# Patent

## Title
Eubacterial tmRNA sequences and uses thereof

## 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a national stage filing under 35 U.S.C. §371 of PCT/US00/08988 filed on 6 Apr. 2000. The present application is also 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.


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. Extaction 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 set 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′ (SEQ ID NO:2);
        
        
        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); and
        
        
        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 dissoved 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)









GGGGGCGGAAAGGATTCGACGGGGTTGACTGCGGCAAAGAGGCATGCCGGGGGGTGGGCACCCG



 


TAATCGCTCGCAAAACAATACTTGCCAACAACAATCTGGCACTCGCAGCTTAATTAAATAAGTT


 


GCCGTCCTCTGAGGCTTCGCCTGTGGGCCGAGGCAGGACGTCATACAGCAGGCTGGTTCCTTCG


 


GCTGGGTCTGGGCCGCGGGGATGAGATCCACGGACTAGCATTCTGCGTATCTTGTCGCTTCTAA


 


GCGCAGAGTGCGAAACCTAAAGGAATGCGACTGAGCATGGAGTCTCTTTTCTGACACCAATTTC


 


GGACGCGGGTTCGATTCCCGCCGCCTCCACCA
















TABLE 2





tmDNA Sequence for Coprothermobacter proteolyticus (60 degrees)
















(SEQ ID NO:10)









GGGGGCGGAAAGGATTCGACGGGGAGTCGGAGCCTTGAGCTGCAGGCAGGGTTGGCTGCCACAC



 


CTTAAAAAGGGTAGCAAGGCAAAAATAAATGCCGAACCAGAATTTGCACTAGCTGCTTAATGTA


 


AGCAGCCGCTCTCCAAACTGAGGCTGCATAAGTTTGGAAGAGCGTCAACCCATGCAGCGGCTCT


 


TAAGCAGTGGCACCAGCTGTTTAAGGGTGAAAAGAGTGGTGCTGGGCAGTGCGGTTGGGCTTCC


 


TGGGCTGCACTGTCGAGACTTCACAGGAGGGCTAAGCCTGTAGACGCGAAAGGTGGCGGCTCGT


 


CGGACGCGGGTTCGATTCCCGCCGCCTCCACCA
















TABLE 3





tmDNA Sequence for Bacteroides thetaiotaomicron (bacteroides/flavobacterium)
















(SEQ ID NO:11)









GGGGCTGATTCTGGATTCGACAGCGGGCAGAAATGGTAGGTAAGCATGCAGTGGGTCGGTAATT



 


TCCACTTAAATCTCAGTTATCAAAACTTTATCTGGCGAAACTAATTACGCTCTTGCTGCTTAAT


 


CGAATCACAGTAGATTAGCTTAATCCAGGCACTAGGTGCCAGGACGAGACATCACTCGGAAGCT


 


GTTGCTCCGAAGCATTCCGGTTCAGTGGTGCAGTAACATCGGGGATAGTCAGAAGCGGCCTCGC


 


GTTTTTGATGAAACTTTAGAGGATAAGGCAGGAATTGATGGCTTTGGTTCTGCTCCTGCACGAA


 


AATTTAGGCAAAGATAAGCATGTAGAAAGCTTATGATTTCCTCGTTTGGACGAGGGTTCAACTC


 


CCGCCAGCTCCACCA
















TABLE 4





tmDNA Sequence for Dictyoglomus thermophilum (70 degrees)
















(SEQ ID NO:12)









GGGGCTGATTCTGGATTCGACAGGGAGTACAAGGATCAAAAGCTGCAAGCCGAGGTGCCGTTAC



 


CTCGTAAAACAACGGCAAAAAAGAAGTGCCAACACAAATTTAGCATTAGCTGCTTAATTTAGCA


 


GCTACGCTCTTCTAACCCGGGCTGGCAGGGTTAGAAGGGTGTCATAATGAGCCAGCTGCCCCTT


 


CCGACTCCCCTAAGGAAGGGAAAGATGTAGGGGATAGGTGCTTACAGAATCCTGCGGGAGGGAG


 


TCTGTAAGTGCCGAAAAGTTAAAACTCCCGCTAAGCTTGTAGAGGCTTTTGATTCTTGCTCTCT


 


GGACGCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 5





tmDNA Sequence for Environmental Sample from Rumenal Fluid
















(SEQ ID NO:13)









ACGCCCTTGTCTCAGACGAGGGCACTCGTTAAAAAGTCTGAAAAGAATAACTGCAGAACCTGTA



 


GCTATGGCTGCTTAATTTAAGGGCAACCCTTGGATCCGCCTCCATCCCGAAGGGGTGGCATCCG


 


AGTCGCAAATCGGGATAGGATGGATCTTGGCAACGAGGAGTACATCCGAAATTTGTCGCTGCTG


 


GCTGAAGCATCGCCGTTCCTCTTTGGGCGTGGCAAGGCAAGATTAAATTCAGAGGATAAGCGTG


 


TAGTAGCGAGTGAGTAGGTGTTTTTGGACGCGGGTTCAAGTCCCGCCATCTCCACCA
















TABLE 6





tmDNA Sequence for Environmental Sample from Sludge
















(SEQ ID NO:14)









GGGGATGTCATGGTTTTGACAGGGAACCAGGAGGTGTGAGATGCATGCCGGAGACGCTGTCCGC



 


TCCGTTATCAAGCAGCAAACAAAACTAATTGCAAACAACAATTACTCCTTAGCAGCGTAAGCAG


 


CTAACGTTCAACCTCTCCGGACCGCCGGGAGGGGATTTGGGCGTCGAAACAGCGCGGACGCTCC


 


GGATAGGACGCCCATAATATCCGGCTAAGACCATGGGTCTGGCTCTCGCGGGTCTGATTGTCTT


 


CCACCGCGCGGGCCGCGATCAAAGACAACTAAGCATGTAGGTTCTTGCATGGCCTGTTCTTTGG


 


ACGCGGGTTCGATTCCCGCCATCTCCACCA
















TABLE 7





tmDNA Sequence for Fibrobacter succinogenes (Fibrobacter)
















(SEQ ID NO:15)









GGGGCTGATTCTGGATTCGACAGGGTTACCGAAGTGTTAGTTGCAAGTCGAGGTCTCAGACGAG



 


GGCTACTCGTTAAAAAGTCTGAAAAAAAATAAGTGCTGACGAAAACTACGCACTCGCTGCCTAA


 


TTAACGGCAACGCCGGGCCTCATTCCGCTCCCATCGGGGTGTACGTCCGGACGCAATATGGGAT


 


AGGGAAGTGTCATGCCTGGGGGCATCTCCCGAGATTTTCTAGGCTGGTCAAACTCCGCGCCGAC


 


CTTCTTGGGCGTGGATAAGACGAGATCTTAAATTCGAAGGGAACACTTGTAGGAACGTACATGG


 


ACGTGATTTTGGACAGGGGTTCAACTCCCGCCAGCTCCA
















TABLE 8





tmDNA Sequence for Fusobacterium mortiferum
















(SEQ ID NO:16)









GGGGCTGATTCTGGATTCGACGGGGTTATGAGGTTATAGGTAGCATGCCAGGATGACCGCTGTG



 


AGAGGTCAACACATCGTTTAGATGGAAACAGAAATTACGCTTTAGCTGCTTAATTAGTCAGCTC


 


ACCTCTGGTTTCTCTCTTCTGTAGGAGAATCCAACCGAGGTGTTACCAATATACAGATTACCTT


 


TAGTGATTTCTCTAAGCTCAAAGGGACATTTTAGAGAATAGCTTCAGTTAGCCCTGTCTGCGGG


 


AGTGATTGTTGCGAAATAAAATAGTAGACTAAGCATTGTAGAAGCCTATGGCGCTGGTAGTTTC


 


GGACACGGGTTCAACTCCCGCCAGCTCCAA
















TABLE 9





tmDNA Sequence for Corynebacterium xerosis (gram +, high G-C content)
















(SEQ ID NO:17)









GGGGCTGATTCTGGATTCGACTTCGTACATTGAGCCAGGGGAAGCGTGCCGGTGAAGGCTGGAG



 


ACCACCGCAAGCGTCGCAGCAACCAATTAAGCGCCGAGAACTCTCAGCGCGACTACGCCCTCGC


 


TGCCTAAGCAGCGACCGCGTGTCTGTCAGACCGGGTAGGCCTCTGATCCGGACCCTGGCATCGT


 


TTAGTGGGGCTCGCTCGCCGACTTGGTCGCAAGGGTCGGCGGGGACACTCACTTGCGACTGGGC


 


CCGTCATCCGGTCATGTTCGACTGAACCGGAGGGCCGAGCAGAGACCACGCGCGAACTGCGCAC


 


GGAGAAGCCCTGGCGAGGTGACGGAGGACCCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 10





tmDNA Sequence for Micrococcus luteus (parfait)
















(SEQ ID NO:18)









GGGGCTATTCTGGATTCGACGGTGTGTGTCGCGTCGGGAGAAGCGGGCCGAGGATGCAGAGTCA



 


TCTCGTCAAACGCTCTCTGCAAACCAATAAGTGCCGAATCCAAGCGCACTGACTTCGCTCTCGC


 


TGCCTGATCAGTGATCGAGTCCGTCACCCCGAGGTCGCTGTCGCCTCGGATCGTGGCGTCAGCT


 


AGATAGCCACTGGGCGTCACCCTCGCCGGGGGTCGTGACGCCGACATCAATCCGGCTGGGTCCG


 


GGTTGGCCGCCCGTCTGCGGGACGGCCAGGACCGAGCAACACCCACAGCAGACTGCGCCCGGAG


 


AAGACCTGGCAACACCTCATCGGACGCGGGTTCAACTCCCGCANTCCCACCA
















TABLE 11





tmDNA Sequence for Mycobacterium smegmatis
















(SEQ ID NO:19)









TCATCTCGGCTTGTTCGCGTGACCGGGAGATCCGAGTAGAGACATAGCGAACTGCGCACGGAGA



 


GGGGCTGATTCCTGGATTCGACTTCGAGCATCGAATCCAGGGAAGCGTGCCGGTGCAGGCAAGA


 


GACCACCGTAAGCGTCGTTGCAACCAATTAAGCGCCGATTCCAATCAGCGCGACTACGCCCTCG


 


CTGCCTAAGCGACGGCTGGTCTGTCAGACCGGGAGTGCCCTCGGCCCGGATCCTGGCATCAGCT


 


AGAGGGACCCACCCACGGGTTCGGTCGCGGGACCTGTGGGGACATCAAACAGCGACTGGGATCG


 


AGCCTCGAGGACATGCCGTAGGACCCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 12





tmDNA Sequence for Bacillus badius
















(SEQ ID NO:20)









GGGGGTGATTCTGGATTCGACAGGGATAGTTCGAGCTTGGGCTGCGAGCCGGAGGGCCGTCTTC



 


GTACCAACGCAAACGCCTAAATATAACTGGCAAAAAAGATTTAGCTTTAGCTGCCTAATATAGG


 


TTCAGCTGCTCCTCCCGCTATCGTCCATGTAGTCGGGTAAGGGGTCCAAACTTAGTGGACTACG


 


CCGGAGTTCTCCGCCTGGGGACAAAGGAAGAGATCAATCAGGCTAGCTGCCCGGACGCCCGTCG


 


ATAGGCAAAAGGAACAGTGAACCCCAAATATATCGACTACGCTCGTAGACGTTCAAGTGGCGTT


 


ATCTTTGGACGTGGGTTCAACTCCCGCCAGCTCCA
















TABLE 13





tmDNA Sequence for Bacillus brevis
















(SEQ ID NO:21)









GGGGGCGGAAAGGATTCGACGGGGATGGTAGAGCATGAGAAGCGAGCCGGGGGGTTGCGGACCT



 


CGTCACCAACGCAAACGCCATTAACTGGCAACAAACAACTTTCTCTCGCTGCTTAATAACCAGT


 


GAGGCTCTCCCACTGCATCGGCCCGTGTGCCGTGGATAGGGCTCAACTTTAACGGGCTACGCCG


 


GAGGCTTCCGCCTGGAGCCAAAGGAAGAAGACCAATCAGGCTAGGTGCCAGGTCAGCGCGTCAC


 


TCCGCGAATCTGTCACCGAAACTCTAAACGAGTGACTGCGCTCGGAGATGCTCATGTATCGCTG


 


TTTTCGGACGGGGGTTCGATTCCCGCCGCCTCACCCA
















TABLE 14





tmDNA Sequence for Bacillus thermoleovorans (50–60 degres)
















(SEQ ID NO:22)









GGGGGCGGAAAGGATTCGACGGGGGTAGGTCGAGCTTAAGCGGCGAGCCGAGGGGGACGTCCTC



 


GTAAAAACGTCACCTAAAGATAACTGGCAAACAAAACTACGCTTTAGCTGCCTAATTGCTGCAG


 


CTAGCTCCTCCCGCCATCGCCCGCGTGGCGTTCGAGGGGCTCATATGGAGCGGGCTACGCCCAA


 


ATCCGCCGCCTGAGGATGAGGGAAGAGACGAATCAGGCTAGCCGCCGGGAGGCCTGTCGGTAGG


 


CGGAACGGACGGCGAAGCGAAATATACCGACTACGCTCGTAGATGCTTAAGTGGCGATGCCTCT


 


GGACGTGGGTTCGATTCCCGCCGCCTCCCCACCA
















TABLE 15





tmDNA Sequence for Clostridium innocuum
















(SEQ ID NO:23)









GGGGGCGGAAAGGATTCGACGGGGATATGTCTGGTACAGACTGCAGTCGAGTGGTTACGTAATA



 


ACCAATTAAATTTAAACGGAAAAACTAAATTAGCTAACCTCTTTGGTGGAAACCAGAGAATGGC


 


TTTCGCTGCTTAATAACCGATATAGGTTCGCAGCCGCCTCTGCATGCTTCTTCCTTGACCATGT


 


GGATGTGCGCGTAAGACGCAAGGGATAAGGAATCTGGTTTGCCTGAGATCAGATTCACGAAAAT


 


TCTTCAGGCACATTCATCAGCGGATGTTCATGACCTGCTGATGTCTTAATCTTCATGGACTAAA


 


CTGTAGAGGTCTGTACGTGGGGCTGTTTCTGGACAGGAGTTCGATTCCCGCCGCCTCACCACCA
















TABLE 16





tmDNA Sequence for Clostridium lentocellum
















(SEQ ID NO: 24)









GGGGGCGGAAAGGATTCGACGGGGGTCACATCTACTGGGGCAGCCATCCGTAGAACGCCGGAGT



 


CTACGTTAAAAGCTGGCACTTAAAGTAAACGCTGAAGATAATTTAGCAATCGCTGCCTAATTAA


 


GGCGCAGTCCTCCTAGGTCTTCCGCAGCCTAGATCAGGGCTTCGACTCGCGGATCCTTCACCTG


 


GCAAAGCTTTGAGCCAACGTGAACACTATGAAGCTACTAAAATCTAGAGCCTGTCTTTGGGCGC


 


TAGATGGAGGGAATGTCAAAACAAAGAATATGATGGTAGAGACCACGCTATATGGGCTTTCGGA


 


CAGGGGTTCGATTCCCGCCGCCTTCACCA
















TABLE 17





tmDNA Sequence for Clostridium perfringens
















(SEQ ID NO: 25)









GGGGCTGATTCTGGATTCGACGGGGGTAAGATGGGTTTGATAAGCGAGTCGAGGGAAGCATGGT



 


GCCTCGATAATAAAGTATGCATTAAAGATAAACGCAGAAGATAATTTTGCATTAGCAGCTTAAT


 


TTAGCGCTGCTCATCCTTCCTCAATTGCCCACGGTTGAGAGTAAGGGTGTCATTTAAAAGTGGG


 


GAACCGAGCCTAGCAAAGCTTTGAGCTAGGAACGGAATTTATGAAGCTTACCAAAGAGGAAGTT


 


TGTCTGTGGACGTTCTCTGAGGGAATTTTAAAACACAAGACTACACTCGTAGAAAGTCTTACTG


 


GTCTGCTTTCGGACACGGGTTCAACTCCCGCCACTCCA
















TABLE 18





tmDNA Sequence for Clostridium stercorarium
















(SEQ ID NO: 26)









GGGGGCGGAAAGGATTCGACGGGGTTATTGAAGCAAGAGTAGCGGGTAGAGGATTCTCGTTGGC



 


CTCTTTAAAAAACGAGAGCTAAAAATAAACGCAAACAACGATAACTACGCTTTAGCTGCTGCGT


 


AAGTAACACGCAGCCCGTCGGCCCCGGGGTTCCTGCGCCTCGGGATACCGGCGTCATCAAGGCA


 


GGGAACCAGCCGGATCAGGCTTCAGGTCCGGTGGGATTTAATGAAGCTACCGACTTATAAAGCC


 


TGTCTCTGGGCGTTATAAGAAGGGAATGTCAAAACAGAGACTGCACCCGGAGAAGCTCTTGTGG


 


ATATGGTTCCGGACACGAGTTCGATTCCCGCCGCCTCCACCA
















TABLE 19





tmDNA Sequence for Enterococcus faecium (sp.)
















(SEQ ID NO: 27)









GGGGCTGATTATGGATTCGACAGGATNGTTGAGCTTGAATTGCGTTTCGTAGGTTACGGCTACG



 


TTAAAACGTTACAGTTAAATATAACTGCTAAAAACGAAAACAATTCTTTCGCTTTAGCTGCCTA


 


AAAACCAGCTAGCGAAGATCCTCCCGGCATCGCCCATGTGCTCGGGTCAGGGTCCTAATCGAAG


 


TGGGATACGCTAAATTTTTCCGTCTGTAAAATTTAGAGGAGCTTACCAGACTAGCAATACAAGA


 


ATGCCTGTCACTCGGCACGCTGTAAAGCGAACCTTTAAATGAGTGTCTATGAACGTAGAGATTT


 


AAGTGGGAATATGTTTTGGACGCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 20





tmDNA Sequence for Heliobacillus mobilis (photosyn/gram +)
















(SEQ ID NO: 28)









GGGGCTGATTCTGGATTCGACGGGGAACGTGTTTGCTTGGGATGCGAGCCGGGTTGCCGCCAGG



 


ACCGTAAAAAGGGCGGAAGGCTTTAATTGCCGAAGATAACTACGCTTTAGCTGCTTAATTGCAG


 


TCTAACCTCTTCTCCTCTGTGCTCTCGGTGAGGATGTAAGGGGTCATTTAAGAGAGCTGGCTTC


 


GACCAATTCTCGGAGGTCCAAGCGAGATTTATCGAGATAGCCTGACCAACGCTCTGTCTGCCGT


 


GCGGAAGGAAGGCGAAATCTAAAACGACAGACTACGCTCGTAGTGTCCTTTGTGGGCATTTCTT


 


CGGACGCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 21





tmDNA Sequence for Heliospirillum gestii
















(SEQ ID NO: 29)









GGGGCTGATTCTGGATTCGACGGGGAACGTGTTTGCTTAGGACGCGAGCCGGGTTGCCGCCAGG



 


ACCGTAAAAAGGGCGGAAGGCTTTAATTGCCGAAGATAACTACGCTTTAGCTGCTTAATTGCAG


 


TCTAACCTCTTCTCCTCTGTGCTCTCGGTGAGGATGTAAGGGGTCATTTAAGAGAGCTGGCTCG


 


AACCAATTCTCGGAGGTTCGGGTAAGACTTATCGAGATAGCCTGACCAACGCTCTGTCTGCCGT


 


GCGGAAGGATGGCGAAATCTAAAACGACAGAATACGCTCGTAGTGTCCTTTGTGGGCATTTCTT


 


CGGACGCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 22





tmDNA Sequence for Lactobacillus acidophilus
















(SEQ ID NO: 30)









GGGGCTGATTCTGGATTCGACAGGCGTAGACCCGCATTGACTGCGGTTCGTAGGTTACGTCTAC



 


GTAAAAACGTTACAGTTAAATATAACTGCAAATAACAAAAATTCTTACGCATTAGCTGCTTAAT


 


TTAGCGCATGCGTTGCTCTTTGTCGGTTTACTCGTGGCTGACACTGAGTATCAACTTAGCGAGT


 


TACGTTTAACTACCTCACCTGAATAGTTGAAAAGAGTCTTAGCAGGTTAGCTAGTCCATACTAG


 


CCCTGTTATATGGCGTTTTGGACTAGTGAAGTTCAAGTAATATAACTATGATCGTAGAGGTCAG


 


TGACGAGATGCGTTTGGACAGCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 23





tmDNA Sequence for Staphylococcus epidermidis
















(SEQ ID NO: 31)









GGGGCTGATTCTGCATTCGACAGGGGTCCCCGAGCTTATTAAGCGTGTGGAGGGTTGGCTCCGT



 


CATCAACACATTTCGGTTAAATATAACTGACAAATCAAACAATAATTTCGCAGTAGCTGCGTAA


 


TAGCCACTGCATCGCCTAACAGCATCTCCTACGTGCTGTTAACGCGATTCAACCCTAGTAGGAT


 


ATGCTAAACACTGCCGCTTGAAGTCTGTTTAGATGAAATATAATCAAGCTAGTATCATGTTGGT


 


TGTTTATTGCTTAGCATGATGCGAAAATTATCAATAAACTACACACGTAGAAAGATTTGTATCA


 


GGACCTCTGGACGCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 24





tmDNA Sequence for Streptococcus faecium
















(SEQ ID NO: 32)









GGGGCTGATTCTGGATTCGACAGGCACAGTTTGAGCTTGAATTGCGTTTCGTAGGTTACGTCTA



 


CGTTAAAACGTTACAGTTAAATATAACTGCTAAAAACGAAAACAACTCTTACGCTTTAGCTGCC


 


TAAAAACAGTTAGCGTAGATCCTCTCGGCATCGCCCATGTGCTCGAGTAAGGGTCTCAAATTTA


 


GTGGGATACGTGACAACTTTCCGTCTGTAAGTTGTTAAAGAGATCATCAGACTAGCGATACAGA


 


ATGCCTGTCACTCGGCAAGCTGTAAAGCGAAACCACAAATGAGTTGACTATGAACGTAGATTTT


 


TAAGTGGCGATGTGTTTGGACGCGGGTTCAACTCCCGCCGTTCCACCA
















TABLE 25





tmDNA Sequence for Thermoanaerobacterium


saccharolyticum (Bacillus/clostridium)
















(SEQ ID NO: 33)









GGGGTAGTAGAGGTAAAAGTAGCGAGCCGAGGTTCCATCTGCTCGTAAAACGGTGGACTTAAAT



 


ATAAACGCAAACGATAATTTAGCTTACGCTGCTTAATTACAAGCAGCCGTTCAACCTTTGATTC


 


CCACATCAAAGGATTGGGCGTCGATTTAGTGGGGAACTGATTTATCAAAGCTTTGAGATAAATC


 


GGATTTTATGAAGCTACCAAAGCAGTTATCCTGTCACTGGGAGAACTGCAGAGGGAATGTCAAA


 


ACAGTGACTGCGCTCGGAGAAGCTTTTACTGTGACACCTTCGGACCGGGGTTCAACTCCCGCCA


 


GCTCCACCA
















TABLE 26





tmDNA Sequence for Mycoplasma fermentans
















(SEQ ID NO: 34)









GGGGCTGATTCTGGATTCGACATGCATTGGGTGATACTAATATCAGTAGTTTGGCAGACTATAA



 


TGCATCTAGGCTTTATAATCGCAGAAGATAAAAAAGCAGAAGAAGTTAATATTTCTTCACTTAT


 


GATTGCACAAAAAATGCAATCACAATCAAACCTTGCTTTCGCTTAGTTAAAAGTGACAAGTGGT


 


TTTAAAGTTGACATTTTCCTATATATTTTAAAATCGGCTTTTAAGGAGAACAGGAGTCTGAAAG


 


GGTTCCAAAAATCTATATTGTTTGCATTTCGGTAGTATAGATTAATTAGAAATGATAAACTGTA


 


AAAAGTATTGGTATTGACTTGGTGTGTGGACTCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 27





tmDNA Sequence for Mycoplasma hyorhinis
















(SEQ ID NO: 35)









GGGGCTGATTCTGGATTCGACATACATAAAAGGATATAAATTGCAGTGGTCTTGTAAACCATAA



 


GACAATTTCTTTACTAAGCGGAAAAGAAAACAAAAAAGAAGATTATTCATTATTAATGAATGCT


 


TCAACTCAATCAAATCTAGCTTTTGCATTTTAAAAAACTAGTAGACCAATTTGCTTCTCACGAA


 


TTGTAATCTTTATATTAGAGAATAGTTAAAAATCTGATCACTTTTTAATGAATTTATAGATCAC


 


AGGCTTTTTTAATCTTTTTGTTATTTTAGATAAAGAGTCTTCTTAAAAATAACTAAACTGTAGG


 


AATTTATATTTAATTATGCGTGGACCCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 28





tmDNA Sequence for Mycoplasma pirum
















(SEQ ID NO: 36)









GGGGAGTCATGGTTTTGACATGAATGATGGACCCATAGAGGCAGTGGGGTATGCCCCTTATAGC



 


TCAAGGTTTAAATTAACCGACAAAACTGACGAAAACGTTGCCGTTGATACAAATTTATTAATCA


 


ACCAACAAGCTCAATTTAACTACGCATTTGCATAGTATAAAAAAATAAATTGTGCTACTCATTG


 


TAATTAGGTTACTAAATTACTTTGTTTTATATAGTCCTGTAACTAGTTCTAGTGATGTCTATAA


 


ACTAGAATGAGATTTATAGACTTATTTGTTGGCGGTTGTGCCATAGCCTAAATCAACAAAGACA


 


ATTTATTTATGGTACTAAACTGTAGATTCTATGATGAAATTATTTGTGGAAACGGGTTCGATTC


 


CCGCCATCTCCACCA
















TABLE 29





tmDNA Sequence for Mycoplasma salivarium
















(SEQ ID NO: 37)









GGGGCTGATTCTGGATTCGACAGGCATTCGATTCATTATGTTGCAGTGGTTTGCAAACCATAAG



 


GCACTAGGCTTTTTTAAACGCAAAAGACCAAAAAACAGAAGATCAAGCAGTTGATCTAGCATTT


 


ATGAATAATTCACAAATGCAATCAAATCTAGTTTTCGCTTAGTAAAATTAGTCAATTTATTATG


 


GTGCTCAACATAATAAATGGTAGTATGAGCTTAATATCATATGATTTTAGTTAATATGATAGGA


 


TTTGTAACTAAACTATGTTATAGAAATTTGTAAATTATATATATGACATAGGAAATTTAATTTA


 


CTAAACTGTAGATGCATAATGTTGAAGATGTGTGGACCGGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 30





tmDNA Sequence for Herpetosiphon aurantiacus
















(SEQ ID NO: 38)









GGGGGCGGAAAGGATTCGACGGGGAGGGCCAATCGTAAGTGGCAAGCCGAGACGCTGAGCCTCG



 


TTAAATCGGCAACGCCATTAACTGGCAAAAACACTTTCCGCGCTCCTGTAGCGCTTGCTGCCTA


 


ATTAAGGCAACACGTCTCTACTAGCCTCAGCCCGATGGGCTTGTAGCGGCGACACTTAGTCGGG


 


TCGCTCCCCTAGTTATGTCTGTGGGCTAGGGGCTAAGATTAACAGGCTGGTCGTGGCCCGCTTT


 


GTCTATCGGGTGGTGCACCGATAAGATTTAATCAATAGACTACGCTTGTAGATGCTTGCGGTTT


 


AACTTTTTGGACGCGGGTTCGATTCCCGCCGCCTCACCACCA
















TABLE 31





tmDNA Sequence for Thermomicrobium roseum


(352 nts, temp. 70 degrees, green non sulfur)















GGGGCTGATTCTGGATTCGACAGGGCCGTAGGTGCGAGGATTGCAGGTCGAGGTCGCCCACGAA


 


CTCGTAAAAAGGGGCAGCCAAGTAACTGGCGAGCGCGAACTCGCTCTGGCTGCGTAATTCACGC


 


AGCCACGTCTGCCCGGACCCTTCCCTGGTGGGTTCGGAGCGGGCGCCGCAAGACCGGGGTGCCC


 


CTGGCCCAAGCGCCGGTGCGGGCCAGGTCAAGCGTGATCCGGCTCGGCTGACCGGGATCCTGTC


 


GGTGGGAGCCTGGCAGCGACAGTAGAACACCGACTAAGCCTGTAGCATATCCTCGGCTGAACGC


 


TCTGGACGCGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:39)
















TABLE 32





tmDNA Sequence for Chlorobium limicola















GGGGCTGATTCTGGATTCGACAGGATACGTGTGAGATGTCGTTGCACTCCGAGTTTCAGCATGG


 


ACGGACTCGTTAAACAAGTCTATGTACCATTAGATGCAGACGATTATTCGTATGCAATGGCTGC


 


CTGATTAGCACAAGTTAACTCAGACGCCATCGTCCTGCGGTGAATGCGCTTACTCTGAAGCCGC


 


CGGATGGCATAACCCGCGCTTGAGCCTACGGGTTCGCGCAAGTAAGCTCCGTACATTCATGCCC


 


GAGGGGCTGTGCGGGTAATTTCTCGGGATAAGGGGACGAACGCTGCTGGCGGTGTAATCGGCCC


 


ACGAAAACCCAATCACCAGAGATGAGTGTGGTGACTGCATCGAGCAGTGTTTTGGACGCGGGTT


 


CAACTCCCGCCAGCTCCACCA (SEQ ID NO:40)
















TABLE 33





tmDNA Sequence for Pirellula staleyi (planctomyces)















GGGGCTGATTCTGGATTCGACCGGATAGCCTGAAGCGAATACGGCGTGCCGTGGTTGATCAGAT


 


GGCCACGTAAAAAGCTGATCACAAACTTAACTGCCGAGAGCAATCTCGCACTTGCTGCCTAACT


 


AAACGGTAGCTTCCGACTGAGGGCTTTAGCCGGAGAGGCCCAAAAGTTGGTCACCAAATCCGGA


 


CCGCCTCGTGCCATGATCGAAACGCACGAGGTCAAAAAAGTTTCGATCTAGTGCAGGGTGTAGC


 


CAGCAGCTAGGCGACAAACTGTGCAAAAATCAAATTTTCTGCTACGCACGTAGATGTGTTCGTG


 


AAAATGTCTCGGGACGGGGGTTCAACTCCCGCCACTCCACCA (SEQ ID NO:41)
















TABLE 34





tmDNA Sequence for Planctomyces limnophilus















GGGGCTGATTCTGGATTCGACAACCTCTCAAGAGGAGCGTGGCCACTATGGGACTCGATTATGT


 


TGAATTCGTCATGGATCTTGAAGAGACCTTCGACATCAAACTGGATGACAAACATTTTTCAGCA


 


GTCAAAACACCACGCGATTTGGCAATCATTATTCGGGATCAATTAGCTGCTGAAGGCAGAATCT


 


GGGATGAATCGAATGCTTTTCGCAAAATCTCGAATTTGAATTGGACGATGTTGCCCGAGTTCCG


 


GATGTGGACTCAAATCAAAAGCTCTCTACCAGTTTCTTTTCACCGACTGCGTCCCAGCACCCGT


 


CTCGTTCAACTCCCGCCANTCCACCA (SEQ ID NO:42)
















TABLE 35





tmDNA Sequence for Planctomyces maris















GGGGCTGATTCTGGATTCGACTGGTTCACCGTATGTTAAGGTGGCGGTGCCGTGGTTGATCAGT


 


TGGCCACGTAAAAAGCTGATCACAATCTAATTGCAAACAAGCAATTTTCAATGGCTGCTTAATA


 


AAAGCAACCCCGGCTTAGGAATCTCTGTCTGAGGAGTCCGACAGCTGGTCACAAAATCAGACTG


 


GTATCAGATCAATGTCCGCTCCGTCTGATACGAGATTCGTGGTGGACTGGTTTCCAACAGGCTC


 


TGTTTATCGTGCCCGAAGAAACGAGACTCAAACGATAAAATATGCACCGTAGAGGCTTTAGCTG


 


AGGGTTCACAGGACGCGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:43)
















TABLE 36





tmDNA Sequence for Alcaligenes eutrophus















GGGGTTGATTCTGGATTCGACGTGGGTTACAAAGCAGTGGAGGGCATACCGAGGACCCGTCACC


 


TCGTTAATCAATGGGAATGCAATAACTGCTAACGACGAACGTTACGCACTGGCCGCTTAATTGC


 


GGCCGTCCTCGCACTGGCTCGCTGACGGGCTAGGGTCGCAAGACCACGCGAGGTCATTTACGTC


 


AGATAAGCTCCGGAAGGGTCACGAAGCCGGGGACGAAAACCTAGTGACTCGCCGTCGTAGAGCG


 


TGTTCGTCCGCGATGCGCCGGTTAAATCAAATGACAGAACTAAGTATGTAGAACTCTCTGTGGA


 


GGGCTTACGGACGCGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:44)
















TABLE 37





tmDNA Sequence for Alcaligenes faecalis (beta proteobacteria)















GGGGGCGGAAAGGATTCGACGGGGGTCAAGAAGCAGCACAGGGCGTGTCGAGCACCAGTACGCT


 


CGTAAATCCACTGGAAAACTATAAACGCCAACGACGAGCGTTTCGCTCTAGCCGCTTAAGGCTG


 


GGCCACTGCACTAATTTGTCTTTGGGTTAGGTAGGGCAACCTACAGCAGTGTTATTTACAAAGA


 


ATCGAATCGGTCTGCGCCACGAAGTCCGGTTCTAAAACTTAGTGGATCGCCAAGGAAAGGCCTG


 


TCAATTGGCATAGTCCAAGGTTAAAACTTAAAATTAATTGACTACACATGTAGAACTGTCTGTG


 


GACGGCTTGCGGACGGGGGTTCGATTCCCGCCGCCTCCACCA (SEQ ID NO:45)
















TABLE 38





tmDNA Sequence for Chromobacterium violaceum (beta-purple)















GGGGCTGATTCTGGATTCGACGGGGGTTGCGAAGCAGATGAGGGCATACCGGGATTTCAGTCAC


 


CCCGTAAAACGCTGAATTTATATAGTCGCAAACGACGAAACTTACGCTCTGGCAGCCTAACGGC


 


CGGCCAGACACTACAACGGTTCGCAGATGGGCCGGGGGCGTCAAAACCCTGTAGTGTCACTCTA


 


CATCTGCTAGTGCTGTTCCGGGTTACTTGGTTCAGTGCGAAATAATAGGTAACTCGCCAAAGTC


 


CAGCCTGTCCGTCGGCGTGGCAGAGGTTAAATCCAAATGACACGACTAAGTATGTAGAACTCAC


 


TGTAGAGGACTTTCGGACGCGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:46)
















TABLE 39





tmDNA Sequence for Hydrogenophaga palleroni (beta-purple)















GGGGCTGATTCTGGATTCGACGTGGGTTCGGACGCGCAGCAGGGCATGTCGAGGTTCTGTCACC


 


TCGTAAATCAGCAGAAAAAAACCAACTGCAAACGACGAACGTTTCGCACTCGCCGCTTAAACAC


 


CGGTGAGCCTTGCAACAGCAGGCCGATGGGCTGGGCAAGGGGGTCGCAAGACCTCCCGGCTGCA


 


AGGTAATTTACATCGGCTGGTTCTGCGTCGGGCACCTTGGCGCAGGATGAGATTCAAGGATGCT


 


GGCTTCCCGTTTAGCGTGCCACTGCGCGACTCGGGCGGCGAGACCCAAATCAGACGGCTACACA


 


TGTAGAACTGCTCGAAAAAGGCTTGCGGACGGGGGTTCAACTCCCGCCAGCTCCACCA


 


(SEQ ID NO:47)
















TABLE 40





tmDNA Sequence for Methylobacillus glycogenes (beta-purple)















GGGGGCGGAAAGGATTCGACGGGGGTTGCAAAGCAGCGCAGGGCATACCGAGGCCTAGTCACCT


 


CGTAAATAAACTAGAACAAGTATAGTCGCAAACGACGAAACTTACGCTCTAGCCGCTTAATCCC


 


GGCTGGACGCTGCACCGAAGGGCCTCTCGGTCGGGTGGGGTAACCCACAGCAGCGTCATTAAGA


 


GAGGATCGTGCGATATTGGGTTACTTAATATCGTATTAAATCCAAGGTAACTCGCCTGCTGTTT


 


GCTTGCTCGTTGGTGAGCATCAGGTTAAATCAAACAACACAGCTAAGTATGTAGAACTGTCTGT


 


GGAGGGCTTGCGGACGGGGGTTCGATTCCCGCCGCCTCACCACCA (SEQ ID NO:48)
















TABLE 41





tmDNA Sequence for Nitrosomonas cryotolerans (beta-purple)















GGGGCTGATTCTGGATTCGACGTGGGTTGCAAAGCAGCGCAGGGCATACCGAGGACCAGAATAC


 


CTCGTAAATACATCTGGAAAAAAATAGTCGCAAACGACGAAAACTACGCTTTAGCCGCTTAATA


 


CGGCTAGCCTCTGCACCGATGGGCCTTAACGTCGGGTCTGGCAACAGACAGCAGAGTCATTAGC


 


AAGGATCGCGTTCTGTAGGGTCACTTTACAGAACGTTAAACAATAGGTGACTCGCCTGCCATCA


 


GCCCGCCAGCTGGCGGTTGTCAGGTTAAATTAAAGAGCATGGCTAAGTATGTAGAACTGTCTGT


 


AGAGGACTTGCGGACGCGGGTTCAACTCCCGCCAGTCCACCA (SEQ ID NO:49)
















TABLE 42





tmDNA Sequence for Pseudomonas testosteroni















GGGGCTGATTCTGGATTCGACGTGGGTTCGGGACCGGTGCGGTGCATGTCGAGCTTGAGTGACG


 


CTCGTAAATCTCCATTCAAAAAACTAACTGCAAACGACGAACGTTTCGCACTCGCCGCTTAATC


 


CGGTGAGCCTTGCAACAGCACGCTAGTGGGCTGGGCAAGGGGGTAGCAATACCTCCCGGCTGCA


 


AGGGAATTTTCATTAGCTGGCTGGATACCGGGCTTCTTGGTATTTGGCGAGATTTTAGGAAGCT


 


GGCTACCCAAGCAGCGTGTGCCTGCGGGGTTTGGGTGGCGAGATTTAAAACAGAGCACTAAACA


 


TGTAGATCTGTCCGGCGAAGGCTTACGGACGCGGGTTCAACTCCCGCCAGCTCCACCA


 


(SEQ ID NO:50)
















TABLE 43





tmDNA Sequence for Ralstonia pickettii (Burkholderia)















GGGGGCGGAAAGGATTCGACGGGGGTTGCGAAGCAGCGGAGGGCATACCGAGGACCCGTCACCT


 


CGTTAATCAATGGGAATGCAATAACTGCTAACGACGAACGTTACGCACTGGCAGCCTAAGGGCC


 


GCCGTCCTCGCACTGGCTCGCTGACGGGCTAGGGTCGCAAGACCAGCGAGGTCATTTACGTCAG


 


ATAAGCTTTAGGTGAGTCACGGGCCTAGAGACGAAAACTTAGTGAATCGCCGTCGTAGAGCGTG


 


TTCGTCCGCGATGCGGCGGTTAAATCAAATGACAGAACTAAGTATGTAGAACTCTCTGTGGAGG


 


GCTTGCGGACGCGGGTTCGATTCCCGCCGCCTCACCACCA (SEQ ID NO:51)
















TABLE 44





tmDNA Sequence for Variovax paradoxus (pseudomonas sp.)















