Chapter 39

Biosynthesis of the Isoprenoid Quinones Menaquinone (Vitamin K₂) and Ubiquinone (Coenzyme Q)

R. MEGANATHAN

INTRODUCTION

Facultatively anaerobic gram-negative bacteria including Escherichia coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) contain the isoprenoid quinones of the benzene and naphthalene series. The structures of these quinones are shown in Fig. 1. According to the IUPAC-IUB recommendations (51), the benzoquinones are termed ubiquinones (Q-n) (structure I in Fig. 1) and the naphthoquinones are termed either menaquinones (MK-n) (structure II in Fig. 1) or demethylmenaquinones (DMK-n) (structure III in Fig. 1). The n refers to the number of prenyl units present in the side chain. Although MK is considered a vitamin (vitamin K₂), Q is not; vitamin K is an essential nutrient (cannot be synthesized by mammals), whereas Q is not an essential nutrient, since it can be synthesized from the amino acid tyrosine.

Fig. 1Structures of quinones found in E.coli and S.typhimurium.

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The major quinones in E. coli are Q-8, MK-8, and DMK-8; minor amounts of Q-1 to Q-7, Q-9, MK-6, MK-7, MK-9, and DMK-7 may also be present (23). The prenyl side chains have all-trans configurations (11). Surprisingly, in contrast to E. coli, whose quinone composition has been extensively investigated, S. typhimurium has not been studied in detail. However, the presence of Q-8 has been reported (32, 33), and it can be assumed that the distribution of other quinones will be identical to that in E. coli. These organisms have neither quinones with one or more of the prenyl residues of the side chain reduced nor MK with more than one methyl group. Methods for the extraction, purification, identification, and analysis of the quinones have been reviewed extensively elsewhere (22, 29, 34, 39, 49, 62, 64, 70, 75, 76, 77, 85, 98, 99, 100, 110, 116, 117, 124).

Most of the information concerning the biosynthesis of MK and Q was obtained with E. coli by using isotopic tracers, isolating mutants, and assaying accumulation of intermediates and enzymes. Because of space limitations, only a general account is given here; for more information, several comprehensive reviews should be consulted (12, 13, 14, 15, 16, 44, 123). Both MK and Q are derived from the shikimate pathway and as such have some common structural features. The quinone nucleus of Q is derived directly from chorismate, whereas that of MK is from chorismate via isochorismate. The prenyl side chain on the nucleus of both is derived from prenyl pyrophosphate (prenyl PP_i), and the methyl groups are derived from S-adenosylmethionine. In addition, MK biosynthesis requires 2-ketoglutarate and ATP, thiamine pyrophosphate (TPP), and coenzyme A (CoA) as cofactors. The biosynthesis of Q under aerobic conditions also requires oxygen, flavoprotein, and NADH. Finally, the Q biosynthetic pathway in prokaryotes differs in several respects from that of eukaryotes (94).

In spite of the fact that both quinones originate from the shikimate pathway, there are several important differences between them.

1. In the formation of the quinoid nuclei, the pathway for Q diverges at chorismate with the loss of a pyruvoyl group by the action of chorismate lyase, resulting in the formation of a benzenoid aromatic acid that is used as the framework on which the rest of the molecule is constructed. MK biosynthesis diverges at isochorismate by the addition of the succinic semialdehyde-TPP anion derived from 2-ketoglutarate; the concerted loss of the pyruvoyl group leads to the formation of the prearomatic compound 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC). This compound is then aromatized to a benzenoid aromatic acid and used as the framework for the construction of the rest of the molecule as shown in Fig. 2.

Fig. 2Formation of Q and MK.

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2. In Q biosynthesis, the prenyl side chain is introduced at an early stage (second step) with retention of the aromatic carboxyl. In MK biosynthesis, in contrast, this side chain is introduced in the next-to-last step and is accompanied by decarboxylation.

3. In MK biosynthesis, all enzymes in the pathway up to prenylation (next-to-last step) are soluble, whereas in Q biosynthesis, all enzymes except the first are membrane bound.

4. In MK biosynthesis, methylation of the carbon of the nucleus is the last step, whereas in Q biosynthesis, the terminal step is the methylation of a hydroxyl group. In addition, in Q biosynthesis, a second O methylation and C methylation take place in the middle portion of the pathway.

5. Q biosynthesis under aerobic conditions requires the introduction of OH groups by reactions involving oxygen; anaerobic Q and MK biosynthesis utilize oxygen atoms derived from water.

MK BIOSYNTHESIS

The MK biosynthetic pathway has been elucidated on the basis of tracer experiments, isolation of mutants blocked at various steps, isolation and identification of intermediates accumulated by the mutants, and enzyme assays. Early isotopic tracer experiments with various bacteria established that methionine and prenyl PP_i contribute to the methyl and prenyl substituents of the naphthoquinone. Early isotopic tracer studies and other work were reviewed by Bentley and Meganathan (15). In 1964, Cox and Gibson (26) observed that [G-¹⁴C]shikimate was incorporated into both MK and Q by E. coli, thus providing the first evidence for the involvement of the shikimate pathway. Chemical degradation of the labeled isolated MK (MK-8) showed that essentially all of the radioactivity was retained in the phthalic anhydride (26, 69). A more complete chemical degradation of the MK derived from labeled shikimate established that all seven carbon atoms were incorporated (21). The remaining three carbon atoms of the naphthoquinone ring were derived from the middle three carbons of 2-ketoglutarate with the loss of both carboxyl groups (20, 102, 103).

These studies established the immediate precursors of the naphthoquinone nucleus of MK as shikimate and the noncarboxyl carbon atoms of 2-ketoglutarate. The methyl and isoprenoid side chains were also shown to be derived from S-adenosylmethionine and an isoprenyl alcohol pyrophosphate ester, respectively. Subsequently, it was shown that the benzenoid aromatic compound o-succinylbenzoate (OSB) (31) and the naphthalenoid aromatic compound 1,4-dihydroxy-2-naphthoate (DHNA) (13, 104) were incorporated into the naphthoquinone ring of MK. This work was confirmed by the demonstration that menB and menA mutants of E. coli excrete OSB and DHNA, respectively, into the culture medium (128). During a study of the biosynthesis of OSB by growing cultures of an E. coli menB mutant, it was demonstrated that C-1 of the glutamate (2-ketoglutarate) was lost and consequently not incorporated into OSB (81). The isotopic labeling pattern is summarized in Fig. 3.

Fig. 3Primary biosynthetic precursors of MK.

