Structure to function of an α-glucan metabolic pathway that promotes Listeria monocytogenes pathogenesis

This article has been updated


Here we employ a ‘systems structural biology’ approach to functionally characterize an unconventional α-glucan metabolic pathway from the food-borne pathogen Listeria monocytogenes (Lm). Crystal structure determination coupled with basic biochemical and biophysical assays allowed for the identification of anabolic, transport, catabolic and regulatory portions of the cycloalternan pathway. These findings provide numerous insights into cycloalternan pathway function and reveal the mechanism of repressor, open reading frame, kinase (ROK) transcription regulators. Moreover, by developing a structural overview we were able to anticipate the cycloalternan pathway's role in the metabolism of partially hydrolysed starch derivatives and demonstrate its involvement in Lm pathogenesis. These findings suggest that the cycloalternan pathway plays a role in interspecies resource competition—potentially within the host gastrointestinal tract—and establish the methodological framework for characterizing bacterial systems of unknown function.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: lmo2446lmo2444 operon function in extracellular CA anabolism.
Figure 2: lmo0178-lmo0184 operon function in cycloalternan (CA) uptake and intracellular catabolism.
Figure 3: Lmo0178 exerts transcriptional control over the lmo0178-lmo0184 operon.
Figure 4: The CA pathway promotes Lm infection and growth on partially hydrolysed starch derivatives.
Figure 5: Atomic model of CA pathway function.

Change history

  • 14 July 2017

    In the PDF version of this article previously published, the year of publication provided in the footer of each page and in the 'How to cite' section was erroneously given as 2017, it should have been 2016. This error has now been corrected. The HTML version of the article was not affected.


  1. 1

    Edwards, A. M. et al. Too many roads not taken. Nature 470, 163–165 (2011).

    CAS  Article  Google Scholar 

  2. 2

    Hermann, J. C. et al. Structure-based activity prediction for an enzyme of unknown function. Nature 448, 775–779 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Konc, J., Hodošček, M., Ogrizek, M., Trykowska Konc, J. & Janežič, D. Structure-based function prediction of uncharacterized protein using binding sites comparison. PLoS Comput. Biol. 9, e1003341 (2013).

    Article  Google Scholar 

  4. 4

    Zhao, S. et al. Discovery of new enzymes and metabolic pathways by using structure and genome context. Nature 502, 698–702 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Vetting, M. W. et al. Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute binding proteins and unbiased panning of metabolomes. Biochemistry 54, 909–931 (2015).

    CAS  Article  Google Scholar 

  6. 6

    Anderson, W. F. Structural genomics and drug discovery for infectious diseases. Infect. Disord. Drug Targets 9, 507–517 (2009).

    CAS  Article  Google Scholar 

  7. 7

    Vazquez-Boland, J. A. et al. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14, 584–640 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Renier, S., Micheau, P., Talon, R., Hébraud, M. & Desvaux, M. Subcellular localization of extracytoplasmic proteins in monoderm bacteria: rational secretomics-based strategy for genomic and proteomic analyses. PLoS ONE 7, e42982 (2012).

    CAS  Article  Google Scholar 

  9. 9

    Suzuki, N. et al. Structural elucidation of the cyclization mechanism of α-1,6-glucan by Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase. J. Biol. Chem. 289, 12040–12051 (2014).

    CAS  Article  Google Scholar 

  10. 10

    Nishimoto, T. et al. Purification and characterization of glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide in Bacillus globisporus C11. Biosci. Biotechnol. Biochem. 66, 1806–1818 (2002).

    CAS  Article  Google Scholar 

  11. 11

    Kim, Y. K., Kitaoka, M., Hayashi, K., Kim, C. H. & Côté, G. L. A synergistic reaction mechanism of a cycloalternan-forming enzyme and a d-glucosyltransferase for the production of cycloalternan in Bacillus sp. NRRL B-21195. Carbohydr. Res. 338, 2213–2220 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Cote, G. L. & Biely, P. Enzymically produced cyclic α-1,3-linked and α-1,6-linked oligosaccharides of d-glucose. Eur. J. Biochem. 226, 641–648 (1994).

    CAS  Article  Google Scholar 

  13. 13

    Watanabe, K., Hata, Y., Kizaki, H., Katsube, Y. & Suzuki, Y. The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 Å resolution: structural characterization of proline-substitution sites for protein thermostabilization. J. Mol. Biol. 269, 142–153 (1997).

    CAS  Article  Google Scholar 

  14. 14

    Kazanov, M. D., Li, X., Gelfand, M. S., Osterman, A. L. & Rodionov, D. A. Functional diversification of ROK-family transcriptional regulators of sugar catabolism in the thermotogae phylum. Nucleic Acids Res. 41, 790–803 (2013).

    CAS  Article  Google Scholar 

  15. 15

    Wurtzel, O. et al. Comparative transcriptomics of pathogenic and non-pathogenic Listeria species. Mol. Syst. Biol. 8, 583 (2012).

    Article  Google Scholar 

  16. 16

    Schumacher, M. A. et al. Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR. EMBO J. 21, 1210–1218 (2002).

    CAS  Article  Google Scholar 

  17. 17

    Wong, J. J., Lu, J., Edwards, R. A., Frost, L. S. & Glover, J. N. Structural basis of cooperative DNA recognition by the plasmid conjugation factor, TraM. Nucleic Acids Res. 39, 6775–6788 (2011).

