 and O2(a1Δg) are identified as dominant sources for oxidative stress. In addition, DNA damage caused by oxidation is found to be an important destruction mechanism.

The nonpathogenic coryneform bacterium, Corynebacterium glutamicum, was isolated as an L-glutamate-overproducing microorganism by Japanese researchers and is currently utilized in various amino acid fermentation processes. L-Glutamate production by C. glutamicum is induced by limitation of biotin and addition of fatty acid ester surfactants and β-lactam antibiotics. These treatments affect the cell surface structures of C. glutamicum. After the discovery of C. glutamicum, many researchers have investigated the underlying mechanism of L-glutamate overproduction with respect to the cell surface structures of this organism. Furthermore, metabolic regulation during L-glutamate overproduction by C. glutamicum, particularly, the relationship between central carbon metabolism and L-glutamate biosynthesis, has been investigated. Recently, the role of a mechanosensitive channel protein in L-glutamate overproduction has been reported. In this chapter, mechanisms of L-glutamate overproduction by C. glutamicum have been reviewed.

Corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. To understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP is found in all three domains of life and plays an important role in the membrane insertion of proteins. SRP RNA is initially transcribed as precursor molecules; however, relatively little is known about its maturation. In C. glutamicum , SRP consists of the Ffh protein and 4.5S RNA lacking an Alu domain. In this study, we found that 3′-to-5′ exoribonuclease, polynucleotide phosphorylase (PNPase), and two endo-type RNases, RNase E/G and YbeY, are involved in the 3′ maturation of 4.5S RNA in C. glutamicum . The mature form of 4.5S RNA was inefficiently formed in Δ rneG Δ pnp mutant cells, suggesting the existence of an alternative pathway for the 3′ maturation of 4.5S RNA. Primer extension analysis also revealed that the 5′ mature end of 4.5S RNA corresponds to that of the transcriptional start site. Immunoprecipitated Ffh protein contained immature 4.5S RNA in Δ pnp , Δ rneG , and Δ ybeY mutants, suggesting that 4.5S RNA precursors can interact with Ffh. These results imply that the maturation of 4.5S RNA can be performed in the 4.5S RNA-Ffh complex. IMPORTANCE Overproduction of a membrane protein, such as a transporter, is useful for engineering of strains of Corynebacterium glutamicum , which is a workhorse of amino acid production. To understand membrane protein biogenesis in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP contains the Ffh protein and SRP RNA and plays an important role in the membrane insertion of proteins. Although SRP RNA is highly conserved among the three domains of life, relatively little is known about its maturation. We show that PNPase, RNase E/G, and YbeY are involved in the 3′ maturation of the SRP RNA (4.5S RNA) in this bacterium. This indicates that 3′ end processing in this organism is different from that in other bacteria, such as Escherichia coli .

Corynebacterium glutamicum is widely used in the industrial production of amino acids. Producer strains are generated by classical random mutagenesis, and therefore have detrimental characteristics caused by unnecessary mutations. Increased alkali sensitivity is one of those undesired characteristics. We found that one of the laboratory strains, AJ12036DeltacspADeltacspB, showed decreased growth under alkaline conditions. To clarify which mutation is responsible for alkali sensitivity, we constructed mutant strains carrying the DeltacspA and/or DeltacspB mutations from wild-type strain ATCC13869. We found that disruption of cspA encoding a mycolyltransferase alone caused increased alkali sensitivity. The DeltacspA mutant also showed increased susceptibility to ethambutol, penicillin, and rifampicin. Disruption of cspB had no effect on alkali sensitivity or drug sensitivity. These results indicate that the mycolate layer is important for alkali sensitivity as well as drug susceptibility in this bacterium.

We found that penicillin-induced glutamate production by Corynebacterium glutamicum is inhibited when a de novo protein synthesis inhibitor, chloramphenicol, is added simultaneously with penicillin. When chloramphenicol was added 4 h after penicillin addition, glutamate production was essentially unaffected. (3)H-Leucine incorporation experiments revealed that protein synthesis continued for 1 h after penicillin addition and then gradually decreased. These results suggest that de novo protein synthesis within 4 h of penicillin treatment is required for the induction of glutamate production. To identify the protein(s) necessary for penicillin-induced glutamate production, proteome analysis of penicillin-treated C. glutamicum cells was performed with two-dimensional gel electrophoresis. Of more than 500 proteins detected, the amount of 13 proteins, including OdhI (an inhibitory protein for 2-oxoglutarate dehydrogenase complex), significantly increased upon penicillin treatment. Artificial overexpression of the odhI gene resulted in the decreased specific activity of the 2-oxoglutarate dehydrogenase complex and increased glutamate production without any triggers. These results suggest that the de novo synthesis of OdhI is the necessary factor for penicillin-induced glutamate overproduction by C. glutamicum. Moreover, continuous glutamate production was achieved by overexpression of odhI without any triggers. Thus, the odhI-overexpressing strain of C. glutamicum can be useful for efficient glutamate production.

We found that Escherichia coli tolC mutants showed increased sensitivity to 5-aminolevulinic acid (ALA), a precursor of porphyrins. The tolC mutant cells grown in the presence of ALA showed a reddish brown color under visible light and a strong red fluorescence under near-UV irradiation. Fluorescence spectrometry and high-performance liquid chromatography analysis showed that the tolC mutant cells grown in the presence of ALA accumulated a large amount of coproporphyrin(ogen) intracellularly. In contrast, the wild-type cells produced coproporphyrin extracellularly. The tolC mutant cells grown in the presence of ALA, which were capable of surviving in the dark, were killed by near-UV irradiation, suggesting that the intracellular coproporphyrin(ogen) renders these cells photosensitive. These results suggest that the TolC-dependent efflux system is involved in the exclusion of porphyrin(ogen)s in E. coli .

S-Benzylisothiourea compound A22 induces coccoid forms in Escherichia coli by inhibiting the function of the actin-like cytoskeletal protein, MreB. The minimum inhibitory concentration of A22 and the minimum concentration to induce coccoid forms for various pathogenic bacteria were determined. At 10 microg/ml, A22 induced coccoid forms in Shigella flexneri but did not inhibit the growth. No alteration of coccoid forms in the Gram-positive bacteria and anaerobic bacteria tested were observed following treatment with A22. To study the relationship between pathogenicity and alterations in bacterial shape, the infectious capacity of A22-induced coccoid S. flexneri was examined using CHO-K1 cells. Invasion of the coccoid cells was significantly reduced, however, no changes in adherence were observed. Using a mutant defective in the type III secretion apparatus, which delivers effectors to the host, we examined the secretion of effectors by A22-induced coccoid S. flexneri. The amount of secreted effectors in the coccoid cells was clearly decreased compared to rod-shaped cells. These results showed that the maintenance of rod-shaped cells by MreB in bacteria was essential for the secretion of effectors via the type III secretion system. Therefore, our results suggest that A22 is a useful lead compound for a novel anti-infectious agent without bactericidal activity and MreB is a candidate target site for development of new anti-infectious agents.

The nonpathogenic coryneform bacterium, Corynebacterium glutamicum, was isolated as an l-glutamate-overproducing microorganism by Japanese researchers and is currently utilized in various amino acid fermentation processes. l-Glutamate production by C. glutamicum is induced by limitation of biotin and addition of fatty acid ester surfactants and β-lactam antibiotics. These treatments affect the cell surface structures of C. glutamicum. After the discovery of C. glutamicum, many researchers have investigated the underlying mechanism of l-glutamate overproduction with respect to the cell surface structures of this organism. Furthermore, metabolic regulation during l-glutamate overproduction by C. glutamicum, particularly, the relationship between central carbon metabolism and l-glutamate biosynthesis, has been investigated. Recently, the role of a mechanosensitive channel protein in l-glutamate overproduction has been reported. In this chapter, mechanisms of l-glutamate overproduction by C. glutamicum have been reviewed.

While studying the mechanism by which high-pressure carbon dioxide treatment (HCT) inactivates bacteria, we found that the efficiency of DNA recovery via phenol extraction was extraordinarily low from E. coli K12 cells that had been subjected to HCT. DAPI staining of the treated cells, however, revealed that nuclear DNA was present. Most DNA from the cells subjected to HCT was probably caught in the denatured protein layer during phenol extraction. The efficiency of DNA recovery from proteinase-treated crude extracts from cells subjected to HCT was high. Crude extracts of E. coli K12 cells that had not undergone HCT were intentionally acidified with acetic acid to pH 5.2 to cause acidic coagulation of cytoplasmic proteins. The efficiency of DNA recovery from the acidified extracts was low. These results suggest that in cells subjected to HCT, cytoplasmic pH is reduced to around pH 5.2, and that nuclear DNA becomes entangled in coagulated cytoplasmic proteins. Acidification of the cytoplasm might be the primary mechanism by which HCT inactivates bacteria.

