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Chlorogenic acid (CGA) is a phenolic compound with well-known antibacterial properties against pathogens. In this study, structural and biochemical characterization was used to show the inhibitory role of CGA against the enzyme of the shikimate pathway, a well-characterized drug target in several pathogens. Here, we report the crystal structures of dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, from Providencia alcalifaciens (PaDHQS), in binary complex with NAD and ternary complex with NAD and CGA. Structural analyses reveal that CGA occupies the substrate position in the active site of PaDHQS, which disables domain movements, leaving the enzyme in an open and catalysis-incompetent state. The binding analyses by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) show that CGA binds to PaDHQS with KD (equilibrium dissociation constant) values of 6.3 μM and 0.5 μM, respectively. In vitro enzyme inhibition studies show that CGA inhibits PaDHQS with a Ki of 235 ± 21 μM, while it inhibits the growth of Providencia alcalifaciens, Moraxella catarrhalis, Staphylococcus aureus, and Escherichia coli with MIC values of 60 to 100 μM. In the presence of aromatic amino acids supplied externally, CGA does not show the toxic effect. These results, along with the observations of the inhibition of the 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) regulatory domain by CGA in our previous study, suggest that CGA binds to shikimate pathway enzymes with high affinity and inhibits their catalysis and can be further exploited for designing novel drug-like molecules. IMPORTANCE The shikimate pathway is an attractive target for the development of herbicides and antimicrobial agents, as it is essential in plants, bacteria, and apicomplexan parasites but absent in humans. The enzymes of shikimate pathway are conserved among bacteria. Thus, the inhibitors of the shikimate pathway act on wide range of pathogens. We have identified that chlorogenic acid targets the enzymes of the shikimate pathway. The crystal structure of dehydroquinate synthase, the second enzyme of the pathway, in complex with chlorogenic acid and enzymatic inhibition studies explains the mechanism of inhibition of chlorogenic acid. These results suggest that chlorogenic acid has a good chemical scaffold and have important implications for its further development as a potent inhibitor of shikimate pathway enzymes.

Shikimate kinase (EC 2.7.1.71) catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP. As the fifth key step in the shikimate pathway for aromatic amino acid biosynthesis in bacteria, fungi, and plants, but not mammals, shikimate kinase represents an attractive target for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here, we report the 1.8-Å crystal structure of Helicobacter pylori shikimate kinase (HpSK). The crystal structure shows a three-layer alpha/beta fold consisting of a central sheet of five parallel β-strands flanked by seven α-helices. An HpSK-shikimate-PO 4 complex was also determined and refined to 2.3 Å, revealing induced-fit movement from an open to a closed form on substrate binding. Shikimate is located above a short 3 10 helix formed by a strictly conserved motif (GGGXV) afterβ 3 . Moreover, several highly conserved charged residues including Asp33 (in a conserved DT/SD motif), Arg57, and Arg132 (interacting with shikimate) are identified, guiding the development of novel inhibitors of shikimate kinase.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.

An 18-kbp Acinetobacter calcoaceticus chromosomal segment contains the pcaIJFBDKCHG operon, which is required for catabolism of protocatechuate, and pobSRA, genes associated with conversion of p-hydroxybenzoate to protocatechuate. The genetic function of the 6.5 kbp of DNA between pcaG and pobS was unknown. Deletions in this DNA were designed by removal of fragments between restriction sites, and the deletion mutations were introduced into A. calcoaceticus by natural transformation. The mutations prevented growth with either quinate or shikimate, growth substrates that depend upon qui gene function for their catabolism to protocatechuate. The location of quiA, a gene encoding quinate-shikimate dehydrogenase, was indicated by its expression in one of the deletion mutants, and the position of the gene was confirmed by determination of its 2,427-bp nucleotide sequence. The deduced amino acid sequence of QuiA confirmed that it is a member of a family of membrane-associated, pyrrolo-quinoline quinone-dependent dehydrogenases, as had been suggested by earlier biochemical investigations. Catabolism of quinate and skikimate is initiated by NAD(+)-dependent dehydrogenases in other microorganisms, so it is evident that different gene pools were called upon to provide the ancestral enzyme for this metabolic step.

A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces collinus. This enzyme, a dimer with subunits of identical M(r) (36,000), exhibits a Km of 1.5 +/- 0.3 microM for NADPH and 25 +/- 3 microM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30 degrees C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained. Ansatrienin A (an antibiotic produced by S. collinus) contains a cyclohexanecarboxylic acid moiety, and it is suggested that the 1-cyclohexenylcarbonyl-CoA reductase described herein catalyzes the final reductive step in the conversion of shikimic acid into this moiety.

