Intracellular ion concentrations and cation-dependent remodelling of bacterial MreB assemblies

Dávid Szatmári,Miklós Nyitrai,Robert C Robinson,Szilvia Barkó,Béla Kocsis,Péter Sárkány,Attila Miseta,Tamás Nagy,Beáta Longauer

doi:10.1038/s41598-020-68960-w

Abstract

Here, we measured the concentrations of several ions in cultivated Gram-negative and Gram-positive bacteria, and analyzed their effects on polymer formation by the actin homologue MreB. We measured potassium, sodium, chloride, calcium and magnesium ion concentrations in Leptospira interrogans, Bacillus subtilis and Escherichia coli. Intracellular ionic strength contributed from these ions varied within the 130–273 mM range. The intracellular sodium ion concentration range was between 122 and 296 mM and the potassium ion concentration range was 5 and 38 mM. However, the levels were significantly influenced by extracellular ion levels. L. interrogans, Rickettsia rickettsii and E. coli MreBs were heterologously expressed and purified from E. coli using a novel filtration method to prepare MreB polymers. The structures and stability of Alexa-488 labeled MreB polymers, under varying ionic strength conditions, were investigated by confocal microscopy and MreB polymerization rates were assessed by measuring light scattering. MreB polymerization was fastest in the presence of monovalent cations in the 200–300 mM range. MreB filaments showed high stability in this concentration range and formed large assemblies of tape-like bundles that transformed to extensive sheets at higher ionic strengths. Changing the calcium concentration from 0.2 to 0 mM and then to 2 mM initialized rapid remodelling of MreB polymers.

Highlights

We measured the concentrations of several ions in cultivated Gram-negative and Gram-positive bacteria, and analyzed their effects on polymer formation by the actin homologue MreB
Ion-selective electrode/colorimetry is able to measure concentrations of free ions, while flame photometry measures the total concentrations of ions, including ions bound to proteins and DNA
To compare these two methods, we measured the intracellular concentration of Na+, K+ and Ca2+ in samples extracted from Escherichia coli using both ion-selective electrode/colorimetry and flame photometry

Summary

Introduction

We measured the concentrations of several ions in cultivated Gram-negative and Gram-positive bacteria, and analyzed their effects on polymer formation by the actin homologue MreB. Important observations were made in three independent studies showing the coupling of the dynamics of MreB to cell-wall synthesis in two evolutionary different species; Gram-negative Escherichia coli[16] and Grampositive Bacillus subtilis[14,15] These results established that MreB polymers rotate around the long axis of the cell on a time scale of minutes. In vitro high salt conditions can cause MreB polymers to stack into stable s heets[27] Such morphologies may be related to the in vivo functions, as MreB structures arranged in helical patterns are required for inner membrane and cell shape stability[28]. The roles of the inherent dynamic properties, remodelling and reorganization of MreB in the cell wall maintenance and cell division still need to be clarified

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