Accumulation of Xanthosine by Auxotrophic Mutants of Bacillus subtilis

Several guanineless mutants derived from Bacillus subtilis IAM 1145 were found to accumulate xanthosine in the culture broth. Further mutation of the guanineless mutants to adenine dependence led to remarkable increase in the accumulation of xanthosine. One of the guanine-adenine doubleless mutants, strain Gu-Ad-3-35, accumulated 8.9g of xanthosine per liter. Xanthosine was isolated in a crystalline form from the culture broth by a procedure involving charcoal treatment and ion-exchange chromatography.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Biodegradation of diphenyl ethers by a copper-resistant mutant ofErwinia sp.

Summary A bacterium tentatively identified as anErwinia sp. was isolated from sewage by enrichment on methanol and lignin. Several mutants developed from this strain were studied for their ability to degrade aromatic ethers. Different concentrations of the chemicals were incubated with the organisms and the degradation was estimated by high-performance liquid chromatography (HPLC). Among these mutants, one isolate,Erwinia sp. strain CU3614, showed resistance to copper ions (>20 mM CuSO₄) and the ability to degrade 4-hydroxydiphenyl ether (4-HDPE), 4-chlorodiphenyl ether (4-CDPE), 4-nitrodiphenyl ether (4-NDPE) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) in the presence of copper ions. Increased concentrations of copper in the medium resulted in higher degradation of 4-HDPE. Further studies with copper-sensitive mutants obtained fromErwinia sp. CU3614 by Tn5 transposon-induced mutagenesis showed a corresponding decrease in the ability to degrade 4-HDPE. These results suggest the presence of a copper-associated activity in the biotransformation of aromatic ethers.     

Abnormal stomatal behavior and root resistance,and hormonal imbalance in three wilty mutants of tomato

Three wilty mutants of tomato, flacca, sitiens, and notabilis, were compared with two control normal cultivars, Rheinlands Ruhm and Lukullus, for concentration of abscisic acid and root resistance to water flow. In addition, the three mutants treated with abscisic acid during development were compared with control mutant plants for stomatal opening and root resistance. The hormonal concentration was estimated by coleoptile assay and gas chromatography. Stomatal opening was estimated by measuring rate of transpiration and by examining leaf imprints. Root resistance was estimated by measuring the amount of exudate from roots of decapitated plants and the difference between the osmotic pressure of the exudate and the root medium. A lower level of abscisic acid was found in all three mutants as compared with the control normal plants. In addition, root resistance to water flow was higher in the three mutants than in the control normal types. All three mutants completely reverted to normal phenotypically, including stomatal and root resistances, when treated with abscisic acid. It has been suggested that the first hormonal change in the mutants is that of abscisic acid, and from it proceed the other changes.This work was supported in part by a research grant from the Ford Foundation (Ford-6, A-VII).     

Pseudomonas corrugata (NRRL B-30409) Mutants Increased Phosphate Solubilization, Organic Acid Production, and Plant Growth at Lower Temperatures

A study for screening and selection of mutants of Pseudomonas corrugata (NRRL B-30409) based on their phosphate solubilization ability, production of organic acids, and subsequent effect on plant growth at lower temperatures under in vitro and in situ conditions was conducted. Of a total 115 mutants tested, two (PCM-56 and PCM-82) were selected based on their greater phosphate solubilization ability at 21°C in Pikovskaya’s broth. The two mutants were found more efficient than wild-type strain for phosphate solubilization activity across a range of temperature from psychotropic (4°C) to mesophilic (28°C) in aerated GPS medium containing insoluble rock phosphate. High-performance liquid chromatography analysis showed that phosphate solubilization potential of wild-type and mutant strains were mediated by production of organic acids in the culture medium. The two efficient mutants and the wild strain oxidized glucose to gluconic acid and sequentially to 2-ketogluconic acid. Under in vitro conditions at 10°C, the mutants exhibited increased plant growth as compared to wild type, indicating their functionality at lower temperatures. In greenhouse trials using sterilized soil amended with either soluble or rock phosphate, inoculation with mutants showed greater positive effect on all of the growth parameters and soil enzymatic activities. To the best of our knowledge, this is the first report on the development of phosphate solubilizing mutants of psychotropic wild strain of P. corrugata, native to the Indian Himalayan region.     

Lipopolysaccharide Defective Mutant of Escherichia coli with Decreased Sensitivity to Colicin E2

Several colicin-sensitivity mutants were isolated from Escherichia coli K-12. The mutants could not form colonies in the presence of colicin E2, but recovered their colony-forming ability on trypsin treatment even after prolonged incubation with the colicin. They showed increased sensitivity to hydrophobic antibiotics and detergents, as well as resistance against P1 and T4 phages, both of which seemed due to structural changes of lipopolysaccharide (LPS). Quantitative analysis by gas-liquid chromatography revealed that the mutant-LPS contained a different stereoisomer of heptose with decreased amounts of neutral sugars (rhamnose, glucose and galactose). LPS extracted from the parental colicin-sensitive strain could neutralize the killing activity of colicin E2 in vitro, but the mutant-LPS could not. The mutant strains retained functional receptor proteins for colicin E2. These observations suggest that LPS plays an important role in the early stage of the interaction of colicin E2 with E. coli cells.     

Erythromycin resistant mutants of Bacillus subtilis

Summary Erythromycin resistant (ery ^r) mutants were isolated from Bacillus subtilis ATCC 6633. The composition of ribosomal proteins were analyzed for thirteen such ery ^r-mutants with chromatography on a carboxymethyl cellulose (CMC) column. The 50s subunit from all of the ery ^r-mutants was found to contain the altered 50d protein. The ribosomes prepared from the ery ^r-mutants did not show in vitro alteration of the ability to combine with erythromycin.     

Nuclear mutations affecting plastoquinone accumulation in maize

We have isolated and characterized two nuclear mutations which affect plastoquinone accumulation in maize. The mutations, hcf103 and hcf114, modify the same genetic locus. Plants homozygous for either mutant allele exhibit reduced PS II electron transport activity, reduced variable chlorophyll fluorescence and reduced delayed fluorescence yield. In these ways, hcf103 and hcf114 resemble previously described PS II mutants which lack stably assembled PS II reaction center complexes. However, unlike most previously described PS II mutants, hcf103 and hcf114 possess stable membrane-associated PS II complexes. Plastoquinone (PQ-9), which performs a variety of redox functions essential to normal non-cyclic electron transport, is severely depleted in the mutants. The lack of PS II electron transport activity is attributed to the absence of PQ-9. This is the first report of mutants deficient in PQ which do not also suffer serious pleiotropic defects.Abbreviations PS II Photosystem II - PQ plastoquinone - Q_A and Q_B primary and secondary stable electron acceptors of PS II - HPLC high pressure liquid chromatography - LDS-PAGE lithium dodecyl sulfate polyacrylamide gel electrophoresis - TLC thin layer chromatography     

Macromolecular Physiology of Plastids

The composition and amount of carotenoid pigments were determined in etiolated seedling leaves of 6 barley (Hordeum vulgare L.) mutants, comprising 1 xantha and 5 tigrina mutants. All mutants had on a mole basis approximately the same content of carotenoids as the wild type. The mutants xan-u²¹, tig-n³², and tig-³³ contained significantly higher amounts of carotenes than the wild type, ranging from 32 to 68% of the total carotenoid content as compared to the 4–8% found in the wild type. In the mutants tig-b²³ and tig-o³⁴, only a slight increase in the amount of carotenes was notable. The carotene content and composition in tig-d¹² was indistinguishable from that of the wild type. The carotenes extracted from xan-u²¹, tig-b²³, tig-n³², tig-³³, and tig-o³⁴ were characterized by adsorption chromatography and spectrophotometry. Mutant xan-u²¹ is in the dark blocked in β-carotene synthesis, and accumulates the aliphatic polyenes: phytofluene, proneurosporene, poly-cis-lycopenes, neo-lycopene and lycopene. The other four mutants synthesize β-carotene, but accumulate in addition various higher saturated carotenes, the main components being ζ-carotene in tig-b²³, a lycopenic pigment in tig-n³² and tig-³³, and lycopene in tig-o³⁴. Accumulation of higher saturated carotenes appears correlated with specific aberrations of the membrane structure in plastids. The regulation of carotene and protochlorophyllide syntheses in etioplasts are closely linked as shown by the single gene mutants which affect both pathways. However, several mutants have been identified which cause defects in protochlorophyllide synthesis only.     

Construction of novel tumor necrosis factor-alpha mutants with reduced toxicity and higher cytotoxicity on human tumor cells

Two tumor necrosis factor-α mutants MT1 (32Trp157Phe) and MT2 (2Lys30Ser-32Trp 157Phe) were constructed by site-directed mutagenesis. These mutants were soluble and over-expressed inE. coli. The purity of purified mutants was above 95% by serial chromatography. The results of Western blot indicated that these mutants could be cross-reactive with monoclonal antibody against native hTNF-α. Compared to parent hTNF-α, the cytotoxicity of these mutants on murine fibrosarcoma L929 cell lines reduced 4–5 orders of magnitude but was equivalent to that of native hTNF-α on human tumor cell lines. The LD50 of mutant MT1 was reduced to 0.34% of wild type and the dose of MT2 that resulted in 30% death of mice reduced to less than 1/700 that of parent hTNF-α.     

Effects of Neurospora nuclease halo (nuh) mutants on secretion of two phosphate-repressible alkaline deoxyribonucleases

Various recently isolated nuh mutants of Neurospora crassa (i.e., mutants which show reduced nuclease haloes on DNA-sorbose plates flooded with HCl) were mapped in several new genes or gene clusters and checked for effects on DNA repair and nuclease secretion. Some of them were found to be sensitive to MMS (methylmethane sulfonate) and sterile in meiosis. Release of nuclease activities into filtrates of liquid cultures was analyzed by DEAE-Sepharose chromatography. In the wild type, three alkaline deoxyribonuclease activities (A, B, and C) can be separated after growth in sorbose minimal media [Fraser, M. J. (1979). Nucleic Acids Res. 6: 231]. When strains were grown in phosphate-free DNA sucrose media, high (200-fold derepressed) DNase levels were found, and crude dialyzed filtrates could be chromatographed. Only two peaks were found, namely, those of DNase A, a Ca²⁺-dependent strand-nonspecific endonuclease, and DNase B, a ss-DNA-specific Mg²⁺-dependent exonuclease. Of the nuh mutants analyzed by one or both of these methods, many resembled the wild type. A few showed poor derepression, since their sorbose filtrates were normal, while profiles from DNA media lacked all peaks. These grew variably in liquid media with organic phosphates and probably produced suppressors, as was regularly found for nuc-2. Other mutants, which lacked specific peaks, gave the same results with both methods. One of these, nuh-7, produced no peaks at all but secreted unusually high amounts of protein.This investigation was supported by Operating Grant A2564 from the National Science and Engineering Research Council of Canada.     

High amylose mutants of rice,Oryza sativa L.

Summary Five mutant lines of rice with increased amylose content in starch granules were identified among floury endosperm mutants. The amylose contents of the mutants ranged from 29.4% to 35.4% and were about twice as high as that of the normal counterpart. Starch properties of the high amylose mutants were analyzed by column chromatography, X-ray diffractometry, photopastegraphy and scanning electron microscopy. The high amylose mutants produced longer unit chains of amylopectin than those of the normal counterpart as well as an increased amount of amylose. A X-ray diffractogram of starch in the mutant was characterized by a type B pattern, while that in the normal counterpart showed a type A pattern which is typical for starches of common cereals. The temperatures at the initiation of gelatinization of the mutants were much higher than that for the normal counterpart. The endosperm cells of the mutant were loosely packed with irregular round-shaped starch granules, whereas those of the normal counterpart were densely packed with polyhedral starch granules. Judging from the results obtained, it was concluded that starch properties of the high amylose mutants of rice were similar to those of the amylose-extender (ae) mutant of maize.     

Lokalisierung von Aminos?ureaustauschen bei Spontanmutanten und nach Fluoruracil-Einbau isolierten Mutanten des Tabakmosaikvirus

Summary Amino acid eschanges within the protein chains of mutants of tobacco mosaic virus (TMV) arisen spontaneously or isolated after incorporation of 5-fluorouracil into the RNA of the TMV strainvulgare, have been localized. To this end, the viral particles were split into protein and RNA, and the protein was digested with trypsin. After their isolation by column and paper chromatography, the tryptic peptides were hydrolyzed and their amino acid compositions were quantitatively determined. Peptides with amino acid differences compared to the strainvulgare were subjected to sequence analyses in order to localize the positions of the amino acid exchanges. The experimental results given in the tables for five mutants will be discussed together with the results from other TMV mutants isolated after nitrous acid treatment and to be described in a following paper.

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Herrn Professor.G. Melchers zum 60. Geburtstag gewidmet.     

Effects of L-Tryptophan on Indoleamines and Catecholamines in Discrete Brain Regions of Wild Type and Lurcher Mutant Mice

The biochemical parameters of the serotoninergic system were examined in wild type mice and Lurcher mutants after chronic treatment (40 days) with the serotonin (5-HT) precursor L-tryptophan (50 mg/kg; i.p.). Tissue contents in 5-HT, dopamine and noradrenaline, as well as some of their metabolites, were measured in frontal cortex, neostriatum, thalamus, brainstem, cerebellum and spinal cord by high-performance liquid chromatography. The tissue levels were used as a biochemical index of the function of the monoamine innervations in this animal model of cerebellar ataxia. The results show that Lurcher mutants retain higher concentrations of L-tryptophan and total indoleamines, but that 5-HT is probably stored in a non-releasable compartment. In the particular case of the hypoplastic cerebellum, the reorganization of 5-HT nerve terminals leads to an accrued indoleamine synthesis, indicating that the Lurcher mutants can accumulate 5-HT, but do not utilize it efficiently in synaptic transmission.     

Peptide analyses of a protein component, 50-8, of 50s ribosomal subunit from erythromycin resistant mutants of Escherichia coli and Escherichia freudii

Summary Chromatographic analyses on a Dowex 50x8 column of tryptic digests of the mutationally altered 50-8 protein component from several erythromycin resistant (ery ^r) mutants of Escherichia coli and Escherichia freundii have been performed. It was found that (1) the difference in the elution profile of the altered components detected with carboxymethyl cellulose column chromatography reflects the difference in their amino acid sequence, (2) the structural change(s) of the 50-8 protein from three E. coli ery ^r mutants examined seems to exist only in the same single peptide fragment and (3) the primary structure of the 50-8(R) protein of E. freundii (ery ^s: wild type) differs from that of E. coli Q13 (ery ^s) and the structural change in 50-8(R) component of E. freundii caused by the ery ^r mutation was found to take place in different peptide fragments from that in which the mutational change of the E. coli 50-8 component occurred.     

Escherichia coli outer membrane protease OmpT confers resistance to urinary cationic peptides

Escherichia coli OmpT, located in the outer membrane, has been characterized as a plasminogen activator, with the ability to hydrolyze protamine and block its entry. In this investigation, a complex of low molecular weight cationic peptides purified from human urine by a combination of membrane ultrafiltration and weak cation exchange chromatography was characterized. The impact of OmpT on E. coli resistance to urinary cationic peptides was investigated by testing ompT knockout strains. The ompT mutants were more susceptible to urinary cationic peptides than ompT⁺ strains, and this difference was abolished by complementation of the mutants with pUC19 carrying the ompT gene. The urinary protease inhibitor ulinastatin greatly decreased the resistance of the ompT⁺ strains. Overall, the data indicate that OmpT may help E. coli persist longer in the urinary tract by enabling it to resist the antimicrobial activity of urinary cationic peptides.     

Comparision between Bacillus subtilis RP24 and its antibiotic-defective mutants

Bacillus subtilis RP24, a promising plant growth-promoting rhizobacterium and a potent biocontrol agent isolated from pigeonpea rhizosphere was mutagenized with ethyl methanesulphonate to study the possible mechanism/s involved in the potential antagonistic properties of the strain. Over 10,000 mutants were screened against the phytopathogenic fungus Macrophomina phaseolina on potato dextrose agar plates to select ten mutants showing partial antagonism as compared to the parent strain and one negative mutant showing no antagonism. The parent strain RP24 was compared with its mutants for the presence of different possible mechanisms behind antagonism. Production of hydrogen cyanide, ammonia, siderophores, and hydrolytic enzymes like lipase, amylase, and protease were detected in all the mutants as well as the parent strain, whereas fungal cell-wall-degrading enzymes, β-1, 3-glucanase and chitosanase were not detected in any of the mutants and the parent strain, indicating that none of these mechanisms was involved in the antagonistic trait of the strain. Two possible mechanisms detected behind the antifungal trait of the strain RP24 were production of antifungal volatiles and extra-cellular diffusible antibiotics. An attempt was made for extraction, partial characterization of the extra-cellular diffusible antifungal metabolite/s by thin layer chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The extracellular, methanol soluble, hydrophobic, ninhydrin-negative, thermostable and pH-stable antifungal metabolites were characterized as cyclic lipopeptides belonging to the iturin group of peptide antibiotics.     

Sterol content and enzyme defects of nystatin-resistant mutants of Neurospora crassa.

Summary Non-saponifiable cell extracts of wild type and sterol mutants of N. crassa were analysed by means of gas-liquid chromatography. The wild-type contained ergosterol and episterol in a 10:1 ratio. None of the mutants was able to synthesize ergosterol. Three of the mutants carry single recessive gene mutations causing blocks in the terminal steps of ergosterol biosynthesis: erg-1 has an inactive ^{8 7} isomerase, erg-2 has an inactive 24(28) hydrogenase, and erg-4 has an inactive C-24 methyl transferase. Some of the mutants accumulated novel sterols as a result of their enzyme defects. The genes erg-1 and erg-2 were mapped close to inl on the right arm of chromosome V.     

Expression of A/B zeins in single and double maize endosperm mutants

Summary Zeins, the major endosperm proteins in maize (Zea mays L.), are deficient in the essential amino acids lysine and tryptophan. Some mutant genes, like opaque-2 (o2) and floury-2 (fl2), reduce the levels of A- and B-zeins, thereby improving maize's nutritional value. Other mutants, such as amylose-extender (ae), floury-1 (fl1), soft starch (h), dull-1 (du), shrunken-1 (sh1), sugary-1 (su1), sugary-2 (su2), and waxy (wx), primarily affect starch composition, but also alter zein composition. We undertook this study to examine the effects of some of these mutant genes on A/B-zein composition and to study the interactions of these genes in double-mutant combinations. Endosperm prolamins were extracted from inbred B37, ten near-isogenic single mutants (ae, du, fl1, fl2, h, o2, sh1, su1, su2, and wx), and most double-mutant combinations. Zeins in these extracts were fractionated by reversed-phase highperformance liquid chromatography (RP-HPLC) into 22–24 peaks. Of the resulting 22 major peaks the areas of 16 (per milligram endosperm) were significantly affected by individual mutant genes relative to the zein composition of the normal inbred. In combination these genes exhibited significant epistatic interactions in regulating the expression of individual A/B zeins. Epistatic interactions were judged to be significant when the amount of a peak in a double mutant differed from the averages for the peak in the two respective single mutants. The o2 gene, alone and in combination with other mutant genes, significantly decreased the amounts of many individual zeins. The effect of the o2 gene was the greatest of all the genes examined. Various clustering techniques were used to see if mutant effects could be grouped; among these was principal component analysis, a multivariate statistical technique that analyzes all peak sizes simultaneously. Three-dimensional scatter graphs were constructed based on the first three principal components. For the single mutants, these showed no relationships to gene actions; for the double mutants, however, this technique showed that four single mutants, o2, sh1, su1 and su2, had the greatest effects on zein composition when combined with each other and with the remaining six single mutants.Presented at the XVI International Congress of Genetics, Toronto, Canada, August 20–27, 1988. The mention of firm names or trade products does not imply that they are endorsed or recommended by the USDA over other brands or similar products not mentioned