# 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 divisional application of U.S. patent application Ser. No. 09/958,206 filed 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 terminator less 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 antibacterial 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 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 Sulfer:
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.
Thermodesulfo bacterium: 
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)
















GGGGGCGGAAAGGATTCGACGGGGTTGACTGCGGCAAAGAGGCATGCCGG



 


GGGGTGGGCACCCGTAATCGCTCGCAAAACAATACTTGCCAACAACAATC


 


TGGCACTCGCAGCTTAATTAAATAAGTTGCCGTCCTCTGAGGCTTCGCCT


 


GTGGGCCGAGGCAGGACGTCATACAGCAGGCTGGTTCCTTCGGCTGGGTC


 


TGGGCCGCGGGGATGAGATCCACGGACTAGCATTCTGCGTATCTTGTCGC


 


TTCTAAGCGCAGAGTGCGAAACCTAAAGGAATGCGACTGAGCATGGAGTC


 


TCTTTTCTGACACCAATTTCGGACGCGGGTTCGATTCCCGCCGCCTCCAC


 


CA (SEQ ID NO:9)

















TABLE 2





tmDNA Sequence for Coprothermobacter proteolyticus



(60 degrees)
















GGGGGCGGAAAGGATTCGACGGGGAGTCGGAGCCTTGAGCTGCAGGCAGG



 


GTTGGCTGCCACACCTTAAAAAGGGTAGCAAGGCAAAAATAAATGCCGAA


 


CCAGAATTTGCACTAGCTGCTTAATGTAAGCAGCCGCTCTCCAAACTGAG


 


GCTGCATAAGTTTGGAAGAGCGTCAACCCATGCAGCGGCTCTTAAGCAGT


 


GGCACCAGCTGTTTAAGGGTGAAAAGAGTGGTGCTGGGCAGTGCGGTTGG


 


GCTTCCTGGGCTGCACTGTCGAGACTTCACAGGAGGGCTAAGCCTGTAGA


 


CGCGAAAGGTGGCGGCTCGTCGGACGCGGGTTCGATTCCCGCCGCCTCCA


 


CCA (SEQ ID NO:10)

















TABLE 3





tmDNA Sequence for Bacteroides thetaiotaomicron



(bacteroides/flavobacterium)
















GGGGCTGATTCTGGATTCGACAGCGGGCAGAAATGGTAGGTAAGCATGCA



 


GTGGGTCGGTAATTTCCACTTAAATCTCAGTTATCAAAACTTTATCTGGC


 


GAAACTAATTACGCTCTTGCTGCTTAATCGAATCACAGTAGATTAGCTTA


 


ATCCAGGCACTAGGTGCCAGGACGAGACATCACTCGGAAGCTGTTGCTCC


 


GAAGCATTCCGGTTCAGTGGTGCAGTAACATCGGGGATAGTCAGAAGCGG


 


CCTCGCGTTTTTGATGAAACTTTAGAGGATAAGGCAGGAATTGATGGCTT


 


TGGTTCTGCTCCTGCACGAAAATTTAGGCAAAGATAAGCATGTAGAAAGC


 


TTATGATTTCCTCGTTTGGACGAGGGTTCAACTCCCGCCAGCTCCACCA


 


(SEQ ID NO:11)

















TABLE 4





tmDNA Sequence for Dictyoglomus thermophilum



(70 degrees)
















GGGGCTGATTCTGGATTCGACAGGGAGTACAAGGATCAAAAGCTGCAAGC



 


CGAGGTGCCGTTACCTCGTAAAACAACGGCAAAAAAGAAGTGCCAACACA


 


AATTTAGCATTAGCTGCTTAATTTAGCAGCTACGCTCTTCTAACCCGGGC


 


TGGCAGGGTTAGAAGGGTGTCATAATGAGCCAGCTGCCCCTTCCGACTCC


 


CCTAAGGAAGGGAAAGATGTAGGGGATAGGTGCTTACAGAATCCTGCGGG


 


AGGGAGTCTGTAAGTGCCGAAAAGTTAAAACTCCCGCTAAGCTTGTAGAG


 


GCTTTTGATTCTTGCTCTCTGGACGCGGGTTCAACTCCCGCCAGCTCCAC


 


CA (SEQ ID NO:12)

















TABLE 5





tmDNA Sequence for Environmental Sample from



Rumenal Fluid
















ACGCCCTTGTCTCAGACGAGGGCACTCGTTAAAAAGTCTGAAAAGAATAA



 


CTGCAGAACCTGTAGCTATGGCTGCTTAATTTAAGGGCAACCCTTGGATC


 


CGCCTCCATCCCGAAGGGGTGGCATCCGAGTCGCAAATCGGGATAGGATG


 


GATCTTGGCAACGAGGAGTACATCCGAAATTTGTCGCTGCTGGCTGAAGC


 


ATCGCCGTTCCTCTTTGGGCGTGGCAAGGCAAGATTAAATTCAGAGGATA


 


AGCGTGTAGTAGCGAGTGAGTAGGTGTTTTTGGACGCGGGTTCAAGTCCC


 


GCCATCTCCACCA (SEQ ID NO:13)

















TABLE 6





tmDNA Sequence for Environmental Sample



from Sludge
















GGGGATGTCATGGTTTTGACAGGGAACCAGGAGGTGTGAGATGCATGCCG



 


GAGACGCTGTCCGCTCCGTTATCAAGCAGCAAACAAAACTAATTGCAAAC


 


AACAATTACTCCTTACCAGCGTAAGCAGCTAACGTTCAACCTCTCCGGAC


 


CGCCGGGAGGGGATTTGGGCGTCGAAACAGCGCGGACGCTCCGGATAGGA


 


CGCCCATAATATCCGGCTAAGACCATGGGTCTGGCTCTCGCGGGTCTGAT


 


TGTCTTCCACCGCGCGGGCCGCGATCAAAGACAACTAAGCATGTAGGTTC


 


TTGCATGGCCTGTTCTTTGGACGCGGGTTCGATTCCCGCCATCTCCACCA


 


(SEQ ID NO:14)

















TABLE 7





tmDNA Sequence for Fibrobacter succinogenes



(Fibrobacter)
















GGGGCTGATTCTGGATTCGACAGGGTTACCGAAGTGTTAGTTGCAAGTCG



 


AGGTCTCAGACGAGGGCTACTCGTTAAAAAGTCTGAAAAAAAATAAGTGC


 


TGACGAAAACTACGCACTCGCTGCCTAATTAACGGCAACGCCGGGCCTCA


 


TTCCGCTCCCATCGGGGTGTACGTCCGGACGCAATATGGGATAGGGAAGT


 


GTCATGCCTGGGGGCATCTCCCGAGATTTTCTAGGCTGGTCAAACTCCGC


 


GCCGACCTTCTTGGGCGTGGATAAGACGAGATCTTAAATTCGAAGGGAAC


 


ACTTGTAGGAACGTACATGGACGTGATTTTGGACAGGGGTTCAACTCCCG


 


CCAGCTCCA (SEQ ID NO: 15)
















TABLE 8





tmDNA Sequence for Fusobacterium mortiferum
















GGGGCTGATTCTGGATTCGACGGGGTTATGAGGTTATAGGTAGCATGCCA



 


GGATGACCGCTGTGAGAGGTCAACACATCGTTTAGATGGAAACAGAAATT


 


ACGCTTTAGCTGCTTAATTAGTCAGCTCACCTCTGGTTTCTCTCTTCTGT


 


AGGAGAATCCAACCGAGGTGTTACCAATATACAGATTACCTTTAGTGATT


 


TCTCTAAGCTCAAAGGGACATTTTAGAGAATAGCTTCAGTTAGCCCTGTC


 


TGCGGGAGTGATTGTTGCGAAATAAAATAGTAGACTAAGCATTGTAGAAG


 


CCTATGGCGCTGGTAGTTTCGGACACCGGTTCAACTCCCGCCAGCTCCAA


 


(SEQ ID NO:16)

















TABLE 9





tmDNA Sequence for Corynebacterium xerosis



(gram +, high G-C content)
















GGGGGTGATTCTGGATTCGACTTCGTACATTGAGCCACGGOAAGCGTGCC



 


GGTGAAGGCTGGAGACCACCGCAAGCGTCGCAGCAACCAATTAAGCGCCG


 


AGAACTCTCAGCGCGACTACGCCCTCGCTGCCTAAGCAGCGACCGCGTGT


 


CTGTCAGACCGGGTAGGCCTCTGATCCGGACCCTGGCATCGTTTAGTGGG


 


GCTCGCTCGCCGACTTGGTCGCAAGGGTCGGCGGGGACACTCACTTGCGA


 


CTGGGCCCGTCATCCGGTCATGTTCGACTGAACCGGAGGGCCGAGCAGAG


 


ACCACGCGCGAACTGCGCACGGAGAAGCCCTGGCGAGGTGACCGAGGACC


 


CGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:17)
















TABLE 10





tmDNA Sequence for Micrococcus luteus (parfait)
















GGGGCTATTCTGGATTCGACGGTGTGTGTCGCGTCGGGAGAAGCGGGCCG



 


AGGATGCAGAGTCATCTCGTCAAACGCTCTCTGCAAACCAATAAGTGCCG


 


AATCCAAGCGCACTGACTTCGCTCTCGCTGCCTGATCAGTGATCGAGTCC


 


GTCACCCCGAGGTCGCTGTCGCCTCGGATCGTGGCGTCAGCTAGATAGCC


 


ACTGGGCGTCACCCTCGCCGGGGGTCGTGACGCCGACATCAATCCGGCTG


 


GGTCCGGGTTGGCCGCCCGTCTGCGGGACGGCCAGGACCGAGCAACACCC


 


ACAGCAGACTGCGCCCGGAGAAGACCTGGCAACACCTCATCGGACGCGGG


 


TTCAACTCCCGCANTCCCACCA (SEQ ID NO:18)
















TABLE 11





tmDNA Sequence for Mycobacterium smegmatis
















TCATCTCGGCTTGTTCGCGTGACCGGGAGATCCGAGTAGAGACATAGCGA



 


ACTCCGCACGGAGAGGGGCTGATTCCTGGATTCGACTTCGAGCATCGAAT


 


CCACGGAAGCGTGCCGGTGCAGGCAAGAGACCACCGTAAGCGTCGTTGCA


 


ACCAATTAAGCGCCGATTCCAATCAGCGCGACTACGCCCTCGCTGCCTAA


 


GCGACGGCTGGTCTGTCAGACCGGGAGTGCCCTCGGCCCGGATCCTGGCA


 


TCAGCTAGAGGGACCCACCCACGCCTTCGCTCGCGGGACCTGTGGGGACA


 


TCAAACAGCGACTGGGATCGAGCCTCGAGGACATGCCGTAGGACCCGGGT


 


TCAACTCCCGCCAGCTCCACCA (SEQ ID NO:19)
















TABLE 12





tmDNA Sequence for Bacillus badius
















GGGGGTGATTCTGGATTCGACAGGCATAGTTCGAGCTTGGGCTGCGAGCC



 


GGAGGGCCGTCTTCGTACCAACGCAAACGCCTAAATATAACTGGCAAAAA


 


AGATTTAGCTTTAGCTGCCTAATATAGGTTCAGCTGCTCCTCCCGCTATC


 


GTCCATGTAGTCGGGTAAGGGGTCCAAACTTAGTGGACTACGCCGGAGTT


 


CTCCGCCTGGGGACAAAGGAAGAGATCAATCAGGCTAGCTGCCCGGACCC


 


CCGTCGATAGGCAAAAGGAACAGTGAACCCCAAATATATCGACTACGCTC


 


GTAGACGTTCAAGTGGCGTTATCTTTGGACGTGGGTTCAACTCCCGCCAG


 


CTCCA (SEQ ID NO:20)
















TABLE 13





tmDNA Sequence for Bacillus brevis
















GGGGGCGGAAAGGATTCGACGGGGATGGTAGAGCATGAGAAGCGAGCCGG



 


GGGGTTGCCGACCTCGTCACCAACGCAAACGCCATTAACTGGCAACAAAC


 


AACTTTCTCTCGCTGCTTAATAACCACTGAGGCTCTCCCACTGCATCGGC


 


CCGTGTGCCCTGGATAGGGCTCAACTTTAACGGGCTACGCCGCAGGCTTC


 


CGCCTGGAGCCAAAGGAAGAAGACCAATCAGGCTAGGTGCCAGGTCAGCG


 


CGTCACTCCGCGAATCTGTCACCGAAACTCTAAACGAGTGACTGCGCTCG


 


GAGATGCTCATGTATCGCTGTTTTCGGACGGGCGTTCGATTCCCGCCGCC


 


TCACCCA (SEQ ID NO:21)

















TABLE 14





tmDNA Sequence for Bacillus thermoleovorans



(50-60 degres)
















GGGGGCGGAAAGGATTCGACGGGGGTAGGTCGAGCTTAAGCGGCGAGCCG



 


AGGGGGACGTCCTCGTAAAAACGTCACCTAAAGATAACTGGCAAACAAAA


 


CTACGCTTTAGCTGCCTAATTGCTGCAGCTAGCTCCTCCCGCCATCGCCC


 


GCGTGGCGTTCGAGGGGCTCATATGGAGCGGGCTACGCCCAAATCCGCCG


 


CCTGAGGATGAGGGAAGAGACGAATCAGGCTAGCCGCCGGGAGGCCTGTC


 


GGTAGGCGGAACGGACGGCGAAGCGAAATATACCGACTACGCTCGTAGAT


 


GCTTAAGTGGCGATGCCTCTGCACGTGGGTTCGATTCCCGCCGCCTCCCC


 


ACCA (SEQ ID NO:22)
















TABLE 15





tmDNA Sequence for Clostridium innocuum
















GGGGGCGGAAAGGATTCGACGGGGATATGTCTGGTACAGACTGCAGTCGA



 


GTGGTTACCTAATAACCAATTAAATTTAAACGGAAAAACTAAATTAGCTA


 


ACCTCTTTGGTGGAAACCAGAGAATGGCTTTCGCTGCTTAATAACCGATA


 


TAGGTTCGCAGCCGCCTCTGCATGCTTCTTCCTTGACCATGTGGATGTGC


 


GCGTAAGACGCAAGGGATAAGGAATCTGGTTTGCCTGAGATCAGATTCAC


 


GAAAATTCTTCAGGCACATTCATCAGCGGATGTTCATGACCTGCTGATGT


 


CTTAATCTTCATGGACTAAACTGTAGAGGTCTGTACGTGGGGCTGTTTCT


 


GGACAGGAGTTCGATTCCCGCCGCCTCACCACCA (SEQ ID NO:23)
















TABLE 16





tmDNA Sequence for Clostridium lentocellum
















GGGGGCGGAAAGGATTCGACGGGGGTCACATCTACTGGGGCAGCCATCCG



 


TAGAACGCCGGAGTCTACGTTAAAAGCTGGCACTTAAAGTAAACGCTGAA


 


GATAATTTAGCAATCGCTGCCTAATTAAGGCGCAGTCCTCCTAGGTCTTC


 


CGCAGCCTAGATCAGGGCTTCGACTCGCGGATCCTTCACCTGGCAAAGCT


 


TTGAGCCAACGTGAACACTATGAAGCTACTAAAATCTAGAGCCTGTCTTT


 


GGGCGCTAGATGGAGGGAATGTCAAAACAAAGAATATGATGGTAGAGACC


 


ACGCTATATGGGCTTTCGGACAGGGGTTCGATTCCCGCCGCCTTCACCA


 


(SEQ ID NO:24)
















TABLE 17





tmDNA Sequence for Clostridium perfringens
















GGGGCTGATTCTGGATTCGACGGGGGTAAGATGGGTTTGATAAGCGAGTC



 


GAGGGAAGCATGGTGCCTCGATAATAAAGTATGCATTAAAGATAAACGCA


 


GAAGATAATTTTGCATTAGCAGCTTAATTTAGCGCTGCTCATGCTTGGTG


 


AATTGGCCAGGGTTGAGAGTAAGGGTCTCATTTAAAAGTGGGGAAGGGAG


 


GGTAGGAAAGCTTTGAGGTAGGAACGGAATTTATGAAGGTTACCAAAGAG


 


GAAGTTTGTGTGTGGACGTTCTCTGAGGGAATTTTAAAAGACAAGAGTAC


 


AGTGGTAGAAAGTGTTACTGGTGTGCTTTCGGAGAGGGCTTGAAGTGGGG


 


CCACTGCA (SEQ ID NO:25)
















TABLE 18





tmDNA Sequence for Clostridium stercorarium
















GGGGGCCGAAAGGATTCGACGGGGTTATTGAAGCAAGAGTAGCGGGTAGA



 


GGATTCTCGTTGGCCTCTTTAAAAAACGAGAGGTAAAAATAAACGCAAAC


 


AACCATAACTACGCTTTAGCTGCTGCGTAAGTAACACGCAGCCCGTCGGC


 


CCCGGGGTTCCTGCGCCTCGGGATACCGGCGTCATCAAGGCAGGGAACCA


 


GCCGGATCAGGCTTCAGGTCCGGTGGGATTTAATGAAGCTACCGACTTAT


 


AAAGCCTGTCTCTGGGCGTTATAAGAAGGGAATGTCAAAACAGAGACTGC


 


ACCCGGAGAAGCTCTTGTGGATATGGTTCCGGACACGAGTTCGATTCCCG


 


CCGCCTCCACCA (SEQ ID NO:26)
















TABLE 19





tmDNA Sequence for Enterococcus faecium (sp.)
















GGGGCTGATTATGGATTCGACAGGATNGTTGAGCTTGAATTGCGTTTCGT



 


AGGTTACGGCTACGTTAAAACGTTACAGTTAAATATAACTGCTAAAAACG


 


AAAACAATTCTTTCGCTTTAGCTGCCTAAAAACCAGCTAGCGAAGATCCT


 


CCCGGCATCGCCCATGTGCTCGGGTCAGGGTCCTAATCGAAGTGGGATAC


 


GCTAAATTTTTCCGTCTGTAAAATTTAGAGGAGCTTACCAGACTAGCAAT


 


ACAAGAATGCCTGTCACTCGGCACGCTGTAAAGCGAACCTTTAAATGAGT


 


GTCTATGAACGTAGAGATTTAAGTGGGAATATGTTTTGGACGCGGGTTCA


 


ACTCCCGCCAGCTCCACCA (SEQ ID NO:27)

















TABLE 20





tmDNA Sequence for Heliobacillus mobilis



(photosyn/gram+)
















GGGGCTGATTCTGGATTCGACGGGGAACGTGTTTGCTTGGGATGCGAGCC



 


GGGTTGCCGCCAGGACCGTAAAAAGGGCGGAAGGCTTTAATTGCCGAAGA


 


TAACTACGCTTTAGCTGCTTAATTGCAGTCTAACCTCTTCTCCTCTGTGC


 


TCTCGGTGAGGATGTAAGGGGTCATTTAAGAGAGCTGGCTTCGACCAATT


 


CTCGGAGGTCCAAGCGAGATTTATCGAGATAGCCTGACCAACGCTCTGTC


 


TGCCGTGCGGAAGGAAGGCGAAATCTAAAACGACAGACTACGCTCGTAGT


 


GTCCTTTGTGGGCATTTCTTCGGACGCGGGTTCAACTCCCGCCAGCTCCA


 


CCA (SEQ ID NO:28)
















TABLE 21





tmDNA Sequence for Heliospirillum gestii
















GGGGCTGATTCTGGATTCGACGGGGAACGTGTTTGCTTAGGACGCGAGCC



0


GGGTTGCCGCCAGGACCGTAAAAAGGGCGGAAGGCTTTAATTGCCGAAGA


 


TAACTACGCTTTAGCTGCTTAATTGCAGTCTAACCTCTTCTCCTCTGTGC


 


TCTCGGTGAGGATGTAAGGGGTCATTTAAGAGAGCTGGCTCGAACCAATT


 


CTCGGAGGTTCGGGTAAGACTTATCGAGATAGCCTGACCAACGCTCTGTC


 


TGCCGTGCGGAAGGATGGCGAAATCTAAAACGACAGAATACGCTCGTAGT


 


GTCCTTTGTGGGCATTTCTTCGGACGCGGGTTCAACTCCCGCCAGCTCCA


 


CCA (SEQ ID NO:29)
















TABLE 22





tmDNA Sequence for Lactobacillus acidophilus
















GGGGCTGATTCTGGATTCGACAGGCGTAGACCCGCATTGACTGCGGTTCG



 


TAGGTTACGTCTACGTAAAAACGTTACAGTTAAATATAACTGCAAATAAC


 


AAAAATTCTTACGCATTAGCTGCTTAATTTAGCCCATGCGTTGCTCTTTG


 


TCGGTTTACTCGTGGCTGACACTGAGTATCAACTTAGCGAGTTACGTTTA


 


ACTACCTCACCTGAATAGTTGAAAAGAGTCTTAGCAGGTTAGCTAGTCCA


 


