Isolation and characterization of a restriction and modification deficient mutant of Brevibacterium lactofermentum

In order to facilitate genetic engineering in amino-acid producing bacteria we have isolated two restriction-deficient Brevibacterium lactofermentum strains. They have been selected for their ability to obtain a high yield of plaques from CL31 phage which was grown on Corynebacterium lilium. These mutant strains do not restrict either phage DNA by transfection or DNA from the shuttle vector pBLA extracted from Escherichia coli by protoplast transformation. These mutants have also lost modification activity. We also report the presence of a restriction modification system in C. lilium ATCC 15990.     

Isolation and characterization of a mutant of Staphylococcus aureus deficient in autolytic activity.

A mutant of Staphylococcus aureus H (RUS3) uas isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The rate of autolysis of whole cells and isolated cell walls of RUS3 was less than 10% of the parent strain. In addition, the ability of the crude soluble enzyme isolated from RUS3 to degrade cell walls was negligible compared with the parent strain. The cell wall composition and the generation time of RUS3 were comparable to the parent strain. Unlike S. aureus H, RUS3 grew in clumps and did not undergo cell wall turnover. Both strains exhibited identical kinetics of killing by penicillin G. This may indicate that autolytic enzymes play a role in cell wall turnover and cell separation, but in S. aureus most of the autolytic activity is unrelated to the lethal effect of cell wall antibiotics.     

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.     

Isolation and characterization of a ribonuclease III deficient mutant of Escherichia coli

Summary A mutant of E. coli K 12 AB301 RNAase ₁₉ ^- , selected for its inability to degrade double-stranded RNA, has been isolated and shown to have less than 1% of RNAase III-activity related to the parental strain.Abbreviations TCA trichloracetic acid - RF replicative form of phage-RNA Enzymes Lysozyme (E.C. 3.2.1.17) - RNAase (E.C. 2.7.7.16) - DNAase (E.C. 3.1.4.5)     

Isolation and characterization of rare earth element-binding protein in roots of maize

Rare earth element-binding protein was isolated from maize, which was grown under greenhouse conditions and characterized in terms of molecular weight, amino acid composition, and ultraviolet absorption. The molecular weight of the maize protein was determined to be 183,000, with two distinct subunits of approximately molecular weights of 22,000 and 69,000, respectively. The protein is particularly rich in asparagine/aspartic acid, glutamine/glutamic acid, glycine, alanine, and leucine and contains 8.0% of covalently bound carbohydrate. The ultraviolet absorption of the protein is low at 280 nm and no change in the adsorption was observed with a change in pH. Compared to the unique features of the metallothioneins with a molecular weight of approximately 10,000, a high cysteine content of 30%, high absorption at 254 nm and a low absorption at 280 nm, and absorption change with pH, the REE-binding protein is unlikely to be plant metallothionein in nature. It was found that an almost twofold greater concentration was found for most of the REEs in the protein isolated from the maize with REE fertilizer use than that without REE fertilizer. This study suggests that the REE-binding protein is a glycoprotein and REEs can be firmly bound with the protein of maize roots.     

Isolation and characterization of outermost layer deficient mutant spores of Bacillus megaterium

Outermost layer deficient mutant spores of Bacillus megaterium ATCC 12872 were isolated by Urografin density gradient centrifugation after mutagenesis with ethyl methanesulfonate. Although the composition of the cortex peptidoglycan was the same as that of the parent spores, three major proteins (48, 36, and 22 K daltons) were missing, suggesting that these proteins are components of the outermost layer. All mutant spores were also found to have very hydrophobic surface by 'salt aggregation test,' which would facilitate selection of such mutants.     

Isolation and characterization of an Escherichia coli mutant deficient in dTMP kinase activity

Escherichia coli LD0181 is sensitive to 15 micrograms of 2',3'-dideoxythymidine per ml. A derivative that was resistant to 40 micrograms of the same chemical per ml at 30 degrees C and that had lost the ability to grow on enriched medium at 42 degrees C was isolated after nitroso-guanidine mutagenesis. This mutant, TD105, produced a dTMP kinase with 25-fold lower specific activity and a 5-fold higher Km for dTMP than the parental strain. The dTMP pool in TD105 was 4.4-fold higher than in the parent. In addition to temperature sensitivity and resistance to 2',3'-dideoxythymidine, the mutant exhibited a hypersensitivity to 5-bromo-2'-deoxyuridine. All three of these phenotypes are cotransducible. The tmk gene was mapped by cotransduction to approximately 30 min on the E. coli map.     

Rate of differentiation and emergence of nodal maize roots

The timing of root production is one of the parameters required for modelling the root system architecture. The objectives of this study are (1) to describe the rate of appearance of adventitious root primordia of maize and their rate of emergence out of the stem; (2) to test equations for the prediction of the rank of the phytomer on which root emergence occurs, in a wide range of field situations.Maize, cultivar Dea, was grown in controlled conditions and in the field in 1987, 1988, 1989 and 1991. Plants were regularly sampled and the following data were recorded: foliar stage, number of root primordia and number of emerged roots per phytomer. Root primordia were counted in transverse thin sections in the stem.At a single plant level, root primordia differentiation occurred sequentially on the successive phytomers, with no overlapping between two phytomers. The same was true for root emergence. Roots belonging to the same phytomer emerged at approximately the same time.At a plant population level, there was a linear relationship between the rank of the phytomer on which root primordia were differentiated and cumulated degree-days after sowing. A linear relationship was also observed between the rank of the phytomer on which roots were emerging and cumulated degree-days or foliar stage. In the range of field situations tested (several years, sowing dates and planting densities), both equations gave an accurate prediction of the timing of root emergence during the plant cycle.     

Isolation and characterization of a mutant of Salmonella typhimurium deficient in a major deoxyribonucleic acid polymerase activity

A mutant of Salmonella typhimurium strain LT2 that is deficient in a major deoxyribonucleic acid (DNA) polymerase activity has been isolated and characterized. This mutant resembles the pol mutants of E. coli in that it has low DNA polymerase activity and it is sensitive to methyl methane sulfonate as well as ultraviolet irradiation. Revertants selected for methyl methane sulfonate resistance are no longer sensitive to ultraviolet irradiation and contain normal DNA polymerase levels. No direct role in replication can be ascribed to this polymerase activity since cells grow well in its absence. In addition, the LT2 plasmid has been shown to exist in the mutant strain.     

Isolation and characterization of a bacteriophage T5 mutant deficient in deoxynucleoside 5''-monophosphatase activity.

A bacteriophage T5 mutant has been isolated that is completely deficient in the induction of deoxynucleoside 5'-monophosphatase activity during infection of Escherichia coli F. The mutant bacteriophage has been shown to be deficient in the excretion of the final products of DNA degradation during infection of E. coli F, and about 30% of the host DNA's thymine residues were reinocorporated into phage DNA. During infection with this mutant, host DNA degradation to trichloroacetic acid-soluble products was normal, host DNA synthesis was shut off normally, and second-step transfer was not delayed. However, induction of early phage enzymes and production of DNA and phage were delayed by 5 to 15 min but eventually reached normal levels. The mutant's phenotype strongly suggests that the enzyme's role is to act at the final stage in the T5-induced system of host DNA degradation by hydrolyzing deoxynucleoside 5'-monophosphates to deoxynucleosides and free phosphate; failure to do this may delay expression of the second-step-transfer DNA.     

Isolation and characterization of a mutant of L1210 murine leukemia deficient in nitrobenzylthioinosine-insensitive nucleoside transport

L1210 mouse leukemia cells exhibit two distinct types of nucleoside transport activity that have similar kinetic properties and substrate specificity, but differ markedly in their sensitivity to the inhibitor nitrobenzylthioinosine (NBMPR) (Belt, J. A. (1983) Mol. Pharmacol. 24, 479-484). It is not known whether these two transport activities are mediated by a single protein or by separate and distinct nucleoside transport proteins. We have isolated a mutant from the L1210 cell line that has lost the NBMPR-insensitive component of nucleoside transport, but retains NBMPR-sensitive transport. In the parental cell line 20-40% of the nucleoside transport activity is insensitive to 1 microM NBMPR. In the mutant, however, uridine and thymidine transport are almost completely inhibited by NBMPR. Consistent with the loss of NBMPR-insensitive transport, the mutant cells can be protected from the toxic effects of several nucleoside analogs by NBMPR. In contrast, the toxicity of the same analogs in the wild type cells is not significantly affected by NBMPR, presumably due to uptake of the nucleosides via the NBMPR-insensitive transporter. On the other hand, NBMPR-sensitive transport in the mutant appears to be unaltered. The mutant is not resistant to cytotoxic nucleosides in the absence of NBMPR and the cells retain the wild type complement of high affinity binding sites for NBMPR. Furthermore, the affinity of the binding site for the inhibitor is similar to that of parental L1210 cells. These results suggest that NBMPR-sensitive and NBMPR-insensitive nucleoside transport in L1210 cells are mediated by genetically distinct proteins. To our knowledge this is the first report of a mutant deficient in NBMPR-insensitive nucleoside transport.     

Isolation and characterization of a transposon mutant of Shewanella putrefaciens MR-1 deficient in fumarate reductase

A transposon mutant, designated CMTn-3, of Shewanella putrefaciens MR-1 that was deficient in fumarate reduction was isolated and characterized. In contrast to the wild-type, CMTn-3 could not grow anaerobically with fumarate as the electron acceptor, and it lacked benzyl viologen-linked fumarate reductase activity. Consistent with this, CMTn-3 lacked a 65 kDa c -type cytochrome, which is the same size as the fumarate reductase enzyme. CMTn-3 retained the wild-type ability to use nitrate, iron(III), manganese(IV) and trimethylamine N -oxide (TMAO) as terminal electron acceptors. The results indicate that the loss of the fumarate reductase enzyme does not affect other anaerobic electron transport systems in this bacterium.     

Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacylglycerol.

The galactolipids monogalactosyl and digalactosyl diacylglycerol occur in all higher plants and are the predominant lipid components of chloroplast membranes. They are thought to be of major importance to chloroplast morphology and physiology, although direct experimental evidence is still lacking. The enzymes responsible for final assembly of galactolipids are associated with the envelope membranes of plastids, and their biochemical analysis has been notoriously difficult. Therefore, we have chosen a genetic approach to study the biosynthesis and function of galactolipids in higher plants. We isolated a mutant of Arabidopsis that is deficient in digalactosyl diacylglycerol by directly screening a mutagenized M2 population for individuals with altered leaf lipid composition. This mutant carries a recessive nuclear mutation at a single locus designated dgd1. Backcrossed mutants show stunted growth, pale green leaf color, reduced photosynthetic capability, and altered thylakoid membrane ultrastructure.     

Aerenchyma formation in maize roots

Maize (Zea mays L.) is generally considered to be a plant with aerenchyma formation inducible by environmental conditions. In our study, young maize plants, cultivated in various ways in order to minimise the stressing effect of hypoxia, flooding, mechanical impedance or nutrient starvation, were examined for the presence of aerenchyma in their primary roots. The area of aerenchyma in the root cortex was correlated with the root length. Although 12 different maize accessions were used, no plants without aerenchyma were acquired until an ethylene synthesis inhibitor was employed. Using an ACC-synthase inhibitor, it was confirmed that the aerenchyma formation is ethylene-regulated and dependent on irradiance. The presence of TUNEL-positive nuclei and ultrastructural changes in cortical cells suggest a connection between ethylene-dependent aerenchyma formation and programmed cell death. Position of cells with TUNEL-positive nuclei in relation to aerenchyma-channels was described.     

Isolation and characterization of an Escherichia coli K-12 mutant deficient in glutaredoxin.

Mutants of Escherichia coli K-12 deficient in glutaredoxin were isolated and partially characterized. The mutants have detectable but significantly reduced glutaredoxin activity in assays of whole cells made permeable with ether as well as in assays of crude extracts coupled to ribonucleotide reductase. In vivo, the mutants appear to be deficient in both sulfate and ribonucleotide reduction, suggesting that in vivo glutaredoxin is the preferred cofactor for ribonucleotide reductase and adenosine 3'-phosphate 5'-phosphosulfate reductase. Complementation of the mutant phenotype by transformants was used to clone the wild-type glutaredoxin allele. The transformants had a high level of glutaredoxin activity and contained a plasmid with an insert that had a restriction endonuclease pattern identical to that predicted by the DNA sequence for glutaredoxin determined by Hoog et al. (J.-O. Hoog, H. von Bahr-Lindstrom, H. Jornvall, and A. Holmgren, Gene 43:13-21, 1986).     

Isolation and characterization of a mutant of Neurospora crassa deficient in general amino acid permease activity.

A mutant of Neurospora crassa (pm-nbg27) was isolated on the basis of its resistance of p-fluoro-phenylalanine on ammonium-deficient Vogel's medium. This mutant was found to be devoid of both conidial and post-conidial (after 180 min of preincubation) transport activity of all amino acids. Genetic analysis of pm-nbg27 by crossing it to wild-type (74A) resulted in the predicted segregants exhibiting transport characteristics of pm-n, pm-b, pm-g, pm-nb, pm-ng, pm-bg and parental types. The above observations confirm the postulated general amino acid permease system as well as a single genetic locus control of that activity.     

Isolation of a recombination deficient Agrobacterium tumefaciens mutant.

Summary The isolation of a recombination deficient (Rec^-) strain of Agrobacterium tumefaciens is described. Strain LBA 4011 was mutagenized with nitrosoguanidine and after segregation 18,000 colonies were replica plated and UV irradiated. Twentytwo UV sensitive strains were isolated and tested for methylmethanesulphonate (MMS) sensitivity. Six of these strains were more MMS-sensitive than LBA 4011. A Ti plasmid that was genetically marked with Tn 1 (Cb^R) was introduced in these strains and the rescue of the Cb^R marker during superinfection with an incompatible cointegrate plasmid Ti::R 702 was determined. One strain exhibited a large reduction in rescue frequency. It is concluded that the latter strain was recombination deficient. This property did not influence the induction of plant tumours.This paper forms part of a Ph.D. Thesis submitted at Leiden University by the first author