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Kwack Kyu Hwan,Lee Jae-Hyung,Moon Ji-Hoi 한국미생물학회 2022 The journal of microbiology Vol.60 No.2
“Antibiotic tolerance” promotes the rapid subsequent evolution of “antibiotic resistance,” however, it is often overlooked because it is difficult to distinguish between tolerant and susceptible organisms. A commensal bacterium S. anginosus subsp. anginosus strain KHUD_S1, isolated from dental biofilm was found to exhibit a high MBC/MIC ratio of 32 against vancomycin. We observed KHUD_S1 cells exposed to vancomycin did not grow but maintained viability. Transmission electron microscope showed KHUD_S1 cells possessed a dense, thick capsule and maintained the cell wall integrity upon vancomycin exposure. To infer the underlying mechanisms of the vancomycin tolerance in KHUD_S1, we performed whole genome sequencing and RNA sequencing. The KHUD_S1 genome carried three genes encoding branching enzymes that can affect peptidoglycan structure through interpeptide bridge formation. Global gene expression profiling revealed that the vancomycin-induced downregulation of carbohydrate and inorganic ion transport/metabolism as well as translation is less prominent in KHUD_S1 than in the vancomycin susceptible strain KHUD_S3. Based on the transcriptional levels of genes related to peptidoglycan synthesis, KHUD_S1 was determined to have a 3D peptidoglycan architecture distinct from KHUD_S3. It was found that, under vancomycin exposure, the peptidoglycan was remodeled through changes in the interpeptide bridge and transpeptidation reactions. Collectively, these features of S. anginosus KHUD_S1, including a dense capsule and differential gene expression in peptidoglycan synthesis, may contribute to vancomycin tolerance. Our results showing the occurrence of vancomycin tolerance amongst oral commensal bacteria highlight the need for considering future strategies for screening of antibiotic tolerance as an effort to reduce antibiotic resistance.
대장균에서 인간의 면역 인터페론을 tac 프로모터를 이용하여 고도로 발현시키는 연구
곽규범,현형환,권병세,김광수 ( Kyu Bum Kwack,Hyung Hwan Hyun,Byung S . Kwon,Kwang Soo Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.3
The cDNA for the structural sequence of mature human mmune-interferon (IFN-γ) was introduced into E. coli high expression vector, pKK223-3,constructed by using tac promoter and rrnBT1T2 transcription terminator region and transformed to host, E. coli JM105, which has lacIq repressor gene on chromosomal DNA, E. coli MM294, and E. coli VMC9, which have laI repressor gene. The resultant ampicillin resistant transformants were analyzed by the immunoblotting and electron microscope. IFN-γ, which cross-reacted with mouse anti human IFN-γ serum, was produced as the soluble and the insoluble inclusion form. Their productions were efficiently regulated in E. coli JM105, but not in E. coli MM294, and E. coli YMC9 by using the IPTG.
High-level Expression of Human Immune Interferon in Escherichia coli by using tac Promoter
곽규범,현형환,권병세,김광수,Kwack, Kyu-Bum,Hyun, Hyung-Hwan,Kwon, Byung-S.,Kim, Kwang-Soo 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.3
인간의 면역 인터페론을 코딩하는 유전자를 tac 프로모터와 rrnBT1T2 전사중지서열을 갖는 프라스미드 pKK223-3에 클론닝하여 체세포 유전자에 lacIq 제한차서열을 갖는 대장균 JM105와 체세포 유전자에 lacIq 제한자서열을 갖는 대장균 MM294와 대장균 YMC9에 각각 형질전환시켰다. 생성된 형질전환주를 immunoblotting과 생물학적 역가분석, 전기영동 그리고 전자현미경을 사용하여 확인했다. 면역 인터페론의 발현은 대장균 JM105에서 효율적으로 조절되었으며, 대장균 MM294와 대장균 YMC9에서는 IPTG의 처리없이도 생산됨을 발견하였다. 또한 면역 인터페론이 용해된 형태와 불용성 상태인 inclusion body로 생성됨을 알았다. 이때 생산성은 $9.7{\times}10^9IU/l$이며 면역 인터페론 단백질이 대장균의 전체 단백질중 약 30%에 해당함을 알았다. The cDNA for the structural sequence of mature human mmune-interferon ($IFN-{\gamma}$) was introduced into E. coli high expression vector, pKK223-3, constructed by using tac promoter and rrnBT1T2 transcription terminator region and transformed to host, E. coli JM105, which has lacIq repressor gene on chromosomal DNA, E. coli MM294, and E. coli VMC9, which have laI repressor gene. The resultant ampicillin resistant transformants were analyzed by the immunoblotting and electron microscope. $IFN-{\gamma}$, which cross-reacted with mouse anti human $IFN-{\gamma}$ serum, was produced as the soluble and the insoluble inclusion form. Their productions were efficiently regulated in E. coli JM105, but not in E. coii MM294, and E. coli YMC9 by using the IPTG.