TACTAGCCCTGTTATATGGCGTTTTGGACTAGTGAAGTTCAAGTAATATA


 


ACTATGATCGTAGAGGTCAGTGACGAGATGCGTTTGGACAGCGGGTTCAA


 


CTCCCGCCAGCTCCACCA (SEQ ID NO:30)
















TABLE 23





tmDNA Sequence for Staphylococcus epidermidis
















GGGGCTGATTCTGCATTCGACAGGGGTCCCCGAGCTTATTAAGCGTGTGG



 


AGGGTTGGCTCCGTCATCAACACATTTCGGTTAAATATAACTGACAAATC


 


AAACAATAATTTCGCAGTAGCTGCGTAATAGCCACTGCATCGCCTAACAG


 


CATCTCCTACGTGCTGTTAACGCCATTCAACCCTAGTAGGATATGCTAAA


 


CACTGCCGCTTGAAGTCTGTTTAGATGAAATATAATCAAGCTAGTATCAT


 


GTTGGTTGTTTATTGCTTAGCATGATGCGAAAATTATCAATAAACTACAC


 


ACGTAGAAAGATTTGTATCAGGACCTCTGGACGCGGGTTCAACTCCCGCC


 


AGCTCCACCA (SEQ ID NO:31)
















TABLE 24





tmDNA Sequence for Streptococcus faecium
















GGGGCTGATTCTGGATTCGACAGGCACAGTTTGAGCTTGAATTGCGTTTC



 


GTAGGTTACGTCTACGTTAAAACGTTACAGTTAAATATAACTGCTAAAAA


 


CGAAAACAACTCTTACGCTTTAGCTGCCTAAAAACAGTTAGCGTAGATCC


 


TCTCCGCATCGCCCATGTGCTCGAGTAAGGGTCTCAAATTTAGTGGGATA


 


CGTGACAACTTTCCGTCTGTAAGTTGTTAAAGAGATCATCAGACTAGCGA


 


TACAGAATGCCTGTCACTCGGCAAGCTGTAAAGCGAAACCACAAATGAGT


 


TGACTATGAACGTAGATTTTTAAGTGGCGATGTGTTTGGACGCGGGTTCA


 


ACTCCCGCCGTTCCACCA (SEQ ID NO:32)

















TABLE 25





tmDNA Sequence for Thermoanaerobacterium



saccharolyticum (Bacillus/clostridium)







GGGGTAGTAGAGGTAAAAGTAGCGAGCCGAGGTTCCATCTGCTCGTAAAA



 


CGGTGGACTTAAATATAAACGCAAACGATAATTTAGCTTACGCTGCTTAA


 


TTACAAGCAGCCGTTCAACCTTTGATTCCCACATCAAAGGATTGGGCGTC


 


GATTTAGTGGGGAACTGATTTATCAAAGCTTTGAGATAAATCGGATTTTA


 


TGAAGCTACCAAAGCAGTTATCCTGTCACTGGGAGAACTGCAGAGGGAAT


 


GTCAAAACACTGACTGCGCTCGGAGAAGCTTTTACTGTGACACCTTCGGA


 


CCGGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:33)
















TABLE 26





tmDNA Sequence for Mycoplasma fermentans
















GGGGCTGATTCTGGATTCGACATGCATTGGGTGATACTAATATCAGTAGT



 


TTGGCAGACTATAATGCATCTAGGCTTTATAATCGCAGAAGATAAAAAAG


 


CAGAAGAAGTTAATATTTCTTCACTTATGATTGCACAAAAAATGCAATCA


 


CAATCAAACCTTGCTTTCGCTTAGTTAAAAGTGACAAGTGGTTTTAAAGT


 


TGACATTTTCCTATATATTTTAAAATCGGCTTTTAAGGAGAACAGGAGTC


 


TGAAAGGGTTCCAAAAATCTATATTGTTTGCATTTCGGTAGTATAGATTA


 


ATTAGAAATGATAAACTGTAAAAAGTATTGGTATTGACTTGGTGTGTGGA


 


CTCGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:34)
















TABLE 27





tmDNA Sequence for Mycoplasma hyorhinis
















GGGGCTGATTCTGGATTCGACATACATAAAAGGATATAAATTGCAGTGGT



 


CTTGTAAACCATAAGACAATTTCTTTACTAAGCGGAAAAGAAAACAAAAA


 


AGAAGATTATTCATTATTAATGAATGCTTCAACTCAATCAAATCTAGCTT


 


TTGCATTTTAAAAAACTACTAGACCAATTTGCTTCTCACGAATTGTAATC


 


TTTATATTAGAGAATAGTTAAAAATCTGATCACTTTTTAATGAATTTATA


 


GATCACAGGCTTTTTTAATCTTTTTGTTATTTTAGATAAAGAGTCTTCTT


 


AAAAATAACTAAACTGTAGGAATTTATATTTAATTATGCGTGGACCCGGG


 


TTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:35)
















TABLE 28





tmDNA Sequence for Mycoplasma pirum
















GGGGAGTCATGGTTTTGACATGAATGATGGACCCATAGAGGCAGTGGGGT



 


ATGCCCCTTATAGCTCAAGGTTTAAATTAACCGACAAAACTGACGAAAAC


 


GTTGCCGTTGATACAAATTTATTAATCAACCAACAAGCTCAATTTAACTA


 


CGCATTTGCATAGTATAAAAAAATAAATTGTGCTACTCATTGTAATTAGG


 


TTACTAAATTACTTTGTTTTATATAGTCCTGTAACTAGTTCTAGTGATGT


 


CTATAAACTAGAATGAGATTTATAGACTTATTTGTTGGCGGTTGTGCCAT


 


AGCCTAAATCAACAAAGACAATTTATTTATGGTACTAAACTGTAGATTCT


 


ATGATGAAATTATTTGTGGAAACGGCTTCGATTCCCGCCATCTCCACCA


 


(SEQ ID NO:36)
















TABLE 29





tmDNA Sequence for Mycoplasma salivarium
















GGGGCTGATTCTGGATTCGACAGGCATTCGATTCATTATGTTGCAGTGGT



 


TTGCAAACCATAAGGCACTAGGCTTTTTTAAACGCAAAAGACCAAAAAAC


 


AGAAGATCAAGCAGTTGATCTAGCATTTATGAATAATTCACAAATGCAAT


 


CAAATCTAGTTTTCGCTTAGTAAAATTAGTCAATTTATTATGGTGCTCAA


 


CATAATAAATGGTAGTATGAGCTTAATATCATATGATTTTAGTTAATATG


 


ATAGGATTTGTAACTAAACTATGTTATAGAAATTTGTAAATTATATATAT


 


GACATAGGAAATTTAATTTACTAAACTGTAGATGCATAATGTTGAAGATG


 


TGTGGACCGGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:37)
















TABLE 30





tmDNA Sequence for Herpetosiphon aurantiacus
















GGGGGCGGAAAGGATTCGACGGGGAGGGCCAATCGTAAGTGGCAAGCCGA



 


GACGCTGAGCCTCGTTAAATCGGCAACGCCATTAACTGGCAAAAACACTT


 


TCCGCGCTCCTGTAGCGCTTGCTGCCTAATTAAGGCAACACGTCTCTACT


 


AGCCTCAGCCCGATGGGCTTGTAGCGGCGACACTTAGTCGGGTCGCTCCC


 


CTAGTTATGTCTGTGGGCTAGGGGCTAAGATTAACAGGCTGGTCGTGGCC


 


CGCTTTGTCTATCGGGTGGTGCACCGATAAGATTTAATCAATAGACTACG


 


CTTGTAGATGCTTGCGGTTTAACTTTTTGGACGCGGGTTCGATTCCCGCC


 


GCCTCACCACCA (SEQ ID NO:38)

















TABLE 31





tmDNA Sequence for Thermomicrobium roseum



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
















GGGGCTGATTCTGGATTCGACAGGGCCGTAGGTGCGAGGATTGCAGGTCG



 


AGGTCGCCCACGAACTCGTAAAAAGGGGCAGCCAAGTAACTGGCGAGCGC


 


GAACTCGCTCTGGCTGCGTAATTCACGCAGCCACGTCTGCCCGGACCCTT


 


CCCTGGTGGGTTCGGAGCGGGCGCCGCAAGACCGGGGTGCCCCTGGCCCA


 


AGCGCCGGTGCGGGCCAGGTCAAGCGTGATCCGGCTCGGCTGACCGGGAT


 


CCTGTCGGTGGGAGCCTGGCAGCGACAGTAGAACACCGACTAAGCCTGTA


 


GCATATCCTCGGCTGAACGCTCTGGACGCGGGTTCAACTCCCGCCAGCTC


 


CACCA (SEQ ID NO:39)
















TABLE 32





tmDNA Sequence for Chiorobium limicola
















GGGGCTGATTCTGGATTCGACAGGATACGTGTGAGATGTCGTTGCACTCC



 


GAGTTTCAGCATGGACGGACTCGTTAAACAAGTCTATGTACCATTAGATG


 


CAGACGATTATTCGTATGCAATGGCTGCCTGATTAGCACAAGTTAACTCA


 


GACGCCATCGTCCTGCGGTGAATGCGCTTACTCTGAAGCCGCCGGATGGC


 


ATAACCCGCGCTTGAGCCTACGGGTTCGCGCAAGTAAGCTCCGTACATTC


 


ATGCCCGAGGGGCTGTGCGGGTAATTTCTCGGGATAAGGGGACGAACGCT


 


GCTGGCGGTGTAATCGGCCCACGAAAACCCAATCACCAGAGATGAGTGTG


 


GTGACTGCATCGAGCAGTGTTTTGGACGCGGGTTCAACTCCCGCCAGCTC


 


CACCA (SEQ ID NO:40)

















TABLE 33





tmDNA Sequence for Pirellula staleyi



(planctomyces)
















GGGGCTGATTCTGGATTCGACCGGATAGCCTGAAGCGAATACGGCGTGCC



 


GTGGTTGATCAGATGGCCACGTAAAAAGCTGATCACAAACTTAACTGCCG


 


AGAGCAATCTCGCACTTGCTGCCTAACTAAACGGTAGCTTCCGACTGAGG


 


GCTTTAGCCGGAGACGCCCAAAAGTTGGTCACCAAATCCGGACCGCCTCG


 


TGCCATGATCGAAACGCACGAGGTCAAAAAAGTTTCGATCTAGTGCAGGG


 


TGTAGCCAGCAGCTAGGCGACAAACTGTGCAAAAATCAAATTTTCTGCTA


 


CGCACGTAGATGTGTTCGTGAAAATGTCTCGGCACGGGGGTTCAACTCCC


 


GCCACTCCACCA (SEQ ID NO:41)
















TABLE 34





tmDNA Sequence for Planctomyces limnophilus
















GGGGCTGATTCTCGATTCGACAACCTCTCAAGAGGAGCGTGGCCACTATG



 


GGACTCGATTATGTTGAATTCGTCATGGATCTTGAAGAGACCTTCGACAT


 


CAAACTGGATGACAAACATTTTTCAGCAGTCAAAACACCACGCGATTTGG


 


CAATCATTATTCGGGATCAATTAGCTGCTGAAGGCAGAATCTGGGATGAA


 


TCGAATGCTTTTCGCAAAATCTCGAATTTGAATTGGACGATGTTGCCCGA


 


GTTCCGCATGTGGACTCAAATCAAAAGCTCTCTACCACTTTCTTTTCACC


 


GACTGCGTCCCAGCACCCGTCTCGTTCAACTCCCGCCANTCCACCA


 


(SEQ ID NO:42)
















TABLE 35





tmDNA Sequence for Planctomyces maris
















GGGGCTGATTCTGGATTCGACTGGTTCACCGTATGTTAAGGTGGCGGTGC



 


CGTGGTTGATCAGTTGGCCACGTAAAAAGCTGATCACAATCTAATTGCAA


 


ACAAGCAATTTTCAATGGCTGCTTAATAAAAGCAACCCCGGCTTAGGAAT


 


CTCTGTCTGAGGAGTCCGACAGCTGGTCACAAAATCAGACTGGTATCAGA


 


TCAATGTCCGCTCCGTCTGATACGAGATTCGTGGTCGACTGGTTTCCAAC


 


AGGCTCTGTTTATCGTGCCCGAAGAAACGAGACTCAAACGATAAAATATG


 


CACCGTAGAGGCTTTAGCTGAGGGTTCACAGGACGCGGGTTCAACTCCCG


 


CCAGCTCCACCA (SEQ ID NO:43)
















TABLE 36





tmDNA Sequence for Alcaligenes eutrophus
















GGGGTTCATTCTGGATTCGACGTGGGTTACAAAGCAGTGGAGGGCATACC



 


GAGGACCCGTCACCTCGTTAATCAATGGGAATGCAATAACTGCTAACGAC


 


GAACGTTACGCACTGGCCGCTTAATTGCGGCCGTCCTCGCACTGGCTCGC


 


TGACGGGCTAGGGTCGCAAGACCACGCGAGGTCATTTACGTCAGATAAGC


 


TCCGGAAGGGTCACGAAGCCGGGCACGAAAACCTAGTGACTCGCCGTCGT


 


AGAGCGTGTTCGTCCGCGATGCCCCGGTTAAATCAAATGACAGAACTAAG


 


TATGTAGAACTCTCTGTCGAGGGCTTACGGACGCGGGTTCAACTCCCCCC


 


AGCTCCACCA (SEQ ID NO:44)

















TABLE 37





tmDNA Sequence for Alcaligenes faecalis



(beta proteobacteria)
















GGGGGCGGAAAGGATTCGACGGGGGTCAAGAAGCAGCACAGGGCGTGTCG



 


AGCACCAGTACGCTCGTAAATCCACTGGAAAACTATAAACGCCAACCACC


 


AGCGTTTCGCTCTAGCCGCTTAAGGCTGGGCCACTGCACTAATTTGTCTT


 


TGGGTTAGGTAGGGCAACCTACAGCAGTGTTATTTACAAAGAATCGAATC


 


GGTCTCCGCCACGAAGTCCGGTTCTAAAACTTAGTGGATCGCCAAGGAAA


 


GGCCTGTCAATTGGCATAGTCCAAGGTTAAAACTTAAAATTAATTGACTA


 


CACATGTAGAACTGTCTGTGGACGGCTTGCGGACGCGGGTTCGATTCCCG


 


CCGCCTCCACCA (SEQ ID NO:45)

















TABLE 38





tmDNA Sequence for Chromobacterium violaceum



(beta-purple)
















GGGGCTGATTCTGGATTCGACGGGGGTTGCGAAGCAGATGAGGGCATACC



 


GGGATTTCAGTCACCCCGTAAAACGCTGAATTTATATAGTCGCAAACGAC


 


GAAACTTACGCTCTGGCAGCCTAACGGCCGGCCAGACACTACAACGGTTC


 


GCAGATGGGCCGGGGGCGTCAAAACCCTGTAGTGTCACTCTACATCTGCT


 


AGTGCTGTTCCGGGTTACTTGGTTCAGTGCGAAATAATAGGTAACTCGCC


 


AAAGTCCAGCCTGTCCGTCGGCGTGGCAGAGGTTAAATCCAAATGACACG


 


ACTAAGTATGTAGAACTCACTGTAGAGGACTTTCCGACGCGGGTTCAACT


 


CCCGCCAGCTCCACCA (SEQ ID NO:46)

















TABLE 39





tmDNA Sequence for Hydrogenophaga palleroni



(beta-purple)
















GGGGCTGATTCTGGATTCGACGTGGGTTCGGACGCGCAGCAGGGCATGTC



 


GAGGTTCTGTCACCTCGTAAATCAGCAGAAAAAAACCAACTGCAAACGAC


 


GAACGTTTCGCACTCGCCGCTTAAACACCGGTGAGCCTTGCAACAGCAGG


 


CCGATGGGCTGGGCAAGGGGGTCGCAAGACCTCCCGGCTGCAAGGTAATT


 


TACATCGGCTGGTTCTGCGTCGGGCACCTTGGCGCAGGATGAGATTCAAG


 


GATGCTGGCTTCCCGTTTAGCGTGCCACTGCGCGACTCGGGCGGCGAGAC


 


CCAAATCAGACGGCTACACATGTAGAACTGCTCGAAAAAGGCTTGCGGAC


 


GGGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:47)

















TABLE 40





tmDNA Sequence for Methylobacillus glycogenes



(beta-purple)
















GGGGGCGGAAAGGATTCGACGGGGGTTGCAAAGCAGCGCAGGGCATACCG



 


AGGCCTAGTCACCTCGTAAATAAACTAGAACAAGTATAGTCGCAAACGAC


 


GAAACTTACGCTCTAGCCGCTTAATCCCGGCTGGACGCTGCACCGAAGGG


 


CCTCTCGGTCGGGTGGGGTAACCCACAGCAGCGTCATTAAGAGAGGATCG


 


TGCGATATTGGGTTACTTAATATCGTATTAAATCCAAGGTAACTCGCCTG


 


CTGTTTGCTTGCTCGTTGGTGAGCATCAGGTTAAATCAAACAACACAGCT


 


AAGTATGTAGAACTGTCTGTGGAGGGCTTGCGGACGGGGGTTCGATTCCC


 


GCCGCCTCACCACCA (SEQ ID NO:48)

















TABLE 41





tmDNA Sequence for Nitrosomonas cryotolerans



(beta-purple)
















GGGGCTGATTCTGGATTCGACGTGGGTTGCAAAGCAGCGCAGGGCATACC



 


GAGGACCAGAATACCTCGTAAATACATCTGGAAAAAAATAGTCGCAAACG


 


ACGAAAACTACGCTTTAGCCGCTTAATACGGCTAGCCTCTGCACCGATGG


 


GCCTTAACGTCGGGTCTGGCAACAGACAGCAGAGTCATTAGCAAGGATCG


 


CGTTCTGTAGGGTCACTTTACAGAACGTTAAACAATAGGTGACTCGCCTG


 


CCATCAGCCCGCCAGCTGGCGGTTGTCAGGTTAAATTAAAGAGCATGGCT


 


AAGTATGTAGAACTGTCTGTAGAGGACTTGCGGACGCGGGTTCAACTCCC


 


GCCAGTCCACCA (SEQ ID NO:49)
















TABLE 42





tmDNA Sequence for Pseudomonas testosteroni
















GCGGCTGATTCTGGATTCGACGTGGGTTCGGGACCGGTGCGGTGCATGTC



 


GAGCTTGAGTGACGCTCGTAAATCTCCATTCAAAAAACTAACTGCAAACG


 


ACGAACGTTTCGCACTCGCCGCTTAATCCGGTGAGCCTTGCAACAGCACG


 


CTAGTGGGCTGGGCAAGGGGGTAGCAATACCTCCCGGCTGCAAGGGAATT


 


TTCATTAGCTGGCTGGATACCGGGCTTCTTGGTATTTGGCGAGATTTTAG


 


GAAGCTGGCTACCCAAGCAGCGTGTGCCTGCGGGGTTTGGGTGGCGAGAT


 


TTAAAACAGAGCACTAAACATGTAGATCTGTCCGGCGAAGGCTTACGGAC


 


GCGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:50)

















TABLE 43





tmDNA Sequence for Raistonia pickettii



(Burkholderia)
















GGGGGCGGAAAGGATTCGACGGGGGTTGCGAAGCAGCGGAGGGCATACCG



 


AGGACCCGTCACCTCGTTAATCAATGGGAATGCAATAACTGCTAACGACG


 


AACGTTACGCACTGGCAGCCTAAGGGCCGCCGTCCTCGCACTGGCTCGCT


 


GACGGGCTAGGGTCGCAAGACCAGCGAGGTCATTTACGTCAGATAAGCTT


 


TAGGTGAGTCACGGGCCTAGAGACGAAAACTTAGTGAATCGCCGTCGTAG


 


AGCGTGTTCGTCCGCGATGCGGCGGTTAAATCAAATGACAGAACTAAGTA


 


TGTAGAACTCTCTGTGGAGGGCTTGCGGACGCGGGTTCGATTCCCGCCGC


 


CTCACCACCA (SEQ ID NO:51)

















TABLE 44





tmDNA Sequence for Variovax paradoxus



(pseudomonas sp.)
















GGGGCTCATTCTGGATTCGACGTGGGTTCGGAGTCGCAGCGGGGCATGTC



 


GAGCTGAATGCGCTCGTAAAACAGATTCAAACAAACTAACTGCAAACGAC


 


GAACGTTTCGCACTCGCTGCTTAATTGCCAGTGAGCCTTGCAACAGTTGG


 


CCGATGGGCTGGGCAAGGGGGTCTGGAGCAATCCTGACCTCCCGGCTGCA


 


AGGATAACTACATGGGCTGGCTCCGATCCGGGTACCTTGGGTCGGGGCGA


 


GAAAATAGGGTACTGGCGTCCGGTTTAGCGTGTGACTGCGCGACTCCGGA


 


AGCGAGACTCAAAACAGATCACTAAACATGTAGAACTGCGCGATGAAGGC


 


TTGCGGACGGGGGTTCAACTCCCGCCAGCTCCACCA


 


(SEQ ID NO:52)

















TABLE 45





tmDNA Sequence for Bdellovibrio bacteriovorus



(delta proteobactene)
















GGGGGCGGAAAGGATTCGACGGGGGTGCTGAAGCATAAGGAGCATACCGG



 


GGCGGATGAGGACCTCGTTAAAAACGTCCACTTTGTAATTGGCAACGATT


 


ACGCACTTGCAGCTTAATTAAGCAGCACGATCAACCTTGTGGTGGTTCCG


 


CACTTGGATTGATCGTCATTTAGGGACCTCGGCGTGTTGGGTTTTCTCCA


 


GCAGACATGCTTAAATTTACTGGGGGAGAGGTCTTAGGGATTTTGTCTGT


 


GGAAGCCCGAGGACCAATCTAAAACACTGACTAAGTATGTAGCGCCTTAT


 


CGTGGATCATTTGCGGACGGGGGTTCGATTCCCGCCGCCTCCACCA


 


(SEQ ID NO:53)

















TABLE 46





tmDNA Sequence for Myxococcus xanthus



(delta proteobacterie)
















GGGGGCGGAAACGATTCGACGGGGGCATTGAAGTTCGAGACGCGTGCCGA



 


GCTTGTCAGGTAGCTCGTAAATTCAACCCGGCAAAGACACAAAAGCCAAC


 


GACAACGTTGAGCTCGCGCTGGCTGCCTAAAAACACCCCATAGTGCGCGG


 


TCCCCCCGCCCTCGGCCTGTGGGGTTGGGACAGACCGTCATAATGCAGGC


 


TGGCTGCCGAGGGTGCCTGGACCCGAGGTGGCGAGATCTTCCCAGGACCG


 


CCTCTGAGTATCCCGTCCGTGGGAGCCTCAGGGACGTAGCAAATCGCGGA


 


CTACGCACGTAGGGTCGAAGAGCGGACGGCTTTCGCACGCGGGTTCGATT


 


CCCGCCGCCTCCACCA (SEQ ID NO:54)
















TABLE 47





tmDNA Sequence for Sulfurospirillum Deleyianum
















GGGGCTGATTCTGGATTCGACAGGAGTAGTTTTAGCTTATGGCTGCATGT



 


CGGGAGTGAGGGTCTTCCGTTACACAACCTTCAAACAATAACTGCTAACA


 


ACAGTAACTATCGTCCTGCTTACGCGCTAGCTGCGTAAGTTTAACAAATA


 


ATGGACTGCTCTCCCCTTTGATGCTATCTTAGGAGGTCTTGGAGAGTATC


 


ATAGATTTGATAGCTATATTACATGAACGCCTTTACATGTAATGAAGTTA


 


AAGGCTCGTTTTGCGTAGTTTTCTGATTGTTGTACGAAGCAAAATTAAAC


 


ACTATCAACAATATCTAAGCATGTAGACGTCATAGGTGGCTATTTTTGGA


 


CTGCGGGTTCAACTCCCGCCAGCTCCACCA (SEQ ID NO:55)
















TABLE 48





tmDNA Sequence for Chromatium vinosum
















GGGGCTGATTCTGGATTCGACGTGGGTCGCGAAACCTAAGGTGCATGCCG



 


AGGTGCGGTTGACCTCGTAAAACCCTCCGCAAACTTATAGTTGCCAACGA


 


CGACAACTACGCTCTCGCTGCTTAATCCCAGCGGGCCTCTGACCGTCACT


 


TGCCTGTGGGCGGCGGATTCCAGGGGTAACCTCACACAGGATCGTGGTGA


 


CGGGAGTCCGGACCTGATCCACTAAAACCTAACGGAATCGCCGACTGATC


 


GCCCTGCCCTTCGGGCGGCAGAAGGCTAAAAACAATAGAGTGGGCTAAGC


 


ATGTAGGACCGAGGGCAGAGGGCTTGCGGACGCGGGTTCAACTCCCGCCA


 


GCTCCACCA (SEQ ID NO:56)

















TABLE 49





tmDNA Sequence for Pseudomonas fluorescens



(gamma proteobacteria)
















GGGGCTGATTCTGGATTCGACGCCGGTTGCGAACCTTTAGGTGCATGCCG



 


AGTTGGTAACAGAACTCGTAAATCCACTGTTGCAACTTTCTATAGTTGCC


 


AATGACGAAACCTACGGGGAATACGCTCTCGCTGCGTAAGCAGCCTTAGC


 


CCTTCCCTCCTGGTACCTTCGGGTCCAGCAATCATCAGGGGATGTCTGTA


 


AACCCAAAGTGATTGTCATATAGAACAGAATCGCCGTGCAGTACGTTGTG


 


GACGAAGCGGCTAAAACTTACACAACTCGCCCAAAGCACCCTGCCCGTCG


 


GGTCGCTGAGGGTTAACTTAATAGACACGGCTACGCATGTAGTACCGACA


 


GCAGAGTACTGGCGGACGCGGGTTCAACTCCCGCCAGCTCCACCA


 


(SEQ ID NO:57)
















TABLE 50





tmDNA Sequence for Borrelia afzeli
















GGGGCTGATTCTGGATTCGACTGAAAATGCTAATATTGTAAGTTGCAAGC



 


AGAGGGAATCTCTTAAAACTTCTAAAATAAATGCAAAAAATAATAACTTT


 


ACAAGTTCAAACCTTGTAATGGCTGCTTAAGTTAGCAOAGAGTTTTGTTG


 


AATTTGGCTTTGAGATTCACTTATACTCTTTTAGACATCGAAGCTTGCTT


 


AAAAATGTTTTCAAGTTGATTTTTAGGGACTTTTATACTTGAGAGCAATT


 


TGGCGGTTTGCTAGTATTTCCAAACCATATTGCTTAGTAAAATACTAGAT


 


AAGCTTGTAGAAGCTTATAGTATTGTTTTTAGGACGCGGGTTCAACTCCC


 


GCCACTCCACCA (SEQ ID NO:58)
















TABLE 51





tmDNA Sequence for Borrelia crociduarae
















GGGGCTGATTCTGGATTCGACTAAGAACTTTAGTAGCATAAATGGCAAGC



 


AGAGTGAATCTCTTAAAACTTCTTTAATAAATGCAAAAAATAATAACTTT


 


ACAAGTTCAGATCTTGTAATGGCTGCTTAATTTAGCAGAGAGTTTTGTTG


 


GATTTTGCTTTGAGGTTCAACTTATACTCTTTAAGACATCAAAGTATGCC


 


TAAAAATGTTTCAAGTTGATTTTTAGGGACCTTTAAACTTGAGAGTAATT


 


TGGTGGTTTGCTTGTTTTCCAAGCCTTATTCCTTTTTCTAAAAATTAGCT


 


AAGCTTGTAGATATTTATGATATTATTTTTAGGACGCGGGTTCAACTCCC


 


GCCAGTTCCACCA (SEQ ID NO:59)
















