Ying Li, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing,# 1 Adrien Boes, Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – review & editing,# 2 , ¤ Yuanyuan Cui, Investigation, Methodology, 1 Shan Zhao, Investigation, Methodology, 1 Qingzhen Liao, Investigation, Methodology, 1 Han Gong, Investigation, Methodology, Validation, 1 Eefjan Breukink, Methodology, Resources, Writing – review & editing, 3 Joe Lutkenhaus, Conceptualization, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing, 4 Mohammed Terrak, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing, 2 ,* and Shishen Du, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing 1 ,*
"1 Department of Microbiology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
2 InBioS-Centre d’Ingénierie des Protéines, Liège University, Liège, Belgium
3 Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands
4 Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, United States of America
Indiana University, UNITED STATES,
The authors have declared that no competing interests exist.
¤Current address: CER Groupe—Immunobiology Laboratory, Novalis Science Park, Aye, Belgium
* E-mail: [email protected] (MT); [email protected] (SD)"
"SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan (PG) glycosyltransferases that form complexes with class B penicillin-binding proteins (bPBPs, with transpeptidase activity) to synthesize PG during bacterial cell growth and division. Because of their crucial roles in bacterial morphogenesis, SEDS proteins are one of the most promising targets for the development of new antibiotics. However, how SEDS proteins recognize their substrate lipid II, the building block of the PG layer, and polymerize it into glycan strands is still not clear. In this study, we isolated and characterized dominant-negative alleles of FtsW, a SEDS protein critical for septal PG synthesis during bacterial cytokinesis. Interestingly, most of the dominant-negative FtsW mutations reside in extracellular loops that are highly conserved in the SEDS family. Moreover, these mutations are scattered around a central cavity in a modeled FtsW structure, which has been proposed to be the active site of SEDS proteins. Consistent with this, we found that these mutations blocked septal PG synthesis but did not affect FtsW localization to the division site, interaction with its partners nor its substrate lipid II. Taken together, these results suggest that the residues corresponding to the dominant-negative mutations likely constitute the active site of FtsW, which may aid in the design of FtsW inhibitors.
DOI: https://dx.doi.org/ 10.1371/journal.pgen.1009993
All phase contrast and fluorescence images were acquired using an Olympus BX53 upright microscopes with a Retiga R1 camera from QImaging, a CoolLED pE-4000 light source and a U Plan XApochromat phase contrast objective lens (100X, 1.45 numerical aperture [NA], oil immersion).
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The pE-4000 Universal Illumination System offers 16 selectable wavelengths from 365 - 770 nm, making it a highly flexible illuminator covering a wide variety of fluorophores
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