A major 30.5-kb cluster of nif and associated genes of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus), a nitrogen-fixing endophyte of sugarcane, was sequenced and analyzed. This cluster represents the largest assembly of contiguous nif-fix and associated genes so far characterized in any diazotrophic bacterial species. Northern blots and promoter sequence analysis indicated that the genes are organized into eight transcriptional units. The overall arrangement of genes is most like that of the nif-fix cluster in Azospirillum brasilense, while the individual gene products are more similar to those in species of Rhizobiaceae or in Rhodobacter capsulatus.Biological nitrogen fixation occurs in species of more than 100 genera distributed among several of the major phylogenetic divisions of prokaryotes (Eubacteria and Archaea) (25). Sequence and mutational analyses of the genes necessary for nitrogen fixation (nif) in many diazotrophs indicate that their products have common structures and functions, while the degree of linkage and arrangement of specific nif and associated genes vary considerably (5,8,17). In addition, nif genes and genes involved in plant invasion and nitrogen fixation effectiveness, such as nod and fix in species of Rhizobiaceae, are often linked.The identification of nitrogen-fixing bacteria with endophytic habitats raises the possibility of a new classification of symbiosis (3). The relationship of a proteobacterial ␣ group member, Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus), with sugarcane represents a promising model system for the study of an association between a monocot and an endophytic nitrogen-fixing bacterium (13,22). The ability of A. diazotrophicus to enhance sugarcane growth has been documented, and while the benefit to plant growth might be due at least in part to the transfer of bacterially fixed N, another plant growth-stimulating factor(s) is indicated, possibly auxin production by A. diazotrophicus (22). Because of its potential agronomic use and unique status as the only diazotrophic species of Acetobacter so far identified, it was of interest to isolate and characterize genes that are involved in nitrogen fixation and regulation.Identification of a major cluster of nif and associated genes. A genomic library of A. diazotrophicus constructed in the widehost-range cosmid pLAFR3 (Tet r ) (4) was transferred by conjugation from Escherichia coli to several different nif mutants of Azotobacter vinelandii (12). Two mutants that yielded Nif ϩ Tet r transconjugants were DJ71 (nifV) and DJ35 (nifE), and several transconjugants of both strains carried the same cosmid, pAD71, the 22-kb insert fragment of which carries nifD through mcpA (Fig.

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