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Formation of Isochorismate (Compound IV → V)

The first cell-free synthesis of OSB starting with chorismate and 2-ketoglutarate in the presence of TPP by E. coli extracts was obtained by Meganathan (78) (Fig. 4). When extracts of E. coli were incubated with 2-[U-¹⁴C]ketoglutarate and chorismate in the presence of TPP, OSB formation was observed (after the conversion of OSB to a dimethyl derivative) by radio-gas chromatography (78). Similarly, cell extracts converted [U-¹⁴C]shikimate and 2-ketoglutarate to [¹⁴C]OSB provided that all the other substrates required for the conversion of shikimate to chorismate were added (82). It had been suggested that isochorismate is a more attractive precursor than chorismate on chemical grounds (31, 44), and evidence in support of this hypothesis was obtained (36, 122).

Fig. 4Pathway for biosynthesis of MK. Each compound in the pathway is identified by a roman numeral, and each reaction step is identified by an arabic numeral. Chemical names, abbreviations, and names of enzymes are shown. III, DMK (maybe initially formed as a quinol); RPP, polyprenyl PPi; PP, PPi; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.

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The conversion of chorismate to isochorismate is mediated by the enzyme isochorismate synthase, which is encoded by the entC gene (92, 118). The product of this reaction, isochorismate, is a common intermediate in the biosynthesis of the siderophore enterobactin and of MK. This dual role raised the question of whether a single isochorismate synthase encoded by the entC gene was responsible for supplying the isochorismate required for both enterobactin and MK. Kaiser and Leistner (54) isolated a Tn10 insertion mutant that was unable to form isochorismate and consequently lost the ability to form both enterobactin and MK.

Previous studies of enterobactin biosynthesis established that the entC gene is regulated by the concentration of iron in the medium (120). At a low iron concentration isochorismate synthase is derepressed, whereas at a high iron concentration, the enzyme is repressed (120). MK biosynthesis, on the other hand, is regulated by oxygen. Aerobically grown cells have low concentrations of MK, whereas anaerobically grown cells have high concentrations (15). It was important to determine how the interaction of iron concentration, aerobiosis, and anaerobiosis regulated isochorismate synthase activity. For the study of this regulation, an operon fusion of the entC gene was constructed, and the expression of β-galactosidase activity was monitored under various conditions. As expected, β-galactosidase activity was fully derepressed at low concentrations of iron and repressed with high iron under both aerobic and anaerobic conditions. Similar results were obtained when the cells were grown anaerobically in the presence of various electron acceptors. These results raised the question of how the organism is able to synthesize MK anaerobically in the presence of high iron when the isochorismate synthase is fully repressed. Similarly, how does the organism prevent the synthesis of MK aerobically under iron-deficient conditions when isochorismate synthase is fully derepressed? These questions were resolved with the demonstration of the anaerobic growth of an entC::Tn5 mutant (92) on glycerol medium with trimethylamine N-oxide, dimethyl sulfoxide, or fumarate as electron acceptor, for all of which MK is known to be obligatory. The entC::Tn5 mutant was able to grow at the same rate as the parent strain in high-iron media. These results provided further support for the existence of a separate isochorismate synthase that is specifically involved in MK biosynthesis and is independent of iron regulation (O. Kwon, M. E. S. Hudspeth, and R. Meganathan, Abstr. 93rd Gen. Meet. Am. Soc. Microbiol., abstr. K-69, p. 272, 1993). As a first step in identifying the gene encoding this second isochorismate synthase, a fragment of DNA 5' to the menD gene was sequenced. This fragment, which is about 650 bp long, has a 33% identity to the entC gene (R. Daruwala, O. Kwon, M. E. S. Hudspeth, and R. Meganathan, Abstr. 94th Gen. Meet. Am. Soc. Microbiol., abstr. K-158, p. 303, 1994). An isochorismate synthase specifically involved in MK biosynthesis was recently identified in Bacillus subtilis (115).

The results just described are in contrast to those of Kaiser and Leistner (54) noted above. It appears that the mutant (PBB1) purported to have been isolated by them was characterized by its inability to grow anaerobically on glycerol-fumarate medium. However, addition of isochorismate to the medium resulted in only slight improvement in growth. This poor response was attributed to the instability of isochorismate. However, the authors’ results failed to support this conclusion, although such support could have been obtained by showing stimulation of growth by a subsequent intermediate of the pathway such as OSB or DHNA. Further, they reported on cloning the entC ⁺ gene and complementation of an entC mutant. Surprisingly, the entC mutant used for complementation was not the PBB1 mutant they had isolated. Instead, they used AN154, which carried an allele designated entC401. This mutation has been shown by detailed genetic and enzymological analysis to be entA, not entC (92, 118).

Formation of SHCHC (Compounds V + VI → VII)

The first evidence for OSB synthesis was obtained by incubating cell extracts of E. coli with chorismate and 2-[U-¹⁴C]ketoglutarate in the presence of TPP. The OSB formed was extracted and converted to a dimethyl derivative and examined by radio-gas chromatography (78). Subsequently, two groups of mutants, designated menC and menD, that were blocked in the formation of OSB and thus required OSB for anaerobic growth were examined for OSB formation. Cell extracts of each mutant alone did not form OSB, but they did so in combination (82). On further examination, it was discovered that extracts of menC mutants accumulated an intermediate that was converted to OSB by extracts of menD mutants (82). This intermediate was highly unstable but could be partially purified by high-pressure liquid chromatography. On mild acid treatment, the intermediate yielded OSB and a degradation product that was identified as o-succinylbenzene. On the basis of these properties in combination with nuclear magnetic resonance data, the intermediate was identified as SHCHC (36).

It has been postulated that the 2-ketoglutarate undergoes TPP-dependent decarboxylation, with the formation of the succinic semialdehyde anion of TPP, and there is evidence that TPP is actually involved in the formation of SHCHC (82, 93, 96). This process is the same as that postulated for the reaction catalyzed by the first enzyme of the 2-ketoglutarate dehydrogenase complex, usually termed E1 (EC 1.2.4.2; oxoglutarate dehydrogenase [lipoamide]). It was initially thought that E1 of the 2-ketoglutarate complex might be supplying the succinic semialdehyde-TPP.

MK functions in a number of anaerobic reactions in E. coli (73), such as reductions of fumarate (40), trimethylamine N-oxide (79), dimethyl sulfoxide (84), and tetrahydrothiophene 1-oxide (83). Since it was known that 2-ketoglutarate dehydrogenase is repressed under anaerobic conditions (3), it was reasoned that the 2-ketoglutarate dehydrogenase complex is probably not involved in the biosynthesis of MK. This assumption was further strengthened by the fact that in a sucA mutant that lacks E1 of the 2-ketoglutarate dehydrogenase complex, OSB formation in vitro was unaffected (82). Further, mutants of E. coli lacking E1 of the 2-ketoglutarate dehydrogenase complex or carrying deletions of the entire complex failed to show an MK deficiency (19, 47). These observations prompted a search for a decarboxylase specifically involved in MK biosynthesis in cell extracts. It was found that a decarboxylase activity can be separated from the 2-ketoglutarate dehydrogenase complex by gel-permeation chromatography on Sepharose CL-6B (74). Alternatively, the 2-ketoglutarate dehydrogenase complex can be precipitated by protamine sulfate, leaving the decarboxylase activity in the supernatant fluid (93). A deletion mutant of E. coli lacking the entire 2-ketoglutarate dehydrogenase complex contains a 2-ketoglutarate decarboxylase (93). The succinic semialdehyde anion of TPP formed by the decarboxylase is believed to react directly with isochorismate (74, 93), and the enzyme responsible for this reaction has been designated SHCHC synthase (93, 97).

The menD gene specifies both SHCHC synthase and 2-ketoglutarate decarboxylase activities. The gene has been cloned, and its sequence has been determined and found to be composed of a single open reading frame (ORF) (93) (see below). The translated protein of this ORF contains the characteristic TPP-binding motif present in all the TPP-requiring enzymes (93). To confirm the validity of the conclusions based on sequence analysis, the protein was purified to homogeneity as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of the protein was 62,000, and the activities of SHCHC synthase and 2-ketoglutarate decarboxylase copurified. The native molecular weight of the protein as determined by gel filtration on Sephacryl S-300 was 190,000. Thus, the protein appears to be a homotrimer. The protein decarboxylated 2-ketoglutarate even in the absence of isochorismate according to assays by either ¹⁴CO₂ release from 2-[1-¹⁴C-]ketoglutarate or the formation of [U-¹⁴C]succinic semialdehyde from 2-[U-¹⁴C]ketoglutarate. Addition of isochorismate to the reaction mixture did not stimulate decarboxylation. In contrast, removal of the pyruvoyl group of isochorismate by SHCHC synthase required the presence of 2-ketoglutarate decarboxylase, presumably because succinic semialdehyde TPP addition and the loss of the pyruvoyl group are concerted (C. Palaniappan and R. Meganathan, unpublished data).

Formation of OSB (Compound VII → VIII)

Dehydration of SHCHC results in formation of the benzenoid aromatic compound OSB. The first evidence for an enzyme was obtained by the demonstration that cell extracts of a menD mutant converted SHCHC (at the time referred to as X) to OSB (82). This enzyme was subsequently designated OSB synthase (97, 106). The gene encoding the enzyme (menC) has been cloned, and its sequence has been determined and amplified (106).

In contrast to these studies, Weische et al. (121) reported an enzyme preparation designated OSB synthase that is capable of converting isochorismic acid and 2-ketoglutaric acid to OSB. Those authors did not consider SHCHC an intermediate in their studies. This enzyme preparation probably contained a mixture of SHCHC synthase–2-ketoglutarate decarboxylase and OSB synthase.

Cyclization of OSB to DHNA (Compound VIII → XI)

Conversion of the benzenoid aromatic OSB to the naphthalenoid aromatic DHNA was demonstrated in E. coli extracts in 1976 (18). The process was dependent on the presence of ATP and CoA. Therefore, it was suggested that an OSB-CoA derivative was formed as an intermediate in the overall reaction (18). With extracts of Mycobacterium phlei, two enzymatic activities were separated (OSB-CoA synthetase and DHNA synthase) (80). The OSB-CoA intermediate was highly unstable and was converted to the spirodilactone form of OSB. Further, during the formation of OSB-CoA, ATP was converted to AMP and PP_i (80). In E. coli, the techniques used for the separation of the two enzymatic activities in M. phlei (protamine sulfate precipitation in the presence of dimethyl sulfoxide) resulted in the loss of enzymatic activity.

Initially, the CoA moiety was suggested to be on the aromatic carboxyl (18, 80), and evidence in favor of this suggestion was obtained (45, 46, 59). However, it was subsequently reported that CoA is in fact located on the aliphatic carboxyl group (60, 61).

A group of E. coli mutants responding to DHNA but not to OSB during anaerobic growth on glycerol-fumarate medium were analyzed for their ability to convert OSB to DHNA in cell extracts in the presence of ATP and CoA (108). None of the four mutants tested formed DHNA when assayed alone. However, when extracts from the four mutants were mixed with each other, one of the mutants complemented the other three and produced DHNA. Hence, extracts from these mutants were assayed for complementation with the OSB-CoA synthetase and DHNA synthase from M. phlei described above. The single mutant that was complemented by OSB-CoA synthetase and that therefore lacked this enzyme was designated menE, and the other three mutants, which were complemented by DHNA synthase, were designated menB (108). Further, extracts of menB mutants form OSB-spirodilactone and excrete the compound into the medium during growth (81). These reactions are summarized in Fig. 5.

Fig. 5Conversion of OSB to DHNA.

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Conversion of DHNA to DMK (Compound XI → III)

The incorporation of DHNA into DMK in intact cells and the conversion of DHNA and farnesyl PP_i to MK-3, DMK-3, or both in cell extracts was first demonstrated by Bentley (13). Subsequently, a membrane-bound DHNA octaprenyltransferase was studied by Shineberg and Young (111). DHNA octaprenyltransferase has many features in common with the 4-hydroxybenzoate octaprenyltransferase involved in Q biosynthesis. Both enzymes are membrane bound, require Mg²⁺, and are relatively nonspecific for the polyprenyl PP_i. The two enzymes probably share a pool of membrane-bound octaprenyl PP_i (111, 129). Demethylmenaquinol is a likely intermediate, although the exact nature of the conversion of DHNA to DMK is not known. Prenylation and decarboxylation of DHNA may occur in concert, since symmetry experiments exclude 1,4-naphthoquinone as a likely intermediate (8). If demethylmenaquinol is an intermediate, then DMK is presumably formed by spontaneous oxidation (17). The biosynthesis of octaprenyl PP_i is the subject of chapter 38 in this volume.

