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. 2016 Jul 13;198(15):2074-88.
doi: 10.1128/JB.00183-16. Print 2016 Aug 1.

YodL and YisK Possess Shape-Modifying Activities That Are Suppressed by Mutations in Bacillus Subtilis mreB and Mbl

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Free PMC article

YodL and YisK Possess Shape-Modifying Activities That Are Suppressed by Mutations in Bacillus Subtilis mreB and Mbl

Yi Duan et al. J Bacteriol. .
Free PMC article

Abstract

Many bacteria utilize actin-like proteins to direct peptidoglycan (PG) synthesis. MreB and MreB-like proteins are thought to act as scaffolds, guiding the localization and activity of key PG-synthesizing proteins during cell elongation. Despite their critical role in viability and cell shape maintenance, very little is known about how the activity of MreB family proteins is regulated. Using a Bacillus subtilis misexpression screen, we identified two genes, yodL and yisK, that when misexpressed lead to loss of cell width control and cell lysis. Expression analysis suggested that yodL and yisK are previously uncharacterized Spo0A-regulated genes, and consistent with these observations, a ΔyodL ΔyisK mutant exhibited reduced sporulation efficiency. Suppressors resistant to YodL's killing activity occurred primarily in mreB mutants and resulted in amino acid substitutions at the interface between MreB and the highly conserved morphogenic protein RodZ, whereas suppressors resistant to YisK occurred primarily in mbl mutants and mapped to Mbl's predicted ATP-binding pocket. YodL's shape-altering activity appears to require MreB, as a ΔmreB mutant was resistant to the effects of YodL but not YisK. Similarly, YisK appears to require Mbl, as a Δmbl mutant was resistant to the cell-widening effects of YisK but not of YodL. Collectively, our results suggest that YodL and YisK likely modulate MreB and Mbl activity, possibly during the early stages of sporulation.

Importance: The peptidoglycan (PG) component of the cell envelope confers structural rigidity to bacteria and protects them from osmotic pressure. MreB and MreB-like proteins are thought to act as scaffolds for PG synthesis and are essential in bacteria exhibiting nonpolar growth. Despite the critical role of MreB-like proteins, we lack mechanistic insight into how their activities are regulated. Here, we describe the discovery of two B. subtilis proteins, YodL and YisK, which modulate MreB and Mbl activities. Our data suggest that YodL specifically targets MreB, whereas YisK targets Mbl. The apparent specificities with which YodL and YisK are able to differentially target MreB and Mbl make them potentially powerful tools for probing the mechanics of cytoskeletal function in bacteria.

Figures

FIG 1
FIG 1
Misexpression of YodL and YisK prevents cell growth on solid medium and causes loss of cell shape in liquid medium. (A) Cells harboring one (1×) or two (2×) copies of Phy-yodL (BAS040 and BAS191) or Phy-yisK (BAS041 and BYD074) were streaked on an LB plate supplemented with 100 μg/ml spectinomycin and, where indicated, 1 mM IPTG or 1 mM IPTG and the denoted concentration of MgCl2. Plates were incubated for ∼16 h at 37°C before image capture. (B) The strains shown in panel A were grown in LB-Lennox medium at 37°C to mid-exponential phase and back-diluted to an OD600 of ∼0.02. Where indicated, 1 mM IPTG or 1 mM IPTG and the denoted concentration of MgCl2 were added. Cells were grown for 1.5 h at 37°C before image capture. Membranes were stained with TMA-DPH. All images were scaled identically.
FIG 2
FIG 2
DNA sequence upstream of yodL and yisK. (A) Putative Spo0A box (underlined) upstream of the yodL start codon. (B) SigH binding motifs (double underline) and putative Spo0A box (underlined) upstream of the yisK start codon.
FIG 3
FIG 3
Expression levels of yodL and yisK promoters during a CH time course experiment. Expression from the putative yodL and yisK promoter regions was monitored in CH medium at 37°C over the time course. The OD600 (A and B) and production of either GFP (C) or beta-galactosidase (D and E) were monitored at 30-min intervals. Membranes were stained with TMA-DPH. All GFP channel images were captured with 1-s exposures and scaled identically to allow for direct comparisons. In this assay, time zero represents the last exponential time point, not the initiation of sporulation. Vertical bars in the graphs represent standard deviations.
FIG 4
FIG 4
Expression from yodL and yisK promoters following sporulation by resuspension. Expression from the putative yodL and yisK promoter regions was monitored in resuspension medium. The production of either GFP (A) or beta-galactosidase (B and C) was monitored at 20-min intervals. Membranes were stained with TMA-DPH. All GFP channel images were captured with 1-s exposures and scaled identically to allow for direct comparisons. Vertical bars in the graphs represent standard deviations.
FIG 5
FIG 5
YodL and YisK comisexpression causes cell lysis. (A) BYD361 (Phy-yodL Phy-yisK) and BYD281 (2× Phy-yodL 2× Phy-yisK) were streaked on an LB plate with 100 μg/ml spectinomycin and, where indicated, 1 mM IPTG or 1 mM IPTG and the denoted concentration of MgCl2. (B) Cells were grown in LB-Lennox medium at 37°C to mid-exponential phase and back-diluted to an OD600 of ∼0.02. Where indicated, 1 mM IPTG or 1 mM IPTG and the denoted concentration of MgCl2 were added. Cells were then grown for 1.5 h at 37°C before image capture. Membranes were stained with TMA-DPH. All images are shown at the same magnification.
FIG 6
FIG 6
YodL and YisK cell-widening activities require MreB and Mbl, respectively. (A) Cells harboring two copies of Phy-yodL in the wild-type (BAS191), ΔponA (BYD176), ΔponA ΔmreB (BYD263), ΔponA Δmbl (BYD259), or ΔponA Δmbl ΔmreBH (BAS249) background were grown at 37°C in LB supplemented with 10 mM MgCl2 to mid-exponential phase. To induce yodL expression, cells were back-diluted to an OD600 of ∼0.02 in LB with 10 mM MgCl2 and IPTG (1 mM) was added. Cells were grown for 1.5 h at 37°C before image capture. Membranes were stained with TMA-DPH. All images are shown at the same magnification. (B) Cells harboring two copies of Phy-yisK in the wild-type (BYD074), ΔponA (BYD175), ΔponA ΔmreB (BYD262), ΔponA Δmbl (BYD258), or ΔponA Δmbl ΔmreBH (BAS248) background were grown at 37°C in LB supplemented with 10 mM MgCl2 to mid-exponential phase. To induce yisK expression, cells were back-diluted to an OD600 of ∼0.02 in LB with 10 mM MgCl2, and IPTG (1 mM) was added. Cells were grown for 1.5 h at 37°C before image capture. Membranes were stained with TMA-DPH. All images are shown at the same magnification.
FIG 7
FIG 7
YisK expression results in cell shortening. (A) Cells harboring two copies of Phy-yisK in a ΔponA Δmbl background (BYD262) were grown at 37°C in LB supplemented with 10 mM MgCl2 to mid-exponential phase. To induce yisK expression, cells were back-diluted to an OD600 of ∼0.02 in LB with 10 mM MgCl2, and IPTG (1 mM) was added. Cells were grown for 1.5 h at 37°C before image capture. Membranes were stained with TMA-DPH. Cell lengths (n = 500 cells/condition) were measured before and after yisK expression and rank-ordered from smallest to largest along the x axis so that the entire population could be visualized without binning. The uninduced population (black) is juxtaposed behind the induced population (semitransparent, gray). The differences in average cell length before and after Phy-yisK induction were statistically significant (P < 0.0001). (B) Cells harboring two copies of Phy-yodL in a ΔponA ΔmreB background (BYD263) were grown, quantitated, and plotted as described above. The differences in average cell length before and after Phy-yodL induction were not statistically significant. (C) Cells harboring two copies of Phy-yisK in a ΔponA Δmbl ΔmreBH background (BAS248) were grown, quantitated, and plotted as described above. The differences in average cell length before and after Phy-yisK induction were statistically significant (P < 0.0001).

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This work was funded by a National Science Foundation grant (1514629) to J.K.H. and by start-up funds from the Center for Phage Technology and the Department of Biochemistry and Biophysics at Texas A&M University.

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