Patent Abstract:
Disclosed is a method for identifying  Streptomyces  species using groEL2 gene that can compensate for drawbacks of conventional methods of morphologic classification and 16S rDNA identification being time-consuming, unfaithful, and expensive, thus enabling to efficiently identify  Streptomyces  species.

Full Description:
FIELD OF THE INVENTION 
     The present invention relates to a method for identifying genus  Streptomyces  using groEL2 gene which comprises the steps of preparing a specific primer capable of amplifying groEL2 gene of all  Streptomyces  species; amplifying groEL2 gene using the primer; sequencing the nucleotide sequence of an amplified product to build a database; and identifying unknown  Streptomyces  species using the database. 
     BACKGROUND OF THE INVENTION 
     Due to the developments of methods for isolating and purifying natural products, more than 10,000 kinds of antibiotics have been isolated from microorganisms. Further, continued studies on a new identification method and technology, discovery of new isolation resources and application of microbial metabolites to veterinary and agricultural industries have contributed to the development of antibiotics using microorganisms. 
     Since mutual antagonism between microorganisms was first observed by Tyndall, various antibiotics have been actively developed. For example, actinomycin was isolated from  S. antibioticus  by Waksman and Woodruff, and streptomycin, from  S. griseus  by Schatz and Wakman based on the discovery of penicillin by Fleming in 1929. As actinomycin and streptomycin were used for treating pulmonary tuberculosis,  Streptomyces  has been regarded as an important microorganism for producing antibiotics in the fields of industry and medical sciences. Since metabolites of  Streptomyces  are very diverse, it can produce various kinds of biologically active substances. Among 10,000 kinds of biologically active substances investigated from microorganisms until now, about two-thirds are found in  Streptomyces . Accordingly, the importance of  Streptomyces  in investigating biologically active substance has been much emphasized.  Streptomyces  has been regarded as one of the most important microorganisms in biomaterial industry. 
       Streptomyces  is one of the most diverse microbial species and possesses many different biologically metabolic activities even in the same species (Anderson A S, Wellington E M. The taxonomy of  Streptomyces  and related genera.  Int. J. Syst. Evol. Microbiol.  2001, 51(3): 797-814). Accordingly, numerous biologically active substances have been developed from  Streptomyces &#39;s metabolites, and infinite possibilities of these substances for applying to agricultural and marine industries (breeding, extermination of damages by blight and harmful insects), environmental industry (disposal of wastes), fine chemical industry (technochemical medicines), food industry (raw materials, additives), semiconductor industry (biosensors) and medicine have been suggested. 
     There have been conducted numerous studies using natural products for the purpose of preventing, alleviating or treating diseases (Emmert E A, Handelsman J. Biocontrol of plant disease: a (Gram−) positive perspective.  FEMS Microbiol. Lett.  1999, 1; 171(1): 1-9; Nielsen J. Metabolic engineering: techniques for analysis of targets for genetic manipulations.  Biotechnol. Bioeng.  1998, 58(2-3): 125-32; Hutchinson C, Colombo A. Genetic engineering of doxorubicin production in  Streptomyces peucetius: J. Ind. Microbiol. Biotechnol.  1999, July; 23(1): 647-652). One of such methods for approaching the purpose is to secure various biological resources. Considering the importance of  Streptomyces  in biological diversity and industrialization possibility, it is expected to play an important role in practical applicability. 
     The international agreement to biological diversity relates to preservation of biological diversity, prolonged utilization and fair distribution of profits obtained from the existing genetic resources, and is interested in the preservation of worldwide biological resources. At the point of becoming worse environmental pollution, it has been regarded as an important matter to secure and prevent domestic microbial resources. The United States has approved a patent right for a microorganism since 1980 and the microorganism has become the subject matter of patent since 1987 in the country. 
     For obtaining a patent right for a microorganism, it is important to analyze exactly the phylogenetical classification of a target microorganism as well as the characteristics of biologically active compounds produced by the microorganism. 
     The current method for screening a new compound from  Streptomyces  has been conducted for the purpose of finding a new compound, but it h as often resulted in finding only already patented compounds. Accordingly, it is preferable to carry out the screening of a new compound after a new species or a new strain of  Streptomyces  is identified, thereby increasing the possibility of discovering new compounds. The classification of  Streptomyces  has been based on a numerical taxonomy via physiological, morphological or biochemical analyses according to the previously discovered phenotypic features. 
     However, there are several obstacles in conducting the numerical taxonomy for  Streptomyces : exact identification of  Streptomyces  requires too much time because there are too many subtypes in  Streptomyces; Streptomyces  has an extremely slow growth rate [cell cycle of  E. coli  (20 min); cell cycle of  Streptomyces  (2-3 hrs)]; and its analytical result is not very reliable. 
     Recently, the numerical taxonomy has been replaced by a molecular taxonomy, which determines a species by analyzing a chronometer molecule showing all bacterial phylogenetic relationship via analyses of nucleotide sequences. Among the chronometer molecules, 16S rDNA molecule has been widely employed for the identification of a microorganism, in particular,  Streptomyces.    
     The method for identifying a microorganism by sequencing analysis of 16S rDNA has been widely employed in place of the numerical taxonomy using the previous phenotypic features. Further, 16S rDNA has also been employed as a target gene of a kit for detecting a microorganism including pathogenic bacteria by a molecular method (e.g., a gene probing kit for detecting a  mycobacterium ). 
     However, there are several drawbacks in the method using 16S rDNA as follows. Although a hypervariable region showing various sequence mutations exists in 16S rDNA, the full-length of 1.5 kb 16S rDNA must be sequenced for the exact identification of a microorganism by comparative sequencing analysis, which is time-consuming and cost-ineffective. This problem raises the problem that too many oligomers should be used to develop a method for identifying a microorganism by a DNA chip in the future. Further, it requires much expense for analyzing the data of 450 kinds or more of species including  Streptomyces . Accordingly, while 16S rDNA database of other strains are established at Genbank, that of total  Streptomyces  species has not been completed except a few species. Besides, it has been reported that 16S rDNA exists in the form of a multi-copy gene in entire chromosomes in some  Streptomyces  species and the nucleotide sequences of these alleles are different from each other, which becomes a critical defect for the identification method using 16S rDNA (Ueda K, Seki T, Kudo T, Yoshida T, Kataoka M. Two distinct mechanisms cause heterogeneity of 16S rRNA.  J. Bacteriol.  1999, January; 181(1): 78-82). Namely, several nucleotide sequences of 16S rDNA exist in one strain, and this raises a technical problem in sequencing analysis. Because it is not possible to directly analyze the nucleotide sequence of a PCR product after PCR amplification of the target gene of  Streptomyces , the amplified product must be cloned into a vector and several clones thus obtained are subjected to sequencing analysis. 
     Due to these problems, it is necessary to select a new chronometer molecule besides 16S rDNA for the identification of  Streptomyces.    
     Potato scab is a pathogenic disease caused by three different  Streptomyces  species of  S. scabiei, S. acidiscabies  and  S. turgidiscabies , with rare exceptions of a few  Streptomyces  species. Of them,  S. scabiei  is the major pathogenic microorganism which is composed of many genetical side groups. 
     Since  Streptomyces  is the most diverse species with a relatively slow growth rate as compared to other microorganisms, it is very difficult to classify  Streptomyces  species by a biochemical or physiological method (Skerman, V. B. D., McGowan, V., Sneath, P. H. A. (ed): Approved Lists of Bacterial Names.  Int. J. Syst. Bacteriol.  1980, 30: 225-420). Therefore, several methods, e.g., a fatty acid analyzing method, DNA-DNA hybridization method and 16S rRNA gene analyzing method, have been developed for identifying a potato scab pathogenic microorganism. Of these methods, the method for analyzing 16S rRNA has an advantage in defining a phylogenetic relationship between microorganisms or identifying an unknown strain and has been effectively used for identifying pathogenic bacteria. However, it is very difficult to exactly classify bacterial strains showing close phylogenetic relationship among them because the nucleotide sequence of 16S rRNA is highly conserved in these strains. Accordingly, there is a need of establishing a method for identifying a potato scab pathogenic microorganism using a new substitute gene for 16S rRNA. To compensate the defect of 16S rRNA analyzing method, there was developed a method for identifying an unknown strain using 16S-23S ITS region as a target gene, a region known to be more hypervariable than 16S rDNA. However, this method is not suitable for the classification and identification of a potato scab pathogenic microorganism because 16S-23S ITS target gene has a few different nucleotide sequences in each individual. Therefore, it has been a long-awaited need to develop a method for identifying a potato scab pathogenic microorganism using a new chronometer molecule as a target gene. 
     The present inventors have therefore endeavored to find a method that meets the above need, and developed a method for identifying  Streptomyces  species using groEL2 gene which comprises the steps of preparing a specific primer for groEL2 gene conserved in all  Streptomyces  species; amplifying groEL2 gene using the primer; sequencing the nucleotide sequence of amplified product to build a database; and identifying unknown  Streptomyces  species using the database. 
     SUMMARY OF THE INVENTION 
     Accordingly, an object of the present invention is to provide a method for identifying  Streptomyces  using groEL2 gene which comprises the steps of preparing a specific primer for groEL2 gene which is capable of amplifying groEL2 gene of all  Streptomyces  species; amplifying groEL2 gene using the primer; sequencing the nucleotide sequence of an amplified product to build a database; and identifying unknown  Streptomyces  species using the database. 
     It is a further object of the present invention to provide a method for identifying a potato scab pathogenic microorganism using the method. 
     In accordance with one aspect of the present invention, there is provided a specific primer capable of amplifying groEL2 gene, which is conserved in all  Streptomyces  species; a groEL2 gene fragment amplified from  Streptomyces ; and a groEL2 gene fragment amplified from a potato scab pathogenic microorganism. 
     In accordance with another aspect of the present invention, there is provided a method for identifying  Streptomyces  species, which comprises the steps of amplifying groEL2 gene using the primer, sequencing the amplified product to build a database, and identifying unknown  Streptomyces  species using the database. 
     It is still another object of this invention to provide a method for identifying a potato scab pathogenic microorganism from  Streptomyces  species. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings which respectively show; wherein 
         FIG. 1  shows the recognition sites of groEL2 specific primers and groEL2 gene fragment amplified using the primers; 
         FIG. 2  shows the result of electrophoresis of 648-bp groEL2 gene fragment amplified from a reference strain of  Streptomyces  species using a primer pair specific for  Streptomyces  species; 
         FIG. 3  shows the result of electrophoresis of 648-bp groEL2 gene fragment amplified from a reference strain of a potato scab pathogenic microorganism using a primer pair specific for  Streptomyces  species; 
         FIG. 4  shows the phylogenetic tree of 40 reference strains of  Streptomyces  species formed by using the nucleotide sequences of 420-bp groEL2 gene fragments; 
         FIG. 5  shows the phylogenetic tree of 40 reference strains of  Streptomyces  species formed by using the polypeptide sequences consisting of 140 amino acids encoded by 420-bp groEL2 gene fragments; 
         FIG. 6  shows the result of identifying 5 non-reference strains by comparing the nucleotide sequences of 420-bp groEL2 fragments; 
         FIG. 7  shows the phylogenetic tree of 40 reference strains of  Streptomyces  species, 15 reference strains of potato scab pathogenic microorganisms and 20 isolated strains formed by using the nucleotide sequences of 420-bp groEL2 fragments; 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides a specific primer capable of amplifying groEL2 gene which is conserved in all  Streptomyces  species; a groEL2 gene fragment of  Streptomyces  amplified by using the primer; and a groEL2 gene fragment of a potato scab pathogenic microorganism amplified by using the primer. 
     The present invention also provides a method for identifying  Streptomyces  species, which comprises amplifying groEL2 gene using the primer, sequencing the nucleotide sequence of an amplified product to build a database, and identifying unknown  Streptomyces  species using the database. 
     Further, the present invention provides a method for identifying a potato scab pathogenic microorganism from  Streptomyces  species. 
     The present invention is described in detail hereunder. 
     In one aspect, the present invention relates to an identification method of genus  Streptomyces  by using groEL2 gene which comprises the steps of preparing a specific primer for groEL2 gene conserved in all  Streptomyces  species; amplifying groEL2 gene using the primer; sequencing the nucleotide sequence of an amplified product to build a database; and identifying  Streptomyces  species using the database. 
     The present invention has employed groEL2 gene encoding groEL2 protein as a new chronometer molecule substitute for 16S rDNA for identifying  Streptomyces  species. groEL2 gene encodes a stress-related protein in bacteria whose function is well conserved both in human and bacteria. Accordingly, groEL2 gene can be regarded as a chronometer molecule that a gene mutation reflects random change involved in cell cycle rather than external selective stress. Namely, it has been thought that the nucleotide sequence of groEL2 gene represents a phylogenic relationship among microorganisms. 
     groEL2 gene employed as a chronometer molecule in the present invention has advantages over the previously employed 16S rDNA as follows: 
     1. In order to exactly identify a bacterial strain by a comparative sequencing analysis using 16S rDNA as a target gene, almost 1.5-kbp of the full-length gene must be sequenced. However, it is possible to precisely identify a bacterial strain by analyzing the nucleotide sequence of only 420-bp or 423-bp of groEL2 gene fragment. This difference can curtail the cost for identifying a bacterial strain several folds. 
     2. The most critical problem of 16S rDNA analyzing method for identifying  Streptomyces  species is that it is impossible to analyze  Streptomyces  species using a direct nucleotide sequencing method since 16S rDNA exists as a multi-copy gene in one individual in some  Streptomyces  species and the multi-copy gene may have different nucleotide sequences. In this case, the nucleotide sequence of 16S rDNA must be indirectly sequenced after a cloning procedure, which leads to several folds higher waste in labor, time and cost than that of a direct nucleotide sequencing method. However, the identification method using groEL2 gene can make up for this defect because it has been reported that groEL2 gene has a single nucleotide sequence in each individual. 
     3. 16S rDNA has hypervariable regions in different lengths, which suggests the presence of a gap in a nucleotide sequence alignment. However, groEL2 gene has an only 420-bp of nucleotide sequence fragment in almost all  Streptomyces  species except a few species. The exceptional species also have a 423-bp of nucleotide sequence fragment wherein only one amino acid, i.e., 3-bp nucleotides, is added. Accordingly, this feature functions as an advantage in a nucleotide sequence alignment or a determination of nucleotide sequence. 
     4. 16S rDNA is not a structural gene, and therefore, does not encode a functional polypeptide. Accordingly, the identification method using 16S rDNA cannot employ the amino acid sequence of the polypeptide encoded by 16S rDNA for identifying a bacterial strain. However, since a functional gene, groEL2, encodes a polypeptide, it is capable of employing not only the nucleotide sequence of groEL2 fragment but also the amino acid sequence of groEL2 protein encoded thereby for identifying a bacterial strain. 
     5. There is a problem of building an individual database of 16S rDNA because the nucleotide sequencing analysis of  Streptomyces  species using 16S rDNA has been sporadically carried out by several different researchers since the middle of 1980. However, since it was discovered that all nucleotide sequence of groEL2 gene analyzed in the present invention is new in Genbank, groEL2 gene has the advantage of building an individual database for classifying  Streptomyces  species. Further, the inventive groEL2 gene has the advantages over rpoB gene disclosed in Korea Patent Laid-open Publication No: 2003-15124 that groEL2 gene shows more variable mutations in the nucleotide sequence and amino acid sequence encoded thereby between two  Streptomyces  species, and therefore, is more favorable as a chronometer molecule for classifying and diagnosing  Streptomyces  species. 
     The inventive method for identifying genus  Streptomyces  by using groEL2 gene is described as follows. 
     The identification method of the present invention comprises the steps of
         1) preparing a specific primer capable of amplifying groEL2 gene of all  Streptomyces  species and amplifying groEL2 gene of target strain using the primer;   2) analyzing the nucleotide sequence of an amplified product; and   3) comparing thus obtained nucleotide sequence with that of a reference strain.       

     In Step 1), to prepare a specific primer for  Streptomyces  species to amplify groEL2 gene, the full-length nucleotide sequences of  S. lividans  and  S. albus  derived groEL2 genes were compared with that of  T. paurometabola  derived groEL2 gene which is phylogenetically close to  Streptomyces  species, and the most highly conserved regions were selected as the recognition sites for forward and reverse primers, respectively. Then, 40 reference strains of  Streptomyces  species were subjected to PCR amplification using the primer pair to confirm whether 648-bp of PCR products are amplified in all target strains. 
     Preferably, the primer has the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. 
     A target strain was subjected to PCR using the  Streptomyces  specific primers to amplify groEL2 gene, and then, the nucleotide sequence of the amplified groEL2 gene fragment was analyzed. At this time, the nucleotide sequence database of groEL2 gene fragments of reference strains comprises the nucleotide sequences of SEQ ID NOs: 3 to 42. 
     When the groEL2 nucleotide sequences of reference and target strains are analyzed and the target strain is determined as a relevant strain by comparing their nucleotide sequences, the nucleotide sequence of a target strain to be subjected to identification was analyzed and added to an alignment database. Then, the nucleotide sequence alignment was carried out again to complete a phylogenetic tree, which resulted in forming a branch close to a relevant strain and determining a species of a target strain from the phylogenetic tree. Further, it was possible to identify a strain to examine whether the target strain shows a 99.8% sequence homology compared with a reference strain. It is due to the fact that the nucleotide sequence variation in same species does not exceed 0.2%. 
     To verify whether the groEL2 database of  Streptomyces  species build in is the present invention is applicable to identify an unknown strain in practice, 5 non-reference strains were subjected to identification by sequencing the groEL2 gene fragment and comparing the nucleotide sequence with that of a reference strain. As a result, it was found that 3 non-reference strains of  S. hygroscopicus  (KCTC 9030, KCTC 9031 and KCTC 9069) had 100%, 99.8% and 99.8% of a sequence homology, respectively, and were located at a position close to a reference strain of  S. hygroscopicus  (KCTC 9782) in the phylogenetic tree. It was also found that 2 non-reference strains of  S. albus  (KCTC 1136 and KCTC 1533) had 99.8% and 100% of a sequence homology, respectively, and were located at a position corresponding to a reference strain of  S. albus  (KCTC 1082) (see  FIG. 6 ). In conclusion, it is important for a chronometer molecule used for the identification of a bacterial strain to have features of intraspecies conservation as well as interspecies variation. The interspecies variation of groEL2 gene has been described above, and the intraspecies conservation of groEL2 gene has been proved by analyzing the nucleotide sequences of 5 non-reference strains. When the nucleotide sequences of 5 non-reference strains were compared with that of a reference strain, they showed a sequence homology ranging from 99.8% to 100%. Further, all 5 non-reference strains can be identified by comparative nucleotide sequence analysis. 
     Meanwhile, the present invention provides a method for identifying a potato scab pathogenic microorganism from  Streptomyces  species, which comprises:
         1) amplifying a groEL2 gene fragment of a target strain by using a specific primer for groEL2 gene of  Streptomyces  species;   2) analyzing the nucleotide sequence of groEL2 gene fragment; and   3) comparing the nucleotide sequence with that of groEL2 gene fragment of a reference strain causing potato scab.       

     15 reference strains to be identified that are well-known as potato scab pathogenic microorganisms were subjected to PCR using the primer to examine whether 648-bp of PCR product is amplified in all strains. 
     A target strain was subjected to PCR using the  Streptomyces  specific primer to amplify groEL2 gene, and then, the nucleotide sequence of the amplified groEL2 gene fragment was analyzed. At this time, the nucleotide sequence database of groEL2 gene fragments of reference strains comprised the nucleotide sequences of SEQ ID NOs: 43 to 61. 
     15 reference strains causing potato scab and 20 isolated strains obtained from Kangwon-do and Jeju-do derived potato scab pathogenic tissues were subjected to sequencing analysis. As a result of comparing the nucleotide sequences of 15 reference strains by multi-alignment, it was found that three strains of  S. scabiei, S. acidiscabies  and  S. turgidiscabies  have different nucleotide sequences from each other and belong to a different group in a phylogenetic tree, respectively. 
     Accordingly, the inventive identification method solves the problems of the previous conventional classification based on the morphological and biochemical tests and 16S rDNA identification method (time-consuming, incorrectness, cost-ineffective, etc.), and therefore, can be effectively used for identifying  Streptomyces  species. 
     This invention is explained in more detail based on the following Examples but they should not be construed as limiting the scope of this invention. 
     REFERENCE EXAMPLE 1 
     40 reference strains consisting of 38  Streptomyces  strains, 1  Rhodococcus  strain and 1  Tsukamurella  strain were obtained from Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB), and subjected to sequencing analysis of groEL2 gene. Further, total 5 non-reference strains of 2 species ( S. hygroscopicus  and  S. albus ) were subjected to comparative analysis of the nucleotide sequence of groEL2 gene (Table 1). 
     
       
         
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 No 
                 Name 
                 Source 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Reference Strain of  Streptomyces   
               
             
          
           
               
                 1 
                 
                   S. acrimycini 
                 
                 KCTC 9679 T   
               
               
                 2 
                 
                   S. aculeolatus 
                 
                 KCTC 9680 T   
               
               
                 3 
                 
                   S. alanosinicus 
                 
                 KCTC 9683 T   
               
               
                 4 
                 
                   S. albireticuli 
                 
                 KCTC 9744 T   
               
               
                 5 
                 
                   S. albofaciens 
                 
                 KCTC 9686 T   
               
               
                 6 
                 
                   S. albogriseolus 
                 
                 KCTC 9675 T   
               
               
                 7 
                 
                   S. alboniger 
                 
                 KCTC 9014 T   
               
               
                 8 
                 
                   S. albus 
                 
                 KCTC 1082 T   
               
               
                 9 
                 
                   S. ambofaciens 
                 
                 KCTC 9111 T   
               
               
                 10 
                 
                   S. aminophilus 
                 
                 KCTC 9673 T   
               
               
                 11 
                 
                   S. anandii 
                 
                 KCTC 9687 T   
               
               
                 12 
                 
                   S. argenteolus 
                 
                 KCTC 9695 T   
               
               
                 13 
                 
                   S. bambergiensis 
                 
                 KCTC 9019 T   
               
               
                 14 
                 
                   S. capillispiralis 
                 
                 KCTC 1719 T   
               
               
                 15 
                 
                   S. carpinesis 
                 
                 KCTC 9128 T   
               
               
                 16 
                 
                   S. catenulae 
                 
                 KCTC 9223 T   
               
               
                 17 
                 
                   S. cellulosae 
                 
                 KCTC 9703 T   
               
               
                 18 
                 
                   S. chartreusis 
                 
                 KCTC 9704 T   
               
               
                 19 
                 
                   S. chattanoogensis 
                 
                 KCTC 1087 T   
               
               
                 20 
                 
                   S. cinnamonensis 
                 
                 KCTC 9708 T   
               
               
                 21 
                 
                   S. cinereoruber 
                 
                 KCTC 9707 T   
               
               
                 22 
                 
                   S. cirratus 
                 
                 KCTC 9709 T   
               
               
                 23 
                 
                   S. coeruleorubidus 
                 
                 KCTC 1743 T   
               
               
                 24 
                 
                   S. collinus 
                 
                 KCTC 9713 T   
               
               
                 25 
                 
                   S. corchorusii 
                 
                 KCTC 9715 T   
               
               
                 26 
                 
                   S. diastaticus 
                 
                 KCTC 9142 T   
               
               
                 27 
                 
                   S. djakartensis 
                 
                 KCTC 9722 T   
               
               
                 28 
                 
                   S. erumpens 
                 
                 KCTC 9729 T   
               
               
                 29 
                 
                   S. fulvissimus 
                 
                 KCTC 9779 T   
               
               
                 30 
                 
                   S. galilaeus 
                 
                 KCTC 1919 T   
               
               
                 31 
                 
                   S. griseochromogenes 
                 
                 KCTC 9027 T   
               
               
                 32 
                 
                   S. griseolus 
                 
                 KCTC 9028 T   
               
               
                 33 
                 
                   S. griseoviridis 
                 
                 KCTC 9780 T   
               
               
                 34 
                 
                   S. humiferus 
                 
                 KCTC 9116 T   
               
               
                 35 
                 
                   S. hygroscopicus 
                 
                 KCTC 9782 T   
               
               
                 36 
                 
                   S. minutiscleroticus 
                 
                 KCTC 9123 T   
               
               
                 37 
                 
                   S. murinus 
                 
                 KCTC 9492 T   
               
               
                 38 
                 
                   S. nodosus 
                 
                 KCTC 9035 T   
               
             
          