Conversion of DMK to MK (Compound III → II)

DMK is converted to MK by the enzyme DMK methyltransferase. As expected, the methyl donor is S-adenosylmethionine. In experiments with whole cells, all three hydrogen atoms of methionine are transferred to DMK (52). The presence of the enzyme S-adenosylmethionine:2-DMK methyltransferase has been shown in cell extracts of E. coli (18). A methyltransferase isolated from M. phlei showed maximal activity toward DMK-3 and DMK-4 (105). A ubiA mutant of E. coli accumulated DMK but not MK. This mutant is believed to be defective in methylation of DMK to MK and may be a double mutant (35). Weissenbach et al. (125) determined the quinone composition of a ubiE mutant. This mutant produced only DMK and no MK. On the basis of these data, it was suggested that the same methylase involved in Q biosynthesis is also involved in the methylation of DMK to MK (125). However, no evidence was provided to rule out the possibility that the ubiE mutant might be a double mutant carrying defects in both Q biosynthesis and DMK methylation.

Recently, a cloned fragment of DNA was found to complement a menA mutant (K. Suvarna, unpublished data). DNA sequence analysis of this fragment located two ORFs. One ORF encoded the prenyltransferase, and the other contained the characteristic S-adenosylmethionine-binding motif (127). Thus, the gene for the methylase appears to be downstream of the menA gene and linked to it (Suvarna, unpublished data).

Genetics of MK Biosynthesis

The first MK-deficient mutant of E. coli was isolated during a search for Q-deficient mutants. After mutagenesis with N-methyl-N '-nitro-N-nitrosoguanidine (NG), colonies unable to use malate as the sole source of carbon and energy were selected (27). Subsequently, this mutation was shown to be cotransducible with metB and argE at frequencies of 30 and 50%, respectively. This mutant was designated menA (87).

In 1975, Young (128) isolated additional MK-deficient mutants during a search for Q-deficient mutants. After mutagenesis with NG, colonies unable to grow on succinate as the sole source of carbon and energy but capable of growing on glucose were isolated. Cell lipids were prepared from these strains and examined for the presence of quinones. Subsequent genetic analysis established that the isolation of men mutants in this manner was fortuitous (128). Among the mutants isolated, those that were unable to attach the isoprenoid side chain and that excreted DHNA into the medium were designated menA. A second group of mutants, designated menB, excreted OSB into the medium (128). Subsequent enzymological analysis revealed that the menB mutants are in fact composed of two groups, menE and menB (108).

From mutants unable to use fumarate as an electron acceptor, Guest (40) identified mutants blocked in the formation of OSB. In addition to menB, these mutants included two other groups, designated menC and menD. Enzyme analyses showed these last two groups to be deficient in OSB synthase and SHCHC synthase, respectively (82). The menB, menC, and menD genes were mapped and located at 49 min on the E. coli chromosome, and the menA gene was mapped at 89 min (7). A transducing phage carrying the men cluster λG68 [λ menCB(D)] was isolated from a pool of λ phages constructed from HindIII digests of E. coli DNA and the corresponding insertion vector (41). Complementation of men mutants showed that this phage contains functional menB and menC genes but only part of the menD gene. The exact location of the menE gene in relation to the menB and menC genes could not be established, since the single menE mutant was leaky (108). However, sequence analysis of the men cluster placed the menE gene downstream of menC (V. Sharma, M. E. S. Hudspeth, and R. Meganathan, unpublished data). Recently, a gene encoding an isochorismate synthase specifically involved in MK biosynthesis was identified and placed upstream of menD as menF (Daruwala et al., Abstr. 94th Gen. Meet. Am. Soc. Microbiol., 1994). The established counterclockwise gene order for the men cluster is purF menF menD menB menC menE gyrA (41; Sharma et al., unpublished data). The sequence of the methyltransferase gene (menG) was determined, and the gene was shown to be adjacent to menA at 89 min with a counterclockwise gene order of glpK menA menG cytR (Suvarna, unpublished data). By sequence analysis the menFDBCE genes are placed at 51 min, and menA and menG are placed at 88.7 min (Fig. 6).

Fig. 6(Top) The men cluster, consisting of the menBCDE genes, was previously assigned to the E.coli linkage map at 49 min. Shown here is the region between 50.8 and 51.3 min on the chromosome, flanked by glpC and nuoN, respectively, and with the men cluster at 51 to 51.2 min. The region is overlapped by Kohara phage ?5H12. The overall correspondence of Kohara restriction sites with sites obtained by DNA sequence analysis place the order of genes and cluster as indicated. The arrow shows the direction of transcription. (Bottom) The menA gene previously assigned to 89 min on the E.coli linkage map was present in the Clarke-Carbon plasmid pLC28?47 (89) encompassing the region glpK and cytR as determined by complementation of a menA mutant. From this plasmid, a 2.0-kb PstI-PstI subclone was isolated. Sequence analysis established the ORF encompassing the menA gene and an additional ORF identified as menG and placed these genes at 88.7 min. The correspondence of menA and menG with Kohara phage ?4?12 is shown. The arrow shows the direction of transcription.

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Some Consequences of Mutations in men Genes

MK plays a major role in anaerobic electron transport (65, 73, 114). The use of fumarate as a terminal electron acceptor has been studied in detail. Mutants of E. coli unable to synthesize MK fail to grow anaerobically on glucose medium unless uracil is added; normally, MK functions as an electron acceptor in the oxidation of dihydroorotate and subsequently transfers the electrons acquired to fumarate (87). It has been reported that men mutants are unable to reduce other electron acceptors like trimethylamine N-oxide (79), dimethyl sulfoxide (84), and tetrahydrothiophene 1-oxide (83). Reduction of these electron acceptors was restored by either OSB or DHNA for a menC mutant and by DHNA for a menB mutant. These results are in agreement with known metabolic blocks. In this connection, the utilization of nitrate as an electron acceptor is unaffected in men mutants (53, 63, 79), since it is a ubiquinone-dependent function.

