The recent discovery of obligate endophytic diazotrophs, like Acetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans colonizing sugar cane roots, stems and leaves, may finally explain the high contributions of BNF to this crop. A. diazotrophicus so far has only been isolated from sugar cane, sweet potatoe and Cameroon grass, all three with high sugar contents and propagated by stem cuttings. Azoarcus spp., another obligate endophytic genus, isolated from Kallar grass (Leptochloa fusca), has been found to establish within wetland rice roots when inoculated. Herbaspirillum seropedicae has been isolated from many graminaceous plants, forage grasses as well as from maize, sorghum and rice, but it does not seem to occur in other plant families. H. rubrisubalbicans so far has only been isolated from sugar cane roots, stems and leaves. These two species, even though clearly identified as two distinct species by DNA homology, are extremely similar and can only be distinguished by growth on n-acetyl-glucosamine (H. seropedicae) and that on meso-erythritol (H. rubrisubalbicans). Transmission of H. seropedicae may occur within seeds or via graminaceous weed plants because even high numbers of this organism inoculated into soil, do not survive for more than one month. Inoculation of endophytic diazotrophs, especially A. diazotrophicus is better achieved by the use of VA mycorrhizae together with bacterial cultures or with spores containing the bacteria. Spores of various VAM species were found to contain this organism and also A. lipoferum and Klebsiella sp. The infection of the root cap zone in micropropagated sugar cane plants with A.diazotrophicus and its colonization of xylem, has been confirmed by optical and electron microscope observations with immunogold-silver-enhanced techniques. Also Herbaspirillum spp. have been localized in the metaxylem after artificial inoculation of sugar cane and sorghum, using the same technique.

Endophytic Occurrence of Diazotrophic Bacteria in Non-Leguminous Crops

The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.

Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense.

Acetobacter diazotrophicus is a nitrogen-fixing bacterium that grows inside sugar cane plant tissue where the sucrose concentration is approximately 10%. The influence of high sugar content on nitrogenase was measured in the presence of oxygen and of nitrogen added in the form of ammonium and amino acids. In all parameters analyzed, 10% sucrose protected nitrogenase against inhibition by oxygen, ammonium, some amino acids, and also to some extent by salt stress. The oxygen concentration at which inhibition occurred increased from 2 kPa in 1% glucose or gluconic acid, to 4 kPa (0.4 atm) in 10% sucrose. Nitrogenase activity was partially inhibited by increased ammonium levels (2.0, 5.0, and 10.0 mM) in the presence of 1% sucrose, but the cells maintained their nitrogenase activity at 10% sucrose. This could be explained by the slow ammonium assimilation by the cells in the presence of high sucrose concentrations, i.e., independent of its concentration between 2 and 10 mM, the assimilation of ammonium was reduced to one-third in cells grown with 10% sucrose. Some amino acids were also tested in the presence of 1 and 10% sucrose. Cells grown in 1% sucrose had their nitrogenase activity reduced by 50–98% in the presence of glutamic acid, glutamine, alanine, asparagine, or threonine, whereas with 10% sucrose, nitrogenase activity was increased by glutamic acid and was reduced by only 61–73% by the other amino acids. The effect of NaCl concentrations (0.0, 0.25, 0.5, 0.75, or 1.0%) was also studied at the two concentrations of sucrose. Nitrogenase activity and growth of A. diazotrophicus, which was visualized by the pellicle formation in semi-solid medium, showed sensitivity even to low NaCl concentrations, which was somewhat relieved at the higher sucrose level. These observations indicate different osmotolerance mechanisms for sucrose and salt.

Effect of high sugar concentration on nitrogenase activity of Acetobacter diazotrophicus.

Gluconacetobacter diazotrophicus (47) (previously known as Acetobacter diazotrophicus [15]) is a strict aerobe and an N2-fixing endophyte originally isolated from sugarcane roots and stems (6). It has been estimated that G. diazotrophicus can fix up to 150 kg of N ha−1 year−1 in sugarcane (2). Such high levels of N2 fixation have not been reported in any other system outside legume-rhizobium symbioses. The bacterium has subsequently been isolated from sweet potato (38), coffee (23), pineapple (44), sorghum (22), finger millet (31), and several other tropical grass species (24).

