Copy number determination, expression analysis of genes potentially involved in replication, and stability assays of pAL1-the linear megaplasmid of Arthrobacter nitroguajacolicus Ru61a

Wagenknecht M, Meinhardt F

Abstract

pAL1 of the actinomycete Arthrobacter nitroguajacolicus Ru61a, a 113-kb catabolic linear megaplasmid of the invertron-type, enables its host to utilize quinaldine (or 4-hydroxyquinaldine) as the sole carbon and energy source. Comparative Southern hybridization and quantitative real-time PCR analyses revealed copy numbers of two for cells grown in minimal medium supplemented with 4-hydroxyquinaldine and four for cells cultivated in Luria-Bertani medium. However, medium-dependent differences in the expression levels of genes potentially involved in plasmid replication were not seen. As replication and maintenance of invertrons may depend on single-stranded DNA-binding proteins (SSBs), ORF39, the gene product of which displaying similarities to SSBs, was knocked out. However, structural stability and replication of pAL1 was not influenced. When plasmid maintenance of LB-grown wild-type cells was assessed, it was seen that after approx. 200 generations almost 80% of the cells were devoid of plasmid, indicating segregational instability of pAL1. (C) 2010 Elsevier GmbH. All rights reserved.

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