Abstract
Bacterial species have diverse cell shapes that enable motility, colonization and virulence. The cell wall defines bacterial shape and is primarily built by two cytoskeleton-guided synthesis machines, the elongasome and the divisome. However, the mechanisms producing complex shapes, like the curved-rod shape of Vibrio cholerae, are incompletely defined. Previous studies have reported that species-specific regulation of cytoskeleton-guided machines enables formation of complex bacterial shapes such as cell curvature and cellular appendages. In contrast, we report that CrvA and CrvB are sufficient to induce complex cell shape autonomously of the cytoskeleton in V. cholerae. The autonomy of the CrvAB module also enables it to induce curvature in the Gram-negative species Escherichia coli, Pseudomonas aeruginosa, Caulobacter crescentus and Agrobacterium tumefaciens. Using inducible gene expression, quantitative microscopy and biochemistry, we show that CrvA and CrvB circumvent the need for patterning via cytoskeletal elements by regulating each other to form an asymmetrically localized, periplasmic structure that binds directly to the cell wall. The assembly and disassembly of this periplasmic structure enables dynamic changes in cell shape. Bioinformatics indicate that CrvA and CrvB may have diverged from a single ancestral hybrid protein. Using fusion experiments in V. cholerae, we find that a synthetic CrvA/B hybrid protein is sufficient to induce curvature on its own, but that expression of two distinct proteins, CrvA and CrvB, promotes more rapid curvature induction. We conclude that morphological complexity can arise independently of cell-shape specification by the core cytoskeleton-guided synthesis machines.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The data supporting the findings of the study are available in this article or on the Princeton DataSpace (https://doi.org/10.34770/b652-mz26). Source data are provided with this paper.
Code availability
The custom MATLAB routines used for processing and analysing microscopy data are freely available from the corresponding author on request.
References
Young, K. D. The selective value of bacterial shape. Microbiol. Mol. Biol. Rev. 70, 660–703 (2006).
Yang, D. C., Blair, K. M. & Salama, N. R. Staying in shape: the impact of cell shape on bacterial survival in diverse environments. Microbiol. Mol. Biol. Rev. 80, 187–203 (2016).
Orr, R. G., Cheng, X., Vidali, L. & Bezanilla, M. Orchestrating cell morphology from the inside out – using polarized cell expansion in plants as a model. Curr. Opin. Cell Biol. 62, 46–53 (2019).
Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006).
Caccamo, P. D. & Brun, Y. V. The molecular basis of noncanonical bacterial morphology. Trends Microbiol. 26, 191–208 (2017).
Taylor, J. A., Sichel, S. R. & Salama, N. R. Bent bacteria: a comparison of cell shape mechanisms in proteobacteria. Annu. Rev. Microbiol. 73, 457–480 (2019).
Typas, A., Banzhaf, M., Gross, C. A. & Vollmer, W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat. Rev. Microbiol. 10, 123–136 (2011).
Yang, X. et al. GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis. Science 355, 744–747 (2017).
Bisson-Filho, A. W. et al. Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division. Science 355, 739–743 (2017).
Ursell, T. S. et al. Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization. Proc. Natl Acad. Sci. USA 111, E1025–E1034 (2014).
Garner, E. C. et al. Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis. Science 333, 222–225 (2011).
Schuech, R., Hoehfurtner, T., Smith, D. J. & Humphries, S. Motile curved bacteria are Pareto-optimal. Proc. Natl Acad. Sci. USA 116, 14440–14447 (2019).
Bartlett, T. M. et al. A periplasmic polymer curves Vibrio cholerae and promotes pathogenesis. Cell 168, 172–185 (2017).
Persat, A., Stone, H. A. & Gitai, Z. The curved shape of Caulobacter crescentus enhances surface colonization in flow. Nat. Commun. 5, 3824 (2014).
Ausmees, N., Kuhn, J. R. & Jacobs-Wagner, C. The bacterial cytoskeleton: an intermediate filament-like function in cell shape. Cell 115, 705–713 (2003).
Charbon, G., Cabeen, M. T. & Jacobs-Wagner, C. Bacterial intermediate filaments: in vivo assembly, organization, and dynamics of crescentin. Genes Dev. 23, 1131–1144 (2009).