Light-responsive gene expression is crucial to photosynthesizing organisms. Here, we studied functions of cis-elements (AU-box and SD sequences) and a trans-acting factor (ribonuclease, RNase) in light-responsive expression in cyanobacteria. The results indicated that AU-rich nucleotides with an AU-box, UAAAUAAA, just upstream from an SD confer instability on the mRNA under darkness. An RNase E/G homologue, Slr1129, of the cyanobacterium Synechocystis sp. strain PCC 6803 was purified and confirmed capable of endoribonucleolytic cleavage at the AU- (or AG)-rich sequences in vitro. The cleavage depends on the primary target sequence and secondary structure of the mRNA. Complementation tests using Escherichia coli rne/rng mutants showed that Slr1129 fulfilled the functions of both the RNase E and RNase G. An analysis of systematic mutations in the AU-box and SD sequences showed that the cis-elements also affect significantly mRNA stability in light-responsive genes. These results strongly suggested that dark-induced mRNA instability involves RNase E/G-type cleavage at the AU-box and SD sequences in cyanobacteria. The mechanical impact and a possible common mechanism with RNases for light-responsive gene expression are discussed.

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.

The Escherichia coli RNase G is known as an endoribonuclease responsible for the 5'-end maturation of 16S rRNA and degradation of several specific mRNAs such as adhE and eno mRNAs. In this study, we found that an RNase G mutant derived from the MC1061 strain did not grow on a glucose minimal medium. Genetic analysis revealed that simultaneous defects of cra and ilvIH, encoding a transcriptional regulator of glycolysis/gluconeogenesis and one of isozymes of acetohydroxy acid synthase, respectively, were required for this phenomenon to occur. The results of additional experiments presented here indicate that the RNase G mutation, in combination with cra mutation, caused the increased production of pyruvic acid from glucose, which was then preferentially converted to valine due to the ilvIH mutation, resulting in depletion of isoleucine. In fact, the rng cra double mutant produced increased amount of pyruvate in the medium. These results suggest that the RNase G mutation could be applied in the breeding of producer strains of pyruvate and its derivatives such as valine.

The Escherichia coli RNase E is an essential endoribonuclease involved in processing and/or degradation of rRNAs, tRNAs, and non-coding small RNAs as well as many mRNAs. It is known that RNase E activity is somehow regulated by an RNA-binding protein Hfq, at least in some cases. We searched for proteins that showed changes in expression in both hfq::cat and rne-1 mutant cells as compared with the wild type, and found that a protein band of 49-kDa decreased in these mutant cells at 42 degrees C, the restrictive temperature for rne-1. N-terminal amino acid sequencing identified it as a mixture of GadA and GadB, two isozymes of glutamate decarboxylase involved in glutamate-dependent acid resistance. The rne-1 mutant as well as the hfq mutant showed decreased survival under acidic conditions (pH 2.5). Hfq is known to regulate the expression of GadA/B in RpoS- and GadY small RNA-dependent ways. We examined the expression of these two regulators in rne-1 mutant cells. In the mutant cells, the induction of GadY was defective at 42 degrees C, but the expression of RpoS was normal. These results indicate that RNase E is required for induction of the glutamate-dependent acid resistance system in a RpoS-independent manner.

We constructed a temperature-sensitive form of the Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The C. glutamicum/Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the E. coli cloning vector pK1, was mutagenized in vitro by treatment with hydroxylamine, and introduced into C. glutamicum cells. A mutant plasmid, which was stably maintained at 25 degrees C but not at 34 degrees C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.

Out of 95,000 commercially available chemical compounds screened by the anucleate cell blue assay, 138 selected hit compounds were further screened. As a result, A189, a 4-aminofurazan derivative was found to inhibit FtsZ GTPase with an IC(50) of 80 mug/ml and to exhibit antibacterial activity against Staphylococcus aureus and Escherichia coli. Light scattering demonstrated that A189 inhibited FtsZ assembly in vitro, and microscopic observation of A189-treated E. coli indicated that A189 perturbed FtsZ ring formation and made bacterial cells filamentous. However, nucleoids staining with DAPI revealed that A189 did not affect DNA replication and chromosome segregation in bacterial filamentous cells. Furthermore, A189 made sulA-deleted E. coli cells filamentous. Taken together, these findings suggest that A189 inhibits FtsZ GTPase activity, resulting in perturbation of FtsZ ring formation, which leads to bacterial cell death.

