Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing

Area of Science:

• Molecular Biology
• Microbiology
• Genomics

Background:

• Mycoplasma identification and classification are crucial for understanding microbial diversity and host-pathogen interactions.
• Previous methods for mycoplasma identification and phylogenetic analysis were often time-consuming or lacked resolution.
• 16S ribosomal DNA (rDNA) sequencing is a standard tool for bacterial taxonomy, but its application to mycoplasmas required optimization.

Purpose of the Study:

• To determine partial 16S ribosomal DNA sequences for 11 previously unsequenced mycoplasmas.
• To establish phylogenetic relationships among these mycoplasmas using DNA sequence information.
• To evaluate automated solid-phase DNA sequencing as a tool for mycoplasma identification and taxonomic studies.

Main Methods:

• Automated solid-phase DNA sequencing was employed for partial 16S ribosomal DNA (rDNA) determination.
• Polymerase chain reaction (PCR) generated a biotinylated DNA fragment (positions 344-939 of E. coli sequence).
• The PCR product was immobilized on streptavidin-coated paramagnetic beads for direct, bidirectional sequencing.

Main Results:

• Phylogenetic analysis revealed an unclassified avian mycoplasma belongs to the Mycoplasma lipophilum cluster.
• Microheterogeneities in rRNA operons of Mycoplasma mycoides subsp. mycoides (SC type) confirmed two distinct rRNA operons.
• Automated sequencing identified Mycoplasma hyorhinis contamination in cell culture, a task difficult for conventional methods.

Conclusions:

• Automated solid-phase DNA sequencing is a powerful and efficient method for mycoplasma identification.
• The technique provides valuable sequence data for establishing accurate phylogenetic relationships among mycoplasmas.
• This approach offers a significant advancement for both routine mycoplasma identification and in-depth taxonomic research.