CA1236037A - Rhizobium vector system - Google Patents
Rhizobium vector systemInfo
- Publication number
- CA1236037A CA1236037A CA000452500A CA452500A CA1236037A CA 1236037 A CA1236037 A CA 1236037A CA 000452500 A CA000452500 A CA 000452500A CA 452500 A CA452500 A CA 452500A CA 1236037 A CA1236037 A CA 1236037A
- Authority
- CA
- Canada
- Prior art keywords
- lac
- rhizobium
- gene
- fraction
- lac operon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/743—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Agrobacterium; Rhizobium; Bradyrhizobium
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
Abstract
Symbiotic genes of Rhizobium bacteria are isolated and identified, for cloning into other cells etc., by a process which involves introducing into the chromosomal DNA of Rhizobium a promoterless lac operon from E. coli. This is accomplished by use of a novel construct of the promoterless lac operon, a transconjugant such as defective mu-d I (kan, lac) phage and a suitable suicide vector such as pGS6. The transconjugant Rhizobia so formed is selected for kanR, to isolate cells which have taken up the lac operon. On a random basis, some of the transconjugants have inserted the lac operon appropriately to a sym gene promoter which now acts as promoter for the lac operon. Now, in the appropriate stimulative environment for the sym gene promoter, the lac Z gene transcription to produce .beta.-galactosidase is initiated instead of the usual sym gene function. The generation of .beta.-galactosidase is readily detected and analysed, so that cells in which the lac operon fraction has entered the sym gene can be identified, cultivated, and their genome DNA analysed to locate the gene.
Description
~3~i~3~
This invention relates to the field of genetic engineering, and more particularly to processes for identifying and modifying the symbiotic genes of bacteria of the Rhizobium species.
Rhizobium bacteria are soil bacteria which have important effects on the propagation and growth of agricultural plants of the Leguminosae type. Rhizobium bacteria enter the root structure of the legume plant by a complex process of endosymbiosis and possess genes coding for nitrogenase, which is involved in the conversion of elemental nitrogen in the soil int~ plant-utilizable ammonia ("nitrogen fixation"). Thus, slow-growing strains of Rhizobium japonicum mediate N-fixation in North America commercial soybean cultivars and hence are of ~reat economic importance. Manipulation of the genes of bacteria such as Rhizobium japonicum offer the possibility of de~ining and controlling the various steps of infection process which leads to nitrogen (N) fixation. However, before symbiotic gen~s involved in N-fixation can be manipulated, enhanced or otherwise modified, they must be identified.
It is an object of the present invention to provide a novel method for identifying the sym~iotic genes of Rhizobium, or isolation thereo.
The lactose operon (or lac operon) of E. coli is a genetic unit of the genome which has been thoroughly studied and reported in the literature. It expresses the enzyme galactosidase in the presence of lactose, which is effective to break down lactose into its constituent monosaccharides glucose and galactose. The lac operon normally comprises a regulator 1, ~3~7 a promoter _, an operator o, and structural genes z, y and a, sequentially arranged in the order i-p-o-z-y-a. Lac Z specifies the enzyme B~galactosidase, and lac y and lac a code for other enzymes. Lac o regulates the transcription of lac genes, and is operative in the presence of lactose, the inducer, which is understood to interact with the regulator or repressor lac i and prevent its interference with the operator lac o. The promoter is the site of RNA polymerase attachment, and is involved in positive control of the lac operon. When the promoter ~ is not active or not present, transcription of the lac Z and other structural genes does not occur. The base sequences of the operator o and promoter p sites have been previously determined and published.
In the present invention, a lac operon containing its lac Z,Y,A genes intact, but missing its promoter, is introduced by use of the bacteriophage mu-d 1 (kan, lac) or any other transposon such as Tn5 and suitable suicide vector plasmids, into the genome of Rhizobium bacteria. This construct carrying the lac operon fraction inserts itself into the Rhizobium cell ~enome on a random basis among the cells tested. In some cases, lac operon fraction enters the plant symbiotic (sym) gene of kh~ Rhiæobium cell and comes under the control of the operator/promoter system of the sym gene. Now, in the a~propriate stimulative environment for the sym gene operator/promoter, the lac Z gene is transcribed to produce ~-galactosidase, instead of the usual ~ gene function. The generation of B-galactosidase is readily detected and analyzedy so that the cells in which the lac operon fraction has entered ~3Çi~3~
~he sym gene are easily identified. These cells can be cultivated and their genom.c DNA isolated and analysed to locate the lac operon fraction therein, so as to identify the sym genes.
The lac operon fraction is suitably introduced into the Rhizobium cell for entry into the genome thereof through the intermediary of a defective mu-d phage and a suicide vector plasmid to which the mu-d I carrying lac has been transposed.
The suicide vector should have the ability for conjugation into the chosen Rhizobium species e.g. Rhizobium japonicum, but should not have the ability to replicate therein. Then, the mu-d I (kan, lac) fraction, once introduced into the Rhizobium cell, can transpose from the vector and incorporate into the genome of the cell. A specific example of a suitable conjugative suicide vector is that designated pGS6 described by G. Selvarej and R. Iyer, 1983, J. Bact. 156, #3, 1292-1300.
As noted above the structure and sequence of the lac operon of E. coli and its constituent parts is well-known and well understood. Most suitably, the present invention utilizes the defective mu phage derivative containing part of the E. coli .l operon, mu-d I (kan, lac) prepared as described by M.J.
Casadaban and J. Chou, "Proceedings of the National Academy of Sciences of U.S.A." tPNAS), vol. 81, pp 535-539 (1984~. This phage may be transposed into the aforementioned suicide vector pGS6 for conjugative transfer into Rhizobium japonicum. The mu-d I (kan, lac) phage carries a selectable antibiotic resistance marker (Kanamycin) to allow for selective cultivation of cells containing it. It can transpose. It contains the lac
This invention relates to the field of genetic engineering, and more particularly to processes for identifying and modifying the symbiotic genes of bacteria of the Rhizobium species.
Rhizobium bacteria are soil bacteria which have important effects on the propagation and growth of agricultural plants of the Leguminosae type. Rhizobium bacteria enter the root structure of the legume plant by a complex process of endosymbiosis and possess genes coding for nitrogenase, which is involved in the conversion of elemental nitrogen in the soil int~ plant-utilizable ammonia ("nitrogen fixation"). Thus, slow-growing strains of Rhizobium japonicum mediate N-fixation in North America commercial soybean cultivars and hence are of ~reat economic importance. Manipulation of the genes of bacteria such as Rhizobium japonicum offer the possibility of de~ining and controlling the various steps of infection process which leads to nitrogen (N) fixation. However, before symbiotic gen~s involved in N-fixation can be manipulated, enhanced or otherwise modified, they must be identified.
It is an object of the present invention to provide a novel method for identifying the sym~iotic genes of Rhizobium, or isolation thereo.
The lactose operon (or lac operon) of E. coli is a genetic unit of the genome which has been thoroughly studied and reported in the literature. It expresses the enzyme galactosidase in the presence of lactose, which is effective to break down lactose into its constituent monosaccharides glucose and galactose. The lac operon normally comprises a regulator 1, ~3~7 a promoter _, an operator o, and structural genes z, y and a, sequentially arranged in the order i-p-o-z-y-a. Lac Z specifies the enzyme B~galactosidase, and lac y and lac a code for other enzymes. Lac o regulates the transcription of lac genes, and is operative in the presence of lactose, the inducer, which is understood to interact with the regulator or repressor lac i and prevent its interference with the operator lac o. The promoter is the site of RNA polymerase attachment, and is involved in positive control of the lac operon. When the promoter ~ is not active or not present, transcription of the lac Z and other structural genes does not occur. The base sequences of the operator o and promoter p sites have been previously determined and published.
In the present invention, a lac operon containing its lac Z,Y,A genes intact, but missing its promoter, is introduced by use of the bacteriophage mu-d 1 (kan, lac) or any other transposon such as Tn5 and suitable suicide vector plasmids, into the genome of Rhizobium bacteria. This construct carrying the lac operon fraction inserts itself into the Rhizobium cell ~enome on a random basis among the cells tested. In some cases, lac operon fraction enters the plant symbiotic (sym) gene of kh~ Rhiæobium cell and comes under the control of the operator/promoter system of the sym gene. Now, in the a~propriate stimulative environment for the sym gene operator/promoter, the lac Z gene is transcribed to produce ~-galactosidase, instead of the usual ~ gene function. The generation of B-galactosidase is readily detected and analyzedy so that the cells in which the lac operon fraction has entered ~3Çi~3~
~he sym gene are easily identified. These cells can be cultivated and their genom.c DNA isolated and analysed to locate the lac operon fraction therein, so as to identify the sym genes.
The lac operon fraction is suitably introduced into the Rhizobium cell for entry into the genome thereof through the intermediary of a defective mu-d phage and a suicide vector plasmid to which the mu-d I carrying lac has been transposed.
The suicide vector should have the ability for conjugation into the chosen Rhizobium species e.g. Rhizobium japonicum, but should not have the ability to replicate therein. Then, the mu-d I (kan, lac) fraction, once introduced into the Rhizobium cell, can transpose from the vector and incorporate into the genome of the cell. A specific example of a suitable conjugative suicide vector is that designated pGS6 described by G. Selvarej and R. Iyer, 1983, J. Bact. 156, #3, 1292-1300.
As noted above the structure and sequence of the lac operon of E. coli and its constituent parts is well-known and well understood. Most suitably, the present invention utilizes the defective mu phage derivative containing part of the E. coli .l operon, mu-d I (kan, lac) prepared as described by M.J.
Casadaban and J. Chou, "Proceedings of the National Academy of Sciences of U.S.A." tPNAS), vol. 81, pp 535-539 (1984~. This phage may be transposed into the aforementioned suicide vector pGS6 for conjugative transfer into Rhizobium japonicum. The mu-d I (kan, lac) phage carries a selectable antibiotic resistance marker (Kanamycin) to allow for selective cultivation of cells containing it. It can transpose. It contains the lac
2, Y, A genes from _. coli from which the lac promoter has been_ ~3~;37 deleted. Thus, after integration o~ the phage into the Rhiæobium genome, transcriptions of the lac Z, Y and A genes can initiate from a site outside the phage. Since ~-galactosidase, the product of lac Z, is easily assayed, transcription from various Rhizobium promoters can be monitored.
In the accompanying drawing:
FIGURE 1 is a diagrammatic representation of the process of integrating the phage into the suicide vector, with simplified restriction maps of the vectors.
FIGURE 2 is a presentation of the experimental results obtained in Example 1 described below:
FIGURE 3 is a representation of the results of testing for enzyme expression, as described in Example 2 below.
By use of the standard genetic techniques, the mu-d I
tkan, lac) phage may be integrated into pGS6 as illustrated in Fig. 1. The integration takes place to the left of the origin o replication (ori). The construct pGS6::mu-d I (kan,lac) is novel, and constitutes a feature of the present invention. Eo coli containing this novel construct can be mated to Rhizobium ~aponicum, and kanamycin resistant Rhizobium colonies selected ~or.
As a result, there are produced a variety of novel Rhizobium japonicum mutant cells or transconjugants which have the lac operon fraction incorporated therein, at various locations in their genome. Indications are that the mu-d I
(kan, lac) integrates into the Rhiæobium chromosome resulting in auxotrophic mutations. They appear to be free of any residual pGS6 sequences, indicating that the rest of the vector ~:3~
suicides. Genomic DNA's from the kanR transconjugants do not appear to hybridize to pGS6. Moreover they contain mu-d I (kan, lac) in different locations, indicating ~hat the mu-d I (kan, lac~ is inserting randomly into the Rhizobium chromosome. In addition, they exhibit different levels of ac Z expression, depending upon where the mu-d I (kan, lac) has integrated.
By examining the kanR transconjugants and looking for increased lac Z expression as evidenced by ~-galactosidase generation when grown under the following conditions, one can indentify Rhizobium genes directly or indirectly involved in the symbiotic process of plant infection, growth etc. For example, ~ne can identify the transconjugants whose lac Z expression is induced/repressed: (1) In the presence of plant (legume) root exudate, (2) In the presence of root extrac~s from uninfected Glycine max, (3) In the presence of nodule extract, (4) Under anaerobic conditions and (5) Growth in the presence of various nitr~gen sources, pH, salinity, etc.
This allows for the identification and isolation as well as the pattern of regulation of those Rhizobium genes that axe specifically induced or repressed (and thus required) for symbiosis with plants leading to nitrogen fixation. Normal, standard genetic techniques can be employed to analyse the ~enome of the transconjugants and locate therein the lac Z,Y,~
genes, whose sequence and structure is well known. Their surrounding genetic environment constitutes the sym gene whose activity has been modified in the respective transconjugant.
