Phosphate sensing microbial gene switch
Genetically engineered bacteria which express RNAs or proteins that produce ammonia upon decreases in phosphate concentrations are disclosed.
1 . A genetically engineered bacterium comprising:
(i) a first gene expression cassette comprising a recombinant DNA molecule comprising a phosphate-sensitive promoter of a bacterial pstS phosphate ABC transporter gene operably linked to DNA encoding a transcript comprising a ribosome binding site (RBS) operably linked to a transcriptional repressor protein coding region,
wherein the phosphate-sensitive promoter comprises a sequence having at least 95% sequence identity to SEQ ID NO: 515 and comprising the sequences of SEQ ID NO: 525, 526, 527, 528, and 529, wherein the RBS comprises SEQ ID NO: 449, and wherein the transcriptional repressor protein coding region encodes a bacterial TetR repressor protein; and
(ii) a second gene expression cassette comprising a recombinant DNA molecule comprising a promoter which is repressed by the transcriptional repressor protein and which is operably linked to a DNA sequence encoding a bacterial glutamine synthetase
wherein the promoter in the second gene expression cassette comprises a Ptet promoter;
wherein the genetically engineered bacterium is of the genus Kosakonia and releases a greater amount of ammonia when grown in liquid culture under microaerobic conditions when inoculated in the presence of 0 mM inorganic phosphate than when grown in liquid culture under microaerobic conditions when inoculated in the presence of 20 mM inorganic phosphate.
2 . The bacterium of claim 1 , wherein the transcriptional repressor protein coding region encodes a bacterial TetR transcriptional repressor protein having at least 95% sequence identity to SEQ ID NO: 516.
3 . The bacterium of claim 1 , wherein the transcriptional repressor protein coding region encodes a bacterial TetR transcriptional repressor protein comprising the amino acid sequence of SEQ ID NO: 516.
4 . The bacterium of claim 1 , wherein the bacterial glutamine synthetase is an Azospirillum glutamine synthetase and the endogenous gene encoding the Kosakonia glutamine synthetase protein contains a null mutation or is deleted.
5 . The bacterium of claim 1 , wherein the bacterial glutamine synthetase comprises a Kosakonia glutamine synthetase.
6 . The bacterium of claim 5 , wherein the Kosakonia glutamine synthetase is encoded by an endogenous Kosakonia gene.
7 . The bacterium of claim 1 , wherein:
(i) the phosphate-sensitive promoter in the first gene expression cassette comprises a Klebsiella variicola pstS phosphate-sensitive promoter having at least 95% sequence identity to SEQ ID NO: 515, the RBS in the first gene expression cassette comprises a BCD22vL RBS encoded by SEQ ID NO: 449, and the transcriptional repressor protein coding region encodes a protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 516; and
(ii) the promoter in the second gene expression cassette comprises a Ptet promoter having at least 95% sequence identity to SEQ ID NO: 32, and the bacterial glutamine synthetase is a glutamine synthetase having at least 95% sequence identity to SEQ ID NO: 229.
8 . A composition comprising the genetically engineered bacterium of claim 1 and an agriculturally acceptable carrier.
9 . The composition of claim 8 , wherein the composition further comprises:
(i) an agriculturally acceptable adjuvant, optionally wherein the adjuvant comprises an adhesive agent, a desiccant, and/or a dispersant;
(ii) a fungicide, an insecticide, a nematicide, a rodenticide, and/or a bacteriocide; and/or
(iii) a fertilizer, optionally wherein the fertilizer comprises nitrogen, phosphorous, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum, and/or selenium.
10 . A plant part or plant propagule which is at least partially coated, imbibed, or mixed with the composition of claim 8 .
11 . An agricultural system comprising:
(i) at least one engineered bacterium of claim 1 ;
(ii) at least one plant growth medium; and
(iii) at least one crop plant, crop plant seed, or crop plant vegetative propagule; wherein the plant growth medium, crop plant, crop seed, and/or crop plant propagule comprise, are at least partially coated, imbibed, and/or are mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier.
12 . The system of claim 11 , wherein the crop plant, seed, or vegetative propagule is an alfalfa, apple, banana, barley, bean, buckwheat, cabbage, cassava, chili, clover, coffee, corn, cotton, cowpea, cucumber, fonio, garlic, herb, lettuce, maize, melon, millet, nut, oat, oilseed rape, olive, onion, orange, sunflower, pea, Phaseolus bean, plantain, potato, quinoa , rice, rye, safflower, sorghum, soybean, sugar beet, sugar cane, sunflower, tangerine, tobacco, tomato, triticale, turnip, wheat, or yam plant, seed, or vegetative propagule.
13 . The system of claim 11 , wherein the plant growth medium comprises soil and/or water, optionally wherein the soil and/or water is non-axenic.
14 . The system of claim 11 , wherein the vegetative propagule comprises a cutting, tuber, or stolon.
15 . A treated plant seed or plant propagule system comprising:
(i) at least one crop plant seed or crop plant vegetative propagule; and
(ii) at least one engineered bacterium of claim 1 ,
wherein the crop plant seed or crop plant propagule are at least partially coated, imbibed, and/or mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier.
16 . The system of claim 15 , wherein the crop plant, seed, or vegetative propagule is an alfalfa, apple, banana, barley, bean, buckwheat, cabbage, cassava, chili, clover, coffee, corn, cotton, cowpea, cucumber, fonio, garlic, herb, lettuce, maize, melon, millet, nut, oat, oilseed rape, olive, onion, orange, sunflower, pea, Phaseolus bean, plantain, potato, quinoa , rice, rye, safflower, sorghum, soybean, sugar beet, sugar cane, sunflower, tangerine, tobacco, tomato, triticale, turnip, wheat, or yam plant, seed, or vegetative propagule.