A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis.

Johana Luhur,Christopher D A Rodrigues,Dena Lyras,Simonetta Gribaldo,Milena Awad,Cécile Morlot,David Z Rudner,Benson Kachappilly,Najwa Taib,Ahmed Mohamed,Helena Chan

doi:10.1371/journal.pgen.1009246

Abstract

How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely suppresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development.

Highlights

Bacterial cell shape is an important characteristic that plays a fundamental role in survival [1,2]
Decades of research have focused on defining the composition and assembly of the multi-layered spore envelope, little is known about how these layers contribute to spore shape
We show that SsdC is an important factor in spore shape development in the monoderm, model organism Bacillus subtilis

Summary

Introduction

Bacterial cell shape is an important characteristic that plays a fundamental role in survival [1,2]. Spore formation is largely restricted to monoderm, Grampositive bacteria, the spore contains a diderm cell envelope, similar to diderm, Gram-negative bacteria. Spore development is an interesting model system for dissecting the similarities and differences of building the cell envelope, and its relationship to shape, in monoderm and diderm bacteria, with potential ramifications for understanding the evolution of the bacterial envelope [6,7,8,9]. We focus on the determinants of spore shape through the characterization of a poorly-characterized Bacillus subtilis protein called SsdC (formerly YdcC), an orthologue of the outer membrane stress response proteins, MucB / RseB found in Gram-negative bacteria [10]

Methods

Results

Discussion

Conclusion