Sequence and characterization of shuttle vectors for molecular cloning in Porphyromonas, Bacteroides, and related bacteria

AbstractThere is a lack of shuttle vectors to be needed for investigations into the genetics ofPorphyromonas gingivalis and related species. To better understand the prevalence of candidates for such tools we have examined multiple strains of black pigmented anaerobes (clinical and laboratory isolates) for plasmids. As no plasmids were found inP. gingivalis strains, we have used the pYH420 plasmid, derived fromP. asaccharolytica, as backbone to construct a shuttle vector in combination with pUC19 fromEscherichia coli. Nucleotide sequence determination of the pYH420 plasmid revealed that that it contained a gene with similarity torep from plasmid pTS1 (isolated fromTreponema denticola) as well as a homolog ofmobA, a member of a gene family found on mobilizable genetic elements found in the genusBacteroides. We constructed the pG106 and pG108 shuttle vectors using parts of the pUC19 and pYH420 vectors. This resulted in a vector with a multiple cloning site (MCS) in thelacZ gene enabling us to perform blue ‐white colony selection. The pG106 and pG108 shuttle vectors are electro‐transformable intoE. coli, P. gingivalis, andB. thetaiotaomicron, where they are stable. We demonstrated that these vectors were suitable in these species for applications of molecular cloning including complementation and gene expression studies. Using the pG108 vector we complement thehcpR mutant strain ofP. gingivalis and rescued its NO2‐ sensitive phenotype. We also performed a gene expression s...
Source: Molecular Oral Microbiology - Category: Microbiology Authors: Tags: ORIGINAL ARTICLE Source Type: research