CA1219233A - Fluorescent siderophore genes and their use - Google Patents

Abstract

FLUORESCENT SIDEROPHORE
GENES AND THEIR USE

ABSTRACT OF THE DISCLOSURE

DNA sequences encoding for plant growth promotant activity have been isolated and introduced into microorganisms. The modified organisms are able to confer plant growth promotant activity analogous to that of the DNA source host. Such modified hosts find use in promoting the growth of root crops by innoculating the rhizosphere with such microorganisms.
E.coli HB101 (pS FL-1) was deposited at the A.T.C.C. on October 8, 1982, and granted accession No. 39206.

Description

2307U-153/REUCl4D

FLUORE S CENT S I DEROPHORE
GENES AND THEIR USE

Genetic evolution has afforded an extraordinary array of biological capabilities in nature. Various organisms and cells achieve these different functions by producing a wide variety of proteins, many of which can in turn produce a wide variety of non-proteinaceous molecules. These naturally occurring compounds can interact to modify their environment in countless ways.
It is known that certain soil microorganisms are beneficial to plant growth through the production of growth hormones, antibiotic substances which ~ill harmful soil microorganisms, and by aiding in the uptake of nutrients by plants. In particular, it has been found that certain fluorescent strains of the genus Pseudomonas enhance root crop production through the production of fluorescent siderophores which confer a competitive advantage on the Pseudomonas and inhibit the growth of competing deleterious microorganisms.
Siderophores are low molecular weight compounds which are capable of sequestering or chelating iron (Fe 3) and acting as transport agents in supplying iron to the microorganism which produce them. While virtually all aerobic and facultative anaerobic microorganisms are able to produce siderophores under low iron stress, fluorescent pseudomonads produce particularly effective siderophores which act to reduce the availability of iron to other microorganisms resulting in the inhibition of diseas~-inducing microorganisms.
It would therefore be desirable to be able to confer on certain beneficial microorganisms the ability to preferentially compete for growth with other deleteri-ous microor~anisms in the root sphere (rhizosphere) of .. ~. - ~

root crops such a~ potatoes, radishes, sugar beets, and the like. In particular, it would be desirable to be able to enhance the siderophorP-producing capability of microorganisms which already display plant growth promoting activity.

A number of papers have been published concern-ing the al:>ility of specific strains of fluorescent Pseudomonas to produce fluorescent sid~rophores and enhance the growth of certain root rops. See, for example, Kloepper and Schroth ~1981), Phytopatholo~y 71:1020-1023; Kloepper, et al. (1980) Curr. Microbiol., 4:317-320; and Kloepper, et al. (1980) Nature, 286:885-886. The structure o:E ferric pseudobactin, a siderophore obtained from a particular strain v fluorescent Pseudomonas, has been determined that T~intze, et al.
(1981) Biochemistry, 20:~446-6457. Other articles of interest include lleyer and Abdallah (1~78~ J. Gen.
Microbiol., 107:319-32~; Meyer and Hornsperger (1978) J. Gen. Mic_obiol./ 107:329~331; and Misaghi, et al.
(1982~ Ph~t~atholo~, 72:33-36.

According to the invention DNA seguences encoding ~or substances which confer enhanced plant growth promotant activity on a microorganism are provided. The DNA sequences can be cloned in a host foreign to the source of the DNA and are capable of imparting plant growth promotant activity to such hosts as well as enhancing such activi ty in the source host itself. DNA sequences, vectors and trans-formants are described.

The subject invention provides for the isola~
tion and utilizatiox~ of DNA segments encoding for plant growth promotant activity (P&PA). By inserting such DNA segments onto an appropriate vector, PGPA can be conferred upon a wide variety of hosts by introducing the vector according to conven-tional techniques. The vector may be a plasmid, phage, or o-ther self-replicating extrachromosomal element which may be used for conjuga-tion, transformation, transduction, or transfection ofthe microorganism host. The hbst may then be grown and cloned, and PGPA clones isolated. The resulting growth promotant microorganisms or subcellular portions or extracts derived therefrom, may be used to treat the rhizosphere of various root crops in order to suppress certain root crop diseases and promote the yrowth of the plant.
In particular, the DNA segments encode for the production of chelating agents which sequester limited multivalent inorganic cations in the soil which are essential nutrients for growth. More particularly, the DNA segments encode for the production of fluores-cent siderophores which are capable of chelating Fe+3.
By introducing the subject DNA sequences to a host, the host is able to preferentially scavenge -the available Fe+3 in the soil, depriving other dele-terious micro-organisms in the soil of this essential nutrient. The deleterious microorganisms will generally either not produce siderophores, produce lesser quantities of siderophores, or produce slderophores having a lesser affinity for iron ~han the`~luorescent siderophores.
The DNA sequence of interest will be about 20kbp in length, or less, usually being greater than _: about 15bp, more usually greater than 30bp. The exact length of the sequence is not critical so long as, when introduced to an appropriate host, the PGPA is expressed.
In order to obtain a DNA sequence encoding for PGPA, a microorganism known to provide for PGPA can be employed. Conveniently, various species of Pseudomonas, such as syringae, fluorscens, and putida, may be employed as a source for the preparation of a gene library, either by random fragmentation of the genome or by synthesis of cDNA from mRNA.
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Various restriction enz~mes may be employed which provide for segments of up to 25kbp by complete or incomplete digestion of the ~ource genome. These fragments may then be cloned. Various vectors may be employed depending on the size of the fragment, the nature of the host, and the like. Plasmids, phages, and cosmids can be employed which allow for insertion of fragments from the genomic library as functional self-replicating extrachromosomal elements in the host.
Such vectors should have convenient restriction sites which allow for insertion of the genomic library frag-ments. Desirably, the vectors should provide a means j for selection and/or screening, typically through antibiotic selection, packaging reguirements, inacti-; 15 vation of a gene, or other means.
Of particular inteEest for cloning is a cosmid vector, more particularly pLAFRl, which has a unique EcoRI site. This vector is a derivative of ~he vector pRK290 (Tcr) that contains the cos sites of phage A for in vitro packaging. It is a broad host range oligocopy vector, having the unique EcoRI site outside Tc gene.
The vector pLAFRl is particularly useful because it selects by packaging for inserts of about 20kb + lOkb in length. The vector is described by Fried~an, et al.
25 (1982~ G _ Vol. 18 pages ~89-296.
While the pLAFRl pla~mid does not provide for selection or screening based on insertional inactiva-tion, the DNA sequences themselves will often provide a : convenient selection technique. The substances produced by P. syringae, P. fluorescens, and P. putida, are fluorescent. By subcloning Tc colonies in a nonfluores-cent host capable of expression, recombinant vectors may be ~elected based on their fluorescence.
Preparation of a cDNA gene library is accom-plished by first isolating the mRNA fraction from thesource microorganism. Care should be taken to inacti-vate the RNases, typically by treatment with VaS04-ribonucleoside complexes. After extracting the total ., ~,, , ~ . ~

;, ~2~L91233 RN~, the mRNA can be separa-ted using a poly~U) or poly (T) chromotography medium which is specific for the poly(A) tail. The cDNA gene library can then be pre-pared using reverse transcriptase in the well known manner.
After cloning and screening, the DNA sequence of in-terest will be introduced into a soil microorganism for eventual population of the rhizophere of the crop being treated. While it will sometimes be possible to introduce the cloning vector directly, it will often be necessary to excise the DNA sequence and insert it onto a vector which is compatible with the contemplated host mi~roorganism.
The DNA sequence encoding for PGPA may be introduced into a wide variety of microorganisms capable of populating the rhizosphere of the various root crops, in particular bacteria and fungi. The choice of host will depend on the availability of a compatible vector, the purpose for introducing the PGPA into the host, and the manner in which the host is to be used.
It is preferred to employ hosts which are PGPA~, in particular hosts in which the wild type produces fluorescent siderophores capable of imparting P~PA, such as P.fluorescens, P.putida and P.syringae. In the latter case, introduction of multiple copies of the DNA
sequence will enhance the P~PA through the greater production of siderophores.
Depending upon the nature o~ the vector, various techniques may be used for introducing the vector carrying the DNA insert into the host. Trans-formation can be achieved in the conventional manner employing calcium precipitated DNA in an appropriate solvent. Transfection may be achieved by contacting the microorganisms with a modified virus, or its DNA in a nutrient medium. Transduction of the microorganisms occurs upon integration of the sequence into the genome.
Conjugation can also be employed, where the plasmid is introdu~ed into one organism, which may then transfer - .:

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the plasmid to a different organism either being capable of mobilization by itself or in conjunction with a mobilizing plasmid. It is particularly desirable that the vector be non-self-transmissible to prevent trans-formation of deleterious microorganisms. Preferably,the DNA sequence will be integrated into the chromosome of any host which is introduced into the soil.
For Pseudomona hosts, derivatives of plasmid RSF1010 such as pKT212 and pKT214 are particularly useful as vectors. The DNA sequences of interest can be inserted at unique Bam ~I and ~ II site, and selection made based on the loss of Tcr.
In isolating the organisms receiving the DNA
sequences of interest, it is desirable to use a PGPA
organism, whereby a resulting clone which is shown to be PGPA is likely to have received a recombinant vector in the cloning. Moreover, when cloning in a nonfluorescent host capable of expressing the fluores-cent gene product, selection can be made based on the fluorescent phenotype. Conveniently, the clones can be screened using a simple technique where colonies plated on an appropriate solid nutrient media are exposed with long-wave ultraviolet light (366nm) which causes colonies expressing the recombinant gene product to fluoresce.
The modified mic~oorganisms of the present invention may be utilized ~fectively in diverse formu-lations, including agronomically-acceptable adjuvants and carriers normally employed for facilitating the . dispersion of active ingredients for agricultural applications. The precise formulation, dosage, mode of application and other variables are chosen to enhance the PGPA in any given application. Thus, the previously described modified microorganisms may be formulated as a suspension or a dispersion, an aqeous or non-aqeous medium, as a dust, as a wetable powder, as an emulsifi-able concentrate, as a granule, or as any of several known types of formulations, depending on the desired mode of application. These compositions may be applied as sprays, dusts or granules to the seeds, seed pieces, roots, plants, soil, or planting site at which activity is desired.

S The following examples illustrate the isola-tion of the siderophore gene~s) and are not intended to limit the lnvention in any way.

EXPERIMENTAL
1. Construction of the DNA Library The DNA from two fluorescent, siderophore-bearing Pseudomonas syrinqae strains designated 31Rl and Cit7 was extracted, purified by two cycles of CsCl-ethidium bromide density gradient centrifugation, and dialyzed against appropriate buffers. The DNA was partially digested wi~h Eco RI and fractionated by sucrose gradient centrifugation in 5-25% neutral sucrose.
The partial digestion employed 0.3 units Eco RI per 1 ~g DNA following the directions of the supplier (Bethesda Research Laboratories, MD) and the reaction 20 stopped after O . 5 hr by heating at 65 C for three minutes. Fractions from the sucrose gradient were analyzed by agarose gel electrophoresis and those rich in fragments in the 18-25kbp range were pooled, enriched by ethanol precipitation, and ligated to the cosmid ~5 vector pLAFRl (Fri~En, et al. (1982) GRne Vol. 18 pages 289-296 supplied by S. Long, Stanford, California) previously linearized with Eco RI.
The cosmid pLAFRl is a derivative of the plasmid pRK290 (Tcr) that contains the cos site of phage lambda for in vitro packaging. pLAFRl includes a single Eco RI insertional site (outside the TCr gene) and selects for inserts of about 20kbp in length.

2. Dexivation o P. syringae 31Rl-?6 A nonfluorescent mutant (designated 31Rl-~6) was derived from P syringae 31Rl by chemical muta-genesis utilizing ethylene methane sulfonate (EMS).

,; .`~ .~' g~33 Strain 31Rl was grown in King's B broth overnight, 0.2 ml seeded into 10 ml fresh King's B broth, and ~rown for four hours to assure log phase growth. Five percent EMS was added to broth, mixed well, and incu-bated with shaking at 25-C for twenty minutes. Cells were then washed two times, resuspended in an equal volume of King's B broth, and placed back in the incu-bator for two hours. Segregated cells were then plated on King's B agar at cell densities of 30-50 colony-forming units/plate. After two days of incubation at25~C, fluorescent colonies were identified by irradia-tion of plates with long-wave ultraviolet light (366nm).
The nonfluorescent mutant 31Rl-26 was detected by observing its lack of fluorescence when irradiated with W light.
To demonstrate that strain 31Rl-26 was deficient in siderophore production, an iron-chelating compound tethylene diamine dihydroxyphenyl acetic acid, 200ppm~
was added to the King's B agar to produce an iron-deficient medium. Strain 31Rl, which produces a sidero-phore, was able to grow on the iron-deficient medium, while nonfluorescent mutant strain 31Rl-26 was not.
The inability of strain 31Rl-26 to grow on iron~deficient medium was reversed upon addition of 10 4M FeC13, confirming that iron starvation was responsible for the lack of growth. ^ ~
Ligation of the DNA fragments in pLAFRl was achieved using T4 DNA lisase following the supplier's _; directions (Bethesda Research Laboratories, MD). A
high ratio of foreign DNA -to linearized pl~FRl was employed to minimize dimerization of the vector. About

3-4 ~g of P. syringae DNA fragments were used per 1 ~g of linearized pLAFRl. The ligation reac-tion was carried out in an appropriately buffered 10 ~1 volume that is heated at 65 C for 5 minutes, 30 minutes at 42 C, followed by two hours at room temperature. ATP was then added to a concentration of 1 mM; 1 unit of T4 DNA

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L9;~33 ligase was added; and the ligation mixture was incubated at 12 C overnight.
In vitro packaging was in accordance with the procedure described by Hohn, M. (In vitro Packaging of 5 A and Cosmid DNA, Wu, ed., Methods in Enzymology, vol. 68, Academic Press, ~ew York, pages 299-309, 1979). Approximately 30 ~1 freeze-thaw (A heads~ and 20 ~1 sonicate (A tails) extracts were combined with 2 ~1, 1 M ATP and 5 ~1 ligated DNA, and the resulting mixture incubated for one hour at room temperature and adjusted to 10 mM MgC12, 10 mM TRIS buffer, pH 7.6.
For transduction, 0.1 ml of the phage stock was mixed with 0.5 ml of E. coli HB101 cells grown to mid-log phase (107-108 cells/ml) in Luria broth supple-mented with 0.4% maltose and incubated for one hour at37 C. Two ml Luria broth was added, and the cells grown for 1.5-2.0 hours at 37 C. Transductants were selected on Luria agar supplemented with 10 mg/ml tetracycline (pLAFRl confers resistance to the antibiotic).

3. Selection of Recombinant Plasmids Havin~
Siderophore Gene(s~
Recombinant pLAFRl plasmids conferring the fluorescent phenotype were selected by mating the transduced HB101 clonës wit~h the nonfluorescent mutant 31Rl-26. Selecti~n can the~ be made based on the acquired fluorescent phenotype. pLAFRl is nonconjuga-tive but mobilizable by the conjugative helper plasmid . pRK2013.
First, single en masse matings of -the HB101 (pLAFRl - P. syrinqae) libraries with the nonfluorescent 31Rl-26 mutant yielded a low frequency of complemented fluorescent transconjugants. These fluorescent trans-conjugants were able to grow on media containing 200 ppm EDDA, as indicated in Table I. However, repeated attempts to isolate the recombinant pLAFRl plasmid from these transconjugants were unsuccessful.

Second, individual c.lones of the HB101 (pLAFR1-_. syrinqae) library s-tored in microtiter plates were utilized as donors in 718 sepa.rate triparental matings with HB101 (pRK2013) and the nonfluorescent mutant 31Rl-26. Two of the 718 separate conjugations resulted in transconjugants which were then fluorescent. Again, repeated attempts to isolate the recombinant plasmid from these transconjugants were unsuccessful, indicating that the cloned fragment complementing this ~qenetic lesion may be incorporated into chromosomal DNA of the transconjugant clone.
The pLAFRl recombinant plasmids were isolated from both B 101 clones which resulted in fluorescent transconjugants. These recombi.nant plasmids were designated pS FL-l and pC FL-l. EcoRI digestion of both plasmids indicated that they were structurally identical and are probably duplicate clones which had been separately stored.

