Auxotrophic mutations related to symbiotic properties of Rhizobium meliloti strain L5-30.

Mutants isolated from effective R. meliloti strain L5-30 which required histidine (his-240), arginine+uracil (arg-55) and cysteine (cys-243, cys-244 and cys-246) showed also loss of effectiveness. Mutant requiring isoleucine+valine (ilv-74) was non-infective. Relation of the metabolic deficiency to the symbiotic properties of these mutants was tested comparing symbiotic response of their prototrophic revertants and transductants. It was found that all revertants and transductants of the strain his-240 were effective which suggests that histidine deficiency was the cause of their ineffectiveness. All revertants and transductants of the cysteine mutants were still ineffective. This result indicates two independent mutations which were not cotransductible. Prototrophic revertants of the mutant arg-55 were ineffective whereas 56.9 percent of transductants appeared effective suggesting close linkage of two mutations. i.e. auxotrophic and the other concerned with symbiotic effectiveness. Though one of 69 prototrophic transductants obtained from the non-nodulating mutant ilv-74 remained non-nodulating, it seems that changes in nodulating ability of the mutant are related to the auxotrophic requirements.     

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.     

Analysis of the pdx-1 (snz-1/sno-1) region of the Neurospora crassa genome: correlation of pyridoxine-requiring phenotypes with mutations in two structural genes

We report the analysis of a 36-kbp region of the Neurospora crassa genome, which contains homologs of two closely linked stationary phase genes, SNZ1 and SNO1, from Saccharomyces cerevisiae. Homologs of SNZ1 encode extremely highly conserved proteins that have been implicated in pyridoxine (vitamin B6) metabolism in the filamentous fungi Cercospora nicotianae and in Aspergillus nidulans. In N. crassa, SNZ and SNO homologs map to the region occupied by pdx-1 (pyridoxine requiring), a gene that has been known for several decades, but which was not sequenced previously. In this study, pyridoxine-requiring mutants of N. crassa were found to possess mutations that disrupt conserved regions in either the SNZ or SNO homolog. Previously, nearly all of these mutants were classified as pdx-1. However, one mutant with a disrupted SNO homolog was at one time designated pdx-2. It now appears appropriate to reserve the pdx-1 designation for the N. crassa SNZ homolog and pdx-2 for the SNO homolog. We further report annotation of the entire 36,030-bp region, which contains at least 12 protein coding genes, supporting a previous conclusion of high gene densities (12,000-13,000 total genes) for N. crassa. Among genes in this region other than SNZ and SNO homologs, there was no evidence of shared function. Four of the genes in this region appear to have been lost from the S. cerevisiae lineage.     

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.     

Mutants of Escherichia coli Lacking Endonuclease I, Ribonuclease I, or Ribonuclease II

To isolate mutants of Escherichia coli K-12 lacking endonuclease I activity (end), a method has been developed which detects, by differential methyl green staining, undegraded deoxyribonucleic acid (DNA) in colonies previously incubated in toluene. This procedure allows isolation of mutant strains in which DNA degradation is reduced. For half of these strains, this defect has been correlated with deficiencies of endonuclease I, ribonuclease I (rns), or ribonuclease II (rne) activities. The enzymatic deficiencies of the other strains remain unknown. An rne mutation is cotransducible with serA (which is located at 56 min on the genetic map). Most end mutations, called endA, are also cotransducible with serA and are located between serA and strA. One end mutation, called endB, is located between purE and trp (i.e., between 13 and 25 min on the genetic map).     

Localized hydroxylamine mutagenesis, and cotransduction of threonine and lysine genes, in Streptomyces venezuelae

A lysate of the generalized transducing phage SV1, grown on the prototrophic type strain 10712 of Streptomyces venezuelae, was mutagenized with hydroxylamine and used to transduce a lysineless auxotroph to lysine independence on supplemented minimal agar. A complex threonine mutant, strain VS95, was isolated from among the transductants and was shown to be carrying at least two different thr mutations. These were about 50% cotransducible with alleles of four independently isolated lysA mutations, as were two other independently isolated threonine mutations, thr-1 and hom-5. The location of thr genes close to lysA occurs in at least three other streptomycetes, but apparently not in Streptomyces coelicolor A3(2), in which the lysA and thr loci are at diametrically opposite locations on the linkage map. This first observation of cotransduction between loci governing the biosynthesis of different amino acids in the genus Streptomyces demonstrates the feasibility of fine-structure genetic analysis by transduction in these antibiotic-producing bacteria.     

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.     

Mapping of rod mutants of Bacillus subtilis

Nine class A salt-dependent rod mutants were mapped on the Bacillus subtilis genome by PBS1-mediated transduction. They are distributed into two small linkage groups designated rod B and rod C; mutations in rod B are over 80% cotransducible with pheA and different mutations in rod C are 12 to 21% cotransducible with hisA. It is established that neither rod B nor rod C is linked by transformation to the other identified rod mutations present in 168-ts-200B and 8332 glu(-). It is hypothesized that salt-dependent mutations are due to enzyme alterations which are corrected by high salt concentrations.     

Mutations affecting the biosynthesis of S-adenosylmethionine cause reduction of DNA methylation in Neurospora crassa.

A temperature-sensitive methionine auxotroph of Neurospora crassa was found in a collection of conditional mutants and shown to be deficient in DNA methylation when grown under semipermissive conditions. The defective gene was identified as met-3, which encodes cystathionine-gamma-synthase. We explored the possibility that the methylation defect results from deficiency of S-adenosylmethionine (SAM), the presumptive methyl group donor. Methionine starvation of mutants from each of nine complementation groups in the methionine (met) pathway (met-1, met-2, met-3, met-5, met-6, met-8, met-9, met-10 and for) resulted in decreased DNA methylation while amino acid starvation, per se, did not. In most of the strains, including wild-type, intracellular SAM peaked during rapid growth (12-18 h after inoculation), whereas DNA methylation continued to increase. In met mutants starved for methionine, SAM levels were most reduced (3-11-fold) during rapid growth while the greatest reduction in DNA methylation levels occurred later. Addition of 3 mM methionine to cultures of met or cysteine-requiring (cys) mutants resulted in 5-28-fold increases in SAM, compared with wild-type, at a time when DNA methylation was reduced approximately 40%, suggesting that the decreased methylation during rapid growth in Neurospora is not due to limiting SAM. DNA methylation continued to increase in a cys-3 mutant that had stopped growing due to methionine starvation, suggesting that methylation is not obligatorily coupled to DNA replication in Neurospora.     

Mutants of Neurospora crassa Deficient in Ornithine-δ-Transaminase

The arg-12(s) mutation of Neurospora causes a partial block in the ornithine transcarbamylase (OTC) reaction. Strains carrying this mutation will use endogenous ornithine, but not exogenous ornithine, as a precursor of arginine. Certain strains carrying arg-12(s) may be used for direct selection of variants able to use exogenous ornithine as an arginine precursor. Among eight such derivatives, six lacked the catabolic enzyme ornithine transaminase (OTA). All six mutations were alleles of a single gene, designated ota, on linkage group III. No mutation affected arginase, the first enzyme in the catabolic pathway with OTA. Strains carrying ota mutations alone are vigorous prototrophs, but, unlike wild-type Neurospora, fail to use ornithine efficiently as a sole nitrogen source. The selection method by which ota mutants arose suggests that OTA is severely competitive with OTC for exogeneous ornithine.     

Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants.

The major DNA-binding protein, or infected-cell protein 8 (ICP8), of herpes simplex virus is required for viral DNA synthesis and normal regulation of viral gene expression. Previous genetic analysis has indicated that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of acting independently as a nuclear localization signal. In this study, we constructed a mutant virus (n11SV) in which the carboxyl-terminal 28 residues of ICP8 were replaced by the simian virus 40 large-T-antigen nuclear localization signal. The n11SV ICP8 localized into the nucleus and bound to single-stranded DNA in vitro as tightly as wild-type ICP8 did but was defective for viral DNA synthesis and viral growth in Vero cells. Two mutant ICP8 proteins (TL4 and TL5) containing amino-terminal alterations could complement the n11SV mutant but not ICP8 gene deletion mutants. Cell lines expressing TL4 and TL5 ICP8 were isolated, and in these cells, complementation of n11SV was observed at the levels of both viral DNA replication and viral growth. Therefore, complementation between n11SV ICP8 and TL4 or TL5 ICP8 reconstituted wild-type ICP8 functions. Our results demonstrate that (i) the carboxyl-terminal 28 residues of ICP8 are required for a function(s) involved in viral DNA replication, (ii) this function can be supplied in trans by another mutant ICP8, and (iii) ICP8 has multiple domains possessing different functions, and at least some of these functions can complement in trans.     

Location of trpR Mutations in the serB-thr Region of Salmonella typhimurium

Tryptophan biosynthesis in Salmonella is controlled by at least one regulatory gene, trpR, which is cotransducible with thr genes and not with the trp operon. Mutations in trpR cause derepression of tryptophan enzyme synthesis and confer resistance to growth inhibition by 5-methyltryptophan. Nineteen trpR mutations were mapped with respect to thrA and serB markers by two-point (ratio) and three-point transduction tests. The results are all consistent with the site order serB80-trpR-thrA59 on the Salmonella chromosome. Very low or undetectable levels of recombination between different trpR mutations have so far prevented the determination of fine structure in the trpR gene. Thirteen other 5-methyltryptophan-resistant mutants previously found not to be cotransducible with either the trp operon or thrA, and designated trpT, were also used in these experiments. Lack of cotransducibility with thrA was confirmed, and no linkage with serB was detected. The nature and location of trpT mutations remain obscure.     

Expression of herpes virus thymidine kinase in Neurospora crassa.

The expression of thymidine kinase in fungi, which normally lack this enzyme, will greatly aid the study of DNA metabolism and provide useful drug-sensitive phenotypes. The herpes simplex virus type-1 thymidine kinase gene ( tk ) was expressed in Neurospora crassa. tk was expressed as a fusion to N.crassa arg-2 regulatory sequences and as a hygromycin phosphotransferase-thymidine kinase fusion gene under the control of cytomegalovirus and SV40 sequences. Only strains containing tk showed thymidine kinase enzyme activity. In strains containing the arg-2 - tk gene, both the level of enzyme activity and the level of mRNA were reduced by growth in arginine medium, consistent with control through arg-2 regulatory sequences. Expression of thymidine kinase in N.crassa facilitated radioactive labeling of replicating DNA following addition of [3H]thymidine or [14C]thymidine to the growth medium. Thymidine labeling of DNA enabled demonstration that hydroxyurea can be used to block replication and synchronize the N.crassa mitotic cycle. Strains expressing thymidine kinase were also more sensitive than strains lacking thymidine kinase to anticancer and antiviral nucleoside drugs that are activated by thymidine kinase, including 5-fluoro-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine and trifluorothymidine. Finally, expression of thymidine kinase in N. crassa enabled incorporation of bromodeoxyuridine into DNA at levels sufficient to separate newly replicated DNA from old DNA using equilibrium centrifugation.     

Linker insertion mutations in the adenovirus preterminal protein that affect DNA replication activity in vivo and in vitro.

Eighteen linker insertion mutants with mutations in the adenovirus precursor to terminal protein (pTP), which were originally constructed and tested in virions by Freimuth and Ginsberg (Proc. Natl. Acad. Sci. USA 83:7816-7820, 1986), were transferred to expression plasmids for assay of the various functions of the isolated pTP. Function was measured by the ability of individual pTP mutant proteins to participate in the initiation of replication from an adenovirus DNA end, by their activity in assays of DNA elongation, and by the intracellular distribution of pTP demonstrated by indirect immunofluorescence. Ten of the 11 mutants that were active in virion formation were also functional in DNA replication reactions in extracts, while 1 had reduced function. Four mutants with mutations that were lethal to virus production were also inactive in DNA replication reactions. These four mutations are probably located at sites required for the function of pTP in DNA synthesis. Three pTP mutants with mutations that were lethal or partially defective with respect to virion formation were active in reactions requiring pTP for initiation and elongation in extracts. All three of these mutant pTPs targeted normally to the nucleus, suggesting a defect after this step in replication. Since pTP has been reported to bind the nuclear matrix, these pTP mutants may have mutations that define sites necessary for binding to this structure. Several mutants with mutations that lie outside the putative nuclear targeting region were aberrantly localized, suggesting either that additional domains are important in nuclear localization or that there are alterations in protein structure that affect nuclear transport for some pTP mutants.     

DNA sequence alterations responsible for the synthesis of thermosensitive VP1 in temperature-sensitive BC mutants of simian virus 40.

The segment of simian virus 40 (SV40) genome which is recognized as the BC domain encodes for the COOH-terminal end of the SV40 major capsid protein VP1. Mutations in this domain lead to the synthesis of a thermosensitive VP1 which fails to assemble mature SV40 at the nonpermissive temperature. We determined the DNA sequences of eight BC mutants and compared them with the DNA sequences of wild-type SV40, polyomavirus, and BK virus. We found that BC11 and BC223 mutations result from changes in nucleotide residues 2367 (A to C) and 2084 (C to T), respectively. The others (i.e., BC208, BC214, BC216, BC217, BC248, and BC274) share the same point mutation at nucleotide 2354 (C to T). These mutations resulted in the following changes: Lys to Thr, His to Tyr, and Pro to Ser at VP1 amino acid residues 290, 196, and 286, respectively.     

Development of auxotrophic agrobacterium tumefaciens for gene transfer in plant tissue culture

Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression. Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nutrient matrix. Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade-24), leucine (leu-27), and cysteine (cys-32). Ade-24 displayed poor T-DNA delivery in a transient expression test delivering GUS from a binary vector, while cys-32 displayed the best ability to deliver DNA of these three auxotrophs. The growth yield of cys-32 on cysteine was assessed to provide a quantitative basis for co-culture nutrient supplementation. The utility of cys-32 for delivering T-DNA to plant tissues is demonstrated, where an 85-fold enhancement in GUS expression over wild-type A. tumefaciens was achieved.