16S RIBOSOMAL-RNA-TARGETED POLYMERASE CHAIN-REACTION AND OLIGONUCLEOTIDE HYBRIDIZATION TO SCREEN FOR AZOARCUS SPP, GRASS-ASSOCIATED DIAZOTROPHS

Phylogenetic analyses after reverse transcriptase sequencing of 16S rR NA of nitrogen-fixing, grass-associated Azoarcus strains confirmed the ir affiliation to the beta subdivision of the Proteobacteria. Strains representing three different species formed a phylogenetically coheren t unit related to Rhodocyclus purpureus, with actual percent similarit ies among the three sequences ranging from 93.1 to 97.3%. Within varia ble regions V2 and V5, we found stretches of sequences considerably co nserved within the genus Azoarcus but differing from most other gram-n egative bacteria, with the specificity being enhanced when different r egions were combined. Genus-specific primers selected from both region s amplified fragments from all but one Azoarcus species in polymerase chain reactions (PCR) but not from any reference strain tested. Primer s of lesser specificity generated fragments from members of all five A zoarcus species as well as from some reference strains. Those unspecif ic amplifications could be differentiated by oligonucleotide hybridiza tion, detecting only fragments generated from Azoarcus strains except strain 6a3, which represents the same group which could not be detecte d by genus-specific PCR. Thus we propose the application of PCR amplif ication with 16S rRNA-targeted, genus-specific primers in combination with hybridization of a 16S rRNA-targeted oligonucleotide to PCR-gener ated fragments as diagnostic tests; this allows an initial screening f or presence of members of the genus Azoarcus.