Éva Vincze

Genetic and Biochemical Analysis of Mutants Affected in Nitrate Reduction in Rhizobium meliloti

Twenty-five mutants unable to utilize nitrate as sole nitrogen source were isolated from Rhizobium meliloti 41. These mutations mapped at four different sites, narA, narB, narC and narD; narB, C and D were located between trp-15 and ade-15 on the chromosome. NarA mutants were affected in assimilatory nitrate reduction but not in‘respiratory’ nitrate reduction and had methyl viologen-coupled nitrate reductase activity. NarB mutants were affected in both assimilatory and ‘respiratory’ nitrate reduction and lacked methyl viologen-coupled nitrate reductase activity. NarC and narD mutants were impaired not only in assimilatory and ‘respiratory’ nitrate reduction but lacked xanthine dehydrogenase activity as well. Acid-treated crude extracts of these two mutant classes were unable to restore NADPH-coupled nitrate reductase activity to the nit-1 mutant of Neurospora crassa, indicating the lack of active molybdenum cofactor. All mutants tested were effective in symbiotic plant tests and had normal nitrogenase activity, indicating that nitrogenase and nitrate reductase do not share the same molybdenum cofactor.

A comparison of three Rhizobium linkage maps

SummaryLinkage maps of R. meliloti 2011 (Rm2011), R. meliloti 41 (Rm41) and R. leguminosarum 300 (R1300), all constructed by means of P1 plasmid-mediated recombination, were compared. Recombination between the two R. meliloti strains occurred at high frequency but was barely detectable in matings between R1300 and Rm41. When co-inheritance data for the three strains were transformed into additive map distances the arrangement of markers showed striking similarities. Each of eight R68.45-primes, carrying different sections of the Rm2011 chromosome, suppressed only those markers of both R1300 and Rm41 which had the same phenotype and map location. Each of these R-primes promoted polarized chromosome transfer in an anticlockwise direction in Rm41, starting from the region corresponding to that carried on the plasmid.

Isolation of an alfalfa histone H3 gene: structure and expression

A histone H3 gene was isolated from a dicotyledonous plant, alfalfa (Medicago sativa). The sequence analysis of this gene revealed no obvious GC preference in its codon usage. Apart from containing most of the typical consensus sequences found in both animal and plant histone genes, the alfalfa H3 gene exhibits distinct structural features such as (1) the unusual location of two GATCC motifs in its 5′ flanking sequence, (2) the existence of a CGCGGATC on the nonsense strand at position −232, (3) the existence of a long palindromic structure, and (4) several polyadenylation signal-like sequences in the 3′ flanking region. There are about 160 copies of histone H3 gene in alfalfa tetraploid genome.Using the alfalfa H3 gene as a probe to study the pattern of histone H3 transcripts in the alfalfa, we found that the H3 RNAs are undetectable in leaves, more in stems than in roots, and highest in somatic embryos. Moreover, the RNA products of H3 genes in all alfalfa tissues tested show unusually long nontranslated region compared to those of animal histone genes. An additional high molecular weight species of H3 transcript was detected only in somatic embryos.

The nucleotide sequence of a nodule-specific gene, Nms-25 of Medicago sativa: its primary evolution via exon-shuffling and retrotransposon-mediated DNA rearrangements

We present the primary structure of a nudule-specific gene, Nms-25 from Medicago sativa L. cultivar Nagyszénási. Analysis of the nucleotide sequence of Nms-25 revealed that this gene shows all the characteristics of an interrupted plant gene consisting of 13 exons and 12 introns. The promoter region of Nms-25 contains the common promoter elements of plant genes as well as motifs which are supposed to be involved in nodule-specific expression. There are two exon-like sequences in the gene named PE1 and PE2 which are not present in the cDNA clones of Medicago sativa cultivar Cardinal. Intron 9 carries a retrotransposon-like element, Tms1, which might be responsible for downstream deletion events in which a heptanucleotide, ATTAGCT, might have been involved. Most of the exons, exept 1, 12 and 13, are similar to each other both in length (54 bp) and sequence (up to 94% sequence similarity). All exons are interrupted by introns in the same phase (type I). It is suggested that exon-shuffling based on illegitimate recombination in which the ATTAGCT motif might have played an active role, and retrotransposon-mediated DNA rearrangements were the primary events in the molecular evolution of the Nms-25 gene. The nucleotide sequence of the genomic clone of Nms-25 from Medicago sativa is available from the EMBL/GenBank/DDBJ Nucleotide Sequence Databases under the accession number X13287.

Molecular cloning of the gene encoding developing seed L-asparaginase from Lupinus angustifolius

A genomic sequence encoding Lupinus angustifolius L-asparaginase has been obtained, and is the first report of this gene from a plant source. The 3.2 kb of DNA sequenced contains a 1136 bp 5′ flanking sequence, four exons and three introns. Intron-exon borders were mapped by comparing the genomic sequence with that of a L. arboreus cDNA. Primer extension analysis revealed transcription start sites 16 bp and 13 bp 5′ of the initiating ATG for L. angustifolius and L. arboreus, respectively. The 5′ flanking region contained sequences associated with seed-specific expression.

Identification and cDNA cloning of a new nodule-specific gene, Nms-25 (nodulin-25) of Medicago sativa.

