TY - JOUR
T1 - Cloning of a functional Salmonella SPI-1 type III secretion system and development of a method to create mutations and epitope fusions in the cloned genes
AU - Wilson, James W.
AU - Nickerson, Cheryl A.
N1 - Funding Information:
We thank Dr. Ed Oaks, Dr. William Picking, and Dr. Wendy Picking for generously providing α-SipC antisera for use in this study. We thank Dr. David H. Figurski for generously providing the plasmid pMAK705 and α-KleA antisera. This work was supported by NASA-Ames Grant NAG 2-1378.
PY - 2006/3/23
Y1 - 2006/3/23
N2 - Bacterial type III secretion systems have significant potential to be harnessed for beneficial purposes including vaccine development, anti-cancer therapies, strategies to counteract harmful bacteria-host interactions, and evolutionary studies. The ability to clone and manipulate type III secretion systems would allow researchers to perform novel experiments that would progress the biotechnological development of the potentially positive uses of these systems. Here, we report the cloning of the entire Salmonella pathogenicity island 1 (SPI-1) type III secretion system on a single DNA fragment that is contained on a self-transmissible plasmid vector for convenient transfer to alternate hosts. We demonstrate that the cloned SPI-1 type III system is functional for secretion and translocation via complementation of an S. typhimurium Δ SPI-1 strain. We also present a convenient method to construct mutations and epitope fusions in the cloned type III genes and demonstrate that the engineered substrate protein fusions are recognized by the cloned type III system. We transferred the cloned SPI-1 type III system into bacterial strains of different genera and found that there is a SPI-1 gene expression defect in these strains. The results describe a novel strategy for cloning and manipulation of bacterial secretion system gene clusters and provide a foundation for future studies to develop the beneficial uses of cloned type III secretion systems.
AB - Bacterial type III secretion systems have significant potential to be harnessed for beneficial purposes including vaccine development, anti-cancer therapies, strategies to counteract harmful bacteria-host interactions, and evolutionary studies. The ability to clone and manipulate type III secretion systems would allow researchers to perform novel experiments that would progress the biotechnological development of the potentially positive uses of these systems. Here, we report the cloning of the entire Salmonella pathogenicity island 1 (SPI-1) type III secretion system on a single DNA fragment that is contained on a self-transmissible plasmid vector for convenient transfer to alternate hosts. We demonstrate that the cloned SPI-1 type III system is functional for secretion and translocation via complementation of an S. typhimurium Δ SPI-1 strain. We also present a convenient method to construct mutations and epitope fusions in the cloned type III genes and demonstrate that the engineered substrate protein fusions are recognized by the cloned type III system. We transferred the cloned SPI-1 type III system into bacterial strains of different genera and found that there is a SPI-1 gene expression defect in these strains. The results describe a novel strategy for cloning and manipulation of bacterial secretion system gene clusters and provide a foundation for future studies to develop the beneficial uses of cloned type III secretion systems.
KW - Bacterial engineering
KW - Broad-host-range
KW - SPI-1
KW - Salmonella
KW - Type III secretion system
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U2 - 10.1016/j.jbiotec.2005.09.005
DO - 10.1016/j.jbiotec.2005.09.005
M3 - Article
C2 - 16253373
AN - SCOPUS:33644541402
SN - 0168-1656
VL - 122
SP - 147
EP - 160
JO - Journal of Biotechnology
JF - Journal of Biotechnology
IS - 2
ER -