US 5,484,723 AGrant
Flavin Reductase Gene From Vibrio Fischeri
Issue Date:1996-01-16
•8 Claims
•2 Drawing Sheets
Abstract
A flavin reductase gene is provided having the nucleotide sequence of SEQ ID NO:1, wherein the sequence is displayed in numbered triplets of capital letters, which numbers proceed sequentially from left to right and from the 5' terminus to the 3' terminus; the sequence of capital letters represent the purine and pyrimidine bases of the nucleotide sequence, as follows: A is adenine; G is guanine; C is cytosine; T is thymine; R is A or G; Y is T or C; N is A, T, C, or G; H is A, C, or T; and M is A or C; wherein triplet number 237 thereof is TAA or TAG or TGA; and wherein further: for triplets numbered 13, 21, 22, 36, 63, 85, 92, 111, 112, 126, 140, 150, 156, 186, 196, and 228, if the 3' nucleotide of a triplet is A or G, then the 5' nucleotide of the triplet is T or C, or if the 3' nucleotide of a triplet is T or C, then the 5' nucleotide of the triplet is C; and if the 5' nucleotide of a triplet is C, then the 3' nucleotide of the triplet is A, T, C, or G, or if the 5' nucleotide of a triplet is T, then the 3' nucleotide of the triplet is A or G; for triplets numbered 19, 46, 56, 103,108, 123, 145, 206, and 214, if the 3' nucleotide of a triplet is T or C, then the 5' nucleotide of the triplet is A or C, or if the 3' nucleotide of a triplet is T or C, then the 5' nucleotide of the triplet is C; and if the 5' nucleotide of a triplet is G, then the 3' nucleotide of the triplet is A, T, C, or G, or if the 5' nucleotide of a triplet is A, then the 3' nucleotide of the triplet is A or G.
Metadata
Assignee
- Chisso Corporation
Inventors
- Shuhei Zenno
- Kaoru Saigo
Application Information
Application Number:US 2665700
Filing Date:1994-06-28
Priority Date:1993-06-28
Art Unit:184
Classifications
IPC:
C12N 902
Field of Search:
435536189;252.3;69.1;172.3;320.123.2
Patent Drawings (2 sheets)
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a flavin reductase gene from a luminescent bacterium Vibrio fischeri, a flavin reductase enzyme, a recombinant vector containing the gene and a bacterium containing the recombinant vector.
2. Description of the Related Art
An enzyme luciferase from a luminescent bacterium forms an oxidized flavin mononucleotide (hereinafter referred to as FMN) and a long chain fatty acid, using as substrates, a reduced flavin mononucleotide, (hereinafter referred to as FMNH.sub.2) and a long chain fatty aldehyde and in the presence of the enzyme, and at that time, catalyzes a blue color-luminescence reaction.
In the cell, the substrate FMNH.sub.2 used in this reaction is supplied from a reduced nicotinamide adenine dinucleotide : flavin mononucleotide (NADH : FMN) reductase and a reduced nicotinamide adenine dinucleotide phosphate: flavin mononucleotide (NADPH : FMN) reductase, and the long chain fatty aldehyde is supplied from a fatty acid reductase complex.
Recently, Spyrou et al. isolated a flavin reductase gene from Escherichia coli and clarified its primary structure and discloses it in [Spyrou, G., Haggard-Ljungquist, E., Krook, M., Jornvall, H., Nilsson, E. & Reichard, P. (1991), J. Bacteriol. 173, 3673-3679].
The present inventors have also already isolated an FMN reductase gene of a luminescent bacterium, Vibrio fischeri and have determined its nucleotide sequence. Further, we have succeeded in expressing the gene in Escherichia coli (Japanese patent application No. Hei 03-351,717). Further, we have isolated a flavin reductase gene from a luminescent bacterium, Xenorhabdus luminescens and have succeeded in its expression in Escherichia coli (Japanese patent application No. Hei 04-279029). This time, we have isolated a novel flavin reductase gene distinct from the above one (the contents corresponding to the present application).
FMNH.sub.2 is easily and instantaneously autoxidized in air and converted into FMN. In order to make the luminescence ability of the bacterial luciferase exhibit up to the most, it is necessary to always continuously supply the FMNH.sub.2 as the substrate, and FMN reductase is most important for attaining the object.
