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Discovery of the Autonomously Replicating Plasmid pMF1 from Myxococcus fulvus and Development of a Gene Cloning System in Myxococcus xanthus

Jing-yi Zhao,^1, Li Zhong,^2, Mei-juan Shen,² Zhi-jie Xia,¹ Qiu-xiang Cheng,² Xia Sun,¹ Guo-ping Zhao,² Yue-zhong Li,^1* and Zhong-jun Qin^2*

State Key Laboratory of Microbial Technology, College of Life Science, Shandong University, Jinan 250100,¹ Shanghai Institute of Plant Physiology, Shanghai Institutes of Biological Science, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China²

Received 16 September 2007/ Accepted 23 January 2008

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies (10⁵ CFU/µg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.

Published ahead of print on 1 February 2008.

J.-Y.Z. and L.Z. contributed equally to this work.