The Organization of the nif Operon

A DNA region containing putative nif genes and belonging to the endosymbiont Burkholderia has been identified and characterized (Minerdi et al. 2001). Screening of the library with Azospirillum brasilense nifHDK genes as the prokaryotic probes led to the identification of a 6413 bp region. Analysis revealed three open reading frames (ORFs) encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs. The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs.

PCR experiments with primers designed on the Burkholderia nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the Gi. margarita endosymbiont. RT-PCR experiments with primers designed on the Burkholderia nifD and nifK

genes and performed on total RNA extracted from germinated spores demonstrate the genes expression during this step of the fungal life cycle.

6. Concluding Remarks

The constant presence of endobacteria in some species of Gigasporaceae through the fungal life cycle and their pattern of distribution in AM species, the origin of which is geographically very far, open intriguing questions on the biology of this association and on the possibility of co-evolution events which have linked the two partners, as in other endosymbioses (Clark et al. 2000). The discovery of relevant genes in the genome of the endosymbiotic Burkholderia (i.e. nitrogen fixation, P-transporter genes) demonstrate that these bacteria possess the molecular mechanisms for playing an active role in the nutrient metabolism, adding a further level of complexity to the nutritional exchanges between plants and mycorrhizal fungi.

7. References

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Bianciotto V et al. (1996) Appl. Environ. Microbiol. 62, 3005-3010

Bianciotto V et al. (2000) Appl. Environ. Microbiol. 66, 4503-4509

Bianciotto V et al. (2001) Mol. Plant Microbe Interactions 14 (2), 255-260

Bonfante P, Perotto S (1995) New Phytol. 130, 3-21

Clark MA et al. (1997) Evolution Int. J. Org. Evolution 54, 517-25

Gianinazzi-Pearson V (2000) In Hoch B (ed), The Mycota ix. pp. 45-61, Springer-Verlag, Berlin

Gillis et al. (1995) Int. J. Syst. Bacterid. 45, 508-516

Harrison MJ (1999) Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 361-389

MaroldaCL (1999) Microbiol. 145, 1509-1517

Minerdi D et al. (2001) Appl. Environ. Microbiol. 67, 725-732

Mosse B (1970) Arch. Mikrobiol. 74, 146-159

Perotto S, Bonfante P (1997) Trends Microbiol. 5, 496-501

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Ruiz-Lozano M, Bonfante P (2000) Microb. Ecol. 39, 137-144

Scannerini S, Bonfante P (1991) In Margulis L, Fester R (eds), Simbiosis as Source of Evolutionary

Innovation: Speciation and Morphogenesis, pp. 273-287, The MIT Press, Cambridge, MA Simon L et al. (1993) Nature 363, 67-68

Smith SE, Read DJ (1997) Mycorrhizal Symbiosis, Academic Press, London, UK van Buuren, ML et al. (1999) Mycol. Res. 103, 955-970 van der Heijden MGA et al. (1998) Nature 396, 69-72

7. Acknowledgements

The research illustrated in this review has been funded by the EU GENOMYCA project, QLK5-CT-2000-01319, the Italian National Council of Research and the National Project Produzione Agricola nella Difesa dell'Ambiente (PANDA).

NITROGEN FIXATION AND INTERACTIONS WITH RICE BY AZOARCUS SP. STRAIN BH72

B. Reinhold-Hurek1, T. Hurek2, D.E. Martin1, A. Sarkar1, S. Wiese1

'Dept of General Microbiology, University Bremen, FB 2, PO Box 33 04 40, D-28334 Bremen, Germany

2Cooperation Laboratory Max-Planck-Institute for Marine Microbiology/ University Bremen, FB 2, PO Box 33 04 40, D-28334 Bremen, Germany

1. Introduction

Plant roots offer a variety of microhabitats for microbial colonization including the rhizosphere soil, the plant surface (rhizoplane) and inner root tissues. Plant-bacteria systems which have recently gained attention are associations between endophytic, nitrogen-fixing bacteria and grasses or cereals (Reinhold-Hurek, Hurek 1998). Well-studied examples are members of the beta-subclass of the Proteobacteria, Azoarcus spp. and Herbaspirillum seropedicae, or a member of the alpha-subclass, Gluconacetobacter diazotrophicus (formerly Acetobacter diazotrophicus). They infect Kallar grass and rice (Hurek et al. 1994), a wide range of grasses and cereals (James et al. 1997, 1998) or sugar cane and coffee plants (James et al. 1994; Jiminez-Salgado et al. 1997), respectively. Surprisingly, rhizobia have also been found to be natural endophytes of non-legumes, e.g. in rice (Yanni et al. 1997; Engelhard et al. 2000); other endophytes were detected in maize (Palus et al. 1996).

2. Plant Colonization

These diazotrophic endophytes have several features in common. Unlike rhizobia, they do not survive well in soil. In most cases they cannot be isolated from soil but only from plant material and are thus ecologically dependent on plants (James, Olivares 1998; Reinhold-Hurek, Hurek 1998). In gnotobiotic culture, it was demonstrated that pure cultures of these bacteria are capable of invading plant roots. Points of invasion are the zone of elongation and differentiation close to the root tip and the emergence points of lateral roots (Hurek et al. 1994; James et al. 1994, 1997). The colonization is mostly intercellular, only rarely intracellular plant cell colonization is observed. However, these plant cells are not viable (Hurek et al. 1994), and there is no evidence for an endosymbiosis in living plant cells (Reinhold-Hurek, Hurek 1998) in contrast to the rhizobium-legume symbiosis.

The major site of endophytic colonization appears to be the root cortex, especially in the aerenchyma of flood-tolerant plants such as Kallar grass (Leptochloa fusca L. (Kunth)) and rice, where large microcolonies of Azoarcus sp. BH72 can be found (Hurek et al. 1994; Egener et al. 1998). However, rarely these endophytes do also invade the stele and penetrate xylem vessels, which were previously thought to be sterile in healthy plants. Xylem colonization may facilitate the systemic infection of plants, the bacteria spreading from roots to shoots of young rice plants (Hurek et al. 1994; Gyaneshwar et al. 2000). For other diazotrophic endophytes (Heraspirillum spp., Gluconacetobacter diazotrophicus), xylem cells appear to be a more frequent colonization site (James, Olivares 1998; Olivares et al. 1997).

Despite the relatively dense colonization (up to almost 108 bacteria per g root dry weight for Azoarcus sp. BH72 in field-grown Kallar grass (Reinhold et al. 1986)), the endophytes do not cause symptoms of plant disease in their host plants. In contrast, in several plants they have been shown to promote plant growth (Hurek et al. 1994; Sevilla et al. 2001). Whether these growth responses are due to bacterial phytohormone production, nitrogen fixation or other mechanisms may vary with the system and is still under investigation.

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