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E R Signer

Publications and source records attributed to E R Signer.

At least 19 recordsLinked to original sources
PubMed1992

Somatic and germinal recombination of a direct repeat in Arabidopsis.

Homologous recombination between a pair of directly repeated transgenes was studied in Arabidopsis. The test construct included two different internal, non-overlapping deletion alleles of npt (neomycin phosphotransferase) flanking an active HPT (hygromycin phosphotransferase) gene. This construct was introduced into Arabidopsis by agrobacterium-mediated transformation with selection for resistance to hygromycin, and two independent single-insert lines were analyzed. Selection for active NPT by resistance to kanamycin gave both fully and partly (chimeric) recombinant seedlings. Rates for one transgenic line were estimated at less than 2 x 10(-5) events per division for germinal and greater than 10(-6) events per division for somatic recombination, a much smaller difference than between meiotic and mitotic recombination in yeast. Southern analysis showed that recombinants could be formed by either crossing over or gene conversion. A surprisingly high fraction (at least 2/17) of the recombinants, however, appeared to result from the concerted action of two or more independent simple events. Some evolutionary implications are discussed.

Alleles↗
PubMed1992

A directional, high-frequency chromosomal mobilization system for genetic mapping of Rhizobium meliloti.

A system for mapping of the Rhizobium meliloti chromosome that utilizes transposon Tn5-Mob, which carries the mobilization site of IncP plasmid RP4 (R. Simon, Mol. Gen. Genet. 196:413-420, 1984), was developed. Insertions of Tn5-Mob that were located at particular sites on the R. meliloti chromosome were isolated and served as origins of high-frequency chromosomal transfer when IncP tra functions were provided in trans. This approach is, in principle, applicable to any gram-negative bacterium in which Tn5 can transpose and into which IncP plasmids can conjugate.

Chromosome Mapping↗
PubMed1991

lpsZ, a lipopolysaccharide gene involved in symbiosis of Rhizobium meliloti.

lpsZ+ is an allele that allows exo (exopolysaccharide-deficient) mutants of Rhizobium meliloti to invade nodules by modifying rhizobial lipopolysaccharide. We have cloned and sequenced the lpsZ gene. The predicted LpsZ protein has a molecular weight of 48,589 and is probably localized in the cytoplasm. A beta-glucuronidase fusion in the lpsZ gene indicates that lpsZ is not regulated by oxygen or nitrogen.

Amino Acid Sequence↗
PubMed1990

Cauliflower mosaic virus P35S promoter activity in Escherichia coli.

We present evidence that the cauliflower mosaic virus promoter P35S can direct expression of the bacterial neomycin phosphotransferase II (NPTII) gene in Escherichia coli. Transcription is initiated at several sites, the major one being located approximately 315 bases upstream of the plant start site. The nucleotide sequence directly preceding this start site is strongly homologous to the prokaryotic promoter consensus sequence. Thus constructs designed for introduction into plants can be expressed in E. coli.

Base Sequence↗
PubMed1990

Temporal and spatial regulation of the symbiotic genes of Rhizobium meliloti in planta revealed by transposon Tn5-gusA.

Tn5-gusA promoter/probe transposons have been constructed that fuse the Escherichia coli gusA reporter gene transcriptionally or translationally with a target promoter. These have been used to monitor expression of Rhizobium meliloti symbiotic genes within alfalfa nodules. Fusions in all 11 nod genes studied show the same pattern of expression: first on the root surface, then throughout the developing nodule, then mainly in the nodule meristem, falling off progressively through the central region, and then disappearing. In contrast, fusions in all five nif genes studied, all four fix genes, and syrM show a second, different pattern: expression beginning later, first throughout the nodule except for the meristem, strongest just behind the meristem, and falling off progressively through the central region. Novel features revealed by these studies include nod expression in the meristem, regulated in planta expression of control genes nodD1 and nodD3, disappearance of nod expression late in organogenesis, and properties of syrM.

Base Sequence↗
PubMed1990

Rhizobium meliloti chromosomal loci required for suppression of exopolysaccharide mutations by lipopolysaccharide.