As described above, this enzyme couples with the bacterial luciferase and catalyzes the reaction of converting FMN as the product of the luciferase reaction into FMNH.sub.2 ; hence when the luciferase is made coexistent with FMN reductase in the reaction system, it is possible to maintain the luminescence reaction. Namely, since the bacterial luciferase turns over many time, luminescence continues as far as a large excess of a long chain aldehyde is present in the reaction system.
As described above, FMN reductase is inevitable for making the best use of the bacterial luciferase, and when its gene is obtained, it is possible to prepare the present enzyme in a large quantity.
Problem to Be Solved by the Invention
The present inventors have made extensive research, and as a result, have isolated a flavin reductase gene from a luminescent bacterium, Vibrio fischeri (ATCC 7744), and have succeeded in clarifying its primary structure, and further have succeeded in preparing Escherichia coli expressing the gene in a large quantity. Thus, the present invention has been completed.
Namely, in view of the above technical situation, the object of the present invention is to provide a flavin reductase gene of luminescent bacterium, Vibrio fischeri, and a flavin reductase, and the object is further to provide a recombinant vector containing the gene and a bacterium containing the recombinant vector.
SUMMARY OF THE INVENTION
Means for Solving the Problem
The present invention has the following constitutions (1) to (8):
(1) The flavin reductase gene having the following nucleotide sequence: ##STR1## wherein the sequence is displayed in numbered triplets of capital letters, which numbers proceed sequentially from left to right and from the 5' terminus to the 3' terminus; the sequence of capital letters represent the purine and pyrimidine bases of the nucleotide sequence, as follows:
A is adenine; G is guanine; C is cytosine; T is thymine;
R is A or G; Y is T or C; N is A, T, C, or G; H is A, C, or T; and M is A or C;
wherein triplet number 237 thereof is TAA or TAG or TGA; and wherein further: for triplets numbered 13, 21, 22, 36, 63, 85, 92, 111, 112, 126, 140, 150, 156, 186, 196, and 228, if the 3' nucleotide of a triplet is A or G, then the 5' nucleotide of said triplet is T or C, or if the 3' nucleotide of a triplet is T or C, then the 5' nucleotide of said triplet is C; and if the 5' nucleotide of a triplet is C, then the 3' nucleotide of said triplet is A, T, C, or G, or if the 5' nucleotide of a triplet is T, then the 3' nucleotide of said triplet is A or G;
for triplets numbered 19, 46, 56, 103, 108, 123, 145, 206, and 214, if the 3' nucleotide of a triplet is T or C, then the 5' nucleotide of said triplet is A or C, or if the 3' nucleotide of a triplet is T or C, then the 5' nucleotide of said triplet is C; and if the 5' nucleotide of a triplet is G, then the 3' nucleotide of said triplet is A, T, C, or G, or if the 5' nucleotide of a triplet is A, then the 3' nucleotide of said triplet is A or G.
(2) The flavin reductase gene according to item (1), which comprises the following sequence: ##STR2##
(3) The flavin reductase gene comprising the following nucleotide sequence: ##STR3##
(4) The flavin reductase enzyme having an amino acid sequence comprising: ##STR4##
(5) The recombinant vector comprising SEQ ID NO:1, as set forth in item (1).
(6) The recombinant vector according to item (5), having a gene having the nucleotide sequence SEQ ID NO:1 of item (1) inserted into a plasmid vector.
(7) The bacterium containing a recombinant vector containing SEQ ID NO:1 of item (1).
(8) The process for producing an enzyme containing an amino acid sequence expressed by SEQ ID NO:6 of item (4), which process comprises cultivating a bacterium modified by a recombinant vector containing a DNA expressed by SEQ ID NO:1 of item (1).
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
FIG. 1 illustrates amino acid sequences (SEQ ID NO:8 and SEQ ID NO:10) having a flavin reductase preserved therein and synthesized oligonucleotide primers.
FIG. 2 illustrates a restriction enzyme map of the enzyme gene of the present invention and a sequence strategy. The arrow marks each indicate the direction in which the nucleotide sequence has been determined. The part indicated by the box corresponds to the structural gene part of the enzyme.
FIG. 3 illustrates the construction steps of the expression vector pf FRI of the present invention containing a flavin reductase gene of the luminescent bacterium. The white box indicates the coding region of the flavin reductase gene. The dotted lines indicate the vector part of pUC13 and the black box indicates the promoter part of lactose operon of Escherichia coli.
Explanation of the symbols:
lacP: promoter of lactose operon
pUC13: plasmid vector
.lambda.VF2: recombinant phage DNA
pVF2: recombinant plasmid
pfFR1: expression plasmid
HpaI; restriction enzyme
SacII: restriction enzyme
SnaBI: restriction enzyme
PacI: restriction enzyme
SacII: restriction enzyme
NspV: restriction enzyme
BglII: restriction enzyme
SalI: restriction enzyme
HindIII: restriction enzyme
XhoI: restriction enzyme
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The constitution and effectiveness of the present invention will be described below.
The enzyme gene of the present invention is characterized by containing a nucleotide chain having a sequence length of 711, expressed by SEQ ID NO:1.
The preferable sequence contains a nucleotide chain expressed by SEQ ID NO:2.
The sequence of DNA having a sequence length of 1221, and expressed by SEQ ID NO:5 can be concretely exemplified.
The kind of the sequence of the gene is Genomic DNA and the gene is isolated from a luminescent bacterium, Vibrio fischeri (ATCC 7744). The sequence codes a protein of a molecular weight of 26067 consisting of 236 amino acids of sequence No. 287 to No. 994 of SEQ ID NO:5, for example.
The enzyme gene product of the present invention has a flavin reducing activity, and for example has an FMN reducing activity.
The enzyme of the present invention is a protein having the amino acid sequence of SEQ ID NO:6, which is derived by translating SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. The protein consists of 236 amino acids, has a molecular weight of 26067 and exhibits a flavin reducing activity in a luminescent bacterium.
The recombinant vector of the present invention contains a DNA expressed by a nucleotide sequence SEQ ID NO:1. Namely, the recombinant vector of the present invention contains a DNA having a nucleotide sequence expressed by the sequence SEQ ID NO:3 and a functional equivalent thereto. The terms "functional equivalent" refer to a DNA fragment which is usable according to an essentially same method in order to obtain an essentially same result, in the production of an enzyme having an FMN reducing activity of a luminescent bacterium by means of an adequate host.
Namely, the DNA fragment refers to a DNA fragment which can code a protein having the same amino acid sequence even when the nucleotide sequence is different, or a DNA fragment which can code a protein having an FMN reducing activity, although there is a certain difference of amino acid sequence accompanying a certain difference of nucleotide sequence. Concretely, it refers to the nucleotide sequence of the sequence SEQ ID NO:1 or the nucleotide sequence of the sequence SEQ ID NO:1 obtained by site-directed mutagenesis.
For example, it refers to the recombinant vector having the DNA fragment containing the nucleotide sequence inserted into a plasmid vector. As such a vector, pUC (C. Yanisch-Perron, J. Vieira & J. Messing, Gene, 33, 110-115 [1985]), pIN III (Y. Masui, J. Coleman, M. Inouye, Experimental Manipulation of Gene Expression (ed. M. Inouye), p. 15, Academic Press [1983], etc. are usable.
FIG. 3 illustrates the construction steps of the recombinant vector (expression vector). Namely, SalI fragment of about 13 kb as an insert from phage DNA .lambda.AVF2 having a reductase gene is inserted into the cleavage site of SalI of pUC13, to obtain a plasmid pVF2, followed by digesting the pVF2 plasmid DNA with XhoI and BglII, recovering the 1.2 kb fragment, and again ligating it to SalI-BamHI cleavage site, to prepare an expression vector pfFR1. As a result, pfFR1 has a flavin reductase gene arranged under the rule of the lac promoter, to thereby express the flavin reductase gene.
The bacterium of the present invention includes a +recombinant DNA containing the nucleotide sequence expressed by the sequence SEQ ID NO:1. The bacterium of the present invention is characterized by producing a protein having a flavin reducing activity.
The production process of the enzyme of the present invention consists in cultivating a bacterium modified by a recombinant vector (expression vector) containing the DNA having a nucleotide sequence expressed by sequence SEQ ID NO:1 to produce a protein containing an amino acid sequence expressed by the sequence SEQ ID NO:6. As the bacterium, Escherichia coli, Bacillus subtilis, etc. are exemplified, and as the medium, LB medium, YT medium, etc. are enumerated.
