WO1999014361A1

WO1999014361A1 - Hybridization probes which differentiate between streptomycetes and maduromycetes

Info

Publication number: WO1999014361A1
Application number: PCT/EP1998/006038
Authority: WO
Inventors: Olga Genilloud; Rafael P. Mellado; Victor Parro; Vicente Rodriguez
Original assignee: Merck Sharp & Dohme De Espana, S.A.E.
Priority date: 1997-09-18
Filing date: 1998-09-16
Publication date: 1999-03-25
Also published as: EP1012346A1; JP2001516590A; CA2303463A1

Abstract

The invention provides a method to differentiate between bacteria from the maduromycetes and the streptomycetes taxa using a specific nucleic acid probe that is complementary to a conserved region of the maduromycetes 16S rRNA genes. The probe permits the rapid detection of DNA coding for the 16S rRNA in a sample differentiating streptomycetes from maduromycetes within the Actinomycetales order. The method is accurate and reproducible.

Description

TITLE OF THE INVENTION

HYBRIDIZATION PROBES WHICH DIFFERENTIATE BETWEEN

STREPTOMYCETES AND MADUROMYCETES

CROSS-REFERENCE TO RELATED APPLICATIONS

Not Applicable.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not Applicable.

REFERENCE TO MICROFICHE APPENDIX

Not Applicable.

FIELD OF THE INVENTION The invention relates to nucleic acid probes derived from the region coding for the mature 16S ribosomal RNA (rRNA) of maduromycetes, which specifically differentiate between streptomycetes and maduromycetes. This invention also relates to assays using these probes.

BACKGROUND OF THE INVENTION

Although much progress have been made in the last decade for the identification of microorganisms, the procedures in use are often still laborious, non-sensitive and not specific. Many of these pitfalls can be overcome by using nucleic acid probes. These nucleic acid probes, made of genomic DNA, plasmids, riboprobes or synthetic oligonucleotides, may target the genomic DNA or certain RNA species present in biological samples. The use of synthetic oligonucleotides as probes is largely preferred because oligonucleotides can be rapidly synthesized in large amounts using chemical methods, have a long shelf- life, and are easily to purify and to label.

Traditional methods for describing the composition of natural microbial communities present difficulties because they are expensive and laborious, and they only permit the identification of those microorganisms that can be cultivated in the laboratory. It is generally considered that less than 20% of the microorganisms in nature have been discovered, and that new culture-independent methods for studying the composition of microbial communities are needed. (Ward, D.M. et al, 1990 Nature, 345: 63-64). The sequence of 16S rRNA, a common but distinctive cellular element, has already been used for this purpose, and has revealed the presence of numerous uncultured microorganisms in natural communities (Ward, D.M. et al., supra; Giovannoni, S.J. et al, 1990, Nature, 345: 60-63; Barry T. et al., 1991 , In PCR Methods and Applications, Cold Spring Harbour, pp 51-56; Mehling, A. et al., 1995, Microbiology, 141: 2139-2147; and Rheims, H. et al., 1996, Microbiology, 142: 2863-2870).

Species -specific probes have been described for a large number of microorganisms. The 16S and 23S rRNA genes are quite often used for probe development since sequences can easily be obtained using described methods and it is known that variable regions exist within these highly conserved genes which can be used for species- specific detection. Universal probes for the detection of bacteria are known (See, for example, Giovannoni, S.J. et al., 1988, /. Bacteriol. 170: 720-726 and Barry, T. et al, 1991, supra). The Actinomycetes are aerobic, gram-positive bacteria which form branching, usually non-fragmenting hyphae and asexual spores borne on aerial mycelia. Data from 16S rRNA sequences has allowed the construction of an evolutionary tree for this order which is quite complex, containing some 37 genera in at least seven groups which include the Streptomycetes and the Maduromycetes. (Goodfellow, M., 1989, "Suprageneric classification of Actinomycetes", In Bergey's Manual of Systematic Bacteriology Vol. 4, Williams and Wilkins pp. 2333-2339).

Currently the art is silent as to a simple yet robust method to discriminate between close groups of actinomycetes which occur in natural isolates, particularly for those bacteria which develop aerial mycelia prior to release of their spores. It would be desirable to have nucleic acid probes which could be used in fast, accurate assays which could differentiate between maduromycetes and streptomycetes. DETAILED DESCRIPTION OF THE INVENTION

This invention also relates to nucleic acid probes which hybridize to nucleic acids encoding a portion of the 16S rRNA of maduromycetes bacteria under hybridization conditions, and which do not hybridize to nucleic acids encoding a portion of 16S rRNA of streptomycetes bacteria under identical hybridization conditions.

