Structural peculiarities of linear megaplasmid, pLMA1, from Micrococcus luteus interfere with pyrosequencing reads assembly

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Micrococcus luteus strains from high-altitude Argentinean wetlands possess linear plasmids (pLMA1, pLMH5, pLMV7) with terminal proteins.
• The pLMA1 plasmid is associated with erythromycin resistance in its host.

Purpose of the Study:

• To perform a detailed characterization of the pLMA1 linear plasmid.
• To understand the genetic makeup and potential functions of pLMA1.

Main Methods:

• Direct sequencing of gel-purified pLMA1 using 454 technology.
• Combined Sanger and 454 sequencing of cloned pLMA1 fragments (23 kb analyzed).
• Bioinformatic analysis to identify sequence repeats, transposase genes, and iteron sequences.

Main Results:

• Direct 454 sequencing of pLMA1 failed due to its complex structure.
• Combined sequencing revealed numerous sequence repeats of varying lengths, explaining the assembly failure.
• Identification of multiple putative transposase genes and a region with five putative iteron sequences potentially involved in replication.

Conclusions:

• The complex repetitive structure of pLMA1 poses challenges for direct high-throughput sequencing.
• pLMA1 harbors genes potentially involved in transposition and replication, contributing to its genetic characteristics.