Diazotrophic bacteria were isolated, in two different years, from the rhizosphere and rhizoplane of coffee (Coffea arabica L.) plants cultivated in Mexico; they were designated as type DOR and type SAd isolates. They showed characteristics of the family Acetobacteraceae, having some features in common with Gluconacetobacter (formerly Acetobacter) diazotrophicus, the only known N2-fixing species of the acetic acid bacteria, but they differed from this species with regard to several characteristics. Type DOR isolates can be differentiated phenotypically from type SAd isolates. Type DOR isolates and type SAd isolates can both be differentiated from Gluconacetobacter diazotrophicus by their growth features on culture media, their use of amino acids as nitrogen sources and their carbon-source usage. These results, together with the electrophoretic mobility patterns of metabolic enzymes and amplified rDNA restriction analysis, suggested that the type DOR and type SAd isolates represent two novel N2-fixing species. Comparative analysis of the 16S rRNA sequences revealed that strains CFN-Cf55T (type DOR isolate) and CFN-Ca54T (type SAd isolate) were closer to Gluconacetobacter diazotrophicus (both strains had sequence similarities of 98.3%) than to Gluconacetobacter liquefaciens, Gluconacetobacter sacchari (similarities < 98%) or any other acetobacteria. Strain CFN-Cf55T exhibited low levels of DNA-DNA reassociation with type SAd isolates (mean 42%) and strain CFN-Ca54T exhibited mean DNA-DNA reassociation of 39.5% with type DOR isolates. Strains CFN-Cf55T and CFN-Ca54T exhibited very low DNA reassociation levels, 7-21%, with other closely related acetobacterial species. On the basis of these results, two novel N2-fixing species are proposed for the family Acetobacteraceae, Gluconacetobacter johannae sp. nov. (for the type DOR isolates), with strain CFN-Cf55T (= ATCC 700987T = DSM 13595T) as the type strain, and Gluconacetobacter azotocaptans sp. nov. (for the type SAd isolates), with strain CFN-Ca54T (= ATCC 70098ST = DSM 13594T) as the type strain.


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