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

Nature Microbiology volume 2, pages 1648–1657 (2017)Cite this article

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An Author Correction to this article was published on 08 January 2018

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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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Fig. 1: Background and introduction to experimental procedures.
Fig. 2: Three-dimensional structured illumination microscopy images of early predation by B. bacteriovorus (pre-labelled with BADA, false-coloured red) on prey E. coli cells after pulse labelling for 10 min with HADA (false-coloured cyan) to show early modification of cell walls.
Fig. 3: Three-dimensional structured illumination microscopy images of early predation by B. bacteriovorus (pre-labelled with BADA, false-coloured green) on prey E. coli imp4213 cells (which are more permeable and thus susceptible to the TADA pre-labelling, false coloured in red) after pulse labelling for 10 min with HADA (false-coloured cyan) to show early modification of cell walls.
Fig. 4: Quantitative and qualitative effects of two l,d-transpeptidases on prey cell wall modifications by FDAAs and their expression profiles.
Fig. 5: Plots showing HADA incorporation in the PG of prey E. coli mutants upon B. bacteriovorus predation and showing the damage by osmotic shock to bdelloplasts formed by B. bacteriovorus Ldt mutants.
Fig. 6: Epifluorescence and 3D-SIM images of the later stages of predation to show PG modification of the growing internal B. bacteriovorus.

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Change history

  • 08 January 2018

    In the original version of this Article, a grant number and acknowledgement were omitted. The Acknowledgements section should have stated that one of the 3D SIM microscopes used for this research was supported by Medical Research Council UK grant (MR/K015753/1) to S. Foster, University of Sheffield, UK, and that the authors thank C. Walther and S. Foster for the access and their kind help with this. This has now been corrected in all versions of the Article.

References

  1. Mainardi, J. L. et al. A novel peptidoglycan cross-linking enzyme for a β-lactam-resistant transpeptidation pathway. J. Biol. Chem. 280, 38146–38152 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Cava, F., de Pedro, M. A., Lam, H., Davis, B. M. & Waldor, M. K. Distinct pathways for modification of the bacterial cell wall by non-canonical d-amino acids. EMBO J. 30, 3442–3453 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Magnet, S. et al. Specificity of l,d-transpeptidases from Gram-positive bacteria producing different peptidoglycan chemotypes. J. Biol. Chem. 282, 13151–13159 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Fura, J. M., Kearns, D. & Pires, M. M. d-amino acid probes for penicillin binding protein-based bacterial surface labeling. J. Biol. Chem. 290, 30540–30550 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gupta, R. et al. The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin. Nat. Med. 16, 466–469 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Peltier, J. et al. Clostridium difficile has an original peptidoglycan structure with a high level of N-acetylglucosamine deacetylation and mainly 3-3 cross-links. J. Biol. Chem. 286, 29053–29062 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lam, H. et al. d-amino acids govern stationary phase cell wall remodeling in bacteria. Science 325, 1552–1555 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Radkov, A. D. & Moe, L. A. Bacterial synthesis of d-amino acids. Appl. Microbiol. Biotechnol. 98, 5363–5374 (2014).

    Article  CAS  PubMed  Google Scholar 

  9. Kuru, E., Tekkam, S., Hall, E., Brun, Y. V. & Van Nieuwenhze, M. S. Synthesis of fluorescent d-amino acids and their use for probing peptidoglycan synthesis and bacterial growth in situ. Nat. Protoc. 10, 33–52 (2015).

    Article  CAS  PubMed  Google Scholar 

  10. Fleurie, A. et al. MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumoniae. Nature 516, 259–262 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Tsui, H. C. et al. Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division of D39. Mol. Microbiol. 94, 21–40 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Pilhofer, M. et al. Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ. Nat. Commun. 4, 2856 (2013).

    Article  PubMed  Google Scholar 

  13. Stolp, H. & Starr, M. P. Bdellovibrio bacteriovorus gen. et sp. n., a predatory, ectoparasitic, and bacteriolytic microorganism. Antonie van Leeuwenhoek  29, 217–248 (1963).

    Article  CAS  Google Scholar 

  14. Sockett, R. E. Predatory lifestyle of Bdellovibrio bacteriovorus. Annu. Rev. Microbiol. 63, 523–539 (2009).

    Article  CAS  PubMed  Google Scholar 

  15. Rittenberg, S. C. & Shilo, M. Early host damage in the infection cycle of Bdellovibrio bacteriovorus. J. Bacteriol. 102, 149–160 (1970).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Abram, D., Castro e Melo, J. & Chou, D. Penetration of Bdellovibrio bacteriovorus into host cells. J. Bacteriol. 118, 663–680 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Abram, D. & Davis, B. K. Structural properties and features of parasitic Bdellovibrio bacteriovorus. J. Bacteriol. 104, 948–965 (1970).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lerner, T. R. et al. Specialized peptidoglycan hydrolases sculpt the intra-bacterial niche of predatory Bdellovibrio and increase population fitness. PLoS Pathog. 8, e1002524 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lambert, C. et al. Ankyrin-mediated self-protection during cell invasion by the bacterial predator Bdellovibrio bacteriovorus. Nat. Commun. 6, 8884 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lambert, C. et al. Interrupting peptidoglycan deacetylation during Bdellovibrio predator-prey interaction prevents ultimate destruction of prey wall, liberating bacterial-ghosts. Sci. Rep. 6, 26010 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Evans, K. J., Lambert, C. & Sockett, R. E. Predation by Bdellovibrio bacteriovorus HD100 requires type IV pili. J. Bacteriol. 189, 4850–4859 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Koval, S. F. et al. Bdellovibrio exovorus sp. nov., a novel predator of Caulobacter crescentus. Int. J. Syst. Evol. Microbiol. 63, 146–151 (2013).

    Article  PubMed  Google Scholar 

  23. Thomashow, M. F. & Rittenberg, S. C. Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: solubilization of Escherichia coli peptidoglycan. J. Bacteriol. 135, 998–1007 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Volle, C. B., Ferguson, M. A., Aidala, K. E., Spain, E. M. & Nunez, M. E. Quantitative changes in the elasticity and adhesive properties of Escherichia coli ZK1056 prey cells during predation by Bdellovibrio bacteriovorus 109J. Langmuir 24, 8102–8110 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Lambert, C., Chang, C. Y., Capeness, M. J. & Sockett, R. E. The first bite-profiling the predatosome in the bacterial pathogen Bdellovibrio. PLoS ONE 5, e8599 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Sanders, A. N. & Pavelka, M. S. Phenotypic analysis of Escherichia coli mutants lacking l,d-transpeptidases. Microbiology 159, 1842–1852 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Magnet, S. et al. Identification of the l,d-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan. J. Bacteriol. 189, 3927–3931 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pilizota, T. & Shaevitz, J. W. Origins of Escherichia coli growth rate and cell shape changes at high external osmolality. Biophys. J. 107, 1962–1969 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pilizota, T. & Shaevitz, J. W. Plasmolysis and cell shape depend on solute outer-membrane permeability during hyperosmotic shock in E. coli. Biophys. J. 104, 2733–2742 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Fenton, A. K., Kanna, M., Woods, R. D., Aizawa, S. I. & Sockett, R. E. Shadowing the actions of a predator: backlit fluorescent microscopy reveals synchronous nonbinary septation of predatory Bdellovibrio inside prey and exit through discrete bdelloplast pores. J. Bacteriol. 192, 6329–6335 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Araki, Y. & Ruby, E. G. A soluble enzyme activity that attaches free diaminopimelic acid to bdelloplast peptidoglycan. Biochemistry 27, 2624–2629 (1988).

    Article  CAS  PubMed  Google Scholar 

  32. Shilo, M. Morphological and physiological aspects of the interaction of bdellovibrio with host bacteria. Curr. Top. Microbiol. Immunol. 50, 174–204 (1969).

    CAS  PubMed  Google Scholar 

  33. Iida, Y. et al. Roles of multiple flagellins in flagellar formation and flagellar growth post bdelloplast lysis in Bdellovibrio bacteriovorus. J. Mol. Biol. 394, 1011–1021 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Eksztejn, M. & Varon, M. Elongation and cell division in Bdellovibrio bacteriovorus. Arch. Microbiol. 114, 175–181 (1977).

    Article  CAS  PubMed  Google Scholar 

  35. Kuru, E. et al. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent d-amino acids. Angew. Chem. 51, 12519–12523 (2012).

    Article  CAS  Google Scholar 

  36. Lambert, C. et al. Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus. Molec. Microbiol. 60, 274–286 (2006).

    Article  CAS  Google Scholar 

  37. Lambert, C. & Sockett, R. Nucleases in Bdellovibrio bacteriovorus contribute towards efficient self-biofilm formation and eradication of pre-formed prey biofilms. FEMS Microbiol. Lett. 320, 109–116 (2013).

    PubMed  PubMed Central  Google Scholar 

  38. Magnet, S., Dubost, L., Marie, A., Arthur, M. & Gutmann, L. Identification of the l,d-transpeptidases for peptidoglycan cross-linking in Escherichia coli. J. Bacteriol. 190, 4782–4785 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Thomason, L. C., Sawitzke, J. A., Li, X., Constantino, N. & Court, D. L. Recombineering: genetic engineering in bacteria using homologous recombination. Curr. Protoc. Mol. Biol. 14, 1–39 (2014).

    Google Scholar 

  41. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Fenton, A. K., Lambert, C., Wagstaff, P. C. & Sockett, R. E. Manipulating each MreB of Bdellovibrio bacteriovorus gives diverse morphological and predatory phenotypes. J. Bacteriol. 192, 1299–1311 (2010).

    Article  CAS  PubMed  Google Scholar 

  43. Ducret, A., Quardokus, E. M. & Brun, Y. V. MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat. Microbiol. 1, 16077 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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. One of the 3D SIM microscopes used for this research was supported by Medical Research Council UK grant (MR/K015753/1) to S. Foster, University of Sheffield, UK, and we thank C. Walther and S. Foster for the access and their kind help with this. 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).

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Author notes

  1. Erkin Kuru & Jonathan Rittichier

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

  2. Adeline Derouaux

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

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

Authors and 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.

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Correspondence to R. Elizabeth Sockett.

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A correction to this article is available online at https://doi.org/10.1038/s41564-017-0087-1.

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

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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.

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Kuru, E., Lambert, C., Rittichier, J. et al. Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation. Nat Microbiol 2, 1648–1657 (2017). https://doi.org/10.1038/s41564-017-0029-y

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