Biological nitrogen fixation

Details Biological nitrogen fixation Biological nitrogen fixation

1992-01-27

2003-04-15

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S069100, C435S091100, C435S252100, C435S320100, C536S023100

Reexamination Certificate

active

06548289

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to genetic engineering of agriculturally useful microorganisms.
Certain naturally occurring microorganisms, e.g., microorganisms of the genus Klebsiella, e.g.,
Klebsiella pneumoniae,
and microorganisms of the genus Rhizobium, e.g.,
R. meliloti
and
R. japonicum,
are capable of converting atmospheric nitrogen into ammonia (“nitrogen fixation”). It has been proposed that the slow-growing soybean-colonizing bacterial species, known for decades as
Rhizobium japonicum,
be reclassified as
Bradyrhizobium japonicum,
to distinguish it from faster growers such as
R. meliloti, R. phaseoli,
etc. Other newly discovered, fast-growing soybean colonizers previously classified as
Rhizobium fredii
are now known and in all the claims as
R. japonicum.
Rhizobium herein refers to all Rhizobium and Bradyrhizobium species.
R. japonicum,
as used herein with reference to the Figures and preferred embodiments, means the slow-growing bacteria now known as
B. japonicum,
and genetic constructions devised therefrom.
K. pneumoniae,
a facultative anaerobe, can fix nitrogen in a free-living state, while Rhizobium and Bradyrhizobium species normally require a symbiotic relationship with leguminous plants.
The transcendent importance of nitrogen fixation in sustaining the biosphere has been recognized for much of the present century. In the last two or three decades the world's human population has outstripped the ability of natural nitrogen fixation processes, spontaneous and biological, to support adequate food production, so that more than 30% of the world's population now depends on artificial nitrogenous fertilizer for its minimal nutrition.
In addition to Rhizobium and Klebsiella species, prokaryotes naturally able to fix nitrogen include obligate anaerobes (e.g.,
Clostridium pasteurianium
), obligate aerobes (e.g.,
Azotobacter vinelandii
), photosynthetic bacteria (e.g.,
Rhodospirillum rubrum
), and some strains of blue-green algae (e.g.,
Anabaena cylindrica
).
A symbiotic relationship can exist between Rhizobium and legumes (e.g., soybeans or alfalfa). Such a relationship begins with host-symbiont recognition and penetration of the root by Rhizobium, and culminates in the differentiation of the bacterium into the nitrogen-fixing “bacteroid” form within the root nodule. It is only in the bacteroid form that nitrogen is fixed by Rhizobium. Rhizobium species exhibit host-range specificity: for example,
R. japonicum
infects soybeans, and
R. meliloti
infects alfalfa.
The plant species commonly used in commercial agriculture cannot fix their own nitrogen unless in symbiotic association with nitrogen fixing microorganisms and are thus reliant, in general, on the addition of nitrogenous fertilizers. However, the symbiotic relationships between legumes and Rhizobium have long been exploited in commercial agriculture. Various strains of Rhizobium are currently sold commercially, to be used as “inoculants” to increase the yields of legume crops such as soybean, alfalfa, and closer. Rhizobial inoculants have been sold in significant volume in the U.S. since 1959, and it has been estimated by the USDA that 50% of the total U.S. acreage of soybean crops and 80% of alfalfa crops are inoculated.
Although rhizobial products are used to such an extent in this country, the existing products are believed not to be very effective in promoting yield increases. One reason for this might be poor competition between the introduced strains and those strains indigenous to the soil.
In nitrogen fixing microorganisms there are genes coding for products involved in the nitrogen fixation pathway. In
K. pneumoniae,
these genes are known as the “nif” genes. Analogous sets of genes are present in other nitrogen fixing species (perhaps arranged differently in each species). The nif genes of
K. pneumoniae
are arranged in sequence in 7-8 operons. One operon contains the structural genes coding for the protein subunits of the major enzyme in the nitrogen fixation pathway, nitrogenase. The nitrogenase operon is composed of a promoter (the nifH promoter); the three subunit structural genes, nifH, nifD, and nifK; and the nifT and nifY genes, of unknown function. Another operon is composed of a promoter and the nifL and nifA genes. The nifA gene encodes a transcriptional activator protein, the nifA protein, which is required for the expression of all operons containing the nif genes, except its own. The nifL gene codes for a protein which renders the nifA transcriptional activator protein nonfunctional, and thus serves to repress nitrogen fixation. (References herein to the nifA gene, the nifL promoter, and the nifH gene and promoter are intended to include DNA derived from
K. pneumoniae,
as well as functionally equivalent DNA derived from any other nitrogen fixing bacteria.)
Buchanan-Wollaston et al., 1981, Nature 294:776 report an investigation of the role of the nifA gene product in the regulation of nif expression. A variety of
Klebsiella pneumoniae
strains were transformed with either of two plasmids constructed to permit constitutive expression of the nifA gene product. In pMC71A the nifA gene was cloned into the tetracycline resistance gene of the plasmid pACYC184 and transcribed from the promoter of the tetracycline resistance gene. In pMC73A, the nifA gene was cloned into the kanamycin resistance gene of the plasmid pACYC177 and transcribed from the promoter of the kanamycin resistance gene. Expression of nifA from these plasmids was tested in a mutant-strain of
K. pneumoniae
which does not express normal nifA activity. Both plasmids were observed to complement the nifA mutation. Constitutive nif expression in the presence of NH4
+
(a negative effector of nif transcription initiation) was also examined by measuring &bgr;-galactosidase activity in a
K. pneumoniae
strain using a genomic fusion of the nifH promoter in reading frame with the lacZ gene.
SUMMARY OF THE INVENTION
The present invention provides a strategy for improving crop yields, involving increasing nitrogen fixation in nitrogen fixing bacteria via genetic engineering.
In general, the invention features a vector for transforming a host microorganism which contains DNA encoding one or more proteins capable of effecting the conversion of atmospheric nitrogen into ammonia in the microorganism, the vector being capable of increasing the capacity of the microorganism to so convert atmospheric nitrogen, the vector including a gene encoding an activator protein capable of activating the transcription of that DNA, the activator protein-encoding gene preferably being under the transcriptional control of an activatable promoter sequence.
Microorganisms transformed with the vector have an improved capacity to fix nitrogen. The activator protein which is normally present in only a limited amount is, by virtue of the vector, produced in a much greater amount which results in increased production of nitrogenase from the nif genes of the host microorganism (although too great an amount of the activator protein can actually be detrimental to plant growth). Thus, even in the presence of a nifL-like repressor protein, there is sufficient overproduction of nifA protein to activate nitrogenase production. Furthermore, the use of an activatable promoter allows for high levels of nifA protein production at the time the host cell initiates nitrogen fixation.
Alternatively, the same effect can be achieved by placing the inserted nifA gene under the transcriptional control of a constitutive promoter, e.g., the promoter of a bacterial gene for kanamycin resistance.
Nitrogen fixing bacteria transformed with a vector of the invention, living in association with legume crops with which the bacteria can live symbiotically, can increase the yields of those crops by virtue of the improved nitrogen fixation provided by the bacteria.
The invention also features a method for stably integrating, by homologous recombination, a DNA sequence into a silent region of the Rhizobium chromosome. This method can

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