           
               
                 Non-reference Strains  Streptomyces   
               
             
          
           
               
                 1 
                 
                   S. hygroscopicus 
                 
                 KCTC 9030 
               
               
                 2 
                 
                   S. hygroscopicus 
                 
                 KCTC 9031 
               
               
                 3 
                 
                   S. hygroscopicus 
                 
                 KCTC 9069 
               
               
                 4 
                 
                   S. albus 
                 
                 KCTC 1136 
               
               
                 5 
                 
                   S. albus 
                 
                 KCTC 1533 
               
             
          
           
               
                 Other Actinomycetes 
               
             
          
           
               
                 1 
                 
                   R. equi 
                 
                 KCTC 9082 
               
               
                 2 
                 
                   T. paurometabola 
                 
                 KCTC 9821 
               
               
                   
               
               
                 Abbreviation: 
               
               
                 KCTC: Korean Collection for Type Cultures 
               
             
          
         
       
     
     REFERENCE EXAMPLE 2 
     Total 15 strains of 7  S. scabiei  strains, 1  S. acidiscabies  strain and 4  S. turgidiscabies  strains known as potato scab pathogenic microorganisms, and 1  S. bottropensis  strain, 1  S. disastatochromogenes  strain and 1  S. neyagawaensis  strain showing a close relationship to the potato scab pathogenic microorganism in a phylogenetic taxonomy were subjected to sequencing analysis of groEL2 gene, and total 20 isolated strains obtained from Kangwon-do and Jeju-do derived potato scab pathogenic tissues were subjected to comparison analysis of the nucleotide sequences of groEL2 gene (Table 2). 
     
       
         
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 No 
                 Name 
                 Source 
                 No 
                 Name 
                 Source 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Potato scab causing reference strains 
               
             
          
           
               
                 1 
                 
                   S. scabiei 
                 
                 ATCC 40173 T   
                 2 
                 
                   S. scabiei 
                 
                 DSMZ 
               
               
                   
                   
                   
                   
                   
                 40961 
               
               
                 3 
                 
                   S. scabiei 
                 
                 DSMZ 40962 
                 4 
                 
                   S. scabiei 
                 
                 IFO 3111 
               
               
                 5 
                 
                   S. scabiei 
                 
                 IFO 13767 
                 6 
                 
                   S. scabiei 
                 
                 IFO 
               
               
                   
                   
                   
                   
                   
                 13768 
               
               
                 7 
                 
                   S. scabiei 
                 
                 IFO 12914 
                 8 
                 
                   S. acidiscabies 
                 
                 ATCC 
               
               
                   
                   
                   
                   
                   
                 49003 T   
               
               
                 9 
                 
                   S. turgidiscabies 
                 
                 ATCC 700248 T   
                 10 
                 
                   S. turgidiscabies 
                 
                 IFO 
               
               
                   
                   
                   
                   
                   
                 16079 
               
               
                 11 
                 
                   S. turgidiscabies 
                 
                 IFO 16080 
                 12 
                 
                   S. turgidiscabies 
                 
                 IFO 
               
               
                   
                   
                   
                   
                   
                 16081 
               
               
                 13 
                 
                   S. bottropenis 
                 
                 IFO 13023 
                 14 
                 
                   S. disastatochromogenes 
                 
                 IFO 
               
               
                   
                   
                   
                   
                   
                 13389 
               
               
                 15 
                 
                   S. neyagawaensis 
                 
                 IFO 3784 
               
             
          
           
               
                 Isolated Potato scab causing strains 
               
               
                 Strains isolated from Kangwon-do 
               
             
          
           
               
                 16 
                 Kangwon-S20 
                 Kangwon-do 
                 17 
                 Kangwon-S27 
                 Kangwon-do 
               
               
                 18 
                 Kangwon-S28 
                 Kangwon-do 
                 19 
                 Kangwon-S32 
                 Kangwon-do 
               
               
                 20 
                 Kangwon-S33 
                 Kangwon-do 
                 21 
                 Kangwon-S34 
                 Kangwon-do 
               
               
                 22 
                 Kangwon-S48 
                 Kangwon-do 
                 23 
                 Kangwon-S51 
                 Kangwon-do 
               
               
                 24 
                 Kangwon-S53 
                 Kangwon-do 
                 25 
                 Kangwon-S56 
                 Kangwon-do 
               
               
                 26 
                 Kangwon-S58 
                 Kangwon-do 
                 27 
                 Kangwon-S59 
                 Kangwon-do 
               
               
                 28 
                 Kangwon-S71 
                 Kangwon-do 
               
             
          
           
               
                 Strains isolated from Jeju-do 
               
             
          
           
               
                 29 
                 Jeju-H11 
                 Jeju-do 
                 30 
                 Jeju-H12 
                 Jeju-do 
               
               
                 31 
                 Jeju-H16 
                 Jeju-do 
                 32 
                 Jeju-H17 
                 Jeju-do 
               
               
                 33 
                 Jeju-H18 
                 Jeju-do 
                 34 
                 Jeju-H19 
                 Jeju-do 
               