Mutants of alfalfa symbiont Rhizobium meliloti SU47 that fail to make extracellular polysaccharide (exo mutants) induce the formation of nodules that are devoid of bacteria and consequently do not fix nitrogen. This Fix- phenotype can be suppressed by an R. meliloti Rm41 gene that affects lipopolysaccharide structure. Here we describe mutations preventing suppression that map at two new chromosomal loci, lpsY and lpsX, present in both strains. Two other lps mutations isolated previously from SU47 also prevented suppression.

Chromatography, Gel↗
PubMed1990

The symbiotic defect of Rhizobium meliloti exopolysaccharide mutants is suppressed by lpsZ+, a gene involved in lipopolysaccharide biosynthesis.

exo mutants of Rhizobium meliloti SU47, which fail to secrete acidic extracellular polysaccharide (EPS), induce Fix- nodules on alfalfa. However, mutants of R. meliloti Rm41 carrying the same exo lesions induce normal Fix+ nodules. We show that such induction is due to a gene from strain Rm41, which we call lpsZ+, that is missing in strain SU47. lpsZ+ does not restore EPS production but instead alters the composition and structure of lipopolysaccharide. In both SU47 and Rm41, either lpsZ+ or exo+ is sufficient for normal nodulation. This suggests that in R. meliloti EPS and lipopolysaccharide can perform the same function in nodule development.

Chromosome Mapping↗
PubMed1990

Rhizobium meliloti suhR suppresses the phenotype of an Escherichia coli RNA polymerase sigma 32 mutant.

sigma 32, the product of the Escherichia coli rpoH locus, is an alternative RNA polymerase sigma factor utilized to express heat shock genes upon a sudden rise in temperature. E. coli K165 [rpoH165(Am) supC(Ts)] is temperature sensitive for growth and does not induce heat shock protein synthesis. We have isolated a locus from Rhizobium meliloti called suhR that allows E. coli K165 to grow at high temperature and induce heat shock protein synthesis. R. meliloti suhR mutants were viable and symbiotically effective. suhR was found to have no DNA or derived amino acid sequence similarity to the genes of previously sequenced sigma factors or other data base entries, although a helix-turn-helix DNA-binding protein motif is present. suhR did not restore the phenotypic defects of delta rpoH E. coli; suppression of the E. coli K165 phenotype is thus likely to involve E. coli sigma 32. Western immunoblots showed that suhR caused an approximately twofold elevation of sigma 32 levels in K165; RNA blots indicated that rpoH mRNA level and stability were not altered. Stabilization of sigma 32 protein and increased rpoH mRNA translation are thus the most probable mechanisms of suppression.

Amino Acid Sequence↗
PubMed1989

Lipopolysaccharide mutants of Rhizobium meliloti are not defective in symbiosis.

Mutants of Rhizobium meliloti selected primarily for bacteriophage resistance fall into 13 groups. Mutants in the four best-characterized groups (class A, lpsB, lpsC, and class D), which map to the rhizobial chromosome, appear to affect lipopolysaccharide (LPS) as judged by the reactivity with monoclonal antibodies and behavior on sodium dodecyl sulfate-polyacrylamide gels of extracted LPS. Mutations in all 13 groups, in an otherwise wild-type genetic background, are Fix+ on alfalfa. This suggests that LPS does not play a major role in symbiosis. Mutations in lpsB, however, are Fix- in one particular genetic background, evidently because of the cumulative effect of several independent background mutations. In addition, an auxotrophic mutation evidently equivalent to Escherichia coli carAB is Fix- on alfalfa.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Interaction of nod and exo Rhizobium meliloti in alfalfa nodulation.

Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation. To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes. Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant. We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen. However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen. In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper. Implications of these results for interaction of nod and exo gene products are discussed.

Genes, Bacterial↗
PubMed1988

Isolation and characterization of Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutations.

