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Applied and Environmental Microbiology, January 2000, p. 54-63, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

Masayuki Inui, Jung Hyeob Roh, Kenneth Zahn, and Hideaki Yukawa*

Research Institute of Innovative Technology for the Earth, Soraku, Kyoto 619-0292, Japan

Received 30 August 1999/Accepted 22 October 1999

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of alpha -proteobacteria.

* Corresponding author. Mailing address: Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu, Soraku, Kyoto 619-0292 Japan. Phone: 81-774-75-2308. Fax: 81-774-75-2321. E-mail: yukawa{at}rite.or.jp.

Applied and Environmental Microbiology, January 2000, p. 54-63, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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