Cell age dependent concentration of Escherichia coli divisome proteins analyzed with ImageJ and ObjectJ.

Norbert O E Vischer,Jolanda Verheul,Kenn Gerdes,Waldemar Vollmer,Marten Postma,Elisa Galli,Bart Van Den Berg Van Saparoea,Tanneke Den Blaauwen,Joen Luirink,Paolo Natale,Miguel Vicente

doi:10.3389/fmicb.2015.00586

Abstract

The rod-shaped Gram-negative bacterium Escherichia coli multiplies by elongation followed by binary fission. Longitudinal growth of the cell envelope and synthesis of the new poles are organized by two protein complexes called elongasome and divisome, respectively. We have analyzed the spatio-temporal localization patterns of many of these morphogenetic proteins by immunolabeling the wild type strain MC4100 grown to steady state in minimal glucose medium at 28°C. This allowed the direct comparison of morphogenetic protein localization patterns as a function of cell age as imaged by phase contrast and fluorescence wide field microscopy. Under steady state conditions the age distribution of the cells is constant and is directly correlated to cell length. To quantify cell size and protein localization parameters in 1000s of labeled cells, we developed ‘Coli-Inspector,’ which is a project running under ImageJ with the plugin ‘ObjectJ.’ ObjectJ organizes image-analysis tasks using an integrated approach with the flexibility to produce different output formats from existing markers such as intensity data and geometrical parameters. ObjectJ supports the combination of automatic and interactive methods giving the user complete control over the method of image analysis and data collection, with visual inspection tools for quick elimination of artifacts. Coli-inspector was used to sort the cells according to division cycle cell age and to analyze the spatio-temporal localization pattern of each protein. A unique dataset has been created on the concentration and position of the proteins during the cell cycle. We show for the first time that a subset of morphogenetic proteins have a constant cellular concentration during the cell division cycle whereas another set exhibits a cell division cycle dependent concentration variation. Using the number of proteins present at midcell, the stoichiometry of the divisome is discussed.

Highlights

Escherichia coli is a Gram-negative rod shaped bacterium that divides by binary fission
Immunolocalization analyzed as function of cell age allowed determination of the concentration of the labeled proteins and revealed that at least the concentration of FtsZ, ZapA, PBP3, FtsN, and PBP5 seem to be cell cycle regulated (Figure 6)
When FtsN is included, it could bind 4 out of the 5 FtsA molecules that are present on an FtsZ protofilament

Summary

Introduction

Escherichia coli is a Gram-negative rod shaped bacterium that divides by binary fission. Large protein complexes that are termed elongasome and divisome synthesize and hydrolyze peptidoglycan during cell elongation and cell division, respectively (Egan and Vollmer, 2013; van der Ploeg et al, 2013) These protein complexes share some of their proteins (Mohammadi et al, 2007; White et al, 2010; van der Ploeg et al, 2013), and many of the proteins have their own enzymatic activities, which categorize the elongasome and divisome as hyperstructures (Norris et al, 2007). These hyperstructures are not assembled and kept stable like the ribosomes, they are rather dynamic and can associate cell cycle dependent. It is relevant for the understanding of the organization of both processes to determine their composition and cellular localization as a function of the bacterial cell division cycle age (cell age)

Methods

Results

Conclusion