GGGGCTGATTCTGGATTCGACGTGGGTTCGGAGTCGCAGCGGGGCATGTCGAGCTGAATGCGCT


 


CGTAAAACAGATTCAAACAAACTAACTGCAAACGACGAACGTTTCGCACTCGCTGCTTAATTGC


 


CAGTGAGCCTTGCAACAGTTGGCCGATGGGCTGGGCAAGGGGGTCTGGAGCAATCCTGACCTCC


 


CGGCTGCAAGGATAACTACATGGGCTGGCTCCGATCCGGGTACCTTGGGTCGGGGCGAGAAAAT


 


AGGGTACTGGCGTCCGGTTTAGCGTGTGACTGCGCGACTCCGGAAGCGAGACTCAAAACAGATC


 


ACTAAACATGTAGAACTGCGCGATGAAGGCTTGCGGACGGGGGTTCAACTCCCGCCAGCTCCACCA


 


(SEQ ID NO:52)
















TABLE 45





tmDNA Sequence for Bdellovibrio bacteriovorus


(delta proteobacterie)















GGGGGCGGAAAGGATTCGACGGGGGTGCTGAAGCATAAGGAGCATACCGGGGCGGATGAGGACC


 


TCGTTAAAAACGTCCACTTTGTAATTGGCAACGATTACGCACTTGCAGCTTAATTAAGCAGCAC


 


GATCAACCTTGTGGTGGTTCCGCACTTGGATTGATCGTCATTTAGGGACCTCGGCGTGTTGGGT


 


TTTCTCCAGCAGACATGCTTAAATTTACTGGGGGAGAGGTCTTAGGGATTTTGTCTGTGGAAGC


 


CCGAGGACCAATCTAAAACACTGACTAAGTATGTAGCGCCTTATCGTGGATCATTTGCGGACGG


 


GGGTTCGATTCCCGCCGCCTCCACCA (SEQ ID NO:53)
















TABLE 46





tmDNA Sequence for Myxococcus xanthus


(delta proteobacterie)
















(SEQ ID NO:54)









GGGGGCGGAAAGGATTCGACGGGGGCATTGAAGTTCGAGACGCGTGCCGAGCTTGTCAGGTAGC



TCGTAAATTCAACCCGGCAAAGACACAAAAGCCAACGACAACGTTGAGCTCGCGCTGGCTGCCT


AAAAACAGCCCATAGTGCGCGGTCCCCCCGCCCTCGGCCTGTGGGGTTGGGACAGACCGTCATA


ATGCAGGCTGGCTGCCGAGGGTGCCTGGACCCGAGGTGGCGAGATCTTCCCAGGACCGGCTCTG


AGTATCCCGTCCGTGGGAGCCTCAGGGACGTAGCAAATCGCGGACTACGCACGTAGGGTCGAAG


AGCGGACGGCTTTCGGACGCGGGTTCGATTCCCGCCGCCTCCACCA
















TABLE 47





tmDNA Sequence for Sulfurospirillum Deleyianum
















(SEQ ID NO:55)









GGGGCTGATTCTGGATTCGACAGGAGTAGTTTTAGCTTATGGCTGCATGTCGGGAGTGAGGGTC



TTCCGTTACACAACCTTCAAACAATAACTGCTAACAACAGTAACTATCGTCCTGCTTACGCGCT


AGCTGCGTAAGTTTAACAAATAATGGACTGCTCTCCCCTTTGATGCTATCTTAGGAGGTCTTGG


AGAGTATCATAGATTTGATAGCTATATTACATGAACGCCTTTACATGTAATGAAGTTAAAGGCT


CGTTTTGCGTAGTTTTCTGATTGTTGTACGAAGCAAAATTAAACACTATCAACAATATCTAAGC


ATGTAGACGTCATAGGTGGCTATTTTTGGACTGCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 48





tmDNA Sequence for Chromatium vinosum
















(SEQ ID NO:56)









GGGGCTGATTCTGGATTCGACGTGGGTCGCGAAACCTAAGGTGCATGCCGAGGTGCGGTTGACC



TCGTAAAACCCTCCGCAAACTTATAGTTGCCAACGACGACAACTACGCTCTCGCTGCTTAATCC


CAGCGGGCCTCTGACCGTCACTTGCCTGTGGGCGGCGGATTCCAGGGGTAACCTCACACAGGAT


CGTGGTGACGGGAGTCCGGACCTGATCCACTAAAACCTAACGGAATCGCCGACTGATCGCCCTG


CCCTTCGGGCGGCAGAAGGCTAAAAACAATAGAGTGGGCTAAGCATGTAGGACCGAGGGCAGAG


GGCTTGCGGACGCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 49





tmDNA Sequence for Pseudomonas fluorescens


(gamma proteobacteria)
















(SEQ ID NO:57)









GGGGCTGATTCTGGATTCGACGCCGGTTGCGAACCTTTAGGTGCATGCCGAGTTGGTAACAGAA



CTCGTAAATCCACTGTTGCAACTTTCTATAGTTGCCAATGACGAAACCTACGGGGAATACGCTC


TCGCTGCGTAAGCAGCCTTAGCCCTTCCCTCCTGGTACCTTCGGGTCCAGCAATCATCAGGGGA


TGTCTGTAAACCCAAAGTGATTGTCATATAGAACAGAATCGCCGTGCAGTACGTTGTGGACGAA


GCGGCTAAAACTTACACAACTCGCCCAAAGCACCCTGCCCGTCGGGTCGCTGAGGGTTAACTTA


ATAGACACGGCTACGCATGTAGTACCGACAGCAGAGTACTGGCGGACGCGGGTTCAACTCCCGC


CAGCTCCACCA
















TABLE 50





tmDNA Sequence for Borrelia afzeli
















(SEQ ID NO:58)









GGGGCTGATTCTGGATTCGACTGAAAATGCTAATATTGTAAGTTGCAAGCAGAGGGAATCTCTT



AAAACTTCTAAAATAAATGCAAAAAATAATAACTTTACAAGTTCAAACCTTGTAATGGCTGCTT


AAGTTAGCAGAGAGTTTTGTTGAATTTGGCTTTGAGATTCACTTATACTCTTTTAGACATCGAA


GCTTGCTTAAAAATGTTTTCAAGTTGATTTTTAGGGACTTTTATACTTGAGAGCAATTTGGCGG


TTTGCTAGTATTTCCAAACCATATTGCTTAGTAAAATACTAGATAAGCTTGTAGAAGCTTATAG


TATTGTTTTTAGGACGCGGGTTCAACTCCCGCCAGTCCACCA
















TABLE 51





tmDNA Sequence for Borrelia crociduarae
















(SEQ ID NO:59)









GGGGCTGATTCTGGATTCGACTAAGAACTTTAGTAGCATAAATGGCAAGCAGAGTGAATCTCTT



AAAACTTCTTTAATAAATGCAAAAAATAATAACTTTACAAGTTCAGATCTTGTAATGGCTGCTT


AATTTAGCAGAGAGTTTTGTTGGATTTTGCTTTGAGGTTCAACTTATACTCTTTAAGACATCAA


AGTATGCCTAAAAATGTTTCAAGTTGATTTTTAGGGACCTTTAAACTTGAGAGTAATTTGGTGG


TTTGCTTGTTTTCCAAGCCTTATTGCTTTTTCTAAAAATTAGCTAAGCTTGTAGATATTTATGA


TATTATTTTTAGGACGCGGGTTCAACTCCCGCCAGTTCCACCA
















TABLE 52





tmDNA Sequence for Borrelia hermsii
















(SEQ ID NO:60)









