Impaired nitrogen fixation and glutamine synthesis in methionine sulfoximine sensitive (MSs) mutants of Rhizobium phaseoli

Summary Random Tn5 mutagenesis of antibiotic-resistant derivatives of Rhizobium phaseoli CFN42 yielded several independent mutants that were sensitive to methionine sulfoximine (MS^s), a specific inhibitor of glutamine synthetase (GS). These MS^s mutants were analyzed for GSI and GSII activities and for their symbiotic properties. Four classes of MS^s mutants have been distinguished. Class I strains are impaired in their synthesis of glutamine and in their symbiotic properties. Class II strains have wild type levels of GSI and GSII activities but have a reduced capacity to fix nitrogen. Class III strains have lost GSII activity, but their symbiotic properties are wild type. In class IV mutants neither glutamine synthesis nor symbiotic properties are affected. Mutants of classes I, III, and IV all have the Tn5 inserted into the chromosome, whereas in class II mutants the Tn5 is located in plasmid p42e, a plasmid different from the previously identified symbiotic plasmid p42d.     

The effects of recessive lethal Notch mutations of Drosophila melanogaster on flavoprotein enzyme activities whose inhibitions cause Notch-like phenocopies

The biochemical action of the Notch locus whose mutants cause morphological aberrations in flies, viz., notches of wings and bristle multiplication, has been analyzed (1) by the addition to the food medium of enzyme inhibitors causing phenocopies of Notch and (2) by comparison of enzyme activity patterns of Notch mutants with different degrees of phenotypic expression. Notch phenocopies were induced by inhibitors of enzyme activities in two biochemical pathways: (1) the de novo pyrimidine synthesis by 5-methylorotate (inhibitor of dihydroorotate dehydrogenase) and (2) the choline shunt by amobarbital (inhibits choline dehydrogenase) and methoxyacetate (inhibits sarcosine dehydrogenase). The inhibition of de novo pyrimidine synthesis prevents the production of deoxyuridine-5-phosphate, the substrate for the synthesis of thymidine-5-phosphate via thymidylate synthase, whereas the inhibition of the choline shunt prevents the production of HCHO groups and glycine, both of which are involved in the synthesis of 5,10-methylenetetrahydrofolate, which is a cofactor of thymidylate synthase. It was already known that the inhibition of the latter enzyme in vivo induces Notch phenocopies. Notch mutants with a strong morphological expression show low enzyme activities for dihydroorotate dehydrogenase and choline dehydrogenase. Both are flavoprotein enzymes linked to the respiratory chain. The correspondence between the low enzyme activities in Notch mutants with a strong morphological expression and the phenocopying effect of antimetabolites on these enzymes in the two biochemical pathways involved strongly suggests that the morphological effects of Notch on flies are a consequence of lowered activities of choline dehydrogenase and dihydroorotate dehydrogenase.     

Pyrimidine auxotrophy and the complementation map of the Rudimentary locus of Drosophila melanogaster

Summary The complementation pattern of twelve rudimentary mutations has been analyzed at two different levels. When analyzed on the basis of complementation for a wing abnormality the mutations can be divided into three groups, each of which is believed to affect the activity of one of the first three enzymes of pyrimidine synthesis (Norby, 1973; Jarry and Falk, 1974; Rawls and Fristrom, 1975). However, when the mutants are analyzed for complementation on the basis of a second phenotype, pyrimidine auxotrophy, the distinction between two of these three groups is not evident. The disparity in the two patterns probably reflects a different threshold of gene activity required for the detection of an auxotrophic phenotype as compared to that at which a wing abnormality is detectable.The biochemical basis of these results is interpreted in light of recent data suggesting that at least the first two enzymes of pyrimidine synthesis are contained within a single multifunctional protein complex (Soderholm et al., 1975).     

Genetic analysis of the structure and function of RNase P fromE. coli

A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations E. coli Escherichia coli - RNase P ribonuclease P     

Temperature sensitive mutations affecting RNA synthesis in Escherichia coli

Summary A streptomycin method has been used for the isolation of mutants with RNA synthesis inhibited at elevated temperature. The method is based on the observation that streptomycin kills bacteria with normal RNA synthesis and does not affect the cells with inhibited synthesis of RNA. This selection method increases the yield of temperature sensitive mutants by a factor 10–20, the amount of mutants with disturbed RNA synthesis is increased 3–5 fold as compared with the method of replicas.Several types of mutants were found among the temperature sensitive strains: those possessing temperature sensitivity of one, two or three types of cellular macromolecules DNA, RNA and protein. The screening among the mutants with affected RNA synthesis revealed a strain ts-19 showing low RNA polymerase activity in cell extracts and partially purified RNA polymerase preparations. The presented evidence suggests that ts-19 mutation affects the structural gene of one of the RNA polymerase subunits.The mapping of the corresponding locus indicated that it was located between the str and thy loci in E. coli K 12 chromosome at a distance of about 20 recombination units from the first locus.     

