Structure of nodules induced by auxotrophic and ineffective mutants of Rhizobium meliloti strain L5-30 requiring cysteine, arginine+uracil and histidine.

Nodules produced by ineffective mutants of R. meliloti strain L5-30 requiring arginine+uracil (arg-55) and cysteine requiring mutants (cys-243, cys-244, cys-246) studied under light microscopy were found to be occupied by bacteria. This indicates on defect in transformation of these mutants into N2 fixing bacteroids. These defects were not associated with auxotrophy. In the nodules induced by histidine requiring mutant (his-240) only few host plant cells were occupied by bacteria. This indicate that his-240 mutant is defective in liberation from the infection thread and its multiplication since supplementation of the plant growth medium with 50 microgram/ml of L-histidine enabled establishment of fully effective association. Prototrophic transductants and revertants were fully effective.     

Chromosomal gene controlling symbiotic nitrogen fixation in Rhizobium meliloti L5-30

Auxotrophic Rhizobium meliloti strain RM 246 carries two independent mutations: in the biosynthesis of cysteine (cys) and symbiotic nitrogen fixation process (fix). These two mutations were mapped by transduction between his-240 and ade-4 markers. Cotransduction frequencies show the following order of genes: his-240 fix-1 cys-246 ade-4.     

Symbiotic properties of adenine requiring mutants of Rhizobium meliloti strain L5-30

From the effective and prototrophic Rhizobium meliloti strain L5-30 two auxotrophic mutants were isolated: RM4 and RM221. These two mutants required adenine and adenine with thiamine for their growth, respectively. Both mutants nodulated lucerne plants ineffectively. Electron microscopic observations of the nodule tissue showed that its cells were not occupied by bacteria. Prototrophic revertants and transductants of these mutants showed high symbiotic effectiveness. It is assumed that adenine or adenine and thiamine requirements made impossible release of bacteria from the infection thread.     

Induction of Symbiotically Defective Auxotrophic Mutants of Rhizobium fredii HH303 by Transposon Mutagenesis

Symbiotically defective auxotrophic mutants were isolated by transposon Tn5 mutagenesis of Rhizobium fredii HH303, a fast-growing microsymbiont of North American commercial soybean cultivars such as Glycine max cv. Williams. Three different Tn5-carrying suicide vectors, pBLK1-2, pSUP1011, and pGS9, were used for mutagenesis with transposition frequencies of 4 x 10, 3 x 10, and 1 x 10, respectively, while the frequency of background mutation resistant to 500 mug of kanamycin per ml was 1 x 10. From 2,600 Tn5-induced mutants, 14 auxotrophic mutants were isolated and classified in seven groups including adenosine (four), aspartate (two), cysteine or methionine (two), isoleucine and valine (two), nicotinic acid (one), pantothenic acid (one), and uracil (two). All the auxotrophs induced nodulation on soybean, but the symbiotic effectiveness of each mutant was different. Three auxotrophs (two cysteine or methionine and one pantothenic acid) formed effective nodules similar to those of the wild type. Three auxotrophs (one nicotinic acid and two aspartate) produced mature nodules like those of the wild type, but the nodules lacked the characteristic pink color inside and were unable to fix nitrogen. Four auxotrophs (two adenosine and two uracil) induced pseudonodules unable to fix nitrogen. The other four auxotrophs repeatedly induced both effective and ineffective nodules, but bacteroids isolated from the effective nodules were prototrophic revertants. The symbiotic phenotype and the degree of effectiveness of the auxotrophic mutants varied with the type of mutation.     