Aerobic endophytic diazotrophs require a high flux of O2 to their respiratory systems to enable an adequate supply of reductant and ATP to support N2 fixation (e.g., see reference 13), yet paradoxically, an excessive flux of O2 to the bacterium can result in an inhibition of nitrogenase activity (14, 21, 26). The inhibition of nitrogenase activity by O2 in aerobic diazotrophs can be reversible or irreversible, depending on the organism and the nature (i.e., duration and severity) of the increase in O2 flux (33, 37, 39). Reversible inhibition of nitrogenase activity (i.e., a temporary “switch-off” of the nitrogenase activity while O2 flux is excessive) can be due to a conformational change in nitrogenase, as seen in Azotobacter (11, 32), to an ADP-ribosylation of dinitrogenase reductase, as seen in the purple nonsulfur bacteria (46) and Azospirillum (49), or to a diversion of electrons from nitrogenase to other reduction pathways, as proposed for Azotobacter (16, 29).

G. diazotrophicus has the ability to fix N2 at ambient atmospheric partial O2 pressures (pO2) (i.e., approximately 20 kPa of O2) in semisolid medium (6) and as colonies on solid medium (10). The ability to fix N2 in colonies on solid medium is especially interesting, as there is evidence that G. diazotrophicus exists in situ in the intercellular spaces of sugarcane vascular tissue as mucoid microcolonies (9). Dong (8) also reported that colony morphology on solid medium and the relative distribution of the bacteria within these highly mucilaginous colony changed with changes in the partial pressure of O2 surrounding the colonies.

Reis and Dobereiner (40) measured nitrogenase activity in liquid cultures of G. diazotrophicus by acetylene reduction in closed batch assays and found that activity was maximal when the culture was at equilibrium with 0.2 kPa of O2 in the gas phase. However, nitrogenase activity of G. diazotrophicus grown in colonies on solid medium in response to changes in atmospheric pO2 has not yet been well characterized. Given that G. diazotrophicus exists in situ as microcolonies adhering to plant cell walls (9), characterization of the response of the bacterium on solid medium to changes in atmospheric pO2 is particularly relevant.

The objective of our study was to characterize the effect of atmospheric pO2 on nitrogenase activity of G. diazotrophicus grown on solid medium using flowthrough gas exchange measurements. Treatments included long-term growth of the bacterium on a range of atmospheric pO2 (10 to 30 kPa) and subsequent rapid changes in atmospheric pO2 in small (5- to 10-kPa) and large (40-kPa) steps. We found that nitrogenase activity by G. diazotrophicus is adaptive to both short-term and long-term changes in atmospheric pO2 and that the bacterium has a switch-off/switch-on mechanism for protection of nitrogenase from rapid changes in atmospheric pO2.

Response of the endophytic diazotroph Gluconacetobacter diazotrophicus on solid media to changes in atmospheric partial O(2) pressure.

Most photosynthetic bacteria are capable of nitrogen fixation and the biochemistry and genetics of nitrogen fixation have been studied extensively in a few of these. Both the traditional, molybdenum-dependent, nitrogenase and the third alternative form of nitrogenase have been found in anoxygenic phototrophs and the properties of these are similar to those in heterotrophic nitrogen fixers. The nif genes for the molybdenum nitrogenase system and the anf genes for the alternative nitrogenase system have been isolated. Nitrogenase activity in some photosynthetic bacteria is regulated by reversible ADP-ribosylation at arg 101 of the dinitrogenase reductase protein, which prevents it from transferring electrons to the dinitrogenase protein. ADP-ribosylation of dinitrogenase is catalyzed by dinitrogenase reductase ADP-ribosyl transferase (DRAT) and the removal of ADP-ribose is performed by dinitrogenase reductase activating glycohydrolase (DRAG). These are encoded by the draT and draG genes, respectively.

The Biochemistry and Genetics of Nitrogen Fixation by Photosynthetic Bacteria

Many N2-fixing organisms can turn off nitrogenase activity in the presence of NH4 + and turn it on again when the NH4 +is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransfer-ase (DRAT). The system can be reactivated when NH4 + is exhausted, by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genus Azospirillum possess the DRAT and DRAG systems (A. lipoferum and A. brasilense), whereas A. amazonense in the same genus lacks DRAT and DRAG. A. amazonense responds to NH4 + but does not exhibit modification of dinitrogenase reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them into A. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function in Acetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N2 at a pH as low as 3.0. A. diazotrophicus does show a rather sluggish response to NH4 +. A level of about 10ΜM NH4 + is required to switch off the system. The response to NH4 + is influenced by the dissolved oxygen concentration (DOC) as has been reported for Azospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa O2 seems optimal for the response in A. diazotrophicus.

Control of nitrogenase in Azospirillum sp.

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Control of nitrogenase in Azospirillum sp.