کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8304299 | 1538404 | 2016 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Subunit orientation in the Escherichia coli enterobactin biosynthetic EntA-EntE complex revealed by a two-hybrid approach
دانلود مقاله + سفارش ترجمه
دانلود مقاله ISI انگلیسی
رایگان برای ایرانیان
کلمات کلیدی
PVDFChrome azurol SCAS assayBACTHNRPSDHBCASHRPDTTIPTG2,3-dihydroxybenzoic acid - 2،3-دی هیدروکسی بنزوئیک اسیدenterobactin - EnterobactinSDS-polyacrylamide gel electrophoresis - الکتروفورز ژل SDS-polyacrylamide gelSDS-PAGE - الکتروفورز ژل پلی آکریل آمیدisopropyl β-D-1-thiogalactopyranoside - ایزوپروپیل β-D-1-thiogalactopyranosideProtein-protein interactions - تعاملات پروتئین-پروتئینFerric uptake regulator - تنظیم کننده جذب آهنFur - خزbacterial two-hybrid - دوبرابر باکتریاییpolyvinylidene difluoride - دی فلوئورید پلی وینیلیدینdithiothreitol - دیتیوتریتولnon-ribosomal peptide synthesis - سنتز پپتید غیر ریبوزومیSiderophore - سیدرفورHorseradish peroxidase - پراکسیداز هوررادیش
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
The siderophore enterobactin is synthesized by the enzymes EntA-F and EntH in the Escherichia coli cytoplasm. We previously reported in vitro evidence of an interaction between tetrameric EntA and monomeric EntE. Here we used bacterial adenylate cyclase two-hybrid (BACTH) assays to demonstrate that the E. coli EntA-EntE interaction occurs intracellularly. Furthermore, to obtain information on subunit orientation in the EntA-EntE complex, we fused BACTH reporter fragments T18 and T25 to EntA and EntE in both N-terminal and C-terminal orientations. To validate functionality of our fusion proteins, we performed Chrome Azurol S (CAS) assays using E. coli entEâ and entAâ knockout strains transformed with our BACTH constructs. We found that transformants expressing N-terminal and C-terminal T18/T25 fusions to EntE exhibited CAS signals, indicating that these constructs could rescue the entEâ phenotype. While expression of EntA with N-terminal T18/T25 fusions exhibited CAS signals, C-terminal fusions did not, presumably due to disruption of the EntA tetramer in vivo. Bacterial growth assays supported our CAS findings. Co-transformation of functional T18/T25 fusions into cyaâE. coli BTH101 cells resulted in positive BACTH signals only when T18/T25 fragments were fused to the N-termini of both EntA and EntE. Co-expression of N-terminally fused EntA with C-terminally fused EntE resulted in no detectable BACTH signal. Analysis of protein expression by Western blotting confirmed that the loss of BACTH signal was not due to impaired expression of fusion proteins. Based on our results, we propose that the N-termini of EntA and EntE are proximal in the intracellular complex, while the EntA N-terminus and EntE C-terminus are distal. A protein-protein docking simulation using SwarmDock was in agreement with our experimental observations.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimie - Volume 127, August 2016, Pages 1-9
Journal: Biochimie - Volume 127, August 2016, Pages 1-9
نویسندگان
Paknoosh Pakarian, Peter D. Pawelek,