Characterization of pNC1, a small and mobilizable plasmid for use in genetic manipulation of Desulfovibrio africanus.

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 (approximately 1.5 x 10(3) CFU/microg 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.