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH7P) synthase (phenylalanine repressible) catalyze similar aldol-type condensations between phosphoenolpyruvate (PEP) and the monosaccharides d-arabinose 5-phosphate (A5P) and d-erythrose 4-phosphate (E4P), respectively (Fig. ​(Fig.1).1). Both enzymes from Escherichia coli are tetrameric, and although their quaternary structures are dissimilar, the monomeric subunits that comprise each enzyme are nearly superimposable (9). As one might expect from this degree of subunit similarity, the facial selectivities of both enzymes from E. coli have been shown to be re-face with respect to the phosphorylated monosaccharide and si-face with respect to PEP, resulting in an (R) configuration at the C-4 position of each product (2, 4, 7, 8). Reports from this laboratory have shown that E. coli DAH7P synthase (encoded by aroG) is capable of utilizing arabinose 5-phosphate, ribose 5-phosphate, and 2-deoxyribose 5-phosphate to synthesize the corresponding eight carbon monosaccharides (14), while E. coli KDO8P synthase is unable to utilize E4P as a substrate (D. L. Howe, G. Y. Sheflyan, and R. W. Woodard, unpublished data). The facial selectivity of DAH7P synthase with regard to both PEP and the alternate substrates listed above is the same as with the natural substrates (16a). FIG. 1 The reactions catalyzed by E. coli KDO8P and DAH7P synthases with their respective substrates and the proposed reaction for the N. gonorrhoeae KDO8P synthase-catalyzed condensation of E4P and PEP. Open arrow, location of newly formed C-C bond. It has recently been reported not only that KDO8P synthase from Neisseria gonorrhoeae utilizes E4P as an alternate substrate but also that the facial selectivity of the enzyme with respect to this monosaccharide is si-face, resulting in an (S) configuration at the C-4 position of the product (16). This suggests that the enzyme from N. gonorrhoeae is distinct from all other reported KDO8P synthases, in terms of both its substrate ambiguity and its stereoselectivity. However, these unusual results were obtained using enzyme isolated directly from N. gonorrhoeae that was neither rigorously purified nor thoroughly biochemically characterized (16). The product of the enzymatic reaction, C7-X (original authors' designation), was neither isolated nor purified, and its stereochemistry was assigned solely on the intensity and rapid production of the chromophore produced in the classic Aminoff assay (periodate-thiobarbituric acid assay) for 3-deoxy-monosaccharides (1, 10). This assay is known to be sensitive to the stereochemistry of the hydroxyl groups of the monosaccharide (11, 18) but is also known to give false-positive results with compounds such as shikimic acid, guanosine, and adenosine, as well as with DNA (6). Since studies in our laboratory are aimed at the elucidation of the similarities and differences between KDO8P and DAH7P synthase, we were intrigued by the reported use of E4P by a KDO8P synthase and decided to further investigate this unusual finding. An open reading frame corresponding to the KDO8P synthase gene (kdsA) was retrieved via a TBLASTN search of the N. gonorrhoeae FA 1090 databank at the University of Oklahoma Advanced Center for Genome Technology (http://www.genome.ou.edu/gono_blast.html) using a fragment of E. coli KDO8P synthase. Standard PCR methodologies were employed to amplify this gene sequence from N. gonorrhoeae DNA. The amplified product was isolated, restricted, and ligated into the expression vector pT7-7 (17). The ligation mixture was used to transform competent E. coli XL1-Blue cells (Stratagene). Plasmids isolated from several transformants were subjected to DNA sequencing, and one plasmid with the expected sequence was isolated (pT7-7-NGKDO). In our sequence, position 119 is threonine; this residue is absolutely conserved in all known KDO8P synthase sequences (M. R. Birck and R. W. Woodard, submitted for publication). KDO8P synthase from N. gonorrhoeae was overexpressed in E. coli BL21(DE3) cells harboring pT7-7-NGKDO and purified, as previously reported, using anion-exchange chromatography (14; W. P. Taylor and R. W. Woodard, submitted for publication). The resultant protein was determined to be >95% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The apparent molecular mass was determined by size-exclusion chromatography and was found to be 88.2 kDa. This value is similar to those reported for the KDO8P synthases from E. coli, Aquifex aeolicus, and Salmonella enterica serovar Typhimurium (3, 12–14; Taylor and Woodard, submitted). The N. gonorrhoeae KDO8P synthase-catalyzed condensation of A5P and PEP was monitored by observing the disappearance of the α,β-unsaturated carbonyl absorbance (λ = 232 nm, ɛ = 2,840 M−1 cm−1) of PEP (14). All reactions were performed in 100 mM Tris-acetate, pH 7.4, at 37°C. The kinetic parameters were determined from progress curves of substrate consumption, as previously described (Taylor and Woodard, submitted), and are as follows: for PEP, Km = 3.1 ± 0.2 μM; for A5P, Km = 8.5 ± 0.1 μM; for both PEP and A5P, kcat = 1.0 ± 0.1 s−1. The Michaelis constants for both PEP and A5P are very similar to those reported for both E. coli and S. enterica serovar Typhimurium KDO8P synthase, although the value for kcat was reduced approximately three- to sixfold (12; Taylor and Woodard, submitted). The effect of divalent metal ions on enzymatic activity was investigated by quantitating the amount of KDO8P produced using the periodate-thiobarbituric acid assay (1, 10, 14). Inclusion of Mn2+ or Zn2+ (1 mM) in the assay mixture had no effect on activity. Preincubation of the enzyme with a 10 mM concentration of either metal prior to reaction led to a small decrease in enzyme activity. Prolonged treatment of purified enzyme with 5 mM EDTA resulted in no loss of enzymatic activity. Purified KDO8P synthase (12 μM) was incubated with 24.3 mM A5P, 23.7 mM PEP, 0.5 mM MnCl2, and 1 mM dithiothreitol in 100 mM Tris-acetate buffer (pH 7.5) in a final volume of 2.0 ml. Even though it is shown in the present study that N. gonorrhoeae KDO8P synthase does not require any metal ion for activity, MnCl2 was included in the reaction mixture in order to mimic the reaction conditions reported by Subramaniam et al. (16). Tris-acetate was substituted for phosphate buffer in the present study since inorganic phosphate has been shown to inhibit E. coli KDO8P synthase (5). Incubation of KDO8P synthase from N. gonorrhoeae with E4P and PEP in 75 mM phosphate buffer (pH 7.0), however, gave no indication of deoxymonosaccharide formation as determined by periodate-thiobarbituric acid analysis. The reaction mixture was incubated at 37°C for 5 h, and then the reaction was quenched by the addition of 10% trichloroacetic acid (2.0 ml). The 3-deoxy-α-keto sugar acid component of the reaction mixture was purified by anion-exchange chromatography as previously described (14). The 500 MHz 1H-nuclear magnetic resonance (NMR) spectrum of the product isolated from the above reaction is shown in Fig. ​Fig.22 and is in complete agreement with that reported previously for 3-deoxy-d-manno-octulosonic acid 8-phosphate obtained from the E. coli KDO8P synthase-catalyzed condensation of A5P and PEP (Fig. ​(Fig.2)2) (2). In a parallel experiment, 33 mM E4P was substituted for A5P and the mixture was subjected to the reaction conditions and purification protocol described above. Periodate-thiobarbituric acid analysis of the quenched enzymatic reaction mixture before chromatography as well as analysis of all column fractions after purification yielded no indication of a keto-deoxy-monosaccharide acid product. 1H-NMR analysis of the column fractions where acidic phosphorylated monosaccharides normally elute (fractions 30 to 40) gave no signals representative of the condensation product (14). Starting materials were recovered in fractions 23 to 27 (E4P) and fractions 35 to 39 (PEP). Thus, we found that KDO8P synthase from N. gonorrhoeae exhibits the same reaction stereochemistry and substrate selectivity observed for E. coli KDO8P synthase (2, 4). FIG. 2 500-MHz 1H-NMR spectra of the product derived from the condensation of A5P and PEP as catalyzed by E. coli KDO8P synthase (X, peak from buffer) (a) and N. gonorrhoeae KDO8P synthase (b). While the present report demonstrates that E4P is not a substrate for N. gonorrhoeae KDO8P synthase, the question remains as to the actual identity of the C7-X compound identified by Subramanian et al. (16). Based on the reported rate of oxidation as well as the fact that C7-X is not a substrate for dehydroquinate synthase, it is tempting to suggest that this compound is indeed 3-deoxy-d-ribo-heptulosonate 7-phosphate. It is known that 2-keto-3-deoxy-6-phosphogluconate aldolase catalyzes the aldol-like condensation of E4P and pyruvic acid to form 3-deoxy-d-ribo-heptulosonate 7-phosphate (15). Alternatively, a minor DAH7P synthase contaminant could prove to be the source of C7-X formation, although this would also require addition to the si-face of E4P, in contrast to the stereochemistry of all other known DAH7P synthase-catalyzed reactions. Efforts are underway in our laboratory to clone, overexpress, and purify N. gonorrhoeae DAH7P synthase in order to address this latter possibility.

Mecillinam, a beta-lactam antibiotic specific to penicillin-binding protein 2 (PBP 2) in Escherichia coli, blocks cell wall elongation and, indirectly, cell division, but its lethality can be overcome by increased levels of ppGpp, the nucleotide effector of the stringent response. We have subjected an E. coli K-12 strain to random insertional mutagenesis with a mini-Tn10 element. One insertion, which was found to confer resistance to mecillinam in relA+ and relA strains, was mapped at 75.5 min on the E. coli map and was located between the promoters and the coding sequence of the aroK gene, which codes for shikimate kinase 1, one of two E. coli shikimate kinases, both of which are involved in aromatic amino acid biosynthesis. The mecillinam resistance conferred by the insertion was abolished in a delta relA delta spoT strain completely lacking ppGpp, and it thus depends on the presence of ppGpp. Furthermore, the insertion increased the ppGpp pool approximately twofold in a relA+ strain. However, this increase was not observed in relA strains, although the insertion still conferred mecillinam resistance in these backgrounds, showing that mecillinam resistance is not due to an increased ppGpp pool. The resistance was also abolished in an ftsZ84(Ts) strain under semipermissive conditions, and the aroK::mini-Tn10 allele partially suppressed ftsZ84(Ts); however, it did not increase the concentration of the FtsZ cell division protein. The insertion greatly decreased or abolished the shikimate kinase activity of AroK in vivo and in vitro. The two shikimate kinases of E. coli are not equivalent; the loss of AroK confers mecillinam resistance, whereas the loss of Arol, does not. Furthermore, the ability of the aroK mutation to confer mecillinam resistance is shown to be independent of polar effects on operon expression and of effects on the availability of aromatic amino acids or shikimic acid. Instead, we conclude that the AroK protein has a second activity, possibly related to cell division regulation, which confers mecillinam sensitivity. We were able to separate the AroK activities mutationally with an aroK mutant allele lacking shikimate kinase activity but still able to confer mecillinam sensitivity.

In the course of sequencing the aroK gene, a number of errors were found in the published sequence. The corrected sequence alters the length of the aroK coding region such that the AroK and AroL proteins are now of comparable length and the homology between them extends the entire length of the two enzymes.

This paper continues the description of transketolase mutants of Escherichia coli ; they are absolutely unable to grow on pentoses, but slightly “leaky” with respect to their aromatic requirement (B. L. Josephson and D. G. Fraenkel, 1969). Several experiments have explored the degree of leakiness and shown it to be low. There is little conversion of radioactive xylose to carbon dioxide. The labeling of ribose in cells grown on [1- 14 C]glucose and [2- 14 C]glucose accords with its origin being chiefly by the oxidative pathway. A mutant lacking both transketolase and gluconate-6-phosphate dehydrogenase has been constructed; it requires supplementation with pentose. Pentoses are inhibitory to growth of transketolase mutants, but high levels of pentose phosphates do not accumulate in this situation. Several experimental results are suggestive of regulation of metabolic flow in the oxidative branch of the hexose monophosphate shunt.

Pretyrosine is an amino acid intermediate of phenylalanine and/or tyrosine biosyntheses in a variety of organisms. A procedure for the isolation of high-quality pretyrosine as the barium salt is described. Stable solutions of ammonium pretyrosine that are suitable for use as substrate in enzyme assays can be prepared in good yield with relatively few purification steps. A triple mutant of Neurospora crassa , bearing genetic blocks corresponding to each initial enzyme step of the three pathway branchlets leading to the aromatic amino acids, accumulates prephenate and pretyrosine. Although the time courses of prephenate and pretyrosine accumulations were found to be parallel in any given experiment, the ratios of the two metabolites varied as much as 100-fold depending upon such variables as carbon source, temperature of growth, accumulation, and especially the presence of aromatic pathway metabolites. Under appropriate nutritional conditions of accumulation, pretyrosine concentrations in excess of 4 mM in culture supernatant fluids were obtained. Strains individually auxotrophic for phenylalanine or tyrosine accumulate lesser amounts of prephenate and pretyrosine. The metabolic blocks of the mutant result in high intracellular levels of prephenate, which is then partially transaminated to pretyrosine. In N. crassa , pretyrosine is a dead-end metabolite since it is not enzymatically converted to phenylalanine or tyrosine. At a mildly acidic pH, pretyrosine is quantitatively converted to phenylalanine in a nonenzymatic reaction.

Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, each possessing a 5-enolpyruvylshikimate 3-phosphate synthase that is sensitive to inhibition by glyphosate [N-(phosphonomethyl)glycine], provide a good cross-section of organisms exemplifying the biochemical diversity of the aromatic pathway targeted by this potent antimicrobial compound. The pattern of growth inhibition, the alteration in levels of aromatic-pathway enzymes, and the accumulation of early-pathway metabolites after the addition of glyphosate were distinctive for each organism. Substantial intracellular shikimate-3-phosphate accumulated in response to glyphosate treatment in all three organisms. Both E. coli and P. aeruginosa, but not B. subtilis, accumulated near-millimolar levels of shikimate-3-phosphate in the culture medium. Intracellular backup of common-pathway precursors of shikimate-3-phosphate was substantial in B. subtilis, moderate in P. aeruginosa, and not detectable in E. coli. The full complement of aromatic amino acids prevented growth inhibition and metabolite accumulation in E. coli and P. aeruginosa where amino acid end products directly control early-pathway enzyme activity. In contrast, the initial prevention of growth inhibition in the presence of aromatic amino acids in B. subtilis was succeeded by progressively greater growth inhibition that correlated with rapid metabolite accumulation. In B. subtilis glyphosate can decrease prephenate concentrations sufficiently to uncouple the sequentially acting loops of feedback inhibition that ordinarily link end product excess to feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by prephenate. The consequential unrestrained entry is an energy-rich substrates into the aromatic pathway, even in the presence of aromatic amino acid end products, is an energy drain that potentially accounts for the inability of end products to fully reverse glyphosate inhibition in B. subtilis. Even in E. coli after glyphosate inhibition and metabolite accumulation were allowed to become fully established, a transient period where end products were capable of only partial reversal of growth inhibition occurred. The distinctive metabolism produced by dissimilation of different carbon sources also profound effects upon glyphosate sensitivity.

A menaquinone mutant (SG1) of Bacillus licheniformis has been isolated by selecting for colonies that are resistant to low levels of kanamycin (1.5 mug/ml) but sensitive to the same concentration of kanamycin in the presence of shikimate (25 mug/ml). The wild type (IU1) contained 0.38 +/- 0.02 nmol of menaquinone-7 (MK-7) per mg (dry weight) of cells when grown +/- shikimate, whereas SG1 had less than 0.01 nmol of MK-7 per mg (dry weight) of cells when grown in the presence of shikimate. SG1 had a generation time of 85 min, as compared to 24 min for IU1 grown +/- shikimate. SG1 doubled with a generation time of 28 min when grown in the presence of shikimate. IU1 consumed O2 at various rates depending on the stage of growth. A triphasic O2 consumption curve with maxima at mid-exponential phase, the transition from exponential to stationary phase, and early stationary phase was found for IU1 +/- shikimate and SG1 + shikimate. SG1 grown without shikimate consumed O2 at a low level (10 to 20% of IU1). Normal respiration could be restored to SG1 8.5 min after shikimate addition, whereas normal growth was not restored until 40 min after shikimate addition. Electron microscopic studies of SG1 and IU1 have indicated a morphological alteration in the mutant. SG1 is a dwarf cell as compared to IU1, when grown without shikimate. However, SG1 grown with shikimate became morphologically indistinguishable from IU1.

3-Deoxy-arabino-heptulosonic acid 7-phosphate synthase, prephenate dehydratase, tryptophan synthase, and 2,3-dihydroxybenzoylserine synthase enzyme activities are derepressed in wild-type Escherichia coli K-12 cells grown on Fe3+-deficient medium. This derepression is reversed when FeSO4 is added to the growth medium. Addition of shikimic acid to the Fe3+-deficient growth medium caused repression of the first three enzyme activities but not of 2,3-dihydroxybenzoylserine synthase activity. Addition of 2,3-dihydroxybenzoic acid to the Fe3+-deficient growth medium has no effect on any of the above-mentioned enzyme activities. The Fe3+ deficiency-mediated derepression of 3-deoxyarabino-heptulosonic acid 7-phosphate synthase activity is due to an elevation of the tyrosine-sensitive isoenzyme; the phenylalanine-sensitive isoenzyme is not derepressed under these conditions.

Mutations in ARO1 and ARO2 genes coding for enzymes involved in the common part of the aromatic amino acid pathway completely block the sporulation of Saccharomyces cerevisiae when in a homozygous state, whereas mutations in all the other genes of the same pathway do not. This effect is not due to the lack of any intermediate metabolite but rather to the accumulation of a metabolite preceding chorismic acid. Shikimic acid or one of its precursors was identified as the possible inhibitor. The presence of the three aromatic amino acids in the sporulation medium restores the ability to undergo meiosis. This seems not to be due to a feedback inhibition of the first enzymes of the pathway but rather to a competition between aromatic amino acids and the inhibitor on a site specific for the meiotic process. The inhibition of sporulation seems to occur at a very early step in meiosis, as indicated by the lack of premeiotic DNA synthesis in aro1 and aro2 mutants.

Daniel, John W. (University of Wisconsin, Madison, Wis.) and Harold P. Rusch . Niacin requirement for sporulation of Physarum polycephalum . J. Bacteriol. 83: 1244–1250. 1962.—The myxomycete Physarum polycephalum undergoes sexual sporulation if exposed to light after 4 days of incubation in the dark on a salts medium containing niacin, niacinamide, or tryptophan. None of these compounds is required for growth. Quinic acid, shikimic acid, intermediates of the kynurenine pathway, diphosphopyridine nucleotide (DPN), and triphosphopyridine nucleotide (TPN) replace niacin but a number of other tryptophan metabolites do not. Analogues of niacin inhibit sporulation when added at the beginning but not at the end of dark incubation with niacin. Folic acid, p -aminobenzoic acid, and p -aminobenzenesulfonamide inhibit sporulation if added at any time during the incubation or illumination periods. Reduced di- or triphosphopyridine nucleotide, but not DPN or TPN, reverse the p -aminobenzoic acid inhibition but do not replace the light requirement or shorten the dark incubation period. Gluconate and 2-ketogluconate also replace niacin. Glucose, pyruvate, malate, and oxalacetate inhibit the niacin-induced sporulation. Iodoacetate and fluoride do not counteract the glucose effect or inhibit sporulation.

Mutants of Escherichia coli K-12 blocked in each of the three enzymatic reactions between chorismate and 2,3-dihydroxybenzoate, in the pathway leading to the iron-sequestering compound enterochelin, have been isolated and biochemically characterized. The three genes concerned (designated entA, entB and entC ) have been shown to be clustered on the chromosome between purE and gal and to be located near minute 14 by cotransduction with the purE, lip , and fep genes. entA, entB , and entC were shown to be the structural genes for 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, 2,3-dihydro-2,3-dihydroxybenzoate synthetase, and isochorismate synthetase, respectively.

Several regulated enzymes involved in aromatic amino acid synthesis were studied in Bacillus subtilis and B. licheniformis with reference to organization and control mechanisms. B. subtilis has been previously shown (23) to have a single 3-deoxy- d - arabino heptulosonate 7-phosphate (DAHP) synthetase but to have two isozymic forms of both chorismate mutase and shikimate kinase. Extracts of B. licheniformis chromatographed on diethylaminoethyl (DEAE) cellulose indicated a single DAHP synthetase and two isozymic forms of chorismate mutase, but only a single shikimate kinase activity. The evidence for isozymes has been supported by the inability to find strains mutant in these activities, although strains mutant for the other activities were readily obtained. DAHP synthetase, one of the isozymes of chorismate mutase, and one of the isozymes of shikimate kinase were found in a single complex in B. subtilis . No such complex could be detected in B. licheniformis . DAHP synthetase and shikimate kinase from B. subtilis were feedback-inhibited by chorismate and prephenate. DAHP synthetase from B. licheniformis was also feedback-inhibited by these two intermediates, but shikimate kinase was inhibited only by chorismate. When the cells were grown in limiting tyrosine, the DAHP synthetase, chorismate mutase, and shikimate kinase activities of B. subtilis were derepressed in parallel, but only DAHP synthetase and chorismate mutase were derepressible in B. licheniformis . Implications of the differences as well as the similarities between the control and the pattern of enzyme aggregation in the two related species of bacilli were discussed.

The growth of a vitamin K-requiring strain of Bacteroides melaninogenicus was promoted by some postulated and proven biosynthetic precursors of bacterial menaquinones, 1,4-dihydroxy-2-naphthoic acid, shikimic acid, chorismic acid, and 4(2′-carboxyphenyl)-4-oxobutyric acid. Growth of the organism with [ 2 ′, 4 - 14 C 2 ]-4(2′-carboxy phenyl)-4-oxobutyric acid as the vitamin K replacement gave rise to a mixture of radioactive menaquinone-9 and menaquinone-10; the dilution factor for this incorporation was 1.8.

Pittard, James (University of Melbourne, Melbourne, Australia), and B. J. Wallace . Gene controlling the uptake of shikimic acid by Escherichia coli . J. Bacteriol. 92: 1070–1075. 1966.—Mutants possessing an impaired ability to take up exogenously supplied shikimic acid were isolated. Mutants showing either a complete or partial loss of this activity were found. The mutations have been mapped on the Escherichia coli chromosome and occur in a single gene, situated near to the cluster of genes concerned with histidine biosynthesis. The uptake of dehydroshikimic acid was also affected by these mutations, suggesting a common mechanism for the uptake of these two related intermediates of aromatic biosynthesis.

Transketolase mutants have been selected after ethyl methane sulfonate mutagenesis ofEscherichia coli. These strains are unable to grow on any pentose and, in addition, require a supplement of aromatic amino acids or shikimic acid for normal growth on any other carbon source. Revertants are normal in both respects and also contain transketolase. Transketolase mutants do not require exogenous pentose for growth. Preliminary genetic mapping of the locus is presented.

l -Histidine and, to a lesser degree, l -phenylalanine at concentrations of 10 −4 m inhibit the growth of leaky mutants (bradytrophs) of Bacillus subtilis that are deficient in the synthesis of p -hydroxyphenylpyruvate, the first intermediate specific to tyrosine synthesis. The inhibition can be overcome by growth factor amounts of l -tyrosine and p -hydroxyphenylpyruvate. Histidine and phenylalanine are capable of inhibiting the synthesis of tyrosine in several ways, and the major physiological effect which results in growth inhibition has not been established. Both l -histidine and l -phenylalanine inhibit the activity of prephenate dehydrogenase at concentrations about 100-fold higher than the inhibitory concentration of l -tyrosine. Histidine also appears to repress the synthesis of prephenate dehydrogenase because a histidine bradytroph growing in histidine-supplemented medium has a twofold lower level of this enzyme than the same cells growing in unsupplemented medium. These same two amino acids also inhibit the growth of a bradytroph deficient in dehydroquinate synthetase, an early enzyme in the pathway of tyrosine, phenylalanine, and tryptophan synthesis. The inhibition is overcome by a combination of tyrosine and phenylalanine. Histidine-resistant derivatives of both the prephenate dehydrogenase and dehydroquinate synthetase-deficient strains, which simultaneously have gained resistance to phenylalanine, have been isolated. Most of these resistant mutants synthesize additional tyrosine compared with the parent strain. One class of resistant mutants excretes tyrosine and has a number of enzymes of aromatic acid synthesis which are no longer repressible by any combination of the aromatic amino acids. Tyrosine inhibits the growth of histidine bradytrophs. Histidine, at growth factor levels, overcomes the inhibition.

A transduction analysis of phenylalanine-responding mutants of Pseudomonas aeruginosa revealed the existence of six unlinked genes. Enzyme assays showed that one gene was involved in the terminal production of phenylalanine (chorismate mutase), and the remaining five genes were involved in the common pathway of aromatic amino acid biosynthesis.

Centrifugation in sucrose density gradients of partially purified extracts from six species of fungi, i.e., Rhizopus stolonifer, Phycomyces nitens, Absidia glauca ( Phycomycetes ), Aspergillus nidulans ( Ascomycetes ), Coprinus lagopus , and Ustilago maydis ( Basidiomycetes ), indicate that the five enzymes catalyzing steps two to six in the prechorismic acid part of the polyaromatic synthetic pathway sediment together. The sedimentation coefficients for these enzymes are very similar in the six species and are comparable to those previously observed for the multienzyme complexes ( arom aggregates) of Neurospora crassa and Saccharomyces cerevisiae . These results are interpreted as indicating the presence in each of these fungi of arom aggregates, presumably encoded by arom gene clusters similar to those in N. crassa and S. cerevisiae . Evidence has also been obtained for the presence in two species ( A. nidulans and U. maydis ) and the absence in the other four species of a second dehydroquinase isozyme which is distinguishable from the synthetic activity on the basis of both thermostability tests and S values. This second dehydroquinase, which is apparently involved in the catabolism of quinic acid via a pathway similar to that in N. crassa , is inducible in A. nidulans (as it is in N. crassa ), but constitutive in U. maydis . These comparative findings are discussed in relation to the organization, evolution, and possible functional relationships of synthetic and catabolic aromatic pathways in fungi.

A series of independentEscherichia coliK eductants has been isolated and tested to determine the extent of their deletions. The deletions cover the P2 prophage in location H, thehisoperon, a suppressor of therecBCphenotype (sbcB), the gene for gluconate-6-phosphate dehydrogenase (gnd), a locus involved in cell wall synthesis (rfb), and in some cases all or part of genes involved in methylgalactoside uptake (mglP). One end of the deletion, the P2 prophage end, appears to be the same for all eductants. The other end, however, can be located before, within, and after themglPlocus.

Montie, Thomas C. (Albert Einstein Medical Center, Philadelphia, Pa.), and Samuel J. Ajl . Selective inhibition by tryptophan analogues of murine toxin synthesis in Pasteurella pestis . J. Bacteriol. 88: 1467–1475. 1964.—Washed-cell suspensions of Pasteurella pestis , avirulent strain “Tjiwidej,” exhibited a preferential inhibition of toxin synthesis relative to total protein formation, when grown in the presence of various tryptophan analogues. Growth was partially inhibited in the presence of methyl analogues. High concentrations of 5-fluorotryptophan induced slight growth-inhibitory effects. However, toxin production was more sensitive to these levels of the analogue. Growth inhibition appeared not to relate to toxin inhibition. Inhibition of toxin synthesis by analogues was reversed by l -tryptophan and indole. Shikimic acid but not anthranilic acid antagonized the action of 4-methyltryptophan on selective toxin synthesis. The formation of tryptophanless protein accounted for continued protein synthesis in tryptophan-depleted cells. Protein resolved by acrylamide gel electrophoresis from crude cell extracts exhibited two toxic protein bands. The synthesis of one toxin-protein band, the less-mobile of the two, appeared to be associated with the membrane fraction of the cell, and was selectively blocked in cells grown in the presence of tryptophan analogues. Cellular tryptophan levels may determine the quantity and quality of proteins made.

In two strains of Bacillus subtilis , menaquinone-7 has been shown to reach maximal cellular concentrations during early stationary phase. These concentration changes closely parallel the previously reported concentration changes in the cytochromes.

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.

Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme.

A multiple aromatic amino acid auxotroph of Bacillus subtilis 168 has been isolated which is unable to synthesize menaquinone-7 (MK-7) unless supplied with shikimic acid (SHK). The mutant, RB163, was isolated by selecting for resistance to low levels (1.5 μg/ml) of kanamycin. Enzymatic and genetic analyses show that the strain is an aroD mutant lacking 5-dehydroshikimate reductase. Under growth conditions in which its MK-7 deficiency is expressed, RB163 is deficient in cytochromes a, b, and c, exhibits low growth yields, and does not sporulate. Genetic analysis indicates that this pleiotropic phenotype is the result of a single genetic event. All phenotypic characteristics are reversible when the mutant is grown under conditions such that MK is synthesized. Comparison of strain RB163 with other aro mutants blocked before SHK (“early-aro” mutants) reveals interesting differences. Most early-aro mutants are cytochrome- and MK-sufficient, sporogenous, and sensitive to kanamycin when grown in the absence of SHK. However, in addition to strain RB163, two other aro mutants were found to show the pleiotropic phenotype. These three mutants have in common, and differ from other early-aro strains in, the inability to synthesize MK. It is suggested that the phenotypically wild-type aro mutants are bradytrophic, allowing enough substrate flow through the common aromatic pathway to satisfy the MK requirement. The pleiotropic mutants are thought to be completely blocked in the common pathway, thus accounting for their inability to synthesize MK.

Phenylalanine inhibited thiazole biosynthesis in a thiamine-regulatory mutant of Escherichia coli , and the inhibition was overcome by tyrosine.

Single step mutants of Bacillus subtilis which required either one or all of the aromatic amino acids for growth were isolated. The relevant gene defect was determined for each mutant by enzyme assays in vitro. A mutant deficient in each enzyme step of aromatic amino acid biosynthesis was found with the exceptions of the shikimate kinase and the phenylalanine and tyrosine transaminases. Representative mutants carrying the defective genes were mapped by deoxyribonucleic acid mediated transformation by reference to the aromatic amino acid gene ( aro ) cluster and, alternately, to any of the other unlinked aro genes. The genes coding for dehydroquinate synthetase, 3-enol pyruvylshikimate 5-phosphate synthetase, one form of chorismate mutase, and prephenate dehydrogenase are linked to the aro cluster. Except for the previously identified linkage between the genes of 3-deoxy- d -arabino heptulosonic acid 7-phosphate synthetase and one species of chorismate mutase, the other genes involved in this pathway are neither linked to the aro cluster nor to each other.

Several aspects of the respiratory physiology of a mutant of Bacillus subtilis deficient in menaquinone-7 (MK-7) and in cytochromes were investigated. The mutant, an aromatic amino acid auxotroph blocked at dehydroshikimate reductase, is unable to synthesize MK-7 unless grown in the presence of the common aromatic amino acid intermediate, shikimate. The inability to synthesize MK-7 prevents the mutant from expressing the normal postexponentialphase cytochrome phenotype. When grown in the presence of shikimate, normal levels of these electron transport components are formed. It was found that the intracellular concentration of MK-7 could be predictably regulated by growing the cells with known concentrations of exogenous shikimate. When the mutant was grown under conditions where MK-7 biosynthesis was severely limited, there was a decrease in oxygen uptake and in membrane-associated reduced nicotinamide adenine dinucleotide (NADH) oxidase and succinate oxidase activity. NADH oxidase, but not succinoxidase, could be restored in membrane preparations by the addition of menadione to the reaction mixture. Reduced-minus-oxidized cytochrome difference spectra indicate that an MK-7 deficiency limits electron flow through the cytochrome chain. Furthermore, oxidation-reduction patterns suggest that MK-7 functions between the primary dehydrogenases and the cytochromes. Although the mutant is asporogenous when grown under conditions where MK-7 biosynthesis is limited, the inability to sporulate does not appear to result from lesions in the electron transport system.