US20090117567A1 - EUBACTERIAL tmRNA SEQUENCES AND USES THEREOF - Google Patents

EUBACTERIAL tmRNA SEQUENCES AND USES THEREOF Download PDF

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US20090117567A1
US20090117567A1 US12/163,276 US16327608A US2009117567A1 US 20090117567 A1 US20090117567 A1 US 20090117567A1 US 16327608 A US16327608 A US 16327608A US 2009117567 A1 US2009117567 A1 US 2009117567A1
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Brice Felden
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention is directed to eubacterial tmDNA sequences and the corresponding tmRNA sequences.
  • the present invention is further directed to alignments of eubacterial tmDNA sequences and use of the sequences and sequence alignments for the development of antibacterial drugs.
  • the present invention is also directed to the use of the sequences for the development of diagnostic assays.
  • Eubacterial tmRNAs (10Sa RNAs) are unique since they function, at least in E. coli , both as tRNA and as mRNA (for a review, see Muto et al., 1998). These ⁇ 360 ⁇ 10% nucleotide RNAs are charged with alanine at their 3′-ends (Komine et al., 1994; Ushida et al., 1994) and also have a short reading frame coding for 9 to 27 amino acids depending on the bacterial species. E.
  • coli tmRNA mediates recycling of ribosomes stalled at the end of terminatorless mRNAs, via a trans-translation process (Tu et al., 1995; Keiler et al., 1996; Himeno et al., 1997).
  • this amino acid tag is co-translationally added to polypeptides synthesized from mRNAs lacking a termination codon, and the added 11 amino acid C-terminal tag makes the protein a target for specific proteolysis (Keiler et al., 1996).
  • tmRNAs have some structural similarities with canonical tRNAs, especially with tRNA acceptor branches.
  • E. coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine, located in the tRNA-like domain of the molecule, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs (Felden et al., 1998).
  • the present invention relates to eubacterial tmDNA sequences and the corresponding tmRNA sequences.
  • the present invention further relates to alignments of eubacterial tmDNA sequences and use of the sequences and sequence alignments for the development of antibacterial drugs.
  • tmDNA sequences were amplified from all species tested apart from those in the alpha- Proteobacteria . This aspect of the invention allowed a more systematical study of the structure and overall distribution of tmRNA within eubacteria
  • alignments are made with the newly isolated tmDNA sequences and previously disclosed tmRNA sequences.
  • the alignments of the tmRNA sequences allow the identification of targets for development of antibacterial drugs.
  • novel tmDNA or tmRNA sequences of the present invention are used to develop diagnostic assays, such as amplification-based assays, for the bacterial species disclosed herein.
  • FIGS. 1A-1B show the effect of the annealing temperature ( FIG. 1A ) and magnesium concentration ( FIG. 1B ) on amplifying eubacterial tmRNA genes from genomic DNAs using PCR.
  • A Varying the annealing temperature from 50° to 70° C. during the PCR amplification of Thermus aquaticus (1).
  • B Varying the magnesium concentration to amplify tmDNA genes from Thermus aquaticus (1), negative effect of increasing the magnesium concentration), Acholeplasma laidlawii (2), positive effect of increasing the magnesium concentration, the upper band is the tmDNA gene) and from Mycoplasma salivarium (3), no discernible effect of magnesium ions in that concentration range).
  • the arrows point toward the 4 novel tmDNA genes that have been sequenced.
  • FIG. 2 shows the distribution of tmDNA sequences within eubacterial genomes.
  • the circled phyla or subgroups contain tmDNA sequences and those shaded are new members of this category.
  • the numbers shown close to each phylum are the 51 tmDNA sequences that have are disclosed herein and the numbers in parenthesis are the 53 tmDNA sequences that were previously known (summarized in Williams, 1999; Wower and Zwieb, 1999).
  • the environmental samples are indicated with a dashed line as their connection to the tree is unknown.
  • the 5 alpha- Proteobacteria in which tmDNA sequences were not detected by PCR analysis are labeled “PCR” and the 3 analyzed by Blast search of the complete, or nearly complete, sequenced genomes are labeled “database”.
  • FIGS. 3A , 3 B and 3 C show the sequence alignment, structural domains and structural features for the tmRNA of several species of Firmicutes.
  • the tmRNA sequences are set forth in SEQ ID NOs:67-87.
  • FIGS. 4A and 4B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Thermophiles.
  • the tmRNA sequences are set forth in SEQ ID NOs:88-99.
  • FIGS. 5A and 5B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Cyanobacteries ( 5 A) and chloroplasts ( 5 B).
  • the tmRNA sequences of the Cyanobacteries are set forth in SEQ ID NOs:100-103, and the tmRNA sequences of the chloroplasts are set forth in SEQ ID NOs:104-108.
  • FIGS. 6A and 6B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Mycoplasmes.
  • the tmRNA sequences are set forth in SEQ ID NOs:109-117.
  • FIGS. 7A-1 , 7 A- 2 , 7 B, 7 C and 7 D show the sequence alignment, structural domains and structural features for the tmRNA of several species of Mesophiles ( 7 A- 1 , 7 A- 2 , 7 C, 7 D) and environmental sludge ( 7 B).
  • the tmRNA sequences of the Mesophiles are set forth in SEQ ID NOs:118-123 and 125-128, and the tmRNA sequence of the environmental sludge is set forth in SEQ ID NO:124.
  • FIGS. 8A and 8B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Actinobacteries ( 8 A) and Spirochaetes ( 8 B).
  • the tmRNA sequences of the Actinobacteries are set forth in SEQ ID NOs:132-136
  • the tmRNA sequences of the Spirochaetes are set forth in SEQ ID NOs:137-142.
  • FIGS. 9A and 9B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Pourpres beta.
  • the tmRNA sequences are set forth in SEQ ID NOs:143-154.
  • FIGS. 10A , 10 B and 10 C show the sequence alignment, structural domains and structural features for the tmRNA of several species of Pourpres gamma.
  • the tmRNA sequences are set forth in SEQ ID NOs:155-169.
  • FIGS. 11A and 11B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Pourpres delta ( 11 A) and Pourpres epsilon ( 11 B).
  • the tmRNA sequences of the Pourpres delta are set forth in SEQ ID NOs:170-172
  • the tmRNA sequences of the Pourpres epsilon are set forth in SEQ ID NOs:173-175.
  • the present invention is directed to eubacterial tmDNA sequences and the corresponding tmRNA sequences.
  • the present invention is further directed to alignments of eubacterial tmDNA sequences and use of the sequences and sequence alignments for the development of antibacterial drugs.
  • the novel eubacterial tmDNA sequences determined in accordance with the present invention are set forth in Tables 1-58, below.
  • the alignment of tmRNA sequences is shown in FIGS. 3A-11B , which also show the structural domains and structural features of the tmRNA.
  • the present invention also includes the tmRNA sequences set forth in these figures to the extent they differ from the sequences set forth in Tables 1-58.
  • sequences represent targets for the development of drugs which may be broadly applicable to many kinds of bacteria, or may be broadly applicable only to a particular genera of phylum of bacteria, or may be specifically applicable to a single species of bacteria.
  • the present invention is further directed to the development of drugs for the therapeutic treatment of bacteria, generically or specifically. Suitable drugs are developed on the basis of the tmRNA sequences as described herein.
  • RNA helices which can be sometimes interrupted by bulges or pseudoknots are either RNA helices which can be sometimes interrupted by bulges or pseudoknots.
  • the RNA helices which are always present are H1, H2, H5 and H6.
  • Helices H1 and H6 are found in all canonical transfer RNAs.
  • H1 and H6 are not good targets for drug development because drugs that would target them will also interfere with the biology of the individual that has a given disease. Consequently, very good candidates for development of drugs for targeting as many bacteria as possible are helices H2 and H5.
  • helices H2 and H5 are critical for the folding of all these tmRNA since both of them connect the two ends of the molecule together. Disruption of either H2 or H5 with a specific drug would lead to inactive tmRNA molecules in vivo.
  • pseudoknots PK1, PK2 and PK3 are always found in the bacterial tmRNAs. Since these pseudoknots are not found in all canonical transfer RNAs, they can also be targeted with specific drugs. Disruption of either PK1 or PK2 or PK3 with a specific drug would lead to inactive tmRNA molecules in vivo.
  • the coding sequence In addition to developing drugs which broadly target many bacteria, drugs are developed which are more genera specific. For trying to target specifically a given bacteria or a complete phylum, the coding sequence (shown in all the alignments) is a very good candidate. Indeed, this region of the RNA is very accessible for DNA antisense binding (such as shown for Escherichia coli ; Matveeva et al., 1997), and thus, is also available for interaction with other drugs. Moreover, the coding sequence is a critical functional domain of the molecule in its quality-control mechanism in cells.
  • the fourth pseudoknot PK4 is made of two smaller pseudoknots called PK4a and PK4b;
  • helix H2 is made of only 4 base-pairs instead of 5 in the other species;
  • pseudoknot PK1 is located upstream (at the 5′-side) of the coding sequence.
  • this pseudoknot is a target for future anti-bacterial drugs.
  • recent data have shown that this PK1 pseudoknot, among all the four pseudoknots within tmRNA gene sequences (sometimes there's only 2 or 3 detectable pseudoknots, depending upon the sequences), is the only one that its correct folding is essential for the biological activity of tmRNA (Nameki et al., 1999; Nameki et al., 2000).
  • alpha-proteobacteria possess tmRNA genes. These genes are permuted and are made in two parts, connected via a processed linker. These tmRNA gene sequences from alpha-proteobacteria were not found in the course of the present invention because usual PCR methods could not amplify them.
  • tmRNA genes are present in all eubacterial genomes, with no exceptions, but are not present in any genomes from archebacteries or eukaryotes, with the exception of some chloroplasts.
  • the very specific location of tmRNA genes within one of the three main kingdoms of life make them ideal targets for the design of novel antibiotics that will, in principle, interfere very weakly with human biochemistry, compared to usual antibiotics.
  • novel antibiotics see Breithaupt (1999).
  • the present invention is also directed to diagnostic assays and kits for the detection of bacterial infection, particularly infections caused by bacterial agents disclosed herein.
  • the coding sequence of each bacterial species is used to design specific primers for use in amplification-based diagnostic assays for infectious diseases.
  • Specific primers are designed in accordance with well known techniques, and such design is readily done by a skilled artisan.
  • Amplification-based diagnostic assays are performed in accordance with conventional techniques well known to skilled artisans.
  • amplification-based assays include, but are not limited to, polymerase chain reaction (PCR) amplification, strand displacement amplification (SDA), ligase chain reaction (LCR) amplification, nucleic acid sequence based amplification (3SR or NASBA) and amplification methods based on the use of Q-beta replicase.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • LCR ligase chain reaction
  • 3SR or NASBA nucleic acid sequence based amplification
  • Drugs which target the sequences described herein are active agents can be formulated in pharmaceutical compositions, which are prepared according to conventional pharmaceutical compounding techniques ( Remington's, 1990).
  • the composition may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, intrathecal, epineural or parenteral.
  • the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/11698.
  • the compound may dissolved in a pharmaceutical carrier and administered as either a solution of a suspension.
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the compounds When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
  • the active agent is preferably administered in an therapeutically effective amount.
  • the actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences (18).
  • targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell-specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, or would otherwise require too high a dosage, or otherwise be unable to enter the target cells.
  • Antisense active agents can also be delivered by techniques described in U.S. Pat. Nos. 5,811,088; 5,861,290 and 5,767,102.
  • Bacterial genomic DNAs were prepared from ⁇ 10 mg freeze-dried cells provided from ATCC (American Type Culture Collection, Virginia, USA). Cell pellets were resuspended in 750 ⁇ L of lysis buffer (50 mM Tris (pH 8.0), 50 mM EDTA and 20% sucrose). 150 ⁇ L of a 10 mg/mL solution of lysozyme was mixed and let stand at room temperature for 15 min. 150 ⁇ L of 1% SDS was added and let stand at room temperature for 15 minutes. Four to five phenol/chloroform extractions were performed, until the sample was clear and there was no interphase.
  • lysis buffer 50 mM Tris (pH 8.0), 50 mM EDTA and 20% sucrose.
  • 150 ⁇ L of a 10 mg/mL solution of lysozyme was mixed and let stand at room temperature for 15 min.
  • 150 ⁇ L of 1% SDS was added and let stand at room temperature for 15 minutes. Four to five phenol/chloroform
  • genomic DNA was precipitated with 1/10 volume of 3M NaOAc (pH 5.5) and 1 volume isopropanol, and stored at ⁇ 20° C. for 2 hours. After centrifugation, the genomic DNAs were washed with 70% ethanol, vacuum-dried and diluted in sterile water to a final concentration of 10 ng/ ⁇ L.
  • primer ser A (based on E. coli tmRNA termini): 5′-GGG GCT GAT TCT GGA TTC GAC-3′ (SEQ ID NO:1) and 5′-TGG AGC TGG CGG GAG TTG AAC-3′; primer set B (based on T. neapolitana tmRNA termini): 5′-GGG GGC GGA AAG GAT TCG ACG-3′ (SEQ ID NO:3) and 5′-TGG AGG CGG CGG GAA TCG AAC-3′; (SEQ ID NO:4) primer set C (based on M.
  • pneumoniae tmRNA termini 5′-GGG GAT GTC ATG GTT TTG ACA-3′ (SEQ ID NO:5) and 5′-TGG AGA TGG CGG GAA TCG AAC-3′; (SEQ ID NO:6) primer set D (based on C. tepidum tmRNA termini): 5′-GGG GAT GAC AGG CTA TCG ACA-3′ (SEQ ID NO:7) and 5′-TGG AGA TGG CGG GAC TTG AAC-3′. (SEQ ID NO:8)
  • Sequences of tmRNA genes were obtained by polymerase chain reaction (PCR) in 25 ⁇ L using 40 ng of genomic DNA per reaction. The following general scheme was utilized for all of the sequences:
  • the various PCR-amplified tmDNA bands were gel purified (5% PAGE), stained (ethidium bromide staining), cut using a sterile razor blade, and shaken over-night (passive elution, using a vibrator) in a 350 ⁇ l solution containing 10 mM Tris-HCl buffer (pH 8.1). The following day, the PCR amplified tmDNAs were ethanol precipitated, washed in 70% ETOH, vacuum dried and the DNA pellets were dissolved in 18 ⁇ l of RNase-DNase free sterile water.
  • Dye terminator sequencing was achieved at the DNA sequencing facility of the Human Genetics Institute. In addition to novel tmRNA sequences that are not available publicly, several tmDNA sequences that were already known have been verified and several sequencing mistakes have been found and corrected (especially for Alcaligenes eutrophus tmRNA).
  • Eubacterial tmDNA was amplified by PCR in accordance with Example 1, using the following conditions.
  • Primer Set B Annealing temp. during PCR: 53° C. for 20 sec; Mg 2+ conc.: 4.5 mM.
  • Primer Set B Annealing temp. during PCR: 55° C. for 30 sec; Mg 2+ conc.: 5.5 mM.
  • Primer Set A Annealing temp. during PCR: 46° C. for 30 sec; Mg 2+ conc.: 4.5 mM.
  • Primer set B Annealing temp. during PCR: 55° C. for 30 sec; Mg 2+ conc.: 4.5 mM.
  • Primer set C Annealing temp. during PCR: 51° C. for 20 sec; Mg2+ conc.: 13.5 mM.
  • Primer set D Annealing temp. during PCR: 50° C. for 30 sec; Mg 2+ conc.: 9.5 mM.
  • Primer set A Annealing temp. during PCR: 51° C.; Mg 2+ conc.: 3.5 mM.
  • Primer set A Annealing temp. during PCR: 52° C.; Mg 2+ conc.: 5.5 mM.
  • Primer set A Annealing temp. during PCR: 50-55° C.; Mg 2+ conc.: 4.5 mM.
  • Primer set A Annealing temp. during PCR: 52° C.; Mg 2+ conc.: 3.5 to 5.5 mM.
  • Primer set A Annealing temp. during PCR: 46° C.; Mg 2+ conc.: 4.5 mM.
  • Primer set A Annealing temp. during PCR: 48 to 52° C.; Mg 2+ conc.: 7.5 mM.
  • Primer set B Annealing temp. during PCR: 55° C.; Mg 2+ conc.: 3.5 to 4.5 mM.
  • Primer set A Annealing temp. during PCR: 46° C. for 30 sec; Mg 2+ conc.: 3.5 mM.
  • Primer set A Annealing temp. during PCR: 44 C for 30 sec; Mg 2+ conc.: 5.5 mM.
  • Primer set A Annealing temp. during PCR: 52° C.; Mg 2+ conc.: 4.5 mM.
  • Primer set B Annealing temp. during PCR: 55° C.; Mg 2+ conc.: 5.5 mM.
  • Primer set B Annealing temp. during PCR: 46° C.; Mg 2+ conc.: 7.5 mM.
  • Primer set B Annealing temp. during PCR: 52° C.; Mg 2+ conc.: 3.5 mM.
  • Primer set A Annealing temp. during PCR: 53° C. for 25 sec; Mg 2+ conc.: 3.5 mM.
  • thermophilic and mesophilic eubacterial tmRNA genes were optimized for both thermophilic and mesophilic eubacterial tmRNA genes.
  • both the magnesium concentration and the annealing temperature were optimized.
  • FIG. 1A a specific amplification of Thermus aquaticus tmDNA was observed with an annealing temperature around 50° C., whereas at higher temperatures there is a gradual decrease in the amount of amplified tmDNA.
  • the magnesium concentration during PCR was critical ( FIG. 1B ), but the annealing temperature could vary from 44° C. to 60° C. without significant effects on the amplification.
  • FIG. 1B shows various effects of increasing the magnesium concentration on the PCR amplification of three novel eubacterial tmDNA genes.
  • Increasing magnesium concentration from 3.5 mM to 5.5 mM has either a negative ( FIG. 1B , panel 1), a positive ( FIG. 1B , panel 2) or no effect on specifically amplifying eubacterial tmDNA genes.
  • tmRNA genes from many eubacteria including known human pathogens were amplified.
  • the PCR was facilitated by sequence conservation at both 5′ and 3′ ends and was performed as described (Williams and Bartel, 1996), with modifications.
  • This study was initiated to collect further sequences from eubacterial tmDNA genes, as well as to test experimentally whether tmDNA genes could be found in all bacterial phyla or subgroups.
  • 51 new tmDNA sequences were determined ( FIG. 2 ), including sequences from members of 8 additional phyla and 1 subgroup (shaded boxes in FIG. 2 ).
  • the 58 new tmDNA sequences are set forth in Tables 1-58.
  • tmDNA sequences could be amplified from all species tested apart from those in the alpha- Proteobacteria.
  • Five genomic DNAs from alpha- Proteobacteria Agrobacterium tumefaciens, Bartonella henselae, Bartonella quintana, Rhodospirillum rubrum and Rickettsia prowazekii ) were extensively checked using various oligonucleotides, annealing temperatures and magnesium concentrations. No specific amplified tmDNA sequences were detected in this subgroup.
  • tmDNA sequences may have largely diverged in the alpha-proteobacterial sub-group compared to other bacterial phyla, and that both PCR methods and Blast searches are missing the relevant sequences.
  • tmRNA is dispensable in E. coli (Ando et al., 1996), it is striking that it has been found in all bacteria tested other than the alpha- Proteobacteria.
  • the alpha- Proteobacteria have undergone reductive evolution. This has been more intensive in one of the two sub-classes than in the other (Gray and Spencer, 1996), but tmRNA sequences have not been found even in the sub-class with the larger genome.
  • the alpha- Proteobacteria and mitochondria are evolutionary relatives (Yang et al., 1985; Andersson et al., 1998).
  • the drastic downsizing in what has become mitochondrial genomes means that it is not reasonable to draw inferences on the relationship between alpha- Proteobacteria and mitochondria based on their mutual apparent absence of tmRNA. It is nevertheless, of interest, that at least some chloroplasts and cyanelle genomes have tmDNA sequences, and the cyanobacteria, with which they are evolutionary related, also have tmRNA.
  • Acidobacterium capsulatum (SEQ ID NO:9) GGGGGCGGAAAGGATTCGACGGGGTTGACTGCGGCAAAGAGGCATGCCGG GGGGTGGGCACCCGTAATCGCTCGCAAAACAATACTTGCCAACAACAATC TGGCACTCGCAGCTTAATTAAATAAGTTGCCGTCCTCTGAGGCTTCGCCT GTGGGCCGAGGCAGGACGTCATACAGCAGGCTGGTTCCTTCGGCTGGGTC TGGGCCGCGGGGATGAGATCCACGGACTAGCATTCTGCGTATCTTGTCGC TTCTAAGCGCAGAGTGCGAAACCTAAAGGAATGCGACTGAGCATGGAGTC TCTTTTCTGACACCAATTTCGGACGCGGGTTCGATTCCCGCCGCCTCCAC CA
  • Fibrobacter succinogenes Fibrobacter ) (SEQ ID NO:15) GGGGCTGATTCTGGATTCGACAGGGTTACCGAAGTGTTAGTTGCAAGTCG AGGTCTCAGACGAGGGCTACTCGTTAAAAAGTCTGAAAAAAAATAAGTGC TGACGAAAACTACGCACTCGCTGCCTAATTAACGGCAACGCCGGGCCTCA TTCCGCTCCCATCGGGGTGTACGTCCGGACGCAATATGGGATAGGGAAGT GTCATGCCTGGGGGCATCTCCCGAGATTTTCTAGGCTGGTCAAACTCCGC GCCGACCTTCTTGGGCGTGGATAAGACGAGATCTTAAATTCGAAGGGAAC ACTTGTAGGAACGTACATGGACGTGATTTTGGACAGGGGTTCAACTCCCG CCAGCTCCA
  • Verrucomicrobium spinosum ( verrucomicrobium ) (SEQ ID NO:66) GGGNNNNATTTGGAATTCGCCGAATGCTAGAAGTGGAGGCTGCATGCCGC GGATGATTCGTTGGCCGCTTTACCAATTCGGATCAAACAACTAAATGCGG ACTCTAACGAGCTTGCCCTCGCCGCTTAATTGACGGTGACGTTCCTCCAG TGAAGTCTGTGAATTGGAGGAGCGACTACTTACAGGCTGGCCAAAAGAGC GGGCGACCGGCCCCAAGGCGAGATCTACAGGCCGCTGGATGGACGGCATC CTGGCAGTAGGAGGCTGGACATCGAGATCAAATNATTGCCTGAGCATGGA GACGCTTTCATAAAGGNGTTCGGACAGGG
  • the newly discovered tmRNA sequences and several known tmRNA sequences were aligned to identify target sites for drug development.
  • the alignments of the sequences are shown in FIGS. 3A-11B .
  • the nucleotides in the tmRNA sequences of these figures exist in several motifs (Felden et al., 1999). These motifs include nucleotides considered to be in RNA helices (Watson-Crick base-pairs GC or AU, or GU Wobble base-pairs). Nucleotides that are in single stranded RNA domains, hence not base-paired. Some nucleotides in the single stranded domains are universally conserved nucleotides.
  • nucleotides are the exceptions to a quasi-sequence conservation in the sequences alignment.
  • nucleotides exist in well established non-canonical structural motifs in RNA structures; for example AG-GA pairs, AA pairs, etc.
  • Some nucleotides are universally conserved Wobble GU base-pairs.
  • H means helices
  • PK means pseudoknot.
  • a pseudoknot is made of the pairing of parts of an RNA-loop with an upstream sequence. Consequently, two helices are made (shown in Felden et al., 1999) for all the 4 pseudoknots PK1 to PK4 for each sequence.
  • the tRNA-like domain as well as the coding sequence, namely the two functional units of the molecule, have also been indicated for each sequence.
  • sequences represent targets for the development of drugs which may be broadly applicable to many kinds of bacteria, or may be broadly applicable only to a particular genera of phylum of bacteria, or may be specifically applicable to a single species of bacteria.
  • RNA helices which can be sometimes interrupted by bulges or pseudoknots are either RNA helices which can be sometimes interrupted by bulges or pseudoknots.
  • the RNA helices which are always present are H1, H2, H5 and H6.
  • Helices H1 and H6 are found in all canonical transfer RNAs.
  • H1 and H6 are not good targets for drug development because drugs that would target them will also interfere with the biology of the individual that has a given disease. Consequently, very good candidates for development of drugs for targeting as many bacteria as possible are helices H2 and H5.
  • helices H2 and H5 are critical for the folding of all these tmRNA since both of them connect the two ends of the molecule together. Disruption of either H2 or H5 with a specific drug would lead to inactive tmRNA molecules in vivo.
  • pseudoknots PK1, PK2 and PK3 are always found in the bacterial tmRNAs. The PK1 structural domain is strictly conserved in the tmRNAs and is located upstream of the coding sequence. Since these pseudoknots are not found in all canonial transfer RNAs, they can also be targeted with specific drugs. Disruption of either PK1 or PK2 or PK3 with a specific drug would lead to inactive tmRNA molecules in vivo.
  • RNA antisense binding which has been shown for Escherichia coli , and thus, is also available for interaction with other drugs.
  • this is a critical functional domain of the molecule in its quality-control mechanism in cells.
  • this coding sequence would be the ideal target to use for designing specific PCR-based diagnostic assays for infection diseases.
  • the fourth pseudoknot PK4 is made of two smaller pseudoknots called PK4a and PK4b;
  • helix H2 is made of only 4 base-pairs instead of 5 in the other species;
  • pseudoknot PK1 is located upstream (at the 5′-side) of the coding sequence.
  • this pseudoknot is a target for future anti-bacterial drugs.
  • recent data have shown that this PK1 pseudoknot, among all the four pseudoknots within tmRNA gene sequences (sometimes there's only 2 or 3 detectable pseudoknots, depending upon the sequences), is the only one that its correct folding is essential for the biological activity of tmRNA (Nameki et al., 1999; Nameki et al., 2000).

Abstract

The present invention is directed to eubacterial tmDNA sequences and the corresponding tmRNA sequences. The present invention is further directed to alignments of eubacterial tmDNA sequences and the use of the sequences and sequence alignments for the development of antibacterial drugs. The present invention is also directed to the use of the sequences for the development of diagnostic assays.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • The present application is division of U.S. patent application Ser. No. 11/329,230 filed on 11 Jan. 2006, which in turn in a division of U.S. patent application Ser. No. 09/958,206 filed on 20 Feb. 2002, now U.S. Pat. No. 7,115,366, which in turn is a national stage filing under 35 U.S.C. §371 of International patent application No. PCT/US00/08988 filed on 6 Apr. 2000, which in turn is related to and claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application Ser. No. 60/128,058 filed on 7 Apr. 1999. Each of these applications is incorporated herein by reference.
  • This application was made with Government support under Grant No. GM 48152, funded by the National Institutes of Health, Bethesda, Md. The Government has certain rights in this invention.
    • BACKGROUND OF THE INVENTION
  • The present invention is directed to eubacterial tmDNA sequences and the corresponding tmRNA sequences. The present invention is further directed to alignments of eubacterial tmDNA sequences and use of the sequences and sequence alignments for the development of antibacterial drugs. The present invention is also directed to the use of the sequences for the development of diagnostic assays.
  • The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
  • Eubacterial tmRNAs (10Sa RNAs) are unique since they function, at least in E. coli, both as tRNA and as mRNA (for a review, see Muto et al., 1998). These ≈360±10% nucleotide RNAs are charged with alanine at their 3′-ends (Komine et al., 1994; Ushida et al., 1994) and also have a short reading frame coding for 9 to 27 amino acids depending on the bacterial species. E. coli tmRNA mediates recycling of ribosomes stalled at the end of terminatorless mRNAs, via a trans-translation process (Tu et al., 1995; Keiler et al., 1996; Himeno et al., 1997). In E. coli, this amino acid tag is co-translationally added to polypeptides synthesized from mRNAs lacking a termination codon, and the added 11 amino acid C-terminal tag makes the protein a target for specific proteolysis (Keiler et al., 1996).
  • Structural analyses based on phylogenetic (Felden, et al., 1996; Williams and Bartel, 1996) and probing (Felden et al., 1997; Hickerson et al., 1998) data have led to a compact secondary structure model encompassing 6 helices and 4 pseudoknots. tmRNAs have some structural similarities with canonical tRNAs, especially with tRNA acceptor branches. E. coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine, located in the tRNA-like domain of the molecule, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs (Felden et al., 1998).
  • Fifty-three tmRNA sequences are now known from both experimental data and Blast searches on sequenced genomes (summarized in Williams, 1999; Wower and Zwieb, 1999). These sequences cover only 10 phyla, less than one third of the known bacterial taxa. It is desired to determine additional tmRNA sequences and to use the tmRNA sequences for drug development.
    • SUMMARY OF THE INVENTION
  • The present invention relates to eubacterial tmDNA sequences and the corresponding tmRNA sequences. The present invention further relates to alignments of eubacterial tmDNA sequences and use of the sequences and sequence alignments for the development of antibacterial drugs.
  • In one aspect of the present invention, an extensive phylogenetic analysis was performed. Fifty-eight new tmDNA sequences including members from nine additional phyla were determined. Remarkably, tmDNA sequences could be amplified from all species tested apart from those in the alpha-Proteobacteria. This aspect of the invention allowed a more systematical study of the structure and overall distribution of tmRNA within eubacteria
  • In a second aspect of the invention, alignments are made with the newly isolated tmDNA sequences and previously disclosed tmRNA sequences.
  • In a third aspect of the invention, the alignments of the tmRNA sequences allow the identification of targets for development of antibacterial drugs.
  • In a fourth aspect of the invention, the novel tmDNA or tmRNA sequences of the present invention are used to develop diagnostic assays, such as amplification-based assays, for the bacterial species disclosed herein.
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIGS. 1A-1B show the effect of the annealing temperature (FIG. 1A) and magnesium concentration (FIG. 1B) on amplifying eubacterial tmRNA genes from genomic DNAs using PCR. A: Varying the annealing temperature from 50° to 70° C. during the PCR amplification of Thermus aquaticus (1). B; Varying the magnesium concentration to amplify tmDNA genes from Thermus aquaticus (1), negative effect of increasing the magnesium concentration), Acholeplasma laidlawii (2), positive effect of increasing the magnesium concentration, the upper band is the tmDNA gene) and from Mycoplasma salivarium (3), no discernible effect of magnesium ions in that concentration range). The arrows point toward the 4 novel tmDNA genes that have been sequenced.
  • FIG. 2 shows the distribution of tmDNA sequences within eubacterial genomes. The circled phyla or subgroups contain tmDNA sequences and those shaded are new members of this category. The numbers shown close to each phylum are the 51 tmDNA sequences that have are disclosed herein and the numbers in parenthesis are the 53 tmDNA sequences that were previously known (summarized in Williams, 1999; Wower and Zwieb, 1999). The environmental samples are indicated with a dashed line as their connection to the tree is unknown. The 5 alpha-Proteobacteria in which tmDNA sequences were not detected by PCR analysis are labeled “PCR” and the 3 analyzed by Blast search of the complete, or nearly complete, sequenced genomes are labeled “database”.
  • FIGS. 3A, 3B and 3C show the sequence alignment, structural domains and structural features for the tmRNA of several species of Firmicutes. The tmRNA sequences are set forth in SEQ ID NOs:67-87.
  • FIGS. 4A and 4B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Thermophiles. The tmRNA sequences are set forth in SEQ ID NOs:88-99.
  • FIGS. 5A and 5B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Cyanobacteries (5A) and chloroplasts (5B). The tmRNA sequences of the Cyanobacteries are set forth in SEQ ID NOs:100-103, and the tmRNA sequences of the chloroplasts are set forth in SEQ ID NOs:104-108.
  • FIGS. 6A and 6B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Mycoplasmes. The tmRNA sequences are set forth in SEQ ID NOs:109-117.
  • FIGS. 7A-1, 7A-2, 7B, 7C and 7D show the sequence alignment, structural domains and structural features for the tmRNA of several species of Mesophiles (7A-1, 7A-2, 7C, 7D) and environmental sludge (7B). The tmRNA sequences of the Mesophiles are set forth in SEQ ID NOs:118-123 and 125-128, and the tmRNA sequence of the environmental sludge is set forth in SEQ ID NO:124.
  • FIGS. 8A and 8B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Actinobacteries (8A) and Spirochaetes (8B). The tmRNA sequences of the Actinobacteries are set forth in SEQ ID NOs:132-136, and the tmRNA sequences of the Spirochaetes are set forth in SEQ ID NOs:137-142.
  • FIGS. 9A and 9B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Pourpres beta. The tmRNA sequences are set forth in SEQ ID NOs:143-154.
  • FIGS. 10A, 10B and 10C show the sequence alignment, structural domains and structural features for the tmRNA of several species of Pourpres gamma. The tmRNA sequences are set forth in SEQ ID NOs:155-169.
  • FIGS. 11A and 11B show the sequence alignment, structural domains and structural features for the tmRNA of several species of Pourpres delta (11A) and Pourpres epsilon (11B). The tmRNA sequences of the Pourpres delta are set forth in SEQ ID NOs:170-172, and the tmRNA sequences of the Pourpres epsilon are set forth in SEQ ID NOs:173-175.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The present invention is directed to eubacterial tmDNA sequences and the corresponding tmRNA sequences. The present invention is further directed to alignments of eubacterial tmDNA sequences and use of the sequences and sequence alignments for the development of antibacterial drugs.
  • The novel eubacterial tmDNA sequences determined in accordance with the present invention are set forth in Tables 1-58, below. The alignment of tmRNA sequences is shown in FIGS. 3A-11B, which also show the structural domains and structural features of the tmRNA. The present invention also includes the tmRNA sequences set forth in these figures to the extent they differ from the sequences set forth in Tables 1-58.
  • The sequences, especially as identified by the sequence alignment, represent targets for the development of drugs which may be broadly applicable to many kinds of bacteria, or may be broadly applicable only to a particular genera of phylum of bacteria, or may be specifically applicable to a single species of bacteria. Thus, the present invention is further directed to the development of drugs for the therapeutic treatment of bacteria, generically or specifically. Suitable drugs are developed on the basis of the tmRNA sequences as described herein.
  • For all the novel tmRNA sequences, as well as with the ones that are already known, there are systematically several structural domains that are always found. These domains can be used as targets for the development of drugs which may be genera specific or which may be eubacteria specific. These domains are either RNA helices which can be sometimes interrupted by bulges or pseudoknots. The RNA helices which are always present are H1, H2, H5 and H6. Helices H1 and H6 are found in all canonical transfer RNAs. Thus, H1 and H6 are not good targets for drug development because drugs that would target them will also interfere with the biology of the individual that has a given disease. Consequently, very good candidates for development of drugs for targeting as many bacteria as possible are helices H2 and H5. Moreover, helices H2 and H5 are critical for the folding of all these tmRNA since both of them connect the two ends of the molecule together. Disruption of either H2 or H5 with a specific drug would lead to inactive tmRNA molecules in vivo. Similarly, pseudoknots PK1, PK2 and PK3 are always found in the bacterial tmRNAs. Since these pseudoknots are not found in all canonical transfer RNAs, they can also be targeted with specific drugs. Disruption of either PK1 or PK2 or PK3 with a specific drug would lead to inactive tmRNA molecules in vivo.
  • In addition to developing drugs which broadly target many bacteria, drugs are developed which are more genera specific. For trying to target specifically a given bacteria or a complete phylum, the coding sequence (shown in all the alignments) is a very good candidate. Indeed, this region of the RNA is very accessible for DNA antisense binding (such as shown for Escherichia coli; Matveeva et al., 1997), and thus, is also available for interaction with other drugs. Moreover, the coding sequence is a critical functional domain of the molecule in its quality-control mechanism in cells.
  • Interestingly, some structural domains are present only in a given bacterial phyla and could be targeted for discovering a drug that will be specific of a phylum, but not of the others. For example:
  • (1) in the cyanobacteria, the fourth pseudoknot PK4 is made of two smaller pseudoknots called PK4a and PK4b;
  • (2) in the mycoplasma, helix H2 is made of only 4 base-pairs instead of 5 in the other species;
  • (3) for two sequences of chlorobium as well as Bacteroides thetaiotaomicron and ppm gingiv., there is an additional domain just downstream of the coding sequence that is unique to them;
  • (4) there is always a stem-loop in the coding sequence of the actinobacteria (Felden et al., 1999); and
  • (5) all the beta proteobacteria possess a sequence insertion in pseudoknot PK2 (shown in the alignment).
  • The novel sequences described herein, when aligned, show that specific structural domains within tmRNA are strictly conserved, as for example pseudoknot PK1 is located upstream (at the 5′-side) of the coding sequence. As previously disclosed, this pseudoknot is a target for future anti-bacterial drugs. Moreover, recent data have shown that this PK1 pseudoknot, among all the four pseudoknots within tmRNA gene sequences (sometimes there's only 2 or 3 detectable pseudoknots, depending upon the sequences), is the only one that its correct folding is essential for the biological activity of tmRNA (Nameki et al., 1999; Nameki et al., 2000).
  • It has recently been discovered that even the alpha-proteobacteria possess tmRNA genes. These genes are permuted and are made in two parts, connected via a processed linker. These tmRNA gene sequences from alpha-proteobacteria were not found in the course of the present invention because usual PCR methods could not amplify them.
  • Recent reports have shown that whereas the gene encoding tmRNA is non-essential in E. coli (does not kill the bacteria when disrupted), it is indeed essential in Neisseria gonorrheae (Huang et al., 2000). Also, tmRNA is directly involved in Salmonella typhymurium pathogenticity (Julio et al., 2000).
  • In summary, tmRNA genes are present in all eubacterial genomes, with no exceptions, but are not present in any genomes from archebacteries or eukaryotes, with the exception of some chloroplasts. The very specific location of tmRNA genes within one of the three main kingdoms of life make them ideal targets for the design of novel antibiotics that will, in principle, interfere very weakly with human biochemistry, compared to usual antibiotics. For a recent review about designing novel antibiotics, see Breithaupt (1999).
  • The present invention is also directed to diagnostic assays and kits for the detection of bacterial infection, particularly infections caused by bacterial agents disclosed herein. In one embodiment, the coding sequence of each bacterial species is used to design specific primers for use in amplification-based diagnostic assays for infectious diseases. Specific primers are designed in accordance with well known techniques, and such design is readily done by a skilled artisan. Amplification-based diagnostic assays are performed in accordance with conventional techniques well known to skilled artisans. Examples of amplification-based assays include, but are not limited to, polymerase chain reaction (PCR) amplification, strand displacement amplification (SDA), ligase chain reaction (LCR) amplification, nucleic acid sequence based amplification (3SR or NASBA) and amplification methods based on the use of Q-beta replicase.
  • Drugs which target the sequences described herein are active agents can be formulated in pharmaceutical compositions, which are prepared according to conventional pharmaceutical compounding techniques (Remington's, 1990). The composition may contain the active agent or pharmaceutically acceptable salts of the active agent. These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, intrathecal, epineural or parenteral.
  • For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/11698.
  • For parenteral administration, the compound may dissolved in a pharmaceutical carrier and administered as either a solution of a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
  • The active agent is preferably administered in an therapeutically effective amount. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences (18).
  • Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell-specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, or would otherwise require too high a dosage, or otherwise be unable to enter the target cells.
  • Antisense active agents can also be delivered by techniques described in U.S. Pat. Nos. 5,811,088; 5,861,290 and 5,767,102.
    • EXAMPLES
  • The present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized.
    • Example 1 Materials and Methods
  • 1. Extraction of Genomic DNA
  • Bacterial genomic DNAs were prepared from ≈10 mg freeze-dried cells provided from ATCC (American Type Culture Collection, Virginia, USA). Cell pellets were resuspended in 750 μL of lysis buffer (50 mM Tris (pH 8.0), 50 mM EDTA and 20% sucrose). 150 μL of a 10 mg/mL solution of lysozyme was mixed and let stand at room temperature for 15 min. 150 μL of 1% SDS was added and let stand at room temperature for 15 minutes. Four to five phenol/chloroform extractions were performed, until the sample was clear and there was no interphase. Two to five μL of a 10 mg/mL solution of RNase DNase-free was added and incubated at room temperature for 30 minutes. After a phenol/chloroform extraction of the enzyme, the genomic DNA was precipitated with 1/10 volume of 3M NaOAc (pH 5.5) and 1 volume isopropanol, and stored at −20° C. for 2 hours. After centrifugation, the genomic DNAs were washed with 70% ethanol, vacuum-dried and diluted in sterile water to a final concentration of 10 ng/μL.
  • 2. Primer Sets for PCR Reactions
  • The following primer sets were used during the PCR:
  • primer ser A (based on E. coli tmRNA termini):
    5′-GGG GCT GAT TCT GGA TTC GAC-3′ (SEQ ID NO:1)
    and
    5′-TGG AGC TGG CGG GAG TTG AAC-3′;
    primer set B (based on T. neapolitana tmRNA
    termini):
    5′-GGG GGC GGA AAG GAT TCG ACG-3′ (SEQ ID NO:3)
    and
    5′-TGG AGG CGG CGG GAA TCG AAC-3′; (SEQ ID NO:4)
    primer set C (based on M. pneumoniae tmRNA
    termini):
    5′-GGG GAT GTC ATG GTT TTG ACA-3′ (SEQ ID NO:5)
    and
    5′-TGG AGA TGG CGG GAA TCG AAC-3′; (SEQ ID NO:6)
    primer set D (based on C. tepidum tmRNA termini):
    5′-GGG GAT GAC AGG CTA TCG ACA-3′ (SEQ ID NO:7)
    and
    5′-TGG AGA TGG CGG GAC TTG AAC-3′. (SEQ ID NO:8)
  • 3. PCR Reaction
  • Sequences of tmRNA genes were obtained by polymerase chain reaction (PCR) in 25 μL using 40 ng of genomic DNA per reaction. The following general scheme was utilized for all of the sequences:
  • (a) 94° C. to 96° C. for 4 min. (first denaturation of genomic DNAs, done only once); then
  • (b) 35 to 40 PCR cycles with 2.5 to 5 Units of Taq DNA polymerase in a 25 μL reaction volume, according to the following scheme (40 ng of genomic DNAs/PCR reaction):
      • 1. denature at 94° to −96° C. for 25 to 30 sec;
      • 2. anneal at 44° to 55° C. for 20 to 30 sec; and
      • 3. extension at 72° C. for 10 sec.
        The magnesium conc. was optimized for each phyla from 3.5 to 13.5 mM.
  • 4. Elution of Amplified DNAs
  • The various PCR-amplified tmDNA bands were gel purified (5% PAGE), stained (ethidium bromide staining), cut using a sterile razor blade, and shaken over-night (passive elution, using a vibrator) in a 350 μl solution containing 10 mM Tris-HCl buffer (pH 8.1). The following day, the PCR amplified tmDNAs were ethanol precipitated, washed in 70% ETOH, vacuum dried and the DNA pellets were dissolved in 18 μl of RNase-DNase free sterile water.
  • 5. DNA Sequencing
  • Six μL of amplified DNAs were added to 3.2 picomoles of the primer that was used in the PCR. To verify the novel tmDNA sequences, each of the two primers were used independently to sequence each of the two PCR-amplified DNA strands. Some tmDNAs were already engineered at their 5′-ends with a T7 promoter, to be able to transcribe directly the tmDNAs into tmRNAs by in vitro transcription.
  • Dye terminator sequencing was achieved at the DNA sequencing facility of the Human Genetics Institute. In addition to novel tmRNA sequences that are not available publicly, several tmDNA sequences that were already known have been verified and several sequencing mistakes have been found and corrected (especially for Alcaligenes eutrophus tmRNA).
    • Example 2 Amplification Reactions for Eubacterial tmDNA
  • Eubacterial tmDNA was amplified by PCR in accordance with Example 1, using the following conditions.
    • Acidobacterium:
  • Primer Set B; Annealing temp. during PCR: 53° C. for 20 sec; Mg2+ conc.: 4.5 mM.
    • Coprothermobacter:
  • Primer Set B; Annealing temp. during PCR: 55° C. for 30 sec; Mg2+ conc.: 5.5 mM.
    • Cytophagales:
  • Primer Set A; Annealing temp. during PCR: 46° C. for 30 sec; Mg2+ conc.: 4.5 mM.
    • Dictyoglomus:
  • Primer set B; Annealing temp. during PCR: 55° C. for 30 sec; Mg2+ conc.: 4.5 mM.
    • Environmental Samples:
  • Sludge DNA
  • Primer set C; Annealing temp. during PCR: 51° C. for 20 sec; Mg2+ conc.: 13.5 mM.
  • Rumenal Fluid DNA
  • Primer set D; Annealing temp. during PCR: 50° C. for 30 sec; Mg2+ conc.: 9.5 mM.
    • Fibrobacter:
  • Primer set A; Annealing temp. during PCR: 51° C.; Mg2+ conc.: 3.5 mM.
    • Firmicutes:
  • Fusobacteria:
  • Primer set A; Annealing temp. during PCR: 52° C.; Mg2+ conc.: 5.5 mM.
  • High G-C:
  • Primer set A; Annealing temp. during PCR: 50-55° C.; Mg2+ conc.: 4.5 mM.
  • Low G-C:
  • Primer sets A or B; Annealing temp. during PCR: 52° C.; Mg2+ conc.: 5.5 to 7.5 mM.
  • Mycoplasmes:
  • Primer set A; Annealing temp. during PCR: 52° C.; Mg2+ conc.: 3.5 to 5.5 mM.
    • Green Non-Sulfur:
  • Primer sets A or B; Annealing temp. during PCR: 46 to 52° C.; Mg2+ conc.: 4.5 mM.
    • Green Sulfur:
  • Primer set A; Annealing temp. during PCR: 46° C.; Mg2+ conc.: 4.5 mM.
    • Planctomycetales:
  • Primer set A; Annealing temp. during PCR: 48 to 52° C.; Mg2+ conc.: 7.5 mM.
    • Proteobacteria:
  • Beta:
  • Primer sets A and/or B; Annealing temp. during PCR: 50° C. for 25 sec; Mg2+ conc.: 3.5 mM.
  • Delta:
  • Primer set B; Annealing temp. during PCR: 55° C.; Mg2+ conc.: 3.5 to 4.5 mM.
  • Epsilon:
  • Primer set A; Annealing temp. during PCR: 46° C. for 30 sec; Mg2+ conc.: 3.5 mM.
  • Gamma:
  • Primer set A; Annealing temp. during PCR: 44 C for 30 sec; Mg2+ conc.: 5.5 mM.
    • Spirochetes:
  • Primer set A; Annealing temp. during PCR: 52° C.; Mg2+ conc.: 4.5 mM.
    • Thermodesulfobacterium:
  • Primer set B; Annealing temp. during PCR: 55° C.; Mg2+ conc.: 5.5 mM.
    • Thermotogales:
  • Primer set B; Annealing temp. during PCR: 46° C.; Mg2+ conc.: 7.5 mM.
    • Deinococcales:
  • Primer set B; Annealing temp. during PCR: 52° C.; Mg2+ conc.: 3.5 mM.
    • Verrucomicrobia:
  • Primer set A; Annealing temp. during PCR: 53° C. for 25 sec; Mg2+ conc.: 3.5 mM.
    • Example 3 Amplification of Eubacterial tmDNA
  • Specific PCR amplification of tmRNA genes was achieved for both thermophilic and mesophilic eubacterial tmRNA genes. For the novel tmDNA genes found in thermophiles, both the magnesium concentration and the annealing temperature (FIG. 1A) were optimized. As shown in FIG. 1A, a specific amplification of Thermus aquaticus tmDNA was observed with an annealing temperature around 50° C., whereas at higher temperatures there is a gradual decrease in the amount of amplified tmDNA. For mesophiles, the magnesium concentration during PCR was critical (FIG. 1B), but the annealing temperature could vary from 44° C. to 60° C. without significant effects on the amplification. FIG. 1B shows various effects of increasing the magnesium concentration on the PCR amplification of three novel eubacterial tmDNA genes. Increasing magnesium concentration from 3.5 mM to 5.5 mM has either a negative (FIG. 1B, panel 1), a positive (FIG. 1B, panel 2) or no effect on specifically amplifying eubacterial tmDNA genes.
  • According to these procedures, tmRNA genes from many eubacteria including known human pathogens were amplified. The PCR was facilitated by sequence conservation at both 5′ and 3′ ends and was performed as described (Williams and Bartel, 1996), with modifications. This study was initiated to collect further sequences from eubacterial tmDNA genes, as well as to test experimentally whether tmDNA genes could be found in all bacterial phyla or subgroups. 51 new tmDNA sequences were determined (FIG. 2), including sequences from members of 8 additional phyla and 1 subgroup (shaded boxes in FIG. 2). The 58 new tmDNA sequences are set forth in Tables 1-58. This brings coverage to a total of 104 sequences in 19 bacterial phyla. Interestingly, tmDNA sequences could be amplified from all species tested apart from those in the alpha-Proteobacteria. Five genomic DNAs from alpha-Proteobacteria (Agrobacterium tumefaciens, Bartonella henselae, Bartonella quintana, Rhodospirillum rubrum and Rickettsia prowazekii) were extensively checked using various oligonucleotides, annealing temperatures and magnesium concentrations. No specific amplified tmDNA sequences were detected in this subgroup. Moreover, no putative tmDNA sequences could be identified (results herein and Williams, 1999) by Blast searches on the 1 fully sequenced (Rickettsia prowazekii) and 2 nearly completed (Caulobacter crescentus and Rhodobacter capsulatus) alpha-proteobacterial genomes (FIG. 2).
  • It cannot be ruled out that tmDNA sequences may have largely diverged in the alpha-proteobacterial sub-group compared to other bacterial phyla, and that both PCR methods and Blast searches are missing the relevant sequences. While tmRNA is dispensable in E. coli (Ando et al., 1996), it is striking that it has been found in all bacteria tested other than the alpha-Proteobacteria. The alpha-Proteobacteria have undergone reductive evolution. This has been more intensive in one of the two sub-classes than in the other (Gray and Spencer, 1996), but tmRNA sequences have not been found even in the sub-class with the larger genome. Based on sequence comparison, the alpha-Proteobacteria and mitochondria are evolutionary relatives (Yang et al., 1985; Andersson et al., 1998). The drastic downsizing in what has become mitochondrial genomes means that it is not reasonable to draw inferences on the relationship between alpha-Proteobacteria and mitochondria based on their mutual apparent absence of tmRNA. It is nevertheless, of interest, that at least some chloroplasts and cyanelle genomes have tmDNA sequences, and the cyanobacteria, with which they are evolutionary related, also have tmRNA.
  • TABLE 1
    tmDNA Sequence for Acidobacterium capsulatum
    (Acidobacterium)
    (SEQ ID NO:9)
    GGGGGCGGAAAGGATTCGACGGGGTTGACTGCGGCAAAGAGGCATGCCGG
    GGGGTGGGCACCCGTAATCGCTCGCAAAACAATACTTGCCAACAACAATC
    TGGCACTCGCAGCTTAATTAAATAAGTTGCCGTCCTCTGAGGCTTCGCCT
    GTGGGCCGAGGCAGGACGTCATACAGCAGGCTGGTTCCTTCGGCTGGGTC
    TGGGCCGCGGGGATGAGATCCACGGACTAGCATTCTGCGTATCTTGTCGC
    TTCTAAGCGCAGAGTGCGAAACCTAAAGGAATGCGACTGAGCATGGAGTC
    TCTTTTCTGACACCAATTTCGGACGCGGGTTCGATTCCCGCCGCCTCCAC
    CA
  • TABLE 2
    tmDNA Sequence for Coprothermobacter proteolyticus
    (60 degrees)
    (SEQ ID NO:10)
    GGGGGCGGAAAGGATTCGACGGGGAGTCGGAGCCTTGAGCTGCAGGCAGG