Having identified and isolated the sym genes, they may be integrated into a foreign genome, to confer a new property on ~236~37 the recipient strain.
- In addition, the product encoded by these genes can be isolated.
- The better understanding of the gene regulators provided by the techniques of the present invention allows the modification of legume seed inoculums for increasing the efficiency of nitrogen fixation.
Among the important sets of Rhizobium genes related to symbiotic nitrogen fixation process, and which may be identified and isolated by the methods of the present invention, may be mentioned the following:
- Rhizobium survival (soil/pH/salinity) - Growth (carbon/nitrogen/phosphorus) - Competition among strains and between other bacteria - Attachment - Host specificity - Infection process - Multiplicity of infection - Systemic regulation in host - Proliferation of infection - Nullification of host defence response(s) - Number of infected cells in nodules S~m~iotic genes related to the efficiency of nitrogen fixation - Duration of nitrogen fixation - Maintenance of bacteriodal state The invention is further described in the following specific illustrative examples. In the examples, microtiter plate and petri-dish assays for ~-galactosidase production from ~23~ 37 Mu-d (lac, kan) transcription fusions of Rhizobium japonicum were conducted as follows:
Microtiter Plate Assays Rhizobial strains containing Mu-d (lac, kan~ were replica-plated [using a multiple inoculator plate (Josey et al., 1979, J. Appl. Bacteriol. 46: 343-350)] into microtiter dishes containing 100 ul/well of PA médiu~ supplemented with 0.2 g/l K~HPO4 (sadowsky and Bohlool, 1983, Appl. Environ.
Microbiol. 46: 906-911), 1.0~ (w/v)`glucose, 50 ug/ml kanamycin sulfate and 10 ug/ml Nalidixic Acid. This medium is referred to a PPM supp. The strains were grown at 28 for three days. In experimen~s designed to test the effect of root exudates on ~-galactosidase production, each well contained 50 ul of 2xPPM
supp and 50 ul of root exudate solution. Root exudates weee prepared ~rom growing 100 sterile seedlings of Glycine max cv Lee in 500 ml of sterile distilled water for seven days. Before use, root exudate solutions were filtered through Whatman #l Eilter paper, and sterilized by filtration through a-0.2 um Millipore Filter~ Fifty ul of enzyme reaction buffer (Sadowsky et al., 1983, Int. J. System. Bacteriol. 33: 716-722) and 5 ul o~ toluene were added to each well and the contents briefly mixed. The cell density of each well was estimated by-mea6uring the absorbance at 630 nm using a Dynatech MR600 Microplate reader. Plates were incubated at 37 for 30 minutes and 75 ul of o-nitrophenyl-B-D- galactopyranoside solution (Sadowsky et al., 1983, Int. J. Syst. Bacteriol. 33: 716-722) was added to each well. The o-nitrophenol pcoduced was measured at 410 nm *Trade Mark ; 8 ~;~;3fi~3~
using the microplate reader~ Enzyme activities were expressed as the ratio of Absorbance 410 nm/Absorbance 630 nm.
Petri D i sh Assay Rhizobial strains containing Mu-d (lac, kan) were replica plated onto the surface of PPM supp. containing 40 ug/ml of X-gal (5-bromo-4-chloro-3-indolyl-s-D-galactoside). For growth in the presence of root exudates, plates were spread-plated with 25X concentrated, filter-sterilized root-exudate to a final concentration of lX. Plates were incubated at 28 for two days and colonies exhibiting a blue color change over the control plates (those without added root exudates) were recorded. Plates were reincubated for a further 24 hours and the results recorded. Colonies turning from blue to white in the presence of root exudates were also recorded.
Example 1 By standard genetic techniques, a vector was constructed of the conjugative suicide vector pGS6 and the ~efective mu phage derivative containing part of the E. coli lac operon, mu-d I (kan, lac), the vector being appropriately radioactively marked. This vector is shown in accompanying Figure 1 and is designated pGS6::mu-d I (kan, lac). The integration took place within the pACYC 177 portion of pGS6 to the left of the origin of replication (ori). E. coli containing this new construct was then mated to Rhizobium japonicum (USDA
201) and kanamycin resistant Rhizobium colonies were selected for.
g ~ ~:3~;0;~
About 1% of the kanamycin resistant transconjugants so formed were unable to grow on minimal media, indicating tht the mu-d I (kan, lac) integrates into the Rhizobium chromosome resulting in auxotrophic mutations.
Genomic DNA's from five randomly-selected kanR
transconjugants were prepared and hybridised to pGS6. None of them showed any hybridization thereto, indicating that they are free of any pGS6 sequences i.e. that the rest of the vector suicides.
Figure 2 illustrates the results of Southern hybridization tests, to determine the nature of the inserts of the lac operon fraction into chromosomal ~NA of Rhizobium japonicum in the transconjugants. After treatment of the total chromosomal DNA fro the transconjugants with restriction enzyme, it is subjected to agarose gel electrophoresis, transferred to nitrocellulose paper and hybridized to the radioactive probe pGS6::mu-d l (kan, lac) by Southern transfer, hence detecting from the radioactive emissions the DNA fractions containing the lac operon. Bands 10, 12, 14, 16 and 18 pertain to the DNA from the five randomly selected kanR transconjugants. These bands show radioactivity from different locations, indicating that the mu-d I (kan, lac) is inserting randomly into the Rhizobium c~romosome as a further control. The different location of the radioactive emission on streaks 10, 12, 14, 16 and 18 indicate that the mu-d I (kan, lac) is inserting randomly into the Rhizobium chromosome.
~236~
Example 2 A variety of the Rhizobium transconjuants prepared according to Example 1 were tested for expression of B-galactosidase. The transconjugants were included separately in wells of a cultivation plate, incubated and then tested for enzyme activity as previously described, using O-nitrophenyl-B-D-galactopyranoside solution as test reagent. The qualitative results are illustrated in Fig. 2, a view in plan of the test wells. These exhibiting the denser appearance or larger dense area are showing greater ~-galactosidase activity, correlatable to ~reater lac Z gene expression. The different levels of lac Z
expression, from the different transconjugants, again indicates that the mu-d I (kan, lac) has integrated randomly into the chromosome of ~. japonicum.
In the accompanying drawing:
FIGURE 1 is a diagrammatic representation of the process of integrating the phage into the suicide vector, with simplified restriction maps of the vectors.
FIGURE 2 is a presentation of the experimental results obtained in Example 1 described below:
FIGURE 3 is a representation of the results of testing for enzyme expression, as described in Example 2 below.
By use of the standard genetic techniques, the mu-d I
tkan, lac) phage may be integrated into pGS6 as illustrated in Fig. 1. The integration takes place to the left of the origin o replication (ori). The construct pGS6::mu-d I (kan,lac) is novel, and constitutes a feature of the present invention. Eo coli containing this novel construct can be mated to Rhizobium ~aponicum, and kanamycin resistant Rhizobium colonies selected ~or.
As a result, there are produced a variety of novel Rhizobium japonicum mutant cells or transconjugants which have the lac operon fraction incorporated therein, at various locations in their genome. Indications are that the mu-d I
(kan, lac) integrates into the Rhiæobium chromosome resulting in auxotrophic mutations. They appear to be free of any residual pGS6 sequences, indicating that the rest of the vector ~:3~
suicides. Genomic DNA's from the kanR transconjugants do not appear to hybridize to pGS6. Moreover they contain mu-d I (kan, lac) in different locations, indicating ~hat the mu-d I (kan, lac~ is inserting randomly into the Rhizobium chromosome. In addition, they exhibit different levels of ac Z expression, depending upon where the mu-d I (kan, lac) has integrated.
By examining the kanR transconjugants and looking for increased lac Z expression as evidenced by ~-galactosidase generation when grown under the following conditions, one can indentify Rhizobium genes directly or indirectly involved in the symbiotic process of plant infection, growth etc. For example, ~ne can identify the transconjugants whose lac Z expression is induced/repressed: (1) In the presence of plant (legume) root exudate, (2) In the presence of root extrac~s from uninfected Glycine max, (3) In the presence of nodule extract, (4) Under anaerobic conditions and (5) Growth in the presence of various nitr~gen sources, pH, salinity, etc.
This allows for the identification and isolation as well as the pattern of regulation of those Rhizobium genes that axe specifically induced or repressed (and thus required) for symbiosis with plants leading to nitrogen fixation. Normal, standard genetic techniques can be employed to analyse the ~enome of the transconjugants and locate therein the lac Z,Y,~
genes, whose sequence and structure is well known. Their surrounding genetic environment constitutes the sym gene whose activity has been modified in the respective transconjugant.