Nucleotide Sequence and Physical Mapping of men Genes

From the Clarke-Carbon ColE1-E. coli gene bank, a plasmid derivative designated pGS22 capable of complementing the menBCD mutants was isolated. A 12.6-kb SalI-HindIII fragment was subcloned into pBR322 to produce a new plasmid, pGS23. This plasmid contained the menBCD genes, as determined by in vivo complementation. However, complementation analysis could not be used to determine whether menE was present in this plasmid because of the leakiness of the single menE mutant available. Strains containing this plasmid produced amplified levels of the menE gene product, OSB-CoA synthetase. Hence, it was concluded that the plasmid pGS23 does contain the menE gene in addition to the menBCD genes (109). Subclones of this men cluster were isolated, and the complete nucleotide sequence was determined (93, 106, 107; Sharma et al., unpublished data). These genes at 49 min have been located on the genome and placed in the counterclockwise gene order of purF menDBCE gyrA. This sequence has been located on the Kohara map. Five ORFs are encoded in the order menD, ORF 241, menB, menC and menE, and are transcribed counterclockwise. The products of the menDBCE ORFs have been biochemically and genetically confirmed by complementation analysis (93, 106, 107; Sharma et al., unpublished data). A putative secondary promoter allows transcription of the menBCE genes (106, 107; Sharma et al., unpublished data). The exact role of ORF 241 is undetermined. During sequence analysis of the men cluster, it was discovered that a region upstream of the 5' end of the menD gene has a 33% identity to the carboxy terminus of the entC protein. This region probably specifies the second isochorismate synthase (menF) (Daruwala et al., Abstr. 94th Gen. Meet. Am. Soc. Microbiol., 1994). The region upstream of the menD gene was cloned into a pUC18 plasmid from the Kohara clone λ5H12. This plasmid, when transformed into an entC mutant, produced amplified levels of the isochorismate synthase, thus establishing that it contains the menF gene.

The menA gene located at 89 min on the E. coli chromosome was subcloned into pUC18 from a Clarke-Carbon ColE1 plasmid, and clones capable of complementing the menA mutation were selected. The complementing plasmid contained a 2.0-kb region between cytR and glpK genes that on sequence analysis revealed two ORFs. One of the ORFs complemented the menA mutant (K. Suvarna, R. Meganathan, and M. E. S. Hudspeth, Abstr. 94th Gen. Meet. Am. Soc. Microbiol., abstr. K-159, p. 303, 1994). The other ORF encoded the methylase gene (menG) (Suvarna, unpublished data).

The physical map of the men genes and its alignment with the Kohara λ clones are shown in Fig. 6. The sizes of the various genes are summarized in Table 1.

Table 1Characteristics of sequenced men gene productsa

MK Biosynthesis in S. typhimurium

In contrast to the extensive work done on the genetics, biochemistry, and molecular biology of MK biosynthesis in E. coli, relatively little has been done with S. typhimurium. Davidson et al. (31a) and Kwan and Barrett (66) isolated mutants blocked in the reduction of trimethylamine N-oxide. Among these mutants they discovered a group that responded to the MK analogs (vitamin K₁ [phylloquinone; 2-methyl-3-phytyl-1,4-naphthoquinone] and vitamin K₅ [2-methyl-4-amino-1-naphthol-HCl]). Mutants that responded to both vitamin K₁ and K₅ were considered analogous to the menBCDE mutants of E. coli, whereas mutants that responded only to vitamin K₁ were considered analogous to menA (66). The men mutants were unable to produce H₂S from thiosulfate. Cross-feeding between the well-characterized E. coli men mutants and the S. typhimurium mutants and their response to the known intermediates (OSB and DHNA) in the restoration of H₂S production led to the identification of menABCD mutants (67).

Functions of MK

Some of the functions of MK are briefly discussed above. A thorough review of the functions is beyond the scope of this chapter. For details of the role of MK in anaerobic respiration, other chapters in this volume and other reviews and papers should be consulted (5, 42, 53, 63, 65, 73, 95, 114, 119, 124).

Q BIOSYNTHESIS

The Q biosynthetic pathway was elucidated largely by Gibson, Cox, Young, and colleagues (25, 38, 39). In 1964, Cox and Gibson observed that [G-14C]shikimate was incorporated into Q, thus establishing that the quinone was derived from the shikimate pathway (26). Mutagenized cultures were screened for colonies unable to grow oxidatively on a reduced substrate, such as malate or succinate, as the sole source of carbon while retaining the ability to grow fermentatively on glucose. Potential mutants were analyzed for the presence or absence of Q (38). By using this procedure, several mutants were isolated; these mutants accumulated sufficient amounts of intermediates so that their structure could be determined by mass spectrometry and magnetic resonance spectrometry (38, 113).

Formation of 4-Hydroxybenzoate (Compound IV → XII)

4-Hydroxybenzoate formation from chorismate is the first committed step in Q biosynthesis (Fig. 7). The reaction is catalyzed by the enzyme chorismate lyase, which is encoded by the ubiC gene (68). The chorismate lyase activity cannot be assayed in wild-type E. coli strains because of the utilization of chorismate along the major pathways to the aromatic amino acids. To circumvent this problem, a strain lacking chorismate mutase-prephenate dehydratase and chorismate mutase-prephenate dehydrogenase activities was used, and the incubation mixture contained l-tryptophan to inhibit anthranilate synthetase. Such a strain contained chorismate lyase activity. When the ubiC mutation was transduced into this strain and the transductant was assayed, it lacked chorismate lyase activity. Consistent with the lack of chorismate lyase, ubiC mutants require 4-hydroxybenzoate for growth on succinate minimal medium (68).

Fig. 7Pathway for biosynthesis of Q in prokaryotes. Each compound and reaction step is identified by a numeral as described in the legend to Fig. 4. The pathway drawn with solid lines is that found under aerobic conditions. Under anaerobic conditions, there are alternative hydroxylases for steps 4, 6, and 8 (dotted arrows). In compound XIII, the chemical numbering system locates the polyprenyl group at C-3; in compound XIV and subsequent intermediates, the prenyl group is assigned to C-2. Compounds XVII, XVIII, and XIX are shown in the quinol form. Some authors draw these structures in the quinone form as well. Abbreviations are as defined in the legend for Fig. 4. Not all the enzymes of the pathway have been identified.

Source:

Chorismate lyase is the only soluble enzyme of the Q biosynthetic pathway. It has been partially purified (J. Lawrence, Ph.D. thesis, Australian National University, Canberra, Australia, 1973).

Recently, the ubiC gene was cloned, and a chorismate lyase-overproducing plasmid was constructed. A strain carrying this plasmid overproduced chorismate lyase 250-fold. A 45-fold-purified homogeneous enzyme was obtained in a single step by using pseudoaffinity chromatography on Cibacron blue 3GA-agarose. This degree of purification yielded an activity about 10,000-fold higher than that found in extracts of the wild-type strain. The purified enzyme had an M _r of 17,000. The K_m was reported to be 9.7 μM and the V _max was determined to be 2.15 μmol of 4-hydroxybenzoate per 30 min. The turnover number was calculated to be 49 min^–1 (88).

