1: Environ Microbiol. 2009 Jan;11(1):149-58. Epub 2008 Sep 22. Role of menaquinone biosynthesis genes in selenate reduction by Enterobacter cloacae SLD1a-1 and Escherichia coli K12. Ma J, Kobayashi DY, Yee N. Department of Environmental Sciences, Rutgers, State University of New Jersey, New Brunswick, NJ, USA. In this study, we investigated the role of menaquinone biosynthesis genes in selenate reduction by Enterobacter cloacae SLD1a-1 and Escherichia coli K12. A mini-Tn5 transposon mutant of E. cloacae SLD1a-1, designated as 4E6, was isolated that had lost the ability to reduce Se(VI) to Se(0). Genetic analysis of mutant strain 4E6 showed that the transposon was inserted within a menD gene among a menFDHBCE gene cluster that encodes for proteins required for menaquinone biosynthesis. A group of E. coli K12 strains with single mutations in the menF, menD, menC and menE genes were tested for loss of selenate reduction activity. The results showed that E. coli K12 carrying a deletion of either the menD, menC or menE gene was unable to reduce selenate. Complementation using wild-type sequences of the E. cloacae SLD1a-1 menFDHBCE sequence successfully restored the selenate reduction activity in mutant strain 4E6, and E. coli K12 menD and menE mutants. Selenate reduction activity in 4E6 was also restored by chemical complementation using the menaquinone precursor compound 1,4-dihydroxy-2-nathphoic acid. The results of this work suggest that menaquinones are an important source of electrons for the selenate reductase, and are required for selenate reduction activity in E. cloacae SLD1a-1 and E. coli K12. Publication Types: Research Support, U.S. Gov't, Non-P.H.S. PMID: 18811645 [PubMed - indexed for MEDLINE] 2: Vaccine. 2008 Aug 18;26(35):4557-62. Epub 2008 Jun 17. Neonatal immune response and serum bactericidal activity induced by a meningococcal conjugate vaccine is enhanced by LT-K63 and CpG2006. Brynjolfsson SF, Bjarnarson SP, Mori E, Del Giudice G, Jonsdottir I. Landspitali, Department of Immunology, Reykjavik, Iceland. Neonates have a poorly developed immune system. Therefore it is important to develop vaccination strategies that induce protective immunity and immunological memory against pathogens early in life. The immunogenicity of a meningococcal serogroup C polysaccharide conjugate (MenC-CRM(197)) was assessed in neonatal mice, and effects of LT-K63 and CpG2006 and immunisation routes were compared. Neonatal mice were primed subcutaneously (s.c.) or intranasally (i.n.) with MenC-CRM(197) with or without LT-K63 or CpG2006 and re-immunised 16 and 30 days later by the same route and formulation. Antibody levels were measured and generation of immunological memory assessed by affinity maturation and kinetics of the Ab response. Serum bactericidal activity (SBA) was measured to evaluate protective efficacy. The second and third dose of MenC-CRM(197) mixed with either LT-K63 or CpG2006 induced a rapid increase in MenC-specific IgG antibodies, to levels higher than elicited by MenC-CRM(197) alone (P<0.01) and in unimmunised mice (P<0.001), indicating efficient generation of memory by priming through both s.c. and i.n. routes. SBA was detected after three s.c. immunisations with MenC-CRM(197) s.c. alone. However, only two doses of MenC-CRM(197)+LT-K63 or MenC-CRM(197)+CpG2006 were needed to induce SBA levels>16. LT-K63 and CpG2006 enhanced neonatal antibody responses, affinity maturation, immunological memory to the conjugate MenC-CRM(197) and protective immunity. These results encourage the development of neonatal vaccination strategies to induce protective immunity and immunological memory against meningococcal disease. Publication Types: Research Support, Non-U.S. Gov't PMID: 18597905 [PubMed - indexed for MEDLINE] 3: J Drug Target. 2005 Sep-Nov;13(8-9):489-98. Protective immune responses to meningococcal C conjugate vaccine after intranasal immunization of mice with the LTK63 mutant plus chitosan or trimethyl chitosan chloride as novel delivery platform. Baudner BC, Verhoef JC, Giuliani MM, Peppoloni S, Rappuoli R, Del Giudice G, Junginger HE. Leiden/Amsterdam Center for Drug Research, University of Leiden, The Netherlands. barbara_baudner@chiron.com Chitosan and its derivative N-trimethyl chitosan chloride (TMC), given as microparticles or powder suspensions, and the non-toxic mucosal adjuvant LTK63, were evaluated for intranasal immunization with the group C meningococcal conjugated vaccine (CRM-MenC). Mice immunized intranasally with CRM-MenC formulated with chitosan or TMC and the LTK63 mutant, showed high titers of serum and mucosal antibodies specific for the MenC polysaccharide. Neither significant differences were observed between microparticle formulations and powder suspensions nor when LTK63 was pre-associated to the delivery system or not. The bactericidal activity measured in serum of mice immunized intranasally with the conjugated vaccine formulated with the delivery systems and the LT mutant was superior to the activity in serum of mice immunized sub-cutaneously. Importantly, intranasal but not parenteral immunization, induced bactericidal antibodies at the nasal level, when formulated with both delivery system and adjuvant. PMID: 16332574 [PubMed - indexed for MEDLINE] 4: J Infect Dis. 2004 Mar 1;189(5):828-32. Epub 2004 Feb 18. Modulation of immune response to group C meningococcal conjugate vaccine given intranasally to mice together with the LTK63 mucosal adjuvant and the trimethyl chitosan delivery system. Baudner BC, Morandi M, Giuliani MM, Verhoef JC, Junginger HE, Costantino P, Rappuoli R, Del Giudice G. Research Center, Chiron Srl, Siena, Italy. Previous work had shown that the immunogenicity of conjugate vaccine against group C meningococci (CRM-MenC) is enhanced when it is delivered intranasally (inl) with mucosal adjuvants, such as mutants of the Escherichia coli enterotoxin (LT), and with delivery systems such as chitosan derivatives. We show, in mice, that the concomitant use of limiting doses of the fully nontoxic LTK63 mutant as a mucosal adjuvant and of the trimethyl derivative of chitosan as a delivery system allows the reduction of each of the components for the induction of antibody and bactericidal responses to CRM-MenC conjugate vaccine delivered inl at titers similar to or higher than those induced by parenteral immunization. These data could affect the design of efficacious mucosal vaccines and their safety. PMID: 14976599 [PubMed - indexed for MEDLINE] 5: Vaccine. 2003 Sep 8;21(25-26):3837-44. The concomitant use of the LTK63 mucosal adjuvant and of chitosan-based delivery system enhances the immunogenicity and efficacy of intranasally administered vaccines. Baudner BC, Giuliani MM, Verhoef JC, Rappuoli R, Junginger HE, Giudice GD. IRIS Research Center, Chiron SpA, Via Fiorentina 1, 53100 Siena, Italy. In this paper we evaluated chitosan microparticles as a vaccine delivery system as well as the mucosal adjuvant LTK63, a nontoxic Escherichia coli enterotoxin (LT) mutant for the intranasal immunization with the group C meningococcal conjugated vaccine (CRM-MenC). Mice receiving intranasally the CRM-MenC vaccine formulated with chitosan microparticles and the LTK63 mutant showed higher titers of systemic and mucosal antibodies specific for the group C meningococcal polysaccharide as compared to those receiving the vaccine subcutaneously. In addition, high bactericidal activity was found in serum samples of mice immunized intranasally with the conjugated vaccine formulated together with the microparticles and the LT mutant. These results demonstrate that the concomitant use of chitosan microparticles and the LTK63 mutant significantly enhances the immunogenicity and the protective efficacy of vaccines given intranasally. PMID: 12922117 [PubMed - indexed for MEDLINE] 6: J Infect Dis. 2002 Nov 1;186(9):1358-61. Epub 2002 Oct 8. Combinations of protein polysaccharide conjugate vaccines for intranasal immunization. Ugozzoli M, Mariani M, Del Giudice G, Soenawan E, O'Hagan DT. Chiron Corporation, Emeryville, California, USA. The ability of 2 mutants of heat-labile Escherichia coli enterotoxin (LTK63 and LTR72) to enhance the immunogenicity of 2 protein polysaccharide conjugate vaccines, Neisseria meningitidis group C (MenC) and Haemophilus influenzae type B (Hib), both of which are conjugated to the nontoxic mutant of diphtheria toxin (CRM197), after intranasal (inl) immunization in mice was evaluated. In addition, the question of whether combining both vaccines in a single formulation with heat-labile E. coli enterotoxin mutants reduced the response to either vaccine was investigated. The results showed that potent serum antibody responses against MenC and Hib could be elicited by inl immunization in combination with the mucosal adjuvants. Moreover, IgA mucosal responses were induced only in animals immunized through the inl route. Finally, the coadministration of 2 conjugate vaccines simultaneously did not adversely affect the responses against either. These studies support the rationale for developing mucosal vaccines, based on combining protein polysaccharide conjugates with heat-labile E. coli enterotoxin mutants, for infants and young children. PMID: 12402209 [PubMed - indexed for MEDLINE] 7: J Biol Chem. 1997 May 9;272(19):12380-3. Identification of a novel gene cluster participating in menaquinone (vitamin K2) biosynthesis. Cloning and sequence determination of the 2-heptaprenyl-1,4-naphthoquinone methyltransferase gene of Bacillus stearothermophilus. Koike-Takeshita A, Koyama T, Ogura K. Bio Research Laboratory, Toyota Motor Corporation, Toyota-cho 1, Toyota, Aichi 471-71, Japan. We recently described the isolation and sequence analysis of a DNA region containing the genes of Bacillus stearothermophilus heptaprenyl diphosphate synthase, which catalyzes the synthesis of the prenyl side chain of menaquinone-7 of this bacterium. Sequence analyses revealed the presence of three open reading frames (ORFs), designated as ORF-1, ORF-2, and ORF-3, and the structural genes of the heptaprenyl diphosphate synthase were proved to consist of ORF-1 (heps-1) and ORF-3 (heps-2) (Koike-Takeshita, A., Koyama, T., Obata, S., and Ogura, K. (1995) J. Biol. Chem. 270, 18396-18400). The predicted amino acid sequence of ORF-2 (234 amino acids) contains a methyltransferase consensus sequence and shows a 22% identity with UbiG of Escherichia coli, which catalyzes S-adenosyl-L-methionine-dependent methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone. These pieces of information led us to identify the ORF-2 gene product. The cell-free homogenate of the transformant of E. coli with an expression vector of ORF-2 catalyzed the incorporation of S-adenosyl-L-methionine into menaquinone-8, indicating that ORF-2 encodes 2-heptaprenyl-1,4-naphthoquinone methyltransferase, which participates in the terminal step of the menaquinone biosynthesis. Thus it is concluded that the ORF-1, ORF-2, and ORF-3 genes, designated heps-1, menG, and heps-2, respectively, form another cluster involved in menaquinone biosynthesis in addition to the cluster of menB, menC, menD, and menE already identified in the Bacillus subtilis and E. coli chromosomes. Publication Types: Research Support, Non-U.S. Gov't PMID: 9139683 [PubMed - indexed for MEDLINE] 8: Gene. 1996 Feb 2;168(1):43-8. Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menE gene from Escherichia coli. Sharma V, Hudspeth ME, Meganathan R. Department of Biological Sciences, Northern Illinois University, DeKalb, 60115, USA. In Escherichia coli, the biosynthesis of the electron carrier menaquinone (vitamin K2) involves at least seven identified enzymatic activities, five of which are encoded in the men cluster. One of these, the conversion of o-succinylbenzoic acid to 1,4-dihydroxy-2-naphthoic acid, requires the formation of o-succinylbenzoyl-CoA (OSB-CoA) as an intermediate. Formation of the intermediate is mediated by OSB-CoA synthetase encoded by the menE locus known to be located either 5' of menB, or 3' of menC. A DNA fragment overlapping the 3' end of menC in shown by enzymatic complementation to elevate OSB-CoA synthetase activity. Nucleotide sequence analysis of the fragment identified a 1.355-kb open reading frame (ORF) which, when deleted at either the 5' or 3' end, failed to generate increased enzymatic activity. The ORF is preceded by a consensus ribosome-binding site, but no apparent sigma-70 promoter. An oppositely transcribed unidentified gene cluster follows the menE ORF. The region 5' of menB contains an an additional ORF of unknown function (orf241) and establishes the order of genes in the men cluster as menD, orf241, menB, menC and menE. All loci are transcribed counter-clockwise. Publication Types: Research Support, U.S. Gov't, P.H.S. PMID: 8626063 [PubMed - indexed for MEDLINE] 9: Gene. 1995 Dec 29;167(1-2):105-9. Structural organization of a Bacillus subtilis operon encoding menaquinone biosynthetic enzymes. Rowland B, Hill K, Miller P, Driscoll J, Taber H. Department of Microbiology, Immunology and Molecular Genetics, Albany Medical College, NY 12208, USA. Menaquinone (MK) is a non-protein component of the Bacillus subtilis (Bs) electron transport chain synthesized from chorismate through a series of MK-specific reactions. The genes encoding biosynthesis of the naphthoquinone ring of MK are clustered at 273 degrees on the Bs chromosome. A 3.9-kb region capable of rescuing men mutants blocked in the early stages of MK biosynthesis was sequenced and found to contain three major open reading frames (ORFs). The first ORF (menF) has a predicted size of 51.8 kDa and 34% amino-acid identity with the isochorismate synthases of Escherichia coli (EntC) and Aeromonas hydrophila (AmoA), ORF2 (menD) a predicted size of 60.2 kDa and 21% identity with MenD of E. coli. ORF3 has a predicted size of 21.4 kDa and 29% identity to triacylglycerol lipase of Psychrobacter immobilis. No sequence corresponding to menC was identified. Plasmid integrational studies of the men gene cluster had suggested the presence of promoters secondary to the previously identified p1 men promoter. Sequence analysis revealed a putative promoter region upstream from ORF3. Publication Types: Research Support, U.S. Gov't, P.H.S. PMID: 8566759 [PubMed - indexed for MEDLINE] 10: J Bacteriol. 1993 Aug;175(15):4917-21. Menaquinone (vitamin K2) biosynthesis: cloning, nucleotide sequence, and expression of the menC gene from Escherichia coli. Sharma V, Meganathan R, Hudspeth ME. Department of Biological Sciences, Plant Molecular Biology Center, Northern Illinois University, DeKalb 60115. The benzenoid aromatic compound o-succinylbenzoic acid is formed by dehydration of the prearomatic compound 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid by the enzyme o-succinylbenzoate synthase, encoded by the menC gene. A 1.3-kb PstI-PvuII fragment was found to complement the menC mutation. The complete nucleotide sequence of this fragment revealed a single open reading frame of 954 bp capable of encoding a 35-kDa protein. A consensus sequence for a ribosomal binding site but no promoter consensus sequences were found. However, the first base of the initiating codon of this open reading frame overlaps the upstream menB gene termination codon, suggesting an operon-like organization for these genes. Consistent with this suggestion, the menB promoter can initiate transcription of the menC gene. Publication Types: Research Support, U.S. Gov't, P.H.S. PMID: 8335646 [PubMed - indexed for MEDLINE] 11: J Bacteriol. 1987 Jun;169(6):2862-5. Tetrahydrothiophene 1-oxide as an electron acceptor for Escherichia coli. Meganathan R, Schrementi J. 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. PMID: 3294808 [PubMed - indexed for MEDLINE] 12: Biochem Biophys Res Commun. 1985 Sep 16;131(2):956-60. Vitamin K (menaquinone) biosynthesis in bacteria: purification and probable structure of an intermediate prior to o-succinylbenzoate. Emmons GT, Campbell IM, Bentley R. The first aromatic intermediate in the menaquinone biosynthetic pathway is o-succinylbenzoate (OSB); it is formed from chorismate/isochorismate and 2-ketoglutarate. Cell-free extracts of menD+ E. coli strains synthesize an intermediate, "X", which is converted to OSB by extracts of menC+ cells. "X" has been purified to near homogeneity by HPLC. On treatment with acid, it yields both OSB and succinylbenzene (SB). This and other data, suggest that "X" has the structure, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (I). PMID: 3902015 [PubMed - indexed for MEDLINE] 13: J Bacteriol. 1983 Feb;153(2):739-46. Thiamine pyrophosphate requirement for o-succinylbenzoic acid synthesis in Escherichia coli and evidence for an intermediate. Meganathan R, Bentley R. Cell-free extracts of various strains of Escherichia coli synthesize the menaquinone biosynthetic intermediate o-succinylbenzoic acid (OSB) when supplied with chorismic acid, 2-ketoglutaric acid, and thiamine pyrophosphate (TPP). To assay for OSB synthesis, 2-[U-14C]ketoglutaric acid was used as substrate, and the synthesized OSB was examined by radiogas chromatography (as the dimethyl ester). [U-14C]Shikimic acid also gave rise to radioactive OSB if the cofactors necessary for enzymatic conversion to chorismic acid were added. Use of 2-[1-14C]ketoglutaric acid does not give rise to labeled OSB. In the absence of TPP during the incubations, OSB synthesis was much reduced; these observations are consistent with the proposed role for the succinic semialdehyde-TPP anion as the reagent adding to chorismic acid. Extracts of cells from menC and menD mutants did not form OSB separately, but did so in combination. There was evidence for formation of a product, X, by extracts of a menC mutant incubated with chorismic acid, TPP, and 2-ketoglutaric acid; X was converted to OSB by extracts of a menD mutant. It appears that the intermediate, X, is formed by one gene product and converted to OSB by the second gene product. Publication Types: Research Support, U.S. Gov't, P.H.S. PMID: 6337125 [PubMed - indexed for MEDLINE] 14: J Bacteriol. 1982 Dec;152(3):1132-7. Characterization of Escherichia coli men mutants defective in conversion of o-succinylbenzoate to 1,4-dihydroxy-2-naphthoate. Shaw DJ, Guest JR, Meganathan R, Bentley R. Four independent menaquinone (vitamin K(2))-deficient mutants of Escherichia coli, blocked in the conversion of o-succinylbenzoate (OSB) to 1,4-dihydroxy-2-naphthoate (DHNA), were found to represent two distinct classes. Enzymatic complementation was observed when a cell-free extract of one mutant was mixed with extracts of any of the remaining three mutants. The missing enzymes in the two classes were identified by in vitro complementation with preparations of OSB-coenzyme A (CoA) synthetase or DHNA synthase isolated from Mycobacterium phlei. Mutants lacking DHNA synthase (and therefore complementing with M. phlei DHNA synthase) were designated menB, and the mutant lacking OSB-CoA synthetase (and therefore complementing with M. phlei OSB-CoA synthetase) was designated menE. The menB mutants produced only the spirodilactone form of OSB when extracts were incubated with [2,3-(14)C(2)]OSB, ATP, and CoA; the OSB was unchanged on incubation with an extract from the menE mutant under these conditions. Experiments with strains lysogenized by a lambda men transducing phage (lambdaG68) and transduction studies with phage P1 indicated that the menB and menE genes form part of a cluster of four genes, controlling the early steps in menaquinone biosynthesis, located at 48.5 min in the E. coli linkage map. Evidence was obtained for the clockwise gene order gyrA....menC- 0000100000 0000110000 0011111000 0000111000 0011111000 0001110000 0000110101 0001111111 0001100000 0000100000 0001101100 0011111000 0011000000 0011000000 0111000111 0111101110 -B-D, where the asterisk denotes the uncertain position of menE relative to menC and menB. The transducing phage (lambdaG68) contained functional menB, menC, and menE genes, but only part of the menD gene, and it was designated lambda menCB(D). Publication Types: Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. PMID: 6754698 [PubMed - indexed for MEDLINE] 15: Mol Gen Genet. 1981;181(3):379-83. Molecular cloning of menaquinone biosynthetic genes of Escherichia coli K12. Guest JR, Shaw DJ. A transducing phage carrying some of the genes (men) defining the early stages of menaquinone biosynthesis was isolated from a pool of recombinant lambda phages that had been constructed from R.HindIII digests of E. coli DNA and the corresponding insertion vector. The lesions of menB and menC mutants were complemented by the phage but menD mutants were transduced either at low frequencies or not at all. This indicates that the transducing phage contains functional menB and menC genes but that only part of the menD gene had been cloned. The phage (lambda G68) was accordingly designated lambda menCB(D). Studies with the transducing phage enabled earlier mapping data (Guest 1979) to be reinterpreted in favour of the gene order nalA....menC..menB..MenD....purF. Restriction analyses established the presence of a bacterial DNA fragment (11.5 kb) linked by a R.HindIII target to the right arm of the lambda genome but fused to the left arm of the vector. Hybridization studies confirmed that the cloned DNA was derived from a larger R.HindIII fragment (21 kb). A physical map of the men region was constructed and some flanking and overlapping fragments were identified. Publication Types: Research Support, Non-U.S. Gov't PMID: 6454056 [PubMed - indexed for MEDLINE] 16: J Gen Microbiol. 