GGGGCTGATTCTGGATTCGACTAAAAACTTTAGTAGCATAAATTGCAAGCAGAGGGAATCTCTT



AAAACTTCTTTAATAAATGCAAGAAATAATAACTTTACAAGTTCAAATCTTGTAATGGCTGCTT


AAATTAGCAGAGAGTTCTGCTGGATTTTGCTTTGAGGTTCAGCTTATACTCTTTTAAGACATCA


AAGCTTGCTTAAAAATATTTCAAGTTGATTTTTAGGGACTTTTAAATTTGAGAGTAATTTGGCG


GTTTGCTAGTTTTTCCAAACCTTATTACTTAAAGAAAACACTAGCTAAGCTTGTAGATATTTAT


GATATTATTTTTAGGACGCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 53





tmDNA Sequence for Borrelia garinii
















(SEQ ID NO:61)









GGGGCTGATTCTGGATTCGACTGAAAATGCGAATATTGTAAGTTGCAGGCAGAGGGAATCTCTT



AAAACTTCTAAAATAAATGCAAAAAATAATAACTTTACAAGCTCAAACCTTGTAATGGCTGCTT


AAGTTAGCAGGGAGTTTCGTTGAATTTGGCTTTGAGGTTCACTTATACTCTTTTCGATATCGAA


GCTTGCTTAAAAATGTTTTCAAGTTAATTTTTAGGGACTTTTGTACTTGAGAGCAATTTGGCGG


TTTGCTAGTATTTCCAAACCATATTGCTTAAGTAAAATGCTAGATAAGCTTGTAGAAGCTTATA


ATATTGTTTTTAGGACGCGGGTTCAACTCCCGCCAGTCCACCA
















TABLE 54





tmDNA Sequence for Thermodesulfobacterium commune (70 degrees)
















(SEQ ID NO:62)









GGGGGCGGAAAGGATTCGACGGGGATAGGTAGGATTAAACAGCAGGCCGTGGTCGCACCCAACC



ACGTTAAATAGGGTGCAAAAACACAACTGCCAACGAATACGCCTACGCTTTGGCAGCCTAAGCG


TGCTGCCACGCACCTTTAGACCTTGCCTGTGGGTCTAAAGGTGTGTGACCTAACAGGCTTTGGG


AGGCTTAATCGGTGGGGTTAAGCCTCCCGAGATTACATCCCACCTGGTAGGGTTGCTTGGTGCC


TGTGACAAGCACCCTACGAGATTTTCCCACAGGCTAAGCCTGTAGCGGTTTAATCTGAACTATC


TCCGGACGCGGGTTCGATTCCCGCCGCCTCCCCACCA
















TABLE 55





tmDNA Sequence for Thermotoga neapolitana (Thermotogales)
















(SEQ ID NO:63)









GGGGGCGGAAAGGATTCGACGGGGATGGAGTCCCCTGGGAAGCGAGCCGAGGTCCCCACCTCCT



CGTAAAAAAGGTGGGAACACGAATAAGTGCCAACGAACCTGTTGCTGTTGCCGCCTAATAGATA


GGCGGCCGTCCTCTCCGGAGTTGGCTGGGCTCCGGAAGAGGGCGTGAGGGATCCAGCCTACCGA


TCTGGGCTCCGCCTTCCGGCCCGGATCGGGAAGGTTCAGGAAGGCTGTGGGAAGCGACACCCTG


CCCGTGGGGGGTCCTTCCCGAGACACGAAACACGGGCTGCGCTCGGAGAAGCCCAGGGGCCTCC


ATCTTCNGACGCGGGTTCGATTCCCGCCACCTCCACCA
















TABLE 56





tmDNA Sequence for Deinococcus proteolyticus
















(SEQ ID NO:64)









GGGGGCGGAAAGGATTCGACGGGGGAACGGAAAGCGCTGCTGCGTGCCGAGGAGCCGTTGGCCT



CGTAAACAAACGGCAAAGCCATTAACTGGCGAAAATAACTACGCTCTCGCTGCTTAAGTGAGAC


AGTGACCACGTAGCCCCGCCTTTGGCGACGTGTGAACTGAGACAAAAGAAGGCTAGCTTAGGTG


AGGTTCCATAGCCAAAAGTGAAACCAAATGGAAATAAGGCGGACGGCAGCCTGTTTGCTGGCAG


CCCAGGCCCGACAATTTAAGAGCAGACTACGCACGTAGATGCACGCTGGATGGACCTTTGGACG


CGGGTTCGATTCCCGCCAGCTCCACCA
















TABLE 57





tmDNA Sequence for Prosthecobacter fusiformis (verrucomicrobia)
















(SEQ ID NO:65)









GGGGCTGATTCTGGATTCGACGGGGAGTACAAGGATCAAAAGCTGCAAGCCGAGGTGCCGTTAC



CTCGTAAAACAACGGCAAAAAAGAAGTGCCAACACAAATTTAGCATTAGCTGCTTAATTTAGCA


GCTACGCTCTTCTAACCCGGGCTGGCAGGGTTAGAAGGGTGTCATAATGAGCCAGCTGCCCCTT


CCGACTCCCCTAAGGAAGGGAAAGATGTAGGGGATAGGTGCTTACAGAATCCTGCGGGAGGGAG


TCTGTAAGTGCCGAAAAGTTAAAACTCCCGCTAAGCTTGTAGAGGCTTTTGATTCTTGCTCTCT


GGACGCGGGTTCAACTCCCGCCAGCTCCACCA
















TABLE 58





tmDNA Sequence for Verrucomicrobium spinosum (verrucomicrobium)
















(SEQ ID NO:66)









GGGNNNNATTTGGAATTCGCCGAATGCTAGAAGTGGAGGCTGCATGCCGCGGATGATTCGTTGG



CCGCTTTACCAATTCGGATCAAACAACTAAATGCGGACTCTAACGAGCTTGCCCTCGCCGCTTA


ATTGACGGTGACGTTCCTCCAGTGAAGTCTGTGAATTGGAGGAGCGACTACTTACAGGCTGGCC


AAAAGAGCGGGCGACCGGCCCCAAGGCGAGATCTACAGGCCGCTGGATGGACGGCATCCTGGCA


GTAGGAGGCTGGACATCGAGATCAAATNATTGCCTGAGCATGGAGACGCTTTCATAAAGGNGTT


CGGACAGGG









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 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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## Claims

1. An isolated nucleic acid sequence selected from the group consisting of the tmDNA sequence for Staphylococcus epidermidis set forth in SEQ ID NO: 31, the complement of said tmDNA sequence, and the tmRNA sequence set forth in SEQ ID NO: 85.

2. A method for diagnosing the presence of Staphylococcus epidermidis comprising determining the presence of a bacterial nucleic acid sequence selected from the group consisting of the tmDNA sequence for Staphylococcus epidermidis set forth in SEQ ID NO: 31, the complement of said tmDNA sequence, and the tmRNA sequence set forth in SEQ ID NO: 85.

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 for Staphylococcus epidermidis set forth in SEQ ID NO: 31.

5. The method of claim 2, wherein the bacterial nucleic acid sequence is the tmDNA sequence for Staphylococcus epidermidis set forth in SEQ ID NO: 31.

6. The method of claim 3, wherein the bacterial nucleic acid sequence is the tmDNA sequence for Staphylococcus epidermidis set forth in SEQ ID NO: 31.