Prenylation of 4-Hydroxybenzoate (Compound XII → XIII)

Prenylation of 4-hydroxybenzoate is carried out by the enzyme 4-hydroxybenzoate octaprenyltransferase, which is encoded by the ubiA gene. The enzyme is membrane bound and requires octaprenyl PP_i and Mg²⁺. (The biosynthesis of octaprenyl PP_i is the subject of chapter 38, and hence is not dealt with here.) In contrast to what happens with wild-type strains, when cell extracts from an aroC mutant were incubated with 4-hydroxybenzoate, 3-octaprenyl-4-hydroxybenzoate and its decarboxylation product 2-octaprenylphenol accumulated. This observation suggests that the reaction was dependent on accumulation of the side chain precursor in the mutant strain, which is unable to synthesize the benzoquinone and naphthoquinone rings of Q and MK, respectively. The mutant cells also accumulated all-trans C₄₀ octaprenol (geranylgeraniol), presumably a breakdown product of the actual side chain precursor (43). In subsequent studies, an aroB strain was used. The enzyme activity and the side chain precursor sedimented at 150,000 × g, thus establishing that the enzyme was membrane bound. The sedimented enzyme required Mg²⁺ for full activity, and cobalt and nickel ions were partially effective. To test for the presence of octaprenyltransferase activity in ubiA mutants, the aroB allele was transduced into a ubiA mutant, and the aroB ubiA double mutants were used. The double mutants lacked the enzyme activity, thus establishing that ubiA is the structural gene for 4-hydroxybenzoate octaprenyltransferase (129).

The specificity of the prenyltransferase for the side chain precursor was investigated with an aroD mutant. The aroD mutant, like the aroC mutant, accumulated octaprenyl PP_i in the membrane during growth because of its inability to form Q and MK rings (see above). When this mutant was grown in the presence of 4-hydroxybenzoate (Q precursor) and OSB (MK precursor) to prevent the accumulation of octaprenyl PP_i, cell extracts incubated with 4-hydroxybenzoate failed to form 3-octaprenyl-4-hydroxybenzoate. However, the same extracts used farnesyl, phytyl, or solanesyl PP_i as an exogenous source of the side chain (35). Thus, the transferase activity is relatively nonspecific with respect to the prenyl donor.

In addition, when aro mutants are grown in the presence of 4-aminobenzoate, the cells accumulate 3-octaprenyl-4-aminobenzoate, thus establishing that the lack of specificity of the transferase extends to the aromatic component as well (35, 43).

Formation of 2-Polyprenylphenol (Compound XIII → XIV)

The conversion of 3-octaprenyl-4-hydroxybenzoate to 2-octaprenylphenol was demonstrated by Cox et al. (28). The enzyme responsible for this conversion was named 3-octaprenyl-4-hydroxybenzoate decarboxylase. The presence of this decarboxylase was also observed by El Hachimi et al. (35). The enzyme activity was absent in ubiD mutants (28).

When cell extracts prepared with a French press were centrifuged at 150,000 × g for 3 h, most of the activity remained in the soluble fraction. The enzyme was purified 24-fold and found to have an M _r of 340,000 (72). For optimal activity, the enzyme required Mn²⁺, washed membranes or an extract of phospholipids, and an unidentified heat-stable factor with a molecular weight of less than 10,000. The reaction was strongly stimulated by dithiothreitol and methanol. Since the substrate of the enzyme 3-octaprenyl-4-hydroxybenzoate is membrane bound and the enzyme is stimulated by phospholipid, the enzyme is thought to function in association with the membrane in vivo (72).

A number of ubiD mutants formed about 20% of the wild-type levels of Q, indicating that the mutants are leaky or that there is another enzyme for the reaction. However, the possibility of an additional carboxy-lyase in the wild-type strains has been questioned (72).

In S. typhimurium, an alternate polyprenyl-4-hydroxybenzoate carboxy-lyase (decarboxylase) specified by the ubiX gene located at 50 min on the linkage map has been described (50); it carries out the same reaction as the ubiD gene product. By sequence homology, a ubiX gene has been found in E. coli (91). It is quite likely that the decarboxylase activity found in the ubiD mutants is due to the ubiX activity.

Hydroxylation Reactions

The three hydroxylation reactions of the pathway are the conversion of 2-octaprenylphenol to 2-octaprenyl-6-hydroxyphenol (compound XIV to XV), the conversion of 2-octaprenyl-6-methoxyphenol to 2-octaprenyl-6-methoxy-1,4-benzoquinol (compound XVI to XVII), and the conversion of 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol to 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinol (OMHMB) (compound XVIII to XIX) (2, 39). The three hydroxyl groups are introduced at positions 6, 4, and 5 of the benzene nucleus (steps 4, 6, and 8, respectively). For convenience, these hydroxylation reactions are considered together, and this description is followed by a consideration of the methylation reactions.

Mutants blocked in each hydroxylation reaction (steps 4, 6, and 8) were isolated and designated ubiB, ubiH, and ubiF, respectively (2, 39). ubiB mutants consistent with the metabolic block being in step 4 accumulate 2-octaprenylphenol (compound XIV) (28, 131). The product of step 4 (compound XV) has never been isolated and characterized. Compound XV may not exist as a free intermediate and may occur as an enzyme-bound intermediate (2, 131). Mutants blocked in the conversion of compound XV to compound XVI (2-octaprenyl-6-methoxyphenol) have never been isolated.

Mutants blocked in the conversion of compound XVI to compound XVII (step 6) were isolated and designated ubiH (131). These ubiH mutants accumulate the intermediate before the block (compound XVI). In addition, these mutants accumulate a high concentration of 2-octaprenylphenol (compound XIV), a distant precursor in the pathway, as well as 2-(hydroxyoctaprenyl) phenol. However, the relevance of the latter compound to Q biosynthesis is unclear (131).

The final hydroxylation reaction in Q biosynthesis involves the conversion of compound XVIII to compound XIX. Mutants blocked in this conversion were isolated and designated ubiF. As expected, the ubiF mutants accumulated compound XVIII, which was isolated and identified (130).

