A Chromosomally Integrated T7 RNA Polymerase Enables T7-Derived Expression in Salmonella enterica without Compromising Virulence
0
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
The T7 RNA polymerase (T7 RNAP) system has revolutionized protein expression in Escherichia coli due to its high transcriptional activity and tight regulation. However, Salmonella enterica, despite its close genetic similarity to E. coli, lacks a T7 RNAP system, limiting the use of T7-based vectors and tools in this pathogen. Establishing a T7-compatible Salmonella strain would enable the seamless application of E. coli-optimized expression systems for studies in a pathogenic context. We engineered S. enterica serovar Typhimurium strain 14028s to stably express T7 RNAP from the chromosome under the control of the lac promoter using the pGRG36 transposon system. The resulting strain (Salmonella-T7) supports robust IPTG-inducible expression of heterologous proteins from T7 promoterdriven vectors, such as the pET series. Salmonella-T7 exhibited growth kinetics comparable to wildtype Salmonella in both rich and minimal media, indicating no detectable fitness cost. Furthermore, macrophage infection assays and murine infection models demonstrated that chromosomal integration of T7 RNAP does not compromise virulence. The engineered Salmonella-T7 strain enables efficient use of T7-based expression systems in S. enterica without affecting bacterial physiology or pathogenicity. This platform provides a valuable tool for studying bacterial pathogenesis as well as applications in synthetic biology and vaccine development.
Abstract
The T7 RNA polymerase (T7 RNAP) system has revolutionized protein expression in Escherichia coli due to its high transcriptional activity and tight regulation. However, Salmonella enterica, despite its close genetic similarity to E. coli, lacks a T7 RNAP system, limiting the use of T7-based vectors and tools in this pathogen. Establishing a T7-compatible Salmonella strain would enable the seamless application of E. coli-optimized expression systems for studies in a pathogenic context. We engineered S. enterica serovar Typhimurium strain 14028s to stably express T7 RNAP from the chromosome under the control of the lac promoter using the pGRG36 transposon system. The resulting strain (Salmonella-T7) supports robust IPTG-inducible expression of heterologous proteins from T7 promoterdriven vectors, such as the pET series. Salmonella-T7 exhibited growth kinetics comparable to wildtype Salmonella in both rich and minimal media, indicating no detectable fitness cost. Furthermore, macrophage infection assays and murine infection models demonstrated that chromosomal integration of T7 RNAP does not compromise virulence. The engineered Salmonella-T7 strain enables efficient use of T7-based expression systems in S. enterica without affecting bacterial physiology or pathogenicity. This platform provides a valuable tool for studying bacterial pathogenesis as well as applications in synthetic biology and vaccine development.