Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – contains exogenous or...
Reexamination Certificate
1999-06-22
2002-11-05
Prouty, Rebecca E. (Department: 1646)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, contains exogenous or...
C435S069100, C435S183000, C435S252300, C435S320100, C435S410000, C536S023200, C800S278000, C800S295000
Reexamination Certificate
active
06475793
ABSTRACT:
TECHNICAL FIELD
This invention relates to a symbiotic plasmid of the broad host-range Rhizobium sp. NGR234 and its use. In particular, this invention relates to the isolation and analysis of the complete sequence of the NGR234 symbiotic plasmid pNGR234a, and the open reading frames (ORFs) identifiable therein as well as the proteins expressible from said ORFs.
BACKGROUND OF THE INVENTION
Together with carbon, hydrogen and oxygen, nitrogen is one of the essential components in organic chemistry. Although it is present in vast quantities in the atmosphere, nitrogen in its diatomic form N
2
remains unassimilable by living organisms. The nitrogen cycle begins by the fixation of nitrogen into ammonia which is chemically more reactive and can be assimilated into the food chain. A large fraction of the total nitrogen fixed every year is produced by microorganisms. Among these, the soil bacteria of the genera Azorhizobium, Bradyrhizobium, Sinorhizobium and Rhizobium, generally referred to as rhizobia, fix nitrogen in symbiotic associations with many plants from the Leguminosae family. This highly specific interaction leads to the formation of specialized root-, and in the case of Azorhizobium, stem-structures called nodules. It is within these nodules that rhizobia differentiate into bacteroids capable of fixing atmospheric nitrogen into ammonia. In turn, ammonia diffuses into the vegetal cells and sustains plant growth even under limiting nitrogen conditions.
The Rhizobium-legume interaction presents many interesting features. Obviously, the possibility of using this symbiosis as an “environmentally friendly” way to provide some of the most important world crops (such as soybean, bean and many other legumes) with fixed nitrogen without using nitrate-rich fertilizers, has important economic consequences. It is also an ideal model to study a non-pathogenic interaction between bacteria and a highly developed, multicellular organism such as the host plant. Furthermore, the various steps involved in the establishment of a functional nitrogen symbiosis, which include some dramatic morphological changes as well as processes of cellular differentiation, require a complex exchange of molecular signals. Despite many decades of studies, it is only recently that the Rhizobium-legume interaction has been partially understood at the molecular level. The establishment of a functional symbiosis can be divided into two major steps as follows.
(A) Rhizosphere Ecology and Modulation
Rhizobia are soil bacteria that proliferate in the rhizosphere of compatible plants, taking advantage of the many compounds released by plant roots. In return it has been shown that the presence of rhizobia in the rhizosphere reduces susceptibility of plants to many root diseases. In the case of low nitrogen levels in the soil, compatible rhizobia can interact with host plants and start the nodulation process (Long, 1989; Fellay et al., 1995; van Rhijn and Vanderleyden, 1995). Molecular signalling between the two partners begins with the release by the plant of phenolic compounds (mostly flavonoids) that induce the expression of nodulation genes (referred to as nod, nol and noe genes). The NodD1 gene product appears to be the central mediator between the plant signal and nodulation gene induction (Bender et al., 1988). It is modified by the binding of flavonoids and acts as a positive regulator on the expression of the remaining nodulation genes. Among them, the nodABC loci encode products responsible for the synthesis of the core structure of lipooligosaccharides called Nod factors (Relić et al., 1994). More nodulation genes are involved in strain-specific modifications of the Nod factors as well as in its secretion. It seems established now that variability in the structure of Nod factors may play a significant role in the determination of the host-range of a given Rhizobium strain, that is in its ability to efficiently nodulate different legumes. For example, the strain
Rhizobium meliloti
can only nodulate Medicago, Melilotus and Trigonella ssp., whereas Rhizobium sp. NGR234 can symbiotically interact with more than 105 different genera of plants, including the non-legume
Parasponia andersonii.
The structure of many Nod factors, their isolation from Rhizobium strains and their commercial application in agriculture have been described (NodNGR-Faktoren: Relić et al., 1994; WO 94/00466; NodRm-Faktoren: WO 91/15496). Secreted Nod factors act in turn as signal molecules that allow rhizobia to enter young root hairs of a host plant, and induce root-cortical cell division that will produce the future nodule. Invaginated rhizobia progress towards the forming nodule within infection threads that are synthesized by the plant cells. Bacteria are then released into the cytoplasm of dividing nodule cells where they differentiate into bacteroids capable of fixing atmospheric nitrogen.
With respect to regulation of the nodulation genes, other regulatory genes with similarities to nodD1 (genes that belong to the lysR family) have been identified in various strains (Davis and Johnston, 1990). The function of these genes, called nodD2, nodD3 or syrM, is only partially understood. Some nodD genes have been described (WO 94/00466; CA 1314249; WO 87/07910; U.S. Pat No. 5,023,180). Also, recombinant DNA molecules including the consensus sequence of the promoters of nodD1-regulated genes, called nod-boxes (Fisher and Long, 1993), have been disclosed (U.S. Pat. Nos. 5,484,718; 5,085,588). Finally, recombinant plasmids with the nodABC genes or, in one case (
Bradyrhizobium japonicum
), a sequence influencing host specificity have been disclosed (U.S. Pat. Nos. 5,045,461; 4,966,847).
(B) Symbiotic Nitrogen Fixation
Inside the nodules, rhizobia differentiate into bacteroids that express the enzymatic complex (nitrogenase) required for the reduction of atmospheric nitrogen into ammonia. The nitrogenase is encoded by three genes nifH, nifD and nifK which are well conserved in nitrogen fixing organisms (Badenoch-Jones et al., 1989). Many additional loci are necessary for functional nitrogenase activity. Those originally identified in
Klebsiella pneumoniae
are known as nif genes, whereas those found only in Rhizobium strains are described as fix genes (Fischer, 1994). Some of these gene products are required for the biosynthesis of cofactors, the assembly of the enzymatic complex or play regulatory and different accessory roles (oxygen-limited respiration, etc.). Many of these genes are less conserved among the various rhizobial strains and in some cases their function is still not fully understood. The high sensitivity of the nitrogenase complex to free oxygen requires a very strict control of most nif and fix gene expression. In this respect, the FixL, FixJ, FixK, NifA and RpoN proteins have been identified in representative Rhizobium species as the major regulatory elements that, in microanaerobic conditions, activate the synthesis of the nitrogenase complex (Fischer, 1994). Recombinant DNA molecules containing nif genes/promoters have been disclosed: nifH promoters of
B. japonicum
(U.S. Pat. No. 5,008,194), nifH and nifD promoter of
R. japonicum
(EP 164245), nifA of
B. japonicum
and
R. meliloti
(EP 339830), nifHDK and hydrogen-uptake (hup) genes of
R. japonicum
(EP 205071).
Many more genetic determinants play a significant role in the Rhizobium-legume symbiosis. Genes (exo, lps and ndv genes) involved in the production of extracellular polysaccharides (EPS), lipopolysaccharides (LPS) and cyclic glucanes of rhizobia play an essential role in the symbiotic interaction (Long et al., 1988; Stanfield et al., 1988). Mutation in these genes negatively influences the development of functional nodules. In this respect, some exopolysaccharides of the NGR234 derivative strain ANU280, have been disclosed (WO 87/06796). Although Nod factors seem to play a key role in the nodulation process, experimental data indicate that other signal molecules produced by the bacterial symbionts are required for functional symbiosis and may pla
Broughton William John
Freiberg Christoph Bernward
Perret Xavier Philippe
Rosenthal Andre
Andre Rosenthal
Prouty Rebecca E.
Rao Manjunath N.
Woodcock & Washburn LLP
LandOfFree
Genomic sequence of Rhizobium sp. NGR 234 symbiotic plasmid does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Genomic sequence of Rhizobium sp. NGR 234 symbiotic plasmid, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Genomic sequence of Rhizobium sp. NGR 234 symbiotic plasmid will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2965346