Another aspect of this invention is a method for detecting the presence of maduromycetes bacteria in a sample comprising: a) contacting the sample with a nucleic acid probe; wherein said probe hybridizes to nucleic acid encoding maduromycetes 16S rRNA, but not to nucleic acids encoding streptomycetes 16S rRNA; b) imposing hybridization conditions, and c) determining if hybridization has occurred. Yet another aspect of this invention is a method of differentiating maduromycetes from streptomycetes in a bacteria sample comprising: a) lysing the bacteria to release bacterial DNA; b) extracting the bacterial DNA; c) contacting the extracted DNA with a probe comprising the sequence of a CNB-ESP probe under hybridizing conditions; and d) determining if hybridization of the probe to the extracted DNA has occurred. A further embodiment of this invention includes a kit for the detection of a bacteria from the maduromycetes group which comprises a probe of from 10 to 250 nucleotides in length which is complementary to or homologous with at least 90% of a nucleic acid sequence comprising maduromycetes nucleic acids corresponding to base pairs 68 to 90 of Streptomyces ambofaciens DNA encoding the mature 16S rRNA molecule.

The kit may additionally comprises reagents, compositions, instructions, disposable hardware and suitable packaging. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a particularly preferred probe of this invention. As used throughout this application and claims, the term "probe" will refer to synthetic or biologically produced nucleic acids, between 10 and 250 base pairs in length which contain specific nucleotide sequences that allow specific and preferential hybridization under predetermined conditions to target nucleic acid sequences, and optionally contain a moiety for detection or for enhancing assay performance. A minimum of ten nucleotides is generally necessary in order to statistically obtain specificity and to form stable hybridization products, and a maximum of 250 nucleotides generally represents an upper limit for length in which reaction parameters can be easily adjusted to determine mismatched sequences and preferential hybridization. Probes may optionally contain certain constituents that contribute to their proper or optimal functioning under certain assay conditions. For examples, probes may be modified to improve their resistance to nuclease degradation (for example, by end-capping), to carry detection ligands (for example fluorescein, 32 P, biotin, etc.) or to facilitate their capture onto a solid support (for example, poly- deoxyadenosine "tails").

"Preferential hybridization" or "hybridizing preferentially" means that hybridization with the intended target nucleic acid results in a hybridization reaction product which is more stable than any hybridization reaction product resulting from hybridization with a non- target nucleic acid under identical conditions. It is well within the skill of the ordinary artisan to compare stability of hybridization reaction products and evaluate which one is more stable, i.e. determine which one has bound "preferentially".

The terms "homology" and "homologous to" are meant to refer to the degree of similarity between to or more nucleic acid sequences and is not meant to imply any taxonomic relatedness between organisms. The degree of similarity is expressed as a percentage, i.e., 90% homology between two sequences will mean that 90% of the bases of the first sequence are identically matched to the bases of the second sequence.

"Specific" means that a nucleotide sequence will hybridize to a predetermined target sequence and will not substantially hybridize to a non-target sequence.

"Specifically discriminate" means that a probe will substantially hybridize to a predetermined target sequence and will not substantially hybridize to a non-target sequence.

"Hybridization" is a process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion to form a double stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another. "Substantial hybridization" means that the amount of hybridization observed will be such that one observing the results would consider the result positive in a clinical setting Data which is considered "background noise" is not substantial hybridization.

"Stringent hybridization conditions" means approximately 35°C to 65°C in a salt solution of approximately 0.9 molar NaCl. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a rule, the stringency of the conditions under which a hybridization is to take place will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art. In designing a probe for identification purposes it is preferred that the probe should be as specific as necessary (i.e., it should not cross-react with undesired nucleic acids) and it should be highly sensitive (i.e. most if not all, strains of the organism to be detected should react with the probe). Hence, the preferred target sequences should have the following characteristics: a) the sequence should be present in the genome of each strain of the microorganism concerned; and b) the evolutionary diversity of the sequence should be such that, on the one hand, there is sufficient sequence-diversity to allow differentiation of the species concerned from other closely related species and, in the other hand, sufficient sequence-conservation to allow detection of the strain of concern with the probe used. Comparison and alignment of the maduromycetes and streptomycetes 16S rDNA sequences present in data bases confirmed the existence of the so-called variable regions that appears in all 16S rDNA of actinomycetes, but also allowed the identification of a particular region within the Actinomadura 16S rDNA, in which the homology with the Streptomyces equivalent sequences seemed to be low. Sequence comparison and analysis were carried out using programs from the UWGCG package (Version 9.0, December, 1996) and the Edit- View 1.0 DNA sequencer viewer (Applied Biosystems). 16S rDNA sequences were obtained from the NCBI Genbank database.

As a result of the sequence comparison, two primers were designed. This first is designated NVR1 : 5' CAC GGA GAG TTT GAT CCT GGC 3' (SEQ.rD.NO: l) which is base pairs 2-12 from the Streptomyces ambofaciens DNA encoding mature 16S rRNA. (Pernodet, J-L., et al, 1989, Gene, 79: 33-46). The second is designated SOR: 5' GTA TTA GAC CCA GTT TCC CGG GC 3' (SEQ.ID.NO.:2) which is maduromycetes DNA that corresponds base pairs 144-176 from the Streptomyces ambofaciens DNA encoding mature 16S rRNA (Pernodet, 1989, supra). Both of these can be used as primers in polymerase chain reaction technology to amplify the DNA region to obtain a preferred probe of this invention designated large quantities of the probe can be generated using known PCR techniques such as those in U.S. Patents 4,683,202 and 4,683,195. A preferred probe CNB-ESP was derived from a highly variable region of the 16S rRNA moelcule. It comprises the sequence 5' GAA AGG CCC TTC GGG GGT ACT CG 3' (SEQ.ID.NO:3), which is maduromycetes DNA corresponding to base pairs 68 to 90 from the Streptomyces ambofaciens DNA encoding mature 16S rRNA. In accordance with this invention, probes similar to CNB-ESP may be made by increasing or decreasing the length of CNB-ESP. For a longer probe, it is preferred that additional nucleotides (either 3' or 5') be those of the corresponding Streptomyces ambofaciens DNA encoding mature 16S rRNA.

One embodiment of this invention is a nucleic acid, designated CNB-ESP, which provides specific binding to the chromosomal DNA encoding the mature part of the 16S rRNA molecule of bacteria from the maduromycetes group and does not substantially hybridize to the equivalent chromosomal region from bacteria belonging to the close related streptomycetes taxa.

The preferred probes of this invention generally contain from at least about 23 nucleotides to about 166 nucleotides (the maximum number of nucleotides of the precursor region plus the mature region plus the regulatory region of the genes coding for the 16S rRNA). More preferably, the probe will contain from about 23 nucleotides to about 166 nucleotides resulting from the PCR amplification of a DNA fragment comprising a DNA sequence hybridizing with the 23 nucleotides long CNB-ESP probe. A particularly preferred probe is given in Figure 1.

The invention also relates to probes for use in hybridization assays, which use an oligonucleotide that is sufficiently complementary to hybridize to a sequence of the chromosomal DNA region encoding the mature 16S rRNA from maduromycetes but is not complementary enough to hybridize to the equivalent region from far related Gram positive bacteria as the bacterium Bacillus subtilis.

A particularly preferred assay in accordance with this invention is a Southern Blot. One probe which can be used for a Southern blot assay is about 23 to 166 bp long, obtained by PCR amplification of the DNA fragment obtained by use of the two primers NVR1 and SOR. The Southern blot, or dot blot assay can be conducted using well known procedures. Generally, it involves the steps of immobilizing a target nucleic acid or population of nucleic acids on a filter such as nitrocellulose, nylon or other derivatized membranes which are readily commercially available. The immobilized nucleic acids are then tested for hybridization under predetermined stingency conditions with the probe of interest. Under stringent conditions probes with nucleotide sequences with greater complementary to the target will exhibit a higher level of hybridization than probes whose sequences have less homology. Hybridization can be detected in a number of ways. For example, the probe can be isotopically labeled with the addition of a

32

P-Phosphorous moiety to the 5 '-end of the oligonucleotide by the conventional polynucleotide kinase reaction. After hybridization has occurred, unhybridized probe is removed by washing. The filters are exposed to x-ray film and the intensity of the hybridization signals is evaluated.

The probes of this invention may be chemically synthesized using commercially available methods and equipment. For example, the solid phase phosphoramidite methods can be used to produce short oligonucleotides between 15 and 30 nucleotides long. For this invention is preferred to chemically synthesize short DNA oligonucleotides using any of the Applied Biosystems DNA Synthesizers, using reagents supplied by the same company. The chemically synthesized oligonucleotides were obtained from Boehringer Mannheim.

Maduromycetes strains which are detectable by the probes of this invention may be obtained from the ATCC collection and are listed in the following table: STRAIN NAME ATCC NUMBER

Actinomadura citrea ATCC 27887

Actinomadura coerulea ATCC 33576

Actinomadura cremea ATCC 33577

Actinomadura spadix ATCC 27298

Actinomadura spiralis ATCC 35114

A ctinomadura fastidiosa ATCC 33516

Actinomadura ferruginea ATCC 35575

Actinomadura helvata ATCC 27295

Actinomadura livida ATCC 33578

Actinomadura madurae ATCC 19425

Actinomadura malachitica ATCC 27888

Actinomadura pusilla ATCC 27296

Actinomadura roseola ATCC 33579

Actinomadura rubra ATCC 27031

Actinomadura salmonea ATCC 33580

Actinomadura viridis ATCC 27103

Microbispora aerata ATCC 15448

Microbispora amethysto genes ATCC 15740

Microbispora diastatica ATCC 33325

Microbispora echinospora ATCC 27300

Microbispora parva ATCC 33326

Microbispora rosea ATCC 12950

Microtetraspora flexuosa ATCC 35864

Microtetraspora fusca ATCC 23058

Microtetraspora glauca ATCC 23057

Microtetraspora niveoalba ATCC 27301

Planobispora rosea ATCC 23866

Streptosporangium roseum ATCC 12428

Streptosporangium vulgare ATCC 33329 The following streptomycetes strains may be used in this invention: Streptomyces ambofaciens, S. antibioticus, S. cinnamonensis, S. coelicolor A3 (2), S. fradiae, S. lividans TK21 , S. nataliensis, S. peucetius, S. violascens and Streptomyces sp. Procedures used for the growth and manipulation of the bacteria related this invention and general DNA manipulation were as described (Hopwood et al, 1985, Genetic Manipulation of Streptomyces. A Laboratory Manual John Innes Foundation. Norwich; and Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York). For DNA extraction, bacteria were grown in LB or YEME media at the ATCC recommended temperatures. Chromosomal DNA was purified from cultures growing in late exponential phase as described for maize (Dellaporta et al, 1985, "Maize DNA Miniprep". Molecular Biology of Plants- a Laboratory Course Manual Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory, N.Y. USA. pp. 36-37) as adapted for Streptomyces (Mehling et al. 1995, Microbiology, 141 :2139-2147).

The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLE 1 Differentiation between streptomycetes and maduromycetes by Southern analysis of genomic DNA To validate the usefulness of the CNB-ESP probe to differentiate between streptomycetes and maduromycetes, the strains of bacteria were obtained and grown until mid logarithmic phase. Genomic DNA was prepared as follows: Approximately 0.5-1. Og mycelia were resuspended in 2 ml lysis buffer (NaCl 0.1 M, EDTA 50 mM, pH 8.0) containing glass beads (3 mm diameter) and the suspension was vortexed for 2 minutes before adding 2 ml of lysis buffer plus 10- 15 mg lysozyme and 50 mg ml^~l RNase DNase-free. The suspension was incubated for 30-80 min. at 37°C. After the addition of 400 ml 10% SDS (w/v), the solution was incubated at 37°C for 15 min. The glass beads were removed and the DNA extracted four times with 1 volume of phenol/chloroform/isoamyl alcohol (25:24: 1) and once more with 1 volume of chloroform. The extracted DNA was ethanol precipitated, dried and resuspended in 500 ml distilled water. 20 mg of the chromosomal DNA extracted from each strain was restricted with 50 units of the endonucleases BamHI or PstI by incubation in the respective buffers as recommended by the supplier (Boehringer Mannheim) at 37°C for 16 h and the samples fractionated by electrophoresis in 0.8% agarose gels. The fractionated DNA fragments were transferred to Hybond N+ membranes (Amersham, pic.) by capillary transfer for 16 h. The DNA immobilized in the solid support was then washed with a hybridization buffer containing 5 x SSC, 5 x Denhardt's solution and 0.5% SDS and allowed to hybridize with 10 pmol of the radioactively labeled CNB-ESP probe in the same buffer for 16 h at 45°C. The solid supports were then washed three times with lx SSC (0.15 M NaCl plus 0.015 M sodium citrate, pH 7.2) and 0.5% SDS at the hybridization temperature. The solid supports were then set to exposure in X-ray films at -70°C prior to be developed. The probe hybridized with all the maduromycetes strains listed in the table above but not with the Streptomyces strains mentioned above. As expected the probe also gave a negative result with low G+C content genomic DNA from the far related bacterium Bacillus subtilis carried as a negative control. The results obtained indicate that CNB- ESP can differentiate between maduromycetes and streptomycetes in hybridization with genomic DNA.

