The wall-less bacterium Spiroplasma poulsonii builds a polymeric cytoskeleton composed of interacting MreB isoforms

Abstract

A rigid cell wall defines the morphology of most bacteria. MreB, a bacterial homologue of actin, plays a major role in coordinating cell wall biogenesis and defining a cells shape. In contrast with most bacteria, the Mollicutes family is devoid of cell wall. As a consequence, many Mollicutes have undefined morphologies. Spiroplasma species are an exception as they robustly grow with a characteristic helical shape, but how they maintain their morphology remains unclear. Paradoxal to their lack of cell wall, the genome of Spiroplasma contains five homologues of MreB (SpMreBs). Since MreB is a homolog of actin and that short MreB filaments participate in its function, we hypothesize that SpMreBs form a polymeric cytoskeleton. Here, we investigate the function of SpMreB in forming a polymeric cytoskeleton by focusing on the Drosophila endosymbiont Spiroplasma poulsonii. We found that in vivo, Spiroplasma maintain a high concentration of all five MreB isoforms. By leveraging a heterologous expression system that bypasses the poor genetic tractability of Spiroplasma, we found that strong intracellular levels of SpMreb systematically produced polymeric filaments of various morphologies. Using co-immunoprecipitation and co-expression of fluorescent fusions, we characterized an interaction network between isoforms that regulate the filaments formation. Our results point to a sub-functionalization of each isoform which, when all combined in vivo, form a complex inner polymeric network that shapes the cell in a wall-independent manner. Our work therefore supports the hypothesis where MreB mechanically supports the cell membrane, thus forming a cytoskeleton. Significance statementBacteria shape is determined by their cell wall. The actin homologue MreB essentially determines shape by organizing cell wall synthesis at the subcellular level. Despite their lack of cell wall, Spiroplasma robustly grow into long helical bacteria. Surprisingly, its genome retains five copies of mreB while it lost genes encoding canonical MreB interactors. We sought to delineate the exact function of Spiroplasma MreBs (SpMreBs). We leveraged in vivo data along with functional studies to systematically investigate MreB polymerization behavior. We uncovered that SpMreBs build into filaments, which structure it determined by a complex interaction network between isoforms. Our results support the hypothesis that MreB can mechanically support the membrane of Spiroplasma, hence acting as a load-bearing cytoskeletal protein.