Cabeen, M. T. et al. Bacterial cell curvature through mechanical control of cell growth. EMBO J. 28, 1208–1219 (2009).
Billini, M., Biboy, J., Kuhn, J., Vollmer, W. & Thanbichler, M. A specialized MreB-dependent cell wall biosynthetic complex mediates the formation of stalk-specific peptidoglycan in Caulobacter crescentus. PLoS Genet. 15, e1007897 (2019).
Wagner, J. K., Galvani, C. D. & Brun, Y. V. Caulobacter crescentus requires RodA and MreB for stalk synthesis and prevention of ectopic pole formation. J. Bacteriol. 187, 544–553 (2005).
Dent, E. W., Gupton, S. L. & Gertler, F. B. The growth cone cytoskeleton in axon outgrowth and guidance. Cold Spring Harb. Perspect. Biol. 3, a001800 (2011).
Brown, J. W. & McKnight, C. J. Molecular model of the microvillar cytoskeleton and organization of the brush border. PLoS ONE 5, e9406 (2010).
Farmer, J. J. III et al. in Bergey’s Manual of Systematics of Archaea and Bacteria, 1–79 (John Wiley & Sons, 2015).
Cowles, K. N. & Gitai, Z. Surface association and the MreB cytoskeleton regulate pilus production, localization and function in Pseudomonas aeruginosa. Mol. Microbiol. 76, 1411–1426 (2010).
Srivastava, P., Demarre, G., Karpova, T. S., McNally, J. & Chattoraj, D. K. Changes in nucleoid morphology and origin localization upon inhibition or alteration of the actin homolog, MreB, of Vibrio cholerae. J. Bacteriol. 189, 7450–7463 (2007).
Cameron, T. A., Anderson-Furgeson, J., Zupan, J. R., Zik, J. J. & Zambryski, P. C. Peptidoglycan synthesis machinery in Agrobacterium tumefaciens during unipolar growth and cell division. mBio 5, e01219–14 (2014).
Howell, M. et al. Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division. Mol. Microbiol. 111, 1074–1092 (2019).
Ouzounov, N. et al. MreB orientation correlates with cell diameter in Escherichia coli. Biophys. J. 111, 1035–1043 (2016).
Dorr, T. et al. Differential requirement for PBP1a and PBP1b in in vivo and in vitro fitness of Vibrio cholerae. Infect. Immun. 82, 2115–2124 (2014).
Bratton, B. P., Shaevitz, J. W., Gitai, Z. & Morgenstein, R. M. MreB polymers and curvature localization are enhanced by RodZ and predict E. coli’s cylindrical uniformity. Nat. Commun. 9, 2797 (2018).
Hussain, S. et al. MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis. eLife 7, e32471 (2018).
Schierle, C. F. et al. The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. J. Bacteriol. 185, 5706–5713 (2003).
Weaver, A. I. et al. Genetic determinants of penicillin tolerance in Vibrio cholerae. Antimicrob. Agents Chemother. 62, e01326-18 (2018).
Taguchi, A., Kahne, D. & Walker, S. Chemical tools to characterize peptidoglycan synthases. Curr. Opin. Chem. Biol. 53, 44–50 (2019).
Battistuzzi, F. U., Feijao, A. & Hedges, S. B. A genomic timescale of prokaryote evolution: insights into the origin of methanogenesis, phototrophy, and the colonization of land. BMC Evol. Biol. 4, 44 (2004).
Jiang, C., Brown, P. J., Ducret, A. & Brun, Y. V. Sequential evolution of bacterial morphology by co-option of a developmental regulator. Nature 506, 489–493 (2014).
Skorupski, K. & Taylor, R. K. Positive selection vectors for allelic exchange. Gene 169, 47–52 (1996).
Edwards, R. A., Keller, L. H. & Schifferli, D. M. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 207, 149–157 (1998).
Bina, X. R., Wong, E. A., Bina, T. F. & Bina, J. E. Construction of a tetracycline inducible expression vector and characterization of its use in Vibrio cholerae. Plasmid 76, 87–94 (2014).
Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).
Bridges, A. A. & Bassler, B. L. The intragenus and interspecies quorum-sensing autoinducers exert distinct control over Vibrio cholerae biofilm formation and dispersal. PLoS Biol. 17, e3000429 (2019).
Sawabe, T. et al. Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov. Front. Microbiol. 4, 414 (2013).
Criscuolo, A. & Gribaldo, S. BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments. BMC Evol. Biol. 10, 210 (2010).
Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010).
Afgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 46, W537–W544 (2018).
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Henikoff, S. & Henikoff, J. G. Amino acid substitution matrices from protein blocks. Proc. Natl Acad. Sci. USA 89, 10915–10919 (1992).
Capra, J. A. & Singh, M. Predicting functionally important residues from sequence conservation. Bioinformatics 23, 1875–1882 (2007).
Desmarais, S. M., Cava, F., de Pedro, M. A. & Huang, K. C. Isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography. J. Vis. Exp. 83, e51183 (2014).
Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).
Acknowledgements
We thank members of the Gitai and Shaevitz laboratories for helpful discussions, the Bina and Bassler laboratories for reagents, and J. Sanfilippo, M. Koch, C. Ellison, J. Shaevitz and T. Silhavy for helpful feedback on the manuscript. This work was supported by a grant (1DP1AI124669 to Z.G.) from the National Institutes of Health, an institutional training grant (T32GM007388 to T.M.B. and E.B.), a grant from the National Science Foundation (PHY-1734030 to B.P.B.) and Graduate Research Fellowships from the National Science Foundation (DGE-2039656 to N.R.M., DGE-2039656 to K.J.C. and DGE-1656466 to T.M.B.).
Author information
Authors and Affiliations
Contributions
N.R.M., E.B., K.J.C. and T.M.B. performed all the experiments with design input from Z.G. N.R.M. and B.P.B. peformed the data and sequence analysis. Z.G. and N.R.M. wrote the manuscript with input from the other authors.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature Microbiology thanks William Margolin, Jie Xiao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 CrvAB sequence alignment and complementation analysis.
a, The crvA and crvB open reading frames drawn to scale at the native locus in V. cholerae. b, Alignment of V. cholerae CrvB amino acid sequence to V. cholerae CrvA by Clustalω49. c–e, Curvature of ΔcrvA (c), ΔcrvB (d) or ΔcrvAB (e) populations expressing an empty vector (EV) or plasmids with the indicated gene(s). Images represent 95th percentile of curvature in respective populations. Scale bars, 1 μm; images within each figure panel are to scale. p-values determined by two-sided Wilcoxon rank sum test; n=300 from three biological replicates.
Extended Data Fig. 2 Quantification of heterologous CrvAB expression.
a, Curvature of populations expressing an empty vector (+EV) or a plasmid with crvA and crvB (+crvAB). p-values determined by two-sided Wilcoxon rank sum test; n = 300 from three biological replicates. b, Cumulative distribution functions of populations in A ± bootstrapped 95% confidence intervals. c, Images represent indicated quantiles from populations in A. Each row corresponds to the species labelled to the left of A. Scale bars are 1μm; images within each species are to scale. a, The crvA and crvB open reading frames drawn to scale at the native locus in V. cholerae. b, Alignment of V. cholerae CrvB amino acid sequence to V. cholerae CrvA by Clustalω49. c-e, Curvature of (c) ΔcrvA, (d) ΔcrvB, or (e) ΔcrvAB populations expressing an empty vector (EV) or plasmids with the indicated gene(s). c-e, Images represent 95th percentile of curvature in respective populations. Scale bars are 1μm; images within each figure panel are to scale. p-values determined by two-sided Wilcoxon rank sum test; n = 300 from three biological replicates.
Extended Data Fig. 3 Morphology of E. coli and V. cholerae after inhibition of PG synthesis machinery.
a, Quantified cell widths of A22-treated populations of E. coli from Fig. 2a. b, Representative images of wild-type and ΔcrvAB V. cholerae after growth with the elongasome inhibitor, A22. c, Quantification of curvature from populations in B. d, Quantification of cell width from populations in B. e, Population curvature and representative images of indicated V. cholerae strains. n = 300 from three biological replicates. b,e, Images represent 95th percentile of curvature in respective populations. c,d, p-values from two-sided Wilcoxon rank sum test; n = 200 from two biological replicates. Scale bars are 1μm; images within each figure panel are to scale.
Extended Data Fig. 4 Functionality, time lapse, and osmotic dissociation of fluorescent CrvA and CrvB fusions.
a, Quantified curvature of populations of V. cholerae expressing the indicated fusion proteins from the native genomic locus. Data from wild type and ΔcrvAB are repeated from Fig. 1a. b, Time lapse images of CrvB-GFP and CrvA-mCherry structures in growing V. cholerae. See Movie S1 for full time lapse. c, Quantified curvature of E. coli populations expressing an empty vector (+EV), a plasmid with crvA and crvB (+crvAB), or the same plasmid with crvA-mCherry and crvB-GFP. Data from +EV and +crvAB are repeated from Fig. 1c. d, Osmotic shock of V. cholerae expressing crvA-GFP or crvB-GFP. a,c, Images were taken 6 h after dilution of saturated overnight cultures. Images represent 95th percentile of curvature in respective populations. c, p-values from two-sided Wilcoxon rank sum test; n = 300 from three biological replicates. Scale bars are 1μm in A-C and 5μm in D; images within each figure panel are to scale.
Extended Data Fig. 5 Quantification and localization of crvACBS in V. cholerae.
a, Schematic of crv locus and representative image of cells expressing the crvACBS chimera tagged with msfGFP (CrvACBS-GFP). b, Quantified curvature of populations from overnight cultures of indicated V. cholerae strains. Data from wild-type, ΔcrvB, and crvACBS are repeated from Fig. 4. p-values from two-sided Wilcoxon rank sum test; n = 300 from three biological replicates. Scale bars are 1μm; images within each figure panel are to scale.
Extended Data Fig. 6 The evolution of CrvA/B/Y and functional study of CrvY.
a, Extended cladogram from Fig. 1b. b, Linear form of extended cladogram in A. c, Phylogeny of all sequenced CrvA, CrvB, and CrvY homologues. d, Curvature of populations of wild-type A. fischeri or ΔcrvY. e, Curvature of populations of V. cholerae ΔcrvAB expressing an empty vector (+EV) or a plasmid with crvY from A. fischeri (+crvY). a,b, Numbers are ‘Clade IDs’ for reference to Supplementary Table 2, which contains full composition of terminal nodes. d, Terminal nodes are placed at the mean ± standard deviation of the sequences collapsed into each node. d,e, Images represent 95th percentile of curvature in respective populations. Scale bars are 1μm; images within each figure panel are to scale. p-values determined by two-sided Wilcoxon rank sum test; n = 300 from three biological replicates.
Extended Data Fig. 7 Comparison of CrvA and CrvB concentrations under inducible expression.
a, Western blots of CrvA-GFP (top) and CrvB-GFP (bottom) quantified in Fig. 5e with molecular weight standards (M). Separate molecular weight standard in top panel is from the same blot as samples to right. Representative of three biological replicates with similar results. b, Western blots of crvA-GFP and crvA-GFP;crvB overexpression from Fig. 6a,b with molecular weight standard (M). Separate left and right panels are from the same blot. Representative of two biological replicates with similar results. c, Representative images from anydrotetracycline (tet) overexpression of CrvA and CrvB-GFP in E. coli. Scale bar is 1μm; all images are to scale. d, Quantification of curvature from populations in C. n.s.=confidence <95%; *=confidence >95%; **=confidence >99%; ***=confidence >99.9% (see Supplementary Table 3 for exact p-values); two-sided Wilcoxon rank sum test with Bonferroni correction; n = 100 from two biological replicates. e, Western blot of crvB-GFP and crvA;crvB-GFP overexpression from (c). Separate molecular weight standard (M) and right panel are from the same blot. Representative of two biological replicates with similar results.
Extended Data Fig. 8 CrvAB overexpression drives early curvature.
a, Curvature and representative images of populations overexpressing CrvA and CrvB two hours after back-dilution into media containing anhydrotetracycline. b, Phase contrast image from A showing the extreme range of curvatures upon overexpression that are not captured by our quantitative analysis. a, p-values calculated from two-sided Wilcoxon rank sum test; n = 300 from three biological replicates. Scale bars are 1μm in A and 5μm in B; images within each figure panel are to scale.
Supplementary information
Supplementary Information
Supplementary Table 1, descriptions of Tables 2 and 3, and captions for Videos 1–4.
Supplementary Table 2
Genomic accession numbers, Crv protein accession numbers, and clade labels for each of 921 Vibrionacae genomes. Clade labels are listed in Extended Data Fig. 6b,c. Excel file with two header rows and 921 data rows. Column names (first header row) and descriptions of the data in each column (second header row) are included for each of the 17 columns.
Supplementary Table 3
Table of P values from statistical tests represented as symbols in figures.
Supplementary Video 1
Time lapse of intact CrvA–mCherry/CrvB–GFP structure in V. cholerae cell from Extended Data Fig. 4b. The fluorescent structure remains intact throughout the time course and remains associated with the curved daughter cell from the initial division. Scale bar, 1 μm.
Supplementary Video 2
Time lapse of CrvA–mCherry/CrvB–GFP structures in V. cholerae filamented on low melting point agarose pads containing 5 μg ml−1 cephalexin. As cells filament, CrvAB structures do not re-localize to regions of high curvature and new CrvAB structures can be seen forming in straight regions of the cell.
Supplementary Video 3
Time lapse of CrvA–mCherry/CrvB–GFP structures in V. cholerae filamented on low melting point agarose pads containing 5 μg ml−1 cephalexin. As cells filament, CrvAB structures do not re-localize to regions of high curvature and new CrvAB structures can be seen forming in straight regions of the cell.
Supplementary Video 4
Time lapse of CrvA–mCherry/CrvB–GFP structures in V. cholerae recapitulating dynamics of population curvature and CrvAB localization observed in time courses from liquid culture. Filled green arrows point to specific CrvA–mCherry/CrvB–GFP structures of interest and outlined green arrows indicate the position of cells before those structures have formed.
Source data
Source Data Fig. 1
Numerical data from Fig. 1a.
Source Data Fig. 2
Numerical data from Fig. 2b.
Source Data Fig. 3
Unprocessed gels.
Source Data Fig. 4
Numerical data from Fig. 4b and c.
Source Data Fig. 5
Unprocessed blot from Fig. 5f and numerical data from Fig. 5a,c–e.
Source Data Fig. 6
Numerical data from Fig. 6b and c.
Source Data Extended Data Fig. 1
Numerical data from Extended Data Fig. 1c and d.
Source Data Extended Data Fig. 2
Numerical data from Extended Data Fig. 2a and b.
Source Data Extended Data Fig. 3
Numerical data from Extended Data Fig. 3a,c–e.
Source Data Extended Data Fig. 4
Numerical data from Extended Data Fig. 4a and c.
Source Data Extended Data Fig. 5
Numerical data from Extended Data Fig. 5b.
Source Data Extended Data Fig. 6
Numerical data from Extended Data Fig. 6d,e.
Source Data Extended Data Fig. 7
Unprocessed blots and numerical data from Extended Data Fig. 7d.
Source Data Extended Data Fig. 8
Numerical data from Extended Data Fig. 8a.
Rights and permissions
About this article
Cite this article
Martin, N.R., Blackman, E., Bratton, B.P. et al. CrvA and CrvB form a curvature-inducing module sufficient to induce cell-shape complexity in Gram-negative bacteria. Nat Microbiol 6, 910–920 (2021). https://doi.org/10.1038/s41564-021-00924-w
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41564-021-00924-w
This article is cited by
-
Evidence of two differentially regulated elongasomes in Salmonella
Communications Biology (2023)
-
Asymmetric peptidoglycan editing generates cell curvature in Bdellovibrio predatory bacteria
Nature Communications (2022)
-
Optimising expression of the large dynamic range FRET pair mNeonGreen and superfolder mTurquoise2ox for use in the Escherichia coli cytoplasm
Scientific Reports (2022)