Symbiotic properties of some auxotrophic mutants of Rhizobium meliloti and of their prototrophic revertants

Summary 1. Auxotrophic mutants of 2 strains ofRhizobium meliloti have been isolated: among them 4 are purine or pyrimidine-dependent (adenine, cytosine), 4 are sulfur amino acid-dependent (methionine, cysteine) and 3 glycine-dependent. 2. All of these mutants induce nodule formation on lucerne (variety ‘Du Puits’). 3. There are associations between nutritional requirements and effectiveness in nitrogen fixation on this host. All the amino acid-dependent mutants are effective. Glycine-dependent mutants are more effective than the wild-type strain, whereas cyste?ne and methionine-dependent mutants are as effective as the wild-type strain. On the other hand, purine and pyrimidine-dependent mutants are ineffective. 4. All the prototrophic revertants of ineffective auxotrophic strains are effective. Some of them are more effective than wild-type strain.     

Transductional analysis of chloramphenicol biosynthesis genes in Streptomyces venezuelae.

Auxotrophs isolated from two chloramphenicol-nonproducing mutants of Streptomyces venezuelae included three requiring pyridoxal (Pxl-), VS248 (cml-11 pdx-2), VS253 (cml-11 pdx-3), and VS258 (cml-12 pdx-4), and one requiring thiosulfate, VS263 (cml-12 cys-28). Results of SV1-mediated transductions were consistent with the relative marker order cys-28-cml-12-cml-11-pdx-2,3,4,5, all of which were cotransducible and must therefore span less than 45 kilobases of DNA, the approximate length of DNA packaged by SV1. cys-28 was also cotransducible with arg-4 and arg-6, but arg and pdx were not cotransducible. Results of crosses with donors carrying any one of 11 cml mutations were consistent with the location of all cml mutations between cys-28 and pdx markers. Also, a new Pxl- auxotroph (pdx-6) and two new Cml- mutants were recovered after localized hydroxylamine mutagenesis of a cys-28 cml+ strain derived from VS263 by transduction.     

Oxidative Metabolism of a Pigmented Mutant of Rhizobium meliloti and its Revertants

Non-infective pigmented mutants of Rhizobium meliloti have been obtained by u.v. irradiation. The majority of colourless revertants of a pigmented mutant obtained by u.v. irradiation nodulated the host plant indicating that pigmentation in rhizobium and ability to nodulate are genetically linked and may have some pleiotrophic effect. All the nodulating revertants and the parent strain oxidized pentoses, hexoses, dihexoses, tricarboxylic acid (TCA) cycle intermediates and related compounds at a higher rate than the non-nodulating revertants and pigmented mutant. Significant differences in the oxidation of pentoses and TCA cycle intermediates in nodulating and non-nodulating revertants indicate that these two energy yielding pathways are operating less efficiently in non-nodulating revertants and suggests that the working of these two pathways may be essential for the nodulation process.     

Symbiotic characteristics of cysteine and methionine auxotrophs of Sinorhizobium meliloti

Twenty one cysteine and 13 methionine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5. The cysteine auxotrophs were sulfite reductase mutants and each of these auxotrophs had a mutation in cysI/cysJ gene. The methionine auxotrophs were metA/metZ, metE and metF mutants. One hundred per cent co-transfer of Tn5-induced kanamycin resistance and auxotrophy from each Tn5-induced auxotrophic mutant indicated that each mutant cell most likely had a single Tn5 insertion. However, the presence of more than one Tn5 insertions in the auxotrophs used in our study cannot be ruled out. All cysteine and methionine auxotrophs induced nodules on alfalfa plants. The nodules induced by cysteine auxotrophs were fully effective like those of the parental strain-induced nodules, whereas the nodules induced by methionine auxotrophs were completely ineffective. The supplementation of methionine to the plant nutrient medium completely restored symbiotic effectiveness to the methionine auxotrophs. These results indicated that the alfalfa host provides cysteine but not methionine to rhizobia during symbiosis. Histological studies showed that the defective symbiosis of methionine auxotrophs with alfalfa plants was due to reduced number of infected nodule cells and incomplete transformation of bacteroids.     

General control of amino acid biosynthesis in mutants of Candida spec. EH 15/D

The general control of amino acid biosynthesis was investigated in Candida spec. EH 15/D, using single and double mutant auxotrophic strains and prototrophic revertants starved for their required amino acids. These experiments show that starvation for lysine, histidine, arginine, leucine, threonine, proline, serine, methionine, homoserine, asparagine, glutamic acid or aspartic acid can result in derepression of enzymes. A correlation was found between the degree of derepression, growth of strains, and concentration of required amino acids. The amino acids pool pattern of mutants and revertants is different from that in the wild type strain.     

Fine-structure genetic map of the cysB locus in Salmonella typhimurium.

A genetic map of the cysB region of the Salmonella typhimurium chromosome was constructed using bacteriophage P22-mediated transduction. Strains bearing delta (supX cysB) mutations were employed to divide this regulatory locus into 12 segments containing a total of 39 single-site mutations. Twenty-five of these single-site mutations were further ordered by reciprocal three-point crosses. The results do not support the concept of multiple cistrons at cysB and suggest that the abortive transductants previously observed in crosses between certain cysB mutants were due to intracistronic complementation. The prototrophic cys-1352 mutation, which causes the constitutive expression of the cysteine biosynthetic enzymes, was found to lie within the cysB region itself. It is bracketed by mutations, which lead to an inability to derepress for these enzymes and result in auxotrophy for cysteine.     

The Auxin Transport Inhibitor N-(1-Naphthyl)phthalamic Acid Elicits Pseudonodules on Nonnodulating Mutants of White Sweetclover

The collection of symbiotic (sym) mutants of white sweetclover (Melilotus alba Desr.) provides a developmental sequence of mutants blocked early in infection or nodule organogenesis. Mutant phenotypes include non-nodulating mutants that exhibit root-hair deformations in response to Rhizobium meliloti, mutants that form ineffective nodules lacking infection threads, and mutants that form infection threads and ineffective nodules. Mutant alleles from both the sym-1 and the sym-3 loci exhibited a non-nodulating phenotype in response to R. meliloti, although one allele in the sym-1 locus formed ineffective nodules at a low frequency. Spot-inoculation experiments on a non-nodulating allele in the sym-3 locus indicated that this mutant lacked cortical cell divisions following inoculation with R. meliloti. The auxin transport inhibitor N-(1-naphthyl)phthalamic acid elicited development of pseudonodules at a high frequency on all of the sweetclover sym mutants, including the non-nodulating mutants, in which the early nodulin ENOD2 was expressed. This suggests that N-(1-naphthyl)phthalamic acid activates cortical cell divisions by circumventing a secondary signal transduction event that is lacking in the non-nodulating sweetclover mutants. The sym-3 locus and possibly the sym-1 locus appear to be essential to early host plant responses essential to nodule organogenesis.     

Mapping of cysteine genes on the chromosome of Pseudomonas aeruginosa PAO

Summary Three loci coding for different steps in the pathway of cysteine biosynthesis have been mapped by R68.45-mediated coconjugation analysis. The cysteine auxotrophic mutants could be subdivided into sulfite and sulfide-requiring mutants. Sulfide-requiring mutants (cysIV group) were localized at a single position between pyrF and pur-67, while sulfite-requiring mutants (cysI and cysII) mapped at two different regions. The cysI group was also localized between pyrF and pur-67, although more distal to pyrF than the cysIV group. This group included the cys-54 marker, which has been mapped previously. The second group of sulfite-requiring mutants, designated as cysII, was cotransducible with hisI and localized at the end of the PAO chromosomal map. This location was also confirmed for the marker cys-59.The marker cys-59 (which was cotransducible with his1) was cotransferred by R68.45-mediated conjugations with both the late marker pur-67 and the early marker ilv-226. As the late marker hisI was positioned at about 60–65 min (Herrmann and Günther, in press) the length of the PAO chromosome was estimated to be about 70 min.     

Genetic Analysis of an Escherichia coli Syndrome

A mutant strain of Escherichia coli that fails to recover from prolonged (72 hr) starvation also fails to grow at 43 C. Extracts of this mutant strain show an increased ribonuclease II activity as compared to extracts of the parental strain, and stable ribonucleic acid is degraded to a larger extent in this strain during starvation. Ts(+) transductants and revertants were tested for all the above-mentioned phenotypes. All the Ts(+) transductants and revertants tested behaved like the Ts(+) parental strain, which suggests that all the observed phenotypes are caused by a single sts (starvation-temperature sensitivity) mutation. The reversion rate from sts(-) to sts(+) is rather low but is within the range of reversion rates for other single-site mutations. Three-point transduction crosses located this sts mutation between the ilv and rbs genes. The properties of sts(+)/sts(-) merozygotes suggested that the Ts(-) phenotype of this mutation is recessive.     

Effectiveness ofRhizobium as modified by mutation for resistance to antibiotics

Many antibiotic-resistant mutants derived from effective strains ofRhizobium — mainlyR. leguminosarum andR. trifolii showed some loss of effectiveness on the homologous host. The frequency and degree of such changes in effectiveness depended on the type of antibiotic-resistance involved in the mutations. Resistance to streptomycin, spiramycin, chloramphenicol, or the tetracycline group of antibiotics was associated with little or no change. Resistance to D-cycloserine, novobiocin, vancomycin, bacitracin or penicillin was accompanied by partial or full loss of effectiveness in about one-half of the mutants. Full loss of effectiveness occurred most frequently among mutants selected for resistance to viomycin or neomycin. In contrast, restoration of the ability for effective symbiosis occurred in prototrophic revertants of a non-nodulating, purine-requiring mutant. Alteration of cell-wall characteristics is suggested as a possible basis for decrease of effectiveness among mutants resistant to those antibiotics, notablyD-cycloserine, which are known to inhibit cell-wall synthesis.This investigation was supported by a Public Health Service research career development award (1-K3-GM-22, 594) and research grant (GM-12131) from the National Institutes of Health.     

Auxotrophic mutants in the selection of the rubomycin producer, Actinomyces coeruleorubidus

A total of 351 auxotrophic mutants with different antibiotic activity, including several mutants with activity higher than that of the parent prototrophic strains were obtained under the effect of gamma-rays from 3 prototrophic strains of Act. coeruleorubidus. It was shown that most of the auxotrophic mutants did not preserve the property of biochemical insufficiency on passages on complete media. A mutant strain 1059-32 with activity 2 times higher than that of the prototrophic strain 2-39 and the parent auxotrophic culture was obtained from the revertants. Requirements in 29 growth factors including 17 amino acids, 4 nitrous bases, 8 vitamins and coenzymes were determined in 46 stable auxotrophic mutants isolated. The effect of the specific and non-specific growth factors on the culture antibiotic production was studied.     

Arginine auxotrophs of Candida albicans deficient in argininosuccinate lyase

Auxotrophic mutants of Candida albicans FC18 were induced by a combination of treatments with nitrous acid and UV irradiation. Arginine (Arg-), histidine (His-) and methionine/cysteine (MetA-) auxotrophs were recovered by this means. The Arg- auxotrophs lacked active argininosuccinate lyase (EC 4.3.2.1), the enzyme catalysing the final step in arginine biosynthesis. Thus the locus may be designated arg-4. The mutant strains bearing this mutation did not form germ tubes unless the germination medium contained arginine.     

Regulatory Mutants at the his1 Locus of Yeast

The his1 gene in Saccharomyces cerevisiae codes for phosphoribosyl transferase, an allosteric enzyme that catalyzes the initial step in histidine biosynthesis. Mutants that specifically alter the feedback regulatory function were isolated by selecting his1 prototrophic revertants that overproduce and excrete histidine. The prototrophs were obtained from diploids homoallelic for his1--7 and heterozygous for the flanking markers thr3 and arg6. Among six independently derived mutant isolates, three distinct levels of histidine excretion were detected. The mutants were shown to be second-site alterations mapping at the his1 locus by recovery of the original auxotrophic parental alleles. The double mutants, HIS1--7e, are dominant with respect to catalytic function but recessive in regulatory function. When removed from this his1--7 background, the mutant regulatory site (HIS1-e) still confers prototrophy but not histidine excretion. To yield the excretion phenotype, the primary and altered secondary sites are required in cis array. Differences in histidine excretion levels correlate with resistance to the histidine analogue, triazoalanine.     

Symbiotic Properties of Antibiotic-Resistant and Auxotrophic Mutants of Rhizobium leguminosarum

A number of auxotrophic and antibiotic-resistant mutants of Rhizobium leguminosarum were isolated and their symbiotic properties examined. Among the auxotrophic mutants tested, only those with an adenine requirement were found to be symbiotically defective and these were all non-infective. Of the antibiotic-resistant mutants isolated, only a minority of the rifampicin-resistant mutants had a modified symbiotic phenotype and this was ineffective. Preliminary evidence suggests that the basis for the resistance in these mutants is a modified DNA-dependent RNA polymerase.     

Isolation and Symbiotic Characteristics of Two Tn5-Derived Phage-Resistant <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> Strains that Nodulate Soybean

Using transponson Tn5 mutagenesis, two transconjugants of Bradyrhizobium japonicum with the properties of both phage resistance and ability to induce nodulation were isolated at the frequency of 0.02%. These transconjugants were tested for their symbiotic performance on soybean cv. JS335 under greenhouse and field conditions. Both phage-resistant mutants induced nodules (nod (+)), but the transconjugant B. japonicum E13 was ineffective in nitrogen fixation (fix (-)). Rhizobiophage presence in the inoculum of phage-resistant mutants did not influence the symbiotic effectiveness. The mixture of wild strain and phage in the inoculum caused reduced symbiotic performance under controlled conditions, while under a field environment phage (100 and 500 mul of approximately 10(8) particles ml(-1)) presence did not have any recognizable effect on increased nodule dry weight, nitrogenase activity, or foliar N(2) content. On the basis of restriction fragment length polymorphism analysis, phage-sensitive, less effective, homologous bradyrhizobia belonging to B. japonicum were detected in root nodules of both inoculated and uninoculated plants. Inoculation of a higher concentration of phage in the inoculum significantly reduced the symbiotic performance, while the lower concentration of phage did not show any effect on phage-susceptible, less effective, homologous bradyrhizobia or, thus, symbiotic efficiency under field conditions. The phage-resistant mutant B. japonicum A49 showed effective symbiosis as efficient as that of the wild strain. Inoculation of phage-resistant mutants with lytic phage may reduce the occupancy of phage-susceptible, ineffective/less effective/mediocre homologous bradyrhizobia strains under natural complex soil conditions.     

Escherichia coli K-12 mutants with increased resistance to ionizing radiation. V. The effect of mutations on spontaneous and radiation mutagenesis

The frequencies of spontaneous mutations (reversions his-4----His+ and forward mutations to rifampicin-, nalidixic acid- or valine-resistance) in radiation-resistant mutants Gamr444 and Gamr445 are much lower than in the wild-type strain AB1157. His+ revertants and rifampicin-resistant mutants Rifr are induced by low doses of gamma-rays more efficiently than in the wild-type. Low doses of UV light only enhanced mutagenic activity in Gamr strains for induction of His+ reversions but not for Rifr mutations. For the wild-type strain the frequencies of His+ and Rifr mutations increase proportionally to the square of dose both of UV light and gamma-rays. For the most radioresistant Gamr444 mutant the frequencies of UV- and gamma-rays-induced Rifr mutations and of gamma-rays-induced reversions increase linearly with the dose. Possible reasons for these anomalies of radiation-induced mutagenesis in Gamr mutants are discussed.