USE OF BACTERIAL LUCIFERASE STRUCTURAL GENES FOR CLONING AND MONITORING GENE EXPRESSION IN MICROORGANISMS AND FOR TAGGING AND IDENTIFICATION OF GENETICALLY ENGINEERED ORGANISMS

Abstract: A host microorganism is genetically and stably modified by the insertion in any of its non-essential chromosomal locations a foreign DNA fragment comprising a non-homologous fragment containing a lux AB gene of a selected bioluminescent bacterium such as V. harveyi such that the nonhomologous fragment is stably reproduced as a part of the chromosome of the cells of the host microorganism as they divide in a growth medium and the gene expression of such microorganism triggers the production of luciferase which, in turn emits light in the presence of aldehyde as a measure of gene expression. Both Gram-positive and Gram-negative bacteria are microorganisms which may be modified as aforesaid. X-ray film can be used to quantify the light being emitted from a microorganism through the use of plural droplets containing the same microorganism each with a known and related cell (or plasmid) count.

Description: USE OF BACTERIAL LUCIFERASE STRUCTURAL GENES FOR CLONING AND MONITORING GENE EXPRESSION IN MICROORGANISMS AND FOR TAGGING AND IDENTIFICATION OF GENETICALLY ENGINEERED ORGANISMS BACKGROUND OF THE INVENTION Microorganism markers are a necessary tool in the study, the manipulation, and the control of gene expression in the fields of research, production and control of microorganisms. This invention relates to the use of the lux AB gene cluster of Vibrio harveyi, a luminescent marine bacterium, as such a marker and/or tag. Naturally occuring microorganisms need to be tagged or marked for monitoring their gene activity whenever that is necessary or desired for whatever reason. Moreover, ways need to be developed to label each genetically engineered organism so its use, identity and fate can be monitored in the field. This would greatly simplify studies of an organism's escape, survival, multiplication and dispersal and may help in tracking the movement of DNA among organisms in the environment. The isolated lux AB gene cluster of ^ harveyi was selected as a marker or tag because it was found to have several advantageous features including an unrestricted host background and an unrestricted growth medium requirement. In this respect, the lux AB genes of ^ harveyi are different from the lac Z gene of Escherichia coli, a commonly used expression marker which, when fused with a promoter, has a restricted host background (i.e. it can be only used in microorganisms with no ability to utilize lactose). Most microorganisms will utilize lactose. Moreover, the cells containing lac Z fusions have to be grown in minimal media (a restricted number of components) in order for the lac Z product (B-galactosidase) to be detected. An especially attractive reason for using the lux AB gene of ^ harveyi as a marker or tag for a microorganism is that it provides for the noninvasive measurement of the presence and absence of the marker through the presence or absence of bioluminescence as a measure of gene expression in vivo. The magnitude of biolu inescence is readily quantizible by new and known light detection bioluminescence is readily quantizible by new and known light detection techniques. The lac Z marker system does not have these capabilities. It uses a chemical substrate the color and intensity of which is the indicator of the reaction rate within the bacterium. As known in the prior art, light is produced in E_ j _ coli cultures that contain seven lux genes cloned from another and different marine bacterium, Vibrio fischeri. (See an article entitled "Measuring Gene Expression with light" by Engebrecht et al. Science Volume 227, March 15, 1985, pages 1345-1347). Therein it states in the abstract: These lux genes were disconnected from their native promoter and inserted into the transposon mini-Mu. The resulting transposon, mini-Mu lux, could induce mutations by insertional inactivation of a target gene, and the lux DNA was oriented to align target gene transcription with that of the lux genes. Genes in Escherichia coli and Vibrio parahaemolyticus were mutagenized, and mutants containing transposon-generated lux gene fusions produced light as a function of target gene transcription. However, the teachings of Engebrecht, et al. have limited application to the marking and tagging of microorganisms because all seven of the lux genes in V. fischeri are required for light production and the large size of this gene cluster increases the chance of its inactivation by mutation. Furthermore, it is not known whether the lux gene cluster of y_ j _ fischeri could reliably function as a measure of activity in microorganisms such as Bradyrhizobium. However, it does so function in E^ coli and it does provide its own substrate for the production of light. A new avenue for research and development in this field was presented by an article entitled "Cloning of the Luciferase Structural Genes from Vibrio harveyi and Expression of Bioluminescence in Escherichia coli" by Baldwin, et al. appearing in Biochemistry, 1984, Vol. 23, Pages 3663-3667. Therein bacterial luciferase in the luminous marine bacterium Vibrio harveyi was discussed as follows: "The DNA encoding the luciferase alpha and beta subunits in the luminous marine bacterium Vibro harveyi (strain 392) is contained within a 4.0-kilobase Hindlll fragment. DNA from V. harveyi was digested with Hindlll, and the resulting fragments were inserted into the Hindlll site of plasmid pBR322. The recombinant plasmids were introduced by transformation into Escherichia coli RR1. The colonies were supplied with n-decanal, the substrate for bioluminescence reaction, and 12 colonies (of ca. 6000 total) were observed to luminesce brightly. One of the recombinant plasmids, pTB7, has been studied in detail. The high level of expression of bioluminescence in pTB7 was the result not of native V^ harveyi promoters but rather of a promoter in pBR322 which is within the tetracycline resistance gene but oriented in the direction opposite to the transcription of the tetracycline gene." The recombinant plasmid vector pTB7 is available from the Agriculture Research Service Patent Collection, Northern Research Center, Peoria, Illinois, 61604 and has been given the designation NRRL B-15231. While the Baldwin et al. article describes the use of the lux AB genes of V. harveyi to select the recombinant plasmid vectors pTB7 in E^ coli as stated, it is clear that it does not teach or show the isolation of those recombinant DNA vectors from the E j _ coli by any means for the purpose of establishing a marker or tag in E^ coli or any other organism as a measure of gene expression. The recombinant plasmid vector pTB7 was isolated only for the purpose of cloning the luciferase gene lux AB. U.S. Patent 4,581,335, inventor Thomas 0. Baldwin, dated April 8, 1986, which relates to the same research as the Baldwin et al. article, supplements the report by suggesting that the plasmid vector pTB7 be transferred to a host E^ coli such as E^ coli P.R1. The transformed E^ coli is given the designation E^_ coli /pTB7 and is deposited at the same place and under the same designation. The type of transformation involved is the transfer of the plasmids pTB7 through and within the cell walls of the E^ coli without insertion of the same into the chromosome of the E^ coli. The plasmid pTB7 replicates outside of the chromosome of E. coli. SUMMARY OF THE PRESENT INVENTION The bioluminescence genes lux AB contained on the plasmid pTB7 in E^ coli RR1 cannot be transferred to any other microorganism, because this plasmid will not replicate in any bacterium, other than E^ coli. The teachings of this invention involve a transfer of the lux AB genes into any Gram-negative microorganism by conjugation using the mob ( ori T) DNA fragment of RP4, and insertion of said genes into the chromosome of the same microorganism, thus providing a genetically stable modification of the same microorganism here and after referred to as "host" or "target organism". When the teachings of the present invention involve the transfer of the lux AB genes to Gram-positive bacteria, a non-conjugal transfer of this DNA is carried out by either calcium-mediated transformation of competent bacterial cells or by the electroporation technique, as referred to below. Prior to the present invention there were no teachings of transferring the lux AB genes to any microorganism other that E^ coli, and no teaching of inserting said genes into the chromosome of any microoganism, including E. coli. As part of the teachings of the present invention the inventors herein were able to modify plasmid vector pTB7 to make what they call a promoter-activated light emission vector herein identified as pPALEOOI . Specifically in the construction of pPALEOOI, a 0.6 kb Sall-Hindlll fragment of pBR322 and a 0.3 kb Eindlll-Sall fragment of V^ harveyi DNA were deleted to form a single promoter-cloning site Sail, located about 130 bp upstream from the translational initiation codon of lux A. Also, the neomycin phosphotransferase gene (Km) was excised from plasmid pREV1000 with Hindlll-Aval and cloned into a Bglll site of pPALEOOI by blunt-end ligation. Even though the P1 promoter of pBR322 was deleted from pPALEOOI , the resulting plasmid did express detectable bioluminescence at about 1% level of that of pTB7. To eliminate this read-through from upstream sequences, trp A transcription terminator (Pharmacia) was ligated into the Xmalll site of pPALEOOI located about 0.3 kb upstream of the promoter-cloning site. The plasmid vector pPALEOOI is a promoterless source of lux AB genes and is used in the present invention to clone a promoter from the selected microorganism of interest, upstream of the lux AB genes to control the luciferase expression. Since pPALEOOI replicates in E^ coli only, the cloning of a promoter from the microorganism of interest is carried out in E_;_ coli as the first step of the present invention. As described hereinafter, a obilizable plasmid vector must ' be especially constructed for its use in the transfer of the promoter lux AB gene fusion from the plasmid vector pPALEOOI to the microorganism of interest. For example, the mobilizable plasmid vector pMR19 was constructed for the specific purpose of inserting a promoter- lux AB gene fusion into the chromosome of B^ aponicum. The plasmid pMR19 contains a 13.5 kb fragment of B^ japonicum chromosome, and on this basis it is capable of inserting any foreign genes placed within said homology, including the above promoter - lux AB gene fusion, specifically into the B^ .japonicum chromosome by double homologous recombination. It is important to note that the 13.5 kb chromosomal homology fragment of pMR19 can be easily removed with the restriction enzyme Kpnl and replaced with a chromosomal fragment of any other Gram-negative bacterium and thus it will mediate the insertion of the bioluminescence genes into any other bacterial chromosome. The teachings of the present invention include the step of excising with Aval the DNA fragment containing a promoter- lux AB gene fusion of all other genes as a part of preparing said fragment to be a part of the carrier vector, e.g. pMR19 for B^ aponicum, for insertion into the chromosome of any target microorganism. Specifically, the carrier vector is prepared by cutting "non-essential" or "silent" DNA pieces out of the chromosome of the target microorganism. * The promoter- lux AB gene fusion is then inserted into this fragment, and the DNA carrying the promoter- lux AB gene fusion is inserted into the chromosome of the target microorganism by homologous recombination. Homologous recombination is a known technique for introducing foreign DNA into the chromosome of a microorganism. For example an article by Legocki, R.P., YUN, a.c. and Szalay, A. 1984 Proc. Natl. Acad. Sci USA 81 pg. 5806-5810 illustrates the insertion of a promoter-marker gene fusion into a Rhizobiuπ chromosome through the use of homologous recombinations. The subsequent transformation of the target microorganism with the pMR19-like plasmid carrying the promoter- lux AB fusion involves two general question. First, what transformation technique should be used to cause the pMR19-like plasmid to cross the cell wall of the target microorganism, and second, will this plasmid replicate episomally or should it be integrated into the chromosome. As is known to those skilled in the art access through the cell wall of the microorganism is gained by (1) soaking the cells in calcium chloride (calcium precipitation); (2) subjecting the cells to electrical shock (electroporation) , or (3) utilizing a mobilizable plasmid following the techniques well known to those skilled in the art and illustrated in an article by Simon, P.. Priefler, U. and Puhler, A; pages 98-106 of a book entitled Molecular Genetics of the Bacteria-Plant Interaction by Springer-Verlag Berlin Heidelberg, 1983 Dr. Alfred Puhler, Editor. The transformation of the promoter- lux AB gene fusion into any host microorganisms such as B^ japonicum for use as a marker or tag of gene expression requires an assured technique'for accessing through the cell wall of the microorganism and a mechanism for a stable insertion of the fragment containing the lux AB fusion into the chromosome of the host microorganism such as B^ japonicum. Accordingly, it is essential to the teachings of the present invention that a mobilizable plasmid (a carrier vector) be especially made for the host (target) microorganism which will pass through the cell wall by conjugation, and through homologous recombination will insert the promoter- lux AE gene fusion stably into the chromosome of the host microorganism. The technology for the construction of such a mobilizable plasmid carrier vector established and known to those skilled in the art has been illustrated by the Legocki et al. and Simon et al. publications. The teaching of this invention is that foreign DNA to be circularly inserted into the chromosome of the host microorganism using homologous recombination is a promoter- lux AB gene fusion, where the promoter comes from the same hose icroorganims of interest, and lux AB genes come from V^ harveyi. Specifically, a mobilizable insertional plasmid such as pMR19 mediates a stable insertion of the promoter- lux AB gene fusion into the procaryotic organism such as B^ aponicum. This plasmid comprises a DNA segment including first and second DNA portions containing DNA homologous to corresponding portion of the chromosome of the microorganism such as B^ japonicum with the first and second DNA portions oriented in relation to each other in the same manner as said homologous chromosomal DNA portions in B^ japonicum and a third DNA portion containing the promoter- lux AB gene fusion located between and covalently bonded to the first and second DNA portion. The mobilization plasmid pMR19 used by the invention herein to insert the lux AB gene fusion into the chromosome of B^ j ponicum using the homologous recombination technique described herein is on patent restricted deposit (Budapest Convention) with the U.S. Department of Agriculture, Northern Regional Research Center, NRRL 1815, North University Street, Peoria, Illinois, 61604, and assigned accession number NRRL B-18081. The mobilization plasmid REV1000 may be used to insert the lux AB gene fusion into the chromosome of stem Rhizobium BTAil using the homologous recombination technique described herein and is on patent restricted deposit (Budapest Convention) with the aforesaid NRRL under accession No. NRRL B—153—19• Both ρMR19 and pREV1000 plasmids contain the mob ( ori T) DNA fragment that can be isolated from RP4 of E^ coli as described in the above mentioned article of Simon et al. (1983). The mob sequences are used in the present invention to establish an efficient conjugal transfer of a plasmid containing the promoter- lux AB gene fusion from Ε^ coli to any Gram-negative bacterium, as exemplified for JB^ aponicum using the plasmid pMR19. In accordance with the present invention, the fragment including the promoter- lux AB gene fusion may be inserted as a marker or tag into the chromosome of any Gram-negative bacterium. Cyanobacteria and photosynthetic microorganisms are other examples. The appropriate chromosomal homology region on this mobilizable plasmid to mediate insertion of the promoter- lux AB gene fusion into the chromosome of a particular microorganism will have to be attached for each separate and distinct microorganism. Conjugal transfer of a mobilizable plasmid vector (herein pMR19 containing the promoter- lux AB gene fusion) from Ξ^ coli to Ε japonicum is a known straightforward procedure that has been described in detail by Jagadish and Szalay Mol Genet. (1984) 196:290-300. Referring to paragraph 2 of page 292 of that paper, the bacterial conjugation for the transfer of lux AB-containing plasmid DNA for insertion into the selected bacterium is accomplished as follows: Donor E^ coli and the recipient bacterium of interest are grown separately in the appropriate media at 30°C and are mixed at approximately equal cell numbers (1 - 1.5 x 1θ9 cells). The mixture is centriifuged and resuspended in 100 ml of the culture medium and 50 ml of this suspension was spotted onto sterile filters (0.45 mm GAG Golman Sciences, Inc.) placed oh the culture medium. Following incubation at 30°C for 24 hours the cells are removed from the filter surface using a sterile toothpick washed in 5 ml of minimal medium and resuspended in 1 ml of fresh minimal medium. Undiluted or serially diluted samples are plated,onto appropriate selective plates for growth of transco jugants. While the transfer of promoter- lux AB gene fusions to host microorganism in the present invention is mediated by mob genes contained e.g. on the plasmid pMR19 via conjugal transfer, and thus is limited to Gram-negative bacteria, it should be clear that the same teachings of this invention apply to Gram-positive bacteria where the other two methods of cell transformation, listed above, should be used. In addition to pPALEOOI , another plasmid vector, known as pFIT001 , has been constructed which contains lux AB genes in accordance with the teachings of the present invention. In pFIT001 , the lux AB genes are expressed consti utively from the P1 promoter of pBR322 plasmid vector. pFIT001 is constructed by modifying plasmid vector pTB7. Specifically: (1) the EcoRI site, located downstream (at the 3' end) of the 4.0 kb jΛ, harveyi DNA fragment in the pBR322 region is inactivated by blunt ending; (2) the Hindlll site located downstream (at the V end) of the 4.0 kb V^ harveyi DNA fragment was inactivated by blunt ending. E. coli carrying derivatives of pFITOOI with DNA inserts in the unique EcoRI site located in lux B form "dark" colonies that are readily distinguished from the biolu inescent or "bright" colonies carrying the original plasmid. Due to its simplicity, the pFITOOI is used as a common cloning vehicle for rapid visualization of plasmid clones carrying inserts. Furthermore, a single Hindlll site in pFITOOI, located between the P1 promoter and lux A, is suitable for testing DNA fragments for the presence of transcriptional terminators based on bioluminescence. While the technical studies described herein were done with the lux AB genes of a biolumenscent bacterium ^ harveyi as made available from the plasmid vector pTB7, it is clear that should another bioluminescent bacterium (such as V^ fischeri ) be of interest and one wished to use the lux AB genes (only) of that other bioluminescent bacterium, one could proceed as described in a publication commented on herein above by Baldwin et al. Biochemistry 1984, 23, 3663. Essentially a plasmid vector (equivalent to pTB7) would have to be cloned in E^ coli containing the lux AB genes of the selected bioluminescent bacterium (such as ^ fischeri, photobacterium, leiognathi, P. phosphoreum and the terrestrial bacterium xenorhobdys) . The control and regulation of gene expression can be readily analyzed in vivo using promoter- lux AB fusions by a variety of simple methods, including a new technique described herein as "luxdot". It is a primary object of the present invention to teach the genetically stable modification of a host microorganism by the insertion in one of its non-essential chromosomal locations of a DNA fragment containing lux AB genes of a selected bioluminescent bacterium such as V^ harveyi fused to a selected promoter isolated from the host microorganism wherein the non-homologous fragment is stably reproduced as a part of the chromosome of the cells of the modified host microorganism as they divide in a growth medium. It is another object of the present invention to teach the genetically stable modification of a host microorganism by the insertion in one of its non-essential chromosomal locations a fragment containing lux AB genes of a selected bioluminescent bacterium such as V^ harveyi fused to a selected promoter isolated from the host microorganism fused to a selected promoter isolated from the host microorganism wherein the non-homologous fragment is stably reproduced as a part of the chromosome of the cells of the modified host microorganism as they divide in a growth medium and the gene expression of such microorganism triggers the production of luciferase which, in turn emits blue-green light in the presence of aldehyde as a measure of gene expression and to serve as a marker or log for the modified microorganism. It is another object of the present invention to teach the genetically stable modification of any host microorganism of the insertion in one of its non-essential locations of a fragment containing lux AB genes of a selected bioluminescent bacterium such as V^ harveyi fused to a selected promoter isolated from the host microorganism wherein the non-homologous fragment is stably reproduced as a part of the chromosome of the cells of the modified host microorganism as they divide in any growth medium known to support growth of the host microorganism in its unmodified form. It is another object of the present invention to teach the genetically stable modification of any host microorganism by the insertion in one of its non-essential chromosomal locations of a fragment containing lux AB genes of a selected bioluminescent bacterium such as ^ harveyi fused to a selected promoter isolated from the host microorganism wherein the non-homologous fragment is stably reproduced as a part of the chromosome of the cells of the modified host microorganism as they divide in a growth medium (normal to the unmodified host microorganism) and the gene expression of such microorganism triggers the production of luciferase which, in turn emits blue-green light in the presence of aldehyde as a measure of gene expression and to serve as a marker or tag for the modified microorganism. It is another object of the present invention to teach the genetically stable modification of a host microorganism by the insertion in one of the non-essential chromosomal locations a fragment containing lux AB genes of a selected bioluminescent bacterium such as V^ harveyi fused to a selected promoter isolated from the host microorganism wherein the non-homologous fragment is stably reproduced as a part of the chromosome of the cells of the modified host microorganism as they divide in a growth medium and the gene expression of such microorganism the production of visible light in the presence of aldehyde as a measure of such activity without the need for drug markers, dyes or other indicators. It is another object of the present invention to teach the genetically stable modification of a host microorganism by the insertion in one of the non-essential chromosomal locations of a fragment containing lux AB genes of a selected bioluminescent bacterium such as V^ harveyi fused to a selected promoter isolated from the host microorganism wherein the non-homologous fragment is stably reproduced as a part of the chromosome of the cells of the modified host microorganism as they divide in a growth medium and the gene expression of such microorganism is monitored by the production of visible light in the presence of aldehyde as a measure of such activity and its. detection does not require any harm or destruction to the living cells or organism. It is another object of the present invention to teach the genetically stable modification of a host microorganism by the insertion in one of the non-essential chromosomal locations of a fragment containing lux AB genes of a selected bioluminescent bacterium such as V^ harveyi fused to a selected promoter isolated from the host microorganism wherein the non-homologous fragment is stably reproduced as a part of the chromosome of the cells of the modified host microorganism as they divide in a growth medium and the gene expression of such microorganism causes the production of visible light in the presence of aldehyde and the intensity of the light as a measure of gene expression can be determined by any one of several simple and rapid methods including: visual assessment by the naked eye, brief exposure to X-ray films, the use of a liquid scintillation counter and the use of in vitro and in vivo luciferase erazyme assays as well as a new and novel light-quantifying method known as luxdot and further described herein. It is another object of the present invention to teach the geneticall stable modification of any replicative DNA forms (bacteriophages, viruses cells and other .Living organisms) by the insertion in one of the non-essentia chromosomal locations a fragment containing lux AB genes of a selecte bioluminescent bacterium such as ^ harveyi fused to a selected promote isolated from the host microorganism wherein the non-homologous fragment i stably reproduced as a part of the chromosomal components of the cells of th modified host microorganisms as they divide ina growth medium and the gen expression of such microorganism causes the production of visible light in th presence of aldehyde and the intensity of the light as a measure of gen expression can be determined for any of the following purposes: 1. The protection and identification as it relates to the modified microorganism. 2. The detection of gene expression signals, including regulatory signals of the modified microorganism. 3. The tracing of tumorous growth and cell migration by tracing a modified microorganism. 4. The tracing of modified soil microorganisms and monitoring this interaction with plants and other organisms. It is still another object of the teachings cf the present invention t provide a modified form of plasmid vector pTB7 to make a promoter activate light emission vector herein identified as pPALEOOI on deposit wit Agricultural Research Service, USDA, Peoria, Illinois 61604 under accessio number NERL B-180S2 where said pPALEOOI has many advantages as a marker or tag because it obtains its promoter from the modified host microorganism int which it is inserted within a. non-essential chromosomal location and th resulting bioluminescence is a measure of the occurance of the microorgan and magnitude of that activation is the measure of promoter strength and gene expression in general. It is still another object of the teachings of the-present invention provide a modified form of plasmid vector pTB7 to make a. common cloni vehicle for rapid visualization of plasmid clones carrying inserts here identified as pFITOOI on deposit with • Agricultural Research Service, USD Peoria, Illinois 61604 under accession number NRRL E-180S0 and furthermo having a single Hindlll sight located between the P1 promoter and lux suitable for testing DNA fragments for the presence of transcription terminators based on bioluminescense. It is an additional object of the teachings of the present invention provide a new and improved method for quantizing the light emitted fr biological material using the exposure of x-ray film from a plurality droplets of said biological material when each of the plural droplets conta a known count of cells (or plasmids) of said biological material and ea related droplet is a diluted sample with a related count of cells or plasmi which is based on the scale which has been selected for the purpose quantizing the magnitude of the light. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A, 1B AND 1C illustrate plasmid vectors pFITOOI, pPALEOOI an pMR19 respectively. Figures 2A, 2B, 2C, 2D and 2E are separate panels illustrating the visua identification of plasmid clones containing inserts using pFITOOI . Figures 3A, 3B, 3C, 3D, and 3E are separate panels illustrating the method used to monitor gene expression by bioluminescence following the teachings o this invention. Figures 4A, 4B, 4C, and 4D are separate panels depicting plura measurements and/or observations as it relates to the insertion of lux A genes into the chromosome of B^ japonicum. Figure 5 illustrates the expression of bioluminescence in soy bean nodule controlled by a constitutively expressed P1 promoter (A) and the symbioticall regulated nif D promoter (B) as monitored by the luxdot assay on x-ray film. DETAILED DESCRIPTION OF THE INVENTION The "Luxdot" system disclosed and taught herein is novel and has h utility. As stated a key teaching herein is that the lux AB genes of harveyi " can be stably inserted in the chromosome of a wide range microorganisms without altering the growth medium composition in which t microorganism will grow and the gene expression of that modified microorgani may be measured by the release of light at a sensitivity level measurable wi femtomoles of luciferase and within a few seconds ( i vivo. ) One of t many ways to measure that intensity of released light is to direct that lig onto x-ray film. The developed film will give a relative measurement of t intensity of the produced light. Since the intensity of the light which produced and described herein above is a measure of the rate of ge expression, it is extremely important that the intensity of the released lig be quantized. The novel technique to accomplish this quantization which described herein has a very high degree of utility and simplicity. The microorganism which has been modified by the stable insertion of promoter- lux AB fusion in a non-essential location within its chromoso following the teachings of the present invention, or one that harbors th gene fusion on a plasmid is grown in liquid media to mid or late log phase a about 1 - 5 ul volume (10? - 10 8 cells) are pelleted by centrifugation. Cells were resuspended in 0.1 ml of water on ice a dilutions were made in water to inhibit cell division. Following t determination of cell numbers with a he ocytometer plural dilutions are ma of the modified microorganism so that the ratio of cells in a 5 ul droplet each dilution is known and then the plural 5 ul droplets of each dilution a placed in an order based on the degree of dilution (or cell copy number) the bottom of an empty plastic Petri dish. A streak of n-decanal is appli to the inner side of the lid and the covered Petri dish is exposed to an X-r film at room temperature. The exposure time can vary between a few secon and several hours, depending on the promoter strength, the copy number of t promoter- lux AB gene fusion and the number of cells in a droplet. The lig intensity recorded by the X-ray film from the plural 5 ul drople representing different dilutions and cell numbers in accordance with t selected scale represents the quantified gene expression with the modifi microorganism as represented by the activation of the promoter lux A3 ge fusion. The selection of 5 ul droplets rather than another volume within limits a matter of choice and the important point is that the droplets from the hi concentration cf potentially light-emitting cells (or plasmids) to the lowe concentration be known in number of cells (or plasmids) and follow t selected scale of the potentially light-emitting cells. The droplets must small enough to maintain themselves as a cohesive drop and form a column droplets of scaled dilutions (and scaled numbers of potentially light-emitti cells or plasmids) . Plural columns of droplets with each column representi a different modified microorganism in plural dilution (or cell ccunt) may arranged as grid in a single Petri dish. A primary teaching of the Luxd system is that each droplet contains a known number of potential light-emitting cells where the light released is quantified by simultaneous sampling plural droplets having cell (or plasmid) counts which are related a selected side where each droplet is placed in a selected location (colu and/or grid) on a flat surface of the Petri dish (or the equivalent) and t X-ray film is placed immediately outside the Petri dish (or its equivalent) record the light emission. If the plural droplets are placed in colum and/or in a grid on the bottom of the Petri dish, the X-ray film may be plac right under the Fetri dish tc record the ii -ht emission from the plur droplets. If the plural droplets are small enough to adhere to the undersi of the Petri dish cover in columns and/or a grid then the Σ-ray film may placed on the top of the Petri dish cover. This system for quantifying t amount of light being emitted from plural droplets of a known number biological cells has broad application. While the system was first used making light measurement based en emissions resulting from the stab insertion of the lux AB genes of ^ harveyi into the chromosomes of a wi range of microorganisms, the Luxdot system cay be used for the quantifyi light emission from all kinds of biological material which emits light as lo as the magnitude of the light is directly related to the number of cells ( plasmids) in the biological material. The understanding of the genetic involvement of Rhizobium spp. in the symbiosis with legumes, leading to the formation of nodules and nitrog fixation, has increased substantially in recent years. The followi publications are cade of reference: (Ruvkun, G.E. & Ausubel, F.K. (198 Proc. Natl. Acad. Sci. USA 77, 191-195.; Banfalvi, A., Sakanyan, V Koncz, C, Kiss, A., Dusha, II. & Kondorosi, A., (1981) Mol. Gen. Gene 184, 318-325.; Frakash, R.K., Schilperoort, R.A. & Nuti, M.P. (1981) Bacteriol. 145, 1129-1136.; Long, S.R., Buike a, W.J. & Ausubel, F. (1982) Nature (London) 298, 485-488. Since the initial identification of t nitrogen fixation ( nif ) and nodulation ( nod ) genes in rhizobia, the promoter regions have been fused to the lac Z gene of E^ coli and measured f expression and regulation by the beta-galactosidase enzyme assay (see Mille J.h. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laborator Cold Spring Harbor, NY), 2nd Ed. p. 352). Both transcriptional a translational fusions were used to detect and quantify the activation of meliloti nifH promoter by the nif A gene product of Klebsiella pneumoni (Sundaresan, V., Jones, J.D.G., Ow, D.W. & Ausubel, F.M. (1983) Natur (London) 301, 728-732.), to monitor the expression of the regulatory nif and gin ALG operons of K^ pneumoniae, and to show that a chromosomall integrated nif E promoter- lac Z fusion is regulated in trans with the nativ nif locus in stem Rhizobium BTAil. Reference is cade to: Dru mond, M. Clements, J. Merrick, M. & Dixon, R. (1983) Nature (London) 301, 302-307.; Ow D.W. δ Ausubel, P.m. (1983) Nature (London) 301, 307-313.; Legocki, R.P. Yun, A.C. & Szalay, A.A. (1984) Proc. Natl. Acad. Sci. USA 81 5806-5810. Recent studies indicated that expression of the R^ meliloti no ABC genes is activated by root exudates and by the nod D gene product, a evidenced by beta-galactosidase measurements using nod C and nod D promoter lac Z gene fusions (Mulligan, J.T. & Long, S.R. (1985) Proc. Natl. Acad Sci. USA 82, 6609-6613). Herein we teach the use of the bacterial luciferase genes lux AB from V harveyi to monitor the activity of procaryotic promoters, including th symbiotically regulated nif D and nif H promoters of Bradyrhizobiu japonicum. The root nodules formed by B^ aponicum transconjugants containin a single copy per genome of a nitrogenase promoter- lux AB fusion emit light of sufficient intensity to be detectable by the dark-adapted eye. Promoter- lux AB fusions can be detected and quantified in vivo with a high signal-to-noise ratio in unrestricted host background without noticeable effect on the growth or viability of the cultures. V. harveyi luciferase is a heterodi er (alpha beta) encoded by two cctranscribe genes ( lux AB) of approximately 2.0 kb in size (Baldwin, T.O., Berends, T., Bunch, T.A., Holzman, T.F., Rauεch, S.K., Sha ansky, L., Treat, M.E. δ Ziegler, M ~ .m. (1984) Biochemistry 23, 3663-3667). A 4-0 kb Hindlll fragment containing the lux AB genes of V j _ harveyi was cloned into pBR322 (pTB7) and shown to be expressed in. E^ coli (Baldwin, T.O., Berends, T., Bunch, T.A.., Holzman, T.F., Eausch, S.K., Shamansky, L., Treat, M.L. δ Ziegler, .M. (1984 Biochemistry 23, 3663-3667.) from the P1 (anti-Tet) promoter (Stuber, D. δ Bujard, E. (1981) Proc. Natl. Acad. Sci. USA 78, 167-171). The nucleotide sequence of lux AB indicated that the alpha and beta subunits have molecular weights of 40,108 and 36,349, respectively, even though their migration in SDS-polyacrylamide gels showed sizes of 42,000 and 37,000 daltons. Reference is made to the following publications: Co n, D.H., Mileham, A.J., Simon, M.I., Nealson, K.H. , Rausch, S.K., Bona , D. & Baldwin, T.O. (1985) J. Bioi. Chem. 260, 6139-6146.; Ziegler, M.M. δ Baldwin, T.O. (1981) Curr. Top. Bioenerg. 12, 65-113. The single active center resides primarily, if not exclusively, on the alpha subunit but both subunits are required for activity (Ziegler, U.K. δ Baldwin, T.O. (1981) Curr. Top. Bioenergy. 12, 65-113.) Bacterial luciferase catalyzes the flavin-mediated hydroxylation of a long-chain aldehyde to yield the carboxylic acid and an excited flavin; the flavin decays to ground state with the concomitant emission of light (lambda max=490nm) . The overall reaction is shown below. [Luciferase] FMNE 2 + 0 2 + RCH0 FMN + RC00H +- H 2 0 + hr490nm Colcnies expressing luciferase genes emit blue-green light upon exposure to aldehyde substrate. The bioluminescence can be detected either visually or by photography with Polaroid films, and it can be quantified by densito etry of X-ray films, with a photo ultiplier photometer, or in a scintillation counter. Strains, Plasmids and Plant Material. Braάyrhizobium ponicum USDA stra 110 was provided by A. Eaglesham (Boyce Thompson Institute). Ξ^ coli strai used were E3101, JA221 , and SM10. Reference is made to the followi publications: Boyer, H.W. & Roulland-Dussoix, D. (1969) J. Mol. 3iol. 4 459-472.; Clarke. L. δ Carbon, J. (1979) Gene 5, 111-126.; Simon, R Priefer, U. & Puhler, A. (1983) in Molecular Genetics of the Bacteria- Pla Interaction, ed. Puhler, A. (Springer, Eerlin), pp. 98-106. Plasmid pMR19 w derived from pJN13 by moving mob genes from the EcoRI site to BamHI, th restoring chloramphenicol resistance, and leaving a single Hindlll si located in the middle of a 13 kb chormosomal fragment from B^ japonicum. Soybean ( Gl cine max, var. Wilkin) seeds were inoculated and plants grown a controlled environment as described by Jagadish and Szalay (Jagadish, M. δ Szalay, A.A. (1984) Mol. Gen. Genet. 196, 290-300). Nodules wer excised, weighed and tested for nitrogen fixation. Plasmid pMRl9 is o deposit as identified herein above. Plasmid Constructions♦ Plasmids pFITOOI and pPALEOOI are derivatives o pBR322. The lux AB gene cluster of Vibrio harveyi was from pTB7, and th restriction sites Hindlll, EcoRI and Sail were made unique as shown in Figur 1. The neo ycin phosphotransferase gene (Km) was excised from pREV1000 1A an 1B with Hindlll-Aval and cloned into a Bg1II site of pPALEOOI by blunt-en ligaticn. In the construction of pPALEOOI, a o.6 kb Sall-HincIII fragment o pBR322 and a 0.3 kb HindIII-Ka1I fragment of ^ harveyi DNA were deleted t form a single promoter-cloning site, Sail, located about 130 bp upstream fro the translational initiation codon of lux A. Even though the P1 promoter o pBR322 was deleted from pPALEOOI , the resulting plasmid expressed detectabl bioluminescence at about 1 the level of pTB7. To eliminate this read-throug from upstream sequences, tr p A transcription terminator (Christie, G.E. Frarnham, P.J. δ Platt, T. (1981) Proc. Natl. Acad. Sci. USA 78 4180-4184) was ligated into the Xmalll site of pPALEOOI located about 0.3 k upstream of the promoter-cloning site. E. coli-Rhizobiuffl Conjugations. Mobilizable plasmids carrying the lux AB genes and the origin of transfer ( ori T or mob ) fragment were conjugated from Z coli strain SM10 to Bradyrhizobiu japonicu strain 110 en TT plates as described in the publication by Jagadish, et al. further identified hereinabove. Transconjugants were selected on Rhizobium minimal media containing antibiotics at 30°C. Detection of Bioluminescent Colonies on Agar Plates. Luciferase activity in an Ξ^ coli or Bradyrhizobium colony in the presence of n-decanal (n-decyl aldehyde, Sigma) vapors was detected visually in a photographic darkroom within 10-60 seconds. A single strea of n-decanal was made on the inside of the Petri dish lid using a Q-tip. For a permanent record, colonies were photographed on Polaroid type 57 film (see legend to Fig. 2) or exposed to an X-ray film. For the latter, colonies ' were first transferred to a sterile nitrocellulose filter as if they were to be processed for colony hybridization. The filter was places on the bottom of a plastic Petri dish, covered with a lid moistened with n-decanal, and directly exposed to X-ray film at room temperature (see also Luxdot Assay). Luxdot Assay. E. coli or Bradyrhizobium. cells were grown in liquid media to mid or late log phase, and about 1-5 ul volumes (10? _ 10 8 cells) were pelleted by centri ugation. Cells were resuspended in 0.1 ml of water on ice, and dilutions were made in water to inhibit cell division. Following the determination of cell number with a hemocyto eter, 5 ul 1 droplets of each dilution were placed on the bottom of an empty Petri dish. A streak of n-decanal was applied to the inner side of the lid and the coovereά Fetri dish was exposed to an X-ray film at room temperature. The exposure time varied between a few seconds and several hours, depending on the promoter strength, the copy number of the promoter- lux AB gene fusion, and the number of cells in a droplet. Luciferase A.ssays In Vivo and In Vitro. Luciferase activity was measured using a photo ultiplier photometer (Eastings, J.W. , Baldwin, T.O. δ Nicoli, M.Z. (1978) Methods Enzymol. 57, 135-152) with the sensitivity of 9.8 x 1C quanta sec -' ' (light unit) -1 , referenced to the liεht standard of Eastings and Weber (Hastings, J.W. δ Weber, G. (1963) J. Opt. Soc. A 53, 1410-1415). The substrate used for both types of assay was a sonicat suspension of 50 ul of n-decanal in 100 ml of 0.9 H NaCl, prepared fres Assays in vivo were carried out by mixing quickly 1 ml of cell culture wi 1ml of the substrate suspension and measuring the peak value of the emitt light. For assays in vitro, cultures (0.5 ml) were centrifuged in Ξppendorf tube and the pellets frozen at -20°C. Cells were resuspended in 50 ul of 10 m EDTA, 1mM DTT, pH 7.0, and sonicated on ice. The reaction mixture consiste of 10ul of the sonicated cell extract, 1 ml of the assay buffer (0.2% 3SA i 50 mM phosphate buffer, pH 7.0), 10 ul of the aldehyde substrate suspension and 1-1.5 ml FMNE (reduced by H with 10% Ft on activated carbon). Measurements of Bioluminescence in a Liquid Scintillation Counter. Absorbance of log phase cultures was first measured at 600 nm to determine th approximate cell number. A 1-10 ul volume of the ' culture (1.5 x 10^ - 1.5 x 1θ5 cells) was pipetted into a scintillation vial containing 1 ml E 0 and 50 ul of the sonicated n-decanal solution, prepared as described above, and the light was measured in the 32p channel. The peak value wa recorded within 1 to 2 minutes, or less if more n-decanal solution was used. Figures 1A and 1B pFITOOI and pPALEOOI respectively plasmid vectors Following the insertion of ^ harveyi DNA (heavy line), the EcoRI and Hindll sites of pBR322 downstream of the cloned fragment were inactivated b blunt-end ligation. The 70 bp nucleotide sequence upstream of the AT indicated in pPALEOOI contains translational stop codons (brackets) in all translational reading fra s. The asterisk at the Xmalll site of pPALEOO indicates that this restriction site has been inactivated by blunt-en instertion of a trp A transcription terminator. B/H and B/A indicate sites o blunt-end ligation of a Eindlll-Aval fragment containing the neomyci phosphotransferase gene (Km) into a Bg1II site of pPALECOI . Figures 2A, 2B, 2C, 2D and 2E provide visual identification of plasmid clones containing inserts " using pFITOOI. E j _ coli strain EE101 clones harboring pFITOOI with and without inserts were grown on an a picillin- containing agar plate and shown photographed (in the panel) . The Figure 2A agar plate was treated with n-decanal and photographed on Polaroid type 57 film in the dark with an object-to-lens distance of 40 cm, 15 minute exposure, and an f-stop of 4.5 (is shown photographed in the panel as Figure 2B). A nitrocellulose replica of the colonies was also exposed to an X-ray film for 15 seconds and are photographed in the panel. Arrowheads in panels Figures 2A, 2B, and 2C indicate some of the dark colonies, i.e. colonies that did not show bioluminescence. The panels in Figure 2D shows an EcoRI digest of plasmid DNA isolated from 3 bright colonies (lanes a-c; no inserts) and 7 dark colonies (lanes ά- , all contain inserts). Total protein isolated from all of the above clones was analyzed in a 15% acrylamide-1% (w/w) bisacrylamide gel containing SDS (Laemmli, U.K. (1970) Nature New Biol. 227, 680-685) (panel E). The arrow indicates the position of the 37 kb beta sub unit of luci erase which is present in the bright colonies (lanes a-c) but absent from the dark colonies (lanes d- ) . The 42kd Q _ subunit comigrates with the E^ coli elongation- factor