               
                 35 
                 Jeju-H20 
                 Jeju-do 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 1 
     Preparation of groEL2 Primer Specific for  Streptomyces  Species 
     Specific forward (STGROF1) and reverse primers (STGROR2) were designed to be capable of amplifying groEL2 gene fragment in all  Streptomyces  species.  S. lividans  (GenBank No. X95971) and  S. albus  (GenBank No. M76658), whose full-length nucleotide sequences of groEL2 gene were already sequenced for other purposes, and  T. paurometabola  (GenBank No. AF352578), which belongs to  Tsukamurella  species closely related to  Streptomyces  species in a phylogenetic tree, were subjected to sequencing analysis, and forward primer STGROF1 (5′-CCATCGCCAAGGAGATCGAGCT-3′: SEQ ID NO: 1) and reverse primer STGROR2 (5′-TGAAGGTGCCRCGGATCTTGTT-3′: SEQ ID NO: 2) that are capable of specifically amplifying all  Streptomyces  species were prepared therefrom ( FIG. 1 ). The primer pair of STGROF1 and STGROR2 is new which has not been used previously for amplifying  Streptomyces  species. 
       FIG. 1  shows the recognition sites of primers employed in the present invention. The inventive primer pair of STGROF1 and STGROR2 was designed to target a total 648-bp of groEL2 gene fragment corresponding to the nucleotide sequence ranging from 161 to 808 in 1623-bp of the full-length groEL2 gene of  S. albus . A forward primer consisting of 22 nucleotides corresponding to the base sequence ranging from 161 to 182 and a reverse primer consisting of 22 nucleotides corresponding to the base sequence ranging from 787 to 808 in the nucleotide sequence of  S. albus  were employed to amplify 648-bp of the groEL2 gene fragment of  Streptomyces  species. The recognition sites of the primers are phylogenetically conserved regions that show a 100% sequence homology to not only  S. lividans  and  S. albus  belong to  Streptomyces  species but also  T. paurometabola  belong to a different group from  Streptomyces  species. 
     EXAMPLE 2 
     Preparation of 420-bp groEL2 Fragment of  Streptomyces  Species 
     1) DNA Extraction 
     DNA was extracted according to a BB/P (Bead beater phenol) method. Cultured cells were harvested and suspended in a TEN buffer solution (Tris-HCl 10 mM, EDTA 1 mM, NaCl 100 mM: pH 8.0). The suspension was re-suspended in the mixture of 100 μl (packing volume) ultrafine magnetobead solution (diameter 0.1 mm; Biospec Products, Bartlesville, Okla., U.S.A.) and 100 μl phenol/chloroform/isopropylalcohol (50/49/1) solution and subjected to shaking for 1 min with a mini beater to disrupt cells. After centrifuging the resulting solution at 12,000 rpm for 5 min, the supernatant (100 μl) was transferred to a new tube and added with 60 μl isopropylalcohol. The tube was then centrifuged at 15,000 rpm for 15 min to produce a pellet. The pellet was washed with 70% ethanol, and then, DNA was recovered in 60 μl TE buffer solution (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA). 
     2) PCR Amplification of groEL2 Gene 
     Forward primer STGROF1 and reverse primer STGROR2 specific for  Streptomyces  species were employed. The PCR reaction solution was prepared by mixing 50 ng of template DNA, 20 pmole each of SRPOF1 and SRPOR2 primers, and AccuPower PCR PreMix (Bioneer, Korea) consisting of 2 units of Taq polymerase, 10 mM dNTP, 10 mM Tris-HCl (pH 8.3) and 1.5 mM MgCl 2 , adjusted to a final volume of 20 μl. The PCR condition consisted of 30 cycles of: 1 min at 95° C. (denaturation), 45 sec at 62° C. (annealing) and 90 sec at 72° C. (extension) after the initial denaturation for 5 min at 95° C., and 5 min at 72° C. (final amplification) (Model 9600 thermocycler, Perkin-Elmer cetus). After PCR was completed, the reaction mixtures were subjected to 1% agarose gel electrophoresis to examine whether 648-bp of PCR product was amplified. 
     As a result of PCR using the primer pair specific for  Streptomyces  species selected above, it was found that 648-bp of groEL2 gene fragments were amplified from all 40 reference strains ( FIG. 2 ). Further, it was found that the inventive primer pair could amplify  Rhodococcus  and  Tsukamurella  species belong to rare actinomycete species as well as  Streptomyces  species. 
     Meanwhile, it was found that the inventive primer pair is capable of amplifying 648-bp of groEL2 gene fragments from 15 potato scab pathogenic reference strains and 20 isolated strains ( FIG. 3 ). 
     3) Purification of PCR Products 
     After 1% agarose gel electrophoresis was completed, gel slices corresponding to 648-bp PCR products of  Streptomyces  reference strains were excised, transferred to a new tube, and subjected to DNA extraction. DNA extraction and purification were carried out using a Qiaex system (Qiagen, Germany). The tube was added with gel dissolving solution QX1 (500 μl) and then incubated at 50° C. for 15 min to completely dissolve the gel. Then, 10 μl of gel bead solution was added thereto and the mixture was kept at 50° C. for 15 min. In the meantime, the tube was subjected to vortexing for 10 sec at intervals of 1 min to spread the bead equally. The reaction mixture was washed once with QX1, twice with QF and dried at 45° C. for 10 min, and then DNA was recovered in 20 μl of TE buffer solution. 
     EXAMPLE 3 
     Automatic Sequencing Analysis of groEL2 Fragment 
     Automatic sequencing analysis was carried out using the gel-eluting product as a target DNA. The reaction mixture was prepared by mixing 60 ng of template DNA, 1.2 pmole of primer and 2 μl of BigDye terminator cycle sequencing kit (PE Appied Biosystems), adjusted to a final volume of 10 μl. The reaction condition consisted of 25 cycles of: 10 sec at 95° C., 10 sec at 60° C. and 4 min at 60° C. (Model 9600 thermocycler, Perkin-Elmer cetus). After the reaction was completed, DNA was extracted according to an ethanol precipitation method. In particular, after 180 μl of distilled water and 10 μl of 3 M sodium acetate were added to the reaction mixture adjusted to a final volume of 200 μl, 2 volumes of 100% ethanol was added thereto, and the mixture was mixed well. The reaction mixture was subjected to centrifugation at 15,000 rpm for 20 min to precipitate DNA. Then, 500 μl of 70% ethanol was added thereto and the precipitated DNA was subjected to centrifugation at 15,000 rpm for 20 min for washing. DNA was recovered using Deionized Formimide (PE Applied Biosystems). Thus purified DNA was heated at 95° C. for 5 min to denature into a single strand DNA and subjected to electrophoresis using an ABI 3100 system for 2.5 hrs to analyze the nucleotide sequence. 
