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Volume 172, Issue 3

Ligand-binding properties of substrate binding proteins of a maltose uptake system in Open Access

Abstract

Glycogen and its breakdown products, maltose and malto-oligosaccharides, are important carbon sources for vaginal bacteria including species. MusEFGKI transport systems for maltose and malto-oligosaccharides have been identified in all species; however, unlike in other species, the operon encodes two substrate-binding proteins (SBPs) (MusE1345, MusE1346, ~60% amino acid identity). Two SBPs could allow binding of additional ligands, providing a competitive advantage to relative to other species with only one SBP. Our objectives were to determine if both genes are expressed in and compare the specificity and affinity of MusE SBPs for glycogen breakdown products. Gene expression analysis showed the presence of a polycistronic transcript spanning both SBP encoding genes; however, transcripts were more abundant, likely due to the presence of an additional promoter identified in the intergenic region. No difference in the relative expression of either gene was observed in isolates grown in media supplemented with glycogen or maltotriose. Predicted structures of both SBPs were highly similar and characteristic of previously characterized maltose-binding proteins. Both proteins had a high affinity for maltose, maltotriose and maltotetraose ( 10 to 10 M) and much lower affinities to maltopentaose and maltohexaose ( 10 to 10 M). Our results demonstrate that the affinities of MusE SBPs for maltose and malto-oligosaccharides are similar under the same experimental conditions.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Gardnerella swidsinskii , glycogen , isothermal titration calorimetry (ITC) , ligand-binding protein , malto-oligosaccharides and vaginal microbiome
Funding
This study was supported by the:
  • Natural Sciences and Engineering Research Council of Canada
    • Principal Award Recipient: JanetHill
© 2026 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2026-03-23
2026-04-15

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References

  1. Coudray MS, Madhivanan P. Bacterial vaginosis—a brief synopsis of the literature. Eur J Obstet Gynecol Reprod Biol 2020; 245:143–148 [View Article]
     [Google Scholar]
  2. Hill JE, Albert AYK. VOGUE Research Group Resolution and cooccurrence patterns of Gardnerella leopoldii, G. swidsinskii, G. piotii, and G. vaginalis within the vaginal microbiome. Infect Immun 2019; 87:e00532-19 [View Article] [PubMed]
     [Google Scholar]
  3. Khan S, Voordouw MJ, Hill JE. Competition among Gardnerella subgroups from the human vaginal microbiome. Front Cell Infect Microbiol 2019; 9:374 [View Article]
     [Google Scholar]
  4. Navarro S, Abla H, Delgado B, Colmer-Hamood JA, Ventolini G et al. Glycogen availability and pH variation in a medium simulating vaginal fluid influence the growth of vaginal Lactobacillus species and Gardnerella vaginalis. BMC Microbiol 2023; 23:186 [View Article] [PubMed]
     [Google Scholar]
  5. Mirmonsef P, Hotton AL, Gilbert D, Gioia CJ, Maric D et al. Glycogen levels in undiluted genital fluid and their relationship to vaginal pH, estrogen, and progesterone. PLoS One 2016; 11:e0153553 [View Article] [PubMed]
     [Google Scholar]
  6. Jenkins DJ, Woolston BM, Hood-Pishchany MI, Pelayo P, Konopaski AN et al. Bacterial amylases enable glycogen degradation by the vaginal microbiome. Nat Microbiol 2023; 8:1641–1652 [View Article] [PubMed]
     [Google Scholar]
  7. Bhandari P, Tingley J, Abbott DW, Hill JE. Glycogen-degrading activities of catalytic domains of α-amylase and α-amylase-pullulanase enzymes conserved in Gardnerella spp. from the vaginal microbiome. J Bacteriol 2023; 205:e0039322 [View Article] [PubMed]
     [Google Scholar]
  8. Schneider E. ABC transporters catalyzing carbohydrate uptake. Res Microbiol 2001; 152:303–310 [View Article] [PubMed]
     [Google Scholar]
  9. Bhandari P, Hill JE. Transport and utilization of glycogen breakdown products by Gardnerella spp. from the human vaginal microbiome. Microbiol Spectr 2023; 11:e0443522 [View Article] [PubMed]
     [Google Scholar]
  10. Henrich A, Kuhlmann N, Eck AW, Krämer R, Seibold GM. Maltose uptake by the novel ABC transport system MusEFGK2I causes increased expression of ptsG in Corynebacterium glutamicum. J Bacteriol 2013; 195:2573–2584 [View Article] [PubMed]
     [Google Scholar]
  11. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R et al. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 2007; 35:W71–4 [View Article] [PubMed]
     [Google Scholar]
  12. Kim A, Fernando C, Hill JE. Extraction of high quality RNA from Gardnerella species. Protocol Exchange 2024 [View Article]
     [Google Scholar]
  13. Xie F, Wang J, Zhang B. RefFinder: a web-based tool for comprehensively analyzing and identifying reference genes. Funct Integr Genomics 2023; 23:125 [View Article] [PubMed]
     [Google Scholar]
  14. Silver N, Best S, Jiang J, Thein SL. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 2006; 7:33 [View Article] [PubMed]
     [Google Scholar]
  15. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett 2004; 26:509–515 [View Article] [PubMed]
     [Google Scholar]
  16. Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004; 64:5245–5250 [View Article] [PubMed]
     [Google Scholar]
  17. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3:RESEARCH0034 [View Article] [PubMed]
     [Google Scholar]
  18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25:402–408 [View Article] [PubMed]
     [Google Scholar]
  19. Abbott DW, Higgins MA, Hyrnuik S, Pluvinage B, Lammerts van Bueren A et al. The molecular basis of glycogen breakdown and transport in Streptococcus pneumoniae. Mol Microbiol 2010; 77:183–199 [View Article] [PubMed]
     [Google Scholar]
  20. Gardner HL, Dukes CD. Haemophilus vaginalis vaginitis: a newly defined specific infection previously classified non-specific vaginitis. Am J Obstet Gynecol 1955; 69:962–976 [PubMed]
     [Google Scholar]
  21. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991; 29:297–301 [View Article] [PubMed]
     [Google Scholar]
  22. Spear GT, French AL, Gilbert D, Zariffard MR, Mirmonsef P et al. Human α-amylase present in lower-genital-tract mucosal fluid processes glycogen to support vaginal colonization by Lactobacillus. J Infect Dis 2014; 210:1019–1028 [View Article] [PubMed]
     [Google Scholar]
  23. Nunn KL, Forney LJ. Unraveling the dynamics of the human vaginal microbiome. Yale J Biol Med 2016; 89:331–337 [PubMed]