    GTTGGCTGCCACACCTTAAAAAGGGTAGCAAGGCAAAAATAAATGCCGAA
    CCAGAATTTGCACTAGCTGCTTAATGTAAGCAGCCGCTCTCCAAACTGAG
    GCTGCATAAGTTTGGAAGAGCGTCAACCCATGCAGCGGCTCTTAAGCAGT
    GGCACCAGCTGTTTAAGGGTGAAAAGAGTGGTGCTGGGCAGTGCGGTTGG
    GCTTCCTGGGCTGCACTGTCGAGACTTCACAGGAGGGCTAAGCCTGTAGA
    CGCGAAAGGTGGCGGCTCGTCGGACGCGGGTTCGATTCCCGCCGCCTCCA
    CCA
  • TABLE 3
    tmDNA Sequence for Bacteroides thetaiotaomicron
    (bacteroides/flavobacterium)
    (SEQ ID NO:11)
    GGGGCTGATTCTGGATTCGACAGCGGGCAGAAATGGTAGGTAAGCATGCA
    GTGGGTCGGTAATTTCCACTTAAATCTCAGTTATCAAAACTTTATCTGGC
    GAAACTAATTACGCTCTTGCTGCTTAATCGAATCACAGTAGATTAGCTTA
    ATCCAGGCACTAGGTGCCAGGACGAGACATCACTCGGAAGCTGTTGCTCC
    GAAGCATTCCGGTTCAGTGGTGCAGTAACATCGGGGATAGTCAGAAGCGG
    CCTCGCGTTTTTGATGAAACTTTAGAGGATAAGGCAGGAATTGATGGCTT
    TGGTTCTGCTCCTGCACGAAAATTTAGGCAAAGATAAGCATGTAGAAAGC
    TTATGATTTCCTCGTTTGGACGAGGGTTCAACTCCCGCCAGCTCCACCA
  • TABLE 4
    tmDNA Sequence for Dictyoglomus thermophilum
    (70 degrees)
    (SEQ ID NO:12)
    GGGGCTGATTCTGGATTCGACAGGGAGTACAAGGATCAAAAGCTGCAAGC
    CGAGGTGCCGTTACCTCGTAAAACAACGGCAAAAAAGAAGTGCCAACACA
    AATTTAGCATTAGCTGCTTAATTTAGCAGCTACGCTCTTCTAACCCGGGC
    TGGCAGGGTTAGAAGGGTGTCATAATGAGCCAGCTGCCCCTTCCGACTCC
    CCTAAGGAAGGGAAAGATGTAGGGGATAGGTGCTTACAGAATCCTGCGGG
    AGGGAGTCTGTAAGTGCCGAAAAGTTAAAACTCCCGCTAAGCTTGTAGAG
    GCTTTTGATTCTTGCTCTCTGGACGCGGGTTCAACTCCCGCCAGCTCCAC
    CA
  • TABLE 5
    tmDNA Sequence for Environmental Sample
    from Rumenal Fluid
    (SEQ ID NO:13)
    ACGCCCTTGTCTCAGACGAGGGCACTCGTTAAAAAGTCTGAAAAGAATAA
    CTGCAGAACCTGTAGCTATGGCTGCTTAATTTAAGGGCAACCCTTGGATC
    CGCCTCCATCCCGAAGGGGTGGCATCCGAGTCGCAAATCGGGATAGGATG
    GATCTTGGCAACGAGGAGTACATCCGAAATTTGTCGCTGCTGGCTGAAGC
    ATCGCCGTTCCTCTTTGGGCGTGGCAAGGCAAGATTAAATTCAGAGGATA
    AGCGTGTAGTAGCGAGTGAGTAGGTGTTTTTGGACGCGGGTTCAAGTCCC
    GCCATCTCCACCA
  • TABLE 6
    tmDNA Sequence for Environmental
    Sample from Sludge
    (SEQ ID NO:14)
    GGGGATGTCATGGTTTTGACAGGGAACCAGGAGGTGTGAGATGCATGCCG
    GAGACGCTGTCCGCTCCGTTATCAAGCAGCAAACAAAACTAATTGCAAAC
    AACAATTACTCCTTAGCAGCGTAAGCAGCTAACGTTCAACCTCTCCGGAC
    CGCCGGGAGGGGATTTGGGCGTCGAAACAGCGCGGACGCTCCGGATAGGA
    CGCCCATAATATCCGGCTAAGACCATGGGTCTGGCTCTCGCGGGTCTGAT
    TGTCTTCCACCGCGCGGGCCGCGATCAAAGACAACTAAGCATGTAGGTTC
    TTGCATGGCCTGTTCTTTGGACGCGGGTTCGATTCCCGCCATCTCCACCA
  • TABLE 7
    tmDNA Sequence for Fibrobacter succinogenes
    (Fibrobacter)
    (SEQ ID NO:15)
    GGGGCTGATTCTGGATTCGACAGGGTTACCGAAGTGTTAGTTGCAAGTCG
    AGGTCTCAGACGAGGGCTACTCGTTAAAAAGTCTGAAAAAAAATAAGTGC
    TGACGAAAACTACGCACTCGCTGCCTAATTAACGGCAACGCCGGGCCTCA
    TTCCGCTCCCATCGGGGTGTACGTCCGGACGCAATATGGGATAGGGAAGT
    GTCATGCCTGGGGGCATCTCCCGAGATTTTCTAGGCTGGTCAAACTCCGC
    GCCGACCTTCTTGGGCGTGGATAAGACGAGATCTTAAATTCGAAGGGAAC
    ACTTGTAGGAACGTACATGGACGTGATTTTGGACAGGGGTTCAACTCCCG
    CCAGCTCCA
  • TABLE 8
    tmDNA Sequence for Fusobacterium mortiferum
    (SEQ ID NO:16)
    GGGGCTGATTCTGGATTCGACGGGGTTATGAGGTTATAGGTAGCATGCCA
    GGATGACCGCTGTGAGAGGTCAACACATCGTTTAGATGGAAACAGAAATT
    ACGCTTTAGCTGCTTAATTAGTCAGCTCACCTCTGGTTTCTCTCTTCTGT
    AGGAGAATCCAACCGAGGTGTTACCAATATACAGATTACCTTTAGTGATT
    TCTCTAAGCTCAAAGGGACATTTTAGAGAATAGCTTCAGTTAGCCCTGTC
    TGCGGGAGTGATTGTTGCGAAATAAAATAGTAGACTAAGCATTGTAGAAG
    CCTATGGCGCTGGTAGTTTCGGACACGGGTTCAACTCCCGCCAGCTCCAA
  • TABLE 9
    tmDNA Sequence for Corynebacterium xerosis
    (gram +, high G-C content)
    (SEQ ID NO:17)
    GGGGCTGATTCTGGATTCGACTTCGTACATTGAGCCAGGGGAAGCGTGCC
    GGTGAAGGCTGGAGACCACCGCAAGCGTCGCAGCAACCAATTAAGCGCCG
    AGAACTCTCAGCGCGACTACGCCCTCGCTGCCTAAGCAGCGACCGCGTGT
    CTGTCAGACCGGGTAGGCCTCTGATCCGGACCCTGGCATCGTTTAGTGGG
    GCTCGCTCGCCGACTTGGTCGCAAGGGTCGGCGGGGACACTCACTTGCGA
    CTGGGCCCGTCATCCGGTCATGTTCGACTGAACCGGAGGGCCGAGCAGAG
    ACCACGCGCGAACTGCGCACGGAGAAGCCCTGGCGAGGTGACGGAGGACC
    CGGGTTCAACTCCCGCCAGCTCCACCA
  • TABLE 10
    tmDNA Sequence for Micrococcus luteus (parfait)
    (SEQ ID NO:18)
    GGGGCTATTCTGGATTCGACGGTGTGTGTCGCGTCGGGAGAAGCGGGCCG
    AGGATGCAGAGTCATCTCGTCAAACGCTCTCTGCAAACCAATAAGTGCCG
    AATCCAAGCGCACTGACTTCGCTCTCGCTGCCTGATCAGTGATCGAGTCC
    GTCACCCCGAGGTCGCTGTCGCCTCGGATCGTGGCGTCAGCTAGATAGCC
    ACTGGGCGTCACCCTCGCCGGGGGTCGTGACGCCGACATCAATCCGGCTG
    GGTCCGGGTTGGCCGCCCGTCTGCGGGACGGCCAGGACCGAGCAACACCC
    ACAGCAGACTGCGCCCGGAGAAGACCTGGCAACACCTCATCGGACGCGGG
    TTCAACTCCCGCANTCCCACCA
  • TABLE 11
    tmDNA Sequence for Mycobacterium smegmatis
    (SEQ ID NO:19)
    TCATCTCGGCTTGTTCGCGTGACCGGGAGATCCGAGTAGAGACATAGCGA
    ACTGCGCACGGAGAGGGGCTGATTCCTGGATTCGACTTCGAGCATCGAAT
    CCAGGGAAGCGTGCCGGTGCAGGCAAGAGACCACCGTAAGCGTCGTTGCA
    ACCAATTAAGCGCCGATTCCAATCAGCGCGACTACGCCCTCGCTGCCTAA
    GCGACGGCTGGTCTGTCAGACCGGGAGTGCCCTCGGCCCGGATCCTGGCA
    TCAGCTAGAGGGACCCACCCACGGGTTCGGTCGCGGGACCTGTGGGGACA
    TCAAACAGCGACTGGGATCGAGCCTCGAGGACATGCCGTAGGACCCGGGT
    TCAACTCCCGCCAGCTCCACCA
  • TABLE 12
    tmDNA Sequence for Bacillus badius
    (SEQ ID NO:20)
    GGGGGTGATTCTGGATTCGACAGGGATAGTTCGAGCTTGGGCTGCGAGCC
    GGAGGGCCGTCTTCGTACCAACGCAAACGCCTAAATATAACTGGCAAAAA
    AGATTTAGCTTTAGCTGCCTAATATAGGTTCAGCTGCTCCTCCCGCTATC
    GTCCATGTAGTCGGGTAAGGGGTCCAAACTTAGTGGACTACGCCGGAGTT
    CTCCGCCTGGGGACAAAGGAAGAGATCAATCAGGCTAGCTGCCCGGACGC
    CCGTCGATAGGCAAAAGGAACAGTGAACCCCAAATATATCGACTACGCTC
    GTAGACGTTCAAGTGGCGTTATCTTTGGACGTGGGTTCAACTCCCGCCAG
    CTCCA
  • TABLE 13
    tmDNA Sequence for Bacillus brevis
    (SEQ ID NO:21)
    GGGGGCGGAAAGGATTCGACGGGGATGGTAGAGCATGAGAAGCGAGCCGG
    GGGGTTGCGGACCTCGTCACCAACGCAAACGCCATTAACTGGCAACAAAC
    AACTTTCTCTCGCTGCTTAATAACCAGTGAGGCTCTCCCACTGCATCGGC
    CCGTGTGCCGTGGATAGGGCTCAACTTTAACGGGCTACGCCGGAGGCTTC
    CGCCTGGAGCCAAAGGAAGAAGACCAATCAGGCTAGGTGCCAGGTCAGCG
    CGTCACTCCGCGAATCTGTCACCGAAACTCTAAACGAGTGACTGCGCTCG
    GAGATGCTCATGTATCGCTGTTTTCGGACGGGGGTTCGATTCCCGCCGCC
    TCACCCA
  • TABLE 14
    tmDNA Sequence for Bacillus thermoleovorans
    (50-60 degres)
    (SEQ ID NO:22)
    GGGGGCGGAAAGGATTCGACGGGGGTAGGTCGAGCTTAAGCGGCGAGCCG
    AGGGGGACGTCCTCGTAAAAACGTCACCTAAAGATAACTGGCAAACAAAA
    CTACGCTTTAGCTGCCTAATTGCTGCAGCTAGCTCCTCCCGCCATCGCCC
    GCGTGGCGTTCGAGGGGCTCATATGGAGCGGGCTACGCCCAAATCCGCCG
    CCTGAGGATGAGGGAAGAGACGAATCAGGCTAGCCGCCGGGAGGCCTGTC
    GGTAGGCGGAACGGACGGCGAAGCGAAATATACCGACTACGCTCGTAGAT
    GCTTAAGTGGCGATGCCTCTGGACGTGGGTTCGATTCCCGCCGCCTCCCC
    ACCA
  • TABLE 15
    tmDNA Sequence for Clostridium innocuum
    (SEQ ID NO:23)
    GGGGGCGGAAAGGATTCGACGGGGATATGTCTGGTACAGACTGCAGTCGA
    GTGGTTACGTAATAACCAATTAAATTTAAACGGAAAAACTAAATTAGCTA
    ACCTCTTTGGTGGAAACCAGAGAATGGCTTTCGCTGCTTAATAACCGATA
    TAGGTTCGCAGCCGCCTCTGCATGCTTCTTCCTTGACCATGTGGATGTGC
    GCGTAAGACGCAAGGGATAAGGAATCTGGTTTGCCTGAGATCAGATTCAC
    GAAAATTCTTCAGGCACATTCATCAGCGGATGTTCATGACCTGCTGATGT
    CTTAATCTTCATGGACTAAACTGTAGAGGTCTGTACGTGGGGCTGTTTCT
    GGACAGGAGTTCGATTCCCGCCGCCTCACCACCA
  • TABLE 16
    tmDNA Sequence for Clostridium lentocellum
    (SEQ ID NO:24)
    GGGGGCGGAAAGGATTCGACGGGGGTCACATCTACTGGGGCAGCCATCCG
    TAGAACGCCGGAGTCTACGTTAAAAGCTGGCACTTAAAGTAAACGCTGAA
    GATAATTTAGCAATCGCTGCCTAATTAAGGCGCAGTCCTCCTAGGTCTTC
    CGCAGCCTAGATCAGGGCTTCGACTCGCGGATCCTTCACCTGGCAAAGCT
    TTGAGCCAACGTGAACACTATGAAGCTACTAAAATCTAGAGCCTGTCTTT
    GGGCGCTAGATGGAGGGAATGTCAAAACAAAGAATATGATGGTAGAGACC
    ACGCTATATGGGCTTTCGGACAGGGGTTCGATTCCCGCCGCCTTCACCA
  • TABLE 17
    tmDNA Sequence for Clostridium perfringens
    (SEQ ID NO:25)
    GGGGCTGATTCTGGATTCGACGGGGGTAAGATGGGTTTGATAAGCGAGTC
    GAGGGAAGCATGGTGCCTCGATAATAAAGTATGCATTAAAGATAAACGCA
    GAAGATAATTTTGCATTAGCAGCTTAATTTAGCGCTGCTCATCCTTCCTC
    AATTGCCCACGGTTGAGAGTAAGGGTGTCATTTAAAAGTGGGGAACCGAG
    CCTAGCAAAGCTTTGAGCTAGGAACGGAATTTATGAAGCTTACCAAAGAG
    GAAGTTTGTCTGTGGACGTTCTCTGAGGGAATTTTAAAACACAAGACTAC
    ACTCGTAGAAAGTCTTACTGGTCTGCTTTCGGACACGGGTTCAACTCCCG
    CCACTCCA
  • TABLE 18
    tmDNA Sequence for Clostridium stercorarium
    (SEQ ID NO:26)
    GGGGGCGGAAAGGATTCGACGGGGTTATTGAAGCAAGAGTAGCGGGTAGA
    GGATTCTCGTTGGCCTCTTTAAAAAACGAGAGCTAAAAATAAACGCAAAC
    AACGATAACTACGCTTTAGCTGCTGCGTAAGTAACACGCAGCCCGTCGGC
    CCCGGGGTTCCTGCGCCTCGGGATACCGGCGTCATCAAGGCAGGGAACCA
    GCCGGATCAGGCTTCAGGTCCGGTGGGATTTAATGAAGCTACCGACTTAT
    AAAGCCTGTCTCTGGGCGTTATAAGAAGGGAATGTCAAAACAGAGACTGC
    ACCCGGAGAAGCTCTTGTGGATATGGTTCCGGACACGAGTTCGATTCCCG
    CCGCCTCCACCA
  • TABLE 19
    tmDNA Sequence for Enterococcus faecium (sp.)
    (SEQ ID NO:27)
    GGGGCTGATTATGGATTCGACAGGATNGTTGAGCTTGAATTGCGTTTCGT
    AGGTTACGGCTACGTTAAAACGTTACAGTTAAATATAACTGCTAAAAACG
    AAAACAATTCTTTCGCTTTAGCTGCCTAAAAACCAGCTAGCGAAGATCCT
    CCCGGCATCGCCCATGTGCTCGGGTCAGGGTCCTAATCGAAGTGGGATAC
    GCTAAATTTTTCCGTCTGTAAAATTTAGAGGAGCTTACCAGACTAGCAAT
    ACAAGAATGCCTGTCACTCGGCACGCTGTAAAGCGAACCTTTAAATGAGT
    GTCTATGAACGTAGAGATTTAAGTGGGAATATGTTTTGGACGCGGGTTCA
    ACTCCCGCCAGCTCCACCA
  • TABLE 20
    tmDNA Sequence for Heliobacillus mobilis
    (photosyn/gram +)
    (SEQ ID NO:28)
    GGGGCTGATTCTGGATTCGACGGGGAACGTGTTTGCTTGGGATGCGAGCC
    GGGTTGCCGCCAGGACCGTAAAAAGGGCGGAAGGCTTTAATTGCCGAAGA
    TAACTACGCTTTAGCTGCTTAATTGCAGTCTAACCTCTTCTCCTCTGTGC
    TCTCGGTGAGGATGTAAGGGGTCATTTAAGAGAGCTGGCTTCGACCAATT
    CTCGGAGGTCCAAGCGAGATTTATCGAGATAGCCTGACCAACGCTCTGTC
    TGCCGTGCGGAAGGAAGGCGAAATCTAAAACGACAGACTACGCTCGTAGT
    GTCCTTTGTGGGCATTTCTTCGGACGCGGGTTCAACTCCCGCCAGCTCCA
    CCA
  • TABLE 21
    tmDNA Sequence for Heliospirillum gestii
    (SEQ ID NO:29)
    GGGGCTGATTCTGGATTCGACGGGGAACGTGTTTGCTTAGGACGCGAGCC
    GGGTTGCCGCCAGGACCGTAAAAAGGGCGGAAGGCTTTAATTGCCGAAGA
    TAACTACGCTTTAGCTGCTTAATTGCAGTCTAACCTCTTCTCCTCTGTGC
    TCTCGGTGAGGATGTAAGGGGTCATTTAAGAGAGCTGGCTCGAACCAATT
    CTCGGAGGTTCGGGTAAGACTTATCGAGATAGCCTGACCAACGCTCTGTC
    TGCCGTGCGGAAGGATGGCGAAATCTAAAACGACAGAATACGCTCGTAGT
    GTCCTTTGTGGGCATTTCTTCGGACGCGGGTTCAACTCCCGCCAGCTCCA
    CCA
  • TABLE 22
    tmDNA Sequence for Lactobacillus acidophilus
    (SEQ ID NO:30)
    GGGGCTGATTCTGGATTCGACAGGCGTAGACCCGCATTGACTGCGGTTCG
    TAGGTTACGTCTACGTAAAAACGTTACAGTTAAATATAACTGCAAATAAC
    AAAAATTCTTACGCATTAGCTGCTTAATTTAGCGCATGCGTTGCTCTTTG
    TCGGTTTACTCGTGGCTGACACTGAGTATCAACTTAGCGAGTTACGTTTA
    ACTACCTCACCTGAATAGTTGAAAAGAGTCTTAGCAGGTTAGCTAGTCCA
    TACTAGCCCTGTTATATGGCGTTTTGGACTAGTGAAGTTCAAGTAATATA
    ACTATGATCGTAGAGGTCAGTGACGAGATGCGTTTGGACAGCGGGTTCAA
    CTCCCGCCAGCTCCACCA
  • TABLE 23
    tmDNA Sequence for Staphylococcus epidermidis
    (SEQ ID NO:31)
    GGGGCTGATTCTGCATTCGACAGGGGTCCCCGAGCTTATTAAGCGTGTGG
    AGGGTTGGCTCCGTCATCAACACATTTCGGTTAAATATAACTGACAAATC
    AAACAATAATTTCGCAGTAGCTGCGTAATAGCCACTGCATCGCCTAACAG
    CATCTCCTACGTGCTGTTAACGCGATTCAACCCTAGTAGGATATGCTAAA
    CACTGCCGCTTGAAGTCTGTTTAGATGAAATATAATCAAGCTAGTATCAT
    GTTGGTTGTTTATTGCTTAGCATGATGCGAAAATTATCAATAAACTACAC
    ACGTAGAAAGATTTGTATCAGGACCTCTGGACGCGGGTTCAACTCCCGCC
    AGCTCCACCA
  • TABLE 24
    tmDNA Sequence for Streptococcus faecium
    (SEQ ID NO:32)
    GGGGCTGATTCTGGATTCGACAGGCACAGTTTGAGCTTGAATTGCGTTTC
    GTAGGTTACGTCTACGTTAAAACGTTACAGTTAAATATAACTGCTAAAAA
    CGAAAACAACTCTTACGCTTTAGCTGCCTAAAAACAGTTAGCGTAGATCC
    TCTCGGCATCGCCCATGTGCTCGAGTAAGGGTCTCAAATTTAGTGGGATA
    CGTGACAACTTTCCGTCTGTAAGTTGTTAAAGAGATCATCAGACTAGCGA
    TACAGAATGCCTGTCACTCGGCAAGCTGTAAAGCGAAACCACAAATGAGT
    TGACTATGAACGTAGATTTTTAAGTGGCGATGTGTTTGGACGCGGGTTCA
    ACTCCCGCCGTTCCACCA
  • TABLE 25
    tmDNA Sequence for Thermoanaerobacterium
    saccharolyticum (Bacillus/clostridium)
    (SEQ ID NO:33)
    GGGGTAGTAGAGGTAAAAGTAGCGAGCCGAGGTTCCATCTGCTCGTAAAA
    CGGTGGACTTAAATATAAACGCAAACGATAATTTAGCTTACGCTGCTTAA
    TTACAAGCAGCCGTTCAACCTTTGATTCCCACATCAAAGGATTGGGCGTC
    GATTTAGTGGGGAACTGATTTATCAAAGCTTTGAGATAAATCGGATTTTA
    TGAAGCTACCAAAGCAGTTATCCTGTCACTGGGAGAACTGCAGAGGGAAT
    GTCAAAACAGTGACTGCGCTCGGAGAAGCTTTTACTGTGACACCTTCGGA
    CCGGGGTTCAACTCCCGCCAGCTCCACCA
  • TABLE 26
    tmDNA Sequence for Mycoplasma fermentans
    (SEQ ID NO:34)
    GGGGCTGATTCTGGATTCGACATGCATTGGGTGATACTAATATCAGTAGT
    TTGGCAGACTATAATGCATCTAGGCTTTATAATCGCAGAAGATAAAAAAG
    CAGAAGAAGTTAATATTTCTTCACTTATGATTGCACAAAAAATGCAATCA
    CAATCAAACCTTGCTTTCGCTTAGTTAAAAGTGACAAGTGGTTTTAAAGT
    TGACATTTTCCTATATATTTTAAAATCGGCTTTTAAGGAGAACAGGAGTC
    TGAAAGGGTTCCAAAAATCTATATTGTTTGCATTTCGGTAGTATAGATTA
    ATTAGAAATGATAAACTGTAAAAAGTATTGGTATTGACTTGGTGTGTGGA
    CTCGGGTTCAACTCCCGCCAGCTCCACCA
  • TABLE 27
    tmDNA Sequence for Mycoplasma hyorhinis
    (SEQ ID NO:35)
    GGGGCTGATTCTGGATTCGACATACATAAAAGGATATAAATTGCAGTGGT
    CTTGTAAACCATAAGACAATTTCTTTACTAAGCGGAAAAGAAAACAAAAA
    AGAAGATTATTCATTATTAATGAATGCTTCAACTCAATCAAATCTAGCTT
    TTGCATTTTAAAAAACTAGTAGACCAATTTGCTTCTCACGAATTGTAATC
    TTTATATTAGAGAATAGTTAAAAATCTGATCACTTTTTAATGAATTTATA
    GATCACAGGCTTTTTTAATCTTTTTGTTATTTTAGATAAAGAGTCTTCTT
    AAAAATAACTAAACTGTAGGAATTTATATTTAATTATGCGTGGACCCGGG
    TTCAACTCCCGCCAGCTCCACCA
  • TABLE 28
    tmDNA Sequence for Mycoplasma pirum
    (SEQ ID NO:36)
    GGGGAGTCATGGTTTTGACATGAATGATGGACCCATAGAGGCAGTGGGGT
    ATGCCCCTTATAGCTCAAGGTTTAAATTAACCGACAAAACTGACGAAAAC
    GTTGCCGTTGATACAAATTTATTAATCAACCAACAAGCTCAATTTAACTA
    CGCATTTGCATAGTATAAAAAAATAAATTGTGCTACTCATTGTAATTAGG
    TTACTAAATTACTTTGTTTTATATAGTCCTGTAACTAGTTCTAGTGATGT
    CTATAAACTAGAATGAGATTTATAGACTTATTTGTTGGCGGTTGTGCCAT
    AGCCTAAATCAACAAAGACAATTTATTTATGGTACTAAACTGTAGATTCT
    ATGATGAAATTATTTGTGGAAACGGGTTCGATTCCCGCCATCTCCACCA
  • TABLE 29
    tmDNA Sequence for Mycoplasma salivarium
    (SEQ ID NO:37)
    GGGGCTGATTCTGGATTCGACAGGCATTCGATTCATTATGTTGCAGTGGT
    TTGCAAACCATAAGGCACTAGGCTTTTTTAAACGCAAAAGACCAAAAAAC
    AGAAGATCAAGCAGTTGATCTAGCATTTATGAATAATTCACAAATGCAAT
    CAAATCTAGTTTTCGCTTAGTAAAATTAGTCAATTTATTATGGTGCTCAA
    CATAATAAATGGTAGTATGAGCTTAATATCATATGATTTTAGTTAATATG
    ATAGGATTTGTAACTAAACTATGTTATAGAAATTTGTAAATTATATATAT
    GACATAGGAAATTTAATTTACTAAACTGTAGATGCATAATGTTGAAGATG
    TGTGGACCGGGGTTCAACTCCCGCCAGCTCCACCA
  • TABLE 30
    tmDNA Sequence for Herpetosiphon aurantiacus
    (SEQ ID NO:38)
    GGGGGCGGAAAGGATTCGACGGGGAGGGCCAATCGTAAGTGGCAAGCCGA
    GACGCTGAGCCTCGTTAAATCGGCAACGCCATTAACTGGCAAAAACACTT
    TCCGCGCTCCTGTAGCGCTTGCTGCCTAATTAAGGCAACACGTCTCTACT
    AGCCTCAGCCCGATGGGCTTGTAGCGGCGACACTTAGTCGGGTCGCTCCC
    CTAGTTATGTCTGTGGGCTAGGGGCTAAGATTAACAGGCTGGTCGTGGCC
    CGCTTTGTCTATCGGGTGGTGCACCGATAAGATTTAATCAATAGACTACG
    CTTGTAGATGCTTGCGGTTTAACTTTTTGGACGCGGGTTCGATTCCCGCC
    GCCTCACCACCA
  • TABLE 31
    tmDNA Sequence for Thermomicrobium roseum
    (352 nts, temp. 70 degrees, green non sulfur)
    (SEQ ID NO:39)
    GGGGCTGATTCTGGATTCGACAGGGCCGTAGGTGCGAGGATTGCAGGTCG
    AGGTCGCCCACGAACTCGTAAAAAGGGGCAGCCAAGTAACTGGCGAGCGC
    GAACTCGCTCTGGCTGCGTAATTCACGCAGCCACGTCTGCCCGGACCCTT
    CCCTGGTGGGTTCGGAGCGGGCGCCGCAAGACCGGGGTGCCCCTGGCCCA
    AGCGCCGGTGCGGGCCAGGTCAAGCGTGATCCGGCTCGGCTGACCGGGAT
    CCTGTCGGTGGGAGCCTGGCAGCGACAGTAGAACACCGACTAAGCCTGTA
    GCATATCCTCGGCTGAACGCTCTGGACGCGGGTTCAACTCCCGCCAGCTC
    CACCA
  • TABLE 32
    tmDNA Sequence for Chlorobium limicola
    (SEQ ID NO:40)
    GGGGCTGATTCTGGATTCGACAGGATACGTGTGAGATGTCGTTGCACTCC
    GAGTTTCAGCATGGACGGACTCGTTAAACAAGTCTATGTACCATTAGATG
    CAGACGATTATTCGTATGCAATGGCTGCCTGATTAGCACAAGTTAACTCA
    GACGCCATCGTCCTGCGGTGAATGCGCTTACTCTGAAGCCGCCGGATGGC
    ATAACCCGCGCTTGAGCCTACGGGTTCGCGCAAGTAAGCTCCGTACATTC
    ATGCCCGAGGGGCTGTGCGGGTAATTTCTCGGGATAAGGGGACGAACGCT
    GCTGGCGGTGTAATCGGCCCACGAAAACCCAATCACCAGAGATGAGTGTG
    GTGACTGCATCGAGCAGTGTTTTGGACGCGGGTTCAACTCCCGCCAGCTC
    CACCA
  • TABLE 33
    tmDNA Sequence for Pirellula staleyi
    (planctomyces)
    (SEQ ID NO:41)
    GGGGCTGATTCTGGATTCGACCGGATAGCCTGAAGCGAATACGGCGTGCC
    GTGGTTGATCAGATGGCCACGTAAAAAGCTGATCACAAACTTAACTGCCG
    AGAGCAATCTCGCACTTGCTGCCTAACTAAACGGTAGCTTCCGACTGAGG
    GCTTTAGCCGGAGAGGCCCAAAAGTTGGTCACCAAATCCGGACCGCCTCG
    TGCCATGATCGAAACGCACGAGGTCAAAAAAGTTTCGATCTAGTGCAGGG
    TGTAGCCAGCAGCTAGGCGACAAACTGTGCAAAAATCAAATTTTCTGCTA
    CGCACGTAGATGTGTTCGTGAAAATGTCTCGGGACGGGGGTTCAACTCCC
    GCCACTCCACCA
  • TABLE 34
    tmDNA Sequence for Planctomyces limnophilus
    (SEQ ID NO:42)
    GGGGCTGATTCTGGATTCGACAACCTCTCAAGAGGAGCGTGGCCACTATG
    GGACTCGATTATGTTGAATTCGTCATGGATCTTGAAGAGACCTTCGACAT
    CAAACTGGATGACAAACATTTTTCAGCAGTCAAAACACCACGCGATTTGG
    CAATCATTATTCGGGATCAATTAGCTGCTGAAGGCAGAATCTGGGATGAA
    TCGAATGCTTTTCGCAAAATCTCGAATTTGAATTGGACGATGTTGCCCGA
    GTTCCGGATGTGGACTCAAATCAAAAGCTCTCTACCAGTTTCTTTTCACC
    GACTGCGTCCCAGCACCCGTCTCGTTCAACTCCCGCCANTCCACCA
  • TABLE 35
    tmDNA Sequence for Planctomyces maris
    (SEQ ID NO:43)
    GGGGCTGATTCTGGATTCGACTGGTTCACCGTATGTTAAGGTGGCGGTGC
    CGTGGTTGATCAGTTGGCCACGTAAAAAGCTGATCACAATCTAATTGCAA
    ACAAGCAATTTTCAATGGCTGCTTAATAAAAGCAACCCCGGCTTAGGAAT
    CTCTGTCTGAGGAGTCCGACAGCTGGTCACAAAATCAGACTGGTATCAGA
    TCAATGTCCGCTCCGTCTGATACGAGATTCGTGGTGGACTGGTTTCCAAC
    AGGCTCTGTTTATCGTGCCCGAAGAAACGAGACTCAAACGATAAAATATG
    CACCGTAGAGGCTTTAGCTGAGGGTTCACAGGACGCGGGTTCAACTCCCG
    CCAGCTCCACCA
  • TABLE 36
    tmDNA Sequence for Alcaligenes eutrophus
    (SEQ ID NO:44)
    GGGGTTGATTCTGGATTCGACGTGGGTTACAAAGCAGTGGAGGGCATACC
    GAGGACCCGTCACCTCGTTAATCAATGGGAATGCAATAACTGCTAACGAC
    GAACGTTACGCACTGGCCGCTTAATTGCGGCCGTCCTCGCACTGGCTCGC
    TGACGGGCTAGGGTCGCAAGACCACGCGAGGTCATTTACGTCAGATAAGC
    TCCGGAAGGGTCACGAAGCCGGGGACGAAAACCTAGTGACTCGCCGTCGT
    AGAGCGTGTTCGTCCGCGATGCGCCGGTTAAATCAAATGACAGAACTAAG
    TATGTAGAACTCTCTGTGGAGGGCTTACGGACGCGGGTTCAACTCCCGCC
    AGCTCCACCA
  • TABLE 37
    tmDNA Sequence for Alcaligenes faecalis
    (beta proteobacteria)
    (SEQ ID NO:45)
    GGGGGCGGAAAGGATTCGACGGGGGTCAAGAAGCAGCACAGGGCGTGTCG
    AGCACCAGTACGCTCGTAAATCCACTGGAAAACTATAAACGCCAACGACG
    AGCGTTTCGCTCTAGCCGCTTAAGGCTGGGCCACTGCACTAATTTGTCTT
    TGGGTTAGGTAGGGCAACCTACAGCAGTGTTATTTACAAAGAATCGAATC
    GGTCTGCGCCACGAAGTCCGGTTCTAAAACTTAGTGGATCGCCAAGGAAA
    GGCCTGTCAATTGGCATAGTCCAAGGTTAAAACTTAAAATTAATTGACTA
    CACATGTAGAACTGTCTGTGGACGGCTTGCGGACGGGGGTTCGATTCCCG
    CCGCCTCCACCA
  • TABLE 38
    tmDNA Sequence for Chromobacterium violaceum
    (beta-purple)
    (SEQ ID NO:46)
    GGGGCTGATTCTGGATTCGACGGGGGTTGCGAAGCAGATGAGGGCATACC
    GGGATTTCAGTCACCCCGTAAAACGCTGAATTTATATAGTCGCAAACGAC
    GAAACTTACGCTCTGGCAGCCTAACGGCCGGCCAGACACTACAACGGTTC
    GCAGATGGGCCGGGGGCGTCAAAACCCTGTAGTGTCACTCTACATCTGCT
    AGTGCTGTTCCGGGTTACTTGGTTCAGTGCGAAATAATAGGTAACTCGCC
    AAAGTCCAGCCTGTCCGTCGGCGTGGCAGAGGTTAAATCCAAATGACACG
    ACTAAGTATGTAGAACTCACTGTAGAGGACTTTCGGACGCGGGTTCAACT
    CCCGCCAGCTCCACCA
  • TABLE 39
    tmDNA Sequence for Hydrogenophaga palleroni
    (beta-purple)
    (SEQ ID NO:47)
    GGGGCTGATTCTGGATTCGACGTGGGTTCGGACGCGCAGCAGGGCATGTC
    GAGGTTCTGTCACCTCGTAAATCAGCAGAAAAAAACCAACTGCAAACGAC
    GAACGTTTCGCACTCGCCGCTTAAACACCGGTGAGCCTTGCAACAGCAGG
    CCGATGGGCTGGGCAAGGGGGTCGCAAGACCTCCCGGCTGCAAGGTAATT
    TACATCGGCTGGTTCTGCGTCGGGCACCTTGGCGCAGGATGAGATTCAAG
    GATGCTGGCTTCCCGTTTAGCGTGCCACTGCGCGACTCGGGCGGCGAGAC
    CCAAATCAGACGGCTACACATGTAGAACTGCTCGAAAAAGGCTTGCGGAC
    GGGGGTTCAACTCCCGCCAGCTCCACCA
  • TABLE 40
    tmDNA Sequence for Methylobacillus glycogenes
    (beta-purple)
    (SEQ ID NO:48)
    GGGGGCGGAAAGGATTCGACGGGGGTTGCAAAGCAGCGCAGGGCATACCG
    AGGCCTAGTCACCTCGTAAATAAACTAGAACAAGTATAGTCGCAAACGAC
    GAAACTTACGCTCTAGCCGCTTAATCCCGGCTGGACGCTGCACCGAAGGG
    CCTCTCGGTCGGGTGGGGTAACCCACAGCAGCGTCATTAAGAGAGGATCG
    TGCGATATTGGGTTACTTAATATCGTATTAAATCCAAGGTAACTCGCCTG
    CTGTTTGCTTGCTCGTTGGTGAGCATCAGGTTAAATCAAACAACACAGCT
    AAGTATGTAGAACTGTCTGTGGAGGGCTTGCGGACGGGGGTTCGATTCCC
    GCCGCCTCACCACCA
  • TABLE 41
    tmDNA Sequence for Nitrosomonas cryotolerans
    (beta-purple)
    (SEQ ID NO:49)
    GGGGCTGATTCTGGATTCGACGTGGGTTGCAAAGCAGCGCAGGGCATACC
    GAGGACCAGAATACCTCGTAAATACATCTGGAAAAAAATAGTCGCAAACG
    ACGAAAACTACGCTTTAGCCGCTTAATACGGCTAGCCTCTGCACCGATGG
    GCCTTAACGTCGGGTCTGGCAACAGACAGCAGAGTCATTAGCAAGGATCG
    CGTTCTGTAGGGTCACTTTACAGAACGTTAAACAATAGGTGACTCGCCTG
    CCATCAGCCCGCCAGCTGGCGGTTGTCAGGTTAAATTAAAGAGCATGGCT
    AAGTATGTAGAACTGTCTGTAGAGGACTTGCGGACGCGGGTTCAACTCCC
    GCCAGTCCACCA
  • TABLE 42
    tmDNA Sequence for Pseudomonas testosteroni
    (SEQ ID NO:50)
    GGGGCTGATTCTGGATTCGACGTGGGTTCGGGACCGGTGCGGTGCATGTC
    GAGCTTGAGTGACGCTCGTAAATCTCCATTCAAAAAACTAACTGCAAACG
    ACGAACGTTTCGCACTCGCCGCTTAATCCGGTGAGCCTTGCAACAGCACG
    CTAGTGGGCTGGGCAAGGGGGTAGCAATACCTCCCGGCTGCAAGGGAATT
    TTCATTAGCTGGCTGGATACCGGGCTTCTTGGTATTTGGCGAGATTTTAG
    GAAGCTGGCTACCCAAGCAGCGTGTGCCTGCGGGGTTTGGGTGGCGAGAT
    TTAAAACAGAGCACTAAACATGTAGATCTGTCCGGCGAAGGCTTACGGAC
    GCGGGTTCAACTCCCGCCAGCTCCACCA
  • TABLE 43
    tmDNA Sequence for Ralstonia pickettii
    (Burkholderia)
    (SEQ ID NO:51)
    GGGGGCGGAAAGGATTCGACGGGGGTTGCGAAGCAGCGGAGGGCATACCG
    AGGACCCGTCACCTCGTTAATCAATGGGAATGCAATAACTGCTAACGACG
    AACGTTACGCACTGGCAGCCTAAGGGCCGCCGTCCTCGCACTGGCTCGCT
    GACGGGCTAGGGTCGCAAGACCAGCGAGGTCATTTACGTCAGATAAGCTT
    TAGGTGAGTCACGGGCCTAGAGACGAAAACTTAGTGAATCGCCGTCGTAG
    AGCGTGTTCGTCCGCGATGCGGCGGTTAAATCAAATGACAGAACTAAGTA
    TGTAGAACTCTCTGTGGAGGGCTTGCGGACGCGGGTTCGATTCCCGCCGC
    CTCACCACCA
  • TABLE 44
    tmDNA Sequence for Variovax paradoxus
    (pseudomonas sp.)
    (SEQ ID NO:52)
    GGGGCTGATTCTGGATTCGACGTGGGTTCGGAGTCGCAGCGGGGCATGTC
    GAGCTGAATGCGCTCGTAAAACAGATTCAAACAAACTAACTGCAAACGAC
    GAACGTTTCGCACTCGCTGCTTAATTGCCAGTGAGCCTTGCAACAGTTGG
    CCGATGGGCTGGGCAAGGGGGTCTGGAGCAATCCTGACCTCCCGGCTGCA
    AGGATAACTACATGGGCTGGCTCCGATCCGGGTACCTTGGGTCGGGGCGA
    GAAAATAGGGTACTGGCGTCCGGTTTAGCGTGTGACTGCGCGACTCCGGA
    AGCGAGACTCAAAACAGATCACTAAACATGTAGAACTGCGCGATGAAGGC
    TTGCGGACGGGGGTTCAACTCCCGCCAGCTCCACCA
  • TABLE 45
    tmDNA Sequence for Bdellovibrio bacteriovorus
    (delta proteobacterie)
    (SEQ ID NO:53)
    GGGGGCGGAAAGGATTCGACGGGGGTGCTGAAGCATAAGGAGCATACCGG
    GGCGGATGAGGACCTCGTTAAAAACGTCCACTTTGTAATTGGCAACGATT
    ACGCACTTGCAGCTTAATTAAGCAGCACGATCAACCTTGTGGTGGTTCCG
    CACTTGGATTGATCGTCATTTAGGGACCTCGGCGTGTTGGGTTTTCTCCA
    GCAGACATGCTTAAATTTACTGGGGGAGAGGTCTTAGGGATTTTGTCTGT
    GGAAGCCCGAGGACCAATCTAAAACACTGACTAAGTATGTAGCGCCTTAT
    CGTGGATCATTTGCGGACGGGGGTTCGATTCCCGCCGCCTCCACCA
  • TABLE 46
    tmDNA Sequence for Myxococcus xanthus
    (delta proteobacterie)
    (SEQ ID NO:54)
    GGGGGCGGAAAGGATTCGACGGGGGCATTGAAGTTCGAGACGCGTGCCGA
    GCTTGTCAGGTAGCTCGTAAATTCAACCCGGCAAAGACACAAAAGCCAAC
    GACAACGTTGAGCTCGCGCTGGCTGCCTAAAAACAGCCCATAGTGCGCGG
    TCCCCCCGCCCTCGGCCTGTGGGGTTGGGACAGACCGTCATAATGCAGGC
    TGGCTGCCGAGGGTGCCTGGACCCGAGGTGGCGAGATCTTCCCAGGACCG
    GCTCTGAGTATCCCGTCCGTGGGAGCCTCAGGGACGTAGCAAATCGCGGA
    CTACGCACGTAGGGTCGAAGAGCGGACGGCTTTCGGACGCGGGTTCGATT
    CCCGCCGCCTCCACCA
  • TABLE 47
    tmDNA Sequence for Sulfurospirillum Deleyianum
    (SEQ ID NO:55)
    GGGGCTGATTCTGGATTCGACAGGAGTAGTTTTAGCTTATGGCTGCATGT
    CGGGAGTGAGGGTCTTCCGTTACACAACCTTCAAACAATAACTGCTAACA
    ACAGTAACTATCGTCCTGCTTACGCGCTAGCTGCGTAAGTTTAACAAATA
    ATGGACTGCTCTCCCCTTTGATGCTATCTTAGGAGGTCTTGGAGAGTATC
    ATAGATTTGATAGCTATATTACATGAACGCCTTTACATGTAATGAAGTTA
    AAGGCTCGTTTTGCGTAGTTTTCTGATTGTTGTACGAAGCAAAATTAAAC
    ACTATCAACAATATCTAAGCATGTAGACGTCATAGGTGGCTATTTTTGGA
    CTGCGGGTTCAACTCCCGCCAGCTCCACCA
  • TABLE 48
    tmDNA Sequence for Chromatium vinosum
    (SEQ ID NO:56)
    GGGGCTGATTCTGGATTCGACGTGGGTCGCGAAACCTAAGGTGCATGCCG
    AGGTGCGGTTGACCTCGTAAAACCCTCCGCAAACTTATAGTTGCCAACGA
    CGACAACTACGCTCTCGCTGCTTAATCCCAGCGGGCCTCTGACCGTCACT
    TGCCTGTGGGCGGCGGATTCCAGGGGTAACCTCACACAGGATCGTGGTGA
    CGGGAGTCCGGACCTGATCCACTAAAACCTAACGGAATCGCCGACTGATC
    GCCCTGCCCTTCGGGCGGCAGAAGGCTAAAAACAATAGAGTGGGCTAAGC
    ATGTAGGACCGAGGGCAGAGGGCTTGCGGACGCGGGTTCAACTCCCGCCA
    GCTCCACCA
  • TABLE 49
    tmDNA Sequence for Pseudomonas fluorescens
    (gamma proteobacteria)
    (SEQ ID NO:57)
    GGGGCTGATTCTGGATTCGACGCCGGTTGCGAACCTTTAGGTGCATGCCG
    AGTTGGTAACAGAACTCGTAAATCCACTGTTGCAACTTTCTATAGTTGCC
    AATGACGAAACCTACGGGGAATACGCTCTCGCTGCGTAAGCAGCCTTAGC
    CCTTCCCTCCTGGTACCTTCGGGTCCAGCAATCATCAGGGGATGTCTGTA
    AACCCAAAGTGATTGTCATATAGAACAGAATCGCCGTGCAGTACGTTGTG
    GACGAAGCGGCTAAAACTTACACAACTCGCCCAAAGCACCCTGCCCGTCG
    GGTCGCTGAGGGTTAACTTAATAGACACGGCTACGCATGTAGTACCGACA
    GCAGAGTACTGGCGGACGCGGGTTCAACTCCCGCCAGCTCCACCA
  • TABLE 50
    tmDNA Sequence for Borrelia afzeli
    (SEQ ID NO:58)
    GGGGCTGATTCTGGATTCGACTGAAAATGCTAATATTGTAAGTTGCAAGC
    AGAGGGAATCTCTTAAAACTTCTAAAATAAATGCAAAAAATAATAACTTT
    ACAAGTTCAAACCTTGTAATGGCTGCTTAAGTTAGCAGAGAGTTTTGTTG
    AATTTGGCTTTGAGATTCACTTATACTCTTTTAGACATCGAAGCTTGCTT
    AAAAATGTTTTCAAGTTGATTTTTAGGGACTTTTATACTTGAGAGCAATT
    TGGCGGTTTGCTAGTATTTCCAAACCATATTGCTTAGTAAAATACTAGAT
    AAGCTTGTAGAAGCTTATAGTATTGTTTTTAGGACGCGGGTTCAACTCCC
    GCCAGTCCACCA
  • TABLE 51
    tmDNA Sequence for Borrelia crociduarae
    (SEQ ID NO:59)
    GGGGCTGATTCTGGATTCGACTAAGAACTTTAGTAGCATAAATGGCAAGC
    AGAGTGAATCTCTTAAAACTTCTTTAATAAATGCAAAAAATAATAACTTT
    ACAAGTTCAGATCTTGTAATGGCTGCTTAATTTAGCAGAGAGTTTTGTTG
    GATTTTGCTTTGAGGTTCAACTTATACTCTTTAAGACATCAAAGTATGCC
    TAAAAATGTTTCAAGTTGATTTTTAGGGACCTTTAAACTTGAGAGTAATT
    TGGTGGTTTGCTTGTTTTCCAAGCCTTATTGCTTTTTCTAAAAATTAGCT
    AAGCTTGTAGATATTTATGATATTATTTTTAGGACGCGGGTTCAACTCCC
    GCCAGTTCCACCA
  • TABLE 52
    tmDNA Sequence for Borrelia hermsii
    (SEQ ID NO:60)
    GGGGCTGATTCTGGATTCGACTAAAAACTTTAGTAGCATAAATTGCAAGC
    AGAGGGAATCTCTTAAAACTTCTTTAATAAATGCAAGAAATAATAACTTT
    ACAAGTTCAAATCTTGTAATGGCTGCTTAAATTAGCAGAGAGTTCTGCTG
    GATTTTGCTTTGAGGTTCAGCTTATACTCTTTTAAGACATCAAAGCTTGC
    TTAAAAATATTTCAAGTTGATTTTTAGGGACTTTTAAATTTGAGAGTAAT
    TTGGCGGTTTGCTAGTTTTTCCAAACCTTATTACTTAAAGAAAACACTAG
    CTAAGCTTGTAGATATTTATGATATTATTTTTAGGACGCGGGTTCAACTC
    CCGCCAGCTCCACCA
  • TABLE 53
    tmDNA Sequence for Borrelia garinii
    (SEQ ID NO:61)
    GGGGCTGATTCTGGATTCGACTGAAAATGCGAATATTGTAAGTTGCAGGC
    AGAGGGAATCTCTTAAAACTTCTAAAATAAATGCAAAAAATAATAACTTT
    ACAAGCTCAAACCTTGTAATGGCTGCTTAAGTTAGCAGGGAGTTTCGTTG
    AATTTGGCTTTGAGGTTCACTTATACTCTTTTCGATATCGAAGCTTGCTT
    AAAAATGTTTTCAAGTTAATTTTTAGGGACTTTTGTACTTGAGAGCAATT
    TGGCGGTTTGCTAGTATTTCCAAACCATATTGCTTAAGTAAAATGCTAGA
    TAAGCTTGTAGAAGCTTATAATATTGTTTTTAGGACGCGGGTTCAACTCC
    CGCCAGTCCACCA
  • TABLE 54
    tmDNA Sequence for Thermodesulfobacterium commune
    (70 degrees)
    (SEQ ID NO:62)
    GGGGGCGGAAAGGATTCGACGGGGATAGGTAGGATTAAACAGCAGGCCGT
    GGTCGCACCCAACCACGTTAAATAGGGTGCAAAAACACAACTGCCAACGA
    ATACGCCTACGCTTTGGCAGCCTAAGCGTGCTGCCACGCACCTTTAGACC
    TTGCCTGTGGGTCTAAAGGTGTGTGACCTAACAGGCTTTGGGAGGCTTAA
    TCGGTGGGGTTAAGCCTCCCGAGATTACATCCCACCTGGTAGGGTTGCTT
    GGTGCCTGTGACAAGCACCCTACGAGATTTTCCCACAGGCTAAGCCTGTA
    GCGGTTTAATCTGAACTATCTCCGGACGCGGGTTCGATTCCCGCCGCCTC
    CCCACCA
  • TABLE 55
    tmDNA Sequence for Thermotoga neapolitana
    (Thermotogales)
    (SEQ ID NO:63)
    GGGGGCGGAAAGGATTCGACGGGGATGGAGTCCCCTGGGAAGCGAGCCGA
    GGTCCCCACCTCCTCGTAAAAAAGGTGGGAACACGAATAAGTGCCAACGA
    ACCTGTTGCTGTTGCCGCCTAATAGATAGGCGGCCGTCCTCTCCGGAGTT
    GGCTGGGCTCCGGAAGAGGGCGTGAGGGATCCAGCCTACCGATCTGGGCT
    CCGCCTTCCGGCCCGGATCGGGAAGGTTCAGGAAGGCTGTGGGAAGCGAC
    ACCCTGCCCGTGGGGGGTCCTTCCCGAGACACGAAACACGGGCTGCGCTC
    GGAGAAGCCCAGGGGCCTCCATCTTCNGACGCGGGTTCGATTCCCGCCAC
    CTCCACCA
  • TABLE 56
    tmDNA Sequence for Deinococcus proteolyticus
    (SEQ ID NO:64)
    GGGGGCGGAAAGGATTCGACGGGGGAACGGAAAGCGCTGCTGCGTGCCGA
    GGAGCCGTTGGCCTCGTAAACAAACGGCAAAGCCATTAACTGGCGAAAAT
    AACTACGCTCTCGCTGCTTAAGTGAGACAGTGACCACGTAGCCCCGCCTT
    TGGCGACGTGTGAACTGAGACAAAAGAAGGCTAGCTTAGGTGAGGTTCCA
    TAGCCAAAAGTGAAACCAAATGGAAATAAGGCGGACGGCAGCCTGTTTGC
    TGGCAGCCCAGGCCCGACAATTTAAGAGCAGACTACGCACGTAGATGCAC
    GCTGGATGGACCTTTGGACGCGGGTTCGATTCCCGCCAGCTCCACCA
  • TABLE 57
    tmDNA Sequence for Prosthecobacter fusiformis
    (verrucomicrobia)
    (SEQ ID NO:65)
    GGGGCTGATTCTGGATTCGACGGGGAGTACAAGGATCAAAAGCTGCAAGC
    CGAGGTGCCGTTACCTCGTAAAACAACGGCAAAAAAGAAGTGCCAACACA
    AATTTAGCATTAGCTGCTTAATTTAGCAGCTACGCTCTTCTAACCCGGGC
    TGGCAGGGTTAGAAGGGTGTCATAATGAGCCAGCTGCCCCTTCCGACTCC
    CCTAAGGAAGGGAAAGATGTAGGGGATAGGTGCTTACAGAATCCTGCGGG
    AGGGAGTCTGTAAGTGCCGAAAAGTTAAAACTCCCGCTAAGCTTGTAGAG
    GCTTTTGATTCTTGCTCTCTGGACGCGGGTTCAACTCCCGCCAGCTCCAC
    CA
  • TABLE 58
    tmDNA Sequence for Verrucomicrobium spinosum
    (verrucomicrobium)
    (SEQ ID NO:66)
    GGGNNNNATTTGGAATTCGCCGAATGCTAGAAGTGGAGGCTGCATGCCGC
    GGATGATTCGTTGGCCGCTTTACCAATTCGGATCAAACAACTAAATGCGG
    ACTCTAACGAGCTTGCCCTCGCCGCTTAATTGACGGTGACGTTCCTCCAG
    TGAAGTCTGTGAATTGGAGGAGCGACTACTTACAGGCTGGCCAAAAGAGC
    GGGCGACCGGCCCCAAGGCGAGATCTACAGGCCGCTGGATGGACGGCATC
    CTGGCAGTAGGAGGCTGGACATCGAGATCAAATNATTGCCTGAGCATGGA
    GACGCTTTCATAAAGGNGTTCGGACAGGG
    • Example 4 Alignment of tmRNA Sequences
  • The newly discovered tmRNA sequences and several known tmRNA sequences were aligned to identify target sites for drug development. The alignments of the sequences are shown in FIGS. 3A-11B. The nucleotides in the tmRNA sequences of these figures exist in several motifs (Felden et al., 1999). These motifs include nucleotides considered to be in RNA helices (Watson-Crick base-pairs GC or AU, or GU Wobble base-pairs). Nucleotides that are in single stranded RNA domains, hence not base-paired. Some nucleotides in the single stranded domains are universally conserved nucleotides. Other nucleotides are the exceptions to a quasi-sequence conservation in the sequences alignment. Several nucleotides exist in well established non-canonical structural motifs in RNA structures; for example AG-GA pairs, AA pairs, etc. Some nucleotides are universally conserved Wobble GU base-pairs.
  • All the gene sequences have been decomposed in several structural domains that have been indicated with names at the top of each block of sequences. These domains are respectively from the 5′-end to the 3′-end of the sequences: H1, H5, H2, PK1, H4, PK2, PK3, PK4, H5 and H6. The bars delineate all the structural domains. H means helices and PK means pseudoknot. A pseudoknot is made of the pairing of parts of an RNA-loop with an upstream sequence. Consequently, two helices are made (shown in Felden et al., 1999) for all the 4 pseudoknots PK1 to PK4 for each sequence. Moreover, the tRNA-like domain as well as the coding sequence, namely the two functional units of the molecule, have also been indicated for each sequence.
  • The sequences, especially as identified by the sequence alignment, represent targets for the development of drugs which may be broadly applicable to many kinds of bacteria, or may be broadly applicable only to a particular genera of phylum of bacteria, or may be specifically applicable to a single species of bacteria.
  • Common Structural Features for Drug Targeting:
  • For all the novel tmRNA sequences, as well as with the ones that are already known, there are systematically several structural domains that are always found. These domains can be used as targets for the development of drugs which may be genera specific or which may be eubacteria specific. These domains are either RNA helices which can be sometimes interrupted by bulges or pseudoknots. The RNA helices which are always present are H1, H2, H5 and H6. Helices H1 and H6 are found in all canonical transfer RNAs. Thus, H1 and H6 are not good targets for drug development because drugs that would target them will also interfere with the biology of the individual that has a given disease. Consequently, very good candidates for development of drugs for targeting as many bacteria as possible are helices H2 and H5. Moreover, helices H2 and H5 are critical for the folding of all these tmRNA since both of them connect the two ends of the molecule together. Disruption of either H2 or H5 with a specific drug would lead to inactive tmRNA molecules in vivo. Similarly, pseudoknots PK1, PK2 and PK3 are always found in the bacterial tmRNAs. The PK1 structural domain is strictly conserved in the tmRNAs and is located upstream of the coding sequence. Since these pseudoknots are not found in all canonial transfer RNAs, they can also be targeted with specific drugs. Disruption of either PK1 or PK2 or PK3 with a specific drug would lead to inactive tmRNA molecules in vivo.
  • Specific Structural Features in Each Phylum that could be Targeted by Drugs:
  • In addition to developing drugs which broadly target many bacteria, drugs are developed which are more genera specific. For trying to target specifically a given bacteria or a complete phylum, the coding sequence (shown in all the alignments) is a very good candidate. Indeed, this region of the RNA is very accessible for DNA antisense binding, which has been shown for Escherichia coli, and thus, is also available for interaction with other drugs. Moreover, this is a critical functional domain of the molecule in its quality-control mechanism in cells. In addition, this coding sequence would be the ideal target to use for designing specific PCR-based diagnostic assays for infection diseases.
  • Interestingly, some structural domains are present only in a given bacterial phyla and could be targeted for discovering a drug that will be specific of a phylum, but not of the others. For example:
  • (1) in the cyanobacteria, the fourth pseudoknot PK4 is made of two smaller pseudoknots called PK4a and PK4b;
  • (2) in the mycoplasma, helix H2 is made of only 4 base-pairs instead of 5 in the other species;
  • (3) for two sequences of chlorobium as well as Bacteroides thetaiotaomicron and ppm gingiv., there is an additional domain just downstream of the coding sequence that is unique to them;
  • (4) there is always a stem-loop in the coding sequence of the actinobacteria (Felden et al., 1999); and
  • (5) all the beta proteobacteria possess a sequence insertion in pseudoknot PK2 (shown in the alignment).
  • The novel sequences described herein, when aligned, show that specific structural domains within tmRNA are strictly conserved, as for example pseudoknot PK1 is located upstream (at the 5′-side) of the coding sequence. As previously disclosed, this pseudoknot is a target for future anti-bacterial drugs. Moreover, recent data have shown that this PK1 pseudoknot, among all the four pseudoknots within tmRNA gene sequences (sometimes there's only 2 or 3 detectable pseudoknots, depending upon the sequences), is the only one that its correct folding is essential for the biological activity of tmRNA (Nameki et al., 1999; Nameki et al., 2000).
  • While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims.
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    • Gray, M. W. and Spencer, D. F. (1996). In Evolution of Microbial Life, Cambridge University Press, pp. 109-126.
    • Hickerson, R. P. et al. (1998). J. Mol. Biol. 279:577-587.
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Claims (6)

1. An isolated nucleic acid sequence selected from the group consisting of the tmRNA sequence for Escherichia coli set forth in SEQ ID NO:165, a tmDNA sequence encoding said tmRNA sequence, and a complement of said tmDNA sequence.
2. A method for diagnosing a bacterial infectious agent comprising determining the presence of a bacterial nucleic acid sequence selected from the group consisting of the tmRNA sequence for Escherichia coli set forth in SEQ ID NO:165, a tmDNA sequence encoding said tmRNA sequence, and a complement of said tmDNA sequence.
3. The method of claim 2, wherein the determination is made by performing an amplification-based assay.
4. The isolated nucleic acid sequence of claim 1, wherein the nucleic acid sequence is the tmDNA sequence encoding the tmRNA sequence for Escherichia coli set forth in SEQ ID NO:165.
5. The method of claim 2, wherein the bacterial nucleic acid sequence is the tmDNA sequence encoding the tmRNA sequence for Escherichia coli set forth in SEQ ID NO:165.
6. The method of claim 3, wherein the bacterial nucleic acid sequence is the tmDNA sequence encoding the tmRNA sequence for Escherichia coli set forth in SEQ ID NO:165.
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