menaquinone-6 and demethylmenaquinone

Topics: Animals; Bacillus subtilis; Bacteria; Escherichia coli; Genes, Bacterial; Intestines; Ketoglutaric Acids; Lactobacillus; Methylation; Mycobacterium; Naphthols; Oxygen; Phenylbutyrates; Prevotella melaninogenica; Shikimic Acid; Staphylococcus aureus; Vitamin K; Vitamin K 2

Whereas the primary actions of β-lactams are well characterized, their downstream effects are less well understood. Although their targets are extracellular, β-lactams stimulate respiration in Escherichia coli leading to increased intracellular accumulation of reactive oxygen species (ROS). Here, we show that β-lactams over a large concentration range trigger a strong increase in ROS production in Enterococcus faecalis under aerobic, but not anaerobic, conditions. Both amoxicillin, to which the bacterium is susceptible, and cefotaxime, to which E. faecalis is resistant, triggers this response. This stimulation of ROS formation depends mainly on demethylmenaquinone (DMK), a component of the E. faecalis respiratory chain, but in contrast to E. coli is observed only in the absence of respiration. Our results suggest that in E. faecalis, β-lactams increase electron flux through the respiratory chain, thereby stimulating the auto-oxidation of reduced DMK in the absence of respiration, which triggers increased extracellular ROS production.

Topics: Amoxicillin; beta-Lactams; Enterococcus faecalis; Escherichia coli; Oxidative Stress; Reactive Oxygen Species; Vitamin K 2

Extracellular electron transfer (EET) in microbial cells is essential for certain biotechnological applications and contributes to the biogeochemical cycling of elements and syntrophic microbial metabolism in complex natural environments. The Gram-positive lactic acid bacterium Enterococcus faecalis, an opportunistic human pathogen, is shown to be able to transfer electrons generated in fermentation metabolism to electrodes directly and indirectly via mediators. By exploiting E. faecalis wild-type and mutant cells, we demonstrate that reduced demethylmenaquinone in the respiratory chain in the bacterial cytoplasmic membrane is crucial for the EET. Heme proteins are not involved, and cytochrome bd oxidase activity was found to attenuate EET. These results are significant for the mechanistic understanding of EET in bacteria and for the design of microbial electrochemical systems. The basic findings infer that in dense microbial communities, such as in biofilm and in the large intestine, metabolism in E. faecalis and similar Gram-positive lactic acid bacteria might be electrically connected to other microbes. Such a transcellular electron transfer might confer syntrophic metabolism that promotes growth and other activities of bacteria in the microbiota of humans and animals.

Topics: Biofilms; Cytochromes; Electricity; Electrochemical Techniques; Electrodes; Electron Transport; Electrons; Enterococcus faecalis; Fermentation; Gram-Positive Bacterial Infections; Humans; Oxidation-Reduction; Vitamin K 2

Active tuberculosis (TB) and latent

Topics: Adenosine Triphosphate; Antitubercular Agents; Biphenyl Compounds; Drug Discovery; Drug Resistance, Bacterial; Humans; Methyltransferases; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Small Molecule Libraries; Vitamin K 2

Aerobic respiration metabolism in Group B Streptococcus (GBS) is activated by exogenous heme and menaquinone. This capacity enhances resistance of GBS to acid and oxidative stress and improves its survival. In this work, we discovered that GBS is able to respire in the presence of heme and 1,4-dihydroxy-2-naphthoic acid (DHNA). DHNA is a biosynthetic precursor of demethylmenaquinone (DMK) in many bacterial species. A GBS gene (gbs1789) encodes a homolog of the MenA 1,4-dihydroxy-2-naphthoate prenyltransferase enzyme, involved in the synthesis of demethylmenaquinone. In this study, we showed that gbs1789 is involved in the biosynthesis of long-chain demethylmenaquinones (DMK-10). The Δgbs1789 mutant cannot respire in the presence of heme and DHNA, indicating that endogenously synthesized DMKs are cofactors of the GBS respiratory chain. We also found that isoprenoid side chains from GBS DMKs are produced by the protein encoded by the gbs1783 gene, since this gene can complement an Escherichia coli ispB mutant defective for isoprenoids chain synthesis. In the gut or vaginal microbiote, where interspecies metabolite exchanges occur, this partial DMK biosynthetic pathway can be important for GBS respiration and survival in different niches.

Topics: Benzoquinones; Biosynthetic Pathways; Heme; Metabolic Networks and Pathways; Naphthols; Streptococcus agalactiae; Vitamin K 2

Quinones are essential building blocks of respiration, a universal process dedicated to efficient harvesting of environmental energy and its conversion into a transmembrane chemiosmotic potential. Quinones differentiate mostly by their midpoint redox potential. As such, γ-proteobacteria such as Escherichia coli are characterized by the presence of demethylmenaquinone (DMK) with an intermediate redox potential between low-potential (menaquinone) and high-potential (ubiquinone) quinones. In this study, we show that demethylmenaquinol (DMKH2) is a good substrate for nitrate reductase A (NarGHI) in nitrate respiration in E. coli. Kinetic studies performed with quinol analogs on NarGHI show that removal of the methyl group on the naphthoquinol ring impacts modestly the catalytic constant but not the KM. EPR-monitored redox titrations of NarGHI-enriched membrane vesicles reveal that endogeneous demethylmenasemiquinone (DMSK) intermediates are stabilized in the enzyme. The measured midpoint potential of the DMK/DMKH2 redox couple in NarGHI (E'm,7.5 (DMK/DMKH2) ~-70mV) is significantly lower than that previously measured for unbound species. High resolution pulsed EPR experiments demonstrate that DMSK are formed within the NarGHI quinol oxidation site. Overall, our results provide the first characterization of a protein-bound DMSK and allows for comparison for distinct use of three quinones at a single Q-site in NarGHI.

Topics: Benzoquinones; Cell Respiration; Electron Spin Resonance Spectroscopy; Escherichia coli; Hydroquinones; Kinetics; Naphthols; Nitrate Reductase; Nitrates; Oxidation-Reduction; Vitamin K 2

Oxygen and oxidative stress have become relevant components in clarifying the mechanism that weakens bacterial cells in parallel to the mode of action of bactericidal antibiotics. Given the importance of oxidative stress in the overall defense mechanism of bacteria and their apparent role in the antimicrobial mode of action, it is important to understand how bacteria respond to this stress at a metabolic level. The aim of this study was to determine the impact of oxygen on the metabolism of the facultative anaerobe Enterococcus faecalis using continuous culture, metabolomics, and (13)C enrichment of metabolic intermediates. When E. faecalis was rapidly transitioned from anaerobic to aerobic growth, cellular metabolism was directed toward intracellular glutathione production and glycolysis was upregulated 2-fold, which increased the supply of critical metabolite precursors (e.g., glycine and glutamate) for sulfur metabolism and glutathione biosynthesis as well as reducing power for cellular respiration in the presence of hemin. The ultimate metabolic response of E. faecalis to an aerobic environment was the upregulation of fatty acid metabolism and benzoate degradation, which was linked to important changes in the bacterial membrane composition as evidenced by changes in membrane fatty acid composition and the reduction of membrane-associated demethylmenaquinone. These key metabolic pathways associated with the response of E. faecalis to oxygen may represent potential new targets to increase the susceptibility of this bacterium to bactericidal drugs.

Topics: Aerobiosis; Anaerobiosis; Enterococcus faecalis; Fatty Acids; Gene Expression Regulation, Bacterial; Metabolomics; Oxygen; Transcriptome; Up-Regulation; Vitamin K 2

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in 'respiratory' electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.

Topics: Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Membrane Proteins; Oxidation-Reduction; Phosphorylation; Protein Kinases; Signal Transduction; Ubiquinone; Vitamin K 2

Two novel Gram-stain-positive bacteria, designated HR08-7(T) and HR08-43(T), were isolated from a sea sediment sample from Rishiri Island, Hokkaido, Japan, and their taxonomic positions were investigated by a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strains HR08-7(T) and HR08-43(T) and the members of the genus Demequina formed a monophyletic cluster with similarity range of 95.5-99.0 %. The peptidoglycan type of strains HR08-7(T) and HR08-43(T) was A4β. The predominant menaquinone of both strains was demethylmenaquinone DMK-9(H(4)) and the major fatty acid was anteiso-C(15 : 0). The DNA G+C contents of strains HR08-7(T) and HR08-43(T) were 64.5 and 62.4 mol%, respectively. The results of phylogenetic analysis and DNA-DNA hybridization, along with differences of strains HR08-7(T) and HR08-43(T) from the recognized Demequina species in phenotypic characteristics, indicate that the two strains merit classification as representatives of two novel species of the genus Demequina, for which the names Demequina flava sp. nov. and Demequina sediminicola sp. nov. are proposed; the type strains are HR08-7(T) (= NBRC 105854(T) = DSM 24865(T)) and HR08-43(T) (= NBRC 105855(T) = DSM 24867(T)), respectively.

Topics: Actinomycetales; Bacterial Typing Techniques; Base Composition; DNA, Bacterial; Fatty Acids; Geologic Sediments; Japan; Molecular Sequence Data; Nucleic Acid Hybridization; Phylogeny; RNA, Ribosomal, 16S; Seawater; Sequence Analysis, DNA; Vitamin K 2; Water Microbiology

A Gram-positive, aerobic, non-motile, non-endospore-forming rod-shaped bacterium with ibuprofen-degrading capacity, designated strain I11(T), was isolated from activated sludge from a wastewater treatment plant. The major respiratory quinone was demethylmenaquinone DMK-7, C18 : 1 cis9 was the predominant fatty acid, phosphatidylglycerol was the predominant polar lipid, the cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid and the G+C content of the genomic DNA was 74.1 mol%. On the basis of 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of strain I11(T) were Patulibacter ginsengiterrae CECT 7603(T) (96.8 % similarity), Patulibacter minatonensis DSM 18081(T) (96.6 %) and Patulibacter americanus DSM 16676(T) (96.6 %). Phenotypic characterization supports the inclusion of strain I11(T) within the genus Patulibacter (phylum Actinobacteria). However, distinctive features and 16S rRNA gene sequence analysis suggest that is represents a novel species, for which the name Patulibacter medicamentivorans sp. nov. is proposed. The type strain is I11(T) ( = DSM 25962(T) = CECT 8141(T)).

Topics: Actinobacteria; Bacterial Typing Techniques; Base Composition; DNA, Bacterial; Fatty Acids; Phosphatidylglycerols; Phylogeny; Portugal; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sewage; Vitamin K 2; Wastewater

The respiratory chain of Escherichia coli contains three quinones. Menaquinone and demethylmenaquinone have low midpoint potentials and are involved in anaerobic respiration, while ubiquinone, which has a high midpoint potential, is involved in aerobic and nitrate respiration. Here, we report that demethylmenaquinone plays a role not only in trimethylaminooxide-, dimethylsulfoxide- and fumarate-dependent respiration, but also in aerobic respiration. Furthermore, we demonstrate that demethylmenaquinone serves as an electron acceptor for oxidation of succinate to fumarate, and that all three quinol oxidases of E. coli accept electrons from this naphtoquinone derivative.

Topics: Aerobiosis; Electron Transport; Escherichia coli; Oxidation-Reduction; Oxygen Consumption; Quinones; Ubiquinone; Vitamin K 2

Ubiquinones (UQs) and menaquinones (MKs) perform distinct functions in Escherichia coli. Whereas, in general, UQs are primarily involved in aerobic respiration, the MKs serve as electron carriers in anaerobic respiration. Both UQs and MKs can accept electrons from various dehydrogenases, and may donate electrons to different oxidases. Hence, they play a role in maintaining metabolic flexibility in E. coli whenever this organism has to adapt to conditions with changing redox characteristics, such as oxygen availability. Here, the authors report on the changes in both the size and the redox state of the quinone pool when the environment changes from being well aerated to one with low oxygen availability. It is shown that such transitions are accompanied by a rapid increase in the demethylmenaquinone pool, and a slow increase in the MK pool. Moreover, in exponentially growing cultures in a well-shaken Erlenmeyer flask, it is observed that the assumption of a pseudo-steady state does not hold with respect to the redox state of the quinone pool.

Topics: Aerobiosis; Anaerobiosis; Carbon; Chromatography, High Pressure Liquid; Escherichia coli; Microbiological Techniques; Oxidation-Reduction; Quinones; Ubiquinone; Vitamin K 2

The intestinal commensal bacterium, Enterococcus faecalis, is unusual among prokaryotic organisms in its ability to produce substantial extracellular superoxide. Transposon mutagenesis, allelic replacement, and electron spin resonance (ESR)-spin trapping showed that superoxide production and generation of derivative hydroxyl radical were dependent on membrane-associated demethylmenaquinone. Extracellular superoxide was generated through univalent reduction of oxygen by reduced demethylmenaquinone. Moreover, extracellular superoxide production was inhibited by exogenous haematin, an essential cofactor for cytochrome bd, and by fumarate, a substrate for fumarate reductase. As integral membrane quinol oxidases, cytochrome bd and fumarate reductase redox cycle demethylmenaquinone, and are necessary for aerobic and anaerobic respiration respectively. A rat model of intestinal colonization demonstrated that conditions exist in the mammalian intestinal tract that permit a mode of respiration for E. faecalis that results in the formation of hydroxyl radical. These results identify and characterize the mechanism by which E. faecalis generates extracellular free radicals.

Topics: Animals; DNA Transposable Elements; Electron Spin Resonance Spectroscopy; Enterococcus faecalis; Intestines; Male; Molecular Sequence Data; Mutagenesis, Insertional; Oxidoreductases; Rats; Rats, Wistar; Sequence Analysis, DNA; Superoxides; Vitamin K 2

Strains of Escherichia coli with mutations in the ubiE gene are not able to catalyze the carbon methylation reaction in the biosynthesis of ubiquinone (coenzyme Q) and menaquinone (vitamin K2), essential isoprenoid quinone components of the respiratory electron transport chain. This gene has been mapped to 86 min on the chromosome, a region where the nucleic acid sequence has recently been determined. To identify the ubiE gene, we evaluated the amino acid sequences encoded by open reading frames located in this region for the presence of sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferases. One open reading frame in this region (o251) was found to encode these motifs, and several lines of evidence that confirm the identity of the o251 product as UbiE are presented. The transformation of a strain harboring the ubiE401 mutation with o251 on an expression plasmid restored both the growth of this strain on succinate and its ability to synthesize both ubiquinone and menaquinone. Disruption of o251 in a wild-type parental strain produced a mutant with defects in growth on succinate and in both ubiquinone and menaquinone synthesis. DNA sequence analysis of the ubiE401 allele identified a missense mutation resulting in the amino acid substitution of Asp for Gly142. E. coli strains containing either the disruption or the point mutation in ubiE accumulated 2-octaprenyl-6-methoxy-1,4-benzoquinone and demethylmenaquinone as predominant intermediates. A search of the gene databases identified ubiE homologs in Saccharomyces cerevisiae, Caenorhabditis elegans, Leishmania donovani, Lactococcus lactis, and Bacillus subtilis. In B. subtilis the ubiE homolog is likely to be required for menaquinone biosynthesis and is located within the gerC gene cluster, known to be involved in spore germination and normal vegetative growth. The data presented identify the E. coli UbiE polypeptide and provide evidence that it is required for the C methylation reactions in both ubiquinone and menaquinone biosynthesis.

Topics: Alleles; Amino Acid Sequence; Base Sequence; Escherichia coli; Escherichia coli Proteins; Genes, Bacterial; Hydroquinones; Methyltransferases; Molecular Sequence Data; Mutation; Open Reading Frames; Point Mutation; Sequence Alignment; Succinates; Succinic Acid; Transformation, Bacterial; Ubiquinone; Vitamin K; Vitamin K 2

The aeration-dependent changes in content of various quinones in Escherichia coli were found to be unaffected by a prokaryotic translation inhibitor chloramphenicol. In addition, this process was shown to be completely intact in cells with mutated fnr, arc and appY loci. It is assumed that E. coli possesses a special system of oxygen-dependent post-transcriptional regulation of the quinone biosynthetic pathways.

Topics: Aerobiosis; Anaerobiosis; Chloramphenicol; Cytochrome b Group; Cytochromes; Electron Transport Chain Complex Proteins; Escherichia coli; Escherichia coli Proteins; Genotype; Oxidoreductases; Quinones; Species Specificity; Ubiquinone; Vitamin K; Vitamin K 2

The mutant strain AN70 (ubiE) of Escherichia coli which is known to lack ubiquinone (Young IG et al. 1971), was analyzed for menaquinone (MK) and demethylmenaquinone (DMK) contents. In contrast to the wild-type, strain AN70 contained only DMK, but no MK. The mutant strain was able to grow with fumarate, trimethylamine N-oxide (TMAO) and dimethylsulfoxide (DMSO), but not with nitrate as electron acceptor. The membranes catalyzed anaerobic respiration with fumarate and TMAO at 69 and 74% of wild-type rates. DMSO respiration was reduced to 38% of wild-type activities and nitrate respiration was missing (less than or equal to 8% of wild-type), although the respective enzymes were present in wild-type rates. The results complement earlier findings which demonstrated a role for DMK only in TMAO respiration (Wissenbach et al. 1990). It is concluded, that DMK (in addition to MK) can serve as a redox mediator in fumarate, TMAO and to some extent in DMSO respiration, but not in nitrate respiration. In strain AN70 (ubiE) the lack of ubiquinone (Q) is due to a defect in a specific methylation step of Q biosynthesis. Synthesis of MK from DMK appears to depend on the same gene (ubiE).

Topics: Anaerobiosis; Dimethyl Sulfoxide; Electron Transport; Escherichia coli; Fumarates; Methylamines; Mutation; Nitrates; Oxidants; Oxidation-Reduction; Vitamin K; Vitamin K 2

The respiratory activities of E. coli with H2 as donor and with nitrate, fumarate, dimethylsulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as acceptor were measured using the membrane fraction of quinone deficient strains. The specific activities of the membrane fraction lacking naphthoquinones with fumarate, DMSO or TMAO amounted to less than or equal to 2% of those measured with the membrane fraction of the wild-type strain. After incorporation of vitamin K1 [instead of menaquinone (MK)] into the membrane fraction deficient of naphthoquinones, the activities with fumarate or DMSO were 92% or 17%, respectively, of the activities which could be theoretically achieved. Incorporation of demethylmenaquinone (DMK) did not lead to a stimulation of the activities of the mutant. In contrast, the electron transport activity with TMAO was stimulated by the incorporation of either vitamin K1 or DMK. Nitrate respiration was fully active in membrane fractions lacking either naphthoquinones or Q, but was less than or equal to 3% of the wild-type activity, when all quinones were missing. Nitrate respiration was stimulated on the incorporation of either vitamin K1 or Q into the membrane fraction lacking quinones, while the incorporation of DMK was without effect. These results suggest that MK is specifically involved in the electron transport chains catalyzing the reduction of fumarate or DMSO, while either MK or DMK serve as mediators in TMAO reduction. Nitrate respiration requires either Q or MK.

Topics: Anaerobiosis; Dimethyl Sulfoxide; Electron Transport; Escherichia coli; Fumarates; Methylamines; Nitrates; Oxidation-Reduction; Oxidoreductases; Vitamin K; Vitamin K 2

Escherichia coli grown with glucose in the absence of added electron acceptors contained 3-4 times more naphthoquinones (menaquinone plus demethylmenaquinone) than in the presence of O2. Presence of electron acceptors resulted in a slight additional increase of the naphthoquinone content. A strain defective in the fnr gene, which encodes the transcriptional activator of anaerobic respiration, showed the same response. With fumarate or dimethyl sulfoxide present, 94% of the naphthoquinones consisted of menaquinone, while with nitrate up to 78% was demethylmenaquinone. With trimethylamine N-oxid as the acceptor the proportion was intermediate. From the donor substrates of anaerobic respiration only glycerol had a significant influence on the ratio of the contents of the 2 quinones. It is concluded that FNR, the gene product of the fnr gene, is not required for anaerobic derepression of naphthoquinone biosynthesis. Menaquinone appears to be involved specifically in the respiration with fumarate or dimethyl sulfoxide, and demethylmenaquinone in nitrate respiration. Both naphthoquinones appear to serve in trimethylamine N-oxide respiration.

Topics: Anaerobiosis; Bacterial Proteins; Dimethyl Sulfoxide; Electron Transport; Escherichia coli; Fumarates; Gene Expression Regulation; Mutation; Oxygen; Vitamin K; Vitamin K 2

Demethylmenaquinone and menaquinone mixtures from some species of enterobacteria were analysed by reverse-phase partition high-performance liquid chromatography. This method allowed clear separation and quantitative determination of these quinone components.

Topics: Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Citrobacter; Enterobacter; Enterobacteriaceae; Enterococcus faecalis; Vitamin K; Vitamin K 2

1,4-Dihydroxy-2-naphthoate:polyprenyltransferase was detected in the membrane fraction from Micrococcus luteus. The specificity of the enzyme ws so tolerant as regards the prenyl-donating substrate that prenyl pyrophosphates ranging in chain length from C15 to C45 were active as substrates. The monophosphate esters were also active, though the reactivities were much lower than those of the corresponding pyrophosphates. The enzyme showed rigorous specificity with respect to the aromatic substrate. Neither 1,4-dihydroxynaphthalene nor its 2-methyl derivative was active at all. 1,4-Dihydroxy-3-methyl-2-naphthoate could be prenylated to afford menaquinone, but the reactivity was much less than that of its demethyl derivative. These results support the view that menaquinone biosynthesis involves the prenylation of 1,4-dihydroxy-2-naphthoate prior to decarboxylation or methylation.

Topics: Alkyl and Aryl Transferases; Cell Membrane; Farnesol; Micrococcus; Naphthols; Polyisoprenyl Phosphates; Sesquiterpenes; Substrate Specificity; Transferases; Vitamin K; Vitamin K 2

Topics: Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Solvents; Vitamin K; Vitamin K 2

Topics: Electron Transport; Haemophilus; NAD; Oxidation-Reduction; Oxygen Consumption; Quinones; Ubiquinone; Vitamin K; Vitamin K 2