The emergence of antibiotic-resistant bacteria often causes serious clinical problems. The TetR family is one of the major transcription factor families that regulate expression of genes involved in bacterial antimicrobial resistance systems. CGL2612 protein is a transcription factor newly identified by genomic DNA analysis on Corynebacterium glutamicum, which belongs to the mycolic acid-containing Actinomycetales, including the well known pathogens Corynebacterium diphtheriae and Mycobacterium tuberculosis. Crystal structure analysis showed that the CGL2612 protein exhibits significant structural similarity to the multidrug resistance (MDR)-related transcription factor QacR from Staphylococcus aureus, despite poor amino acid sequence similarity between these proteins. Binding DNA sequence analysis of CGL2612 protein using the systematic evolution of ligands by the exponential enrichment (systematic evolution of ligands by exponential enrichment, or SELEX) method revealed that this protein is a new member of the TetR family, which regulates expression of the immediately upstream gene, cgl2611, probably encoding a major facilitator superfamily permease. Subsequent functional analyses confirmed a function of the CGL2612 as a transcriptional repressor responsible for the antimicrobial resistance system in C. glutamicum. The strategy used in the present study is one of the most convenient and powerful methods to analyze functionally unknown transcription factors, and the results obtained here will contribute to our understanding of the drug resistance mechanism not only in C. glutamicum but also in the related bacteria, C. diphtheriae and M. tuberculosis.

The 2-arylvinyl moiety in 1-(3-chlorophenyl)-3-(4-piperidyl)-5-[(E)-2-(5-chloro-1H-indol-3-yl)vinyl]pyrazole 2, which has previously shown improved DNA gyrase inhibition and target-related antibacterial activity, was transformed to other groups and the in vitro antibacterial activity of the synthesized compounds was evaluated. Many of the 5-[(E)-2-arylvinyl]pyrazoles synthesized in this study exhibited potent antibacterial activity against quinolone-resistant clinical isolates of Gram-positive bacteria with minimal inhibitory concentration values equivalent to those against susceptible strains.

The endoribonuclease RNase E, encoded by the essential gene rne, plays a major role in cellular RNA metabolism, i.e. maturation of functional RNAs such as rRNA and tRNA, degradation of many mRNAs and processing of the ftsZ mRNA which encodes the essential cell division protein FtsZ. RNase E function is somehow regulated by the RNA binding protein Hfq. We found that temperature-sensitive colony formation of a rne-1 mutant was partially suppressed by introduction of a hfq::cat mutation. Neither accumulation of rRNA and tRNA(Phe) precursors nor incomplete processing of ftsZ mRNA in the rne-1 mutant was rescued by the hfq::cat mutation. However, the amount of FtsZ protein that was decreased in the rne-1 mutant was recovered up to a level similar to that of wild-type cells by the hfq::cat mutation. Overproduction of Hfq inhibited cell division because of decreased expression of FtsZ. Artificial expression of the FtsZ protein from a plasmid-borne ftsZ gene partially suppressed the temperature-sensitivity of the rne-1 mutant. These results suggest that the decreased level of FtsZ is, at least in part, responsible for the inviability of RNase E-deficient cells.

SummaryFaithful 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 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.

The 4-piperidyl moiety and the pyrazole ring in 1-(3-chlorophenyl)-5-(4-phenoxyphenyl)-3-(4-piperidyl)pyrazole 2 , which has previously shown improved DNA gyrase inhibition and target-related antibacterial activity, were transformed to other groups and the in vitro antibacterial activity of the synthesized compounds was evaluated. The selected pyrazole, oxazole and imidazole derivatives showed moderate inhibition against DNA gyrase and topoisomerase IV with similar IC 50 values (IC 50 = 9.4–25 μg/mL). In addition, many of the pyrazole, oxazole and imidazole derivatives synthesized in this study exhibited potent antibacterial activity against quinolone-resistant clinical isolates and coumarin-resistant laboratory isolates of Gram-positive bacteria with minimal inhibitory concentration values equivalent to those against susceptible strains.

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.