EFTu and its presence in lanes a-c was detected using antibodies to purified luciferase (see also Fig. 4C). Lane cant.: protein isolated from Ξ^ coli EB101 containing pBR322, mw: molecular weight standards. Figures 3A,3B,3C and 3D illustrate the methods used to monitor gene expression by bioluminescence. The P1 promoter- lux AB gene fusion of pFITOOI was opened at the Hindlll site, different DNA fragments known to be devoid of transcription termination signals inserted, and the bioluminescence of cells carrying each construction determined. The panel in Figure 3A shows a BamfiT digest of: pFITOOI (a), pFITOOI carrying different DNA fragments (b-e; see text for description of fragments), pFITOOI carrying a 28 bp trp A transcription terminator (f) and molecular weight markers (g). Luxdot assay performed on E^ coli containing the above constructs (panel of Fig. 3B, 90 sec exposure) shows that cells carrying pBR322 do not express bioluminescence (cont.) while the expression of lux AB from the P1 promoter (a) is attenuated to varying degrees by different DNA fragments (b-e) and inhibited to about 5% level (see panel C) by the trp A transcription terminator (f). Panel C sho a part of the same luxdot pattern after a 30 second exposure to X-ray fil Relative densitometric scanning data, shown below each dot, and the liqu scintillation counter data, shown in the bar graph, are expressed percentage of the activity measured for pFITOOI . The bar graph in panel shows the results of the luciferase enzyme assays performed in vitro (op bars) and in vivo (dotted bars) on the same clones. The numbers refer light units per 2.4 x 10? cells, with one light unit equal to 9.8 x 1θ9 quanta/sec. Fig. 4 shows measurements of acetylene reduction (A) and bioluminescen (B) in soybean nodules formed by the wild-type strail of B japonicum (op bars) and by the transco ugant strain containing the nif D promoter- lux gene fusion (hatched bars). Soybeans were transferred to nitrogen-rich medi at day 18 after inoculation and both assays were performed every 12 hours f 4 days. Panel C shows an SDS-polyacrylamide gel containing total bactero proteins from nature wild-type (a) and bioluminescent (b) nodules (Coomass blue stain). After the addition of nitrate, bacteroids were isolated every hours (panel D, lanes 0-84 hr) and their total protein extrac electrophoresed, transferred to nitrocuellulose, and reacted with an antiser against purified luciferase. Antigen-IgG complexes were visualized using goat anti-rabbit serum conjugated with peroxidase Boehringer Mannheim), w bacteroids from wild-type nodules, alpha and beta: subunits of luciferase. Fig. 5 illustrates espression of bioluminescence in soybean nodule coontrolled by a constitutively expressed P.1 promoter (A) and t symbiotically regulated nif D promoter (B), as monitored by the luxdot ass on X-ray film. Five ul droplets of total nodule extracts (0.1g nodule per 0. ml of the assay buffer, Materials and Methods) were exposed to X-ray film the presence of n-decanal vapors. The increase in luxdot intensity in panel is due to an increase of the bacteroid number from about IxlO^/mg tissue on day 7 to approximately 1.5x10 1( Vmg on day 14, as determined in a hemocytometer.. Panel C shows nitrogen fixation activity in the bioluminescen nodules measured prior to their homogenization. Visual Identification of Plasmid Clones Containing Inserts Using oFITOOI . Identification of bacterial clones carrying DNA inserts has generally been accomplished by targeting inserts into a gene encoding either antibiotic resistance or a marker enzyme (e.g. lac Z) and subsequent screening for antibiotic sensitivity or a phenotype (e.g., blue/white colonies on X-gal plates), or through a miniprep isolation and electrophoretic analysis of plasmid DNA from individual clones. The use of the vector pFITCCI allowed us to detect easily by eye all colonies carrying inserts and distinguish them from colonies containing the parent plasmid. The PI promoter- lux AB gene fusion was found to be constitutively expressed in all E coli and E^ aponicum strains testes. To demonstrate its use in a rapid identification of colonies containing plasmids with inserts, the pFITOOI plasmid was linearized with EcoRI and ligated to about 30% insertion frequency (Dugaiczyk, A., Boyer, H.W. δ Goodman, E.M. (1975) J. Mol. Biol. 96, 171-184) with a small population of DNA fragments previously digested with EcoRI. Since the EcoRI site of pFITOOI is located in the lux AB gene (Fig. 1a), which is known to be essential for bioluminescence, the presence of any inserts will result in dark transformants. Figures 2A shows the pFITOOI-containing transformants of E^ coli strain HB101 on an agar plate. Following treatment of the same plate with n-decanal vapors, bioluminescent colonies were photographed in the dark (Fig. 2E) , and subsequently exposed for about 15 seconds to an X-ray film (Fig. 2C). The arrowheads in panels A, B and C indicate the presence of several colonies that do not emit light. All dark colonies analyzed were shown to carry an EcoRI insert in the pFITOOI vector (Fig. 2D, lanes d-j), while no inserts were found in any of the bioluminescent colonies (Fig. 2D, lanes a-c). Direct evidence for the lack of luciferase in the dark colonies was provided by protein analysis on polyacrylamide gels containing SDS (Fig. 2E) . Measurement of Promoter Strength In Vivo: Luxdot Assay. The use of the plasmid vector pFITOOI has enabled us not only to visually identify plasmid clones containing inserts, but also to detect and quantify changes in the rate of gene expression in intact E^ coli or Bradyrhizobium cells. To alter the rate of luciferase expression from the F1 promoter of pFITCOI , dif erent DNA frag ents free of transcriptional terminators were inserted into the regio separating the promoter from lux AB, and the resultant clones were analyze for bioluminescence. Four DNA fragments representing central crotions o selected structural genes were purified by electroelution (Yang, R.C.A., Lis J. δ Wu, R. (1979) Methods Enzy ol. 68, 176-182) and blunt-end ligated int the Hindlll site of pFITOOI. The following fragments were used: a 0.6 k BamHI-NruI fragment of the tetracycline resistance gene of pBR322 (Fig. 3A lane b), a 0.85 kb Aval fragment of stem Rhizobium BTAi chromosome isolate from pRL11H (Legocki, R.P., Eaglesham, A.R.J. δ Szalay, A.A. (1982) i Molecular Genetics of the Bacteria-Plant Interaction, ed. Puhler, A (Springer, Berlin), pp. 210-219) (lane c), a 0.5 kb PvuII fragment of the la Z gene isolated from pXJ003 (lane d), and a 1.1 kb Bg1II-Sa1I fragmen containing promoterless NPTII gene isolated from transposon Tr.5 of pSUP101 (lane e). Droplets of 5 ul dilutions of E^ coli cells transformed with th above plasmids were spotted on the bottom of an empty Petri dish and expose to an X-ray film in the presence of n-decanal vapors. The luxdot patter shown in Fig. 3B was obtained after a 90 second exposure to an X-ray film It is apparent that the intensity of light produced by the uninterrupted P promoter- lux AB gene fusion (lane a) is attenuated to varying degrees by th insertion of different DNA fragments (lanes b-e), and that the transcriptiona signal can be efficiently blocked by the trp A transcription terminator (lan f). The observed differences in luciferase levels are likely to be due t changes in the 5' region cf mRNA's and their translatibility, rather than due to a decreased promoter strength. However, when the ?1 promoter of pFITOOI was replaced with other promoter signals, including a synthetic lac promoter (Marians, K.J., Wu, R., Stawinski, J., Hozumi, T., δ Narang, S.A. (1976) Nature 263, 744-748), tac promoter (deBoer, H.A., Comstock, L.J. δ Vasser, M. (1983) Proc. Natl. Acad. Sci. USA 80, 21-25), and a B^ japonicum promoter library expressable in E^ coli, a wide variation in luxdot intensity was observed that could be related directly to promoter strength (data not shown) . Similar results to those in E^ coli were obtained in Bradyrhizobium japonicum 110, using the P1 promoter- lux AB gene fusions on a wide hose range plasmid pSUP106, however, dots of comparable intensity to those shown in Fig. 3B were observed only after about 30 minute exposure to X-ray film. Our data indicated that a luxdot assay can be quantified very simply by two methods, and that the results obtained are in agreement with the luciferase enzyme assays in vivo and in vitro. Figure 3C shows a part of the same luxdot pattern generated by 1.6 x 1θ cells in each 5 ul droplet, followed by a 30 second exposure to X-ray film. The film was scanned (Quick Scan RδD, Eelena Laboratories), and data computed using an Apple He computer with an Applegration II program. Numbers shown under the luxdot pattern in Fig. 3C represent the relative strength of lux expression in each clone with reference to the original pFITOOI construct (100% activity), used as an nternal standard. Based on the luciferase assay carried out in vitro for the clone selected as an internal standard, all relative numbers obtained from a luxdot pattern can be expressed in light units (1 light unit = 9.8 x 1θ9 quanta/sec). The diagram in Fig. 3C illustrates luciferase expression in the above clones as measured by a liquid scintillation counter, with reference to the same internal standard of pFITOOI . The results obtained were in consistent with those from the luxdot scanning technique, and could also be related to the actual light units using the selected clone as the internal standard. To measure luciferase activity directly, all of the above clones were subjected to the enzyme assays in vitro and in vivo, and the results expressed in light units (Fig. 3D). It is apparent that all measurements presented in Fig. 3, including the simple scintillation counting method, are in good agreement with the results of the enzyme assay in vitro, and that using just one clone as an internal standard it is possible to convert all data to the actual enzyme units. Bioluminescence in Soybean Nodules: Expression of Luciferase Genes ' from nifP and nifH Promoters of Bradyrhizobium japonicum. The measurements of nitrogen fixation in free-living diazotrophs and in odules have been based almost uniquely on the acetylene reduction assay, in which the conversion of acetylene to ethylene by nitrogenase is determined by gas chro atography (Hardy, R.W.F., Holsten, R.D., Jackson, E.K. δ Burns, R.C. (1968) Plant Physiol. 43, 1185-1207). We have previously shown in stem nodules of Aeschynomene scabra that a chromosomally integrated nif H promoter- lac Z gene fusion in the stem Rhizobium bacteroids is activated in trans with the nati nif region. We now report that the activity of nif D and nif H nitrogena promoters of Bradyrhizobium japonicum can be measured in a single root nodu of soybean using liquid scintillation counter, based on bioluminescence. The nif D promoter region of 3^ japonicum was excised from pAY6 as a 0.8 CLal fragment containing approximately o.3 kb of the II- terminal region a C5 kb of the 5' upstream sequences. The nif H promoter region was located a 0.75 kb SMal-XhoI fragment of pAY8 containing approximately 0.3 kb of t N-terminal region and 0.45 kb of the 5' upstream sequences. The nif D and n H promoter-containing fragments were purified by electroelution, and eac blunt-end ligated into the Sail site of pPALEOOI. As indicated in Fig. plasmid pPALEOOI carries a 70 bp sequence of V^ harveyi containing translati stop codons (brackets) in all three reading frames prior to the ATG codon lux A. This sequence will block any translational fusion upstream of lux without causing transcriptional termination. The nif D and nif H promoter- lux AB fusions were excised from pPALEO with Aval (see Fig. 1) and placed in the middle of a 13 kb fragment of japonicum chromosome, located on a mobilizable plasmid pMR19 (Materials a Methods). Bradyrhizobium transconjugants were selected in the presence kanamycin, and colonies containing a single copy of ' the promoter fusion p genome were identified as "double cross-overs" by DNA hybridization, demonstrated previously in stem Rhizobium BTAil . No bioluminescence w detected in free-living transconjugants containing the nif promoter-luci eras gene fusions, as judged by extended exposures to X-ray film. For plant tests, soybeans were divided into three inoculation groups wild-type, nif D promoter - lux AB fusion, and nif H promoter- lux AB fusion Eeginning at day 18 after inoculation, single nodules were removed from roots weighed, and measured for acetylene reduction in a 0.5 cc volume of 10% (v/v acetylene. Each nodule was homogenised in 200 ul of the luciferase assa buffer using a small mortar and pestle, and total extracts were examined fo bioluminescence in a liquid scintillation counter. Figure 4A shows tha nodules formed by the wild- type 3^ .-aponicum and those formed b transcon ugants containing the nif D promoter- lux AB fusion fix nitrogen a approximately equal rates. About 36 hr after addition of nitrogen-rich mediu (Sum erfield, R. J. Dart, P.J., Huxley, P. ., Eaglesham, A.R.J., Minchin F.R. δ Day, J.M. (1977) Exp. Agric, 13, 129-142.), which is known t inhibit expression of nif genes in rhizobia, a rapid decline in nitroge fixation was observed. Measurements of bioluminescence performed on the sam tissues (Fig. 4B) showed high levels of luciferase activity in nodule containing the nif D promoter- lux A3 fusion and a similar decline of biolu inesce at 60 hr. There was n bioluminescence detected at any stage in the wild-type nodules. It was not fully understood why the decline in bioluminescence did no coincide with the decline in nitrogen fixation, but rather the two event occured 24 hr apart. In contrast to the nitrogenase complex, the bacteria luciferase from ^ harveyi has been described as a relatively stable protein eg. its structure and enzymatic activity were not affected by repeate freezing and thawing (Eastings, J.W., Baldwin, T.O. δ Nicoli, M.Z. (1978 Methods Enzymol. 57, 135-152), and thus it was possible that this protein ha a lone half-life in B^ japonicum bacteroids. To determine levels of th luciferase protein in nodules treated with nitrate, bacteroids were isolate at 12 hr intervals, and their total protein extracts analyze electrophoreticaliy. Figure 4C shows that the amoung of luciferase i bioluminescent nodules is very small and that its presence cannot be detecte by staining. A nitrocellulose replica reacted with antibodies agains purified luciferase (Fig. 4D) indicates virtually the same level of th enzyme between 0 and 48 hr following the addition of nitrate, and a stead decrease in the amount of luciferase polypeptides in nodules may indeed b very low, and that the stability of this protein may cause the observed dela in the decline of bioluminescence. To determine if in the early stages of nodule development the expression o nitrogenase is in fact accompanied by the expression of bioluminescence, tota extracts of two types of nodules were compared using the luxdot assay: nodule formed by L_ japonicum containing a single chromosomal copy of a P1 promoter lux A3 fusion, and nodules equipped with the nif D promoter- lux A3 fusion. It is clear that bioluminescence controlled by the constitutively expressed promoter is detectable even in very young nodules (7 days after inoculatio Fig. 5A) , whereas expression of bioluminescence is nodules containing the n D promoter- lux AB fusion occurs only at 3-4 days later (Fig. 5B), and concides with the nitrogenase activity (Fig. 5C). The initiation of nitrog fixation in soybean nodules at day 10 of the symbiosis is consistent wi previous publications (Eergersen, F.J. & Goodchild, D.J. (1973) Australi J. Bio. Sci. 26, 729-741), and the appearance of bioluminescence at th time shows that the nif D promoter- lux AB fusion is activated coordinatel with the native nif locus of B_ j _ japonicum. It is interesting to note that while bioluminescence in total extracts fro wild-type nodules showed on the average 30-40 cpm per nodule, the same siz nodule containing the nif D promoter- lux AB fusion showed typically a pea value of as much as 6 x 10? cpm. Bioluminescence in these nodules in the presence of n-decanal vapors was visible to the naked eye, but only iif th tissue was cut open (see also Discussion). Similar results to those shown i Fig. 4 were obtained for nodules containing the nif H promoter- lux AB fusio (data not shown). It is also noteworthy that extracts from a single nodule containing on copy of lux AB per bacteroid yielded as much as 2.0 - 9.0 x 10^ cpm in a liquid scintillation counter without addition of exogenous aldehyde. Thes results indicated the presence in soybean nodules cf aldehyde(s) that ca serve as substrate for the bioluminescence reaction. The levels o bioluminescence are two orders of magnitude lower than those with n-decana added exogenously, but nevertheless, the observation is worthy of furthe study. Bacterial luciferase genes lux AB from ^ harveyi may become an extremel useful tool for studying procaryotic gene expression. The lux AB gene system appears to have several distinct advantages over the lac Z system. First, th luciferase appears to function in any host background and in any growth mediu tested. Second, the activity of the enzyme can be visually detected in viv within a few seconds of exposure to aldehyde vapor, and permanent records useful for quantitation can be made quickly by either direct exposure to X-ra film or by photographic methods. Finally, the range of sensitivity o bioluminescence systems is unparalleled by systems that utilize chromogeni substrates. With a simple photomultiplier photometer (Mitchell, G.W " . an Blastings, J.W. (1971) Anal. Bioche . 39, ' 243- 250), it is possible t detect and quantify light from as little as a few femtomoles of luciferase corresponding to picogra amounts of enzyme. Using E^ coli strain SM1 transformed with pFITOOI , the luxdot assay has allowed us to detect as few a 1θ4 cells after 15 minute exposure to X-ray film. One feature unique to the bacterial luciferase system is that the substrate for the reaction n-decanal, is a volatile aldehyde whose vapors rapidly penetrate cel membranes and stimulate bioluminescence in vivo. It is thus possible t detect and quantify the luciferase activity in living cultures withou perturbing the living system. This cannot be accomplished using the firefl luci erase system from Photinus pyralis which utilizes firefly luciferi (De¥et, J.R., Wood, K.V. , Eelinski, D.R. δ DeLuca, M. (1985) Proc. Natl Acad. Sci. USA.82, 7870-7873). Luciferin does not readily pass membranes and may require some carrier such as DMSO to facilitate permeability, an hence its use appears to be restricted to measurements in vitro usin homogenized extracts. Although a comparison between the lac Z gene of E^ coli and the lux AB gen cluster of V^ harveyi as gene expression markers indicates some importan advantages to the latter, both systems use the indirect approach of measurin gene expression via enzyme activity (beta- galactosidase and luciferase respectively) , that may not reflect quantitatively the transcriptiona events. Nevertheless, the validity of a general approach using promoterles lac Z or NPTII (neomycin phosphotransferase) genes to quantify promote strength through enzyme assays, has been widely documented using plasmids phage olucules and chromosomally integrated gene fusions. The use of pFITOOI and pPALEOOI plasmid vectors presented here is based o the fusion of lux AB genes with constitutively expressed or symbioticall regulated promoter regions. In contrast to the recently described expressio vector Mini-Mu lux (Engebrecht, J. , Simon, 11. , & Silver an, M. (1985) Scienc 227, 1345-1347) that involves the use of the entire 7 gene lux cluster of fischeri in E^ coli, the above constructs harbor luciferase structural gen only and require addition of the aldehyde substrate exogenously. Measurements of bioluminescence in soybean nodules demonstrated that t expression of a single copy per B^ japonicum genome of the nif D or nif promoter- lux AB fusion can be detected in a liquid scintillation count using only a fraction of a single nodule extract. Parallel measurements nitrogen fixation and bioluminescence carried out on the same nodules prior and after treatment of plants with nitrate suggested that the expression nitrogenase and luciferase genes in these nodules is controlled coordinatel It is important to note that while the beta-galactosidase enzyme ass performed on stem nodules involved a significant plant backgroun measurements of Bradyrhizobium bioluminescence in soybean nodules we completely unaffected by the presence of plant tissue. The lack of pla background to the luciferase system in nodules was apparent during t measurement of bioluminescence in extracts from the wild-type nodules and t nodules formed by bradyrhizobium transconjugants that contained lux AB gen fused to the nif D or nif H promoter regions in the opposit (wron orientation. Nodules from both plant groups showed normal nitrogen fixati activities, but virtually no bioluminescence. As mentioned hereinabove, bioluminescence in nodules containing nif promoter- lux AB fusion was strong enough to be visible to the dark-adapt eye, but only if the nodules were cut open. This may be due to two facto related to nodule structure. The light generated by bacteroids, located the central cortex, could be blocked by several uninfected cell layers of t peripheral cortex and/or the luciferase reaction, known to require oxygen, enhanced upon exposure of bacteroids to atmospheric oxygen. The discussion herein focuses on the isolation of symbiotically regulate promoters of B_ ; _ japonicum using promoter- lux AB gene fusions. However, du to the high sensitivity and overall simplicity of the bacterial luciferas assay, it may be possible to detect and monitor activities of even wea promoter signals in single nodules throughout the development of the symbioti process, as well as in very early stages of nodulation. Rhizobia carrying constitutively expressed luciferase genes will also be extremely useful for competition and localization analysis of nodule initiation. The foregoing description has been directed to particular embodiments of the invention in accordance with the requirements of the Patent Statutes for the purposes of illustration and explanation. It will be apparent, however, to those skilled in this art that many modifications and changes will be possible without department from the scope and spirit of the invention. It is intended that the following claims be interpreted to embrace all such modifications and changes.

Claims: We claim : -3 3- 1. A host microorganism genetically and stably modified by the insertion any of its non-essential chromosomal locations of a foreign DNA fragme comprising, a non-homologous fragment containing lux AB genes of a select bioluminescent bacterium wherein the nonhomologous fragment is stab reproduced as a part of the chromosome of the cells of the host microorgani as they divide in a growth medium. 2. A host microorganism genetically and stably modified by the insertion any of its non-essential chromosomal locations of a foreign DNA fragme comprising a non-homologous fragment containing lux AB genes of ^ harve wherein the nonhomologous fragment is stably reproduced as a part of t chromosome of the cells of the host microorganism as they divide in a grow medium. 3. The host microorganism of Claim 1 wherein that microorganism is japonicum. 4« The host microorganism of Claim 1 wherein that microorganism is ste Rhizobium. 5. The host microorganism of Claim 1 wherein the gene expression of suc microorganism triggers the production of luciferase which, in turn emits ligh as a measure of gene expression and to serve as a marker or tag for th modified microorganism. 6. The host microorganism of Claim 2 wherein the gene expression of suc microorganism triggers the production cf luciferase which, in turn emit blue-green light in the presence of aldehyde as a measure of gene expressio and to serve as a marker or tag for the modified microorganism. 7. The host microorganism of Claim 6 wherein the growth medium is a mediu known to support growth of the host microorganism in its unmodified form. 8. A new and inspired method for quantizing light being emitted fro biological material comprising the following steps: Placing a plurality of droplets of a selected biological material whic emits light as a measure of gene expression on a surface onto which each o said droplets will adhere and maintain itself; each of said droplets containing a known count of replicable units of sai biological material and each related droplet containing a related known coun of replicable units"which is based en the scale which has been selected fo the purpose of quantifying the magnitude of the light being emitted from th selected biological material; placing an x-ray film adjacent to the said plural related droplets fo sampling the relative intensity of the light emitted from said plura droplets; developing said x-ray film for the purpose of inspecting and measuring th intensity of light from each of the said related droplets for the purpose o quantising the light emission replicable units of biological material. 9. The method of Claim 8 wherein the droplets of the selected biologica material which emits light as a measure of gene expression .s maintained in liαuid medium during the measuring process such that the replicable units said biological material are not replicating. 10. The method of Claim 8 wherein more than one different selected samples biological material which emit light as a measure of gene expression are e quantified by placing plural and progressively diluted droplets in rows s by side en a surface in the form of a grid. 11. The method of tagging a selected gram positive procaryotic microorgan using bacterial luciferase genes lux AB from V^ harveyi comprising: select the procaryotic microorganism to be tagged, transfering by electroporation calcium precipitation into said microorganism the chromosomal DNA fragm from the selected ricrcorganism placing said modified procaryo microorganism in a growth media and exposing it to aldehyde vapor resulting bioluminescence in vivo providing an identification of the prese of said procaryotic microorganisms. 12. The method of tagging a selected gram negative procaryotic microorgan using bacterial luciferase genes lux A3 from V^ harveyi comprising: select the procaryotic microorganism to be tagged, transfering by conjugation i said microorganism by a mobilizable plasmid vector carrying the chromoso DNA fragment from the selected microorganism, of the promoter- lux AB ge fusions placing said modified procaryotic microorganism in a growth media a exposing it to aldehyde vapor the resulting bioluminescence in vivo providi an identification of the presence of said procaryotic microorganisms. 13- A plasmid vector pFITOOI as deposited, NRRL B-18080. 14. A plasmid vector pPALEOOI as deposited, NRRL E-18082. 15. A plasmid vector pMR19 as deposited, NRRL B-180S1. 16. The method of tagging a microorganims comprising the insertion of foreign DNA fragment in the form of a non-homologous fragment containing l AB genes of a selected- bioluminescent bacterium into a non-essenti chromosomal location of the microorganism.

Inventors: BALDWIN THOMAS O, LEGOCKI MISUK, LEGOCKI ROMAN P, SZALAY ALADAR A, BALDWIN, THOMAS, O., LEGOCKI, MISUK, LEGOCKI, ROMAN, P., SZALAY, ALADAR, A.
Applicants: BOYCE THOMPSON INST PLANT RES, TEXAS A & M UNIV SYS, TEXAS A & M UNIVERSITY SYST
Application year: 1987
Main classification: C12Q1
IPC codes: C12N9, C12N15, C12Q1