Having identified and isolated the sym genes, they may be integrated into a foreign genome, to confer a new property on ~236~37 the recipient strain.
- In addition, the product encoded by these genes can be isolated.
- The better understanding of the gene regulators provided by the techniques of the present invention allows the modification of legume seed inoculums for increasing the efficiency of nitrogen fixation.
Among the important sets of Rhizobium genes related to symbiotic nitrogen fixation process, and which may be identified and isolated by the methods of the present invention, may be mentioned the following:
- Rhizobium survival (soil/pH/salinity) - Growth (carbon/nitrogen/phosphorus) - Competition among strains and between other bacteria - Attachment - Host specificity - Infection process - Multiplicity of infection - Systemic regulation in host - Proliferation of infection - Nullification of host defence response(s) - Number of infected cells in nodules S~m~iotic genes related to the efficiency of nitrogen fixation - Duration of nitrogen fixation - Maintenance of bacteriodal state The invention is further described in the following specific illustrative examples. In the examples, microtiter plate and petri-dish assays for ~-galactosidase production from ~23~ 37 Mu-d (lac, kan) transcription fusions of Rhizobium japonicum were conducted as follows:
Microtiter Plate Assays Rhizobial strains containing Mu-d (lac, kan~ were replica-plated [using a multiple inoculator plate (Josey et al., 1979, J. Appl. Bacteriol. 46: 343-350)] into microtiter dishes containing 100 ul/well of PA médiu~ supplemented with 0.2 g/l K~HPO4 (sadowsky and Bohlool, 1983, Appl. Environ.
Microbiol. 46: 906-911), 1.0~ (w/v)`glucose, 50 ug/ml kanamycin sulfate and 10 ug/ml Nalidixic Acid. This medium is referred to a PPM supp. The strains were grown at 28 for three days. In experimen~s designed to test the effect of root exudates on ~-galactosidase production, each well contained 50 ul of 2xPPM
supp and 50 ul of root exudate solution. Root exudates weee prepared ~rom growing 100 sterile seedlings of Glycine max cv Lee in 500 ml of sterile distilled water for seven days. Before use, root exudate solutions were filtered through Whatman #l Eilter paper, and sterilized by filtration through a-0.2 um Millipore Filter~ Fifty ul of enzyme reaction buffer (Sadowsky et al., 1983, Int. J. System. Bacteriol. 33: 716-722) and 5 ul o~ toluene were added to each well and the contents briefly mixed. The cell density of each well was estimated by-mea6uring the absorbance at 630 nm using a Dynatech MR600 Microplate reader. Plates were incubated at 37 for 30 minutes and 75 ul of o-nitrophenyl-B-D- galactopyranoside solution (Sadowsky et al., 1983, Int. J. Syst. Bacteriol. 33: 716-722) was added to each well. The o-nitrophenol pcoduced was measured at 410 nm *Trade Mark ; 8 ~;~;3fi~3~
using the microplate reader~ Enzyme activities were expressed as the ratio of Absorbance 410 nm/Absorbance 630 nm.
Petri D i sh Assay Rhizobial strains containing Mu-d (lac, kan) were replica plated onto the surface of PPM supp. containing 40 ug/ml of X-gal (5-bromo-4-chloro-3-indolyl-s-D-galactoside). For growth in the presence of root exudates, plates were spread-plated with 25X concentrated, filter-sterilized root-exudate to a final concentration of lX. Plates were incubated at 28 for two days and colonies exhibiting a blue color change over the control plates (those without added root exudates) were recorded. Plates were reincubated for a further 24 hours and the results recorded. Colonies turning from blue to white in the presence of root exudates were also recorded.
Example 1 By standard genetic techniques, a vector was constructed of the conjugative suicide vector pGS6 and the ~efective mu phage derivative containing part of the E. coli lac operon, mu-d I (kan, lac), the vector being appropriately radioactively marked. This vector is shown in accompanying Figure 1 and is designated pGS6::mu-d I (kan, lac). The integration took place within the pACYC 177 portion of pGS6 to the left of the origin of replication (ori). E. coli containing this new construct was then mated to Rhizobium japonicum (USDA
201) and kanamycin resistant Rhizobium colonies were selected for.
g ~ ~:3~;0;~
About 1% of the kanamycin resistant transconjugants so formed were unable to grow on minimal media, indicating tht the mu-d I (kan, lac) integrates into the Rhizobium chromosome resulting in auxotrophic mutations.
Genomic DNA's from five randomly-selected kanR
transconjugants were prepared and hybridised to pGS6. None of them showed any hybridization thereto, indicating that they are free of any pGS6 sequences i.e. that the rest of the vector suicides.
Figure 2 illustrates the results of Southern hybridization tests, to determine the nature of the inserts of the lac operon fraction into chromosomal ~NA of Rhizobium japonicum in the transconjugants. After treatment of the total chromosomal DNA fro the transconjugants with restriction enzyme, it is subjected to agarose gel electrophoresis, transferred to nitrocellulose paper and hybridized to the radioactive probe pGS6::mu-d l (kan, lac) by Southern transfer, hence detecting from the radioactive emissions the DNA fractions containing the lac operon. Bands 10, 12, 14, 16 and 18 pertain to the DNA from the five randomly selected kanR transconjugants. These bands show radioactivity from different locations, indicating that the mu-d I (kan, lac) is inserting randomly into the Rhizobium c~romosome as a further control. The different location of the radioactive emission on streaks 10, 12, 14, 16 and 18 indicate that the mu-d I (kan, lac) is inserting randomly into the Rhizobium chromosome.
~236~
Example 2 A variety of the Rhizobium transconjuants prepared according to Example 1 were tested for expression of B-galactosidase. The transconjugants were included separately in wells of a cultivation plate, incubated and then tested for enzyme activity as previously described, using O-nitrophenyl-B-D-galactopyranoside solution as test reagent. The qualitative results are illustrated in Fig. 2, a view in plan of the test wells. These exhibiting the denser appearance or larger dense area are showing greater ~-galactosidase activity, correlatable to ~reater lac Z gene expression. The different levels of lac Z
expression, from the different transconjugants, again indicates that the mu-d I (kan, lac) has integrated randomly into the chromosome of ~. japonicum.
Claims (3)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for identifying sym genes in Rhizobium bacteria, which comprises:
introducing into the genomes of a plurality of Rhizobium bacterial cells a vector comprising a lac operon fraction from E. coli, said fraction containing the lac Z gene but omitting the lac promoter, a phage Mu-dI (kan, lac) and a suicide vector pGS6;
isolating and cultivating those of the Rhizobium bacterial cells into which said lac operon fraction has been successfully incorporated;
subjecting selected ones of said Rhizobium cells containing the lac operon fraction to predetermined symbiotic environmental conditions;
and determining the .beta.-galactosidase production from said cells under the predetermined symbiotic environmental conditions.
introducing into the genomes of a plurality of Rhizobium bacterial cells a vector comprising a lac operon fraction from E. coli, said fraction containing the lac Z gene but omitting the lac promoter, a phage Mu-dI (kan, lac) and a suicide vector pGS6;
isolating and cultivating those of the Rhizobium bacterial cells into which said lac operon fraction has been successfully incorporated;
subjecting selected ones of said Rhizobium cells containing the lac operon fraction to predetermined symbiotic environmental conditions;
and determining the .beta.-galactosidase production from said cells under the predetermined symbiotic environmental conditions.
2. Novel mutant bacterial cells of the Rhizobium species, said cells being characterized by the presence in the genome of a lac operon fraction from E. coli, said fraction containing the lac 2 gene but omitting the lac promoter, said fraction having been inserted into said bacterial cells in a novel vector pGS6::Mu-dI (kan, lac).
3. The novel vector pGS6::Mu-d I (kan, lac) illustrated in Figure 1 of the accompanying drawings.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000452500A CA1236037A (en) | 1984-04-19 | 1984-04-19 | Rhizobium vector system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000452500A CA1236037A (en) | 1984-04-19 | 1984-04-19 | Rhizobium vector system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1236037A true CA1236037A (en) | 1988-05-03 |
Family
ID=4127704
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000452500A Expired CA1236037A (en) | 1984-04-19 | 1984-04-19 | Rhizobium vector system |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1236037A (en) |
-
1984
- 1984-04-19 CA CA000452500A patent/CA1236037A/en not_active Expired
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