Reconstitution and functional analysis of multi-enzyme complexes involved in Escherichia coli peptidoglycan synthesis
Lead Research Organisation:
Newcastle University
Department Name: Inst for Cell and Molecular Biosciences
Abstract
The shape and size of a bacterial cell is determined by the shape and size of the peptidoglycan sacculus, which forms an exoskeleton that is essential for the osmotic stability of the cell. Peptidoglycan is present in most bacterial species and its biosynthesis is the target of widely used, safe and effective antibiotics. Although many of the enzymes involved in peptidoglycan metabolism have been characterised, surprisingly little is known about how they co-operate to enlarge the peptidoglycan during growth. We will attempt to reconstitute and characterise the multi-enzyme complexes responsible for peptidoglycan synthesis in E. coli. The enlargement of the stress-bearing peptidoglycan sacculus requires the oligomerization of the lipid II precursor and its insertion into existing peptidoglycan by transglycosylation and transpeptidation. Various synthetic and hydrolytic enzymes are involved, and in previous studies we obtained evidence that the synthases and other proteins involved function as components of multi-enzyme complexes. We aim to reconstitute such complexes in vitro in their native forms from the purified components. With the help of a functional peptidoglycan synthesis assay involving the structural analysis of the resulting peptidoglycan, we will study the conditions required for the formation of complexes and the role of each protein. In particular, we will determine the substrate specificity of the monofunctional transpeptidases, and aim to identify and characterise as yet unknown protein-protein interactions involving the peptidoglycan synthases. Ultimately these studies will lead to an understanding of the molecular mechanisms involved in the enlargement of the peptidoglycan layer during cell growth. Because these reactions are specific to bacteria, the inhibition of the peptidoglycan synthesis represents an excellent target for antimicrobial chemotherapy. Knowledge of the molecular details of peptidoglycan synthesis will be crucial for the development of novel antibiotics required to treat drug-resistant pathogens.
Technical Summary
The enlargement of the predominantly single-layered peptidoglycan (murein) sacculus during growth of Escherichia coli is a complex process involving peptidoglycan synthases, hydrolases and structural proteins. A fail-safe cell wall growth mechanism is essential because even a temporal weakening of the stress-bearing sacculus is likely to result in cell lysis and death. In addition, the incorporation of new wall material has to occur in such a way that the rod-shape of E. coli is maintained throughout the growth cycle. E. coli is known to encode six peptidoglycan synthases (penicillin-binding proteins or PBPs) and 12 known autolysins. The latter are periplasmic peptidoglycan hydrolases participating in cell growth and division. It is not known how these enzymes co-operate to insert the lipid II precursor at the cell wall growth sites. There is, however, evidence that the enlargement of the peptidoglycan layer is performed by multi-enzyme complexes containing a combination of synthases and other proteins. In the current proposal, we aim to unravel the mechanism(s) of peptidoglycan growth by studying the interactions and the reactions of the peptidoglycan synthases. Interactions involving peptidoglycan synthases will be studied in vivo and in vitro, and the reactions of the synthases as well as the activity of reconstituted synthases complexes and other cell division/elongation proteins will be studied in vitro.
Organisations
- Newcastle University (Collaboration, Lead Research Organisation)
- University of Amsterdam (Collaboration)
- Yale University (Collaboration)
- IBS Grenoble (Collaboration)
- Fred Hutchinson Cancer Research Center (FHCRC) (Collaboration)
- Utrecht University (Collaboration)
- University of Washington (Collaboration)
People |
ORCID iD |
Waldemar Vollmer (Principal Investigator) |
Publications
Banzhaf M
(2012)
Cooperativity of peptidoglycan synthases active in bacterial cell elongation.
in Molecular microbiology
Bisicchia P
(2011)
Acquisition of VanB-type vancomycin resistance by Bacillus subtilis: the impact on gene expression, cell wall composition and morphology.
in Molecular microbiology
Sycuro LK
(2012)
Multiple peptidoglycan modification networks modulate Helicobacter pylori's cell shape, motility, and colonization potential.
in PLoS pathogens
Typas A
(2010)
Regulation of peptidoglycan synthesis by outer-membrane proteins.
in Cell
Vollmer W
(2010)
Architecture of peptidoglycan: more data and more models.
in Trends in microbiology
Vollmer W
(2010)
Microbial Glycobiology
Description | •Significant new knowledge generated (see publications) •Important new research questions opened up |
Exploitation Route | N/A (there was no Pathway to Impact Statement) |
Sectors | Pharmaceuticals and Medical Biotechnology |
Description | Basic research in bioscience/microbiology. |
Impact Types | Cultural Societal |
Description | Christine Jacobs-Wagner |
Organisation | Yale University |
Department | Yale Comprehensive Cancer Center |
Country | United States |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Christine Jacobs-Wagner on bacterial cell envelope. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in cell envelope of different bacteria, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 31209025 PMID: 27506799 PMID: 25470067 PMID: 23469030 PMID: 22505677 PMID: 20497503 PMID: 20435724 PMID: 20023035 PMID: 19279668 PMID: 17501919 PMID: 17501913 |
Description | Eefjan Breukink |
Organisation | Utrecht University |
Department | Rudolf Magnus Institute |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Eefjan Breukink on bacterial cell wall proteins. We contribute our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributes their expertise in peptidoglycan enzymes and precursor synthesis, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 30504892 PMID: 30233559 PMID: 30093673 PMID: 30046664 PMID: 30044025 PMID: 28233869 PMID: 27709766 PMID: 27257764 PMID: 26370943 PMID: 25951518 PMID: 24821816 PMID: 22606933 PMID: 22487093 PMID: 21472954 PMID: 21183073 PMID: 17938168 PMID: 16840781 PMID: 16154998 |
Description | Jean-Pierre Simorre |
Organisation | IBS Grenoble |
Country | France |
Sector | Private |
PI Contribution | This is a long-term scientific collaboration with Jean-Pierre Simorre on bacterial cell walls and proteins. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in NMR spectroscopy for the analysis of bacterial cell walls and protein structure determination, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 30713527 PMID: 30046664 PMID: 30044025 PMID: 30031884 PMID: 25951518 PMID: 24954617 PMID: 24821816 PMID: 24691651 PMID: 24493340 PMID: 22192687 PMID: 20681725 PMID: 18393418 |
Description | Joe Gray |
Organisation | Newcastle University |
Department | Newcastle University Medical School |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Joe Gray on bacterial cell walls and proteins. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in mass spectrometry for the analysis of bacterial cell walls and proteins, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 31916938 PMID: 31209025 PMID: 30713527 PMID: 30102748 PMID: 30066424 PMID: 29311645 PMID: 28974693 PMID: 28581639 PMID: 27506799 PMID: 26537571 PMID: 26010014 PMID: 25424554 PMID: 24292151 PMID: 18996994 |
Start Year | 2007 |
Description | Joseph Mougous |
Organisation | University of Washington |
Department | Division of Medical Genetics |
Country | United States |
Sector | Academic/University |
PI Contribution | Expertise in bacterial cell wall. Experiments to determine peptidoglycan composition and enzymatic assays of peptidoglycan enzymes. |
Collaborator Contribution | Expertise in a the molecular biology of a variety of bacterial species, and in bacterial secretion systems. Experimental work on bacterial secretion systems. |
Impact | Scientific publications: PMID: 28696203 PMID: 25470067 PMID: 23878199 PMID: 22813741 PMID: 22607806 PMID: 21776080 |
Start Year | 2011 |
Description | Nina Salama |
Organisation | Fred Hutchinson Cancer Research Center (FHCRC) |
Country | United States |
Sector | Academic/University |
PI Contribution | Expertise in bacterial cell wall biochemistry and analysis. We performed peptidoglycan analysis in Helicobacter pylori, and measured the activities of cell wall enzymes. |
Collaborator Contribution | Expertise in the biology of Helicobacter. Performed experiments with Helicobacter cells. |
Impact | Scientific publications: PMID: 20510929 PMID: 22457625 PMID: 24112477 PMID: 31916938 PMID: 31036730 |
Start Year | 2009 |
Description | Rick Lewis |
Organisation | Newcastle University |
Department | Newcastle University Medical School |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Rick Lewis on bacterial cell wall proteins. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in x-ray crystallography for the analysis for protein structure determination, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 27862967 PMID: 27329754 PMID: 27257764 PMID: 25403286 PMID: 24954617 PMID: 24909784 PMID: 22432711 PMID: 21964069 PMID: 19074383 |
Start Year | 2007 |
Description | Tanneke den Blaauwen |
Organisation | University of Amsterdam |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Tanneke den Blaauwen on cell envelope biogenesis in E. coli. We contribute our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in bacterial cell biogenesis and cellular localization of proteins, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 30233559 PMID: 26124755 PMID: 25951518 PMID: 23387922 PMID: 22606933 PMID: 21472954 PMID: 21183073 PMID: 20545860 PMID: 16803586 |