We have previously found that a pyrazole derivative 1 possesses antibacterial activity and inhibitory activity against DNA gyrase and topoisomerase IV. Here, we synthesized new pyrazole derivatives and found that 5-[(E)-2-(5-chloroindol-3-yl)vinyl]pyrazole 16 possesses potent antibacterial activity and selective inhibitory activity against bacterial topoisomerases. Many of the synthesized pyrazole derivatives were potent against clinically isolated quinolone- or coumarin-resistant Gram-positive strains and had minimal inhibitory concentration values against these strains equivalent to those against susceptible strains.

Some rod-shaped bacteria, including Escherichia coli, exhibit cell filamentation without septum formation under high-hydrostatic-pressure conditions, indicating that the cell-division process is affected by hydrostatic pressure. The effects of elevated pressure on FtsZ-ring formation in E. coli cells were examined using indirect immunofluorescence microscopy. Elevated pressure of 40 MPa completely inhibited colony formation of E. coli cells under the cultivation conditions used, and the cells exhibited obviously filamentous shapes. In the elongated cells, normal cell-division processes appeared to be inhibited, because no FtsZ rings were observed by indirect immunofluorescent staining. In addition, it was observed that hydrostatic pressure dissociated the E. coli FtsZ polymers in vitro. These results suggest that high hydrostatic pressure directly affects cell survival and morphology through the dissociation of the cytoskeletal frameworks.

In this study, we designed and synthesized novel 5-vinylpyrazole analogues by decreasing the lipophilicity of the parent compounds 1a,b; 3-[(3-methoxycarbonyl)cyclohexylaminomethyl]indazoles while keeping the van der Waals interaction with the lipophilic area of DNA gyrase B. The selected compound 8bb exhibited good antibacterial activity against staphylococci and enterococci, including multi-drug resistant strains.

In this study, we report the design, synthesis and structure-activity relationships of novel indazole derivatives as DNA gyrase inhibitors with Gram-positive antibacterial activity. Our results show that selected compounds from this series exhibit potent antibacterial activity against Gram-positive bacteria including multi-drug resistant strains that is methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE).

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.

To improve the efficiency of sterilization by high hydrostatic pressure treatment (HPT), it is desirable to know the biochemical process of bacteria most sensitive to the treatment. We investigated growth properties after release from HPT of exponentially growing Escherichia coli K-12 cells. We observed growth retardation after treatment (30 min at 37 degrees C) above 75 MPa. Long filamentous cells of about eight times normal cell length were observed at 90 min growth after treatment at 75 MPa. In the subsequent period the filamentous cells divided into normal-sized cells. recA and sulA mutant strains also formed filamentous cells, indicating that filamentation was SulA-independent. Nucleoids segregated normally in the filamentous cells. Only one FtsZ ring (or none) was detected at possible division sites in the elongated cells. Western blotting analysis demonstrated that the amount of FtsZ protein was not affected by the treatment. GTP-dependent in vitro polymerization of either FtsZ protein in E. coli crude extract or purified FtsZ protein, however, was sensitive to HPT. These facts suggest that HPT at 75 MPa denatures a fraction of FtsZ molecules, and that these denatured molecules interfere with the polymerization of functional FtsZ, resulting in the significantly reduced number of FtsZ rings.

Bone-resorbing osteoclasts exhibit polarized morphological structures such as actin rings, clear zones, and ruffled borders. To gain insight into the mechanism of bone-resorbing activity of osteoclast and to discover new types of anti-resorptive agents, we have screened for natural compounds that inhibit the bone-resorbing activity of osteoclast-like multinucleated cells (OCLs). Destruxin B (DestB) and E (DestE), cyclodepsipeptides, were found to inhibit pit formation without affecting osteoclast differentiation and survival. Destruxins reversibly induced morphological changes in OCLs in a dose-dependent manner (DestB, 0.2-1 microM; DestE, 0.01-0.05 microM) and inhibited pit formation. Destruxin-induced morphological changes were accompanied by disruption of the actin rings in OCLs. The formation of actin rings in OCLs after adhesion was also inhibited by destruxins. Electron microscopical analysis revealed that destruxin-treated OCLs on dentine slices have no prominent clear zones and ruffled borders. The effective concentrations of destruxins on the morphological changes were almost the same as those that inhibited bone resorption in organ culture system. These results suggest that the anti-resorptive effects of destruxins result from induction of a disorder of the morphological structures in polarized OCLs.

