Method for identification and detection of microorganisms using gyrase gene as an indicator

With the nucleotide sequence of gyr B, it is possible to classify or identify an unidentified microorganism strain quickly and accurately. Furthermore, PCR primers for monitoring a specific microorganism, which are needed in risk assessment in various bioprocesses, can be designed easily. Also, changes in mycelial tufts can be accurately monitored.

DETAILED DESCRIPTION OF THE INVENTION Hereinbelow, the present invention will be described in detail. The method for identification and detection of a microorganism of the present invention is a method for identifying and detecting of a microorganism based on the nucleotide sequence of a gyrB gene. The nucleotide sequence data of a gyrB gene may be obtained from a DNA fragment amplified by PCR (one fragment method), or may be obtained from two fragments having a mutually overlapping portion (two fragments method). In order to identify and detect a microorganism with high accuracy, a nucleotide sequence of approx. 1 kb DNA fragment is required. In 1994, Yamamoto and Harayama filed a patent application for an invention relating to primers to amplify a gyrB fragment, which is a gene encoding the B subunit of DNA gyrases (Japanese Patent Application Laid-Open (Kokai) No. 1995-213299). However, the subsequent study has clarified that, according to the disclosure of the above application alone, the identification and detection of a microorganism may be difficult for the following reasons: (1) It is difficult to determine a nucleotide sequence by a one-time sequencing reaction. (2) Some strains are inefficiently amplified with only the known primer sequences. (3) In some cases, it is difficult to determine a nucleotide sequence, since parE, being a homologous gene of gyrB, is amplified together with gyrB. In other cases, an incorrect identification of a microorganism is performed, since parE is selectively amplified. To overcome the above-stated problems, in addition to the primer sequences disclosed by Yamamoto and Harayama in 1995, the present invention provides: primer sequences conserved among gyrB genes of a large number of bacteria; primer sequences specific for gyrB genes, not appearing in parE genes, or primer sequences appearing in both gyrB and parE genes, which allow easy distinction of an amplified parE fragment from an amplified gyrB fragment; and a method for specific amplification of a gyrB gene by the combined use of the above primer sequences. To overcome the above-stated problems, sometimes it may be more preferable to amplify separately two DNA fragments having a mutually overlapping portion, and to determine the nucleotide sequence of each fragment, followed by connecting the obtained two sequences before using the data. In order to amplify the two fragments, DNA obtained from the microorganism (which also contains various DNAs as well as gyrB gene DNA) may be used as is, as a template (“one step—two fragments method”), but as another option, after performing PCR using primers capable of specifically amplifying a gyrB gene DNA, the obtained amplified products may be used as a template (“two steps—two fragments method”). Primers used for the above three types of DNA amplification methods (“one fragment method”, “one step—two fragments method” and “two steps—two fragments method”) are shown in FIG. 1 . The FIGURE shows the amino acid sequence of each GyrB of 3 kinds of strains, Bacillus subtilis 168, Escherichia coli K-12 and Pseudomonas putida PRS2000, and (a)-(l) in the FIGURE respectively correspond to amino acid sequences (a)-(l) shown as