IDENTIFICATION OF GRASS-ASSOCIATED AND TOLUENE-DEGRADING DIAZOTROPHS,AZOARCUS SPP, BY ANALYSES OF PARTIAL 16S RIBOSOMAL DNA-SEQUENCES

The genus Azoarcus includes nitrogen-fixing, grass associated strains as well as denitrifying toluene degraders. In order to identify and gr oup members of the germs Azoarcus, phylogenetic analysis based on part ial sequences of 165 rRNA genes (165 rDNAs) is proposed. 165 rRNA-targ eted PCR using specific primers to exclude amplification in the majori ty of other members of the beta subclass of the class Proteobacteria w as combined with direct sequencing of the PCR products. Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp. sequences and for analysis of the compl ete 165 rDNA sequence. These three species formed a phylogenetically c oherent group with representatives of two other Azoarcus species which were subjected to 165 rRNA sequencing in this study. This group was r elated to Rhodocyclus purpureus and Thauera selenatis. New isolates an d also sequences of so far uncultured bacteria from roots of Kallar gr ass were assigned to the genus Azoarcus as well. Also, strains degradi ng monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates . All representative members of the five:: species harboring rhizosphe ric bacteria were able to form N2O from nitrate and showed anaerobic g rowth on malic acid with nitrate but not on toluene. In order to visua lize different Azoarcus spp. by whole-cell in situ hybridizations, we generated 165 rRNA-targeted, fluorescent probes by in vitro transcript ion directly from PCR products which spanned the variable region V2. H ybridization was species specific for Azoarcus communis and Azoarcus i ndigens. The proposed scheme of phylogenetic analysis of PCR-generated 165 rDNA segments will facilitate studies on ecological distribution, host range, and diversity of Azoarcus spp. and may even allow rapid i dentification of uncultured strains from environmental DNAs.