Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 79, Issue 1, Pages 23-31Publisher
ELSEVIER
DOI: 10.1016/j.mimet.2009.07.013
Keywords
Desulfovibrio africanus; Plasmid; Mobilization; Partitioning
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Funding
- U.S. Department of Energy, Environmental Remediation Science Program (ERSP)
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To develop a vector system that facilitates genetic manipulation in Desulfovibrio species, we screened native sulfate-reducing bacteria for small plasmids. A self-replicating plasmid was discovered in Desulfovibrio africanus SR-1. Sequence analysis of this 8568-bp plasmid (pNC1) revealed a G+C content of 47.2% and nine open reading frames. This plasmid has a copy number of six. Compatible hosts include D. africanus and Pseudomonas aeruginosa PA14. Genetic characterization of pNC1 revealed that 53.6% of the plasmid contains genes associated with replication, mobilization, and partitioning. The 1123-bp replicon is composed of a rep gene and four 22-bp iterons. The mobilization operon is composed of three genes with a putative 144-bp oriT The partitioning operon is composed of parA and parB with a downstream parS. We report the construction of a small pNC1-based cloning vector which transforms D. africanus at high frequencies (similar to 1.5 x 10(3) CFU/mu g DNA), is mobilizable at high transfer frequency (4.8 x 10(-4) transconjugants/donor), and is stably maintained under non-selective pressure. This study provides a potential host-vector system for Desulfovibrio gene functional analyses. (C) 2009 Elsevier B.V. All rights reserved.
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