follows: (a) (Pro or Ser)-(Ala or Thr)-(Ala, Val or Leu)-Glu or Asp)-(Val or Thr)-(Ile or Val)-(Met, Leu or Phe)-Thr-(Val, Gln or Ile)-Leu-His-Ala-Gly-Gly-Lys-Phe-(Asp or Gly)-(Asp, Gly, Asn or Ser)-(Ser, Lys, Gly, Asp or Asn) &lsqb;SEQ ID NO.69&rsqb; (b) Gly-Gly-Thr-His &lsqb;SEQ ID NO.70&rsqb; (c) (Ile or Leu)-Met-Thr-Asp-Ala-Asp-Val-Asp-Gly-(Ala or Ser)-His-Ile-Arg-Thr-Leu &lsqb;SEQ ID NO.71&rsqb; (d) Arg-Lys-Arg-Pro-(Gly or Ala)-Met-Tyr-Ile-Gly-(Ser or Asp)-Thr &lsqb;SEQ ID NO.72&rsqb; (e) Gln-(Thr or Pro)-(Lys or Asn)-(Thr, Asp, Gly, Lys, Ser, Phe or Tyr)-Lys-Leu &lsqb;SEQ ID NO.73&rsqb; (f) (Tyr or Phe)-Lys-Gly-Leu-Gly-Glu-Met-Asn-(Ala or Pro) &lsqb;SEQ ID NO.74&rsqb; (g) Val-Glu-Gly-Asp-Ser-Ala-Gly-Gly-Ser &lsqb;SEQ ID NO.75&rsqb; (h) Lys-(His or Val)-Pro-Asp-Pro-(Gln or Lys)-Phe &lsqb;SEQ ID NO.76&rsqb; (i) Leu-Pro-Gly-Lys-Leu-Ala-Asp-Cys-(Ser or Gln)-(Ser or Glu)-(Lys or Arg)-Asp-Pro-(Ala or Ser) &lsqb;SEQ ID NO.77&rsqb; (j) Gln-Leu-(Trp or Arg)-(Glu or Asp)-Thr-Thr-(Met or Leu)-(Asp or Asn)-Pro &lsqb;SEQ ID NO.78&rsqb; (k) Ala-(Lys or Arg)-(Lys or Arg)-Ala-Arg-Glu &lsqb;SEQ ID NO.79&rsqb; (l) Phe-Thr-Asn-Asn-Ile-(Pro or Asn)-(Thr or Gln) &lsqb;SEQ ID NO.80&rsqb;. Two primers used for the “one fragment method” include the ones which are synthesized based on a single pair of amino acid sequences selected from the group consisting of sequence pairs (a) and (f), (a) and (j), (d) and (c), (d) and (f), and (d) and (j). The gyrB gene of members of proteobacteria has an approx. 500 bp insertion sequence located between a sequence pair (c) and (f), and another sequence pair (c) and (j), on the other hand, parE which is a homologous gene thereof does not have such sequence (Kato, J.-i., Nishimura, Y., Imamura, R., Niki, H., Hiraga, S., and Suzuki, H. New topoisomerase essential for chromosome segregation in E. coli Cell 63, 393-404 (1990)). Accordingly, in a case where two primers synthesized based on amino acid sequence pairs (a) and (f), (a) and (j), (d) and (f), or (d) and (j) are used for identification and detection of a microorganism belonging to proteobacteria, DNA fragments of gyrB genes can easily be separated from those of parE genes by electrophoresis and so on, since the length of the amplified DNA fragments of the two types of genes is different. Two primers used for the “one step—two fragments method” include the following amino acid sequence combinations: 1 Amplified fragment 1 Amplified fragment 2 Combination 1 sequences (d) and (e) sequences (h) and (c), sequences (h) and (f), or sequences (h) and (g) Combination 2 sequences (a) and (i) sequences (k) and (c), sequences (k) and (f), or sequences (k) and (g) Combination 3 sequences (a) and (i) sequences (l) and (c), sequences (l) and (f), or sequences (l)and (g) Among amino acid sequences in the above table, sequences (i), (e), (h), (k) and (l) do not exist in ParE, and in many cases, are specific for GyrB. For example, these sequences are determined to be specific for GyrB in the strains shown in the following table. 2 Sequence Strain (i) Leclercia adecarboxylata GTC 1267 Pseudoalteromonas sp. A316 Gordonia amarae DSM 46078 Rhodococcus koreensis JCM 10743 Shigella dysenteriae GTC 786 Salmonella typhi P1 Sphingomonas sp. MBIC 5538 Serratia ficaria GTC 343 Alteromonas macleodii MBIC 1375 Vibrio fluvialis GTC 315 (e) Mycobacterium tuberculosis KPM T21 Bacteroides fragilis Acholeplasma laidlawii PG-8B Bacillus cereus JCM 2152 Treponema denticola ATCC 35405 Streptococcus pneumoniae 7785 Arthrobacter oxidans IFO 12138 Porphyromonas asaccharolytica JCM 6326 Myxococcus xanthus ER-15 Streptomyces coelicolor A3 (2) (h) Pseudomonas putida MBIC 5295 Pseudoalteromonas sp. MBIC 3307 Vibrio hollisae 89A 1962 Aeromonas hydrophila P3 Photobacterium histaminum JCM 8968 Escherichia coli W3110 Bacillus anthracis Pasteur &num;2 Streptococcus pneumoniae 7785 Acinetobacter calcoacelicus ATCC 23055 Acholeplasma laidlawii PG-8B (k) Pseudomonas putida MBIC 5295 Pseudoalteromonas sp. MBIC 3307 Vibrio hollisae 89A 1962 Citrobacter sp. MAM-1 Comamonas terrigena IAM 12052 Salmonella typhimurium Acinetobacter junii SEIP 14. 81 Legionella pneumophila ATCC 33152 Escherichia coli W3110 Photobacterium histaminum JCM 8968 (l) Leclercia adecarboxylata GTC 1267 Pseudoalteromonas sp. A316 Pseudomonas sp. MBIC 5390 Marinobacter sp. MBIC 4911 Shigella dysenteriae GTC 786 Salmonella typhi P1 Sphingomonas sp. MBIC 5538 Caulobacter sp. GTC 1043 Sinorhizobium fredi ATCC 35423 Comamonas sp. GTC 866 Accordingly, by performing PCR with the combined use of the above primers, the gyrB gene DNA only can be specifically amplified. Two primers used for the “two steps—two fragments method” include the following amino acid sequence combinations: 3 2 nd amplification step 1 st amplification step fragment 1 fragment 2 Combination 1 sequences (a) and (c) sequences (a) and (e) sequences (b) and (c) Combination 2 sequences (a) and (f) sequences (a) and (e) sequences (b) and (f) Combination 3 sequences (d) and (t) sequences (d) and (e) sequences (b) and (f) Among amino acid sequences in the above table, sequence (b) exists in GyrB of all known bacteria, and so primers synthesized based on this sequence can be applied for a wide range of microorganisms. Since sequence (b) consists of 4 amino acid residues, when PCR is performed using, as a template, DNA obtained from the microorganism with primers synthesized based on the sequence, there is a possibility to amplify DNA fragments totally irrelevant to gyrB genes. However, for the “two steps—two fragments method”, amplified gyrB gene DNA fragments are used as a template, and so the above non-specific amplification can be avoided. Since sequence (f) is conserved in GyrB genes of many bacteria, primers synthesized based on this sequence can be applied for a wide range of microorganisms. Since sequence (f) exists also in ParE, when PCR is performed using this sequence, not only gyrB gene DNA fragments, but also parE gene DNA fragments are amplified. However, the amplification of parE gene DNA fragments can be prevented by performing PCR using primers synthesized based on sequence (e) specific for GyrB. “Primers synthesized based on sequences (a)-(l)” mean primers encoding all or a part of amino acid sequences (a)-(l), and having a length sufficient to specifically hybridize to a specific site of a template DNA. The nucleotide sequences of primers synthesized based on sequences (a)-(l) and amino acid sequences encoded by these nucleotide sequences are shown in the following table: 4 Sequence Amino acid sequence Nucleotide sequence (a) SEQ ID NO: 26 SEQ ID NO: 25 SEQ ID NO: 30 SEQ ID NO: 29 SEQ ID NO: 54, 55, 56, 57 SEQ ID NO: 53 (b) SEQ ID NO: 34 SEQ ID NO: 33 SEQ ID NO: 36, 37 SEQ ID NO: 35 (c) SEQ ID NO: 28 SEQ ID NO: 27 SEQ ID NO: 32 SEQ ID NO: 31 SEQ ID NO: 42 SEQ ID NO: 41 (d) SEQ ID NO: 46, 47 SEQ ID NO: 45 (e) SEQ ID NO: 39, 40 SEQ ID NO: 38 (f) SEQ ID NO: 44 SEQ ID NO: 43 (g) SEQ ID NO: 49 SEQ ID NO: 48 (h) SEQ ID NO: 63, 64 SEQ ID NO: 62 (i) SEQ ID NO: 59 SEQ ID NO: 58 (j) SEQ ID NO: 66, 67, 68 SEQ ID NO: 65 (k) SEQ ID NO: 51, 52 SEQ ID NO: 50 (l) SEQ ID NO: 61 SEQ ID NO: 60 Microorganisms applicable to the identification and detection method of the present invention include bacteria, yeast, Fungus, archaebacteria and the like. 
