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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.

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.

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 .

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.

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.

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.

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.

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.

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.

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.

Corynebacterium glutamicum is known to excrete large amounts of L-glutamic acid upon treatment by penicillin. However, the mechanism of L-glutamate overproduction by penicillin treatment is still unknown. A 5.3-kb HindIII fragment was isolated by directly introducing the C. glutamicum (Brevibacterium lactofermentum) ATCC 13869 gene library into the temperature-sensitive Escherichia coli murE mutant and selecting temperature resistant clones. Two open reading frames (ORFs) were found in this fragment: (1) murE, encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase, and (2) ftsI, encoding septum-peptidoglycan synthetase, one of the targets of penicillin (penicillin-binding protein 3). Both ORFs were involved in peptidoglycan synthesis. Proteins were synthesized from the C. glutamicum murE and ftsI genes, 55 kDa and 73 kDa respectively, in an in vitro protein synthesis system, using E. coli S30 extracts.

Background Escherichia coli rng gene (previously called cafA) encodes a novel RNase, named RNase G, which is involved in the 5′ end-processing of 16S rRNA. In rng mutant cells, a precursor form of 16S rRNA, 16.3S rRNA, is accumulated. Here we report a role of RNase G in the in vivo mRNA metabolism. Results We found that rng::cat mutant strains overproduced a protein of about 100 kDa. N-terminal amino acid sequencing of this protein showed that it was identical to the fermentative alcohol dehydrogenase, the product of the adhE gene located at 28 min on the E. coli genetic map. The level of adhE mRNA was significantly higher in the rng::cat mutant strain than that in its parental strain, while such differences were not seen in other genes we examined. A rifampicin-chase experiment revealed that the half-life of adhE mRNA was 2.5-fold longer in the rng::cat disruptant than in the wild-type. Conclusion These results indicate that, in addition to rRNA processing, RNase G is involved in in vivo mRNA degradation in E. coli.

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 Ca2+. These results suggest the possible role of calcium ion in the process of chromosome partitioning in E. coli.

We found that the Escherichia coli cafA::cat mutant accumulated a precursor of 16S rRNA. This precursor migrated to the same position with 16.3S precursor found in the BUMMER strain that is known to be deficient in the 5′ end processing of 16S rRNA. Accumulation of 16.3S rRNA in the BUMMER mutant was complemented by introduction of a plasmid carrying the cafA gene. The mutant type cafA gene cloned from the BUMMER strain had a 11-bp deletion in its coding region. A small amount of the mature 16S rRNA was still formed in the cafA::cat mutant. This residual activity was found to be due to RNase E encoded by the rne/ams gene by rifampicin-chase experiments of the cafA::cat ams1 double mutant. These results indicated that the cafA gene encodes a novel RNase responsible for processing of the 5′ end of 16S rRNA.

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.

Taking advantage of highly conserved domains present in the ftsZ genes from Escherichia coli, Rhizobium meliloti, and Bacillus subtilis, we designed degenerate oligonucleotides (oligos) corresponding to these regions. These oligos were used as primers in PCR in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA. The PCR product was used as a probe to recover genomic fragments from a lambda library of Br. flavum MJ233. The complete nucleotide sequence (nt) of the cloned 4.2-kb EcoRI fragment containing the ftsZ homolog from Br. flavum MJ233 indicated that the deduced gene product of the Br. flavum ftsZ homolog is composed of 438 amino acids (aa) with a deduced molecular weight of 46.9 kDa. This size of molecular weight was also confirmed by the in vitro protein synthesis assay. Comparison of this aa sequence to the corresponding sequences from E. coli, Rh. meliloti, B. subtilis, and Streptomyces coelicolor revealed a high degree of conservation and suggested that the Br. flavum ftsZ homolog has a putative GTP binding motif and a GTP hydrolizing region. Expression of Br. flavum ftsZ gene in E. coli, JM109 inhibited its cell division, leading to filamentation. This suggested that the Br. flavum ftsZ product competed with the E. coli ftsZ product.

The Corynebacterium glutamicum NCgl1221 mechanosensitive channel mediates L-glutamate secretion by sensing changes in membrane tension caused by treatments such as biotin limitation and penicillin. The NCgl1221 protein has an N-terminal domain (1-286 a.a.) homologous to the Escherichia coli MscS and a long C-terminal domain (287-533 a.a.) of unknown function. In order to investigate the role of the C-terminal domain in L-glutamate secretion, we constructed a series of C-terminally truncated mutants of NCgl1221. We found that the N-terminal domain, homologous to E. coli MscS, retained the ability to cause L-glutamate secretion in response to the treatment. Electrophysiological analysis confirmed that the N-terminal domain mediated L-glutamate secretion. 3D homology modeling has suggested that the N-terminal domain of NCgl1221 has an extra loop structure (221-232 a.a.) that is not found in most other MscS proteins. The mutant NCgl1221, deleted for this loop structure, lost the ability to secrete L-glutamate. In addition, we found that mutant NCgl1221 lacking the C-terminal extracytoplasmic domain (420-533 a.a.) produced L-glutamate without any inducing treatment. These results suggest that the N-terminal domain is necessary and sufficient for the excretion of L-glutamate in response to inducing treatment, and that the C-terminal extracytoplasmic domain has a negative regulatory role in L-glutamate production.

The gene encoding a 51 kDa polypeptide of Porphyromonas gingivalis 381 was isolated by immunoblotting using an antiserum raised against P. gingivalis alkaline phosphatase. DNA sequence analysis of a 2.5 kb DNA fragment containing a gene encoding the 51 kDa protein revealed one complete and two incomplete ORFs. Database searches using the FASTA program revealed significant homology between the P. gingivalis 51 kDa protein and the MurC protein of Escherichia coli, which functions in peptidoglycan synthesis. The cloned 51 kDa protein encoded a functional product that complemented an E. coli murC mutant. Moreover, the ORF just upstream of murC coded for a protein that was 31% homologous with the E. coli MurG protein. The ORF just downstream of murC coded for a protein that was 17% homologous with the Streptococcus pneumoniae penicillin-binding protein 2B (PBP2B), which functions in peptidoglycan synthesis and is responsible for antibiotic resistance. These results suggest that P. gingivalis contains a homologue of the E. coli peptidoglycan synthesis gene murC and indicate the possibility of a cluster of genes responsible for cell division and cell growth, as in the E. coli mra region.

Normal-sized anucleate cells were observed in the cultures of a delta hns mutant strain. Even in nucleate cells, some populations showed irregular intracellular localization of the nucleoids. The delta hns mutant showed reduced ploidy, although initiation of chromosome replication was essentially synchronous as defined by flow cytometric analysis. These results indicate that the delta hns mutant is defective in the mechanisms of chromosome partitioning and chromosome replication.

Corynebacterium glutamicum has one RNase E/G ortholog and one RNase J ortholog but no RNase Y. We previously reported that the C. glutamicum NCgl2281 gene encoding the RNase E/G ortholog complemented the rng::cat mutation in Escherichia coli but not the rne-1 mutation. In this study, we constructed an NCgl2281 knockout mutant and found that the mutant cells accumulated 5S rRNA precursor molecules. The processing of 16S and 23S rRNA, tRNA, and tmRNA was normal. Primer extension analysis revealed that the RNase E/G ortholog cleaved at the -1 site of the 5' end of 5S rRNA. However, 3' maturation was essentially unaffected. These findings showed that C. glutamicum NCgl2281 endoribonuclease is involved in the 5' maturation of 5S rRNA. This is the first report showing the physiological function of the RNase E/G ortholog in bacteria having one RNase E/G and one RNase J but no RNase Y.

Background The TolC outer membrane channel is a key component of several multidrug resistance (MDR) efflux pumps driven by H+ transport in Escherichia coli. While tolC expression is under the regulation of the EvgA-Gad acid resistance regulon, the role of TolC in growth at low pH and extreme-acid survival is unknown. Methods and Principal Findings TolC was required for extreme-acid survival (pH 2) of strain W3110 grown aerobically to stationary phase. A tolC deletion decreased extreme-acid survival (acid resistance) of aerated pH 7.0-grown cells by 105-fold and of pH 5.5-grown cells by 10-fold. The requirement was specific for acid resistance since a tolC defect had no effect on aerobic survival in extreme base (pH 10). TolC was required for expression of glutamate decarboxylase (GadA, GadB), a key component of glutamate-dependent acid resistance (Gad). TolC was also required for maximal exponential growth of E. coli K-12 W3110, in LBK medium buffered at pH 4.5–6.0, but not at pH 6.5–8.5. The TolC growth requirement in moderate acid was independent of Gad. TolC-associated pump components EmrB and MdtB contributed to survival in extreme acid (pH 2), but were not required for growth at pH 5. A mutant lacking the known TolC-associated efflux pumps (acrB, acrD, emrB, emrY, macB, mdtC, mdtF, acrEF) showed no growth defect at acidic pH and a relatively small decrease in extreme-acid survival when pre-grown at pH 5.5. Conclusions TolC and proton-driven MDR efflux pump components EmrB and MdtB contribute to E. coli survival in extreme acid and TolC is required for maximal growth rates below pH 6.5. The TolC enhancement of extreme-acid survival includes Gad induction, but TolC-dependent growth rates below pH 6.5 do not involve Gad. That MDR resistance can enhance growth and survival in acid is an important consideration for enteric organisms passing through the acidic stomach.

The sopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli. In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied. [alpha-32P]ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs. In contrast, no ATP binding activity was detected for the SopB protein. The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA. The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein. However, the SopB protein alone failed to stimulate the ATPase activity.

Single and multiple mutations in the mreB, mreC, and mreD genes of Escherichia coli, which are involved in rod shape determination and the accompanied change in the cell's mecillinam resistance, caused an increase in the quantity of septum-peptidoglycan synthetase penicillin-binding protein (PBP)-3, and more general peptidoglycan synthetase PBP-1B. The mreD mutant, but not the mreB and mreC mutants, showed a decreased growth which could be suppressed by deletion of the total mreBCD-orfEF area on the chromosome. The mre mutations additionally caused a complex nature increase/decrease of several other cytoplasmic and membrane protein quantities. These results suggest that the mreB, mreC, and mreD genes are involved in a complex regulatory mechanism in the process of cell growth, division and shape determination.

The murG gene located in the mra region at 2min on the Escherichia coli chromosome map was found to be responsible for UDP-GlcNAc: undecaprenyl-pyrophosphoryl-N-acetylmuramoyl-pentapeptide N-acetylglucosaminyl transferase activity in lipid cycle reactions of cell wall peptidoglycan synthesis. murG was concluded to be the structural gene for this enzyme from the following findings: (1) Expression of GlcNAc-transferase activity by the murG gene seemed to be thermosensitive when the chromosome carried an amber mutation in murG and a thermosensitive sup mutation. (2) Overproduction of the murG product caused an increase of GlcNAc-transferase activity. Incidentally, chemically phosphorylated polyprenol was shown to function as a carrier in lipid cycle reactions in peptidoglycan synthesis in E. coli.

The Corynebacterium glutamicum NCgl2281 gene encodes an RNase E/G family endoribonuclease having an additional N-terminal domain of unknown function. In this study, we constructed plasmids expressing the full length (FL) and the N-terminally truncated form (DeltaN) of NCgl2281 and examined their complementation ability as to Escherichia coli rng::cat and rne-1 mutations. Both FL- and DeltaN-NCgl2281 rescued the defects caused by the rng::cat mutation, i.e., accumulation of 16S rRNA precursor, overproduction of the AdhE protein, and growth inhibition on M9 glucose medium. On the other hand, they did not complement the rne-1 mutation. These results indicate that the C. glutamicum NCgl2281 endoribonuclease is functionally more closely related to the E. coli RNase G than to RNase E.

Amplification of the mraY gene, previously called open reading frame Y (ORF-Y, 1,080 bp), at 2 min in the chromosome map of Escherichia coli enhanced the activity of UDP-N-acetylmuramoyl-pentapeptide: undecaprenyl-phosphate phospho-N-acetylmuramoyl-pentapeptide transferase (EC 2.7.8.13). This enzyme catalyzes the formation of undecaprenyl-pyrophosphoryl-N-acetylmuramoyl-pentapeptide from UDP-N-acetylmuramoyl-pentapeptide and undecaprenyl-phosphate, the first step in the lipid cycle reactions in biosynthesis of bacterial cell wall peptidoglycans. The enhanced enzyme activity was sensitive to tunicamycin, and the amino tunicamycin-sensitive N-acetylglucosamine-1-phosphate transferase of Saccharomyces cerevisiae. Very probably mraY is the structural gene for the above enzyme.

The oriC region of Escherichia coli constructs a specific complex to associate with the outer membrane fraction (oriC complex). The oriC complex was periodically formed before as well as in the short period after initiation of DNA replication. Using the DNA extracted from outer membrane fractions of the cells just after initiation as a probe, the whole E. coli genomic library was assayed by plaque hybridization. DNA regions that hybridized with the probe corresponded to about a 100-kb length of chromosome DNA which included oriC. In addition, clones located in a counterclockwise direction from oriC were more preferentially hybridized among these positive clones. Thus, we conclude that the oriC region is a unique locus of the chromosome that binds to the outer membrane at the time of initiation of chromosome replication.

