PROIMI   05436
PLANTA PILOTO DE PROCESOS INDUSTRIALES MICROBIOLOGICOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The linear plasmid, pLMA1, from Micrococcus luteus: Structural peculiarities interfere with pyrosequencing reads assembly
Autor/es:
WAGENKNECHT, MARTIN; DIB, JULIÁN RAFAEL; THÜRNER, ANDREA; DANIEL, ROLF; FARÍAS, MARIA EUGENIA; MEINHARDT, FRIEDHELM
Lugar:
Dubrovnic
Reunión:
Workshop; Mass Spectrometry in Biotechnology & Medicine; 2011
Resumen:
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: 4045
[3] Wagenknecht et al. (2010) Biotechnology Letters 32: 18531862