1979 Dec;115(2):259-71. Anaerobic growth of Escherichia coli K12 with fumarate as terminal electron acceptor. Genetic studies with menaquinone and fluoroacetate-resistant mutants. Guest JR. Fifteen independent menaquinone biosynthesis mutants (men) of Escherichia coli K12, selected for their inability to use fumarate as terminal electron acceptor, were investigated. Two nutritionally distinct groups were detected. The major group (13 mutants) responded to 1,4-dihydroxy-2-naphthoate (DHN), 2-succinylbenzoate (SB) and its dilactone, whereas the minor group (2 mutants) only responded to DHN. DHN was at least five times more effective than SB but it inhibited growth at concentrations greater than 10 microM. For anaerobic growth on glucose minimal medium the auxotrophs responded to much lower concentrations of DHN and SB and these intermediates could be replaced by uracil. Anaerobic growth tests showed that glycerol, formate and H2 are good substrates for E. coli when fumarate is the ultimate electron acceptor but growth with lactate or with fumarate alone is poor. All 15 men mutations were located between glpT and purF at approximately 49 min in the E. coli linkage map. Cotransduction frequencies with relevant markers were: nalA (21%), glpT (35%) and purF (15%). The presence of at least three genetically distinct classes (menC and menD, SB-requirers; menB, DHN-requirers) was indicated using abortive transduction as a complementation test and three-factor genetic analysis. The relative orientation nalA...menC-(D,B)...purF was indicated. Fluoroacetate-resistant mutants were isolated and four different classes were identified: ack, lacking acetate kinase; pta, lacking phosphotransacetylase; facA, lacking both of these activities; and facB, which retained both of these enzyme activities. Some of the pta mutants and all of the facA mutants failed to grow on media containing fumarate as terminal electron acceptor or anaerobically on glucose minimal medium. All four types had genetic lesions clustered between the men and purF sites. Average cotransduction frequencies with relevant markers were: nalA (4%), men (27 to 35%) and purF (71 to 80%). PMID: 393800 [PubMed - indexed for MEDLINE] 17: J Bacteriol. 1977 Jun;130(3):1038-46. Menaquinone biosynthesis: mutants of Escherichia coli K-12 requiring 2-succinylbenzoate. Guest JR. Two independent mutants of Escherichia coli K-12, selected for their inability to grow anaerobically with fumarate as the terminal electron acceptor, were shown to be deficient in menaquinone biosynthesis. In both cases, exogenously supplied 2-succinylbenzoate promoted normal anaerobic growth on a lactate plus fumarate medium. Anaerobic growth of the mutants on glucose minimal medium was impaired but could be restored to normal by adding either uracil or 2-succinylbenzoate. The addition of 2-succinylbenzoate (but not uracil) permitted the synthesis of menaquinone and demethylmenaquinone by both mutants. The menaquinone content of the parental strain grown on lactate plus fumarate was three times greater than observed after growth on glucose. Transduction studies with phage P1 showed that the two mutations are very closely linked and probably affect the same gene, menC, which is cotransducible with nalA (23%), glpT (51%), and purF (8 to 14%). The gene order nalA-nrdA-glpTA-menC-purF was indicated. The results were consistent with 2-succinylbenzoate being an intermediate in menaquinone biosynthesis and show that the gene designated menC (located at 48.65 min of the E. coli chromosome) is involved in the conversion of chorismate to 2-succinylbenzoate. It was also concluded that menaquinone is essential for electron transport to fumarate in E. coli. PMID: 324971 [PubMed - indexed for MEDLINE]