Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori
Abstract
Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. The helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod H. pylori. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape.
Data availability
The MATLAB scripts used to reconstruct cell surfaces and perform the geometric enrichment analyses are publicly available under a BSD 3-clause license at https://github.com/PrincetonUniversity/shae-cellshape-public and archived at https://doi.org/10.5281/zenodo.1248978.
Article and author information
Author details
Funding
National Institutes of Health (R01 AI136946)
- Nina Reda Salama
National Science Foundation (DGE-1256082)
- Jennifer A Taylor
- Kris M Blair
Department of Defense (National Defense Science & Engineering Graduate Fellowship (NDSEG))
- Jennifer A Taylor
GO-MAP (Graduate Opportunity Program Research Assistantship Award)
- Sophie R Sichel
National Science Foundation (PHY-1734030)
- Benjamin P Bratton
- Josh W Shaevitz
Glenn Centers for Aging Research
- Benjamin P Bratton
National Institutes of Health (R21 AI121828)
- Benjamin P Bratton
- Josh W Shaevitz
National Institutes of Health (GM113172)
- Michael S VanNieuwenhze
National Institutes of Health (U01 CA221230)
- Catherine L Grimes
- Nina Reda Salama
National Institutes of Health (T32 CA009657)
- Kris M Blair
National Institutes of Health (T32 GM95421)
- Sophie R Sichel
National Institutes of Health (T32 GM008550)
- Kristen E DeMeester
National Institutes of Health (P30 CA015704)
- Nina Reda Salama
National Center for Research Resources (Stanford Imaging Award Number 1S10OD01227601)
- Nina Reda Salama
Wellcome (101824/Z/13/Z)
- Waldemar Vollmer
National Science Foundation (DGE-0718124)
- Jennifer A Taylor
The funders had no role in study design, datacollection and interpretation, or the decision to submit the work for publication. The opinions, findings, and conclusions or recommendationsexpressed in this material contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR,the National Institutes of Health, the Department of Defense, or the National Science Foundation.
Copyright
© 2020, Taylor et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.