TABLE I
COMPLEMENTATION OF NONFLUORÆSCENT MUT~NT OF
PSEUDOMONAS SYRINGAE WITH CLONED DNA FRAGMENT

~~ Growth on King's B
- ~ EDDA EDDA+lO 6M
Fluorescence ~200ppm) FeC13 . . _ _ . _ . P. syrin-qae - 3lRl + -~ +
30 P. syrinqae Cit 7 ~ +
P. syrinqae 31Rl-26 - _ +
P. ~
35 31Rl-26 (pS FL-l) + + +
P. syrin~ae 31Rl-26 (pC FL-l) + + +

---- __ It is apparent from -the above results that PGPA can be transferred to hosts which have not pre-viously had this capability. Moreover, the introduction of said DNA fragments into PGPA hosts to obtain in-creased expression of PGPA products through the use ofa multiple copy vector is also possible. Thus, organisms capable of colonizing a wide variety of soil conditions can be modified so as to provide for PGPA in new envi-ronments and/or with higher efficiency.
Although the foregoing invention has been described in some detail by way of illustration and ; example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (12)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A microorganism having enhanced plant growth promoting activity as a result of in vitro introduction into said microorganism, or into a parent of said microorganism, a DNA sequence encoding for the ability to utilize a multivalent inorganic cation in the soil in pre-ference to deleterious microorganisms, said DNA sequence being expressed by a gene present in pS FL-l (ATCC
Accession No. 39206).

2. A microorganîsm according to claim 1, wherein said DNA sequence is incorporated on an extra-chromosomal element.

3. A microorganism according to claim 1, wherein said DNA sequence is integrated into the chromosome.

4. A microorganism as in claim 1, wherein said DNA sequence encodes for the production of a siderophore which confers the preferential utilization of Fe+3.

5. A method for promoting the growth of root crops, said method comprising introducing a growth promo-tant microorganism into the rhizosphere, said microorganism having been modified by introduction in vitro of a DNA
sequence which confers the ability to utilize multivalent inorganic cation in the soil in preference to phytopathoge-nic microorganisms, said DNA sequence being expressed by a gene present in pS FL-1 (ATCC Accession No. 39206).

6. A method as in claim 5, wherein the microorganism has been modified by the introduction of a gene system which produces a siderophore capable of sequestering Fe+3 so that said Fe+3 is unavailable to the phytopathogenic microorganisms.

7. A method for modifying a microorganism to enhance its ability to compete with other microorganisms for limited Fe+3 nutrient, said method comprising intro ducing to said modified microorganism a gene system capable of producing a siderophore which sequesters at least a por-tion of the available Fe+3, making said portion unavailable to said other microorganisms, said gene system being expressed by a gene present in pS FL-1 (ATCC Accession No.
39206).

8. A method as in claim 7, wherein said gene system is derived from a fluorescent strain of P. fluorscens, P. putida, or P. syringae.

9. A microorganism modified by the method of claim 7.

10. A microorganism as in claim 9, selected from the group consisting of fluorescent strains of Pseudomonas.

11. A method for conferring on a host the abi-lity to produce a fluorescent siderphore capable of sequestering Fe+3, said method comprising:

screening a gene library obtained from a fluorescent strain of Pseudomonas to obtain a DNA sequence which produces the siderophore;
incorporating said DNA sequence onto a vector capable of replication and expression in the host; and introducing the vector to the host.
12 . A DNA sequence encoding for the production of a fluorescent siderophore and having a length of about 20kbp or less, said DNA sequence being expressed by a gene present in pS FL-1 (ATCC Accession No. 39206).
13. A DNA sequence as in claim 12, wherein the DNA is derived from the chromosomal DNA of a fluorescent strain of Pseudomonas.
14. A vector containing the DNA sequence of

claim 12.