EXAMPLE 2 Differentiation between streptomycetes and maduromycetes by Southern analysis of PCR amplified DNA Chromosomal DNA from all the strains used in Example 1 was extracted as described and the extracted DNA was used for PCR amplification using primers NVRl 5'- CAC GGA GAG TTT GAT CCT GGC 3' (SEQ.ID.NO.:l) and SOR (5'-GTA TTA GAC CCA GTT TCC CGG GC 3' (SEQ.ID.NO.:2). Approximately 0.5-1.0 mg genomic DNA template was used with 280 ng of each primer in a final reaction volume of 100 ml of a incubation buffer containing 16.6 mM (NH4)2S04, 67 mM Tris-HCl (pH 8.8), 0.1 % Tween-20 and 1 mM MgCl2- Amplifications were performed in automated thermocyclers by incubation at 95°C (5 min) followed by 30 cycles of incubation at 95°C (1 min), 55°C (1 min) and 72°C (1 min) in the presence of one unit of EcoTaq polymerase (Ecogen), plus a final elongation cycle of 10 min at 72°C.

The resulting about 166 bp long amplified DNA fragments were fractionated by electrophoresis in 1.5% agarose gels. The fractionated DNA fragments were transferred to Hybond N+ membranes (Amersham, pic.) by capillary transfer for 16 h. The DNA immobilized in the solid support was then washed with a hybridization buffer containing 5 x SSC, 5 x Denhardt's solution and 0.5% SDS and set to hybridize with 10 pmol. of the radioactively labeled CNB-ESP probe in the same buffer for 16 h at 45°C. The solid supports were then washed three times with lx SSC (0.15 M NaCl plus 0.015 M sodium citrate, pH 7.2) and 0.5% SDS at the hybridization temperature. The solid supports were then set to exposure in X-ray films at -70°C prior to be developed.

The probe hybridized with all the about 166 bp long PCR amplified fragments from all the maduromycetes strains listed in the Table above but not with Streptomyces strains mentioned above. The results obtained indicate that CNB-ESP can differentiate between maduromycetes and streptomycetes in hybridization with genomic DNA.

Claims

WHAT IS CLAIMED:

1. A nucleic acid probe which hybridizes to a nucleic acid encoding a portion of 16S rRNA of maduromycetes bacteria under hybridization conditions, and which does not hybridize to nucleic acids encoding a portion of 16S rRNA of streptomycetes bacteria under identical hybridization conditions.

2. A probe according to Claim 1 which is DNA.

3. A probe according to Claim 2 which is between 10- 250 base pairs.

4. A probe according to Claim 3 which is complementary to or homologous with at least 90% of a nucleic acid sequence comprising a maduromycetes nucleic acid corresponding to base pairs 68 - 90 of Streptomycetes ambofaciens DNA encoding mature 16S rRNA.

5. A probe according to Claim 3 comprising

5'-GAA AGG CCC TTC GGG GGT ACT CG-3' (SEQ.ID. NO.:3).

6. A probe according to Claim 4 which is Figure 1.

7. A method for detecting the presence of maduromycetes bacteria in a sample comprising: a) contacting the sample with a nucleic acid probe, wherein said probe hybridizes to nucleic acid encoding maduromycetes 16S rRNA, but not to nucleic acids encoding streptomycetes 16S rRNA; b) imposing hybridization conditions; and c) determining if hybridization has occurred.

8. A method according to Claim 7 further comprising the step of lysing bacteria in the sample prior to step a).

9. A method according to Claim 8 wherein the probe is a radioactively labeled probe.

10. A method according to Claim 9 wherein hybridization conditions are stringent hybridization conditions.

11. A method for differentiating between maduromyces bacteria and streptomyces bacteria in a sample comprising: a) immobilizing nucleic acids from putative maduromycetes and/or streptomyces bacteria; b) contacting the immobilized nucleic acids with a probe, wherein said probe hybridizes to nucleic acids encoding maduromycetes 16S rRNA but not to nucleic acids encoding streptomycetes 16S rRNA; and wherein said probe is labeled; c) imposing hybridization conditions; and d) detecting hybridization of the probe to maduromycetes nucleic acid.

12. A method according to Claim 11 wherein said probe comprises nucleic acids selected from the group consisting of: a) nucleic acids comprising SEQ.ID.NO.:3; b) nucleic acids comprising a 166 bp amplification product made by PCR amplification using primers SEQ.ID.NO.: l and SEQ. ID. NO.2, and c) nucleic acids shown in Figure 1.