Menaquinone (Vitamin K2) Biosynthesis: Localization and Characterization of the menA Gene from Escherichia coli

A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of a menA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from the orf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into the orf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.     

Shewanella oneidensis MR-1 Uses Overlapping Pathways for Iron Reduction at a Distance and by Direct Contact under Conditions Relevant for Biofilms

We developed a new method to measure iron reduction at a distance based on depositing Fe(III) (hydr)oxide within nanoporous glass beads. In this “Fe-bead” system, Shewanella oneidensis reduces at least 86.5% of the iron in the absence of direct contact. Biofilm formation accompanies Fe-bead reduction and is observable both macro- and microscopically. Fe-bead reduction is catalyzed by live cells adapted to anaerobic conditions, and maximal reduction rates require sustained protein synthesis. The amount of reactive ferric iron in the Fe-bead system is available in excess such that the rate of Fe-bead reduction is directly proportional to cell density; i.e., it is diffusion limited. Addition of either lysates prepared from anaerobic cells or exogenous electron shuttles stimulates Fe-bead reduction by S. oneidensis, but iron chelators or additional Fe(II) do not. Neither dissolved Fe(III) nor electron shuttling activity was detected in culture supernatants, implying that the mediator is retained within the biofilm matrix. Strains with mutations in omcB or mtrB show about 50% of the wild-type levels of reduction, while a cymA mutant shows less than 20% of the wild-type levels of reduction and a menF mutant shows insignificant reduction. The Fe-bead reduction defect of the menF mutant can be restored by addition of menaquinone, but menaquinone itself cannot stimulate Fe-bead reduction. Because the menF gene encodes the first committed step of menaquinone biosynthesis, no intermediates of the menaquinone biosynthetic pathway are used as diffusible mediators by this organism to promote iron reduction at a distance. CymA and menaquinone are required for both direct and indirect mineral reduction, whereas MtrB and OmcB contribute to but are not absolutely required for iron reduction at a distance.     

The growth and EPA synthesis ofShewanella oneidensis MR-1 and expectation of EPA biosynthetic pathway

Shewanella oneidensis MR-1 has the ability to inhale certain metals and chemical compounds and exhale these materials in an altered state; as a result, this microorganism has been widely applied in bioremediation protocols. However, the relevant characteristics of cell growth and biosynthesis of PuFAs have yet to be thoroughly investigated. Therefore, in this study, we have attempted to characterize the growth and fatty acid profiles ofS. oneidensis MR-1 under a variety of temperature conditions. The fastest growth ofS. oneidensis MR-1 was observed at 30°C, with a specific growth rate and doubling time of 0.6885 h⁻¹ and 1.007 h. The maximum cell mass of this microorganism was elicited at a temperature of 4°C. The eicosapentaenoic acid (EPA) synthesis ofS. oneidensis MR-1 was evaluated under these different culture temperatures.S. oneidensis MR-1 was found not to synthesize EPA at temperatures in excess of 30°C, but was shown to synthesize EPA at temperatures below 30°C. The EPA content was found to increase with decreases in temperature. We then evaluated the EPA biosynthetic pathway, using a phylogenetic tree predicted on 16s rRNA sequences, and the homology of ORFs betweenS. oneidensis MR-1 andShewanella putrefaciens SCRC-2738, which is known to harbor a polyketide synthase (PKS)-like module. The phylogenetic tree revealed that MR-1 was very closely related to bothMoritella sp., which is known to synthesize DHA via a PKS-like pathway, andS. putrefaciens, which has been reported to synthesize EPA via an identical pathway. The homology between the PKS-like module ofS. putrefaciens SCRC-2738 and the entire genome ofS. oneidensis MR-1 was also analyzed, in order to mine the genes associated with the PKS-like pathway inS. oneidensis MR-1. A putative PKS-like module for EPA biosynthesis was verified by this analysis, and was also corroborated by the experimental finding thatS. oneidensis MR-1 was able to synthesize EPA without the expression of dihomo-γ-linoleic acid (DGLA) and arachidonic acid (AA) formed during EPA synthesis via the FAS pathway.     

Tetrahydrothiophene 1-oxide as an electron acceptor for Escherichia coli.

Escherichia coli used tetrahydrothiophene 1-oxide (THTO) as an electron acceptor for anaerobic growth with glycerol as a carbon source; the THTO was reduced to tetrahydrothiophene. Cell extracts also reduced THTO to tetrahydrothiophene in the presence of a variety of electron donors. Chlorate-resistant (chl) mutants (chlA, chlB, chlD, and chlE) were unable to grow with THTO as the electron acceptor. However, growth and THTO reduction by the chlD mutant were restored by high concentrations of molybdate. Similarly, mutants of E. coli that are blocked in the menaquinone (vitamin K2) biosynthetic pathway, i.e., menB, menC, and menD mutants, did not grow with THTO as an electron acceptor. Growth and THTO reduction were restored in these mutants by the presence of appropriate intermediates of the vitamin K biosynthetic pathway.     

Electron donors and the quinone involved in dimethyl sulfoxide reduction inEscherichia coli

Escherichia coli can use dimethyl sulfoxide (DMSO) as an electron acceptor during anaerobic growth on the oxidizable substrate, glycerol. During growth, the DMSO is reduced to dimethyl sulfide (DMS). For the reduction of DMSO, NADH, formate, lactate, reduced benzyl viologen, reduced methyl viologen, and dithionite can serve as electron donors. The terminal reductase and the dehydrogenases linking the various electron donors to the electron transport chain were found to be membrane bound. Chlorate-resistant mutants (chl) were unable to grow and reduce DMSO. However, in the case of thechlD mutant, growth and DMSO reduction can be restored by growth in the presence of high concentrations of molybdate. Mutants ofE. coli blocked in menaquinone (vitamin K₂) biosynthesis—menB, menC, andmenD—were unable to grow with DMSO as an electron acceptor, even though the terminal reductase is present in these mutants. Both growth and DMSO reduction could be restored in these mutants by growth in the presence of the menaquinone intermediates,o-succinylbenzoate and 1,4-dihydroxy-2-naphthoate, depending on the metabolic block of the mutant. Thus menaquinone is involved in electron transport during DMSO reduction.     

Inability of men mutants of Escherichia coli to use trimethylamine-N-oxide as an electron acceptor

Abstract Mutants of Escherichia coli blocked in the biosynthesis of menaquinone were unable to use trimethylamine- N -oxide as an electron acceptor in complex medium containing glucose. Trimethylamine- N -oxide reduction and the consequent in-crease in growth could be restored in a menC mutant by either o -succinylbenzoic acid (OSB) or 1,4-dihydroxy-2-naphthoic acid (DHNA). In the case of a menB mutant, growth could not be restored by OSB; but addition of DHNA resulted in the restoration of growth to the full extent. These results are consistent with the metabolic blocks in the respective men mutants.     

Identification and analysis of the Shewanella oneidensis major oxygen-independent coproporphyrinogen III oxidase gene

Shewanella oneidenesis MR-1 is a facultative anaerobe that can use a large number of electron acceptors including metal oxides. During anaerobic respiration, S. oneidensis MR-1 synthesizes a large number of c cytochromes that give the organism its characteristic orange color. Using a modified mariner transposon, a number of S. oneidensis mutants deficient in anaerobic respiration were generated. One mutant, BG163, exhibited reduced pigmentation and was deficient in c cytochromes normally synthesized under anaerobic condition. The deficiencies in BG163 were due to insertional inactivation of hemN1, which exhibits a high degree of similarity to genes encoding anaerobic coproporphyrinogen III oxidases that are involved in heme biosynthesis. The ability of BG163 to synthesize c cytochromes under anaerobic conditions, and to grow anaerobically with different electron acceptors was restored by the introduction of hemN1 on a plasmid. Complementation of the mutant was also achieved by the addition of hemin to the growth medium. The genome sequence of S. oneidensis contains three putative anaerobic coproporphyrinogen III oxidase genes. The protein encoded by hemN1 appears to be the major enzyme that is involved in anaerobic heme synthesis of S. oneidensis. The other two putative anaerobic coproporphyrinogen III oxidase genes may play a minor role in this process.     

A class C radical S‐adenosylmethionine methyltransferase synthesizes 8‐methylmenaquinone

The membranous quinone/quinol pool is essential to the majority of life forms and has been widely used as an important biomarker in microbial taxonomy. In the anaerobic world, the most important quinones are menaquinone (MK) and a methylated form of MK, designated methylmenaquinone (MMK), which is anticipated to serve specifically in low‐potential electron transport chains involved in anaerobic respiration. However, it has remained unclear how MMK is generated. Here, we show that a novel enzyme homologous to class C radical SAM methyltransferases (RSMTs) synthesizes MMK using MK as substrate. Such enzymes, termed either MenK or MqnK, are present in MMK‐producing bacteria (and some archaea) that possess either the classical MK biosynthesis pathway (Men) or the futalosine pathway (Mqn). An mqnK deletion mutant of the model Epsilonproteobacterium Wolinella succinogenes was unable to produce MMK₆ but its formation was restored upon genomic complementation using either the native mqnK gene or menK from the human gut bacterium Adlercreutzia equolifaciens or Shewanella oneidensis. Moreover, any of the menK genes enabled Escherichia coli cells to produce MMK₈ and a methylated form of 2‐demethylmenaquinone₈ (DMK₈). The results expand the knowledge on quinone synthesis and demonstrate an unprecedented function for a class C RSMT‐type enzyme in primary cell metabolism.     

Current Production and Metal Oxide Reduction by Shewanella oneidensis MR-1 Wild Type and Mutants

Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.     

Methods for imaging Shewanella oneidensis MR-1 nanofilaments

Nanofilament production by Shewanella oneidensis MR-1 was evaluated as a function of lifestyle (planktonic vs. sessile) under aerobic and anaerobic conditions using different sample preparation techniques prior to imaging with scanning electron microscopy. Nanofilaments could be imaged on MR-1 cells grown in biofilms or planktonically under both aerobic and anaerobic batch culture conditions after fixation, critical point drying and coating with a conductive metal. Critical point drying was a requirement for imaging nanofilaments attached to planktonically grown MR-1 cells, but not for cells grown in a biofilm. Techniques described in this paper cannot be used to differentiate nanowires from pili or flagella.     

Mutants of an electrogenic bacterium <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1 with increased reducing activity

The mutants of Shewanella oneidensis MR-1 resistant to fosfomycin, a toxic analogue of phosphoenolpyruvate, were obtained. The mutants exhibited increased reducing activity and higher rates of lactate utilization. A correlation was shown between the rates of metabolism of oxidized substrates and the rate of reduction of methylene blue, a mediator of electron transport. The mutants of S. oneidensis MR-1 may be used in microbial fuel cells for intensification of energy production from organic compounds.     

Type 1 fimbriation is negatively regulated by cyclic AMP and its receptor proteinvia conjugative plasmid F inEscherichia coli K-12

Expression of fimbriation was studied inEscherichia coli K-12 CA8000 HfrH, and itscya, crp and MS2 resistant mutants. The cells of cya⁺ crp⁺ parent strain were observed to be flagellated bacilli, lacking fimbriae, unable to agglutinate erythrocytes and deficient in ability to produce surface pellicle during growth in stationary culture. The cells ofcya andcrp mutants were observed to be cocci or coccobacilli devoid of flagella, having haemagglutinating activity, fimbriated and capable of producing surface pellicle in stationary cultures. The fimbriation and haemagglutinating activities were lower incya mutants grown with cAMP supplementation. Thecya andcrp mutants produced relatively small, smooth and compact colonies consisting mostly of fimbriated cells, like those of earlier described Fimσ mutants. Thecya ⁺ crp⁺ MS2 resistant mutant produced large sized colonies like those of parent but was deficient in conjugal donor ability. It resembledcya andcrp mutants in haemagglutinating and fimbriation properties. Thecya andcrp mutants have been earlier shown to be deficient in several Tra functions including conjugal donor ability. It is concluded thatEscherichia coli K-12 cells express fimbriation when Tra functions of F-plasmid carried in them are not expressed either due to deficiency of active cAMP-receptor protein complex or mutation in F-plasmid or when F-plasmid is absent.     

Enhancement of 1,4-Dihydroxy-2-Naphthoic Acid Production by Propionibacterium freudenreichii ET-3 Fed-Batch Culture

The production of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch culture of Propionibacterium freudenreichii ET-3. DHNA is a precursor of menaquinone (MK) and is transformed to MK by combination with an isoprenoid unit. We found that ET-3 stopped MK production and increased DHNA production in an anaerobic fed-batch culture by maintaining the lactose concentration at approximately zero. The maximum DHNA concentration observed in the anaerobic fed-batch culture was markedly higher than the maximum DHNA concentration observed in an anaerobic batch culture. Moreover, MK or DHNA production was affected by the lactose feeding rate; this suggests that lactose metabolism participates in the syntheses of these products. On the other hand, accumulation of propionate was found to inhibit DHNA production in the fed-batch culture. Based on the fact that ET-3 increases DHNA production in an aerobic culture by consuming propionate, we carried out a cultivation experiment in which an anaerobic fed-batch culture was switched to an anaerobic batch culture and found that the DHNA production was increased to a greater extent than the DHNA production in an anaerobic fed-batch culture. These results suggest that DHNA production by ET-3 is markedly influenced by carbon source limitation and the oxygen supply.     

Decolorization and detoxification of a sulfonated triphenylmethane dye aniline blue by Shewanella oneidensis MR-1 under anaerobic conditions

In this work, the extracellular decolorization of aniline blue, a sulfonated triphenylmethane dye, by Shewanella oneidensis MR-1 was confirmed. S. oneidensis MR-1 showed a high capacity for decolorizing aniline blue even at a concentration of up to 1,000 mg/l under anaerobic conditions. Maximum decolorization efficiency appeared at pH?7.0 and 30 °C. Lactate was a better candidate of electron donor for the decolorization of aniline blue. The addition of nitrate, hydrous ferric oxide, or trimethylamine N-oxide all could cause a significant decline of decolorization efficiency. The Mtr respiratory pathway was found to be involved into the decolorization of aniline blue by S. oneidensis MR-1. The toxicity evaluation through phytotoxicity and genotoxicity showed that S. oneidensis MR-1 could decrease the toxicity of aniline blue during the decolorization process. Thus, this work may facilitate a better understanding on the degradation mechanisms of the triphenylmethane dyes by Shewanella and is beneficial to their application in bioremediation.     

Shewanella oneidensis MR‐1 mutants selected for their inability to produce soluble organic‐Fe(III) complexes are unable to respire Fe(III) as anaerobic electron acceptor

Summary Recent voltammetric analyses indicate that Shewanella putrefaciens strain 200 produces soluble organic‐Fe(III) complexes during anaerobic respiration of sparingly soluble Fe(III) oxides. Results of the present study expand the range of Shewanella species capable of producing soluble organic‐Fe(III) complexes to include Shewanella oneidensis MR‐1. Soluble organic‐Fe(III) was produced by S. oneidensis cultures incubated anaerobically with Fe(III) oxides, or with Fe(III) oxides and the alternate electron acceptor fumarate, but not in the presence of O₂, nitrate or trimethylamine‐N‐oxide. Chemical mutagenesis procedures were combined with a novel MicroElectrode Screening Array (MESA) to identify four (designated Sol) mutants with impaired ability to produce soluble organic‐Fe(III) during anaerobic respiration of Fe(III) oxides. Two of the Sol mutants were deficient in anaerobic growth on both soluble Fe(III)‐citrate and Fe(III) oxide, yet retained the ability to grow on a suite of seven alternate electron acceptors. The rates of soluble organic‐Fe(III) production were proportional to the rates of iron reduction by the S. oneidensis wild‐type and Sol mutant strains, and all four Sol mutants retained wild‐type siderophore production capability. Results of this study indicate that the production of soluble organic‐Fe(III) may be an important intermediate step in the anaerobic respiration of both soluble and sparingly soluble forms of Fe(III) by S. oneidensis.     

Hydrogen Metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of ΔhydA, ΔhyaB, and ΔhydA ΔhyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.     

NADH dehydrogenases drive inward electron transfer in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a promising chassis organism for microbial electrosynthesis because it has a well-defined biochemical pathway (the Mtr pathway) that can connect extracellular electrodes to respiratory electron carriers inside the cell. We previously found that the Mtr pathway can be used to transfer electrons from a cathode to intracellular electron carriers and drive reduction reactions. In this work, we hypothesized that native NADH dehydrogenases form an essential link between the Mtr pathway and NADH in the cytoplasm. To test this hypothesis, we compared the ability of various mutant strains to accept electrons from a cathode and transfer them to an NADH-dependent reaction in the cytoplasm, reduction of acetoin to 2,3-butanediol. We found that deletion of genes encoding NADH dehydrogenases from the genome blocked electron transfer from a cathode to NADH in the cytoplasm, preventing the conversion of acetoin to 2,3-butanediol. However, electron transfer to fumarate was not blocked by the gene deletions, indicating that NADH dehydrogenase deletion specifically impacted NADH generation and did not cause a general defect in extracellular electron transfer. Proton motive force (PMF) is linked to the function of the NADH dehydrogenases. We added a protonophore to collapse PMF and observed that it blocked inward electron transfer to acetoin but not fumarate. Together these results indicate a link between the Mtr pathway and intracellular NADH. Future work to optimize microbial electrosynthesis in S. oneidensis MR-1 should focus on optimizing flux through NADH dehydrogenases.     

A partial metabolic pathway enables group b streptococcus to overcome quinone deficiency in a host bacterial community

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 Δ gbs1789 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.     

The outer membrane protein Omp35 affects the reduction of Fe(III), nitrate,and fumarate by <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1

Background  

Shewanella oneidensis MR-1 uses several electron acceptors to support anaerobic respiration including insoluble species such as iron(III) and manganese(IV) oxides, and soluble species such as nitrate, fumarate, dimethylsulfoxide and many others. MR-1 has complex branched electron transport chains that include components in the cytoplasmic membrane, periplasm, and outer membrane (OM). Previous studies have implicated a role for anaerobically upregulated OM electron transport components in the use of insoluble electron acceptors, and have suggested that other OM components may also contribute to insoluble electron acceptor use. In this study, the role for an anaerobically upregulated 35-kDa OM protein (Omp35) in the use of anaerobic electron acceptors was explored.     

Uridine phosphorylase from Shewanella oneidensis MR-1: Heterological expression, regulation, transcription, and properties

A DNA fragment containing a promoter-operator and structural parts of the uridine phosphorylase gene from Shewanella oneidensis MR-1 was cloned. Cross-heterological expression of the udp genes from Sh. oneidensis MR-1 and Escherichia coli under the control of authentic regulatory regions is shown. The UDP protein accumulates in an active form in the cytoplasmic fraction of cells. The recombinant UDP protein from Sh. oneidensis MR-1 obtained by heterological expression was isolated and characterized. E. coli udp gene promoter activity was observed during heterological expression in Sh. oneidensis MR-1 cells under both aerobic and anaerobic conditions.