Annamaria Grifoni

Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for microorganisms

We report the rapid generation of DNA probes for several Azospirillum strains. This method does not require any knowledge of the genetics and/or the molecular biology of the organism (genome) to be investigated. The procedure is based on the generation of random amplified polymorphic DNA (RAPD) fingerprints using primers with an embedded restriction site. The amplification product(s) peculiar to one strain or common to two or more strains can be purified, cloned, sequenced and used as molecular probes in hybridization experiments for the detection and identification of microorganisms. We have tested this methodology in the nitrogen‐fixing bacterium Azospirillum by amplyfing the total DNA extracted from several Azospirillum strains. We have used amplification bands with different specificity as molecular probes in hybridization experiments performed on amplified DNA. Results obtained have demonstrated the usefulness of this methodology for Azospirillum. Its use in microbial ecology studies as a general strategy to generate specific DNA probes is also discussed.

Phylogenetic Studies of the Genus Azospirillum

The 16S rDNA of seventeen Azospirillum strains, fourteen of which assigned to one of known A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum species, and other three of uncertain taxonomic collocation, was sequenced and analysed after polymerase chain reaction amplification, in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. Moreover the use of histidine biosynthetic genes and of anonymous sequences generated by RAPD technique to infer phylogenetic relationships among Azospirillum strains is also described. The relationships among the five species, as revealed by DNA sequence data, appeared to be congruent with other molecular and phenotypic results, showing that the genus Azospirillum constitutes a phylogenetically separate entity in which A. brasilense and A. lipoferum are very close species, whereas the species A. amazonense is the most divergent.

Cloning and Analysis of an Azospirillum brasilense Iteron and hslUV Operon Containing Region

A 3 kb DNA region from Azospirillum brasilense strain SPF94, located upstream of the histidine biosynthetic cluster, was cloned and analysed. The 5′ region of this sequence contained two putative ORF sharing a very high degree of sequence homology with the Escherichia coli hslUV heat-shock operon.

Phylogeny of the genus based on 16S rDNA sequence

The 16S rDNA of 17 strains of Azospirillum, 14 assigned to one of the known species A. amazonense A. brasilense A. halopraeferens A. irakense and A. lipoferum, and the other three of uncertain taxonomic position, was sequenced after polymerase chain reaction amplification and analysed in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. The phylogenetic analysis confirms that the genus Azospirillum constitutes a phylogenetically separate entity within the a subclass of Proteobacteria and that the five species are well defined. A. brasilense and A. lipoferum are closely related species and form one cluster together with A. halopraeferens; the pair of species A. amazonense and A. irakense forms a second cluster in which Rhodospirillum centenum is also placed.

Regulation of the nifHDK Genes Transcription in Azospirillum Brasilense

The regulation of transcription of the structural genes for nitrogenase of Azospirilium brasilense was investigated. The function of two regions controlling the transcription of the nifH gene was studied. Data obtained suggested a mechanism of nif regulation in Azospirillum different from that described in Klebsiella pneumonlae. The effect of different nitrogen sources both on nitrogenase activity and on the transcription of the nifHPK cluster was also studied.