Analysis of Tn916-induced mutants ofClostridium acetobutylicum altered in solventogenesis and sporulation

Summary The conjugative transposon Tn916 was used for mutagenesis ofClostridium acetobutylicum ATCC 824. Tetracycline-resistant mutants were screened for loss of granulose synthesis and five classes of granulose mutants, that contained single transposon insertions, were identified on the basis of altered solvent production. Class 1 mutants did not make acetone or butanol, lacked activity of enzymes induced during solventogenesis, and did not sporulate, indicating that they are regulatory mutants. The class 2 mutant strains also did not produce acetone but did form small amounts of butanol and ethanol while the class 3 mutants produced low amounts of all solvents. Class 4 and 5 mutants produced essentially the same or higher amounts of solvents than the parent strain. Transposon insertions in the class 1 mutants were used as markers for in vitro synthesis of flanking chromosomal DNA using Tn916-specific primers. The DNA fragments were labeled to produce specific probes. Transposon insertion sites in the chromosomes of 13 different class 1 regulatory mutants were compared by hybridization of the specific probes to Southern blots of restriction endonuclease-digested parental chromosomal DNA. Insertions in two mutants appeared to be, in the same region of the chromosome. These results predict, that multiple regulatory elements are required to induce solvent production and sporulation.     

Physiological characteristics and virulence of auxotrophic and morphological mutants of Beauveria Brongniartii (sacc.) petch (= Beauveria tenella)

Various mutants have been obtained of B. brongniartii, a hyphomycete pathogenic for insects. Both one pigment less and two blastospore negative mutants retained their initial virulence. Thus neither the characteristic parasitic blastospore morphology nor the synthesis of the pigment appeared to be required for pathogenesis. Analysis of avirulent mutants indicated that chitinolytic activity may be essential for fungal penetration of the cuticle.     

GENETIC BLOCKS IN THE THIAMINE SYNTHESIS OF THE ANGIOSPERM ARABIDOPSIS,,

Five thiamine-requiring mutants were obtained at two loci. Two are blocked in the synthesis of the pyrimidine part of the vitamin, the other three have lost the ability to make the thiazole moiety. None of the tested substances suggested as possible or likely precursors of the pyrimidine or the thiazole components of thiamine displayed any activity in the mutants. These conditional lethals responded to remarkably small supplements of thiamine. The pyrimidine-requiring mutants utilized to some extent the anti-vitamin neopyrithiamine. The thiazole-less mutants grew on basal media supplemented only with the analog, oxythiamine. Thiamine deficiency, irrespective of the position of the genetic block in the synthesis, results in a characteristic anomaly of pigmentation. The position of the py locus in the second linkage group has been determined. Allelic complementation has not been detected. The frequency of mutations affecting thiamine synthesis appears about the same in Arabidopsis as in fungi. The general frequency of reparable genetic lesions is, however, one to two orders of magnitude lower in Arabidopsis than that in fungi or bacteria.     

Internal reinitiation of translation in polar mutants of the trpB gene of Salmonella typhimurium.

Summary The trpB gene of S. typhimurium codes for the bifunctional component II subunit of the AS-PRT complex which catalyzes the first two steps of tryptophan biosynthesis. It has previously been shown that the amino-terminal 40% of the component II molecule possesses the catalytic sites determining glutamine amidotransferase (GAT) activity, demonstrable indirectly by complementation with component I, the product of trpA, in the synthesis of anthranilic acid from chorismic acid and glutamine (AS activity), while its carboxy-terminal 60% possesses the catalytic sites determining anthranilate-PRPP phosphoribosyl transferase (PRT) activity, demonstrable by direct enzymatic assay. Here we further demonstrate the functional independence of the two regions of the component II subunit by providing evidence for the existence of monofunctional (GAT^-, PRT⁺) carboxy-terminal restart fragments of component II in certain chain terminating trpB mutants. Nonsense and frameshift mutants of the operator-proximal portion (region 1) of trpB have been found to grow well in media supplemented with anthranilic acid, implying the presence of PRT activity in the cell. Analysis of extracts of these strains has demonstrated the presence of low, but variable levels of PRT activity, but no GAT activity. Correlation of the map location of these mutations with the intensity of their polar effects on the expression of operator-distal genes suggests the existence of at least two gradients or units of polarity within region 1. Furthermore, in double mutant polarity tests, multiplicatiove polar effects were found in certain region 1 trpB-trpB double mutants strains. Taken together, these results lead us to conclude that at least two sites for reinitiation of translation exist within region 1 of trpB which can be activated by the presence of a nearby chain terminating codon. Such reinitation leads to the synthesis of labile carboxy-terminal restart fragments of component II which possess PRT function, but lack GAT function.     

Partial characterization of 5-fluoropyrimidine-resistant mutants of Neurospora crassa

Summary The primary lesion in a number of 5-fluoropyrimidine resistant mutants of Neurospora crassa has been identified. ud-1 mutants, previously designated fdu-2, are deficient in nucleoside uptake and show extensive intragenic complementation. uc-4 mutants lack uracil phosphoribosyl transferase with no complementation between 23 alleles. udk mutants lack uridine kinase activity. fdu-2 mutants affect the repression of the first two de novo pyrimidine biosynthetic enzymes, have no detectable uridine kinase activity and show decreased uridine uptake. Accordingly, fdu-2 may be involved in the regulation of pyrimidine uptake, salvage and de novo synthesis.Supported by S.R.C. grant GR/A/64655F. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

Sugar repression in the methylotrophic yeast Hansenula polymorpha studied by using hexokinase-negative, glucokinase-negative and double kinase-negative mutants

Two glucose-phosphorylating enzymes, a hexokinase phosphorylating both glucose and fructose, and a glucose-specific glucokinase were electrophoretically separated in the methylotrophic yeastHansenula polymorpha. Hexokinase-negative mutants were isolated inH. polymorpha by using mutagenesis, selection and genetic crosses. Regulation of synthesis of the sugar-repressed alcohol oxidase, catalase and maltase was studied in different hexose kinase mutants. In the wild type and in mutants possessing either hexokinase or glucokinase, glucose repressed the synthesis of maltase, alcohol oxidase and catalase. Glucose repression of alcohol oxidase and catalase was abolished in mutants lacking both glucose-phosphorylating enzymes (i.e. in double kinase-negative mutants). Thus, glucose repression inH. polymorpha cells requires a glucose-phosphorylating enzyme, either hexokinase or glucokinase. The presence of fructose-phosphorylating hexokinase in the cell was specifically needed for fructose repression of alcohol oxidase, catalase and maltase. Hence, glucose or fructose has to be phosphorylated in order to cause repression of the synthesis of these enzymes inH. polymorpha suggesting that sugar repression in this yeast therefore relies on the catalytic activity of hexose kinases.     

Indole-3-butyric acid synthesis in ecotypes and mutants of Arabidopsis thaliana under different growth conditions

Although IBA is a naturally occurring auxin, its role in plant development is still under debate. In this study a set of Arabidopsis mutants was used to analyze the biosynthesis of IBA in vitro. The mutants chosen for this study can be classified as: (1) involvement in auxin metabolism, transport or synthesis (amt1, aux1, ilr1, nit1, rib1, sur1, trp1-100); (2) other hormones possibly involved in the regulation of IBA synthesis (aba1, aba3, eto2, fae1, hls1, jar1); (3) photomorphogenesis (det1, det2, det3); and (4) root architecture (cob1, cob2, scr1). In addition, two transgenic lines overexpressing the IAA glucose synthase (iaglu) gene from maize were analyzed. The ecotypes No-0 and Wassilewskija showed the highest IBA synthetase activity under control conditions, followed by Columbia, Enkheim and Landsberg erecta. In the mutant lines IBA synthetase activity differed in most cases from the wild type, however no particular pattern of up- or down-regulation, which could be correlated to their possible function, was found. For rib1 mutant seedlings it was tested whether reduced IBA synthetase activity correlates with the endogenous IBA levels. Free IBA differed only depending on the culture conditions, but gave no clear correlation with IBA synthetase activity compared to the wild type. Since drought and osmotic stress as well as abscisic acid (ABA) application enhanced IBA synthesis in maize, it was tested whether IBA synthetase from Arabidopsis is also inducible by drought stress conditions. This was confirmed for the two ecotypes Col and Ler which showed different IBA synthetase activity when cultivated with various degrees of drought stress. IBA synthetase was also determined in photomorphogenic mutants under different light regimes. Induction of IBA synthetase in det1 and det3 plants was found under short day plus a red light pulse or in the dark, respectively. The results are discussed with respect to the functions of the mutated genes.     

The redundant aminotransferases in lysine and arginine synthesis and the extent of aminotransferase redundancy in Escherichia coli

Aminotransferases can be redundant or promiscuous, but the extent and significance of these properties is not known in any organism, even in Escherichia coli. To determine the extent of redundancy, it was first necessary to identify the redundant aminotransferases in arginine and lysine synthesis, and then complement all aminotransferase‐deficient mutants with genes for all aminotransferases. The enzymes with N‐acetylornithine aminotransferase (ACOAT) activity in arginine synthesis were ArgD, AstC, GabT and PuuE; the major anaerobic ACOAT was ArgD. The major enzymes with N‐succinyl‐l ,l ‐diaminopimelate aminotransferase (SDAP‐AT) activity in lysine synthesis were ArgD, AstC, and SerC. Seven other aminotransferases, when overproduced, complemented the defect in a triple mutant. Lysine availability did not regulate synthesis of the major SDAP‐ATs. Complementation analysis of mutants lacking aminotransferases showed that the SDAP‐ATs and alanine aminotransferases were exceptionally redundant, and it is proposed that this redundancy may ensure peptidoglycan synthesis. An overview of all aminotransferase reactions indicates that redundancy and broad specificity are common properties of aminotransferases.     

Analysis of Cyanothece sp. BH68K mutants defective in aerobic nitrogen fixation

The unicellular cyanobacterium, Cyanothece sp. BH68K, is capable of performing both oxygen-sensitive nitrogen fixation and oxygenic photosynthesis within a single cell. To understand the oxygen protection mechanisms of nitrogenase, mutants defective in nitrogen fixation (Nif^-) were isolated by use of diethyl sulfate as a mutagen. Out of 24 mutants screened, 6 mutants could not express nitrogenase activity under aerobic conditions, but expressed activity under anaerobic conditions (Fox^-); 4 mutants showed no activity under both aerobic and anaerobic conditions (Fix^-); and the remaining mutants were impaired in both aerobic and anaerobic nitrogenase activity (Imp). Respiratory oxygen consumption and photosynthetic oxygen evolution were analyzed in the wild-type and in two Fox^- mutants. In the wild-type the appearance of high aerobic nitrogenase activity was correlated with an increase in dark respiration, whereas no such increase was seen in the Fox^- mutants. We propose that in Fox^- mutants, respiratory oxygen consumption plays an important role in maintaining aerobic nitrogenase activity.     

Regulation of methyl beta-galactoside permease activity in pts and crr mutants of Salmonella typhimurium

Summary We have studied the regulation of the synthesis and activity of a major galactose transport system, that of methyl -galactoside (MglP), in mutants of Salmonella typhimurium. Two classes of mutation that result in a (partially) defective phosphoenolpyruvate: sugar phosphotransferase system (PTS) interfere with MglP synthesis. pts mutations, which eliminate the general proteins of the PTS Enzyme I and/or HPr and crr mutations, which result in a defective glucose-specific factor III^Gle of the PTS, lead to a low MglP activity, as measured by methyl -galactoside transport. In both ptsH,I, and crr mutants the amount of galactose binding protein, one of the components of MglP, is only 5%–20% of that in wild-type cells, as measured with a specific antibody. We conclude that synthesis of MglP is inhibited in pts and crr mutants. Once the transport system is synthesized, its transport activity is not sensitive to PTS sugars (i.e., no inducer exclusion occurs). The defect in pts and crr mutants with respect to MglP synthesis can be relieved in two ways: by externally added cyclic adenosine 3, 5-monophosphate (cAMP) or by a mutation in the cAMP binding protein. The conclusion that MglP synthesis is dependent on cAMP is supported by the finding that its synthesis is also defective in mutants that lack adenylate cyclase. pts and crr mutations do not affect growth of S. typhimurium on galactose, however, since the synthesis and activity of the other major galactose transport system, the galactose permease (GalP), is not sensitive to these mutations. If the galactose permease is eliminated by mutation, growth of pts and crr mutants on low concentrations of galactose becomes very slow due to inhibited MglP synthesis. Residual growth observed at high galactose concentrations is the result of yet another transport system with low affinity for galactose.     

Terminal synthesis of xanthommatin in Drosophila melanogaster. I. Roles of phenol oxidase and substrate availability

Eye color mutants of Drosophila melanogaster are known which block the conversion of 3-hydroxykynurenine to xanthommatin. It has been proposed that this reaction depends on the presence of 3-hydroxykynurenine and a redox system maintained by phenol oxidase activity. The mutants st and ltd lack throughout development detectable amounts of 3-hydroxykynurenine or its metabolic derivatives. When the substrate is fed or injected, these mutants fail to form xanthommatin even though phenol oxidase activity is normal. The mutant cd accummulates excessive amounts of 3-hydroxykynurenine, has normal phenol oxidase activity, but is also deficient in xanthommatin formation. Mutants are also known which lack phenol oxidase activity but nevertheless form xanthommatin. It is concluded that the proposed relationship between 3-hydroxy-kynurenine and phenol oxidase activity is not sufficient to explain the in vivo synthesis and regulation of synthesis of xanthommatin in Drosophila. The bearing of these findings on the actual mode of synthesis is discussed.Supported by PHS 1029 and NSF GB-4539.     

Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis

Laccase, EC 1.10.3.2 or p-diphenol:dioxygen oxidoreductase, has been proposed to be involved in lignin synthesis in plants based on its in vitro enzymatic activity and a close correlation with the lignification process in plants. Despite many years of research, genetic evidence for the role of laccase in lignin synthesis is still missing. By screening mutants available for the annotated laccase gene family in Arabidopsis, we identified two mutants for a single laccase gene, AtLAC15 (At5g48100) with a pale brown or yellow seed coat which resembled the transparent testa (tt) mutant phenotype. A chemical component analysis revealed that the mutant seeds had nearly a 30% decrease in extractable lignin content and a 59% increase in soluble proanthocyanidin or condensed tannin compared with wild-type seeds. In an in vitro enzyme assay, the developing mutant seeds showed a significant reduction in polymerization activity of coniferyl alcohol in the absence of H₂O₂. Among the dimers formed in the in vitro assay using developing wild-type seeds, 23% of the linkages were β-O-4 which resembles the major linkages formed in native lignin. The evidence strongly supports that AtLAC15 is involved in lignin synthesis in plants. To our knowledge, this is the first genetic evidence for the role of laccase in lignin synthesis. Changes in seed coat permeability, seed germination and root elongation were also observed in the mutant.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

A carbon catabolite repression mutant of Saccharomyces cerevisiae with elevated hexokinase activity: Evidence for regulatory control of hexokinase PII synthesis

Summary Mutants were investigated that had elevated hexokinase activity and had been isolated previously as resistant to carbon catabolite repression (Zimmermann and Scheel 1977). They were allele tested with mutant strains of Lobo and Maitra (1977), which had defects in one or more of the genes coding for glucokinase and unspecific hexokinases. It was shown, that the mutation abolishing carbon catabolite repression had occured in a gene that was not allelic to any of the structural genes coding for hexokinases. This indicated that a regulatory defect was responsible for elevated hexokinase activity. This agreed with observations that hexokinase activities were like wild-type during growth on non-fermentable carbon sources in hex2 mutants. Recombination between the mutant allele hex2 and mutant alleles hxk1 and hxk2, coding for hexokinase PI and PII respectively, clearly demonstrated that only hexokinase PII was elevated in hex2 mutants. When hex2 mutant cells grown on YEP ethanol were shifted to YEP glucose media, hexokinase activity increased after 30min. This increase depended on de novo protein synthesis. hex2 mutants provide evidence, that carbon catabolite repression and synthesis of hexokinase PII are under common regulatory control.     

Characterisation of mutants of Escherichia coli K12, selected by resistance to streptozotocin

Summary From cultures of sensitive bacteria, treated with the antibiotic streptozotocin, two classes of resistant mutants can be isolated: 1) mutants, resistant under all the conditions tested to even the highest doses of the antibiotic. These are either pleiotropicdefective, pts-mutants, or more frequently, mutants lacking a transport system (enzyme II^Nag-complex of the PEP-dependent phosphotransferase system) encoded by the gene nagE. This gene is inducible by N-acetyl-glucosamine and seems to be part of the nag operon. The transport system in question is responsible for the uptake of N-acetyl-glucosamine, of D-glucosamine and of streptozotocin; 2) conditional resistant mutants which are unable to energize or to synthesize the streptozotocin transport system under certain growth conditions but do have the transport activity under other conditions. These include a) mutants auxotrophic for amino acids, vitamins, or nucleotides, b) mutants negative or sensitive to carbohydrates in the medium, and c) mutants with defects in energy metabolism such as PEP synthesis.