Characterization of a Major Cluster ofnif,fix, and Associated Genes in a Sugarcane Endophyte,Acetobacter diazotrophicus

Abstract

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.

The fixABCX Genes in Rhodospirillum rubrum Encode a Putative Membrane Complex Participating in Electron Transfer to Nitrogenase

“…R. rubrum strains were routinely grown photoheterotrophically at 30°C in the minimal medium described by Ormerod et al (40) with 40 mM malate and 28 mM NH 4 Cl as the carbon and nitrogen sources, respectively (Nϩ). Diazotrophic growth was achieved by sparging nitrogen-free medium aseptically with 95% N 2 -5% CO 2 (NϪ) (31). nif-derepressing conditions were also achieved by growing R. rubrum with 27 mM glutamate as the nitrogen source.…”

History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience

“…The organization of the nif, fix and mop genes is known, however the mechanisms involved in their regulation still needs further study (Lee et al 2000). Because of its endophytic life style, this bacterium has been used as a vector to express heterologous genes of interest.…”

Analysis and Functional Annotation of an Expressed Sequence Tag Collection for Tropical Crop Sugarcane

“…Libraries were also generated from a mixture of calli tissues subjected to cold or heat treatment. Two libraries (AD1 and HR1) were constructed with plantlets inoculated with Gluconacetobacter diazotroficans or Herbaspirilum diazotroficans, both of which are endophytic nitrogen-fixing bacteria that colonize sugarcane tissues (Lee et al 2000). From each cDNA library, between 3000 and 14,000 clones were sequenced.…”