Localization and interactions of teichoic acid synthetic enzymes in Bacillus subtilis

Abstract

The thick wall of gram-positive bacteria is a polymer meshwork composed predominantly of peptidoglycan (PG) and teichoic acids, both of which have a critical function in maintenance of the structural integrity and the shape of the cell. In Bacillus subtilis 168 the major teichoic acid is covalently coupled to PG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradation over the lateral wall has been shown to occur in a helical pattern. However, the spatial organization of WTA assembly and its relationship with cell shape and PG assembly are largely unknown. We have characterized the localization of green fluorescent protein fusions to proteins involved in several steps of WTA synthesis in B. subtilis: TagB, -F, -G, -H, and -O. All of these localized similarly to the inner side of the cytoplasmic membrane, in a pattern strikingly similar to that displayed by probes of nascent PG. Helix-like localization patterns are often attributable to the morphogenic cytoskeletal proteins of the MreB family. However, localization of the Tag proteins did not appear to be substantially affected by single disruption of any of the three MreB homologues of B. subtilis. Bacterial and yeast two-hybrid experiments revealed a complex network of interactions involving TagA, -B, -E, -F, -G, -H, and -O and the cell shape determinants MreC and MreD (encoded by the mreBCD operon and presumably involved in the spatial organization of PG synthesis). Taken together, our results suggest that, in B. subtilis at least, the synthesis and export of WTA precursors are mediated by a large multienzyme complex that may be associated with the PG-synthesizing machinery.

FIG. 1.

Overview of WTA biosynthesis. The biosynthetic steps in the formation of WTA precursors are shown. Arrows indicate catalytic steps, and the enzymes responsible are shown. The precursor is assembled at the cytoplasmic membrane prior to transport by TagGH and linkage to the PG. The question mark indicates an unknown enzymatic reaction.

FIG. 2.

Localization of the ABC transporter TagGH. Fluorescence images of cells expressing GFP-TagG (A and B), GFP-TagH (C and D) in a wild type background or in the absence of Mbl (E and H), MreB (F and I), or MreBH (G and J) are shown. Cells were grown to mid-exponential stage at 30°C in CH medium (or CH supplemented with SMM for the mutants [see text]) with 5 mM K₂HPO₄/KH₂PO₄ (pH 7.5) and 0.5% xylose and imaged on wet agarose slides. Panels B and D show 2D deconvolutions of panels A and C, respectively. Panels E to J show 2D deconvolutions of raw images that are not included in the figure. In panels A and C, white arrowheads represent ongoing division sites and white arrows indicate regions of reduced fluorescence between the division septum and the lateral CW. White arrowheads in B and D indicate the absence of TagGH from polar regions. Bar, 5 μm.

FIG. 3.

Localization of TagO, TagB, and TagF. Fluorescence images of cells expressing TagO-GFP (A), TagB-GFP (B), and TagF-GFP (C) are shown. Panels D to F show 2D deconvolutions of the images in panels A to C, respectively. Cells were grown to mid-exponential stage at 30°C in CH medium with 5 mM K₂HPO₄/KH₂PO₄ (pH 7.5) and imaged on wet agarose slides. Expression of the fusion proteins was under control of the native promoters. Bar, 5 μm.

FIG. 4.

Two-hybrid interaction assays. (A and B) Y2H interactions. Matrices of Y2H interactions occurring between TagABDFGHO (A) and between TagABDFGHO and MreCD (B) are shown. The indicated proteins were expressed as baits (BD, Gal4 BD fusion) and/or as preys (AD, Gal4 AD fusion). Pairs of independent diploid yeast cells (1 and 2) expressing the various BD/AD combinations were arrayed as indicated and subjected to selection for expression of the HIS3 interaction reporter as described in Materials and Methods. The empty bait and prey vectors (BD and AD) were used to detect self-activation and as negative controls for interaction (−). The BD-TagB fusion protein exhibited a strong self-activation phenotype (a). Positive interactions are boxed or circled in red. (C and D) B2H interactions. Matrices of bacterial two-hybrid interactions occurring between TagABDEFGHO (C) and crosses of TagABDEFGHO with MreC and MreD (D) are shown. C refers to a fusion to the C terminus of T18 in the high-copy pUT18C vector. Other fusions are made to the C terminus of T25 (low-copy pKT25 vector) or to the N terminus of T18 (high-copy pUT18 vector). Transformations were carried out and plates incubated at 30°C for a maximum of 36 h.