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 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 ‐sensitive phenotype. We also performed a gene expression study using the P‐glow BS2 fluorescent reporter gene and the ahpC promoter in B. thetaiotaomicron.

中文翻译：

---

用于在卟啉单胞菌，拟杆菌和相关细菌中进行分子克隆的穿梭载体的序列和表征。

对于牙龈卟啉单胞菌和相关物种的遗传学研究，缺少穿梭载体。为了更好地了解使用这种工具的人的普遍性，我们检查了多种黑色色素厌氧菌菌株（临床和实验室分离株）的质粒。由于没有质粒中发现的牙龈卟啉菌的菌株，我们已经使用了pYH420质粒，衍生自P. asaccharolytica，作为主链构建的穿梭载体中从与pUC19的组合大肠杆菌。对pYH420质粒的核苷酸序列测定表明，该质粒含有与质粒pTS1 rep（分离自密螺旋体）的rep相似的基因。）以及MobA的同源物，该基因家族的一个成员是属于拟杆菌属的可动员遗传元件。我们使用部分pUC19和pYH420载体构建了pG106和pG108穿梭载体。这导致在lacZ基因中具有多克隆位点（MCS）的载体，使我们能够进行蓝白菌落选择。pG106和pG108穿梭载体可电转化为大肠杆菌，牙龈卟啉单胞菌和B. thetaiotaomicron，它们在哪里稳定。我们证明了这些载体适用于这些物种中的分子克隆应用，包括互补和基因表达研究。使用pG108载体，我们补充了牙龈卟啉单胞菌的hcpR突变株，并拯救了其敏感表型。我们还使用了P-glow BS2荧光报告基因和thetaiotaomicron中的ahpC启动子进行了基因表达研究。