The dcw cluster of genes involved in cell division and cell wall synthesis from the piezophilic deep-sea bacterium Shewanella violacea was isolated and characterized. It comprises 15 open reading frames, of which the organization is mraZ-mraW-ftsL-ftsI-murE-murF-mraY-murD-ftsW-murG-murC-ftsQ-ftsA-ftsZ-envA, in that order. To analyze transcription upstream from the ftsZ gene, Northern blot and primer extension analyses were performed. The results showed that gene expression is not pressure dependent. Western blot analysis showed that the FtsZ protein is equally expressed under several pressure conditions in the range of atmospheric (0.1 MPa) to high (50 MPa) pressures. Using immunofluorescence microscopy, the FtsZ ring was observed in the center of cells at pressure conditions of 0.1 to 50 MPa. These results imply that the FtsZ protein function is not affected by elevated pressure in this piezophilic bacterium.

To propose candidates for the prevention or treatment of osteoporosis, we have screened compounds naturally in food for their ability to regulate the differentiation and function of osteoclasts. One of the major green tea flavonoids, (-)-epigallocatechin-3-gallate (EGCG), was found to induce apoptotic cell death of osteoclast-like multinucleated cells after 24 h treatment in a dose-dependent manner (25-100 microM), whereas osteoblasts were not affected. In the present study, we report for the first time a novel cell-death-inducing mechanism triggered by EGCG. The induction of apoptosis by EGCG was suppressed by pretreatment of catalase or calcitonin. It was also suppressed by Fe(III) and Fe(II) chelators. Furthermore, EGCG promoted the reduction of Fe(III) into Fe(II), and the combination of EGCG/Fe(III)/H(2)O(2) induced single-strand DNA breakage in a cell free system. These results indicate that the Fenton reaction is primarily involved in EGCG-induced osteoclastic cell death.

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.

No one has, as yet, addressed the relationship between the nature of the outer membrane and cell division. kdsA encodes 3-deoxy-D-manno-octulosonic acid (KDO) 8-phosphate synthetase which catalyses the first step in the synthesis of KDO, the linker between lipid A and oligosaccharide of lipopolysaccharide (LPS). Seven temperature-sensitive mutants containing missense mutations in kdsA were affected in the production of KDO and all mutants stopped dividing at 41 degrees C and formed filaments with either one or no FtsZ ring. All observed defects were reversed by the plasmid-borne wild-type kdsA gene. Western blotting analysis, however, demonstrated that the amount of FtsZ protein was not affected by the mutation. The mutants were more susceptible to various hydrophobic materials, such as novobiocin, eosin Y and SDS at 36 degrees C. Methylene blue, however, restored kdsA mutant growth. Plasmid-borne wild-type msbA, encoding a lipid A transporter in the ABC family, partially suppressed kdsA mutation. A mutation of lpxA, functioning at the first stage in lipid A biosynthesis, inhibited both cell division and growth, producing short filaments. These results indicate that the instability of the outer membrane, caused by the defect in KDO biosynthesis, affects FtsZ-ring formation.

Escherichia coli RNase G, encoded by the rng gene, is involved in the processing of 16S rRNA and degradation of the adhE mRNA encoding a fermentative alcohol dehydrogenase. In a search for the intracellular target RNAs of RNase G other than the 16S rRNA precursor and adhE mRNA, total cellular proteins from rng+ and rng::cat cells were compared by two-dimensional gel electrophoresis. The amount of enolase encoded by the eno gene reproducibly increased two- to three-fold in the rng::cat mutant strain compared with the rng+ parent strain. Rifampicin chase experiments showed that the half-life of the eno mRNA was some 3 times longer in the rng::cat mutant than in the wild type. These results indicate that the eno mRNA was a substrate of RNase G in vivo, in addition to 16S rRNA precursor and adhE mRNA.

Escherichia coli RNase G encoded by the rng gene is involved in degradation of adhE mRNA. Overproduction of the AdhE protein by rng mutants was found to depend on the genetic background of strains derived from DC272 (adhC81) or MC1061. We found that DC272 carried a point mutation in the Cra-binding site of the adhE promoter. The Cra protein encoded by the cra gene is known to act as a repressor of adhE. P1-phage-mediated transduction and lacZ fusion analysis with the mutant adhE promoter confirmed that this mutation is responsible for overproduction. On the other hand, Southern hybridization revealed that MC1061 had a 0.85-kb deletion of the cra gene. Overproduction of AdhE in the MC1061 background was reversed to the wild-type levels by introduction of a plasmid carrying the cra(+) gene. These results indicated that expression of the adhE gene was regulated transcriptionally by Cra and posttranscriptionally by RNase G.