The procedures of isolating the gene and identifying it, important in the present invention will be described by way of Examples.
EXAMPLE 1
Isolation of a flavin reductase gene (fre) of Vibrio fischeri:
A luminescent bacterium, Vibrio fischeri (ATCC 7744), was cultivated with shaking in a medium of Photobacterium at 26.degree. C. overnight, followed by centrifugal separation at 10,000 rpm, to collect the bacteria, and suspending the bacterial cells in Tris.HCl-EDTA buffer (hereinafter referred to as TE buffer).
The resulting suspension was treated with lyzozyme at 37.degree. C. for one hour, followed by adding sodium dodecylsulfate (hereinafter abbreviated to SDS), subjecting the mixture to proteinase K treatment at 50.degree. C. for 3 hours, three times carrying out phenol treatment, precipitating with ethanol, drying, dissolving in TE buffer, again subjecting to proteinase K treatment, three times treating with phenol, precipitating with ethanol and recovering genome DNA.
Using synthesized oligonucleotide primers FRE3 and FRE4 shown in FIG. 1, which are portions preserved in the coding regions of Escherichia coli and the flavin reductase enzyme gene (fre) of a luminescent bacterium, Xenorhabdus luminescens (ATCC 29999), the fre gene was amplified using Vibrio fischeri as a template, according to PCR method [Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis and H. A. Erlic (1988), Science 239 487] and the DNA fragment 10 was inserted into the cleavage site of Hinc II of pUC 8 plasmid DNA [Hanna, Z., Fregeau, C., Prefontaine, G., Brousseau, R. (1984), Gene, 30 247].
Determination of nucleotide sequence:
The gene was confirmed to be Vibrio fischeri fre gene according to the method of [Hattori, M. & Sasaki, Y. (1986), Anal. Biochem. 152 232]. From this plasmid DNA, DNA fragment of the fre gene portion was cut off with HindIII/EcoRi, and was used as a probe DNA for screening of fre gene having a complete chain length. The .sup.32 P labeling of the probe DNA was carried out according to the random priming method [Feinberg, A. & Vogelstein, B. (1983), Anal. Biochem. 132 6].
EXAMPLE 2
Preparation of .lambda. phage library of the luminescent bacterium gene:
The luminescent bacterium, Vibrio fischeri (ATCC 7744) was cultivated under shaking in a Photobacterium medium at 26.degree. C. overnight, followed by subjecting the bacterium to centrifugal separation at 10,000 rpm to collect the bacterium, suspending the bacterial cells in Tris HCl.EDTA buffer (hereinafter referred to as TE buffer), treating the suspension with lyzozyme at 37.degree. C. for one hour, adding sodium dodecylsulfate (hereinafter abbreviated to SDS), subjecting the mixture to protainase K treatment at 50.degree. C. for 3 hours, three times carrying out phenol treatment, precipitating with ethanol, drying, dissolving in TE buffer, again subjecting to proteinase K treatment, three times treating with phenol and precipitating with ethanol, to recover genome DNA.
A restriction enzyme, Sau3AI of 10 units, was reacted with the genome DNA (50 .mu.g) at 37.degree. C., followed by dispersing each portion of the reaction product at the respective reaction times of 5, 10, 20, 30, 45, 60, 90 and 120 minutes, adding EDTA (ethylenediaminetetracetic acid) to terminate the reaction, and subjecting the respective portions to agarose gel electrophoresis to confirm the degree of the partial decomposition of the genome DNA, combining together the reaction solutions of the respective times into one solution, and precipitating with ethanol to recover the precipitate.
The precipitate was dissolved in a small quantity of TE buffer, followed by subjecting the solution to agarose electrophoresis, recovering a fraction of 9 to 23 kilobases (Kb) by means of an electric elution procedure, electrophoretically eluting the DNA of the 9 to 23 Kbs fraction from an agarose gel containing the fraction of 9 to 23 Kbs into a dialysis tube, three times treating with phenol, precipitating with ethanol, and dissolving the precipitate in a TE buffer so as to give about 200 ng/.mu.l.
