Amplified ribosomal DNA restriction analysis for the characterization of Azotobacteraceae: a contribution to the study of these free-living nitrogen-fixing bacteria.
A 16S rRNA gene-based fingerprinting method was developed for the identification of Azotobacteraceae and tested onto 48 soil isolates and 28 reference strains belonging to the free-living nitrogen-fixing bacterial group and to the most common species found in soil samples. According to this method, the 16S rRNA gene was amplified using universal primers for Eubacteria and PCR products were subsequently digested with RsaI, HhaI, HpaII, FnuDII, and AluI. The analysis of the restriction profiles obtained showed that the method is able to define a unique species-specific phylotype (SSP) for each of the eight Azotobacteraceae species tested. Cluster analysis was successfully employed for the identification of members of the family Azotobacteraceae, being assignation into species of the isolates confirmed by means of partial 16S rRNA gene sequencing.
Aquilanti
Lucia
L
Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy. anmicro@univpm.it
Mannazzu
Ilaria
I
Papa
Roberto
R
Cavalca
Lucia
L
Clementi
Francesca
F
D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D020459:Genes, rRNA; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation; D010802:Phylogeny; D011548:Pseudomonadaceae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000235:genetics; D021521:Ribotyping; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D012988:Soil Microbiology
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D019003:Deoxyribonuclease HpaII; C024669:endodeoxyribonuclease AluI; C039566:CGCG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases; C040158:GTAC-specific type II deoxyribonucleases