Das Bhabatosh, Kumari Reena, Pant Archana, Sen Gupta Sourav, Saxena Shruti, Mehta Ojasvi, Nair Gopinath Balakrish
Centre for Human Microbial Ecology, Translational Health Science and Technology Institute, Gurgaon, Haryana, India
Centre for Human Microbial Ecology, Translational Health Science and Technology Institute, Gurgaon, Haryana, India.
J Bacteriol. 2014 Dec;196(23):4071-80. doi: 10.1128/JB.01966-14. Epub 2014 Sep 15.
CTXΦ, a filamentous vibriophage encoding cholera toxin, uses a unique strategy for its lysogeny. The single-stranded phage genome forms intramolecular base-pairing interactions between two inversely oriented XerC and XerD binding sites (XBS) and generates a functional phage attachment site, attP(+), for integration. The attP(+) structure is recognized by the host-encoded tyrosine recombinases XerC and XerD (XerCD), which enables irreversible integration of CTXΦ into the chromosome dimer resolution site (dif) of Vibrio cholerae. The dif site and the XerCD recombinases are widely conserved in bacteria. We took advantage of these conserved attributes to develop a broad-host-range integrative expression vector that could irreversibly integrate into the host chromosome using XerCD recombinases without altering the function of any known open reading frame (ORF). In this study, we engineered two different arabinose-inducible expression vectors, pBD62 and pBD66, using XBS of CTXΦ. pBD62 replicates conditionally and integrates efficiently into the dif of the bacterial chromosome by site-specific recombination using host-encoded XerCD recombinases. The expression level of the gene of interest could be controlled through the PBAD promoter by modulating the functions of the vector-encoded transcriptional factor AraC. We validated the irreversible integration of pBD62 into a wide range of pathogenic and nonpathogenic bacteria, such as V. cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Gene expression from the PBAD promoter of integrated vectors was confirmed in V. cholerae using the well-studied reporter genes mCherry, eGFP, and lacZ.
CTXΦ是一种编码霍乱毒素的丝状弧菌噬菌体，它采用独特的溶原策略。单链噬菌体基因组在两个反向排列的XerC和XerD结合位点（XBS）之间形成分子内碱基配对相互作用，并产生用于整合的功能性噬菌体附着位点attP(+)。attP(+)结构被宿主编码的酪氨酸重组酶XerC和XerD（XerCD）识别，这使得CTXΦ能够不可逆地整合到霍乱弧菌的染色体二聚体解离位点（dif）。dif位点和XerCD重组酶在细菌中广泛保守。我们利用这些保守特性开发了一种广宿主范围的整合表达载体，该载体可以使用XerCD重组酶不可逆地整合到宿主染色体中，而不会改变任何已知开放阅读框（ORF）的功能。在本研究中，我们利用CTXΦ的XBS构建了两种不同的阿拉伯糖诱导型表达载体pBD62和pBD66。pBD62有条件地复制，并通过使用宿主编码的XerCD重组酶进行位点特异性重组，有效地整合到细菌染色体的dif中。通过调节载体编码的转录因子AraC的功能，可以通过PBAD启动子控制目的基因的表达水平。我们验证了pBD62在多种致病性和非致病性细菌中的不可逆整合，如霍乱弧菌、河流弧菌、副溶血性弧菌、大肠杆菌、肠炎沙门氏菌和肺炎克雷伯菌。使用经过充分研究的报告基因mCherry、eGFP和lacZ在霍乱弧菌中证实了整合载体的PBAD启动子的基因表达。
