The L-arabinose-resistance test with Salmonella typhimurium strain SV3 selects forward mutations at several ara genes.

A new assay has been described for mutagenicity testing using an L-arabinose-sensitive strain of Salmonella typhimurium. The test strain SV3 and several L-arabinose-resistant mutants selected therefrom are characterized in the present study by 3 different criteria: inhibition of growth by L-arabinose, accumulation of keto-sugars, and activities of the enzymes involved in L-arabinose catabolism. Strain SV3 (ara-531) shows high levels of inducible L-arabinose isomerase (EC 5.3.1.4) and L-ribulokinase (EC 2.7.1.16) activities, but is deficient in L-ribulose-5-phosphate 4-epimerase (EC 5.1.3.4), the enzyme encoded in Escherichia coli by gene D in the araBAD operon. Addition of L-arabinose to SV3 growing in glycerol or casamino acids stops growth. D-Glucose only partially reverses this inhibition. Reversion of the ara-531 mutation restores different levels of epimerase activity and resistance to L-arabinose. However, the great majority of the L-arabinose-resistant mutants do not utilize L-arabinose. The physiological and enzymatic properties of these L-arabinose non-utilizing mutants suggest that L-arabinose resistance is due to forward mutations in at least 3 other genes, araA, araB and araC, blocking steps prior to L-ribulose 5-phosphate accumulation.     

Regulation of the L-arabinose catabolic pathway in Aerobacter aerogenes

Three enzymes of the l-arabinose catabolic pathway in Aerobacter aerogenes, l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, are specifically induced in the presence of l-arabinose. Mutants constitutive for kinase activity are also constitutive for the isomerase and 4-epimerase activities, suggesting that these three enzymes are coordinately controlled in A. aerogenes. l-Ribulokinase activity can still be induced in the presence of l-arabinose in an isomerase-deficient strain of A. aerogenes. Since l-arabinose is not converted to l-ribulose in such a strain, it appears that l-arabinose must be the inducer of l-ribulokinase, as well as the coordinately controlled isomerase and 4-epimerase. As in the metabolism of l-arabinose, growth of A. aerogenes on l-arabitol also requires a 4-epimerase for the conversion of l-ribulose-5-phosphate to d-xylulose-5-phosphate. However, loss of ability to metabolize l-arabinose, due to a deficiency in 4-epimerase synthesis in the presence of l-arabinose, does not affect growth on l-arabitol. In addition, synthesis of the 4-epimerase associated with l-arabitol metabolism is not accompanied by l-arabinose isomerase or l-ribulokinase synthesis. These results suggest either the existence of two different l-ribulose-5-phosphate 4-epimerases in A. aerogenes, or of two different regulatory mechanisms for the control of the same epimerase.     

Sugar catabolism in Aquaspirillum gracile

Aquaspirillum (Spirillum) gracile is one of the few spirilla that cause acidification of the medium when cultured with sugars. Acidic reactions have been reported only for d-glucose, d-galactose, and l-arabinose, and the mode of attack of these sugars has not been previously investigated. The soluble portion of extracts of glucose-cultured cells of A. gracile ATCC 19624 was found by spectrophotometric methods to contain enzyme activities characteristic of the Entner-Doudoroff and Embden-Meyerhof-Parnas pathways. No activity for 6-phosphogluconate dehydrogenase (EC 1.1.1.44) was detected. Pyridine nucleotide-linked dehydrogenase activities for l-arabinose and d-galactose (EC 1.1.1.46 and EC 1.1.1.48) occurred in the soluble fraction of cells cultured with either sugar. Glucose-cultured cells contained not only glucokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activities but also glucose dehydrogenase (EC 1.1.1.47) activity. Enzymes capable of oxidizing gluconate were not detectable, but gluconokinase (EC 2.7.1.12) activity was present. Paper chromatographic analysis of the spent culture supernatant media from glucose-cultured cells indicated an accumulation of gluconic acid, and this was confirmed by enzymatic methods. Evidence is presented for the production of d-galactonic and l-arabonic acids in cultures containing d-galactose or l-arabinose, respectively.     

d-Sedoheptulose-7-phosphate: d-Glyceraldehyde-3-phosphate Glycolaldehydetransferase and d-Ribulose-5-phosphate 3-Epimerase Mutants of a Bacillus Species

Determination of enzyme activities on the non-oxidative section of the pentose phosphate pathway in d-ribose-forming mutants of a Bacillus species revealed that two strains, which were isolated as shikimic acid-requiring mutants, lacked d-sedoheptulose-7-phosphate: d-glyceraldehyde glycolaldehydetransferase (EC 2.2.1.1) and one strain, which was isolated as d-gluconate-non-utilizing mutant, lacked d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1). These three strains were also found to have a kind of pleiotropic property, hardly growing on d-glucose.     

Studies on sugar isomerases of Lactobacillus sp.: Whole cell immobilization of d-glucose isomerase

A new soil isolate of Lactobacillus sp. grown in Yamanaka medium under submerged conditions showed the presence of d-glucose, d-xylose and d-ribose isomerases in washed cell suspension and cell free extracts. d-Xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) and d-ribose isomerase (d-ribose ketol-isomerase, EC 5.3.1.20) activities reached a maximum in 48 h of growth and then declined. d-Glucose isomerase (d-glucose 6-phosphate isomerase, d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) activity was maximum after 72 h and remained constant for ～120 h of growth. d-Glucose isomerase activity increased with the increase in number of generations of culture and reached a maximum in 5–6 generations, whereas d-xylose and d-ribose isomerase activities decreased. The washed and starved whole cells could be heat treated and immobilized on the rough surface of glass rods or glass slides using acetone treatment. The heat treated immobilized cells showed only the presence of d-glucose isomerase activity and showed no d-xylose and d-ribose isomerase activities. d-Glucose isomerase activity of heat treated immobilized cells was inhibited less by sorbitol, mannitol, sodium arsenate, cysteine and calcium ions than the free d-glucose isomerase activity in fresh untreated washed whole cells and cell free extracts. EDTA inhibition had the same effect for both forms. Ca²⁺inhibition could be reversed by adding Mg²⁺ions.     

Methylglyoxal-mediated growth inhibition in an Escherichia coli cAMP receptor protein mutant

Under certain growth conditions, some strains of Escherichia coli accumulate toxic levels of methylglyoxal. This report characterizes a strain which synthesizes a mutant cAMP receptor protein in an adenylate cyclase deletion background. When cultured in glucose 6-phosphate minimal medium, this strain (222) was prematurely growth arrested due to methylglyoxal production; growth inhibition did not occur when the strain was grown in glucose minimal medium. A comparison of a variety of enzyme and cofactor levels in the related strains 222 (mutant) and 225 (wild-type) grown on either glucose or glucose 6-phosphate medium was carried out. The only difference found that might explain an increase in methylglyoxal accumulation was an elevated level of phosphofructokinase in strain 222 grown on glucose 6-phosphate. Since this enzyme activity probably limits hexose phosphate metabolism, it is suggested that growth inhibition in strain 222 may be due to increased production of triose phosphate, some of which is converted to methylglyoxal.     

The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

Characterization of Arabidopsis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase enzymes and mutants revealed highly complex metabolite-mediated feedback regulation of the plant shikimate pathway.     

Construction and Characterization of Viable Deletion Mutants of Simian Virus 40 Lacking Sequences near the 3′ End of the Early Region

Five viable deletion mutants of simian virus 40 (SV40) were prepared and characterized. These mutants lack 15 to 60 base pairs between map positions 0.198 and 0.218, near the 3′ end of the early region of SV40 and extend further into the body of the A gene, encoding the large T antigen, than previously described deletion mutants. These mutants were isolated after transfection of monkey kidney CV-1p cells with full-sized linear DNA prepared by partial digestion of form I SV40 DNA with restriction endonucleases HinfI or MboII, followed by removal of approximately 25 base pairs of DNA from the 5′ termini using λ-5′-exonuclease and purification of the DNA in agarose gels. Based on camparisons of the DNA sequence of SV40 and polyoma virus, these mutations map in the 19% of the SV40 A gene that shares no homology with the A gene of polyoma virus. The mutations exist in two different genetic backgrounds: the original set of mutants (dl2401 through dl2405) was prepared, using as a parent SV40 mutant dl862, which has a deletion at the single HpaII site (0.725 map unit). A second set (dl2491 through dl2495) contains the same deletions in a wild-type SV40 (strain SV-S) background. Relative to wild-type SV40, the original mutants showed reduced rates of growth, lower yields of progeny virus and viral DNA, and smaller plaque size; in these properties the mutants resembled parental dl862, although mutant progeny yields were usually lower than yields of dl862, suggesting a possible interaction between the two deletions. The second set of mutants had growth properties and progeny yields similar to those of wild-type SV40; however, Southern blotting experiments indicated that viral DNA replication proceeds at a slightly reduced rate. All of the mutants transformed mouse NIH/3T3 cells and mouse embryo fibroblasts at the same frequency as wild-type SV40. Mutants dl2402, dl2492, and dl2405 consistently produced denser and larger foci in both types of cells. All mutants directed the synthesis of shortened large T antigens. Adenovirus helper function was retained by all mutants.     

Control of amino sugar metabolism in Escherichia coli and isolation of mutants unable to degrade amino sugars

1. Growth of Escherichia coli on glucosamine results in an induction of glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] and a repression of glucosamine 6-phosphate synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16); glucose abolishes these control effects. 2. Growth of E. coli on N-acetylglucosamine results in an induction of N-acetylglucosamine 6-phosphate deacetylase and glucosamine 6-phosphate deaminase, and in a repression of glucosamine 6-phosphate synthetase; glucose diminishes these control effects. 3. The synthesis of amino sugar kinases (EC 2.7.1.8 and 2.7.1.9) is unaffected by growth on amino sugars. 4. Glucosamine 6-phosphate synthetase is inhibited by glucosamine 6-phosphate. 5. Mutants of E. coli that are unable to grow on N-acetylglucosamine have been isolated, and lack either N-acetylglucosamine 6-phosphate deacetylase (deacetylaseless) or glucosamine 6-phosphate deaminase (deaminaseless). Deacetylaseless mutants can grow on glucosamine but deaminaseless mutants cannot. 6. After growth on glucose, deacetylaseless mutants have a repressed glucosamine 6-phosphate synthetase and a super-induced glucosamine 6-phosphate deaminase; this may be related to an intracellular accumulation of acetylamino sugar that also occurs under these conditions. In one mutant the acetylamino sugar was shown to be partly as N-acetylglucosamine 6-phosphate. Deaminaseless mutants have no abnormal control effects after growth on glucose. 7. Addition of N-acetylglucosamine or glucosamine to cultures of a deaminaseless mutant caused inhibition of growth. Addition of N-acetylglucosamine to cultures of a deacetylaseless mutant caused lysis, and secondary mutants were isolated that did not lyse; most of these secondary mutants had lost glucosamine 6-phosphate deaminase and an uptake mechanism for N-acetylglucosamine. 8. Similar amounts of (14)C were incorporated from [1-(14)C]-glucosamine by cells of mutants and wild-type growing on broth. Cells of wild-type and a deaminaseless mutant incorporated (14)C from N-acetyl[1-(14)C]glucosamine more efficiently than from N[1-(14)C]-acetylglucosamine, incorporation from the latter being further decreased by acetate; cells of a deacetylaseless mutant showed a poor incorporation of both types of labelled N-acetylglucosamine.     

Levels of enzymes of the pentose phosphate pathway in Pachysolen tannophilus Y-2460 and selected mutants

The compositions of intracellular pentose phosphate pathway enzymes have been examined in mutants of Pachysolen tannophilus NRRL Y-2460 which possessed enhanced D-xylose fermentation rates. The levels of oxidoreductive enzymes involved in converting D-xylose to D-xylulose via xylitol were 1.5–14.7-fold higher in mutants than in the parent. These enzymes were still under inductive control by D-xylose in the mutants. The D-xylose reductase activity (EC 1.1.1.21) which catalyses the conversion of D-xylose to xylitol was supported with either NADPH or NADH as coenzyme in all the mutant strains. Other enzyme specific activities that generally increased were: xylitol dehydrogenase (EC 1.1.1.9), 1.2–1.6-fold; glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 1.9–2.6-fold; D-xylulose-5-phosphate phosphoketolase (EC 4.1.2.9), 1.2–2.6-fold; and alcohol dehydrogenase (EC 1.1.1.1), 1.5–2.7-fold. The increase of enzymatic activities, 5.3–10.3-fold, occurring in D-xylulokinase (EC 2.7.1.17), suggested a pivotal role for this enzyme in utilization of D-xylose by these mutants. The best ethanol-producing mutant showed the highest ratio of NADH- to NADPH-linked D-xylose reductase activity and high levels of all other pentose phosphate pathway enzymes assayed.     

Utilization of gluconate by Escherichia coli. Induction of gluconate kinase and 6-phosphogluconate dehydratase activities

1. A mutant of Escherichia coli, devoid of phosphopyruvate synthetase, glucosephosphate isomerase and 6-phosphogluconate dehydrogenase activities, grew readily on gluconate and inducibly formed an uptake system for gluconate, gluconate kinase and 6-phosphogluconate dehydratase while doing so. 2. This mutant also grew on glucose 6-phosphate and inducibly formed 6-phosphogluconate dehydratase; however, the formation of the gluconate uptake system and gluconate kinase was not induced under these conditions. 3. The use of the Entner–Doudoroff pathway for the dissimilation of 6-phosphogluconate, derived from either gluconate or glucose 6-phosphate, by this mutant was also demonstrated by the accumulation of 2-keto-3-deoxy-6-phosphogluconate (3-deoxy-6-phospho-l-glycero-2-hexulosonate) from both these substrates in a similar mutant that also lacked phospho-2-keto-3-deoxygluconate aldolase activity. 4. Glucose 6-phosphate inhibits the continued utilization of fructose by cultures of the mutants growing on fructose, as it does in wild-type E. coli. 5. The mutants do not use glucose for growth. This is shown to be due to insufficiency of phosphopyruvate, which is required for glucose uptake.     

Specific inactivation of glucose metabolism from eucaryotic cells by pentalenolactone

Pentalenolactone, an antibiotic related to the class of the sesquiterpene-lactones and produced by the strain Streptomyces arenae Tü-469, inhibits specifically the glucose metabolism by inactivation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD⁺ oxidoreductase (phosphorylating) EC 1.2.1.12). The sensitivity of several eucaryotic cell-systems for pentalenolactone was shown under in vivo conditions. The glycolytic as well as the gluconeogenetic pathway of mammalian cells can be completely inhibited with low concentration of the antibiotic. In all cases, the minimum inhibitory concentration is dependent on cell density. The inhibitory effect in vivo and in vitro does not seem to be species-specific. In erythrocytes from rats, in Ehrlich-ascites tumor cells and in Plasmodium vinckei infected erythrocytes from mice glycolysis can be inhibited with concentrations of 18–90 μM pentalenolactone. In hepatocytes, glycolysis as well as gluconeogenesis is prevented by the same concentrations. In contract to these results, in yeast the inhibition depends on growth conditions. The inhibition in glucose medium is cancelled by precultivation on acetate-containing medium.     

Glycerol catabolism in wild-type and mutant strains ofPseudomonas aeruginosa

Glycerol uptake, glycerol kinase (EC 2.7.1.30) and glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) activities are specifically induced during growth ofPseudomonas aeruginosa PAO on either glycerol or glycerol-3-phosphate. Mutants of strain PAO unable to grow on both glycerol and glycerol-3-phosphate were isolated. Mutant PFB 121 was deficient in an inducible, membrane-bound, pyridine nucleotide-independent, glycerol-3-phosphate dehydrogenase activity and PFB 82 was deficient in glycerol uptake and glycerol kinase and glycerol-3-phosphate dehydrogenase activities. Each mutant spontaneously reverted to wild phenotype, which indicates that each contained a single genetic lesion. These results demonstrate that membrane-bound, inducible glycerol-3-phosphate dehydrogenase is required for catabolism of both glycerol and glycerol-3-phosphate and provide suggestive evidence for a single regulatory locus that controls the synthesis of glycerol uptake, glycerol kinase, and glycerol-3-phosphate dehydrogenase inP. aeruginosa.     

"In vivo" detection of L-arabinose-binding protein, CRM-negative mutants

An "in vivo" assay for the detection of mutants negative to CRM (cross-reacting material) is described. l-Arabinose-negative mutants of Escherichia coli B/r were grown on Casamino Acids-l-arabinose plates to which a 3-ml agar layer, containing antiserum to the l-arabinose-binding protein (ABP), had been applied. After incubation and partial lysis of the clones "in situ," the plates were refrigerated for 36 hr, rinsed of colonial growth with water, and observed for the presence or absence of an immune precipitation. ABP-minus and l-arabinose regulator (araC)-minus mutants do not produce a precipitin reaction. l-Arabinose isomeraseless (EC 5.3.1.4; araA), kinaseless (EC 2.7.1.16; araB), and epimeraseless (EC 5.1.3.a; araD) mutants produce precipitin reactions. Mutants of E. coli B/r generated by treatment of the wild type with ethyl methane sulfonate or ultraviolet irradiation were isolated, tested for l-arabinose uptake, and screened for the presence or absence of ABP by the described assay. The applications of such an assay are discussed.     

Glucosamine metabolism in Drosophila virilis salivary glands: Ontogenetic changes of enzyme activities and metabolite synthesis

In Drosophila virilis salivary glands the in vitro activities of enzymes involved in the glucosamine pathway were examined during the third larval instar and in the prepupa. While glutamine-fructose-6-phosphate aminotransferase (EC 5.3.1.19) becomes inactive at the time of puparium formation, glucosamine-6-phosphate isomerase (EC 5.3.1.10) and glucosamine-6-phosphate N-acetyltransferase (EC 2.3.1.3) show maximal activities in the prepupal gland. The activity of UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) may also decrease prior to puparium formation. Incubation of larval and prepupal glands in medium containing [³H]glucose + [¹⁴C]-uridine or [¹⁴C]glucosamine and subsequent separation of intermediates of the glucosamine pathway by chromatographic procedures reveal that the capacity of the glands to incorporate the isotopes into these intermediates decreases significantly at the time of puparium formation. The results suggest that in D. virilis salivary glands the formation of aminosugars is mainly controlled by the activities of the two enzymes glutamine-fructose-6-phosphate aminotransferase and UDP-N-acetylglucosamine pyrophosphorylase.     

Inhibition of the β-carbonic anhydrases from Mycobacterium tuberculosis with C-cinnamoyl glycosides: Identification of the first inhibitor with anti-mycobacterial activity

A small series of C-cinnamoyl glycoside containing the phenol moiety was tested for the inhibition of the three Mycobacterium tuberculosis β-carbonic anhydrases (CAs, EC 4.2.1.1) with activities in the low micromolar range detected. The compounds were also tested for the inhibition of growth of M. tuberculosis H₃₇Rv strain, leading to the identification of (E)-1-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosyl)-4-(3-hydroxyphenyl)but-3-en-2-one (1) as the first carbonic anhydrase inhibitor with anti-tubercular activity.     

The Hypocrea jecorina (syn. Trichoderma reesei) lxr1 gene encodes a d-mannitol dehydrogenase and is not involved in l-arabinose catabolism

The Hypocrea jecorina LXR1 was described as the first fungal l-xylulose reductase responsible for NADPH dependent reduction of l-xylulose to xylitol in l-arabinose catabolism. Phylogenetic analysis now reveals that LXR1 forms a clade with fungal d-mannitol 2-dehydrogenases. Lxr1 and the orthologous Aspergillus nigermtdA are not induced by l-arabinose but expressed at low levels during growth on different carbon sources. Deletion of lxr1 does not affect growth on l-arabinose and l-xylulose reductase activity remains unaltered whereas d-mannitol 2-dehydrogenase activities are reduced. We conclude that LXR1 is a d-mannitol 2-dehydrogenase and that a true LXR1 is still awaiting discovery.     

Characteristics of a Binding Protein-Dependent Transport System for sn-Glycerol-3-Phosphate in Escherichia coli That Is Part of the pho Regulon

The ugp-dependent transport system for sn-glycerol-3-phosphate has been characterized. The system is induced under conditions of phosphate starvation and in mutants that are constitutive for the pho regulon. The system does not operate in membrane vesicles and is highly sensitive toward osmotic shock. The participation of a periplasmic binding protein in the transport process can be deduced from the isolation of transport mutants that lack the binding protein. As with other binding protein-dependent transport systems, this protein appears to be necessary but not sufficient for transport activity. The isolation of mutants has become possible by selection for resistance against the toxic analog 3,4-dihydroxybutyl-1-phosphonate that is transported by the system. sn-Glycerol-3-phosphate transported via ugp cannot be used as the sole carbon source. Strains have been constructed that lack alkaline phosphatase and glycerol kinase. In addition, they are constitutive for the glp regulon and contain high levels of glycerol-3-phosphate dehydrogenase. Despite the fact that these strains exhibit high ugp-dependent transport activity for sn-glycerol-3-phosphate they are unable to grow on it as a sole source of carbon. However, when cells are grown on an alternate carbon source, ¹⁴C label from [¹⁴C]sn-glycerol-3-phosphate appears in phospholipids as well as in trichloroacetic acid-precipitable material. The incorporation of ¹⁴C label is strongly reduced when sn-glycerol-3-phosphate is the only carbon source. In the presence of an alternate carbon source, this inhibition is relieved, and sn-glycerol-3-phosphate transported by ugp can be used as the sole source of phosphate.     

Role of Gne and GalE in the virulence of Aeromonas hydrophila serotype O34

The mesophilic Aeromonas hydrophila AH-3 (serotype O34) strain shows two different UDP-hexose epimerases in its genome: GalE (EC 3.1.5.2) and Gne (EC 3.1.5.7). Similar homologues were detected in the different mesophilic Aeromonas strains tested. GalE shows only UDP-galactose 4-epimerase activity, while Gne is able to perform a dual activity (mainly UDP-N-acetyl galactosamine 4-epimerase and also UDP-galactose 4-epimerase). We studied the activities in vitro of both epimerases and also in vivo through the lipopolysaccharide (LPS) structure of A. hydrophila gne mutants, A. hydrophila galE mutants, A. hydrophila galE-gne double mutants, and independently complemented mutants with both genes. Furthermore, the enzymatic activity in vivo, which renders different LPS structures on the mentioned A. hydrophila mutant strains or the complemented mutants, allowed us to confirm a clear relationship between the virulence of these strains and the presence/absence of the O34 antigen LPS.     

Pathways of ultraviolet mutability in Saccharomyces Cerevisiae. IV. The relation between canavanine toxicity and ultraviolet mutability to canavanine resistance.

Seven umr mutants of Saccharomyces cerevisiae which had reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were tested for sensitivity to L-canavanine relative to one wild-type UMR strain and one slightly UV-sensitive but phenotypically umr⁺ strain (mutant 306). Relative UV mutation resistance was estimated by dividing the UV fluence needed to yeild a particular induced mutation frequency by that needed to reach the same frequency in the genotypic wild-type strain. The umr5 and umr6 strains were especially sensitive to canavanine growth inhibition, while umr1 was no more sensitive than either wild type; umr2, umr3, umr4, a umr7, and α umr7 were equally sensitive to an intermediate degree. Incubation at 30°C of wildtype cells plated on canavanine-selective agar for increasingly longer times before UV irradiation resulted in decreasing UV mutation frequencies (reduced to 50% in 1.6 h). All umr strains tested in this way lost UV mutability faster than wild type, including mutant 306, umr1 (not sensitive to growth inhibition), and umr6 (very sensitive to growth inhibition). Cells were grown to stationary phase in YEDP growth medium and assayed for arginine and tryptophan transport into the cell. The umr6 strain, which had weak UV mutation resistance but high sensitivity to canavanine growth inhibition, transported arginine and tryptophan at essentially wild-type levels. The umr1 strain, however, which had moderate UV mutation resistance and normal canavanine toxicity, transported both amino acids at rates tenfold higher than wild type. The data suggest that increased canavanine toxicity does not necessarily lead to defective mutability at CAN1, and that mutational deficiency cannot result solely from increased canavanine toxicity. Although exposure to canavanine was shown to block mutation fixation and/or expression, it is suggested that the degree of growth inhibition is not strictly correlated with the degree of mutation resistance.