3D Microscopy of Rod-Shaped Bacteria Reveals Roles of MreB in Diameter Control and Center-Line Curvature

Abstract

Over the past few years, our lab has developed a method for precisely determining the shape of bacterial cells in 3D by fluorescence microscopy. We use this method to measure the position of MreB in relation to the cell surface. To ensure that our measurements of fluorescent MreB reflect untagged MreB as nearly as possible, we have integrated our fusion at the native locus. This construct has unperturbed mass doubling times and proper rod-like shape. The shape and localization measurements that we report here are measured as snapshots from hundreds to a few thousand cells per condition.MreB, a membrane-binding structural homolog of actin, is one of the key players in properly patterning growth of the bacterial cell wall and thereby the shape of the cell. In one series of experiments, we show that the helical pitch of MreB filaments in E. coli is highly correlated to the diameter of the cell. This correlation holds for E. coli whose diameter has been altered by treatment with sub-lethal doses of the MreB targeting drug A22 and for single point mutants in MreB.Additionally, MreB polymers show a clear preference for regions of negative Gaussian curvature. We hypothesize that cells use this geometric sensing mechanism to straighten their centerlines [Ursell et al., PNAS 2014]. When this curvature preference is abolished (E. coli MreB A158T), cells are more frequently branched. In the smaller, comma shaped bacterium Caulobacter crescentus, the curvature enrichment profile shows a plateau region at low positive Gaussian curvature. This plateau enables Caulobacter cells to grow with their characteristic comma shape instead of as straight rods.