The DNA of the above fraction of 9 to 23 Kbs was subjected to a ligating reaction with T4 DNA ligase at 16.degree. C. overnight, to EMBL3 phage DNA cleaved by a restriction enzyme BamHI in advance and treated by an alkaline phosphotase. The ligase reaction solution was mixed with a packaging extraction solution, followed by reacting the mixture at 22.degree. C. for 2 hours to prepare a recombinant phage. This phage was made a gene library.
EXAMPLE 3
Isolation of a flavin reductase gene having a complete long chain:
The titer of the gene library prepared in Example 2 was measured, followed by scattering the gene library described above so that 10,000 phages per one plate could form plaques, cultivating at 37.degree. C. overnight, allowing the resulting culture to stand at 4.degree. C. for 2 hours, and transferring phages to two nylon membrane filters against the respective plates.
The filter was denatured, followed by neutralization and then ultraviolet-rays irradiation. Further, it was placed in a hybridization solution {20 ml of 6.times.SET buffer [20.times.SET buffer: NaCl (3 M), Tris-HCl (pH 8.0) (0.6M) and EDTA (0.04M)], 10.times.Denhardt's solution (calf serum albumin, polyvinyl pyrrolidone, and Ficoll, each 0.2% solution), 0.1% SDS and salmon sperm DNA (heat-denatured, 50 .mu.g/ml)}, followed by keeping the temperature at 68.degree. C. for one hour.
Further, the solution was replaced, followed by keeping the temperature at 68.degree. C. for one hour, adding a probe of .sup.32 P-labeled Vbrio fischeri fre, hybridizing at 65.degree. C. overnight, discarding the solution, washing the filter with a 2.times.SET buffer, shaking with 0.2.times.SET buffer at 65.degree. C. for 20 minutes, twice repeating this procedure, air-drying; subjecting to autoradiography, overlaying the filter on a developed X-ray film, and photographing the position of ink marker on the film.
A positive phage (clone) whose signal was identified to overlie on two films made from one plate, was obtained from about 1,000 recombinant phages. This clone was named .lambda.VF2. This insert DNA was inserted into the SalI site of pUC13 plasmid.
Using a synthetic oligonucleotide primer (20 mer) prepared based upon the nucleotide sequence of PCR clone, and using the above plasmid DNA as a template, the nucleotide sequence was determined according to the dideoxy method (Hattori, M. & Sasaki, Y. (1986), Anal. Biochem. 152, 232).
Further, a synthetic primer was newly prepared based upon the nucleotide sequence, and its nucleotide sequence was determined in the same manner. FIG. 2 illustrates its restriction map and the strategy of the sequence determination. The determined nucleotide sequence was as shown in the sequence SEQ ID NO:3, and it was considered that the sequence had a length of 1221 bp and coded a protein consisting of 236 amino acids of nucleotide Nos. 287 to 994 and having a molecular weight of 26067.
EXAMPLE 4
Construction of expression vector of flavin reductase gene and preparation of its transformed strain (see FIG. 3):
DNA of a recombinant plasmid pVF2 was digested with XhoI and BglII, followed by recovering a fragment of 1.2 Kb, subjecting the fragment to a ligation reaction to the SalI/Bam.sup.HI site of plasmid pUC13, with T4 DNA ligase transforming a portion of the reaction solution into Escherichia coli D1210 strain, preparing a plasmid DNA from the transformed strain and choosing an insert DNA of 1.2 Kb.
The plasmid was named pfFR1. This pfFR1 was in the form wherein a flavin reductase gene was arranged under the rule of the promoter of lactose operon (lac) and was constructed so as to express the flavin reductase.
EXAMPLE 5
Preparation of flavin reductase and measurement of its activity:
A solution (0.25 ml) obtained by cultivating the above transformed strain overnight was inoculated in an LB liquid medium (10 ml) containing ampicillin (0.1 mg/ml), followed by cultivating under shaking at 37.degree. C. for 2 hours, adding isopropyl-.beta.-D(-)-thiogalactopyranoside (hereinafter abbreviated to IPTG) so as to give a final concentration of 1 mM and further cultivating for 3 hours.
The resulting culture solution (3.0 ml) subjected to IPTG derivation treatment was subjected to centrifugal separation at 10,000 rpm, followed by removing the supernatant, suspending the resulting bacterial cells in a buffer solution (0.75 ml) of 50 mM potassium phosphate containing 1 mM dithiothreitol, subjecting the suspension to ultrasonic crushing and subjecting to centrifugal separation at 4.degree. C. for 30 minutes, to make the supernatant a cell extraction liquid. The enzymatic flavin reducing activity of the cell extraction liquid was measured. The results are shown in Table 1.
Flavin reductase activity Was measured according to the method of Jablonski, E. & DeLuca, M. (1977), Biochemistry, 16, 2932. The protein concentration was determined using a protein assay kit made by Bio-Rad Co., Ltd., and according to dye-binding method (Bradford., M. M. (1976), Anal. Biochem. 72, 248-254).
As seen from the results of Table 1, the values of the reductase activities of FMN and riboflavin of pfFR1 were higher by a number of one cipher than those of pUC13. This gene was confirmed to code the flavin reductase.
Effectiveness of the Invention:
The enzyme gene of the present invention was isolated as a gene of enzyme having FMN reducing activity of a luminescent bacterium, Vibrio fischeri By employing a suitable host such as Escherichia coli, it is possible to prepare a large quantity of the enzyme protein from the Escherichia coli.
By introducing its expression vector into a suitable host such as Escherichia coli, it is possible to prepare an organism or microorganism expressing an enzyme having an FMN reducing activity of the luminescent bacterium, in a large quantity, and further, by extracting the enzyme from the organism having introduced the gene, it is possible to prepare the reductase in a large quantity.
Due to the above-mentioned functions, the reductase amplifies the luminescent reaction of the bacterial luciferase, can be applied to a number of measurement methods, and is useful for example as diagnostic medicine or inspection medicine.
Claims
What we claim is:
1. A flavin reductase gene having the nucleotide sequence of SEQ ID NO:1, wherein the sequence is displayed in numbered triplets of capital letters, which numbers proceed sequentially from left to right and from the 5' terminus to the 3' terminus; the sequence of capital letters represent the purine and pyrimidine bases of the nucleotide sequence, as follows: A is adenine; G is guanine; C is cytosine; T is thymine; R is A or G; Y is T or C; N is A, T, C, or G; H is A, C, or T; and M is A or C; wherein triplet number 237 thereof is TAA or TAG or TGA; and wherein further: for triplets numbered 13, 21, 22, 36, 63, 85, 92, 111, 112, 126, 140, 150, 156, 186, 196, and 228, if the 3' nucleotide of a triplet is A or G, then the 5' nucleotide of said triplet is T or C, or if the 3' nucleotide of a triplet is T or C, then the 5' nucleotide of said triplet is C; and if the 5' nucleotide of a triplet is C, then the 3' nucleotide of said triplet is A, T, C, or G, or if the 5' nucleotide of a triplet is T, then the 3' nucleotide of said triplet is A or G; for triplets numbered 19, 46, 56, 103, 108, 123, 145, 206, and 214, if the 3' nucleotide of a triplet is T or C, then the 5' nucleotide of said triplet is A or C, or if the 3' nucleotide of a triplet is T or C, then the 5' nucleotide of said triplet is C; and if the 5' nucleotide of a triplet is G, then the 3' nucleotide of said triplet is A, T, C, or G, or if the 5' nucleotide of a triplet is A, then the 3' nucleotide of said triplet is A or G.
2. The flavin reductase gene according to claim 1, wherein said gene has the nucleotide sequence of SEQ ID NO:3.
3. A flavin reductase gene having the nucleotide sequence of SEQ ID NO:5.
4. A flavin reductase enzyme having the amino acid sequence of SEQ ID NO:6.
5. A recombinant vector comprising the nucleotide sequence of SEQ ID NO:1, as set forth in claim 1.
6. The recombinant vector according to claim 5, wherein said SEQ ID NO:1 is inserted into a plasmid vector.
7. A bacterium having a recombinant vector comprising the flavin reductase gene according to claim 1.
8. A process for producing an enzyme having the amino acid sequence of SEQ ID NO:6, comprising cultivating a bacterium according to claim 7.
Non-Patent Literature (2)
- Zenno et al. 1994 J. Bacteriology 176: 3544-3551.
- Spyrou et al. 1991 J. Bacteriology 173: 3673-3679.