Title: Materials and methods for the enhancement of effective root nodulation in legumes
Abstract: The subject invention relates to compounds and compositions which induce transcripts of the nolA gene in nitrogen-fixing bacteria, such as Bradyrhizobium japonicum. Novel bacterial strains which are insensitive too NolA, soil inoculants comprising NolA insensitive bacteria and/or nolA inducers, and methods of increasing nitrogen fixation in legumes are also disclosed.
Patent Number: 6,855,536 Issued on 02/15/2005 to Loh, et al.
| Inventors:
|
Loh; John T. (Knoxville, TN);
Stacey; Gary (Knoxville, TN)
|
| Assignee:
|
University of Tennessee Research Corp., Inc. (Knoxville, TN)
|
| Appl. No.:
|
909735 |
| Filed:
|
July 20, 2001 |
| Current U.S. Class: |
435/252.2; 435/244; 435/252.1; 435/253.6; 504/117 |
| Intern'l Class: |
A01N 063//00 |
| Field of Search: |
435/252.1,244,252.2,253.6
47/58.1
504/117
|
References Cited [Referenced By]
U.S. Patent Documents
| 4535061 | Aug., 1985 | Chakrabarty.
| |
| 5173424 | Dec., 1992 | Stacey.
| |
| 5432079 | Jul., 1995 | Johansen et al.
| |
| 5695541 | Dec., 1997 | Kosanke.
| |
| 5916029 | Jun., 1999 | Smith.
| |
| Foreign Patent Documents |
| 09224654 | Sep., 1997 | JP.
| |
| WO 99/27786 | Jun., 1999 | WO.
| |
Other References
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strains nodulating groundnut", Plant and Soil, 163:177-187, Kluwer
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two-component signal-transduction systems in Gram-positive bacteria," Mol.
Microbiol. 24(5):895-904, Blackwell Science Ltd.
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Rosemeyer et al. [1998] "luxl- and luxR-Homologous Genes of Rhizobium etli
CNPAF512 Contribute to Synthesis of Autoinducer Molecules and Nodulation
of Phaseolus vulgaris," J. Bacteriol. 180(4):815-821, American Society for
Microbiology.
Sadowsky et al. [1991] "The Bradyrhizobium japonicum noIA gene and its
involvement in the genotype-specific nodulation of soybeans," Proc. Natl.
Acad Sci. USA 88:637-641.
Thome and Williams [1999] "Cell Density-Dependent Starvation Survival of
Rhizobium leguminosarum bv. phaseoli: Identification of the Role of an
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Bacteriol. 181(3):981-990, American Society for Microbiology.
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Chloroform Soluble and is not Required for Effective Nodulation," J.
Batceriol. 162(3):1079-1082, American Society for Microbiology.
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strains nodulating groundnut", Plant and Soil, 163:177-187, Kluwer
Academic Publishers, Netherlands.
Wijffelman et al. [1983] "Repression of Small Bacteriocin Excretion in
Rhizobium Leguminosarum and Rhizobium trifolii by Transmissible Plasmids,"
Mol. Gen. Genet. 192: 171-176, Springer-Verlag.
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Bradyrhizobium japonicum by Organic Acids," Mol. Plant-Microbe Interact.
9(5):424-428, The American Phytopathological Society.
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japonicum,"Mol. Gen. Genet. 214:420-424, Springer-Verlag.
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Int11(11):1119-1129, The American Phytopathological Society.
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Rhizobium leguminosarum Biovar viciae," J. Bacteriol. 174-4026-4035,
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Bradyrhizobium japonicum," Mol. Plant-Microbe Interact. 7(65):596-602, The
American Phytopathological Society.
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repression of the nodABC operon," Mol. Microbiol. 27(5):1039-1050,
Blackwell Science Ltd.
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Family of Cell Density-Responsive Transcriptional Regulators," J.
Bacteriol. 176(2):269-275, American Society for Microbiology.
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Activates Agrobacterium Ti Plasmid Conjugal Transfer in the Presence of a
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the noIA gene of Bradyrhizobium japonicum," Mol. Plant-Microbe Interact
9(7):625-635, The American Phytopathological Society.
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NC92 Contains Two nodD Genes Involved in the Repression of nodA and a noIA
Gene Required for the Efficient Nodulation of Host Plants," J. Bacteriol.
178(10):2757-2766, American Society for Microbiology.
Gray et al. [1996] "Cell-to-Cell Signaling in the Symbiotic Nitrogen-Fixing
Bacterium Rhizobium leguminosarum: Autoinduction of a Stationery Phase and
Phizosphere-Expressed Genes," J. Bacteriol. 178(2):372-376, American
Society for Microbiology.
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dependent regulation of gene expression innpathogenic and non-pathogenic
bacteria," Antonie van Leeuwenhoek 74:199-210, Kluwer Academic Publishers,
Netherlands.
Rosemeyer et al. [1998] "luxi- and luxR-Homologous Genes of Rhizobium etli
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of Phaseolus vulgaris," J. Bacteriol. 180(4):815-821. American Society for
Microbiology.
|
Primary Examiner: Lankford, Jr.; Leon B.
Attorney, Agent or Firm: Saliwanchik, Lloyd & Saliwanchik
Goverment Interests
The subject invention was made with government support under a research
project supported by The National Science Foundation Grant No. IBN-972828
1. The government may have certain rights in this invention.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to United States Provisional Application
60/219,509, filed Jul. 20, 2000, hereby incorporated by reference in its
entirety, including the disclosure, figures, and tables.
Claims
We claim:
1. A method of more efficiently nodulating a target plant species
comprising producing a nodulation inoculant comprising culturing at least
one strain of Bradyrhizobium japonicum in a liquid medium wherein the
medium comprises a sufficient amount of iron to reduce the amount of Cell
Density Factor (CDF) expression and then applying said inoculant to a
target plant, parts of a target plant, or seeds thereof.
2. The method according to claim 1, wherein said at least one strain is
Bradyrhizobium japonicum USDA 110.
3. The method according to claim 1, wherein said at least one strain is
Bradyrhizobium japonicum USDA 123.
4. A method of more efficiently nodulating a target plant species
comprising the steps of:
a) producing a nodulation inoculant by culturing at least one strain of
Bradyrhizobium japonicum in a liquid medium wherein the medium comprises a
sufficient amount of iron to reduce the amount of Cell Density Factor
(CDF) expression;
b) incorporating said nodulation inoculant into soil; and
c) and then applying seed or plant material of said target plant to said
soil.
5. The method according to claim 4, wherein said at least one strain is
Bradyrhizobium japonicum USDA 110.
6. The method according to claim 4, wherein said at least one strain is
Bradyrhizobium japonicum USDA 123.
7. A method of improving the efficiency of soil inoculation comprising
producing a nodulation inoculant by culturing at least one strain of
Bradyrhizobium japonicum in a liquid medium wherein the medium comprises a
sufficient amount of iron to reduce the amount of Cell Density Factor
(CDF) expression, applying said inoculant soil, and growing plants in said
soil.
8. The method according to claim 7, wherein said at least one strain is
Bradyrhizobium japonicum USDA 110.
9. The method according to claim 7, wherein said at least one strain is
Bradyrhizobium japonicum USDA 123.
Description
BACKGROUND OF THE INVENTION
Leguminous plants, such as soybeans, are able to fix nitrogen from the
atmosphere due to a symbiotic relationship between the plants and bacteria
which dwell in nodules formed in the roots of the plants. Specifically,
soil bacteria that are members of the family Rhizobiaceae, are capable of
infecting plants and inducing highly differentiated root nodule structures
within which atmospheric nitrogen is reduced to ammonia by the bacteria.
The host plant utilizes the ammonia as a source of nitrogen. The symbiotic
root nodule bacteria are classified in several separate genera, including
Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium.
Legume nodulation by rhizobia exhibits some species specificity.
Bradyrhizobium species include the commercially important soybean
nodulating strains B. japonicum (i.e., strains USDA 110 and 123),
promiscuous rhizobia of the cowpea group, and B. parasponia (formerly
Parasponia rhizobium) which nodulates the non-legume Parasponia, as well
as a number of tropical legumes, including cowpea and siratro. The most
important agricultural host of B. japonicum is soybean (Glycine max), but
this bacterium will nodulate a few other legumes (e.g., cowpea and
siratro). Fast growing rhizobia include, among others, Rhizobium etli,
Sinorhizobium meliloti (formerly Rhizobium meliloti), and Rhizobium
leguminosarum biovar trifolii, which nodulate bean, alfalfa, and clover,
respectively. These Rhizobium species generally display a narrow host
range. However, Rhizobium sp. NGR234 has the ability to nodulate over 100
genera of legumes. Sinorhizobium fredii (formerly Rhizobium fredii), is
phylogenetically distinct from B. japonicum, but has the ability to
nodulate Glycine soja (a wild soybean species), G. max cv. Peking, and a
few other soybean cultivars.
There are currently about 70,000,000 acres of soybean grown in the United
States. An inoculant industry exists to sell B. japonicum to farmers for
incorporation into the soil during soybean planting. The use of these
inoculants is intended to enhance the efficiency of nitrogen fixation.
Unfortunately, for most of the United States, inoculation has been shown
to be ineffective. Therefore, the inoculant industry remains relatively
small (approximately $20-30 million per year). Indeed, at present,
inoculation is only recommended for newly planted fields (i.e., those not
planted with soybeans previously) and fields that have been out of
production for over three years.
The primary reason for the inefficiency of soil inoculation is the presence
of competing extant B. japonicum in soil. When a field has been producing
soybean for more than one season, there is a build up of the B. japonicum
populations in soil. These bacteria are highly competitive since they have
adapted to their soil environment. Hence, when the inoculant is added, the
indigenous soil B. japonicum strains compete and win the battle to
nodulate the plant. The result is that, in many cases, less than 1% of the
nodules formed on the planted soybean are due to the inoculant added.
Therefore, even if a high-yielding B. japonicum strain is used as the
inoculant, the farmer does not see the yield increase due to the fact that
the inoculant has not found its way into the plant.
In the major soybean growing areas of the Midwest, the most competitive
population of B. japonicum is that of serogroup 123. If improvement in the
nitrogen fixing capacity of the soybean-Bradyrhizobium symbiosis through
application of superior strains is to be realized, then the difficult
problem of competition from indigenous populations (such as serogroup 123)
will have to be solved.
Significant efforts have been made to understand and alter the
competitiveness of indigenous Bradyrhizobia. For example, attempts to
alter soybean nodule occupancy ratios of indigenous versus applied
Bradyrhizobia have been reported. However, such alterations were only
achieved by using ultra-high, economically infeasible rates of the applied
strain. In a seven year study, Dunigan et al. [Agron. J. 76: 463-466
(1984)] demonstrated that the inoculant strain USDA 110 eventually formed
the majority of nodules after high rates of application in the first 2
years (serogroup 123 was not among the indigenous population). However,
the tenacious competitive ability of serogroup 123 appears not to be
related to numbers per se and when normal rates of inoculant are applied
the indigenous serogroup 123 population can still form up to 95% of the
nodules on soybean.
The formation of nodules on leguminous plants involves a complex exchange
and recognition of diffusible signals between the plant and the bacterial
symbiont. A key plant signal are the flavonoids which trigger the
induction of the bacterial nodulation genes (Day et al. [2000] In:
Prokaryotic Nitrogen Fixation: A Model System for the Analysis of a
Biological Process, ed. Triplett, E., Horizon Scientific Press, Norfolk,
England, pp 385-414).
Nodulation genes of Bradyrhizobium and Rhizobium strains affect the early
stages of nodule formation including host-bacterium recognition, infection
and nodule development. Wild type strains of Bradyrhizobium species
display some variation in these early nodulation steps which is reflected
in differences in relative rates of initiation of nodulation and
ultimately in differences in competitiveness between strains for nodule
occupancy. For example, B. japonicum USDA 123 is believed to be more
competitive for nodulation than B. japonicum USDA 110. Strains which
initiate infection and nodules earlier will occupy a greater portion of
the nodules on a given plant. Improving the competitiveness of a specific
Bradyrhizobium is an important part of the development of improved
inoculants for legumes. A more effective Bradyrhizobium strain must be
able to out-compete the indigenous rhizobia population for nodule
occupancy in order for their improved qualities to impact on the
inoculated legume. Therefore, there is a significant need for an
inoculating composition and/or an inoculating method which would improve
competitiveness of a selected inoculant strain.