     The sequencing analysis was carried out in one direction using the forward primer STGROF1, and accordingly, the groEL2 gene fragment (420-bp or 423-bp) in the 648-bp of full-length nucleotide sequence was determined. 
     The PCR product was purified according to the method described above, and subjected to automatic sequencing analysis without going through a cloning process. A 420-bp of fragment corresponding to the nucleotide sequence ranging from 232 to 631 in the full-length groEL2 gene of  S. albus  was sequenced as shown in  FIG. 1 . As a result, the nucleotide sequences of all 420-bp fragments amplified from 40 reference strains and 35 potato scab causing strains were determined without a certain ambiguous result (if several copies of a target gene exist in a chromosome and their nucleotide sequences are different from each other, it is impossible to determine the exact nucleotide sequence since the nucleotide sequence at the other position maybe overlapped with that of the correct position in a direct sequencing analysis). 
     As a result of comparing the nucleotide sequences in multi-alignment, all 40-reference strains had a nucleotide sequence of their own which are different from each other. Namely, they showed interspecies variation. For a certain gene to be targeted in identifying a bacterial strain, it is a prerequisite that the interspecies variation be preserved among species. It was found that the inventive identification method met the requirement. 
     Further, except 3 strains ( S. ambofaciens, S. erumpens  and  S. murinus ) having 423-bp of fragment wherein 1 amino acid, i.e., 3-bp (GCG), was inserted at the 301 st  residue based on the full-length groEL2 nucleotide sequence of  S. albus , all the nucleotide sequences of 37 reference strains encoded 420-bp of groEL2 gene fragment without insertion or deletion in the multi-alignment. Namely, there was no gap in the multi-alignment. 16S rDNA shows a gap at a high frequency in the alignment. It has been reported that the gap makes an error in building an entire phylogenetic tree since the gap is apt to be analyzed by removing all the aligned nucleotides corresponding to that region during the multi-alignment, it is likely. Accordingly, the result described above demonstrated the superiority of the inventive groEL2 gene for identifying a bacterial strain. 
     As a result of multi-alignment using a polypeptide encoded by 420-bp of groEL2 gene fragment which consists of 140 amino acids that corresponds to the region ranging from the 78 th  to the 217 th  residues in the amino acid sequence of full-length groEL2 protein of  S. albus , it was found that all 37 reference strains encoded the polypeptide consisting of 140 amino acids except 3 strains of  S. ambofaciens, S. erumpens  and  S. murinus  having an insertion of alanine at the 101 st  residue in the amino acid sequence of full-length groEL2 protein of  S. albus  which encodes a polypeptide consisting of 141 amino acids. Further, it was found that 33 alleles existed in 40 reference strains based on the sequence homology of amino acid. These results suggested that the polypeptide encoded thereby as well as the nucleotide sequence of groEL2 gene were efficiently used for the identification of  Streptomyces  species different from 16S rDNA which does not encode any polypeptides. 
     Meanwhile, the results for identifying potato scab causing strains among  Streptomyces  species were as follows. 
     As a result of comparing the nucleotide sequences of 15 potato scab causing reference strains, it was found that three different species of  S. scabiei, S. acidiscabies  and  S. turgidiscabies  known as potato scab pathogenic microorganisms had their own nucleotide sequences different from each other and belonged to a group different from each other in the phylogenetic tree. It was also found that  S. scabiei  significantly represents various genotypes in the phylogenetic tree different from other two strains that showed closely related genotypes. These results coincided with the previous report that these species are composed of diverse genotypes. Namely, as a result of comparing the sequence homology of each 420-bp groEL2 nucleotide sequence of 7 reference strains belong to  S. scabiei , they showed a sequence homology ranging from 88.9 to 100%.  S. scabiei  was divided into 4 groups based on the phylogenetic tree made by using the sequence homology. Group I included two reference strains of ATCC 49173T and DSMZ 40962 that showed a 100% sequence homology; Group II, two reference strains of IFO 12914 and IFO 3111 that show a 98.1% sequence homology; Group III, two reference strains of IFO 13767 and IFO 13768 that show a 100% sequence homology; and Group IV, one reference strain of DSMZ 40961. It was found that while 7 reference strains of  S. scabiei  showed interspecies variation, 4 reference strains (ATCC 700248T, IFO 16079, IFO 16080 and IFO 16081), which belong to  S. turgidiscabies , showed a 100% sequence homology with each other. 
     EXAMPLE 4 
     Arrangement and Homology Analysis of groEL2 Nucleotide Sequence and Preparation of Phylogenetic Tree 
     The nucleotide sequences (420-bp or 423-bp) of groEL2 gene fragments of 40  Streptomyces  reference strains analyzed by an automatic sequencing method were subjected to multi-alignment using a Megalign program of DNAstar software to build a groEL2 database. Once 420-bp of the nucleotide sequences were translated into a polypeptide consisting of 140 amino acids in the Megalign program, the translated amino acids were subjected to multi-alignment according to a Clustal method stored in the Megalign program. Then, 140 amino acids thus aligned were converted into 420 nucleotides to build a database for identifying actinomycete species. Sequence homology to each nucleotide sequence of 40 strains was analyzed by applying the aligned database to a sequence distance method stored in the Megalign program. 
     After the multi-alignment of nucleotide sequence, sequence homology of 40 reference strains were examined according to the method described above. As a result, all the reference strains showed a different in sequence homology with each other. As a result of analyzing the sequence homology of 38  Streptomyces  species, they showed a sequence homology ranging from 88.4% (between  S. griseolus  and  S. albus ) to 99.1% (between  S. humiferus  and  S. ambofaciens ). 
