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.
Castañeda-Carrión
I Nydia
IN
Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA.
Whiteley
Marvin
M
Krumholz
Lee R
LR
D001482:Base Composition; D003227:Conjugation, Genetic; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D045284:Desulfovibrio africanus / Q000235:genetics*; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D016366:Open Reading Frames; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial