Article

Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation

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Abstract

Modification of essential bacterial peptidoglycan (PG)-containing cell walls can lead to antibiotic resistance; for example, β-lactam resistance by l,d-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG-labelling approach utilizing timed pulses of multiple fluorescent d-amino acids, we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall, l,d-transpeptidaseBd-mediated d-amino acid modifications strengthening prey PG during Bdellovibrio invasion, and a zonal mode of predator elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division.

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Acknowledgements

We thank D. Kearns and his laboratory (Indiana University, USA) for facilities and hospitality to culture B. bacteriovorus, A. Lovering (University of Birmingham, UK) for insights and assistance with the alignment of l,d-transpeptidase protein sequences in B. bacteriovorus, T. Pilizota (University of Edinburgh, UK) for advice on osmotic stress conditions, and R. Lowry (University of Nottingham, UK) for assistance in image acquisition. This work was supported by BBSRC grant [BB/M010325/1] to C.L., a Leverhulme Trust (UK) Research Leave Fellowship RF-2013-348 to R.E.S., NIH GM113172 grant to M.V.N. and Y.V.B. and R35GM122556 and GM51986 to Y.V.B. A.De. was supported by an EMBO long-term fellowship, and W.V. was supported by funds from the Wellcome Trust (101824/Z/13/Z).

Author information

Author notes

    • Erkin Kuru
    •  & Jonathan Rittichier

    Present address: Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA

    • Adeline Derouaux

    Present address: Xpress Biologics, Tour GIGA B34 (+3), Avenue de l’Hôpital, 11, B-4000, Liège (Sart-Tilman), Belgium

  1. Erkin Kuru and Carey Lambert contributed equally to this work.

Affiliations

  1. Molecular and Cellular Biochemistry Department, Indiana University Bloomington, Bloomington, IN, 47405, USA

    • Erkin Kuru
  2. School of Life Sciences, Nottingham University, Queen’s Medical Centre, Nottingham, NG7 2UH, UK

    • Jonathan Rittichier
    •  & Michael VanNieuwenhze
  3. Department of Chemistry, Indiana University Bloomington, Bloomington, IN, 47405, USA

    • Adrien Ducret
    •  & Yves V. Brun
  4. Department of Biology, Indiana University Bloomington, Bloomington, IN, 47405, USA

    • Carey Lambert
    • , Rob Till
    •  & R. Elizabeth Sockett
  5. The Centre for Bacterial Cell Biology, Baddiley Clark Building, Medical School, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX, UK

    • Adeline Derouaux
    • , Joe Gray
    • , Jacob Biboy
    •  & Waldemar Vollmer
  6. Bases Moléculaires et Structurales des Systèmes Infectieux, IBCP, Université Lyon 1, CNRS, UMR 5086, 7 passage du Vercors, 69367, Lyon Cedex 07, France

    • Adrien Ducret

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Contributions

E.K. and R.E.S. conceived the study and carried out the experiments along with C.L. using reagents constructed by M.V.N. and J.R., and bacterial strains constructed by R.T. and A. De. J.G. and J.B. performed muropeptide analysis in the laboratory of W.V. A. Du. wrote code and aided C.L. and E.K. with image analysis. Y.V.B. provided microscopy facilities and with M.V.N. and W.V. provided helpful comments. E.K., C.L. and R.E.S. wrote the manuscript with input and comments from the other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to R. Elizabeth Sockett.

Electronic supplementary material

  1. Supplementary Information

    Supplementary Figures 1–9, Supplementary Tables 1–3, Supplementary References.

  2. Life Sciences Reporting Summary

  3. Supplementary Video 1

    3D-project of 3D-SIM scan of BADA-labelled Bdellovibrio cells (false coloured in red) preying upon E. coli cells pulse-labelled with HADA (false coloured in cyan), 15 minutes post-mixing.