The origin of the oxygen atoms of Q from aerobically grown E. coli was studied by using ¹⁸O labeling. Cultures were grown under strictly aerobic conditions with succinate as a carbon source and a defined atmosphere containing ¹⁸O_2. The mass spectrum of Q isolated from such cells showed several prominent peaks with m/z values differing from those of normal Q by +6. Thus, three atoms of ¹⁸O had been incorporated; they were located at positions 4, 5, and 6(2).

The nature of the hydroxylations has been investigated. The effect of cytochrome P-450 inhibitors on the synthesis of ¹⁴C-labeled Q-8 from 2-octaprenyl-[U-¹⁴C]phenol was investigated in an aroB mutant of E. coli. SKF-524A and metapyrene markedly inhibited the reaction, whereas 7,8-benzoflavone and carbon monoxide failed to inhibit the reaction(58). A hemA mutant defective in the biosynthesis of cytochromes was able to synthesize ¹⁴C-labeled Q-8 from 2-octaprenyl-[¹⁴C]phenol, thus ruling out the involvement of the cytochrome P-450 monooxygenase system (57). Thus, flavin-linked monooxygenases are involved in these reactions (57), and the three genes ubiB, ubiH, and ubiF encode the aerobic monooxygenases.

E. coli strains grown anaerobically on glycerol-fumarate form appreciable quantities of Q (50 to 70% of aerobic-growth levels). A number of ubi ⁺ strains were grown anaerobically with fumarate as an electron acceptor and examined for the presence of intermediates of the aerobic pathway of Q biosynthesis. All strains accumulated high levels of octaprenylphenol (XIV) under anaerobic conditions. In contrast, this compound does not accumulate in significant quantities in aerobically grown cells. Strains carrying ubiA, ubiD, and ubiE, when grown anaerobically, remained deficient in Q (1). Since the ubiA mutant is blocked in the prenylation of 4-hydroxybenzoic acid, no Q intermediates accumulated in the cells. The ubiD and ubiE mutants lack the carboxy-lyase and 2-octaprenyl-6-methoxy-1,4-benzoquinol methyltransferase, respectively, of the aerobic pathway. Hence, they accumulated 3-octaprenyl-4-hydroxybenzoate (XIII) and 2-octaprenyl-6-methoxy-1,4-benzoquinol (XVII), respectively, under anaerobic conditions. These observations established that octaprenyltransferase, carboxy-lyase, and 2-octaprenyl-6-methoxy-1,4-benzoquinol methyltransferase are common to both aerobic and anaerobic pathways and that compounds XII, XIII, and XVII are also involved in the anaerobic biosynthesis of Q. As expected, the ubiA and ubiD mutants did not accumulate 2-octaprenylphenol (XIV) anaerobically. Furthermore, the ubiE, ubiF, ubiG, and ubiH mutants accumulated 2-octaprenylphenol (XIV) at the same levels as the wild-type strain, implicating 2-octaprenylphenol as an intermediate in anaerobic Q biosynthesis. A ubiG mutant studied accumulated compound XIX as expected. Further, the ubiG mutants, being leaky, formed about 10% of wild-type levels of Q both aerobically and anaerobically, thus establishing the involvement of compound XIX in Q biosynthesis (1).

In contrast to the ubi mutants discussed above, ubiB, ubiH, and ubiF mutants grown anaerobically were able to synthesize Q, providing evidence that specific hydroxylases are involved in the anaerobic pathway (1).

Methylation Reactions

The methylation reactions of the pathway are the conversion of 2-octaprenyl-6-hydroxyphenol to 2-octaprenyl-6-methoxy-phenol (compound XV → XVI), the conversion of 2-octaprenyl-6-methoxy-1,4-benzoquinol to 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol (compound XVII → XVIII), and the conversion of OMHMB to ubiquinol (compound XIX → XX). The methylation reactions (steps 5, 7, and 9) alternate with the three hydroxylations, introducing methyl groups at the 6-OH, at the ring C-3, and at the 5-OH group, respectively. The C-methyl and the two O-methyl groups are derived from methionine (52), with S-adenosylmethionine as the actual methyl donor.

Mutants blocked in the O methylation of 6-OH are not known (discussed above under Hydroxylation Reactions). However, mutants blocked in the methylation of C-3 and 5-OH were isolated and designated ubiE and ubiG respectively (113, 130). The ubiE mutants did not form Q and accumulated compound XVII (130). The isolation and characterization of the product of the methylation, compound XVIII, was achieved with a ubiF mutant (130) and is discussed above under Hydroxylation Reactions. The ubiG mutants are blocked in the O methylation of the 5-OH and hence accumulate OMHMB (XIX), which has been isolated and characterized (71). The enzyme encoded by the ubiG gene has been designated S-adenosylmethionine:OMHMB methyltransferase (71). The enzyme was released from the cells with a Sorvall Rabi cell fractionator. When the cell extract was centrifuged at 30,000 × g and then at 150,000 × g, 98% of the enzyme remained in the supernatant fluid. The enzyme was purified 14-fold by protamine sulfate treatment, ammonium sulfate precipitation, and then column chromatography on Bio-Gel A-5m. The enzyme had an M _r of 50,000 and showed an absolute requirement for dithiothreitol and divalent metal ion (optimally, Zn²⁺). The sequence of the ubiG gene has been determined; the gene is capable of encoding a protein of 26.5 kDa (127). Thus, the protein appears to be a homodimer with two subunits. The enzyme can use either purified or membrane-bound OMHMB as substrate. The enzyme is not tightly bound to the membrane but nevertheless interacts efficiently with membrane-bound OMHMB (71).

Regulation of ubiG gene expression has been studied by using a fusion between ubiG and lacZ on a plasmid. The expression of ubiG was higher aerobically than anaerobically, and glucose decreased transcription. Further, the expression of ubiG was lower in cya and crp mutants than in the wild-type strain. The presence of cyclic AMP increased the expression of ubiG in the wild type and the cya mutant but not in the crp mutant (37).

