Deciphering the Role of a SLOG Superfamily Protein YpsA in Gram-Positive Bacteria

Abstract

Bacteria adapt to different environments by regulating cell division and several conditions that modulate cell division have been documented. Understanding how bacteria transduce environmental signals to control cell division is critical in understanding the global network of cell division regulation. In this article we describe a role for Bacillus subtilis YpsA, an uncharacterized protein of the SLOG superfamily of nucleotide and ligand-binding proteins, in cell division. We observed that YpsA provides protection against oxidative stress as cells lacking ypsA show increased susceptibility to hydrogen peroxide treatment. We found that the increased expression of ypsA leads to filamentation and disruption of the assembly of FtsZ, the tubulin-like essential protein that marks the sites of cell division in B. subtilis. We also showed that YpsA-mediated filamentation is linked to the growth rate. Using site-directed mutagenesis, we targeted several conserved residues and generated YpsA variants that are no longer able to inhibit cell division. Finally, we show that the role of YpsA is possibly conserved in Firmicutes, as overproduction of YpsA in Staphylococcus aureus also impairs cell division.

Keywords: Bacillus subtilis; FtsZ; GpsB; NAD; SLOG; cell division; oxidative stress.

Figure 1

(A) Left: Cartoon representation of ypsA gene neighborhood in Firmicutes, not to scale. The genes that encode protein products containing a domain of unknown function DUF1798 are named as such. Right: Phylogenetic tree of the YpsA family, key branches with >70% bootstrap support are denoted with yellow circles. Reproducible clades within the family are color-coded according to their phyletic distribution and labeled with names and representative conserved domain architectures and gene neighborhoods. For these genome context depictions, colored polygons represent discrete protein domains within a protein, while boxed arrows represent individual genes within a neighborhood. Each context is labeled with NCBI accession and organism name, separated by an underscore. For gene neighborhoods, the labeled gene contains the YpsA domain. Abbreviations: A/G_cyclase, adenylyl/guanylyl cyclase. (B) Multiple sequence alignment of the YpsA family of proteins. Secondary structure and amino acid biochemical property consensus are provided on the top and bottom lines, respectively. Black arrows at top of alignment denote positions subject to site-directed mutagenesis. Sequences are labeled to left with NCBI accession and organism name separated by vertical bars. Gene names from the text are provided after organism name. Selected members of the YpsA clade, which associate with GpsB, are enclosed in a purple box. YpsA and YpsA-like YoqJ are highlighted in orange. Alignment coloring and consensus abbreviations as follows: b, big and gray; c, charged and blue; h, hydrophobic and yellow; l, aliphatic and yellow; p, polar and blue; s, small and green; u, tiny and green. The conserved aromatic position in the first loop, abbreviated ‘a', and the conserved negatively-charged position in the second helix, abbreviated ‘-', are both colored in red with white lettering to distinguish predicted, conserved positions located within the active site pocket.

Figure 2

YpsA plays a role in oxidative stress response. (A) Disc diffusion assay with lawns made of WT (PY79) or a strain lacking ypsA (RB42) treated with blank disc and 1 M H₂O₂ are shown. (B) Fluorescence micrographs showing cells of WT (PY79), ΔypsA (RB42), and ΔypsA complemented with a copy of inducible ypsA or ypsA-gfp at an ectopic locus (RB160 or RB161) grown for 1 h with or without 1 mM H₂O₂, 250 uM IPTG, and stained with FM4-64 (membrane, red). White arrows indicate aberrantly shaped cells and vesicle-like structures. (C) Fluorescence micrographs of RB221 strain that produces YpsA-GFP under the control of its native promoter imaged at mid-log (OD600 = 0.5) phase with or without H2O2 treatment. Yellow arrowheads indicate YpsA-GFP foci. Scale bar: 1 μm.

Figure 3

Elevated production of YpsA or YpsA-GFP leads to inhibition of cell division. (A–D) Morphology of cells containing inducible ypsA (GG82) or ypsA-gfp (GG83) grown in the absence of inducer IPTG (A,C) or in the presence of inducer for 1 h (B,D). (E–H) Cell morphology of strains lacking gpsB and containing either inducible ypsA (RB43) or ypsA-gfp (RB44) grown in the absence (E,G) or presence (F,H) of inducer. (I) Quantification of cell lengths of cells shown in (A–D). Time-lapse micrographs of ypsA-gfp expressing cells (GG83) at time intervals indicated at the bottom. White and orange arrows follows different foci that are mobile. DIC (gray) and fluorescence of membrane dye (FM4-64; red), GFP (green) are shown. Scale bar: 1 μm.

Figure 4

YpsA inhibits FtsZ ring assembly. (A) Fluorescence micrographs of cells that either constitutively produce FtsZ-GFP in otherwise wild type strain (PE92; top panel) and cells that constitutively produce FtsZ-GFP and additionally harbor a copy of inducible ypsA (RB15) grown in the absence (middle panel) or presence of inducer IPTG are shown. Fluorescence of FM4-64 membrane dye (red) and GFP (green) are shown. (B) Cellular morphologies of cells that constitutively produce FtsZ-GFP (PE92) and cells that additionally contain a copy of inducible ypsA-mCherry (RB97) grown in the absence (middle panel) or presence of inducer are shown. DIC (gray) and fluorescence of GFP (green) and mCherry (red) are shown. (C) Cells of GG82 (inducible ypsA) and RB15 (inducible ypsA + constitutively expressed ftsZ-gfp) were imaged after grown in the presence of inducer for 1 h (top panels) or 3 h after the removal of inducer (bottom panels). Scale bars: 1 μm.

Figure 5

Growth rate-dependent inhibition of cell division. (A) Fluorescence micrographs of cells of WT, inducible ypsA (GG82) or ypsA-gfp (GG83) were grown in CH medium in the absence of any supplements (top panels), with 1% glucose supplementation (middle panels), or with 1% sucrose supplementation (bottom panels). (B) WT, GG82, or GG83 strains described above were grown in LB medium at 37°C (top panels) or 22°C (bottom panels). Fluorescence of membrane dye (FM4-64; red), GFP (green) are shown. Scale bar: 1 μm.

Figure 6

Site-directed mutagenesis reveals key residues in YpsA. (A) Cell morphologies of YpsA-GFP (WT; GG83) and GFP-fusions of G42A (RB119), E44Q (RB115), G53A (RB120), E55Q (RB116), W45A (RB35), W57A (RB26), and W87A (RB37) are shown. The cells were grown either in the absence (left panels) or presence (right panels) of inducer IPTG. Fluorescence of membrane stain FM4-64 (red) and GFP (green) are shown. Scale bar: 1 μm. (B) Production of GFP-tagged YpsA variants were detected by immunoblot of cell extracts of strains shown in (A) grown in the presence of inducer using anti-GFP and corresponding anti-SigA (loading control) antisera.

Figure 7

Production of YpsA^SA inhibits cell division in S. aureus. (A) Fluorescence micrographs of wild type (SH1000; left) or transposon-disrupted ypsA (RB162; right) strains. (B) Morphologies of SH1000 cells harboring empty vector (pEPSA5) in the absence of presence of inducer. (C) SH1000 cells harboring plasmid encoded xylose-inducible copy of ypsA^SA (pRB36) grown in the absence (left; 37°C) or presence of inducer grown at 37°C (second panel), or 3 h after removal of inducer (third panel), or when grown at 22°C (fourth panel) are shown. Arrow indicates a cell dividing unevenly. Cell membrane were visualized using FM4-64 dye (red). Scale bar: 1 μm.