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RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic discovery for the peptidoglycan polymerase pathway

Nature Microbiology volume 2, Article number: 16253 (2017) Cite this article

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A Corrigendum to this article was published on 30 January 2017

Abstract

The bacterial cell wall is a highly conserved essential component of most bacterial groups. It is the target for our most frequently used antibiotics and provides important small molecules that trigger powerful innate immune responses. The wall is composed of glycan strands crosslinked by short peptides. For many years, the penicillin-binding proteins were thought to be the key enzymes required for wall synthesis. RodA and possibly other proteins in the wider SEDS (shape, elongation, division and sporulation) family have now emerged as a previously unknown class of essential glycosyltranferase enzymes, which play key morphogenetic roles in bacterial cell wall synthesis. We provide evidence in support of this role and the discovery of small natural product molecules that probably target these enzymes. The SEDS proteins have exceptional potential as targets for new antibacterial therapeutic agents.

The bacterial cell wall is an ancient structure that was probably developed early in the evolution of cells and may have contributed to the explosive bacterial radiation that occurred about 3–4 billion years ago1. The wall is essential for bacterial viability and an important target for antibiotics2. The major conserved component of the wall is peptidoglycan (PG), which comprises long strands of alternating amino sugars crosslinked by peptide bridges. The key enzymes needed for assembly of the wall are glycosyltransferases (GTase), which generate the glycan strands and transpeptidases (TPase), which generate the crosslinks3. Penicillin-binding proteins (PBPs), first identified because they bind β-lactam antibiotics that prevent the TPase reaction, were considered to be the key enzymes in PG assembly because one major class (class A) also carry a GTase domain capable of synthesizing PG strands. A second major class of PBPs (class B) lack the GTase domain. Curiously, the class B PBPs more frequently display genetic essentiality than the class A PBPs. Furthermore, their mutant phenotypes suggest that they play specialized roles in morphogenesis; thus, mutants of rod-shaped bacteria such as Escherichia coli can be specifically affected in either cell elongation (for example, pbpA gene) or cell division (ftsI gene)4. Bacillus subtilis has two redundant elongation enzymes, PBP2A and PBPH (ref. 5), and an essential division enzyme, PBP2B (ref. 6).

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Figure 1: Use of MOE sensitivity to screen for missing GTase candidate genes.
Figure 2: Evidence for RodA as a candidate for the missing GTase.
Figure 3: Isolation and characterization of a putative inhibitor of the missing GTase.

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Acknowledgements

This work was funded by grants from the BBSRC (BB/G015902/1, to R.A.D., J.E. and A.G.), ERC (670980, to J.E., Y.K. and K.E.) and the Wellcome Trust (WT098374AIA, to J.E. and L.J.W.) and core resources in Demuris Ltd. The authors thank R. Emmins, who constructed some of the mutants, D. Roberts for help with microfluidic microscopy and R. Sweet for help with the large-scale fermentation run.

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Author notes

  1. Kaveh Emami, Aurelie Guyet and Yoshikazu Kawai: These authors contributed equally to this work.

Authors and Affiliations

  1. The Centre for Bacterial Cell Biology, Baddiley-Clark Building, Medical School, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX, UK

    Kaveh Emami, Aurelie Guyet, Yoshikazu Kawai, Ling J. Wu, Richard A. Daniel & Jeff Errington

  2. Demuris Ltd, Newcastle Biomedicine Bio-Incubators, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK

    Jenny Devi, Nick Allenby & Jeff Errington

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  1. Kaveh Emami
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Contributions

K.E., A.G., Y.K., J.D., L.J.W. and R.A.D. carried out the experiments. All authors contributed to the experimental design and concepts, and that all authors contributed to the text. J.E. wrote the main text with contributions from all other authors.

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Correspondence to Richard A. Daniel or Jeff Errington.

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Competing interests

J.E. is a director and shareholder at Demuris Ltd. N.A. and J.D. are employees at Demuris Ltd.

Supplementary information

Supplementary Information

Legends for all Supplementary Material and Supplementary Figures 2–5. (PDF 795 kb)

Supplementary Figures and Tables

Supplementary Figure 1 and Supplementary Tables 1 and 2. (PDF 845 kb)

Supplementary Video 1

Effect of compound 654/A (30 μl) on growth and morphology of the Δ4 strain. Approximately 3 hours after addition of 654/A to Δ4 cells in the microfluidic device, cell growth slows down significantly and extensive cell lysis occurs. (AVI 19450 kb)

Supplementary Video 2

Untreated control for growth and morphology of the Δ4 strain. In the absence of compound 654/A, Δ4 cells continue their normal growth up to full confluency. (AVI 39740 kb)

Supplementary Video 3

Effect of compound 654/A (30 μl) on growth and morphology of the wild type. Wild-type cells show normal growth at relatively low concentrations of 654/A. (AVI 34112 kb)

Supplementary Video 4

Effect of compound 654/A (70 μl) on growth and morphology of the wild type. At relatively high concentrations of 654/A, extensive lysis of wild-type cells occurs, similar to that of the Δ4 cells (see Supplementary Video 1). (AVI 25887 kb)

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Emami, K., Guyet, A., Kawai, Y. et al. RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic discovery for the peptidoglycan polymerase pathway. Nat Microbiol 2, 16253 (2017). https://doi.org/10.1038/nmicrobiol.2016.253

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