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Wall proficient E. coli capable of sustained growth in the absence of the Z-ring division machine

Nature Microbiology volume 1, Article number: 16091 (2016) Cite this article

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Abstract

The peptidoglycan cell wall is a major protective external sheath in bacteria and a key target for antibiotics1. Peptidoglycan is present in virtually all bacteria, suggesting that it was probably present in the last bacterial common ancestor2. Cell wall expansion is orchestrated by cytoskeletal proteins related to actin (MreB) and tubulin (FtsZ)3. FtsZ is a key essential player in a highly organized division machine that directs an invaginating annulus of cell wall peptidoglycan. The recent discovery that cell-wall-less bacteria (L-forms) can grow and divide independently of FtsZ4,5, provided a means of generating an ftsZ null mutant of Escherichia coli. Remarkably, we have been able to isolate variants of E. coli that lack FtsZ but are capable of efficient growth in a walled state. Genetic analysis reveals that a combination of mutations is needed for this phenotype. Importantly, the suppressive mutations lead to a major cell shape change, from the normal cylindrical shape to a branched and bulging, ramified shape, which we call ‘coli-flower’. The results highlight the versatility of bacterial cells and illustrate possible evolutionary routes leading to the emergence of specialized bacteria, such as pathogenic Chlamydia or aquatic Planctomycetes, that lack FtsZ but retain the cell wall6–8.

Unexpectedly, after prolonged incubation of a plate bearing an ftsZ-deleted strain, several colonies appeared, presumably containing cell variants capable of growth (Supplementary Fig. 1a). After confirming that the colonies were variants of the original strain rather than contaminants, we tested their properties and found that they were of two general classes. The first class had resumed growth as L-forms, presumably by acquiring mutations that block cell wall synthesis, and these were not studied further. The second class of mutants (CFL; coli-flower), however, appeared to comprise walled cells, and several lines of evidence suggested that these were mutants capable of walled growth in the absence of FtsZ. First, unlike L-forms, the CFL mutants were able to grow in either the absence or the presence of osmotic stabilizers (Supplementary Fig. 1b,c, right and left panels), although additional magnesium was needed to stabilize the CFL cells (Supplementary Fig. 1b,c, middle and right panels, Supplementary Fig. 1e,f and Supplementary Movie 1). Second, the CFL mutants grew in liquid media (Supplementary Fig. 1d), whereas E. coli L-forms are unable to proliferate under these conditions5. Finally and in contrast to L-forms, the CFL mutant strains were highly sensitive to β-lactam antibiotics (Fig. 1c and Supplementary Fig. 1g).

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Figure 1: Isolation and characterization of walled E. coli ftsZ-less strains.
Figure 2: Time-lapse imaging of a CFL strain grown in the complete absence of FtsZ.
Figure 3: PBP1B activity inactivation is required for the transition to a CFL mode of proliferation in the absence of FtsZ.
Figure 4: PBP1B activity inhibits CFL proliferation by restoration of cylindrical cell shape morphology in the absence of FtsZ.

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Acknowledgements

The authors thank T. Mignot for hosting parts of the work in his laboratory, W. Vollmer for the gift of the plasmid pBAD33-lpoB and comments on the manuscript, H. Strahl and L. Espinosa for discussions and D.W. Adams for critical reading of the manuscript. This work was funded by European Research Council Advanced Investigator Grants (nos 250363 and BH141574) to J.E.

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Authors and Affiliations

  1. Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Richardson Road, Newcastle upon Tyne NE2 4AX, UK

    Romain Mercier, Yoshikazu Kawai & Jeff Errington

  2. Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, CNRS-Aix-Marseille University, 31 Chemin Joseph Aiguier, 13009 Marseille, France

    Romain Mercier

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  1. Romain Mercier
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Contributions

R.M. designed and performed the experiments. R.M. and Y.K. analysed the data. R.M. and J.E. wrote the manuscript.

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Correspondence to Romain Mercier or Jeff Errington.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Data 1–4, Supplementary Figures 1–8, Supplementary Tables 1–3, Supplementary References (PDF 7045 kb)

Supplementary Video 1

Time-lapse series showing ΔftsZsup5 cells growing on NA 20mM Mg2+, from which the panels in Figure 2b were obtained. Phase contrast images were acquired automatically every minute for about 279 min. Scale bar is 3 μm. (AVI 1117 kb)

Supplementary Video 2

Time-lapse series showing ΔftsZsup1 cell growing on NA 20mM Mg2+, from which the panels in Figure 2b were obtained. Phase contrast images were acquired automatically every minute for about 279 min. Scale bar is 3 μm. (AVI 2475 kb)

Supplementary Video 3

Time-lapse series showing ΔftsZsup7 cell growing on NA 20mM Mg2+. Phase contrast images were acquired automatically every minute for about 109 min. Scale bar is 3 μm. (AVI 810 kb)

Supplementary Video 4

Time-lapse series showing ΔftsZsup7 cell dividing on NA 20mM Mg2+, from which the panels in Figure 2b were obtained. Phase contrast images were acquired automatically every minute for about 69 min. Scale bar is 3 μm. (AVI 297 kb)

Supplementary Video 5

Time-lapse series showing ΔftsZsup7 cell dividing on NA + 20mM Mg2+, from which the panels in Figure 2c were obtained. Phase contrast images were acquired automatically every minute for about 90 min. Scale bar is 3 μm. (AVI 419 kb)

Supplementary Video 6

Time-lapse series showing RM445 (ΔftsZ/Δlpp/ΔlpoBsup) cell, bearing the plasmid pBAD33-lpoB cell growing on NA + 20mM Mg2+ref with glucose (lpp expression repressed). Phase contrast images were acquired automatically every minute for about 209 min. Scale bar is 3 μm. (AVI 3149 kb)

Supplementary Video 7

Time-lapse series showing RM445 (ΔftsZ/Δlpp/ΔlpoBsup), bearing the plasmid pBAD33-lpoB cell growing on NA + 20mM Mg2+ with lpoB expression induced (arabinose), from which the panels in Figure 3d were obtained. Phase contrast images were acquired automatically every minute for about 209 min. Scale bar is 3 μm. (AVI 3477 kb)

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Mercier, R., Kawai, Y. & Errington, J. Wall proficient E. coli capable of sustained growth in the absence of the Z-ring division machine. Nat Microbiol 1, 16091 (2016). https://doi.org/10.1038/nmicrobiol.2016.91

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