 PREFERRED EMBODIMENTS OF THE INVENTION 
 EXAMPLE 1 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 25 and 27 (corresponding to the amino acid sequences of SEQ ID NOS: 26 and 28, respectively) as primers and DNA from Bacteroides vulgatus IFO 14291 strain as a template. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 1 and 2, respectively. The PCR amplification conditions were as described below. 5 PCR amplification conditions: 96° C. 1 min; 48° C. 1 min; 72° C. 2 min: 3 cycles 96° C. 1 min; 48° C. 1 min; 72° C. 2 min: 3 cycles 96° C. 1 min; 48° C. 1 min; 72° C. 2 min: 30 cycles Total: 36 cycles Primer concentration 1 &mgr;M each dATP 200 &mgr;M each Template DNA <1 &mgr;g/100 &mgr;l AmpliTaq ™ and the supplied PCR Buffer (Perkin Elmer) were used. 
 EXAMPLE 2 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 29 and 31 (corresponding to the amino acid sequences of SEQ ID NOS: 30 and 32, respectively) as primers and DNA from Mycobacterium simiae KPM 1403 strain as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 3 and 4, respectively. 
 EXAMPLE 3 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 33 and 27 (corresponding to the amino acid sequences of SEQ ID NOS: 34 and 28, respectively) as primers and DNA from Chitinophaga pinensis DSM 2588 strain as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 5 and 6, respectively. 
 EXAMPLE 4 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 25 and 35 (corresponding to the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 36 or 37, respectively) as primers and DNA from Flavobacterium aquatile IAM 12316 strain as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 7 and 8, respectively. 
 EXAMPLE 5 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 29 and 38 (corresponding to the amino acid sequences of SEQ ID NO: 30 and SEQ ID NO: 39 or 40, respectively) as primers and DNA from Mycobacterium asiaticum ATCC 25274 strain as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 9 and 10, respectively. 
 EXAMPLE 6 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 41 and 43 (corresponding to the amino acid sequences of SEQ ID NOS: 42 and 44, respectively) as primers and DNA from Cytophaga lytica IFO 16020 strain as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 11 and 12, respectively. 
 EXAMPLE 7 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 45 and 48 (corresponding to the amino acid sequences of SEQ ID NO: 46 or 47 and SEQ ID NO: 49, respectively) as primers and DNA from Synechococcus sp. PCC 6301 strain as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 13 and 14, respectively. 
 EXAMPLE 8 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 53 and 62 (corresponding to the amino acid sequences of SEQ ID NO: 54, 55, 56 or 57 and SEQ ID NO: 63 or 64, respectively) as primers and DNA from Caulobacter crescentus ATCC 15252 strain as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 15 and 16, respectively. 
 EXAMPLE 9 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 53 and 58 (corresponding to the amino acid sequences of SEQ ID NO: 54, 55, 56 or 57 and SEQ ID No: 59, respectively) as primers and DNA from Pseudomonas putida ATCC 17484 strain as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 17 and 18, respectively. 
 EXAMPLE 10 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 65 and 50 (corresponding to the amino acid sequences of SEQ ID NO: 66, 67 or 68 and SEQ ID NO: 51 or 52, respectively) as primers and DNA from Synechococcus sp. PCC 6301 strain as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 19 and 20, respectively. 
 EXAMPLE 11 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 60 and 31 (corresponding to the amino acid sequences of SEQ ID NOS: 61 and 32, respectively) as primers and DNA from Caulobacter crescentus ATCC 15252 strain as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 21 and 22, respectively. 
 EXAMPLE 12 A PCR was performed using oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOS: 25 and 43 (corresponding to the amino acid sequences of SEQ ID NOS: 26 and 44, respectively) as primers and DNA from an unidentified strain MBIC 1544 as a template. The PCR amplification conditions were the same as in Example 1. The nucleotide sequence of the amplified DNA fragment and the amino acid sequence deduced therefrom are shown in SEQ ID NOS: 23 and 24, respectively. This nucleotide sequence was compared with the nucleotide sequence database possessed by the applicant. As a result, the unidentified strain MBIC 1544 was identified as Cytophaga lytica. 
 EFFECT OF THE INVENTION With the nucleotide sequence of gyr B determined by the pre sent invention, it is possible to classify or identify an unidentified microorganism strain quickly and accurately. Besides, according to the present invention, PCR primers for monitoring a specific microorganism which are needed in risk assessment in various bioprocesses can be designed easily. Also, the present invention enables highly accurate monitoring of changes in mycelial tufts.