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Structure of rrn operons in pathogenic non-cultivable treponemes: sequence but not genomic position of intergenic spacers correlates with classification of Treponema pallidum and Treponema paraluiscuniculi strains.

Cejková D, Zobaníková M, Pospísilová P, Strouhal M, Mikalová L, Weinstock GM, Smajs D - J. Med. Microbiol. (2012)

Bottom Line: Other than these deletions, only 17 heterogeneous sites were found within the entire region (excluding the 16S-23S intergenic spacer region encoding tRNA-Ile or tRNA-Ala).The pattern of nucleotide changes in the rrn operons corresponded to the classification of treponemal strains, whilst two different rrn spacer patterns (Ile/Ala and Ala/Ile) appeared to be distributed randomly across species/subspecies classification, time and geographical source of the treponemal strains.It is suggested that the random distribution of tRNA genes is caused by reciprocal translocation between repetitive sequences mediated by a recBCD-like system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, Building A6, 62500 Brno, Czech Republic.

ABSTRACT
This study examined the sequences of the two rRNA (rrn) operons of pathogenic non-cultivable treponemes, comprising 11 strains of T. pallidum ssp. pallidum (TPA), five strains of T. pallidum ssp. pertenue (TPE), two strains of T. pallidum ssp. endemicum (TEN), a simian Fribourg-Blanc strain and a rabbit T. paraluiscuniculi (TPc) strain. PCR was used to determine the type of 16S-23S ribosomal intergenic spacers in the rrn operons from 30 clinical samples belonging to five different genotypes. When compared with the TPA strains, TPc Cuniculi A strain had a 17 bp deletion, and the TPE, TEN and Fribourg-Blanc isolates had a deletion of 33 bp. Other than these deletions, only 17 heterogeneous sites were found within the entire region (excluding the 16S-23S intergenic spacer region encoding tRNA-Ile or tRNA-Ala). The pattern of nucleotide changes in the rrn operons corresponded to the classification of treponemal strains, whilst two different rrn spacer patterns (Ile/Ala and Ala/Ile) appeared to be distributed randomly across species/subspecies classification, time and geographical source of the treponemal strains. It is suggested that the random distribution of tRNA genes is caused by reciprocal translocation between repetitive sequences mediated by a recBCD-like system.

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A schematic representation of the treponemal rrn operons consisting of 16S–23S–5S rRNA genes, intergenic regions and a 212 bp DNA sequence upstream of 16S rRNA gene. The positions of the 33 and 17 bp deletions in the non-TPA strains are shown. Please note that both spacer patterns of the 16S–23S ISR encoding either tRNA-Ile or tRNA-Ala were present among TPA and TPE strains in the rrn1 and rrn2 regions. Green symbols represent tRNA-Ile, whilst red symbols represent tRNA-Ala. Small coloured arrows in green and purple represent primers used in the clinical samples examined.
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f1: A schematic representation of the treponemal rrn operons consisting of 16S–23S–5S rRNA genes, intergenic regions and a 212 bp DNA sequence upstream of 16S rRNA gene. The positions of the 33 and 17 bp deletions in the non-TPA strains are shown. Please note that both spacer patterns of the 16S–23S ISR encoding either tRNA-Ile or tRNA-Ala were present among TPA and TPE strains in the rrn1 and rrn2 regions. Green symbols represent tRNA-Ile, whilst red symbols represent tRNA-Ala. Small coloured arrows in green and purple represent primers used in the clinical samples examined.

Mentions: The composition of 16S–23S ISR sequences in the rrn1 and rrn2 operons, encoding either tRNA-Ile or tRNA-Ala, was determined by another nested PCR. In the first step, each clinical isolate was tested in four parallel reactions with the following primer pairs (Fig. 1 and Table S3): RNA1Fb and RNA1-tRNA-Ile (first reaction), RNA1Fb and RNA2-tRNA-Ala (second reaction), RNA2Fc and RNA1-tRNA-Ile (third reaction) and RNA2Fc and RNA2-tRNA-Ala (fourth reaction). Using these primer sets, the PCR products revealed the position (rrn1 or rrn2) and composition (tRNA-Ile or tRNA-Ala) of the amplified rrn operon. In the second step of the nested PCR, the PCR product of the rrn1 (from the first and second reactions) region was amplified using TP0225-6aF and TP0225-6bR primers, whilst the PCR product of the rrn2 (from the third and fourth reactions) region was amplified with RNA2Fa and TP0225-6bR. The second step was not specific for the Ile/Ala or Ala/Ile rrn spacer pattern but improved the sensitivity of detection of the PCR product from the first step. Each PCR contained 0.4 µl 10 mM dNTP mix, 2 µl 10× ThermoPol Reaction buffer (New England BioLabs), 0.1 µl each primer (100 pmol µl−1), 0.1 µl Taq DNA polymerase (5000 U ml−1; New England BioLabs), 1 µl test sample and 16.3 µl PCR-grade water, giving 20 µl in total. PCR amplification was performed using a GeneAmp 9800 thermocycler (Applied Biosystems) with the following cycling conditions: 94 °C for 5 min; 40 cycles of 94 °C for 60 s, 72 °C for 20 s and 72 °C for 150 s; and a final extension at 72 °C for 10 min. The second step of the nested PCR used the same conditions but a lower annealing temperature of 67 °C.


Structure of rrn operons in pathogenic non-cultivable treponemes: sequence but not genomic position of intergenic spacers correlates with classification of Treponema pallidum and Treponema paraluiscuniculi strains.

Cejková D, Zobaníková M, Pospísilová P, Strouhal M, Mikalová L, Weinstock GM, Smajs D - J. Med. Microbiol. (2012)

A schematic representation of the treponemal rrn operons consisting of 16S–23S–5S rRNA genes, intergenic regions and a 212 bp DNA sequence upstream of 16S rRNA gene. The positions of the 33 and 17 bp deletions in the non-TPA strains are shown. Please note that both spacer patterns of the 16S–23S ISR encoding either tRNA-Ile or tRNA-Ala were present among TPA and TPE strains in the rrn1 and rrn2 regions. Green symbols represent tRNA-Ile, whilst red symbols represent tRNA-Ala. Small coloured arrows in green and purple represent primers used in the clinical samples examined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3755535&req=5

f1: A schematic representation of the treponemal rrn operons consisting of 16S–23S–5S rRNA genes, intergenic regions and a 212 bp DNA sequence upstream of 16S rRNA gene. The positions of the 33 and 17 bp deletions in the non-TPA strains are shown. Please note that both spacer patterns of the 16S–23S ISR encoding either tRNA-Ile or tRNA-Ala were present among TPA and TPE strains in the rrn1 and rrn2 regions. Green symbols represent tRNA-Ile, whilst red symbols represent tRNA-Ala. Small coloured arrows in green and purple represent primers used in the clinical samples examined.
Mentions: The composition of 16S–23S ISR sequences in the rrn1 and rrn2 operons, encoding either tRNA-Ile or tRNA-Ala, was determined by another nested PCR. In the first step, each clinical isolate was tested in four parallel reactions with the following primer pairs (Fig. 1 and Table S3): RNA1Fb and RNA1-tRNA-Ile (first reaction), RNA1Fb and RNA2-tRNA-Ala (second reaction), RNA2Fc and RNA1-tRNA-Ile (third reaction) and RNA2Fc and RNA2-tRNA-Ala (fourth reaction). Using these primer sets, the PCR products revealed the position (rrn1 or rrn2) and composition (tRNA-Ile or tRNA-Ala) of the amplified rrn operon. In the second step of the nested PCR, the PCR product of the rrn1 (from the first and second reactions) region was amplified using TP0225-6aF and TP0225-6bR primers, whilst the PCR product of the rrn2 (from the third and fourth reactions) region was amplified with RNA2Fa and TP0225-6bR. The second step was not specific for the Ile/Ala or Ala/Ile rrn spacer pattern but improved the sensitivity of detection of the PCR product from the first step. Each PCR contained 0.4 µl 10 mM dNTP mix, 2 µl 10× ThermoPol Reaction buffer (New England BioLabs), 0.1 µl each primer (100 pmol µl−1), 0.1 µl Taq DNA polymerase (5000 U ml−1; New England BioLabs), 1 µl test sample and 16.3 µl PCR-grade water, giving 20 µl in total. PCR amplification was performed using a GeneAmp 9800 thermocycler (Applied Biosystems) with the following cycling conditions: 94 °C for 5 min; 40 cycles of 94 °C for 60 s, 72 °C for 20 s and 72 °C for 150 s; and a final extension at 72 °C for 10 min. The second step of the nested PCR used the same conditions but a lower annealing temperature of 67 °C.

Bottom Line: Other than these deletions, only 17 heterogeneous sites were found within the entire region (excluding the 16S-23S intergenic spacer region encoding tRNA-Ile or tRNA-Ala).The pattern of nucleotide changes in the rrn operons corresponded to the classification of treponemal strains, whilst two different rrn spacer patterns (Ile/Ala and Ala/Ile) appeared to be distributed randomly across species/subspecies classification, time and geographical source of the treponemal strains.It is suggested that the random distribution of tRNA genes is caused by reciprocal translocation between repetitive sequences mediated by a recBCD-like system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, Building A6, 62500 Brno, Czech Republic.

ABSTRACT
This study examined the sequences of the two rRNA (rrn) operons of pathogenic non-cultivable treponemes, comprising 11 strains of T. pallidum ssp. pallidum (TPA), five strains of T. pallidum ssp. pertenue (TPE), two strains of T. pallidum ssp. endemicum (TEN), a simian Fribourg-Blanc strain and a rabbit T. paraluiscuniculi (TPc) strain. PCR was used to determine the type of 16S-23S ribosomal intergenic spacers in the rrn operons from 30 clinical samples belonging to five different genotypes. When compared with the TPA strains, TPc Cuniculi A strain had a 17 bp deletion, and the TPE, TEN and Fribourg-Blanc isolates had a deletion of 33 bp. Other than these deletions, only 17 heterogeneous sites were found within the entire region (excluding the 16S-23S intergenic spacer region encoding tRNA-Ile or tRNA-Ala). The pattern of nucleotide changes in the rrn operons corresponded to the classification of treponemal strains, whilst two different rrn spacer patterns (Ile/Ala and Ala/Ile) appeared to be distributed randomly across species/subspecies classification, time and geographical source of the treponemal strains. It is suggested that the random distribution of tRNA genes is caused by reciprocal translocation between repetitive sequences mediated by a recBCD-like system.

Show MeSH
Related in: MedlinePlus