The occurrence in Azospirillum brasilense of genes that code for exopolysaccharide (EPS) synthesis was investigated through complementation studies of Rhizobium meliloti Exo- mutants. These mutants are deficient in the synthesis of the major acidic EPS of Rhizobium species and form empty, non-nitrogen-fixing root nodules on alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We demonstrated that the exoC mutation of R. meliloti could be corrected for EPS production by several cosmid clones of a clone bank of A. brasilense ATCC 29145. However, the EPS produced differed in structure from the wild-type R. meliloti EPS, and the symbiotic deficiency of the exoC mutation was not reversed by any of these cosmid clones. The exoB mutation could be corrected not only for EPS production but also for the ability to form nitrogen-fixing nodules on alfalfa by one particular cosmid clone of A. brasilense. Tn5 insertions in the cloned DNA were isolated and used to construct Azospirillum mutants with mutations in the corresponding loci by marker exchange. It was found that these mutants failed to produce the wild-type high-molecular-weight EPS, but instead produced EPSs of lower molecular weight.

Genes↗
PubMed1988

All nod genes of Rhizobium meliloti are involved in alfalfa nodulation by exo mutants.

Nodulation of alfalfa by exoB mutants of Rhizobium meliloti occurred without root hair curling or infection thread formation. nod exoB double mutants had the same nodulation deficiency as single nod mutants. Therefore, all the known nod genes are involved in nodule induction by exoB mutants, which apparently occurs via intercellular invasion.

Chromosome Deletion↗
PubMed1986

Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives.

Two derivatives of the prokaryotic transposon Tn5 were constructed in vitro. In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin. In Tn5-235, the Escherichia coli beta-galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter. Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti. When a Tn5-derivative is introduced into an R. meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons. In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations. We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage phi M12. In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac+ phenotype. Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative.

Anti-Bacterial Agents↗
PubMed1986

Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes.

Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.

DNA Transposable Elements↗
PubMed1985

Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation.

Spontaneous mutants at a new symbiotic locus in Rhizobium meliloti SU47 are resistant to several phages and are conditionally insensitive to a monoclonal antibody to the bacterial surface, apparently because they are deficient in a wild-type exopolysaccharide. On alfalfa, the mutants do not curl root hairs, but penetrate the epidermis directly, forming nodules that contain no visible infection threads or "bacteroids," have a few bacteria in superficial intercellular spaces only and not within the nodule cells, and fail to fix nitrogen (Fix-). Evidently, infection threads are not essential for cell proliferation and nodule formation, which are here induced by a bacterial signal at a distance and uncoupled from the bacterial differentiation that normally goes on as well.

Antibodies, Monoclonal↗
PubMed1985

Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules.

By screening with the fluorescent stain Calcofluor, we have isolated 26 independent transposon Tn5 insertion mutants of Rhizobium meliloti that are deficient in the production of a known extracellular polysaccharide (Exo-). The mutants belonged to six distinct genetic groups based on the ability of their Exo- phenotype to be complemented by different recombinant plasmids from a R. meliloti clone bank. With few exceptions, all of the mutants formed ineffective (non-nitrogen-fixing) nodules on alfalfa. For all but one group, the complementing plasmids restored effective nodulation. These results establish a firm and extensive correlation between the ability of Rhizobium to produce a particular polysaccharide and symbiotic proficiency. The ineffective nodules appeared to contain no bacteroids and to form without shepherds' crooks or infection threads; this symbiotic phenotype matches that described for a set of independently isolated mutants that belong phenotypically and genetically to the group B exopolysaccharide mutants described previously [Finan et al. (1985) Cell 40, 869-877]. Apparently the exopolysaccharide, although not required for nodule formation, is involved in wild-type nodule invasion.

DNA, Recombinant↗
PubMed1984

General transduction in Rhizobium meliloti.

General transduction by phage phi M12 in Rhizobium meliloti SU47 and its derivatives is described. Cotransduction and selection for Tn5 insertions which are closely linked to specific loci were demonstrated. A derivative of SU47 carrying the recA::Tn5 allele of R. meliloti 102F34 could be transduced for plasmid R68.45 but not for chromosomally located alleles. Phage phi M12 is morphologically similar to Escherichia coli phage T4, and restriction endonuclease analysis indicated that the phage DNA was ca. 160 kilobases in size.

Bacteriophages↗