TABLE 52





tmDNA Sequence for Borrelia hermsii
















GGGGCTGATTCTGGATTCGACTAAAAACTTTAGTAGCATAAATTGCAAGC



 


AGAGGGAATCTCTTAAAACTTCTTTAATAAATGCAAGAAATAATAACTTT


 


ACAAGTTCAAATCTTGTAATGGCTGCTTAAATTAGCAGAGAGTTCTGCTG


 


GATTTTGCTTTGAGGTTCAGCTTATACTCTTTTAAGACATCAAAGCTTGC


 


TTAAAAATATTTCAAGTTCATTTTTAGGGACTTTTAAATTTGAGAGTAAT


 


TTGGCGGTTTCCTAGTTTTTCCAAACCTTATTACTTAAAGAAAACACTAG


 


CTAAGCTTCTAGATATTTATGATATTATTTTTAGGACGCGGGTTCAACTC


 


CCGCCAGCTCCACCA (SEQ ID NO:60)
















TABLE 53





tmDNA Sequence for Borrelia garinii
















GGGGCTGATTCTGGATTCGACTGAAAATGCGAATATTGTAAGTTGCAGGC



 


AGAGGGAATCTCTTAAAACTTCTAAAATAAATGCAAAAAATAATAACTTT


 


ACAAGCTCAAACCTTGTAATGGCTGCTTAAGTTAGCAGGGAGTTTCGTTG


 


AATTTGGCTTTGAGGTTCACTTATACTCTTTTCGATATCGAAGCTTGCTT


 


AAAAATGTTTTCAAGTTAATTTTTAGGGACTTTTGTACTTGAGAGCAATT


 


TGGCGGTTTGCTAGTATTTCCAAACCATATTGCTTAAGTAAAATGCTAGA


 


TAAGCTTGTAGAAGCTTATAATATTGTTTTTAGGACGCGGGTTCAACTCC


 


CGCCAGTCCACCA (SEQ ID NO:61)

















TABLE 54





tmDNA Sequence for Thermodesulfobacterium commune



(70 degrees)
















GGGGGCGGAAAGGATTCGACGGGGATAGGTAGGATTAAACAGCAGGCCGT



 


GGTCGCACCCAACCACGTTAAATAGGGTGCAAAAACACAACTGCCAACGA


 


ATACGCCTACGCTTTGGCAGCCTAAGCGTGCTCCCACGCACCTTTAGACC


 


TTGCCTGTGGGTCTAAAGGTGTGTGACCTAACAGGCTTTGGGAGGCTTAA


 


TCCGTGGGGTTAAGCCTCCCGAGATTACATCCCACCTGGTAGGGTTGCTT


 


GGTGCCTGTGACAAGCACCCTACGAGATTTTCCCACAGGCTAAGCCTGTA


 


GCGGTTTAATCTGAACTATCTCCGGACGCGGGTTCGATTCCCGCCGCCTC


 


CCCACCA (SEQ ID NO:62)

















TABLE 55





tmDNA Sequence for Thermotoga neapolitana



(Thermotogales)
















GGGGGCGGAAAGGATTCGACGGGGATGGAGTCCCCTGGGAAGCGACCCGA



 


GGTCCCCACCTCCTCGTAAAAAAGGTGGGAACACGAATAAGTGCCAACGA


 


ACCTGTTGCTGTTGCCGCCTAATAGATAGGCGGCCGTCCTCTCCGGAGTT


 


GGCTGGGCTCCGGAAGAGGGCGTGAGGGATCCAGCCTACCGATCTCCCCT


 


CCGCCTTCCGGCCCGGATCCGGAAGGTTCAGCAAGGCTGTGGGAAGCGAC


 


ACCCTCCCCGTGGGGGGTCCTTCCCGACACACGAAACACGGGCTGCGCTC


 


GGAGAAGCCCAGGGGCCTCCATCTTCNGACGCGGGTTCGATTCCCGCCAC


 


CTCCACCA (SEQ ID NO:63)
















TABLE 56





tmDNA Sequence for Deinococcus proteolyticus
















GGGGGCGGAAAGGATTCGACGGGGGAACGGAAAGCGCTGCTGCGTGCCGA



 


GGAGCCGTTGGCCTCGTAAACAAACGGCAAAGCCATTAACTGGCGAAAAT


 


AACTACGCTCTCGCTGCTTAAGTGAGACAGTGACCACGTAGCCCCGCCTT


 


TGGCGACGTGTGAACTGAGACAAAAGAAGGCTAGCTTAGGTGAGGTTCCA


 


TAGCCAAAAGTGAAACCAAATGGAAATAAGGCGGACGGCAGCCTGTTTGC


 


TGGCAGCCCAGGCCCCACAATTTAAGAGCAGACTACGCACGTAGATGCAC


 


GCTGGATGGACCTTTGGACGCGGGTTCGATTCCCGCCAGCTCCACCA


 


(SEQ ID NO:64)

















TABLE 57





tmDNA Sequence for Prosthecobacter fusiformis



(verrucomicrobia)
















GGGGCTGATTCTGGATTCGACGGGGAGTACAAGGATCAAAAGCTGCAAGC



 


CGAGGTGCCGTTACCTCGTAAAACAACGGCAAAAAAGAAGTGCCAACACA


 


AATTTAGCATTAGCTGCTTAATTTAGCAGCTACGCTCTTCTAACCCGGGC


 


TGGCAGGGTTAGAAGGGTGTCATAATGAGCCAGCTGCCCCTTCCGACTCC


 


CCTAAGGAAGGGAAAGATGTAGGGGATAGGTGCTTACAGAATCCTGCGGG


 


AGGGAGTCTGTAAGTGCCGAAAAGTTAAAACTCCCGCTAAGCTTGTAGAG


 


GCTTTTGATTCTTGCTCTCTGGACGCGGGTTCAACTCCCGCCAGCTCCAC


 


CA (SEQ ID NO:65)

















TABLE 58





tmDNA Sequence for Verrucomicrobium spinosum



(verrucomicrobium)
















GGGNNNNATTTGGAATTCGCCGAATGCTAGAAGTGGAGGCTGCATGCCGC



 


GGATGATTCGTTGGCCGCTTTACCAATTCGGATCAAACAACTAAATGCGG


 


ACTCTAACGAGCTTGCCCTCGCCGCTTAATTGACGGTGACGTTCCTCCAG


 


TGAAGTCTGTGAATTGGAGGAGCGACTACTTACAGGCTGGCCAAAAGAGC


 


GGGCGACCGGCCCCAAGGCGAGATCTACAGGCCGCTGGATGGACGGCATC


 


CTGGCAGTAGGAGGCTGGACATCGAGATCAAATNATTGCCTGAGCATGGA


 


GACGCTTTCATAAAGGNGTTCGGACAGGG (SEQ ID NO:66)









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 tmRNA sequence for Staphylococcus aureus set forth in SEQ ID NO:86, and the coding sequence thereof consisting of nucleotides 94-129 of SEQ ID NO:86.

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 Staphylococcus aureus set forth in SEQ ID NO: NO:86, and the coding sequence thereof consisting of nucleotides 94-129 of SEQ ID NO:86.

3. The method of claim 2, wherein the determination is made by performing an amplification-based assay.