The linear plasmid, pLMA1, from Micrococcus luteus: Structural peculiarities interfere with pyrosequencing reads assembly

WAGENKNECHT, MARTIN; DIB, JULIÁN RAFAEL; THÜRNER, ANDREA; DANIEL, ROLF; FARÍAS, MARIA EUGENIA; MEINHARDT, FRIEDHELM

Dubrovnic

Workshop; Mass Spectrometry in Biotechnology & Medicine; 2011

Introduction Microbial linear plasmids are a rather widespread class of extrachromosomal DNA elements. These replicons frequently occur in Gram-positive bacteria, such as streptomycetes and rhodococci [1]. Recently, we isolated the first linear plasmids (pLMA1, pLMH5, and pLMV7) from different strains of Micrococcus luteus [2]. The bacteria originate from Argentinean high-altitude wetlands – pristine and extreme environments that are characterized by, e.g., high arsenic concentration and UV radiation. Accordingly, above micrococci display high heavy metal and UV tolerance and, unexpectedly, resistance against a number of antibiotics. To check if such attributes are plasmid-encoded and to verify a suggested link between erythromycin resistance and pLMA1 [2], we decided to determine exemplarily the nucleotide sequence of pLMA1. Methods pLMA1 DNA was isolated using pulsed-field gel electrophoresis [2]. Cloning of pLMA1 restriction fragments, PCR amplifications, and Southern blotting were done according to standard protocols. The sequencing was performed employing Sanger and 454 pyrosequencing technology (Roche, Mannheim). Obtained sequences were analyzed by applying standard bioinformatic tools. Results When isolated pLMA1 DNA was subjected to 454 pyrosequencing attempts to assemble the obtained reads encountered insurmountable obstacles [3]. Combined Sanger/454 sequencing of cloned pLMA1 fragments, covering 23 kb of the 110-kb-comprising plasmid, enabled us to identify a large number of repetitive sequences, thus, providing an explanation for the failure in the reads assembly. Moreover, numerous putative transposase encoding genes were identified. Recently, we have determined the entire pLMA1 nucleotide sequence using solely the Sanger technology. The, yet preliminary, plasmid sequence revealed several open reading frames (ORF) with the potential to confer resistance to heavy metals. We also identified an ORF that codes for a putative rRNA methylase presumably conveying the observed erythromycin resistance. Conclusions The high number of ORFs involved in transposition suggests that pLMA1 is a very flexible element. Moreover, the genetic equipment provided by pLMA1 presumably allows its host to survive the extreme environmental conditions. References [1] Meinhardt and Klassen (2007) Microbial linear plasmids, Microbiology Monographs, vol. 7 [2] Dib et al. (2010) Plasmid 63: 40–45 [3] Wagenknecht et al. (2010) Biotechnology Letters 32: 1853–1862