We examined formation of an oriC-membrane complex through the chromosome replication cycle by dot-blot hybridization using an oriC plasmid as a probe. In a wild-type culture synchronized for chromosome replication, oriC complex formation was observed periodically and transiently corresponding to the replication initiation event. Prior to initiation of replication the oriC complex was recovered in the outer membrane fraction as well as at the time of initiation of replication. Moreover, periodic formation of the oriC complex was observed even when further initiation of replication was suppressed by culturing an initiation ts mutant at the restrictive temperature. Similar periodic formation of the oriC complex was also observed when DNA elongation was inhibited by addition of nalidixic acid to the culture. However, the second periodic peak did not appear when rifampicin or chloramphenicol was added. Cells which formed the oriC complex at the restrictive temperature could immediately initiate chromosome replication when the cells were transferred to the permissive temperature. We conclude that the oriC region of Escherichia coli forms a specific complex periodically just before and at the time of initiation of chromosome replication and that oriC complex formation is a prerequisite for initiation of chromosome replication.

Determination of nucleotide sequence of a 2.8kb DNA fragment involving the murG and murC genes has completed the sequencing of the total 12kb mra region at 2min on the Escherichia coli chromosome map, which functions in the growth and division of the cell. Product proteins of the genes in the mra region have also been identified, of which the MurC and MurG proteins are reported here. Considerable homologies were found in the deduced amino acid sequences of four ligases, products of the murC, murD, murE and murF genes in the mra region. These synthesize UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid in peptidoglycan synthesis. The MurG protein, also involved in the cell growth of E. coli, showed considerable homology of the deduced amino acid sequence with that of a peptide coded for by an open reading frame in the spoVE-ftsZ region of the B. subtilis chromosome.

The cold-sensitive fcsA29 mutation of Escherichia coli was found to be a new type of cold-sensitive allele of the polA gene encoding DNA polymerase I, caused by an Asp(116)--Asn change in the 5'--3' exonuclease domain. The fcsA29 mutant showed typical polA mutant phenotypes such as UV sensitivity and unacceptability of recA mutation. Cold-sensitive growth of the mutant was suppressed by introduction of a sulA mutation, indicating that cell filamentation was due to the SOS response.

We investigated the mode of action of ES-1273, a novel DNA gyrase inhibitor obtained by optimization of ES-0615, which was found by screening our chemical library using anucleate cell blue assay. ES-1273 exhibited the same antibacterial activity against S. aureus strains with amino acid change(s) conferring quinolone- and coumarin-resistance as that against a susceptible strain. In addition, ES-1273 inhibited DNA gyrase supercoiling activity, but not ATPase activity of the GyrB subunit of DNA gyrase. Moreover, ES-1273 did not induce cleavable complex. These findings demonstrate that the mechanism by which ES-1273 inhibits DNA gyrase is different from that of the quinolones or the coumarins. Preincubation of DNA gyrase and substrate DNA prevented inhibition of DNA gyrase supercoiling activity by ES-1273. ES-1273 antagonized quinolone-induced cleavage. In electrophoretic mobility shift assay, no band representing DNA gyrase-DNA complex was observed in the presence of ES-1273. Taken together, these results indicate that ES-1273 prevents DNA from binding to DNA gyrase. Furthermore, our results from surface plasmon resonance experiments strongly suggest that ES-1273 interacts with DNA. Therefore, the interaction between ES-1273 and DNA prevents DNA from binding to DNA gyrase, resulting in inhibition of DNA gyrase supercoiling. Interestingly, we also found that ES-1273 inhibits topoisomerase IV and human topoisomerase IIalpha, but not human topoisomerase I. These findings indicate that ES-1273 is a type II topoisomerase specific inhibitor.

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 sigma(D)-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.

The actin-like MreB cytoskeletal protein and RNA polymerase (RNAP) have both been suggested to provide the force for chromosome segregation. Here, we identify MreB and RNAP as in vivo interaction partners. The interaction was confirmed using in vitro purified components. We also present convincing evidence that MreB and RNAP are both required for chromosome segregation in Escherichia coli. MreB is required for origin and bulk DNA segregation, whereas RNAP is required for bulk DNA, terminus, and possibly also for origin segregation. Furthermore, flow cytometric analyses show that MreB depletion and inactivation of RNAP confer virtually identical and highly unusual chromosome segregation defects. Thus, our results raise the possibility that the MreB–RNAP interaction is functionally important for chromosome segregation.

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.

Transcriptional initiation sites of the ostA gene involved in organic solvent sensitivity in Escherichia coli were found by primer extension analysis. Two transcriptional initiation sites were newly identified at -133 and -48 nucleotides from the initiation codon of ostA, but the previously reported sigmaE-dependent one at -227 could not be detected. No heat-inducible expression of ostA was observed by Northern blotting analysis, indicating that the contribution of sigmaE-dependent transcription was very small if any. SigmaD-dependent promoter-like sequences were found just upstream of the newly identified transcriptional initiation sites by computer-aided analysis. Deletion analysis of ostA-lacZ fusions demonstrated that these two promoters contributed almost equally to the constitutive expression of the ostA gene.

Corynebacterium glutamicum mutant KY9707 was originally isolated for lysozyme-sensitivity, and showed temperature-sensitive growth. Two DNA fragments from a wild-type C. glutamicum chromosomal library suppressed the temperature-sensitivity of KY9707. These clones also rescued the lysozyme-sensitivity of KY9707, although partially. One of them encodes a protein of 382 amino acid residues, the N-terminal domain of which was homologous to RNase HI. This gene suppressed the temperature-sensitive growth of an Escherichia coli rnhA rnhB double mutant. We concluded that this gene encodes a functional RNase HI of C. glutamicum and designated it as rnhA. The other gene encodes a protein of 707 amino acid residues highly homologous to RecG protein. The C. glutamicum recG gene complemented the UV-sensitivity of E. coli recG258::kan mutant. KY9707 showed increased UV-sensitivity, which was partially rescued by either the recG or rnhA gene of C. gluamicum. Point mutations were found in both recG and rnhA genes in KY9707. These suggest that temperature-sensitive growth, UV-sensitivity, and probably lysozyme-sensitivity also, of KY9707 were caused by mutations in the genes encoding RNase HI and RecG.

The Streptomyces coelicolor gene SCC88.10c encodes a protein (RNase ES) which is homologous to endoribonucleases in the RNase E/G family. We expressed S. coelicolor RNase ES as a 6 x His-tagged protein in an Escherichia coli mutant carrying a rng (which encodes RNase G) or a rne (which encodes RNase E) mutation to study whether S. coelicolor RNase ES is able to complement these mutations in host E. coli cells. The results clearly indicated that the S. coelicolor RNase ES can partially abrogate either the rng::cat or rne-1 mutation, as measured by the ability to suppress the several aberrant phenotypes resulting from the rng or rne mutation. Thus, S. coelicolor RNase ES appears to have the dual ability to supplant the functions of both RNase G and RNase E in E. coli.

The FtsZ protein is a GTPase that is essential for cell division. We have cloned, sequenced, and expressed the FtsZ (PgFtsZ) gene from the Porphyromonas gingivalis, an oral, anaerobic, rod-shaped bacterium implicated in progressive periodontal disease. The PgFtsZ gene consisted of 1374 bp and coded for an acidic protein with a calculated molecular mass of 50,253 Da. The deduced amino acid sequence exhibited a significant homology with E. coli FtsZ (54% identical residues). Like other prokaryotic FtsZs, PgFtsZ possessed the clear motifs for GTP binding (GGGTGTG) and hydrolysis (NLDFADV). When PgFtsZ was overexpressed in E. coli, cell division was inhibited. Recombinant PgFtsZ was purified to homogeneity and characterized. The purified PgFtsZ exhibited GTPase activity even in the absence of Mg2+, and completely retained its activity with EDTA. Furthermore, Na+ and K+ ions inhibited its GTPase activity in a dose-dependent manner. These results suggest that PgFtsZ contains an atypical GTPase activity that has not been previously described.

The elongation of Escherichia coli cells, in response to high hydrostatic pressure growth, is a unique property of this bacterium. Our high pressure observation microscope system (HPOMS) has provided direct observation of living E. coli elongated cells under high pressure. To study elongation mechanisms of E. coli under high pressure, the formation of FtsZ ring which was an early event of cell division was examined by immuno-fluorescence microscopy (IFM), and the chromosomal DNA was also detected by DAPI staining. The FtsZ-ring formation and the condensed nucleoid structure was observed in the filamentous cells when the cells were fixed after releasing the pressure. Surprisingly, no FtsZ ring was observed in whole filamentous cells, when the cells were fixed without decompression. Also the chromosomal DNA was segregated but not condensed at the same condition. These results suggested that the cell division of E. coli was inhibited by high hydrostatic pressure at early stage in the cell cycle before the FtsZ-ring formation during cultivation, however, cells could grow continuously without septum formation, chromosomal DNA condensation.