A new nodule-specific gene, Nms-25 (nodulin-25), was identified in cDNA clones isolated from a nodule-specific cDNA library of Medicago sativa. The first transcript of this gene appeared 9 days after inoculation of the roots with Rhizobium meliloti. The time of expression and the quantity of the transcripts of the Nms-25 gene was similar to that of leghemoglobin genes suggesting a similar regulation. A protein of 246 amino acids could be deduced from a full-length cDNA clone. The first 24 amino acids at the N-terminal end of this protein formed a signal sequence which might direct membrane transport into the peribacteroid space. Using different predictive methods the signal sequence cleaved protein was tentatively predicted to be a water-soluble enzyme, but not hydrolase.

Characterization and Cloning of Two Rhizobium leguminosarum Genes Coding for Glutamine Synthetase Activities

We have demonstrated that Rhizobium leguminosarum strain LPR1105 contains a heat stable and a heat labile glutamine synthetase (EC 6.3.1.2) activity similar to those described for other Rhizobiaceae. Most of the activity is heat stable when this strain is grown on glutamine as sole nitrogen source, but most is heat labile when grown on nitrate. Using a gene bank of R. leguminosarum DNA we have isolated two clones, which code for heat stable (p7D9) and heat labile (p4F7) glutamine synthetase activity, by complementing the glutamine auxotrophy of Klebsiella pneumoniae glnA mutants. Cross-hybridization of p7D9 with a fragment of the glnA gene of K. pneumoniae was observed, but no cross-hybridization between p7D9 and p4F7 was found. Since these two regions hybridize to genomic DNA of R. leguminosarum they are probably the structural genes for GSI and GSII, and the availability of these genes will make it possible to test this hypothesis. Clone p4F7 complements an ntrC+ but not an ntrC K. pneumoniae glnA mutant, suggesting that the ntrC gene is required for the complementation of the glutamine auxotrophy by this plasmid.

A phosphate group at the cos ends of phage lambda DNA is not a prerequisite for in vitro packaging: an alternative method for constructing genomic libraries using a new phasmid vector, λpGY97

It is shown here that the phosphate groups at the cos ends of phage lambda DNA are not a prerequisite for in vitro packaging. Molecules with phosphatase-treated cos ends are packaged in vitro as efficiently as native lambda DNA. This observation can be used for an alternative strategy to improve the efficiency of gene library construction, since cos-cos ligation decreases in vitro encapsidation and infectivity. Dephosphorylated cos ends and a new phasmid vector lambda pGY97 have been used to construct a representative gene bank of alfalfa in a Mcr- (5-methylcytosine restriction deficient) Escherichia coli host strain. These recombinant clones can be propagated as phages or more conveniently as plasmids in recA- E. coli, to prevent possible homologous recombination events between repetitive sequences of the insert that would otherwise interfere with clone stability. The 5-19-kb inserts can be easily recloned as plasmids from the recombinant phasmids with simple EcoRI digestion and re-ligation. This observation also implies that the construction of gene libraries in cosmid vectors can be made more efficient if cos-cos ligates were cleaved by lambda terminase just before in vitro packaging.

Repression of the L-asparaginase gene during nodule development in Lupinus angustifolius

Upon the establishment of an effective nitrogen-fixing symbiosis in amide-transporting plants the enzymatic activity and transcript levels of L-asparaginase are dramatically decreased. This decrease in L-asparaginase activity is essential for the correct functioning of the Rhizobium-legume symbiosis in lupin in which asparagine, synthesized from recently fixed nitrogen, is exported to aerial parts of the plant for use in growth and development. Concomitant with this decrease in L-asparaginase transcript a DNA-binding protein was detected in the nodules. This binding protein was not detectable in ineffective nodules, in nodules treated with nitrate, or in root tips, mature roots, developing flowers or developing seeds. The DNA-binding activity was shown to interact with a 59 bp sequence proximal to the transcription start site. Within this sequence a CTAAAAT direct repeat and a ACTGT/TGTCA incomplete inverted repeat were implicated in the binding of protein to the DNA by DNase I protection experiments. Competitive binding studies with synthesized binding sites were consistent with the CTAAAAT/TGTCA sequence pair proximal to the transcription start site having the highest affinity for the DNA-binding protein. We postulate that this DNA-binding protein is associated with repression of L-asparaginase gene expression in mature lupin root nodules.

The trans-Acting Protein Interacting with the DNA Motif Proximal to the Transcriptional Start Site of Plant L-Asparaginase Is Bacterial Sarcosine Oxidase

A trans-acting protein interacting with a specific sequence motif proximal to the transcriptional start site of the L-asparaginase promoter has been observed previously (E. Vincze, J. M. Reeves, E. Lamping, K. J. F. Farnden, and P. H. S. Reynolds, Plant Mol. Biol. 26:303-311, 1994). Gel retardation experiments in which protein extracts of Mesorhizobium loti and developing nodules were used suggested a bacterial origin for the repressor binding protein (rep2037). Nodulation tests were performed by using different Fix(-) Tn5 mutants of M. loti. Analyses of these mutants revealed a correlation between the presence of Mesorhizobium in the nodule-like structures and the ability of nodule protein extracts to bind the repressor binding domain (RBD). Through the use of mutated RBD sequences, the RBD sequence was identified as CTAAAAT. The repressor protein was isolated from M. loti NZP2037 by multiple chromatographic procedures and affinity separation by using concatemers of RBD attached to magnetic beads. Sequencing of the recovered protein resulted in identification of the repressor protein as the sarcosine oxidase alpha subunit. This was confirmed by expression of the gene encoding the M. loti alpha subunit of sarcosine oxidase in Escherichia coli. When the expressed peptide was bound to RBD, the gel retardation result was identical to the result obtained with rep2037 from M. loti strain NZP2037.