In the Bradyrhizobium japonicum-soybean symbiosis, several key regulatory
components have been identified in the regulation of bacterial nodulation
genes. Two of these, i.e., a LysR regulator, NodD.sub.1 and a two
component regulatory system, NodWV are known to positively activate the B.
japonicum nodulation genes in response to the plant produced
isoflavonoids, genistein and daidzein. A third regulatory component (i.e.,
NolA) is a MerR type regulator (Sadowsky et al. [1991] Proc. Natl. Acad
Sci. USA 88:637-641) that possesses the unique capacity to exist in three
functionally distinct forms (i.e., NolA.sub.1, NolA.sub.2 and NolA.sub.3)
(Loh et al. [1999] J. Bacteriol . 181:1544-1554). These polypeptides are
derived from alternative translation of three in-frame initiation codons.
Induction of the B. japonicum nolA gene leads to the subsequent repression
of the nodulation genes in this bacterium. The products of the nodulation
genes are required for soybean nodulation. Thus, these plant compounds, by
inducing nolA expression, lead eventually to an inhibition of nodulation.
NolA.sub.1 is required for the expression of both NolA.sub.2 and
NolA.sub.3. Two transcriptional (P1 and P2) start sites have been
identified (Loh et al. [1999] J. Bacteriol. 181:1544-1554). Transcription
from P1 results in the formation of an mRNA encoding NolA.sub.1.
NolA.sub.1 then regulates transcription from P2, resulting in the
expression of both NolA.sub.2 and NolA.sub.3.
Although NolA is involved in the negative control of the nodulation genes
(Dockendorff, T. C., J. Sanjuan, P. Grob, and G. Stacey [1994] Mol.
Plant-Microbe Interact. 7:596-602), current information suggests that NolA
does not act directly to repress nod gene expression. This view is
supported by the observation that while expression of NolA from a
multicopy plasmid resulted in a reduction of nod gene expression,
interposon mutations to the nolA gene did not lead to elevated levels of
nod gene expression (Garcia, M. L., J. Dunlap, J. Loh, and G. Stacey
[1996] Mol. Plant-Microbe Interact 9:625-635). In fact, NolA appears to
positively regulate the expression of NodD.sub.2, the latter of which has
been shown to be a repressor of the nod genes in Rhizobium spp. NGR234,
Bradyrhizobium spp. (Arachis) NC92 and Bradyrhizobium japonicum (Garcia,
M. L., J. Dunlap, J. Loh, and G. Stacey [1996] Mol. Plant-Microbe Interact
9:625-635; Gillette, W. K. and G. H. Elkan [1996] J. Bacteriol .
178:2757-2766; and Fellay, R., M. Hanin, G. Montorzi, J. Frey, C.
Freiberg, W. Golinowski et al. [1998] Mol. Microbiol. 27:1039-1050.
Therefore, NolA affects repression indirectly, through the control of
nodD.sub.2 expression.
Cell-cell signaling plays a large role in the ability of bacteria to
respond and adapt to a particular environment. Regulatory systems that
control gene expression in response to population density (i.e., quorum
sensing) govern such bacterial phenotypes as bioluminescence, antibiotic
production, plasmid conjugal transfer and the synthesis of virulence
factors in both plant and animal pathogens (Hardman, A. M. et al. [ 1998]
Antonie van Leeuwenhoek 74:199-210). Quorum sensing involves the
recognition of self-produced signal compounds, which function to regulate
the expression of genes when threshold levels of these signals have
accumulated in cultures of a sufficiently high population density. hi
Gram-negative bacteria, the best studied of these signals are N-Acyl
homoserine-lactones (AHL) (Fuqua, W. C. et al. [1994] J. Bacteriol
176:269-275). In Gram-positive bacteria, an equivalent role is played by
various posttranslationally-modified peptides (Kleerebezem, M. et al.
[1997] Mol. Microbiol. 24:895-904). Several AHL compounds have been
identified from rhizobia, including Rhizobium leguminosarum biovars
viciae, trifoli and phaseoli, Rhizobium etli, and Rhizobium meliloti
(Thorne and Williams [1999] J. Bacteriol. 181:981-990; Cha et al. [1998]
Mol. Plant Microbe Int. 11:1119-1129; Gray et al. [1996] J. Bacterial.
178:372-376; Rosemeyer et al. [1998] J. Bacteriol. 180:815-821; VanBrussel
et al. [1985] J. Bacteriol. 162:1079-1082; and Wijffelman et al. [1983]
Mol. Gen. Genet. 192:171-176). In a few cases, these autoinducers have
been implicated in the nodulation process. For example, the small AHL
molecule produced by R. leguminosarum by. viciae is required for the
expression of the rhiABC operon, which is important for rhizosphere growth
and nodulation efficiency (Cubo et al. [1992] J. Bacteriol.
174:4026-4035). In R. etli, mutations that disrupt AHL synthesis resulted
in decreased nodule numbers on host plants (Rosemeyer et al. [1998] J.
Bacteriol. 180:815-821). Therefore, AHL-mediated quorum sensing may play
an important role in the symbiotic process. To date, no quorum-sensing
compound has been identified from the soybean symbiont Bradyrhizobium
japonicum.
The current invention addresses the inefficiency of soil inoculation due to
the presence of competing indigenous B. japonicum in soil and provides
novel compounds and compositions which increase the efficiency of
nodulation in target plants. Specifically, field inoculants comprising
high-yielding NolA insensitive B. japonicum and nolA inducers address the
long standing obstacle of inefficient nodulation due to indigenous B.
japonicum strains.
BRIEF SUMMARY OF THE INVENTION
The subject invention provides materials and methods to improve nitrogen
fixation in leguminous plants. In a preferred embodiment of the subject
invention, the improvement in nitrogen fixation is achieved by providing
an inoculant of nitrogen-fixing bacteria which, when applied according to
the subject invention, have a competitive advantage over indigenous
strains.
In a specific embodiment, the subject invention provides compounds and
compositions which induce transcription of the nolA gene in
nitrogen-fixing bacteria, such as Bradyrhizobium japonicum. By applying
these NolA inducers to the situs of indigenous B. japonicum it is possible
to induce transcription of the nolA gene in indigenous bacteria, thereby
reducing the ability of these bacteria to initiate nodulation.
A further aspect of the subject invention is the identification of novel
bacteria which are insensitive to NolA. In a preferred embodiment of the
subject invention, these NolA insensitive microbes can be applied to
legumes in conjunction with the NolA inducers of the subject invention.
The NolA inducers inhibit the indigenous bacteria but do not adversely
affect the nodulation capabilities of the NolA insensitive (NolA.sup.INS)
inoculant bacteria. This gives the inoculant bacteria a competitive
advantage compared to the indigenous bacteria.
A further aspect of the subject invention relates to nucleic acids,
expression cassettes, and vectors which encode the NolA inducer compounds
of the subject invention. These genetic materials can be used to
efficiently produce the inducer compounds. The inducer compounds can be
produced in recombinant hosts including plants. Thus, one aspect of the
subject invention concerns plants having polynucleotides which encode
compounds which induce transcription of the nolA gene in nitrogen-fixing
bacteria.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1--HPLC reverse phase chromatography (C18) of soybean seedlings
extract (SSGE) fractions. Peaks collected as IND-1 and IND-2 were active
on B. japonicum nolA-lacZ fusions.
FIG. 2A--Structure of IND-1. FIG. 2B-Effect of phthalic acid
bis-(2-ethyl-hexyl) ester on nolA expression. B. japonicum cells harboring
a nolA-lacZ fusion were treated with increasing concentrations of the
phthalate derivate, and the level of nolA expression determined.
FIG. 3--Effect of phosphatidyl inositol extract on nolA expression.
Phosphatidyl inositol samples were either treated or untreated with 100
.mu.g/ml chitinase (Sigma Chemical Co.).
FIG. 4--Analysis of soybean phosphatidyl-inositol extracts. FIG. 4A-Reverse
phase comparison of extracts that had been untreated or treated with
chitinase (100 .mu.g/ml). FIG. 4B--Effect of chitinase on the ability of
peak 9 to induce nolA expression.
FIG. 5-FIG. 5A depicts population density dependent expression of
nolA.sub.1,2,3 -lacZ and nodD.sub.2 -lacZ. B. japonicum cultures harboring
either nolA.sub.1,2,3 -lacZ, nodD.sub.2 -lacZ or npt-lacZ were grown to
various population densities and the .beta.-galactosidase activity of
these fusions determined. Percent maximum activity is
[.beta.-galactosidase activity/maximal .beta.-galactosidase activity of
fusion].times.100%. FIG. 5B--Inducer of nolA expression is population
density dependent. Conditioned medium was obtained from B. japonicum
cultures grown to various population densities and used to induce a B.
japonicum strain harboring a nolA.sub.1,2,3 -lacZ fusion. Standard
deviation was less than 10%.
FIG. 6--The inducibility of nod gene expression as a function of initial
population density.
FIG. 7--Comparison of nodY-lacZ expression in a USDA110 and BjB3 (nolA
mutant). B. japonicum cultures were grown to various population densities
and the ability of 0.05 .mu.M genistein to induce nodY expression
determined. The fold induction is presented. The uninduced levels of
nodY-lacZ expression in USDA110 and BjB3 were 4.+-.1 and 3.+-.1,
respectively. Standard deviation was less than 10%.
FIG. 8--Effect of IND-1 on genistein induction of a nodY-lacZ expression in
B. japonicum. B. japonicum cells harboring a nodY-lacZ fusion were
incubated with increasing amounts and the ability of this compound to
affect nod gene expression determined.
FIG. 9--Effect of IND-1 on nolA.sub.1,2,3, nolA.sub.1, nolA.sub.2 and
nolA.sub.3 expression.
FIG. 10--Effect of quorum factor (i.e., conditioned medium) and IND-1 on
the ability of B. japonicum strain USDA110 to nodulate soybean. B.
japonicum cells were untreated (left) or incubated in conditioned medium
or IND-1 for 1 h, and then inoculated onto soybean plant (107 cells per
root). The number of nodules (.+-.standard error) was determined 21 days
post-inoculation, both above the mark (i.e., upper zone), or below the
mark (new tissue) at the time of inoculation (n=number of plants per
treatment).
FIG. 11A-C--The expression of nodD.sub.2 -lacZ and nolA-lacZ fusions as a
function of B. japonicum culture density was examined (FIG. 11A).
NolA.sub.1 expression is cell-density dependent and required for
NodD.sub.2 expression (FIG. 11B). The ability of the conditioned medium to
induce the nolA fusions was population density dependent with little or no
induction of the fusions observed using conditioned medium derived from
cultures of A.sub.600 <0.2 (FIG. 11C).
FIG. 12--HPLC isolation of Cell Density Factor (Quorum Factor) from B.
japonicum conditioned medium (concentrated approximately 10-fold). Quorum
factor containing material was applied to a C18 column (Phenomenex, Inc.,
Torrance, Calif.) and eluted with a methanol gradient (0-100%) at a flow
rate of 1 mL per minute. Cell density factor was demonstrated to be a
potent inducer of nolA expression.
FIG. 13--FIG. 13 provides a graphical depiction of the invention.
FIGS. 14A-B illustrate the effect of FeCl.sub.3 on nolA-lacZ expression. B.
japonicum cells harboring a nolA-lacZ fusion were treated with increasing
concentrations of FeCl.sub.3 for five hours and the level of nolA
expression was determined (FIG. 14A). FIG. 14B demonstrates an increase in
the expression of a nodY-lacZ fusion protein when cells are grown in the
presence of iron. B. japonicum cells containing a nodY-lazZ fusion were
induced for five hours with 0.025 .mu.M genistein in the presence or
absence of 500 .mu.M FeCl.sub.3.
FIG. 15 shows the effect of bis-(2-ethyl-hexyl) ester phthlate (BEHP) on
nodule occupancy by the NwsB mutant. Different ratios of B. japonicum
USDA110 and B. japonicum NwsB mutant were innoculated on soybean plants
grown in growth pouches. At the time of innoculation, the root tip (RT)
mark was noted on the outside of the pouches. Nodules were extracted 21
days post innoculation and the extracts were plated on RDY plates. Single
colonies were picked and tested for streptomycin resistance (a marker for
the NwsB mutant). (A) is above RT at time of innoculation; (B) below RT at
time of innoculation.
FIG. 16 shows a mutant selection scheme for the isolation of B. japonicum
mutants that nodulate in the presence of inhibitory concentrations of
BEHP.
FIGS. 17-18 illustrates the expression of CDF or quorum factor-like
molecules in a variety of other bacteria.
DETAILED DISCLOSURE OF THE INVENTION
The subject invention provides materials and methods for promoting the
growth of leguminous plants by enhancing the efficiency of root nodulation
by nitrogen-fixing bacteria. This enhancement of nodulation efficiency is
achieved by providing high-performing inoculant bacteria with a
competitive advantage over indigenous bacteria.
Although indigenous bacteria are typically excellent competitors for
forming root nodules, they are typically less efficient at
nitrogen-fixation than inoculant bacteria. Therefore, in order for the
inoculant bacteria to be capable of exerting their excellent
nitrogen-fixing effects, they must first be able to out-compete the
indigenous bacteria in order to form root nodules. Advantageously, the
subject invention provides materials and methods which enable the
inoculant bacteria to establish root nodules, even in the presence of
indigenous bacteria.
In one aspect, the present invention provides isolated novel compounds
which induce transcription of the nolA gene. These compounds are,
collectively, referred to as nolA inducers. In soybean extracts, HPLC
analysis of the compounds revealed at least two active compounds, referred
to herein as IND-1 and IND-2. IND-1 has been identified as phthalic acid
bis-(2-ethyl-hexyl) ester and is able to induce nolA . IND-1 has been
identified as a contaminant of solvents used in the extraction process;
however, phthalic acid bis-(2-ethyl-hexyl) ester is a potent inducer of
nolA . IND-2 is a plant-produced NolA inducer that can be isolated
according to the methods disclosed herein.
In addition to the plant-derived NolA inducers, the instant invention also
provides isolated novel compounds produced by B. japonicum which induce
nolA expression. These novel compounds may also be referred to as
bacterial nolA inducers. The bacterial nolA inducer appears to be produced
in a density dependent manner in batch culture and may be referred to as a
"quorum sensing" molecule or cell density factor (CDF). Quorum sensing
molecules regulate the expression of genes, such as nolA , in response to
bacterial population density. The bacterial nolA inducer is insensitive to
heat treatment and appears to have a molecular weight of less than 3,000
Da.
Compositions comprising one or more nolA inducers and a carrier are also
taught according to the subject invention. NolA inducers include chemical
compounds, plant-derived NolA inducers, and bacterial-derived NolA
inducers. By way of example, compositions having a NolA inducer include
commercially available soybean phosphatidyl inositol extracts, conditioned
medium obtained from cultured B. japonicum, commercially available soybean
extracts, or compositions having IND-1 (or isomers, analogs, or homologs
thereof), IND-2, or CDF. The compositions may, optionally, include one or
more NolA.sup.INS mutants.
Carriers useful in formulation of the compositions of the invention are
well known to those skilled in the art and include those described in
detail in a number of sources which are well known and readily available
to those skilled in the art. Also contemplated as carriers are
agricultural materials such as soil additives. Non-limiting examples of
such additives include peat, soil conditioners, chemical fertilizers, and
organic fertilizers (such as chicken or cow manure).
The present invention also provides bacterial cells which are insensitive
to the effects of the nolA inducers. These bacterial cells are referred to
as NolA.sup.INS mutants. An exemplary NolA.sup.INS mutant has been
isolated and will be deposited with the American Type Tissue Culture
[10801 University Blvd., Manassas, Va. 20110-2209].
Other NolA.sup.INS mutants include bacterial cells in which the gene or
genes encoding the nolA inducer has been inactivated. Inactivation of the
gene or genes encoding nolA inducers may be accomplished by deletion of
all, or a portion, of the gene or genes encoding the nolA inducer,
insertion of nucleic acid sequences within gene or genes encoding the nolA
inducer or inactivation of transcriptional control sequences operably
linked to nolA inducers. Alternatively, the nolA inducer gene may be
inactivated by mutation or deletion of ribosome binding sites. Mutation or
deletion of translation initiation sites may also be used to inactivate
the nolA gene. Methods of site directed mutagenesis in Gram negative
bacteria, such as Rhizobia, are well known to those skilled in the art.
NolA insensitive strains can be isolated using a variety of selection
procedures. For example, since NolA inducers inhibit nodulation, one can
select for NolA insensitive B. japonicum mutants by inoculating plants
with a mutated population in the presence of the NolA inducer (e.g.,
IND-1, IND-2, or CDF, or quorum sensing factor). Bacteria isolated from
nodules that form rapidly on the soybean roots would be presumptive
mutants that were insensitive to the inhibitory effects of the nolA
inducers. These mutants could then be confirmed by directly testing the
ability of the inducers to activating transcription of nolA (e.g., using
either Northern hybridization or measuring nolA-lacZ expression).
Similarly, since nolA expression increases with culture age, plating of
mutated B. japonicum cells (containing the nolA-lacZ fusion) on medium
containing X-GAL (5-bromo-4-chloro-3- indolyl-.beta.-D-galactoside) allows
one to distinguish the blue, NolA expressing, and white, NolA
non-expressing,cells. This system has been used to isolate and select
mutants that are insensitive to the quorum sensing inducer that is
expressed in the colonies after prolonged growth (i.e., cells remaining
white).
This same selection scheme can also be used to isolate B. japonicum mutants
that lack the ability to produce quorum sensing factor. These mutants
should also appear white after prolonged growth. These mutants can also be
selected by plating a mutated population of B. japonicum and then
overlaying these colonies with soft agar (0.4%) containing a B. japonicum
strain with the nolA-lacZ fusion and X-GAL. Mutants defective in
production of the quorum sensing factor will not induce the nolA-lacZ
fusion in the overlay, while those still producing the factor will rapidly
induce the fusion resulting in a blue color.
The subject invention advantageously provides methods of increasing
nitrogen fixation in plants by applying a nodulation inoculant having
NolA.sup.INS mutants and one or more nolA inducers to plants. In a
preferred embodiment, the plants are legumes; in a more preferred
embodiment, the plants are soybeans. The inoculant contains NolA.sup.INS
mutants in amounts effective to induce nodulation in the plant and amounts
of one or more nolA inducers sufficient to induce the activity of the nolA
gene. Methods of preparing inoculants, or coating seeds with inoculants,
suitable for use in the present invention are well known in the art and
include those taught in U.S. Pat. Nos. 4,535,061, 5,173,424, 5,695,541,
and 5,916,029 hereby incorporated by reference in their entireties.
The subject invention also provides methods of producing a nodulation
inoculant containing reduced amounts of quorum factor (CDF). These
improved nodulation inoculants are produced by adding iron to cultures
containing nodulating bacterial cells. As used herein, a nodulation
inoculant includes any bacterial species that nodulates a plants.
Nodulation inoculants produced according to these methods contain lower
amounts of quorum factor (CDF) as compared to nodulation inoculants not
grown in the presence of iron, and are able to more efficiently nodulate
target plant species (as compared to indigenous nodulating bacterial cells
or nodulation inoculants not grown in the presence of iron).
The subject invention further provides methods of reducing the production
of cell density factor or quorum factor in a nodulation inoculant or a
method of increasing the nodulation efficiency of a nodulation inoculant
comprising the addition of iron to medium containing the nodulation
inoculant. Iron is added in amounts sufficient to suppress the production
of cell density factor or quorum factor.
In some embodiments of the above-identified methods, the iron is in the
form of compounds containing Fe.sup.3+. One embodiment provides iron in
the form of FeCl.sub.3. As would be apparent to one skilled in the art,
nodulation inoculants can be prepared by culturing the bacterial cells in
any size container. For example, the cells can be cultured in a fermenter,
batch cultured, cultured on solid medium, cultured in standard culture
flasks, or cultured in test tubes.
In various embodiments, iron is added to the culture medium at various
stages of bacterial growth in amounts sufficient to suppress the
production of CDF or quorum factor. Thus, iron can be added to nodulation
inoculants in lag, early exponential, exponential, late exponential, early
stationary, or stationary growth phase. In other embodiments, the iron can
be added to the culture medium prior to the addition of an inoculant
starter culture; alternatively, iron can be added to the starter culture
and this admixture then added to the culture medium. Iron can also be
added to the culture medium and the starter culture. Various embodiments
of the invention provide for the addition of at least about 0.05 .mu.M or
at least about 0.1 .mu.M of iron. Other embodiments provide for the
addition of iron in concentrations of at least about 1 .mu.M, 10 .mu.M,
100 .mu.M, or at least about 1 mM. Iron concentrations that ranges from
0.5 .mu.M to 1M can be also be used in the practice of the instant
invention. In some embodiments, the iron has a concentration that ranges
from 1 .mu.M to 500 mM. Other embodiments provide iron concentrations that
range from 10 .mu.M to 250 mM or from 100 .mu.M to 100 mM. Alternatively,
iron can be added in a range of 500 .mu.M to 50 mM, 750 .mu.M to 5 mM, or
about 1 mM. Each of these ranges is to be construed as providing written
support of an iron concentration ranges falling within the range. For
example, the range of 100 .mu.M to 100 mM is also to be construed as
providing written support for a ranges such as 300 .mu.M to 50 mM, 400
.mu.M to 10 mM, or 500 .mu.M to 1 mM. Furthermore, as would be apparent to
the skilled artisan, aseptic or sterile techniques can be utilized in the
practice of the invention.
In some embodiments, the nodulation inoculant comprises a single species or
strain of nodulating bacteria. Other embodiments provide for the
combination of different species of nodulating bacteria. Thus, combination
of at least two different species of nodulating bacteria can be used in
the practice of the disclosed inventions. In some embodiments, the
nodulating bacteria is one or more species or strain of Bradyrhizobium.
Other non-limiting examples of inoculants that can be produced according
to the instant invention include Parasponia rhizobium (now identified as
B. parasponia), Rhizobium leguminosarum biovars viciae, trifolii and
phaseoli, Rhizobium sp. NGR234, B. japonicum USDA 110 and 123, Rhizobium
etli, Sinorhizobium meliloti, Rhizobium leguminosarum spp., or those
listed in FIGS. 17-18.
The subject invention also provides for methods of screening organisms or
extracts for the production of IND-1, IND-2, CDF (quorum factor), or
CDF-like molecules. In this method, extracts or culture supernatants are
analyzed for their ability to modulate nolA-lacZ, nodY-lacZ, nodC-lacZ, or
nodD-lacZ fusions in transformed host cells. For example, where such
molecules are present in the extract or supernatant, nolA expression is
induced. In contrast, very little induction is observed with samples where
no IND-1, IND-2, CDF (quorum factor), or CDF-like molecules are present.
Conditioned medium from organisms to be tested for the presence of CDF or
CDF-like molecules can also be used in the subject screening methods.
A further aspect of the subject invention relates to polynucleotides
encoding nolA inducers of the subject invention. The polynucleotide
sequence encoding the nolA inducers may, optionally, be operably linked to
transcriptional control sequences. As is apparent to one of ordinary skill
in the art, the disclosed inducers may be encoded by multiple
polynucleotide sequences because of the redundancy of the genetic code. It
is well within the skill of a person trained in the art to create these
alternative DNA sequences encoding the same, or essentially the same,
proteins. As used herein, reference to "essentially the same" sequence
refers to sequences which have amino acid substitutions, deletions,
additions, or insertions which do not materially affect biological
activity of the inducers of the invention (namely the ability to induce
nolA). Fragments of the inducers which retain the ability to induce nolA
expression are also included in this definition.
The polynucleotides of the subject invention include vectors and expression
cassettes. The vectors and expression cassettes may contain
transcriptional control sequences which are operably linked to
polynucleotide sequences encoding the nolA inducers of the instant
invention. The vectors and expression cassettes of the invention may
further include selectable markers.
The subject invention also provides transformed plant cells and transgenic
plants which have one or more polynucleotide sequences which encode
plant-derived or bacterial-derived nolA inducers. The polynucleotide
sequences encode compounds which induce the expression of nolA, thereby
reducing nodulation in plants by susceptible bacteria. Methods of
transforming cells with polynucleotide sequences, vectors, or expression
cassettes which encode nolA are well known to those skilled in the art.
Plants and plant cells may be transformed by, for example,
electroporation, Agrobacterium transformation, engineered plant virus
replicons, electrophoresis, microinjection, micro-projectile bombardment,
micro-LASER beam-induced perforation of cell wall, or simply by incubation
with or without polyethylene glycol (PEG).
The method of increasing nitrogen fixation in plants, to which the
NolA.sup.INS mutants are applied, may be practiced in transgenic plants
which express the nolA inducer and non-transgenic plants which
constitutively express the nolA inducer; this method may involve the
application of compositions having NolA.sup.INS mutants (bacterial cells)
directly to the roots of transgenic plants having polynucleotides encoding
a nolA inducer. The compositions having NOlA.sup.INS mutants may,
optionally, further include one or more nolA inducers. In one embodiment,
the roots may be wounded to enable the NolA.sup.INS bacterial cells to
penetrate the roots more quickly and easily; however, wounding of the
roots is not required. In a preferred embodiment, the plants are legumes.
More preferably, the plants are soybeans.
The present invention also provides methods of reducing or inhibiting the
nodulation activity of indigenous B. japonicum by adding a composition
having one or more nolA inducers of the invention to soil. In this aspect
of the invention, NolA.sup.INS bacterial cells may, optionally, be
included in the composition. The soil to which these compositions are
added include active and fallow fields.
To facilitate understanding of the invention, a number of terms are defined
below. All publications, patents and patent applications cited herein,
whether supra or infra, are hereby incorporated by reference in their
entirety to the extent that the reference is not inconsistent with the
teachings provided herein. As used in this specification and the appended
claims, the singular forms "a," "an" and "the" include plural references
unless the context clearly dictates otherwise.
As used herein, the term "transgenic plants" refers to plants (monocots or
dicots), having plant cells in which heterologous polynucleotides, such as
those encoding plant or bacterial nolA inducers, are expressed as the
result of manipulation by the hand of man.
As used herein, the term "peptide" refers to a polymer of amino acids and
does not refer to a specific-length of the product; thus, polypeptides,
oligopeptides, and proteins are included within the definition of peptide.
This term also does not refer to, or exclude, post expression
modifications of the peptide, for example, glycosylations, acetylations,
phosphorylations and the like. Included within the definition are, for
example, peptides containing one or more analogs of an amino acid
(including, for example, unnatural amino acids, etc.), polypeptides with
substituted linkages, as well as other modifications known in the art,
both naturally occurring and non-naturally occurring.
The terms "purified" and "isolated" indicate that the molecule is present
in the substantial absence of other molecules of the same type. The term
"purified" as used herein preferably means at least 75% by weight, more
preferably at least 85% by weight, more preferably still at least 95% by
weight, and most preferably at least 98% by weight, of molecules of the
same type are present.
The terms "purified" and "isolated", when referring to a polynucleotide,
nucleotide, or nucleic acid, indicate a nucleic acid the structure of
which is not identical to that of any naturally occurring nucleic acid or
to that of any fragment of a naturally occurring genomic nucleic acid
spanning more than three separate genes. The term therefore covers, for
example, (a) a DNA which has the sequence of part of a naturally occurring
genomic DNA molecules but is not flanked by both of the coding or
non-coding sequences that flank that part of the molecule in the genome of
the organism in which it naturally occurs (e.g., DNA excised with a
restriction enzyme); (b) a nucleic acid incorporated into a vector or into
the genomic DNA of a prokaryote or eukaryote in a manner such that the
resulting molecule is not identical to any naturally occurring vector or
genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a
fragment produced by polymerase chain reaction (PCR), or a restriction
fragment; and (d) a recombinant nucleotide sequence that is part of a
hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded
from this definition are nucleic acids present in mixtures of (i) DNA
molecules, (ii) transfected cells, and (iii) cell clones, e.g., as these
occur in a DNA library such as a cDNA or genomic DNA library.
The term "polynucleotide" as used herein refers to a polymeric form of
nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
This term refers only to the primary structure of the molecule and thus
includes double- and single-stranded DNA and RNA. It also includes known
types of modifications, for example, labels which are known in the art,
methylation, "caps", substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications, such
as those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) and with charged
linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those
containing pendant moieties, such as proteins (including for e.g.,
nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),
those with intercalators (e.g., acridine, psoralen, etc.), those
containing chelators (e.g., metals, radioactive metals, boron, oxidative
metals, etc.), those containing alkylators, those with modified linkages
(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of
the polynucleotide.
"Operably linked" refers to a juxtaposition wherein the components so
described are in a relationship permitting them to function in their
intended manner. A control sequence "operably linked" to a coding sequence
is ligated in such a way that expression of the coding sequence is
achieved under conditions compatible with the control sequences.
As used herein, the term "expression cassette" refers to a molecule
comprising at least one coding sequence operably linked to a control
sequence which includes all nucleotide sequences required for the
transcription of cloned copies of the coding sequence and the translation
of the mRNAs in an appropriate host cell. Expression cassettes can
include, but are not limited to, cloning vectors, specifically designed
plasmids, viruses or virus particles. The cassettes may further include an
origin of replication for autonomous replication in host cells, selectable
markers, various restriction sites, a potential for high copy number and
strong promoters.
By "vector" is meant any genetic element, such as a plasmid, phage,
transposon, cosmid, chromosome, virus etc., which is capable of
replication when associated with the proper control elements and which can
transfer gene sequences between cells. Thus, the term includes cloning and
expression vehicles, as well as viral vectors.
In order to provide a means of selecting transformed plant or bacterial
cells, the vectors for transformation will typically contain a selectable
marker gene. Marker genes are expressible DNA sequences which express a
polypeptide which resists a natural inhibition by, attenuates, or
inactivates a selective substance. Examples of such substances include
antibiotics and, in the case of plant cells, herbicides. Suitable marker
genes for use in this invention are well known to those skilled in the
art.
It is also contemplated that a particular amino acid sequence of NolA may
be encoded by more than one polynucleotide sequence. It may be
advantageous to produce nucleotide sequences possessing a substantially
different codon usage. Codons can be selected to increase the rate at
which expression of the peptide occurs in a particular prokaryotic or
eukaryotic expression host in accordance with the frequency with which
particular codons are utilized by the host. Other reasons for
substantially altering the nucleotide sequence without altering the
encoded amino acid sequence include the production of RNA transcripts
having more desirable properties, such as a longer half-life, than
transcripts produced from the naturally occurring sequence.
MATERIALS AND METHODS
Bacterial Stains, plasmids and culture conditions. For routine bacterial
growth, B. japonicum cells were maintained on RDY medium (So, J. -S. et
al. [1987] Mol. Gen. Genet. 207:15-23). Bacteria were grown in minimal
medium (Bergensen, F. J. [1961] Aust. J. Biol. Sci. 14:349-360) for
.beta.-galactosidase activity. As required, antibiotics were used at the
following concentrations, Cm (30 .mu.g/ml), Sm (100 .mu.g/ml), Sp (100
.mu.g/ml), Tc (100 .mu.g/ml). The B. japonicum strains used in this study
were Bj110-42, BJAlac12, BJAlac23, BJAlac13 and BJ110-1248-1, ZB977, SL101
and Bj110-573. These strains harbored the following translational fusions;
BJ110-1248-1 (nodD.sub.2 -lacZ, plasmid pRJ1248, Dockendorff et al. [1994]
Mol. Plant-Microbe Interact. 7:596-602), ZB977 (nodY-lacZ, plasmid pZB32,
Banfalvi et al. [1988] Mol. Gen. Genet. 214:420-424), SL101 (npt-lacZ,
Yuen, J. P.-Y and G. Stacey [1996] Mol. Plant-Microbe Interact.
9:424-428), and Bj110-573 (nodC-lacZ, chromosomally integrated fusion,
Dockendorff et al. [1994] Mol. Plant-Microbe Interact 7:596-602). Strains
Bj110-42, BJAlac23, BJAlac12, BJAlac13 harbored nolA-lacZ translational
fusions encoded on plasmids pBGALac1, pNMAlac23, pNMAlac13, pNMAlac12
respectively (Garcia et al. [1996] Mol. Plant-Microbe Interact. 9:625-635;
Loh et al. [1999] J. Bacteriol. 181:1544-1554). Plasmid pNMAlac23
contained mutations to ATG2 and ATG3 of nolA and allowed the specific
expression of NolA.sub.1 -lacZ. In contrast, plasmids pNMAlac13 (mutations
to ATG1 and ATG3) and pNMAlac12 (mutation to ATG1 and ATG3, Loh et al.
[1999] J. Bacteriol. 181:1544-1554) only expressed NolA.sub.2 -lacZ and
NolA.sub.3 -lacZ, respectively.
Following are examples which illustrate procedures for practicing the
invention. These examples should not be construed as limiting. All
percentages are by weight and all solvent mixture proportions are by
volume unless otherwise noted. All references, publications, and patents
cited herein are hereby incorporated by reference in their entireties.
EXAMPLE 1
Identification of NolA Inducers from Plant Extracts
While many of the nodulation genes of B. japonicum are induced by the plant
flavonoids genistein and daidzein, these compounds and a variety of other
flavonoids failed to induced nolA expression. NolA expression was,
however, induced by plant extracts. Analysis of these extracts, using
Reverse Phase HPLC, have identified the presence of two distinct compounds
(IND-1 and IND-2) that are capable of inducing nolA (FIG. 1).
IND-1 has been identified as a phthalic acid bis-(2-ethyl-hexyl) ester
(FIG. 2A). To confirm the activity of this compound, phthalic acid
bis-(2-ethyl-hexyl) ester was chemically synthesized and shown to be able
of inducing nolA expression (FIG. 2B). This compound is a strong inducer
of NolA expression and inhibits nodulation.
Initial analysis of IND-2 indicates that its activity is sensitive to
chitinase treatment. IND-2 may also be purified from soybean extracts,
particularly commercially available soybean phosphatidylinositol extracts
(available from companies such as Sigma Chemical Co., St. Louis, Mo.).
When tested, the commercially available phosphatidylinositol soybean
extracts (Sigma) were found to be capable of inducing nolA expression
(FIG. 3). Moreover, these extracts were also sensitive to chitinase
treatment. As shown in FIGS. 4A-4B, we have identified a peak (i.e. peak
9) after Reverse Phase HPLC, that is both capable of inducing nolA and
sensitive to chitinase. Materials containing IND-1 and IND-2 were applied
to a C18 column (Phenomenex, Inc., Torrance, Calif.) and eluted with a
methanol gradient (0-100%) at a flow rate of 1 mL per minute.
EXAMPLE 2
NolA Inducer from B. japonicum
Typically, B. japonicum cells are found in high population density in
commercial inoculants. To determine if the bacterial nolA inducer was
present in commercial inoculant, three soybean inoculants from two
commercially available sources were extracted with butanol, and these
extracts analyzed for their ability to induce the nolA-lacZ fusions. As
shown in Table 1, nolA expression was induced significantly by the B.
japonicum inoculant extracts. In contrast, very little induction was
observed with samples where no B. japonicum were present (i.e., peat
alone).
EXAMPLE 3
Quorum Control Mechanism for NolA Induction
NolA expression is population density dependent; its expression is low at a
low population density, and significantly higher in more dense cultures
(FIG. 5). This quorum control of nolA expression appears to be regulated
by a compound that is secreted and accumulates in the culture medium.
Addition of this compound (i.e. conditioned medium) to B. japonicum
cultures grown to a low population density greatly increases the
expression of nolA (Table 2). Consistent with the fact that nolA regulates
nodD.sub.2, the levels of nodD.sub.2 expression not only showed a similar
population density dependence (FIG. 6, Table 2), but were also found to be
affected by the addition of the nolA inducer.
As shown in Tables 2-3 and FIG. 11C, an inducer of nolA-lacZ and nodD.sub.2
-lacZ expression was detected in conditioned medium from B. japonicum
cultures grown to a high population density. This inducer was capable of
inducing transcription of both fusions when added to B. japonicum cultures
at 10 .mu.l/ml. The ability of the conditioned medium to induce the nolA
fusions was population density dependent with little or no induction of
the fusions observed using conditioned medium derived from cultures of
A.sub.600 <0.2 (FIG. 11C). Significant induction was observed with
conditioned medium from cultures of A.sub.600 =0.5, reaching a maximum at
A.sub.600 >1.0. The bacterially derived inducer within the conditioned
medium is insensitive to heat. The nolA inducer present in the conditioned
medium was lost after dialysis using a membrane with a cutoff at 3 kDa
(Fisher Scientific, Pittsburgh, Pa.).
EXAMPLE 4
Population Density Dependence of Nod Gene Expression
Nod gene induction by genistein is population-density dependent. It has
been observed that optimal gene expression occurs at very low population
densities (i.e., A.sub.600 <0.05, Yuen, J. P.-Y. and G. Stacey [1996]
Mol. Plant-Microbe Interact. 9:424-428). In order to examine this
observation in a systematic way, Bj110-573 cells (containing a
chromosomally integrated nodC-lacZ fusion, Dockendorff, T. C. et al.
[1994] Mo