    CAS  Article  Google Scholar 

  18. 18

    Bréchemier-Baey, D., Domínguez-Ramírez, L. & Plumbridge, J. The linker sequence, joining the DNA-binding domain of the homologous transcription factors, Mlc and NagC, to the rest of the protein, determines the specificity of their DNA target recognition in Escherichia coli. Mol. Microbiol. 85, 1007–1019 (2012).

    Article  Google Scholar 

  19. 19

    Brechemier-Baey, D., Dominguez-Ramirez, L., Oberto, J. & Plumbridge, J. Operator recognition by the ROK transcription factor family members, NagC and Mlc. Nucleic Acids Res. 43, 361–372 (2015).

    CAS  Article  Google Scholar 

  20. 20

    Gopal, S. et al. Maltose and maltodextrin utilization by Listeria monocytogenes depend on an inducible ABC transporter which is repressed by glucose. PLoS ONE 5, e10349 (2010).

    Article  Google Scholar 

  21. 21

    Hibbing, M. E., Fuqua, C., Parsek, M. R. & Peterson, S. B. Bacterial competition: surviving and thriving in the microbial jungle. Nat. Rev. Microbiol. 8, 15–25 (2010).

    CAS  Article  Google Scholar 

  22. 22

    Mashburn, L. M., Jett, A. M., Akins, D. R. & Whiteley, M. Staphylococcus aureus serves as an iron source for Pseudomonas aeruginosa during in vivo coculture. J. Bacteriol. 187, 554–566 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Miethke, M. & Marahiel, M. A. Siderophore-based iron acquisition and pathogen control. Microbiol. Mol. Biol. Rev. 71, 413–451 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Macromol. Crystallogr. A 276, 307–326 (1997).

    CAS  Article  Google Scholar 

  25. 25

    McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D 61, 458–464 (2005).

    Article  Google Scholar 

  26. 26

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  Google Scholar 

  27. 27

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Painter, J. & Merritt, E. A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D 62, 439–450 (2006).

    Article  Google Scholar 

  29. 29

    Eschenfeldt, W. H., Lucy, S., Millard, C. S., Joachimiak, A. & Mark, I. D. A family of LIC vectors for high-throughput cloning and purification of proteins. Methods Mol. Biol. 498, 105–115 (2009).

    CAS  Article  Google Scholar 

  30. 30

    Côté, G. L. & Sheng, S. Penta-, hexa-, and heptasaccharide acceptor products of alternansucrase. Carbohydr. Res. 341, 2066–2072 (2006).

    Article  Google Scholar 

  31. 31

    Kelley, L. A. & Sternberg, M. J. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363–371 (2009).

    CAS  Article  Google Scholar 

  32. 32

    Eschenfeldt, W. H. et al. New LIC vectors for production of proteins from genes containing rare codons. J. Struct. Funct. Genomics 14, 135–144 (2013).

    CAS  Article  Google Scholar 

  33. 33

    Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    CAS  Article  Google Scholar 

  34. 34

    Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Jones, D. T., Taylor, W. R. & Thornton, J. M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282 (1992).

    CAS  Google Scholar 

  36. 36

    Tamura, K. et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739 (2011).

    CAS  Article  Google Scholar 

  37. 37

    Luan, C. H., Light, S. H., Dunne, S. F. & Anderson, W. F. Ligand screening using fluorescence thermal shift analysis (FTS). Methods Mol. Biol. 1140, 263–289 (2014).

    CAS  Article  Google Scholar 

  38. 38

    Port, G. C. & Freitag, N. E. Identification of novel Listeria monocytogenes secreted virulence factors following mutational activation of the central virulence regulator, PrfA. Infect. Immun. 75, 5886–5897 (2007).

    CAS  Article  Google Scholar 

  39. 39

    Bruno, J. C. Jr & Freitag, N. E. Constitutive activation of PrfA tilts the balance of Listeria monocytogenes fitness towards life within the host versus environmental survival. PLoS ONE 5, e15138 (2010).

    Article  Google Scholar 

  40. 40

    Auerbuch, V., Lenz, L. L. & Portnoy, D. A. Development of a competitive index assay to evaluate the virulence of Listeria monocytogenes actA mutants during primary and secondary infection of mice. Infect. Immun. 69, 5953–5957 (2001).

    CAS  Article  Google Scholar 

Download references


The Center for Structural Genomics of Infectious Diseases has been funded with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Department of Health and Human Services, under contract nos. HHSN272200700058C and HHSN272201200026C (to W.F.A.). This work was supported by NIH grants R01 AI083241 and AI083241-03S1 (to N.E.F.) and F32 AI 115954 (to L.A.C.). Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research meprogram (grant no. 085P1000817). This work was supported by the Northwestern University High Throughput Analysis Laboratory, the Northwestern University Keck Biophysics Facility and a Cancer Center Support Grant (NCI CA060553). The authors thank C.-H. Luan, G. Minasov, Z. Wawrzak and B. Xayarath for assisting with experiments and S. Almo and G. Côté for providing reagents.

Author information




S.H.L., L.A.C., N.E.F. and W.F.A. designed experiments. S.H.L., L.A.C. and A.S.H. performed experiments. S.H.L. wrote the manuscript.

Corresponding author

Correspondence to Wayne F. Anderson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Figures 1–10, Supplementary Tables 1–3 and Supplementary References (PDF 1402 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Light, S., Cahoon, L., Halavaty, A. et al. Structure to function of an α-glucan metabolic pathway that promotes Listeria monocytogenes pathogenesis. Nat Microbiol 2, 16202 (2017).

Download citation

Further reading


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing