Bacteriophage T4 baseplate components. III. Location and properties of the bacteriophage structural thymidylate synthetase.

Two T4D thymidylate synthetase (td) temperature-sensitive mutants have been isolated and characterized. Both mutants produce heat-labile phage particles. This observation supports the view that this viral-induced protein is a phage structural component. Further, antiserum to td has been shown to block a specific step in tail plate morphogenesis. The results indicated that the td protein is largely covered by the T4D tail plate gene 11 protein. Since the phageinduced dihydrofolate reductase (dfr) also is partially covered by the gene 11 protein, it appears that td was adjacent to the tail plate dfr. This location has been confirmed by constructing a T4D mutant which is dfrtstdts and showing that these two tail plate constituents interact and give altered physical properties to the phage particles produced. A structural relationship for the tail plate folate, dfr, and td has been reported.     

Bacteriophage Tail Components: II. Dihydrofolate Reductase in T4D Bacteriophage

The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants, wh1 and wh11, have been examined. Various lines of evidence support the view that the product of the wh locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced "early" enzyme is a component of the phage coat.     

Bacteriophage T4 baseplate components. I. Binding and location of the folic acid.

Two different proteins with high affinities for the pteridine ring of folic acid have been used to determine the location of this portion of the folate molecule in the tail plate of T4D and other T-even bacteriophage particles. The two proteins used were (i) antibody specific for folic acid and (ii) the folate-binding protein from bovine milk. Both proteins were examined for their effect on various intact and incomplete phage particles. Intact T2H was weakly inactivated by the antiserum but not by the milk protein. No other intact T-even phage, including T4D, was affected by these two proteins. When incomplete T4D particles were exposed in an in vitro morphogenesis system, it was found that neither of the two proteins affected either the addition of the long tail fibers to fiberless particles or the addition of tail cores to tail plates. On the other hand, these two proteins specifically blocked the addition of T4D gene 11 product to the bottom of T4D baseplates. After the addition of the gene 11 protein, these two reagents did not inhibit the further addition of the gene 12 protein to the baseplate. It can be concluded that the phage folic acid is a tightly bound baseplate constituent and that the pteridine portion of the folic acid is largely covered by the gene 11 protein.     

Bacteriophage T4 mutants deficient in alteration and modification of the Escherichia coli RNA polymerase

An extensive screening of coliphage T4 mutants has revealed two distinct classes defective, respectively, in the two sequential phage-induced phosphorylations of the host RNA polymerase, alteration and modification. The existence of these mutants proves that T4-specified functions are involved in both processes. The viabilities of these mutants demonstrate that neither alteration nor modification is essential for growth in Escherichia coli B/r. Physiological studies after infection of E. coli B/r have failed to reveal any abnormalities of phage deficient in alteration or modification. Both mutants normally inhibit host protein and stable RNA synthesis and normally express all classes of T4 genes. Thus, these specific phage-induced structural changes in the host RNA polymerase are not fundamental to the control of gene expression during T4 development. Alteration and modification may be required for growth in some strains of E. coli and hence be selectively advantageous because they extend the normal host range of the phage.Alteration appears to be catalyzed by a T4 function injected with the DNA. A polypeptide of molecular weight 61,000, which is probably cleaved during morphogenesis from a precursor of molecular weight 79,000, is missing in phage particles of alteration-deficient strains and may be the phage activity so injected. The T4 gene involved in alteration is named alt.Modification is controlled by a T4-replicative gene that has been mapped into a region of about 500 base-pairs between genes 39 and 56. These mapping data show that the defect in α modification defines a new T4 gene, named mod.     

Function of T4D Structural Dihydrofolate Reductase in Bacteriophage Infection

Various properties of the bacteriophage structural dihydrofolate reductase (DFR) have been examined to determine its function during phage infection. It has been found that a binding site for reduced nicotinamide adenine dinucleotide phosphate (NADPH), most likely on the DFR present in the phage tail plate, is required for phage viability. Attachment of adenosine diphosphoribose, an analogue of NADPH, to this site prevents phage adsorption and injection. This adenosine diphosphoribose inhibition can be competitively reversed by the addition of NADPH or oxidized nicotinamide adenine dinucleotide phosphate. It is suggested that, during phage infection, the host bacterial cell might leak compounds functionally similar to the pyridine nucleotides. These compounds have been shown to nonenzymatically change the conformation of the phage tail plate DFR which is apparently necessary for successful injection.     

Bacteriophage T4 Virion Baseplate Thymidylate Synthetase and Dihydrofolate Reductase

Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td⁺, whereas particles produced after infection with td⁻ had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from td^ts to td⁺ simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both td^ts and frd^ts genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frd^ts mutants to frd⁺ not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles.     

Bacteriophage-induced functions in Escherichia coli K (lambda) infected with rII mutants of bacteriophage T4

Rutberg, Blanka (Karolinska Institutet, Stockholm, Sweden), and Lars Rutberg. Bacteriophage-induced functions in Escherichia coli K(lambda) infected with rII mutants of bacteriophage T4. J. Bacteriol. 91:76-80. 1966.-When Escherichia coli K(lambda) was infected with rII mutants of phage T4, deoxycytidine triphosphatase, one of the phage-induced early enzymes, was produced at initially the same rate as in r(+)-infected cells. Deoxyribonuclease activity was one-third to one-half of that of r(+)-infected cells. This lower deoxyribonuclease activity was observed also in other hosts or when infection was made with rI or rIII mutants. Presence of chloramphenicol did not allow a continued synthesis of phage deoxyribonucleic acid in rII-infected K(lambda). No phage lysozyme was detected nor was any antiphage serum-blocking antigen found in rII-infected K(lambda). It is suggested that the rII gene is of significance for the expression of phage-induced late functions in the host K(lambda).     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates I. Identification of T4D Gene 28 Product in the Tail Plug

The T4D bacteriophage gene 28 product is a component of the central plug of the tail baseplate, as shown by the following two independent lines of evidence. (i) A highly sensitive method for radioactive labeling of only tail baseplate plug components was developed. These labeled plug components were incorporated by a complementation procedure into new phage particles and were analyzed by radioautography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three new structural proteins were found in addition to the three known tail plug proteins (i.e., gP29, gP27, and gP5). One of the three newly identified components had a molecular weight of 24,000 to 25,000 and appeared to be a product of T4D gene 28. (ii) Characterization of mutants of Escherichia coli bacteriophage T4D which produced altered gene 28 products also indicated that the gene 28 product was a viral tail component. T4D 28^ts phage particles produced at the permissive temperature had altered heat labilities compared with parent T4D particles. We isolated a single-step temperature revertant of T4D 28^ts and found that it produced phage particles which phenotypically resembled the original T4D particles. Since the properties of the phage baseplate components usually determine heat lability, these two changes in physical stability after two sequential single mutations in gene 28 supported the other evidence that the gene 28 product was a viral baseplate component. Also, compared with parent T4D particles, T4D 28^ts and T4D 28am viral particles adsorbed at different rates to various types of host cells. In addition, T4D 28^ts particles exhibited a different host range than parent T4D particles. This T4D mutant formed plaques with an extremely low efficiency on all E. coli K-12 strains tested. We found that although T4D 28^ts particles adsorbed rapidly and irreversibly to the E. coli K-12 strains, as judged by gene rescue experiments, these particles were not able to inject their DNA into the E. coli K-12 strains. On the other hand, the T4D 28^ts revertant had a plating efficiency on E. coli K-12 strains that was quite similar to the plating efficiency of the original parent, T4D. These properties of phage particles containing an altered gene 28 product supported the analytical finding that the gene 28 product is a structural component of the central plug of the T4D tail baseplate. They also indicated that this component plays a role in both host cell recognition and viral DNA injection.     

Inactivation of T4D Bacteriophage by Antiserum Against Bacteriophage Dihydrofolate Reductase

Antiserum was prepared against highly purified T4D bacteriophage-induced dihydrofolate reductase (DFR). This serum not only inactivated the enzyme but also inactivated all strains of T4D examined. T6 was inactivated to a lesser extent, and T2L, T2H, and T5 were unaffected by the antiserum. The phage-killing power of the serum could be blocked by prior incubation with partially purified T4D dfr obtained from host cells unable to make phage structural proteins. These observations confirm earlier results that the phage dfr is a structural component of the phage particle, and they offer new evidence on the manner in which this enzyme in incorporated into the tail structure.     

Microbial Degradation of Corrinoids VI. Reduction of Hydroxocobalamin by Cell-free Particles from Pseudomonas rubescens

Cell-free particles from Pseudomonas rubescens have been shown to reduce hydroxocobalamin to vitamin B(12r). The particles are unable to reduce the B(12r) to B(12s). The reduction of hydroxocobalamin is dependent upon reduced nicotinamide adenine dinucleotide and is stimulated by flavin adenine dinucleotide. Cobinamide and diaquocobinamide were reduced at 25 and 10%, respectively, of the rate of hydroxocobalamin. Cyanocobalamin, coenzyme B(12), pseudovitamin B(12), and diaquopseudocobalamin were not reduced. Reduced nicotinamide adenine dinucleotide phosphate and flavin mononucleotide were not active. Diaphorase and xanthine oxidase activity were not present in the particulate fraction.     

Lambda-repressed mutants of bacteriophage T5. II. Physiological characterization.

Physiological properties of bacteriophage T5 gene A1 mutants, whose growth is inhibited in λ lysogens, and designated T5 lr, have been studied. In the presence of λ gene rex, which is responsible for lr growth inhibition, gene A1 product is synthesized and functional. However, several physiological defects were observed: phage DNA synthesis is inhibited; late phage-induced proteins are synthesized in markedly decreased amounts after a delay of about 15 minutes; phage DNA transfer into the host goes beyond the first-step transfer fragment but, in most bacteria, is interrupted after penetration of about 55% of the genome. Relationships between these different defects are discussed.     

6-Aminonicotinamide-resistant mutants of Salmonella typhimurium

Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD.     

The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae.

We have constructed a dihydrofolate reductase mutant (dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfr1 mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfr1 mutants. The inability of the mutants to metabolize FA suggests that the DFR1 gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfr1 strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfr1 strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFR1 gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfr1 mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfr1 strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.     

Mutants of Neurospora deficient in nicotinamide adenine dinucleotide (phosphate) glycohydrolase.

A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance (in heterocaryons) and in vitro mixing experiments did not provide evidence that nada mutations alter activators or inhibitors of NAD(P)ase. Thus, the nada gene appears to specify only the structure of N. crassa NAD(P)ase.     

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD(+), thereby indicating ethanol as one of the substrates of BmADH.     

Obligatory biosynthesis of L-tyrosine via the pretyrosine branchlet in coryneform bacteria.

Species of coryneform bacteria (Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes) utilize pretyrosine [beta-(1-carboxy-4-hydroxy-2,5-cyclohexadien-1-yl) alanine] as an intermediate in L-tyrosine biosynthesis. Pretyrosine is formed from prephenate via the activity of at least one species of aromatic aminotransferase which is significantly greater with prephenate as substrate than with either phenylpyruvate or 4-hydroxyphenylpyruvate. Pretyrosine dehydrogenase, capable of converting pretyrosine to L-tyrosine, has been partially purified from all three species. Each of the three pretyrosine dehydrogenases is catalytically active with either nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as cofactors. The Km values for nicotinamide adenine dinucleotide phosphate in C. glutamicum and B. flavum are 55 microM and 14.2 microM, respectively, and corresponding Km values for nicotinamide adenine dinucleotide are 350 microM and 625 microM, respectively. The molecular weights of pretyrosine dehydrogenase in C. glutamicum and in B. flavum are both about 158,000, compared with 68,000 moleculr weitht in B. ammoniagenes. In all three species the enzyme is not feedback inhibited by L-tyrosine. Results obtained with various auxotropic mutants, which were used to manipulate internal concentrations of L-tyrosine, suggest that pretyrosine dehydrogenase is expressed constitutively. Pretyrosine dehydrogenase is quite sensitive to p-hydroxymercuribenzoic acid, complete inhibition being achieved at 10 to 25 microM concentrations. This inhibition is readily reversed by thiol reagents such as 2-mercaptoethanol. Coryneform organisms, like species of blue-green bacteria, appear to lack the 4-hydroxyphenylpyruvate pa thway of L-tyrosine synthesis altogether. The loss of pretyrosine dehydrogenase in extracts prepared from a tyrosine auxotroph affirms the exclusive role of pretyrosine dehydrogenase in L-tyrosine biosynthesis. Other reports in the literature, in which the presence in these organisms of prephenate dehydrogenase is described, appear to be erroneous.     

Enzymatic Synthesis of l-Carnitine by Reduction of an Achiral Precursor: the Problem of Reduced Nicotinamide Adenine Dinucleotide Recycling

Synthesis of l-carnitine has been carried out by the enzymatic reduction of the carbonyl group of the achiral precursor 3-dehydrocarnitine with the oxidized nicotinamide adenine dinucleotide-linked carnitine dehydrogenase. Various enzymatic or chemical systems have been tested to regenerate the reduced nicotinamide adenine dinucleotide oxidized in the reduction of 3-dehydrocarnitine. Because of the instability of this compound in aqueous solutions, it was added by continuous feeding as a rate-limiting constituent in the reaction mixture. Under these conditions, conversion yields of 95% were achieved with the glucose plus glucose dehydrogenase system. A total number of 530 reduced nicotinamide adenine dinucleotide recyclings was obtained with this system for a production of 45 g of l-carnitine per liter. The stabilities of the oxidized nicotinamide adenine dinucleotide and the reduced nicotinamide adenine dinucleotide have been determined at various pH values. In view of these results, several possible strategies for enzymatic syntheses with the reduced nicotinamide adenine dinucleotide as a regenerable coenzyme are discussed.     

Identification of bacteriophage T4D gene products 26 and 51 as baseplate hub structural components

Products of two bacteriophage T4D genes, 26 and 51, both known to be essential for the formation of the central hub of the phage tail baseplate, have been partially characterized chemically, and their biological role has been examined. The gene 26 product was found to be a protein with a molecular size of 41,000 daltons and the gene 51 product a protein of 16,500 daltons. The earlier proposal (L. M. Kozloff and J. Zorzopulos, J. Virol. 40:635-644), from observations of a 40,000-dalton protein in labeled hubs, that the gene 26 product is a structural component of the baseplate, has been confirmed. The gene 51 product, not yet detected in phage particles, appears from indirect evidence also to be a structural component of the baseplate hub. These current conclusions about the gene 26 and 51 products are based on properties of T4 mutant particles containing altered gene 26 or 51 products and include (i) changes in heat lability, (ii) changes in adsorption rates, and (iii) changes in plating efficiencies on different hosts, and with the results of previous isotope incorporation experiments indicate that T4 particles contain three copies of the gene 26 product and possibly one or at most two copies of the gene 51 product. Properties of these mutant particles indicate that the gene 26 product, together with the other hub components such as the gene 28 product, plays a critical role in phage DNA injection into the host cell, whereas the 51 product seems essential in initiating baseplate hub assembly.