DivIVA-mediated polar localization of ComN, a posttranscriptional regulator of Bacillus subtilis

Abstract

ComN (YrzD) is a small, 98-amino-acid protein recently shown to be involved in the posttranscriptional control of the late competence comE operon in Bacillus subtilis. We show here that ComN localizes to the division site and cell poles in a DivIVA-dependent fashion. Yeast two-hybrid and glutathione S-transferase pulldown experiments showed that ComN interacts directly with DivIVA. ComN is not essential for the polar assembly of the core competence DNA uptake machinery. Nevertheless, polar localization of ComN should play some role in competence acquisition because delocalization of ComN leads to a small reduction in competence efficiency. We found that ComN promotes the accumulation of its target comE mRNA to septal and polar sites. Thus, we speculate that localized translation of ComE proteins may be required for efficient competence development. Our results underscore the versatility of DivIVA as a promoter of the differentiation of bacterial poles and demonstrate that the repertoire of polarly localized molecules in B. subtilis is broad, including a regulator of gene expression and its target mRNA. Moreover, our findings suggest that mRNA localization may play a role in the subcellular organization of bacteria.

Fig 1

ComN localizes to division sites and cell poles. Strain FG916, bearing a GFP-ComN fusion, was grown to mid-log phase (OD₆₀₀ = 0.3 to 0.5) in LB medium supplemented with 0.5% xylose and imaged as described in Materials and Methods. Note that GFP-ComN localizes both to midcell bands (new septa), as well as to cell poles (old septa). Arrows point to examples of cells in which GFP-ComN is retained at the cell poles. Bar, 5 μm.

Fig 2

ComN is a late recruit to the division site and colocalizes with DivIVA. (A) Colocalization of FtsZ and ComN. Strain FG925, expressing FtsZ-mCherry (FtsZ-mCh) and GFP-ComN fusions, was grown to mid-log phase (OD₆₀₀ = 0.3 to 0.5) in LB medium supplemented with 0.5% xylose and 100 μM IPTG and imaged as described in Materials and Methods. The arrow marks a Z-ring that is not decorated with ComN. These presumably represent young division complexes. An asterisk marks a ComN ring that is not decorated with FtsZ. These should correspond to septa in the final phase of cytokinesis, when FtsZ has already left the division site. Bar, 5 μm. (B) Colocalization of ComN and DivIVA. Strain FG1254, expressing DivIVA-GFP and ComN-mCherry (ComN-mCh), was grown to mid-log phase (OD₆₀₀ = 0.3 to 0.5) in LB medium supplemented with 0.5% xylose and 100 μM IPTG. The overlay image shows that ComN and DivIVA exhibit essentially perfect colocalization.

Fig 3

DivIVA is required for ComN localization. The GFP-ComN fusion was introduced into different mutants and localization was assayed in cells grown to mid-log phase. In the case of ftsZ, which is an essential gene, a depletion strain was used and grown for at least five generations in the absence of ftsZ expression before being imaged. For each mutant, a panel corresponding to the GFP-ComN image (gray) and a panel with the overlay between the GFP-ComN channel (green) and membrane channel (red) are shown. ftsZ (FG985), minD (FG932), minJ (FG994), minJ minD (FG1007), divIVA (FG1253), and divIVA minD (FG931) mutant strains are shown as indicated. Scale bar, 5 μm.

Fig 4

ComN and DivIVA interact. (A) Yeast two-hybrid assay. The yeast strain harboring the GAL4 activation domain fused to ComN (AD-ComN) and the GAL4 DNA-binding domain fused to DivIVA (BD-DivIVA) was able to activate transcription of the reporter genes ADE and HIS and grow on SC medium lacking adenine and histidine. Strains containing the AD-ComN fusion and the unfused DNA-binding domain (BD) or the BD-ComN fusion and the unfused activation domain (AD) did not activate transcription and were unable to grow in the absence of adenine and histidine. BD-DivIVA and AD-DivIVA was used as a positive control. (B) Pulldown assay. Total extract from cells expressing either GST or GST-DivIVA (TE) was incubated with glutathione-Sepharose, the flowthrough fraction was collected (F1) and, after washing, the total extract of cells expressing His-ComN (F2) was loaded. The resin was washed again, and the bound fraction (B) was eluted with glutathione. Proteins in each of the fractions were electrophoresed, transferred, and visualized by Western blotting with monoclonal anti-His IgG2a. His-ComN is only pulled down by GST-DivIVA (rightmost lane). ComN in the middle lane represents total extract of cells expressing His-ComN and serves as a size reference.

Fig 5

Mutation in comN does not affect the pattern or frequency of division. Fluorescence micrographs of membrane-stained wild-type (PY79) and comN mutant (FG1076) bacteria. Strains were grown to mid-log phase in LB medium at 37°C and imaged as described in Materials and Methods. Bar, 5 μm.

Fig 6

(A) Colocalization of ComGA and ComN. Strain FG1109, bearing ComGA-CFP and GFP-ComN fusions, was grown to competence by the two-step procedure described in Materials and Methods. Cells were harvested 1.5 h after being diluted into SpC medium and imaged. In the overlay, GFP-ComN is pseudocolored green, and ComGA-CFP is pseudocolored red. (B) ComGA-CFP localization in the absence of ComN. Strain FG1117, bearing the ComGA-CFP fusion and the comN::cat deletion, was grown to competence and visualized as described for panel A. Bar, 5 μm. (C) Frequency of comGA foci in wild-type and comN mutant cells.

Fig 7

ComN mediates localization of comE mRNA. Strains bearing an IPTG-inducible MS2-GFP fusion (amyE::P_spank-ms2d-gfp) and tagged versions of the comE mRNA (comEA-6×BS and comEC-6×BS) were grown in competence medium and imaged as described in Materials and Methods. The IPTG concentration used for all samples was 50 μM. (A and A′) comEA-6×BS localization in a wild-type background (strain AB229); (B and B′) comEC-6×BS localization in a wild-type background (strain AB230); (C and C′) comEC-6×BS localization in a comN mutant background (strain AB235); (D and D′) comEC-6×BS localization in a divIVA minD mutant background (strain AB239). The accumulation of comE mRNA in septa and new poles are marked with arrows, and accumulation in old poles is marked with arrowheads. Scale bar, 5 μm.