Molecular imaging of glycan chains couples cell-wall polysaccharide architecture to bacterial cell morphology - PubMed (original) (raw)

Molecular imaging of glycan chains couples cell-wall polysaccharide architecture to bacterial cell morphology

Robert D Turner et al. Nat Commun. 2018.

Abstract

Biopolymer composite cell walls maintain cell shape and resist forces in plants, fungi and bacteria. Peptidoglycan, a crucial antibiotic target and immunomodulator, performs this role in bacteria. The textbook structural model of peptidoglycan is a highly ordered, crystalline material. Here we use atomic force microscopy (AFM) to image individual glycan chains in peptidoglycan from Escherichia coli in unprecedented detail. We quantify and map the extent to which chains are oriented in a similar direction (orientational order), showing it is much less ordered than previously depicted. Combining AFM with size exclusion chromatography, we reveal glycan chains up to 200 nm long. We show that altered cell shape is associated with substantial changes in peptidoglycan biophysical properties. Glycans from E. coli in its normal rod shape are long and circumferentially oriented, but when a spheroid shape is induced (chemically or genetically) glycans become short and disordered.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Fig. 1

Direct visualisation of glycan strand arrangement in the E. coli polymer envelope by AFM. a 3D representation of a peptidoglycan fragment mounted on poly-

l

-ornithine (see annotations). b Higher resolution image of boxed region in a. Long glycan chains are visible. c Zoom of marked boxed region in b showing side-by-side chains. d Zoom of marked boxed region in b showing overlapping chains

Fig. 2

Fig. 2

Mapping and quantification of glycan chain arrangement. a Example AFM image data. b Gradient orientation map of a. Inset: Polar histogram of data shown in b, overlaid with fitted ellipse. Dividing the long axis of the ellipse by the short yields a dimensionless parameter which reflects orientation order, with a higher number indicating an increased tendency to order. c Location of pores identified in the example image a. Inset: distribution of pore areas. d Peptidoglycan at pole: Large scan with bacterial shape outlined (dotted line). The instability inherent in imaging overlapping polymer leaflets is apparent in the top half of the image (height range 20 nm). e Smaller scan (acquired subsequently at lower scan speed) of region marked in d (height range 4 nm). f Enlargement of region marked in e. Many glycan strands running approximately along the circumferential axis of the cell are clearly visible (height range 3.5 nm). Inset: polar histogram. g Peptidoglycan from cylinder: Large scan of a different sacculus to d (height range 7.5 nm). h Smaller scan (acquired subsequently at lower scan speed) of region marked in g (height range 4 nm). i Enlargement of region marked in h. Many glycan strands running approximately along the circumferential axis of the cell are clearly visible (height range 4 nm). Inset: polar histogram

Fig. 3

Fig. 3

Size exclusion chromatography and AFM imaging of isolated glycan strands. a Size exclusion chromatography trace of glycan chains from E. coli (MG1655). b AFM images of glycan chains from fraction shown in a (each panel adjusted for best contrast). c Sums of extracted ion counts (EIC) for positively charged adducts of the major peptidoglycan sugar-peptide monomer and dimer from material digested with either Cellosyl alone (black bars), or ATL amidase, then Cellosyl (white bars). Amidase treatment removes the cross-linking peptides, substantially reducing the abundance of sugar-peptide adducts and freeing the glycan chains

Fig. 4

Fig. 4

Peptidoglycan characteristics in spheroid E. coli. a Optical phase contrast microscopy image of E. coli bacterial cells grown in media containing 10 μg/ml A22. b Optical phase contrast microscopy image of E. coli bacterial cells lacking the gene encoding MreB. c Image of peptidoglycan from E. coli grown in media containing A22, and associated polar histogram. Glycan strands appear less orientationally ordered and this is reflected in the more circular shape of the polar histogram. d Image of peptidoglycan from E. coli lacking the gene encoding MreB, and associated polar histogram. Glycan strands again appear less orientationally ordered. e Size exclusion chromatography trace of glycan chains from E. coli grown in media containing 10 μg/ml A22 (black line). Chains from bacteria grown in this way are shorter than for bacteria grown in unmodified media (see trace in grey for comparison). f Size exclusion chromatography trace of glycan chains from E. coli lacking the gene encoding MreB (black line). Chains from this genetically modified bacterial strain are shorter than those of unmodified bacteria (grey line)

Fig. 5

Fig. 5

Conceptual diagrams. a Peptidoglycan from rod-shaped E. coli (MG1655) is not crystalline yet has quantifiable orientational order with chains likely to be in a circumferential direction. This corresponds to the direction of maximum stress in the cylindrical part of the cell (stress is isotropic at the poles). It contains glycans up to 200 nm long. b Peptidoglycan from roughly spherical E. coli (lacking MreB or treated with A22) is much less ordered and has shorter glycan chains. Stress is isotropic in this case

Similar articles

Cited by

References

    1. Meeske AJ, et al. SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature. 2016;537:634–638. doi: 10.1038/nature19331. - DOI - PMC - PubMed
    1. van Heijenoort J. Peptidoglycan hydrolases of Escherichia coli. Microbiol. Mol. Biol. Rev. 2011;75:636–663. doi: 10.1128/MMBR.00022-11. - DOI - PMC - PubMed
    1. Turner RD, Vollmer W, Foster SJ. Different walls for rods and balls: the diversity of peptidoglycan. Mol. Microbiol. 2014;91:862–874. doi: 10.1111/mmi.12513. - DOI - PMC - PubMed
    1. Dufrêne YF, et al. Imaging modes of atomic force microscopy for application in molecular and cell biology. Nat. Nanotechnol. 2017;12:295–307. doi: 10.1038/nnano.2017.45. - DOI - PubMed
    1. Hayhurst EJ, Kailas L, Hobbs JK, Foster SJ. Cell wall peptidoglycan architecture in Bacillus subtilis. Proc. Natl Acad. Sci. USA. 2008;105:14603–14608. doi: 10.1073/pnas.0804138105. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources