Identification of Escherichia coli ubiB, a gene required for the first monooxygenase step in ubiquinone biosynthesis

It was recently discovered that the aarF gene in Providencia stuartii is required for coenzyme Q (CoQ) biosynthesis. Here we report that yigR, the Escherichia coli homologue of aarF, is ubiB, a gene required for the first monooxygenase step in CoQ biosynthesis. Both the P. stuartii aarF and E. coli ubiB (yigR) disruption mutant strains lack CoQ and accumulate octaprenylphenol. Octaprenylphenol is the CoQ biosynthetic intermediate found to accumulate in the E. coli strain AN59, which contains the ubiB409 mutant allele. Analysis of the mutation in the E. coli strain AN59 reveals no mutations within the ubiB gene, but instead shows the presence of an IS1 element at position +516 of the ubiE gene. The ubiE gene encodes a C-methyltransferase required for the synthesis of both CoQ and menaquinone, and it is the 5' gene in an operon containing ubiE, yigP, and ubiB. The data indicate that octaprenylphenol accumulates in AN59 as a result of a polar effect of the ubiE::IS1 mutation on the downstream ubiB gene. AN59 is complemented by a DNA segment containing the contiguous ubiE, yigP, and ubiB genes. Although transformation of AN59 with a DNA segment containing the ubiB coding region fails to restore CoQ biosynthesis, transformation with the ubiE coding region results in a low-frequency but significant rescue attributed to homologous recombination. In addition, the fre gene, previously considered to correspond to ubiB, was found not to be involved in CoQ biosynthesis. The ubiB gene is a member of a predicted protein kinase family of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member. The possible protein kinase function of UbiB and Abc1 and the role these polypeptides may play in CoQ biosynthesis are discussed.     

The distal heme center in Bacillus subtilis succinate:quinone reductase is crucial for electron transfer to menaquinone

Succinate:quinone reductases are membrane-bound enzymes that catalyze electron transfer from succinate to quinone. Some enzymes in vivo reduce ubiquinone (exergonic reaction) whereas others reduce menaquinone (endergonic reaction). The succinate:menaquinone reductases all contain two heme groups in the membrane anchor of the enzyme: a proximal heme (heme b(P)) located close to the negative side of the membrane and a distal heme (heme b(D)) located close to the positive side of the membrane. Heme b(D) is a distinctive feature of the succinate:menaquinone reductases, but the role of this heme in electron transfer to quinone has not previously been analyzed. His28 and His113 are the axial ligands to heme b(D) in Bacillus subtilis succinate:menaquinone reductase. We have individually replaced these His residues with Leu and Met, respectively, resulting in assembled membrane-bound enzymes. The H28L mutant enzyme lacks succinate:quinone reductase activity probably due to a defective quinone binding site. The H113M mutant enzyme contains heme b(D) with raised midpoint potential and is impaired in electron transfer to menaquinone. Our combined experimental data show that the heme b(D) center, into which we include a quinone binding site, is crucial for succinate:menaquinone reductase activity. The results support a model in which menaquinone is reduced on the positive side of the membrane and the transmembrane electrochemical potential provides driving force for electron transfer from succinate via heme b(P) and heme b(D) to menaquinone.     

Extracellular S100A4 stimulates the migration rate of astrocytic tumor cells by modifying the organization of their actin cytoskeleton

An overview of the present knowledge about succinate:quinone oxidoreductase in Paracoccus denitrificans and Bacillus subtilis is presented. P. denitrificans contains a monoheme succinate:ubiquinone oxidoreductase that is similar to that of mammalian mitochondria with respect to composition and sensitivity to carboxin. Results obtained with carboxin-resistant P. denitrificans mutants provide information about quinone-binding sites on the enzyme and the molecular basis for the resistance. B. subtilis contains a diheme succinate:menaquinone oxidoreductase whose activity is dependent on the electrochemical gradient across the cytoplasmic membrane. Data from studies of mutant variants of the B. subtilis enzyme combined with available crystal structures of a similar enzyme, Wolinella succinogenes fumarate reductase, substantiate a proposed explanation for the mechanism of coupling between quinone reductase activity and transmembrane potential.     

Reactivity of the Bacillus subtilis succinate dehydrogenase complex with quinones.

The succinate dehydrogenase isolated from Bacillus subtilis was found to catalyze the oxidation of succinate with hydrophilic quinones. Either naphthoquinones or benzoquinones served as acceptors. The enzyme activity increased with the redox potential of the quinone. The highest turnover number was commensurate with that of the bacterial succinate respiration in vivo. The succinate dehydrogenase was similarly active in fumarate reduction with quinols. The highest activity was obtained with the most electronegative quinol. The fumarate reductase isolated from Wolinella succinogenes catalyzed succinate oxidation with quinones and fumarate reduction with the corresponding quinols at activities similar to those of the B. subtilis enzyme. Succinate oxidation by the lipophilic quinones, ubiquinone or vitamin K-1, was monitored as cytochrome c reduction using proteoliposomes containing succinate dehydrogenase together with the cytochrome bc1 complex. The activity with ubiquinone or vitamin K-1 was commensurate with the succinate respiratory activity of bacteria or of the bacterial membrane fraction. The results suggest that menaquinone is involved in the succinate respiration of B. subtilis, although its redox potential is unfavorable.     

Alternative hydroxylases for the aerobic and anaerobic biosynthesis of ubiquinone in Escherichia coli.

The synthesis of ubiquinone under anaerobic conditions was examined in a variety of strains of Escherichia coli K12. All were shown to synthesize appreciable quantities of ubiquinone 8 when grown anaerobically on glycerol in the presence of fumarate. Under these conditions, ubiquinone 8 was in most cases the principal quinone formed, and levels in the range 50--70% of those obtained aerobically were observed. Studies with mutants blocked in the various reactions of the aerobic pathway for ubiquinone 8 synthesis established that under anaerobic conditions three alternative hydroxylation reactions not involving molecular oxygen are used to derive the C-4, -5, and -6 oxygens of ubiquinone 8. Thus, mutants blocked in either of the three hydroxylation reactions of the aerobic pathway (ubiB, ubiH, or ubiF) are each able to synthesize ubiquinone 8 anaerobically, whereas mutants lacking the octaprenyltransferase (ubiA), carboxy-lyase (ubiD), or methyltransferases (ubiE or ubiG) of the aerobic pathway remain blocked anaerobically. The demonstration that E. coli possesses a special mechanism for the anaerobic biosynthesis of ubiquinone suggests that this quinone may play an important role in anaerobic metabolism.     

Characterization and Genetic Analysis of Mutant Strains of Escherichia coli K-12 Accumulating the Ubiquinone Precursors 2-Octaprenyl-6-Methoxy-1,4-Benzoquinone and 2-Octaprenyl-3-Methyl-6-Methoxy-1,4-Benzoquinone

The ubiquinone precursors, 2-octaprenyl-6-methoxy-1,4-benzoquinone and 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone, were isolated from ubiquinone-deficient mutants of Escherichia coli and identified by nuclear magnetic resonance and mass spectrometry. Mutants accumulating 2-octaprenyl-6-methoxy-1,4-benzoquinone and 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone were shown to carry mutations in genes designated ubiE and ubiF, respectively. The ubiE gene was shown to be cotransducible with metE (minute 75) and close to two other genes concerned with ubiquinone biosynthesis. The ubiF gene was located close to minute 16 by cotransduction with the lip, gltA, and entA genes.     

Isolation and characterization of Escherichia coli mutants affected in aerobic respiration: the cloning and nucleotide sequence of ubiG. Identification of an S-adenosylmethionine-binding motif in protein, RNA, and small-molecule methyltransferases.

We report the isolation and characterization of a mutant of Escherichia coli unable to grow aerobically on non-fermentable substrates, except for very slow growth on glycerol. The mutant contains cytochrome oxidases o and d, and grows anaerobically with alternative electron acceptors. Oxygen consumption rates of cell-free extracts were low relative to activities in an isogenic control strain, but were restored in vitro by adding ubiquinone-1 to cell-free extracts. Transformation with a cloned 2.8 kb ClaI-EcoRV fragment of chromosomal DNA restored the ability of this mutant (AN2571) to grow on succinate and also restored cellular quinone levels in this strain. The plasmid also complemented a previously isolated ubiG mutant (AN151) for aerobic growth on succinate. The nucleotide sequence revealed a 0.7 kb portion of gyrA. Unidirectional nested deletions from this fragment and complementation analysis identified an open reading frame encoding a protein with a predicted molecular mass of 26.5 kDa. This gene (ubiG) encodes the enzyme 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase, which catalyses the terminal step in the biosynthesis of ubiquinone. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by three palindromic unit sequences. Comparison of the inferred amino acid sequence of UbiG with the sequence of other S-adenosylmethionine (AdoMet)-dependent methyltransferases reveals a highly conserved AdoMet-binding region. The cloned 2.8 kb fragment also contains a sequence encoding the C-terminus of a protein with 42-44% identity to fungal acetyl-CoA synthetases.     

Sulfide oxidation in gram-negative bacteria by expression of the sulfide-quinone reductase gene of Rhodobacter capsulatus and by electron transport to ubiquinone.

The oxidation of sulfide was studied in recombinant bacteria expressing the sulfide-quinone reductase gene (sqr) from Rhodobacter capsulatus. Sulfide was oxidized by the Escherichia coli strain W3110 harboring the sqr construct (pKKSQ) under anaerobic conditions and nitrate was utilized as a terminal electron acceptor. Following the oxidation, elemental sulfur and nitrite were produced as the final reaction products. This activity was retained in the membrane preparation and was sensitive towards antimycin A, stigmatellin, and azide. As a consequence of the ubiquinone deficiency, this activity was markedly decreased. In additon, by recovery of ubiquinone, the oxidation was also restored to rates similar to those of the wild-type strain. These results indicate that sulfide oxidation in this strain occurs via the quinone pool in vivo, and that this sulfide-quinone reductase (SQR) in particular utilizes ubiquinone as a more appropriate electron acceptor than menaquinone or demetylmenaquinone. To our knowledge, this is the first study to show a direct interaction between SQR and ubiquinone in cells. When expressed in Pseudomonas putida and Rhizobium meliloti, the SQR conferred on these organisms the ability to oxidize sulfide as well as E. coli in vivo.     

Esre: a novel essential non-coding RNA in Escherichia coli

YigP gene (GeneID: 948915) locates between ubiquinone biosynthetic genes ubiE and ubiB in Escherichia coli. GeneBank annotates yigP as a putative protein-coding gene. In this study, we found a new essential sRNA gene, esre, locates within the region of yigP. The E. coli strain with inactive esre must rely on a complementary plasmid to survive. Moreover, RACE experiments showed esre encodes an RNA molecule of 252 nt. Further experiments revealed esre gene is immune to frame shift mutations and the function of esre depends mostly on the RNA secondary structure, which are typical traits of sRNA. Since it is difficult to predict the target of an essential sRNA, more research is needed to reveal the function and mechanism of esre.     

Ubiquinone （Coenzyme Q） Biosynthesis in Chlamydophila pneumoniae AR39： Identification of the ubiD Gene

Ubiquinone is an essential electron carrier in prokaryotes.Ubiquinone biosynthesis involves atleast nine reactions in Escherichia coli.3-octaprenyl-4-hydroxybenzoate decarboxylase (UbiD) is an importantenzyme on the pathway and deletion of the ubiD gene in E.coli gives rise to ubiquinone deficiency in vivo.A protein from Chlamydophila pneumoniae AR39 had significant similarity compared with protein UbiDfrom E.coli.Based on this information,the protein-encoding gene was used to swap its counterpart inE.coli,and gene expression in resultant strain DYC was confirmed by RT-PCR.Strain DYC grew usingsuccinate as carbon source and rescued ubiquinone content in vivo,while ubiD deletion strain DYD did not.Results suggest that the chlamydial protein exerts the function of UbiD.     

Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli.

The biosynthetic origin of the oxygen atoms of ubiquinone 8 from aerobically grown Escherichia coli was studied by 18O labeling. An apparatus was developed which allowed the growth of cells under a defined atmosphere. Mass spectral analysis of ubiquinone 8 from cells grown under highly enriched 18O2 showed that three oxygen atoms of the quinone are derived from molecular oxygen. It was established that the molecular oxygen is incorporated into the two methoxyl groups (at C-5 and C-6) and one of the carbonyl positions of the ubiquinone molecule by demonstrating that only one of the incorporated oxygens will exchange with water under acidic conditions that specifically catalyze the exchange of carbonyl, but not methoxyl, oxygens. That the C-4 carbonyl oxygen is derived from molecular oxygen was shown by the incorporation of three atoms of 18O2 into ubiquinone 8 biosynthesized from added 4-hydroxybenzoic acid. Comparison of ubiquinone 8 and menaquinone 8 from E. coli grown under 18O2 confirmed that the labeled carbonyl oxygen of the [18O2]ubiquinone 8 is incorporated biosynthetically and not by chemical exchange in the cell. It is concluded that the three hydroxylation reactions involved in the pathway for the aerobic biosynthesis of ubiquinone are all catalyzed by monooxygenases. The implications of this study for the anaerobic biosynthesis of ubiquinone 8 in E coli are discussed.     

Role of quinones in electron transport to oxygen and nitrate in Escherichia coli. Studies with a ubiA− menA− double quinone mutant

A ubiA^? menA^? double quinone mutant of Escherichia coli K12 was constructed together with other isogenic strains lacking either ubiquinone or menaquinone. These strains were used to study the role of quinones in electron transport to oxygen and nitrate. Each of the four oxidases examined (NADH, d-lactate, α-glycerophosphate and succinate) required a quinone for activity. Ubiquinone was active in each oxidase system while menaquinone gave full activity in α-glycerophosphate oxidase, partial activity in d-lactate oxidase but was inactive in NADH and succinate oxidation. The aerobic growth rates, growth yields and products of glucose metabolism of the quinone-deficient strains were also examined. The growth rate and growth yield of the ubi⁺ menA^? strain was the same as the wild-type strain, whereas the ubiA^? men⁺ strain grew more slowly on glucose, had a lower growth yield (30% of wild type) and accumulated relatively large quantities of acetate and lactate. The growth of the ubiA^? menA^? strain was even more severely affected than that of the ubiA^? men⁺ strain.Electron transport from formate, d-lactate, α-glycerophosphate and NADH to nitrate was also highly dependent on the presence of a quinone. Either ubiquinone or menaquinone was active in electron transport from formate and the activity of the quinones in electron transport from the other substrates was the same as for the oxidase systems. In contrast, quinones were not obligatory carriers in the anaerobic formate hydrogenlyase system. It is concluded that the quinones serve to link the various dehydrogenases with the terminal electron transport systems to oxygen and nitrate and that the dehydrogenases possess a degree of selectivity with respect to the quinone acceptors.     

Role of quinones in the branch of the Escherichia coli respiratory chain that terminates in cytochrome o.

The role of quinones in the cytochrome o branch of the Escherichia coli respiratory chain was investigated by using mutant strains lacking the cytochrome d terminal oxidase complex. The only cytochromes present were cytochrome b556 and the cytochrome o complex, consisting of cytochrome b555-b562. Mutant strains missing ubiquinone, menaquinone, or both were constructed in the cytochrome d-minus (cyd) background. The steady-state levels of cytochrome b reduction were examined and compared in these strains to assess the effects of the quinone deficiencies. The data clearly show that a ubiquinone deficiency results in a lower level of cytochrome b reduction in the steady state. The data are consistent with a simple model in which ubiquinone is placed on the dehydrogenase side of all the cytochromes in this branch of the respiratory chain. There is no evidence from these experiments for a role of quinones in the respiratory chain at any site besides this one.     

Identification of a ubiG-like gene involved in ubiquinone biosynthesis from Chlamydophila pneumoniae AR39

AIMS: To investigate if one hypothetical protein from Chlamydophila pneumoniae AR39 exerts UbiG-like function by complementary experiments. METHODS AND RESULTS: Proteins UbiG have a signature S-adenosylmethionine-binding motif compared with other methyltransferases. Probing with the conserved motif, one hypothetical protein from C. pneumoniae AR39 was proposed to be a UbiG-like protein. The protein encoding the gene was used to swap its counterpart in Escherichia coli, and its expression in resultant strain DYCG was confirmed by RT-PCR. Strain DYCG grew on succinate as a carbon source, and rescued ubiquinone content in vivo, while the ubiG deletion strain DYK did not. CONCLUSIONS: Results indicate that the putative protein from C. pneumoniae exerts a UbiG-like function involved in ubiquinone biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of the ubiG-like gene will facilitate research on ubiquinone biosynthesis and aerobic respiration in the genus Chlamydophila owing to the important function of ubiquinone in vivo.     

Comparison of catalytic activity and inhibitors of quinone reactions of succinate dehydrogenase (Succinate-ubiquinone oxidoreductase) and fumarate reductase (Menaquinol-fumarate oxidoreductase) from Escherichia coli.

Escherichia coli succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate reductase (QFR) are excellent model systems to understand the function of eukaryotic Complex II. They have structural and catalytic properties similar to their eukaryotic counterpart. An exception is that potent inhibitors of mammalian Complex II, such as thenoyltrifluoroacetone and carboxanilides, only weakly inhibit their bacterial counterparts. This lack of good inhibitors of quinone reactions and the higher level of side reactions in the prokaryotic enzymes has hampered the elucidation of the mechanism of quinone oxidation/reduction in E. coli Complex II. In this communication DT-diaphorase and an appropriate quinone are used to measure quinol-fumarate reductase activity and E. coli bo-oxidase and quinones are used to determine succinate-quinone reductase activity. Simple Michaelis kinetics are observed for both enzymes with ubiquinones and menaquinones in the succinate oxidase (forward) and fumarate reductase (reverse) reactions. The comparison of E. coli SQR and QFR demonstrates that 2-n-heptyl 4-hydroxyquinoline-N-oxide (HQNO) is a potent inhibitor of QFR in both assays; however, SQR is not sensitive to HQNO. A series of 2-alkyl-4,6-dinitrophenols and pentachlorophenol were found to be potent competitive inhibitors of both SQR and QFR. In addition, the isolated E. coli SQR complex demonstrates a mixed-type inhibition with carboxanilides, whereas the QFR complex is resistant to this inhibitor. The kinetic properties of SQR and QFR suggest that either ubiquinone or menaquinone operates at a single exchangeable site working in forward or reverse reactions. The pH activity profiles for E. coli QFR and SQR are similar showing maximal activity between pH 7.4 and 7.8, suggesting the importance of similar catalytic groups in quinol deprotonation and oxidation.     

Mutant Strains of Escherichia coli K-12 Unable to Form Ubiquinone

A strain of Escherichia coli was isolated which was unable to form ubiquinone. This mutant was obtained by selecting strains unable to grow on malate as sole source of carbon. Such strains were further screened by examination of the quinone content of cells grown on a glucose medium. A mutant unable to form vitamin K was also isolated by this procedure. A genetic analysis of the ubiquinoneless strain showed that it possessed two mutations affecting ubiquinone biosynthesis.     

Identity of the quinone in Bacillus alcalophilus.

Every Bacillus species so far examined contains menaquinone as the sole quinone. In contrast, the alkalophilic Bacillus alcalophilus has been reported to be unusual in containing ubiquinone rather than menaquinone. In this communication, we demonstrate that B. alcalophilus, like all the other bacilli, contains menaquinone as the only quinone.     

Cross-Complementation Study of the Flagellar Type III Export Apparatus Membrane Protein FlhB

The bacterial type III export apparatus is found in the flagellum and in the needle complex of some pathogenic Gram-negative bacteria. In the needle complex its function is to secrete effector proteins for infection into Eukaryotic cells. In the bacterial flagellum it exports specific proteins for the building of the flagellum during its assembly. The export apparatus is composed of about five membrane proteins and three soluble proteins. The mechanism of the export apparatus is not fully understood. The five membrane proteins are well conserved and essential. Here a cross-complementation assay was performed: substituting in the flagellar system of Salmonella one of these membrane proteins, FlhB, by the FlhB ortholog from Aquifex aeolicus (an evolutionary distant hyperthermophilic bacteria) or a chimeric protein (AquSalFlhB) made by the combination of the trans-membrane domain of A. aeolicus FlhB with the cytoplasmic domain of Salmonella FlhB dramatically reduced numbers of flagella and motility. From cells expressing the chimeric AquSalFlhB protein, suppressor mutants with enhanced motility were isolated and the mutations were identified using whole genome sequencing. Gain-of-function mutations were found in the gene encoding FlhA, another membrane protein of the type III export apparatus. Also, mutations were identified in genes encoding 4-hydroxybenzoate octaprenyltransferase, ubiquinone/menaquinone biosynthesis methyltransferase, and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, which are required for ubiquinone biosynthesis. The mutations were shown by reversed-phase high performance liquid chromatography to reduce the quinone pool of the cytoplasmic membrane. Ubiquinone biosynthesis could be restored for the strain bearing a mutated gene for 4-hydroxybenzoate octaprenyltransferase by the addition of excess exogenous 4-hydroxybenzoate. Restoring the level of ubiquinone reduced flagella biogenesis with the AquSalFlhB chimera demonstrating that the respiratory chain quinone pool is responsible for this phenomenon.     

Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis sfpo and Escherichia coli entD genes.

In response to iron deprivation, Bacillus subtilis secretes a catecholic siderophore, 2,3-dihydroxybenzoyl glycine, which is similar to the precursor of the Escherichia coli siderophore enterobactin. We isolated two sets of B. subtilis DNA sequences that complemented the mutations of several E. coli siderophore-deficient (ent) mutants with defective enterobactin biosynthesis enzymes. One set contained DNA sequences that complemented only an entD mutation. The second set contained DNA sequences that complemented various combinations of entB, entE, entC, and entA mutations. The two sets of DNA sequences did not appear to overlap. AB. subtilis mutant containing an insertion in the region of the entD homolog grew much more poorly in low-iron medium and with markedly different kinetics. These data indicate that (i) at least five of the siderophore biosynthesis genes of B. subtilis can function in E. coli, (ii) the genetic organization of these siderophore genes in B. subtilis is similar to that in E. coli, and (iii) the B. subtilis entD homolog is required for efficient growth in low-iron medium. The nucleotide sequence of the B. subtilis DNA contained in plasmid pENTA22, a clone expressing the B. subtilis entD homolog, revealed the presence of at least two genes. One gene was identified as sfpo, a previously reported gene involved in the production of surfactin in B. subtilis and which is highly homologous to the E. coli entD gene. We present evidence that the E. coli entD and B. subtilis sfpo genes are interchangeable and that their products are members of a new family of proteins which function in the secretion of peptide molecules.