Mechanical Genomics Identifies Diverse Modulators of Bacterial Cell Stiffness

Abstract

Bacteria must maintain mechanical integrity to withstand the large osmotic pressure differential across the cell membrane and wall. Although maintaining mechanical integrity is critical for proper cellular function, a fact exploited by prominent cell-wall-targeting antibiotics, the proteins that contribute to cellular mechanics remain unidentified. Here, we describe a high-throughput optical method for quantifying cell stiffness and apply this technique to a genome-wide collection of ∼4,000 Escherichia coli mutants. We identify genes with roles in diverse functional processes spanning cell-wall synthesis, energy production, and DNA replication and repair that significantly change cell stiffness when deleted. We observe that proteins with biochemically redundant roles in cell-wall synthesis exhibit different stiffness defects when deleted. Correlating our data with chemical screens reveals that reducing membrane potential generally increases cell stiffness. In total, our work demonstrates that bacterial cell stiffness is a property of both the cell wall and broader cell physiology and lays the groundwork for future systematic studies of mechanoregulation.

Figure 1. GRABS methodology enables high-throughput measurements of cellular mechanical properties

A) Growth curves of E. coli BW25113 embedded in 0.25%–5% agarose gels and liquid medium (n = 3) monitored with a plate reader; cell growth diminishes as gel stiffness increases. Solid lines and shaded areas represent the smoothed average and one standard deviation, respectively. Inset: schematic of a bacterial cell growing in agarose. Of the forces acting on the cell, F_gel is proportional to the stiffness of the agarose gel, F_cw is proportional to the composite stiffness of the cell envelope including the membranes and peptidoglycan layer, and F_turgor represents the turgor pressure across the cell membrane. B) Maximal growth rate of an embedded E. coli culture and gel Young’s modulus (Tuson et al., 2012) as a function of gel stiffness. Solid lines and shaded areas represent the average and one standard deviation (n ≥ 3 for each concentration), respectively. C) Schematic of the GRABS assay. A microwell is filled with 1% agarose or liquid medium, cells are pipetted into the well, and cells are mixed using a magnetic stirrer until homogenously dispersed. The stirrer is then removed and the agarose is left to solidify. In the 96-well microplate, wells in columns 1–6 are filled with 150 L of 1% agarose and columns 7–12 are filled with liquid medium. Grey circles in columns 1–6 represent magnetic stirrers. D) Example growth curves for wild-type E. coli BW25113 (black) and mutants with GRABS score = 0 (wild-type-like, green), > 0 (increased stiffness, red), and < 0 (decreased stiffness, blue). Wild-type growth curves in liquid are represented by dotted lines, and growth curves of cells in agarose are represented by dashed lines. Many mutants with a GRABS score < 0 have the same growth rate as wild-type cells in liquid and a reduced growth rate in 1% agarose. A GRABS score > 0 typically occurs when a mutant has a decreased growth rate in liquid and a growth rate similar to that of wild-type cells in agarose.

Figure 2. Cell-stiffness modulators encompass a large range of cellular functions

A) Maximal growth rates in liquid and agarose. Blue and red ovals represent the mean growth rates ± one standard deviation for BW25113 and ΔmrcB, respectively (n ≥ 5 independent experiments); the gray oval represents the distribution of values for the Keio collection. B) The 46 genes with GRABS score < −0.15 (n = 41) or > 0.3 (n = 5) come from 18 Clusters of Orthologous Groups (COGs). Several genes (ΔrecA, ΔtolR, ΔtolB, ΔwcaG, ΔfliA) are assigned to two or more orthologous groups, which gives rise to n = 53 total assignments. C) Top: distribution of GRABS scores. Bottom: GRABS hits in (B). The average GRABS score and standard deviation across ≥ 6 replicates from multiple independent experiments are shown. Genes are colored according to their COG assignment. Genes with ≥ 2 COG assignments are colored accordingly. E. coli BW25113 has a GRABS score of 0 by definition.

Figure 3. Microfluidic bending assay and elongation rate measurements of embedded cells yield relative stiffness measurements consistent with GRABS scores

A) Representative images of filamentous wild-type cells and ΔmrcB cells in a microfluidic cell-bending device. Blue and black lines indicate the configuration of cells in the absence and presence of fluid flow, respectively. Deflection was measured as the difference between the position of the cell tip in the flow direction in the absence and presence of fluid flow. Scale bar: 10 μm. B) Young’s moduli for sulA-induced (left) and aztreonam-treated (right) cells were determined by fitting deflection data (Extended Experimental Procedures) from a microfluidic-based bending assay. Error bars indicate 95% confidence interval to the fit (Extended Experimental Procedures). Data are colored according to each gene’s assignment to Clusters of Orthologous Groups, as in Figure 2B (n ≥ 130 cells, two independent experiments).

Figure 4. GRABS analysis of cell-wall synthesis mutants relates biochemical activities in vitro to changes in embedded growth in vivo

A) Crystal structure of PBP1b (PDB ID: 3VMA) highlighting residues used for mutational analysis via GRABS. N-terminal trans-membrane α-helix, residues 64–87 (orange); UvrB Domain 2 Homolog (UB2H) domain, residues 109–200 (gold); glycosyltransferase (GT) domain, residues 203–367 (green); transpeptidase (TP) domain, residues 444–736 (pink). Other residues are colored in black. Inset: mutated residues in the UB2H domain. B) OD at t = 8 h of agarose-embedded E. coli BW25113 cells and ΔmrcB cells expressing various mrcB alleles in trans. WT, wild-type. Error bars represent one standard deviation above and below the mean. * indicates a point mutation in the corresponding domain of PBP1b (UB2H, glycosyltransferase (GT), and transpeptidase (TP)). n ≥ 5 independent experiments. C) Growth curves of agarose-embedded ΔlpoB cells and cells harboring mutations in the 6UB2H domain, E233Q (glycosyltransferase), and S510A (transpeptidase) of mrcB. Shaded regions represent one standard deviation above and below mean growth curves. * indicates a point mutation in the corresponding domain of mrcB. n ≥ 5 independent experiments.

Figure 5. GRABS score generally increases under CCCP treatment

GRABS score with and without CCCP, a proton ionophore that reduces membrane potential. Solid circles, representing GRABS score for the dimethyl sulfoxide (DMSO) control, are generally at lower values than the open circles, which represent GRABS score in the presence of 2 μg/mL CCCP. Genes are colored according to their assignment to Clusters of Orthologous Groups. Solid lines and shaded areas represent the average and 95% confidence interval to a linear fit, respectively, of n ≥ 4 independent experiments.