     [Google Scholar]
  24. Bhandari P, Tingley JP, Palmer DRJ, Abbott DW, Hill JE. Characterization of an α-glucosidase enzyme conserved in Gardnerella spp. isolated from the human vaginal microbiome. J Bacteriol 2021; 203:e0021321 [View Article] [PubMed]
     [Google Scholar]
  25. Boos W, Shuman H. Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation. Microbiol Mol Biol Rev 1998; 62:204–229 [View Article] [PubMed]
     [Google Scholar]
  26. Ferreira MJ, Sá-Nogueira I de. A multitask ATPase serving different ABC-type sugar importers in Bacillus subtilis. J Bacteriol 2010; 192:5312–5318 [View Article] [PubMed]
     [Google Scholar]
  27. Marion C, Aten AE, Woodiga SA, King SJ. Identification of an ATPase, MsmK, which energizes multiple carbohydrate ABC transporters in Streptococcus pneumoniae. Infect Immun 2011; 79:4193–4200 [View Article]
     [Google Scholar]
  28. Jagadeesan R, Dash S, Palma CSD, Baptista ISC, Chauhan V et al. Dynamics of bacterial operons during genome-wide stresses is influenced by premature terminations and internal promoters. Sci Adv 2025; 11:eadl3570 [View Article] [PubMed]
     [Google Scholar]
  29. Muscariello L, Vastano V, Siciliano RA, Sacco M, Marasco R. Expression of the Lactobacillus plantarum malE gene is regulated by CcpA and a MalR-like protein. J Microbiol 2011; 49:950–955 [View Article] [PubMed]
     [Google Scholar]
  30. Chen S, Oldham ML, Davidson AL, Chen J. Carbon catabolite repression of the maltose transporter revealed by X-ray crystallography. Nature 2013; 499:364–368 [View Article]
     [Google Scholar]
  31. Daus ML, Grote M, Schneider E. The MalF P2 loop of the ATP-binding cassette transporter MalFGK2 from Escherichia coli and Salmonella enterica serovar typhimurium interacts with maltose binding protein (MalE) throughout the catalytic cycle. J Bacteriol 2009; 191:754–761 [View Article] [PubMed]
     [Google Scholar]
  32. de Boer M, Gouridis G, Vietrov R, Begg SL, Schuurman-Wolters GK et al. Conformational and dynamic plasticity in substrate-binding proteins underlies selective transport in ABC importers. Elife 2019; 8:e44652 [View Article] [PubMed]
     [Google Scholar]
  33. Berntsson RPA, Smits SHJ, Schmitt L, Slotboom DJ, Poolman B. A structural classification of substrate-binding proteins. FEBS Lett 2010; 584:2606–2617 [View Article] [PubMed]
     [Google Scholar]
  34. Bhat AH, Nguyen MT, Das A, Ton-That H. Anchoring surface proteins to the bacterial cell wall by sortase enzymes: how it started and what we know now. Curr Opin Microbiol 2021; 60:73–79 [View Article] [PubMed]
     [Google Scholar]
  35. Marraffini LA, Dedent AC, Schneewind O. Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiol Mol Biol Rev 2006; 70:192–221 [View Article] [PubMed]
     [Google Scholar]
  36. Bhattacharya D, Zhang R, Yu W. Protein trafficking across the cell envelope of gram-positive bacteria. J Bacteriol 2025; 207:e0010025 [View Article] [PubMed]
     [Google Scholar]
  37. Alberts B, Johnson A, Lewis J, Raff M, Roberts K et al. Membrane proteins. In Molecular Biology of the Cell, 4th ed Garland Science; 2002 https://www.ncbi.nlm.nih.gov/books/NBK26878/
     [Google Scholar]
  38. Clarke RJ. Electrostatic switch mechanisms of membrane protein trafficking and regulation. Biophys Rev 2023; 15:1967–1985 [View Article] [PubMed]
     [Google Scholar]
  39. Miller DM, Olson JS, Pflugrath JW, Quiocho FA. Rates of ligand binding to periplasmic proteins involved in bacterial transport and chemotaxis. J Biol Chem 1983; 258:13665–13672 [PubMed]
     [Google Scholar]
  40. Quiocho FA. Carbohydrate-binding proteins: tertiary structures and protein-sugar interactions. Annu Rev Biochem 1986; 55:287–315 [View Article] [PubMed]
     [Google Scholar]
  41. Spurlino JC, Lu GY, Quiocho FA. The 2.3-A resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis. J Biol Chem 1991; 266:5202–5219 [View Article]
     [Google Scholar]
  42. Corrêa DHA, Ramos CHI. The use of circular dichroism spectroscopy to study protein folding, form and function. AJBR 2009; 3:164–173
     [Google Scholar]
  43. Wei Y, Thyparambil AA, Latour RA. Protein helical structure determination using CD spectroscopy for solutions with strong background absorbance from 190 to 230nm. Biochim Biophys Acta 2014; 1844:2331–2337 [View Article]
     [Google Scholar]
  44. Gilardi G, Mei G, Rosato N, Agro AF, Cass AE. Spectroscopic properties of an engineered maltose binding protein. