PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

Abstract

An efficient electroporation procedure for Vibrio vulnificus was designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 10(6) transformants per μg DNA were achieved. The vector stably replicated in both V. vulnificus and Escherichia coli and was also successfully introduced into Vibrio parahaemolyticus and Vibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and the vvhBA hemolysin operon were inserted into the vector and functionally expressed in Vibrio and E. coli.

The diverse genetic switch of enterobacterial and marine telomere phages

“…The VP58.5 immB region harbors 2 genes with prophage repressor activity To study the activity of the regulatory proteins in Vibrio, the immB genes were inserted into the shuttle vector pVv3 containing the lac promoter 19 and introduced into V. parahaemolyticus strain 37.5 by electroporation. Thereafter, transformants were infected with phage VP58.5.…”

“…19 After molecular cloning in E. coli K12, the construct was cleaved with BpiI, which cuts ORF44 twice. Following this, the chloramphenicol acetyltransferase gene (cat) of pBR329 was amplified by PCR and inserted into ORF44.…”

“…Upon transformation of E. coli K12 the construct was isolated from a chloramphenicol resistant colony. Thereafter, it was introduced into V. parahaemolyticus 37.5 by electroporation applying the protocol of Klevanskaa et al 19 Transformants harboring the recombinant plasmid were infected with VP58.5. Lysates were purified and plated on V. parahaemolyticus 37.5.…”

“…19 To accomplish this, the regulatory genes were amplified by PCR using phage DNA as template. The forward and reverse primers contained embedded restriction sites for NdeI and HindIII (pMS470D8Cat) or BamHI and HindIII (pVv3), respectively.…”

Improved Method for Transformation of Vibrio vulnificus by Electroporation

“…One of the greatest obstacles hindering molecular analyses of V. vulnificus pathogenesis is the lack of efficient methods for introduction of foreign DNA into this bacterium (Gulig, Tucker, Thiaville, Joseph, & Brown, 2009;Klevanskaa, Bier, Stingl, Strauch, & Hertwig, 2014;McDougald, Simpson, Oliver, & Hudson, 1994). Attempts to transform V. vulnificus by chemical transformation or electroporation have met limited success (Mc-Dougald et al, 1994).…”