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

There is a lack of shuttle vectors to be needed for investigations into the genetics of Porphyromonas 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 in P. gingivalis strains, we have used the pYH420 plasmid, derived from P. asaccharolytica, as backbone to construct a shuttle vector in combination with pUC19 from Escherichia coli. Nucleotide sequence determination of the pYH420 plasmid revealed that that it contained a gene with similarity to rep from plasmid pTS1 (isolated from Treponema denticola) as well as a homolog of mobA, a member of a gene family found on mobilizable genetic elements found in the genus Bacteroides. 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 the lacZ gene enabling us to perform blue-white colony selection. The pG106 and pG108 shuttle vectors are electro-transformable into E. coli, P. gingivalis, and B. 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 the hcpR mutant strain of P. gingivalis and rescued its NO2- sensitive phenotype. We also performed a gene expression study using the P-glow BS2 fluorescent reporter gene and the ahpC promoter in B. thetaiotaomicron.