Localization of Q Biosynthetic Enzymes in E. coli

As discussed above, not all the enzymes involved in Q biosynthesis have been studied in cell extracts. Among the enzymes studied, chorismate pyruvate-lyase (step 1) is a cytoplasmic enzyme, and 4-hydroxybenzoate octaprenyltransferase (step 2) is firmly membrane bound. The other two enzymes that have been studied, 3-octaprenyl-4-hydroxybenzoate carboxy-lyase and OMHMB methyltransferase, are considered to be normally associated with the membrane (71, 72). The association of enzymes with membrane is supported by the isolation of a 2-octaprenyl-[U-¹⁴C]phenol (compound XIV)-charged enzyme complex with an M _r of 2 × 10⁶ and containing at least 12 proteins with M _rs ranging from 40,000 to 80,000 from cells grown anaerobically on glycerol-fumarate medium in the presence of 4-hydroxy-[U-¹⁴C]benzoate. When this complex was incubated with S-adenosylmethionine, NADH, NADPH, Mg²⁺, and a cytoplasmic enzyme with an M _r of about 20,000 (probably a methyltransferase) (55) in the presence of oxygen, all of the ¹⁴C-labeled phenol was converted to Q (56). This complex therefore contains the oxygen-dependent Q-8 biosynthetic apparatus. In anaerobically grown cells, this apparatus, which is charged with 2-octaprenyl phenol, may be kept in a standby position. When oxygen becomes available, Q-8 biosynthesis can be effectively turned on. Since this complex was isolated without detergent treatment, it was thought to have broken from the membrane as a distinct and native domain. This complex contains, in addition to a high level of 2-octaprenylphenol and low levels of Q, phospholipid and other membrane proteins (55, 56).

ubi Genes of E. coli and Their Organization

The isolation of E. coli mutants with defects in Q biosynthesis has depended on the inability of such strains to grow on oxidizable substrates such as succinate or malate while retaining the ability to grow on the fermentable substrate glucose (38). A step-by-step detailed procedure has been described elsewhere (25). Extensive studies by Gibson and Young (38, 39) established the following locations for the ubi genes: ubiA and ubiC, 92 min; ubiB, ubiD, and ubiE, 87 min; ubiH, 63 min; ubiX, 50 min; ubiG, 48 min; and ubiF, 15 min (7, 101). The new locations of these genes according to nucleotide sequencing are shown in Fig. 7. The assignment of ubiX is based on sequence homology with the ubiX gene of S. typhimurium (91). The ubiX gene of S. typhimurium specifies a second polyprenyl 4-hydroxybenzoate carboxy-lyase (50) that apparently carries out the same reaction as the ubiD gene product (28, 72). However, a number of E. coli gene maps erroneously refer to the ubiX gene product as polyprenyl p-hydroxybenzoate carboxylase (7, 101) rather than polyprenyl p-hydroxybenzoate carboxy-lyase (50).

Some Consequences of Mutations in ubi Genes

An E. coli mutant resistant to the antibiotic and antitumor agent phleomycin was isolated. The mutant was also resistant to bleomycin, unable to grow on succinate as the sole source of carbon, and resistant to the lethal effects of heating at 52°C. The Suc^– phenotype and mapping data led to the conclusion that the mutant was defective in the ubiF gene. To confirm the observed properties, known ubiA, ubiD, and ubiF mutants were compared with the newly isolated mutants. They exhibited the same properties (24).

A mutant showing partial resistance to streptomycin was defective in the ubiF gene. Membranes of this strain accumulated 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol but not Q. A previously characterized ubiF mutant showed reduced uptake of gentamicin. At present, no evidence implicates Q in aminoglycoside antibiotic uptake, and these observations are attributed to the general impairment of respiratory capacity (86).

Mutations in the Q biosynthetic pathway (ubiD, ubiB, and ubiG) lead to a lack of flagellar synthesis and motility (10, 48). An ubiA men ⁺ strain was motile anaerobically and nonmotile aerobically, whereas mutants blocked in Q and MK were nonmotile under both aerobic and anaerobic conditions. Thus, a functional electron transport system appears to be essential for motility and flagellar synthesis.

Mutants lacking Q, MK, or both quinones have been isolated, and the role of quinones in electron transport to oxygen and nitrate has been studied (119).

Nucleotide Sequence Analysis of the ubi Genes

Complete information on the Q biosynthetic genes is not available as it is for the MK biosynthetic genes. The sequence of the ubiC, ubiG, and ubiX genes and the relative molecular masses of the proteins have been determined. The sequence of the ubiA gene has been reported by a number of groups. However, the relative molecular mass of the protein has not been determined. Sequences of the ubiBDE genes were determined as part of the E. coli genome project. Although the ORF encoding the ubiB gene and the size of this ORF were determined, the two ORFs in the ubiD and ubiE region could not be unambiguously assigned. The sequences of the ubiH and ubiF genes have yet to be determined. The available information on the ubi genes is summarized in Table 2.

Table 2Characteristics of sequenced ubi gene products

Q Biosynthesis in S. typhimurium

Compared to what is available for E. coli, very little information is available on the biosynthesis of Q in S. typhimurium. Most of this information was obtained while studying other physiological processes. Ames and colleagues (4, 6) isolated a number of mutants defective in the high-affinity histidine transport system. Some of these mutants produced 25% of the wild-type level of Q and showed growth stimulation in the presence of p-hydroxybenzoic acid. Initially, this deficiency was attributed to the deletion of one or more of the ubi genes located at 50 min on the chromosome, and the deficient gene(s) was designated ubiX.

Subsequently, it was shown that the ubiX mutants were defective in flagellation and that a functional Q biosynthetic pathway was essential for flagellation (9). Further biochemical analysis of the ubiX mutant revealed that phenotypically, it resembled the previously described ubiD mutants in producing 20 to 35% of the wild-type level of Q and being deficient in the decarboxylation of 3-octaprenyl-4-hydroxbenzoate (50). The relation between the ubiD and ubiX mutants is discussed above for E. coli.

Studies of pyrimidine metabolism led to the isolation of a mutant dependent on carbamoylaspartate for resistance to 5-fluorouracil. This mutant was deficient in Q biosynthesis and accumulated the Q biosynthetic precursor 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol; hence, it was designated ubiF (132).

Functions of Q

A thorough review of the functions of Q is beyond the scope of this chapter. The role of Q in bacterial motility is briefly discussed above. For the role of Q in aerobic electron transport and in nitrate respiration, other reviews should be consulted (5, 42, 53, 95, 133).

ACKNOWLEDGMENTS

This chapter is dedicated to Ronald Bentley, who introduced me to the world of quinones. I wish him a long, productive, and happy retirement. I thank my colleague M. E. S. Hudspeth for critical reading of the manuscript, and I thank past and present members of my research group: M. E. S. Hudspeth, V. Sharma, C. Palaniappan, K. Suvarna, R. Daruwala, O. Kwon, and H. McHugh. Last but not least, my thanks go to Freyja Altepeter for her cheerful and enthusiastic processing of the manuscript. Research in my laboratory was supported by Public Health Service grants GM42137-01 and GM50262 from the National Institutes of Health.