Patterns of mitochondrial DNA instability in Brassica campestris cultured cells

We previously showed that the mitochondrial DNA (mtDNA) of a Brassica campestris callus culture had undergone extensive rearrangements (i.e. large inversions and a duplication) relative to DNA of the control plant [54]. In this study we observed that after continued growth, the mtDNA of this culture continues to change, with rearranged forms amplifying and diminishing to varying proportions. Strikingly similar changes were detected in the mtDNA profiles of a variety of other long- and short-term callus and cell suspension lines. However, the proportions of parental (‘unrearranged’) and novel (‘rearranged’) forms varied in different cultured cell mtDNAs. To address the source of this heterogeneity, we compared the mtDNA organization of 28 individual plants from the parental seed stock. With the exception of one plant containing high levels of a novel plasmid-like mtDNA molecule, no significant variation was detected among individual plants and therefore source plant variation is unlikely to have contributed to the diversity of mitochondrial genomes observed in cultured cells. The source of this culture-induced heterogeneity was also investigated in 16 clones derived from single protoplasts. A mixed population of unrearranged and rearranged mtDNA molecules was apprent in each protoclone, suggesting that the observed heterogeneity in various cultures might reflect the genomic composition of each individual cell; however, the induction of an intercellular heterogeneity subsequent to the protoplast isolation was not tested and therefore cannot be ruled out. The results of this study support our earlier model that the rapid structural alteration of B. campestris mtDNA in vitro results from preferential amplification and reassortment of minor pre-existing forms of the genome rather than de novo rearrangement. Infrequent recombination between short dispersed repeated elements is proposed as the underlying mechanism for the formation of these minor mtDNA molecules.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43428/1/11103_2004_Article_BF00017914.pd

Functional organization of the glnB-glnA cluster of Azospirillum brasilense.

The functional organization of the glnB-A cluster of Azospirillum brasilense, which codes for the PII protein and glutamine synthetase, respectively, was studied with the aid of lacZ fusions, deletion mapping, site-directed mutagenesis, and complementation. It was shown previously by mRNA mapping that the cluster contains two tandemly organized promoters, glnBp1 and glnBp2, of the sigma 70 and sigma 54 types, respectively, upstream of glnB and a third unidentified promoter upstream of glnA. Data obtained with lacZ fusions in the wild-type strain confirmed that cotranscription of glnBA and transcription of glnA alone were oppositely regulated by the cell N status. Quantification of promoter activities showed a high level of transcription from glnBp1p2 and a low level from glnAp under conditions of nitrogen limitation. The opposite situation prevails under conditions of nitrogen excess. As a consequence, PII polypeptide synthesis is increased under conditions of nitrogen fixation, which strongly suggests that PII plays an important role under these conditions. Null mutant strains of glnB, ntrB-ntrC, nifA, and point mutant strains in glnA were analyzed. NtrB and NtrC are not involved in the regulation of glnBA expression, in contrast to PII and glutamine synthetase. Glutamine synthetase probably acts by modulating the intracellular N status, and PII acts by modifying the properties of an unidentified regulator which might be a functional homolog of NtrC. In addition, a Nif- null mutant strain of glnB was characterized further. A Nif+ phenotype was restored to the strain by nifA from Klebsiella pneumoniae but not by nifA from A. brasilense. This mutant strain is not impaired in NifA synthesis, which is relatively independent of the growth conditions in A. brasilense. It is therefore most likely that PII is required for NifA activation under conditions of nitrogen fixation. Deletion mapping and site-directed mutagenesis showed glnAp was located within a 45-bp DNA fragment upstream of the mRNA start site, dissimiar to previously described consensus sites for sigma factors

Partial characterization of nif genes from the bacterium Azospirillum amazonense

Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A. brasilense. Our work suggests that A. brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A. amazonense. This is the first analysis revealing homology between A. brasilense nif genes and the A. amazonense genome. Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A. amazonense and related genes of A. brasilense and other bacteria. However, the A. amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes) differed from related regions of A. brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria. The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A. amazonense was shown. Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A. amazonense and can distinguish this species from A. brasilense

Regulation of 'nif' Gene Expression and Nitrogen Metabolism in 'Azospirillum'

The 'Azospirillum' genus comprises nitrogen-fixing bacteria which have been isolated from the roots of numerous grasses, including cereals, sugar cane and forage grasses. Among the five species known, 'Azospirillum brasilense' is the best documented in terms of genetics and regulation of nitrogen fixation and ammonia assimilation. Several key regulatory and structural genes have been characterized, including 'nifA', 'ntrBC', 'glnA', 'glnB' and 'rpoN'. Regulation of their expression has been studied at the free-living state and in association with wheat. A model of regulation is reported and the challenge of engineering nitrogen-fixing associations is also discussed

Regulation of nif gene expression and nitrogen metabolism in Azospirillum

International audienceThe Azospirillum genus comprises nitrogen-fixing bacteria which have been isolated from the roots of numerous grasses, including cereals, sugar cane and forage grasses. Among the five species known, Azospirillum brasilense is the best documented in terms of genetics and regulation of nitrogen fixation and ammonia assimilation. Several key regulatory and structural genes have been characterized, including nifA, ntrBC, glnA, glnB and rpoN. Regulation of their expression has been studied at the free-living state and in association with wheat. A model of regulation is reported and the challenge of engineering nitrogen-fixing associations is also discussed

Characterisation of the glnK-amtB operon and the involvement of AmtB in methylammonium uptake in Azorhizobium caulinodans

International audienceThis work reports the characterisation of the Azorhizobium caulinodans amtB gene, the deduced protein sequence of which shares similarity to those of several ammonium transporters. amtB is located downstream from glnK, a glnB-like gene. It is cotranscribed with glnK from an NtrC- and sigma54-dependent promoter. glnK and amtB insertion mutant strains have been isolated. Methylammonium uptake was assayed in these strains and in other mutant strains in which the regulation of nitrogen metabolism is impaired. Our data suggest that the AmtB protein is an ammonium transporter, which is mainly regulated by NtrC in response to nitrogen availability