     Accordingly, it was found that there was sequence heterogeneity ranging from 0.9% to 11.6% among  Streptomyces  species. From these results, it was confirmed that the inventive groEL2 gene has a higher interspecies variation thus being regarded as the most important feature of a target gene for identifying a bacterial strain than 16S rDNA which showed 3% and less of sequence heterogeneity between  Streptomyces  species. When the nucleotide sequences of 38  Streptomyces  reference strains were compared with those of  R. equi  and  T. paurometabola , they showed 85.5% (between  S. anandii  and  R. equi ) and less of a sequence homology. 
     As a result of examining the sequence homology of polypeptides encoded by above 420-bp groEL2 fragments of 38  Streptomyces  species, they showed a sequence homology ranging from 91.4% (between  S. griseolus  and  S. albus ) to 100%. When the amino acid sequences of polypeptides derived from 38  Streptomyces  species were compared with those of  R. equi  and  T. paurometabola , they showed 87.9% (between  S. anandii  and  R. equi ) or less of a sequence homology. 
     A phylogenetic relationship between each species was analyzed from a phylogenetic tree, which was built by using the MEGA software. The aligned 420-bp nucleotide sequences of 40 strains were analyzed by a Neighbor-Joining method based on a Juke-Cantor distance measuring method and a pair wise detection method to build a phylogenetic tree. Bootstrap analysis was carried out by 100 replications. 
     The sequence homology and phylogenetic tree of polypeptides encoded by groEL2 gene fragments were analyzed by translating 420-bp of the nucleotide sequences into 140 amino acids using the Megalign program and multi-aligning the amino acid sequences according to the Clustal method stored in the Megalign program. 
     The aligned nucleotide sequences of 40 strains were subjected to build a Neighbor-Joining phylogenetic tree using the Mega software described above. As a result, it was found that all the 40 strains had a nucleotide sequence of their own which are different from each other and 40 kinds of characteristic fragments. Further, it was found that 38 strains of  Streptomyces  species formed an individual group as against  R. equi  and  T. paurometabola  ( FIG. 4 ). 
     As a result of multi-aligning the polypeptides according to the Clustal method in the Megalign program, it was found that 33 alleles coding different polypeptides from each other among 40 reference strains formed the same number of fragment. Similarly, 33 fragments formed an individual group as against  R. equi  and  T. paurometabola  ( FIG. 5 ). 
     EXAMPLE 5 
     Identification of Non-reference Strains by Comparative Nucleotide Sequence Analysis Using a Reference Strain Database 
     As shown in Table 1, total 5 non-reference strains of 3  S. hygroscopicus  (KCTC 9030, KCTC 9031 and KCTC 9069) and 2  S. albus  (KCTC 1136 and KCTC 1533) were subjected to identification. The non-reference strains were identified by the following steps of: analyzing the nucleotide sequences of 420-bp groEL2 fragments of each strain according to the method described above; inputting the analyzed nucleotide sequences into the Megalign program of DNAstar software; conducting multi-alignment described above; and preparing a phylogenetic tree according to the Neighbor-Joining method of Mega software. 
     To examine whether the reference strain database (55 strains consisting of 40 reference strains of  Streptomyces  species and 15 reference strains of potato scab pathogenic microorganisms) can be applied to the identification of a bacterial strain in practice, total 5 non-reference strains of 3  S. hygroscopicus  (KCTC 9030, KCTC 9031 and KCTC 9069) and 2  S. albus  (KCTC 1136 and KCTC 1533); and total 20 potato scab pathogenic microorganisms of 13 strains isolated from Kangwon-do and 7 strains isolated from Jeju-do described in Table 2 were subjected to comparative analysis of the nucleotide sequence of groEL2 gene. 
     As a result, 3 strains of  S. hygroscopicus  (KCTC 9030, KCTC 9031 and KCTC 9069) showed a sequence homology of 100%, 99.8% and 99.8%, respectively, and were located at a position corresponding to  S. hygroscopicus  (KCTC 9782; reference strain) in the phylogenetic tree ( FIG. 6 ). Further, 2 strains of  S. albus  (KCTC 1136 and KCTC 1533) showed a sequence homology of 99.8% and 100%, respectively, and were located at a position corresponding to  S. albus  (KCTC 1082; reference strain) ( FIG. 6 ). 
     In addition, it was found that all 20 isolated strains belonged to a potato scab pathogenic group consisting of  S. scabiei, S. acidiscabies  and  S. turgidiscabies.  11 isolated strains (9 strains isolated from Kangwon-do [Kangwon-S20, Kangwon-S28, Kangwon-S32, Kangwon-S33, Kangwon-S34, Kangwon-S53, Kangwon-S56, Kangwon-S58 and Kangwon-S59] and 2 Jeju-do isolated strains [Jeju-H11 and Jeju-H16]) out of total 20 strains (55%) belonged to  S. scabiei . Coinciding with the previous report,  S. scabiei  was identified at the highest frequency in the present invention. It was confirmed that these species belong to three groups (Group I, III and IV) among four groups of  S. scabiei.  7 strains of Kangwon-S28, Kangwon-S32, Kangwon-S33, Kangwon-S53, Kangwon-S56, Kangwon-S58 and Jeju-H16 showed a sequence homology ranging from 98.8% to 100% at higher frequency and belonged to Group I. 3 strains (Kangwon-S20, Kangwon-S59 and Jeju-H11) out of them showed a sequence homology ranging from 99.5% to 100% and belonged to Group II. Kangwon-S34 strain showed a sequence homology of 99.3% with the reference strain DSM 40961 and belonged to Group IV ( FIG. 7 ). 
     5 strains [1 strain isolated from Kangwon-do (Kangwon-S71), 4 Jeju-do strains isolated (Jeju-H12, Jeju-H17, Jeju-H18 and Jeju-H20)] out of 20 strains showed a sequence homology ranging from 96.9% to 100% at 25% of isolation frequency and were identified as  S. scabiei . Further, 4 strains [3 strains isolated from Kangwon-do (Kangwon-S27, Kangwon-S48 and Kangwon-S51), 1 strain isolated from Jeju-do (Jeju-H19)] showed a sequence homology of 100% with each other at 20% of isolation frequency and were identified as  S. turgidiscabies.    
     While the embodiments of the subject invention have been described and illustrated, it is obvious that various changes and modifications can be made therein without departing from the spirit of the present invention which should be limited only by the scope of the appended claims.

Technology Classification (CPC): 2