Luca Turbanti

Cloning of the histidine, pyrimidine and cysteine genes of Azospirillum brasilense: Expression of pyrimidine and three clustered histidine genes in Escherichia coli

SummaryA gene bank from Azospirillum brasilense, Sp6 strain, was constructed in Escherichia coli in plasmid pRK290 and was used to identify Azospirillum genes. Clones carrying the his1, his2, pyr and cys1 genes were identified by genetic complementation and the expression of A. brasilense his1, his2 and pyr genes in E. coli was demonstrated. By E. coli complementation experiments, a cluster of three genes for the histidine biosynthetic pathway in A. brasilense has also been found, suggesting the existence of an operon-like unit.

Cell-recycle batch fermentation using immobilized cells of flocculent Saccharomyces cerevisiae wine strains

Five, highly flocculeng strains of Saccharomyces cerevisiae, isolated from wine, were immobilized in calcium alginate beads to optimize primary must fermentation. Three cell-recycle batch fermentations (CRBF) of grape musts were performed with the biocatalyst and the results compared with those obtained with free cells. During the CRBF process, the entrapped strains showed some variability in the formation of secondary products of fermentation, particularly acetic acid and acetaldehyde. Recycling beads of immobilized flocculent cells is a good approach in the development and application of the CRBF system in the wine industry.

Mutants of Azospirillum brasilense resistant to methylammonium

One hundred and twenty-nine mutants of Azospirillum brasilense strain Sp6, resistant to methylammonium, were isolated. Three of the mutants were found to be able to reduce acetylene in the presence of 4 mM ammonium or 120mM methylammonium, concentrations which strongly reduced the nitrogenase activity of the parental strain. Under N2-fixing conditions, two mutants failed to switch off nitrogenase when NH4Cl was added. Moreover, the three mutants showed a reduced capacity to incorporate [14C]methylammonium. The level of glutamine synthetase activity found in the mutants was not reduced as compared to that of the parental strain. All of the data indicate an impairement in the mechanism of ammonium uptake by the bacterial cell.

Isolation and Characterization of a Glutathione-overproducing, Captan-resistant Mutant of Azospirillum brasilense

The fungicide Captan inhibited the growth of Azospirillum brasilense at a concentration of 25 μg ml-1. When cysteine and glutathione were added to the medium they removed the toxicity of the fungicide. A spontaneous mutant was isolated which was able to grow and fix nitrogen in the presence of 100 μg Captan ml-1. Characterization of the mutant indicated very high levels of glutathione and glutathione transferase activity as compared to the parent strain. The role of these cellular components in the mechanism of resistance to Captan is discussed; the involvement of a selenium-independent glutathione peroxidase appears essential to Captan resistance in the mutant strain.

Construction of a Gene Bank of Azospirillum Brasilense

A gene bank of DNA from A.brasilense, strain Sp6, has been constructed in the single EcoRI and BgIII sites of plasmid pRK290. The vector was able to be transferred by conjugation from E.coli to A.brasilense by using the helper plasmid pRK2013 at a frequency of about 10−3per recipient cell. Clones carrying his and pyr genes have been identified by genetic complementation; the expression of A.brasilense in E.coli has been demonstrated. Keywords: gene bank, cloning, conjugation.

Cloning of Azospirillum Brasilense Sequences Promoting Transcription in Escherichia Coli

Some genes of A.brasilense can be expressed in E.coli. We take advantage of this property for the isolation of A.brasilense promoter sequences. DNA fragment of A.brasilense SP6 were cloned into the BamHI site of plasmid pKK232–8 upstream the chloramphenicol resistance (CAT) gene which lacks its own promoter. Some of the Azospirillum DNA sequences selected for the ability to promote CAT gene transcription have been further characterized either for the level of resistance to chloramphenicol and for sequence analysis.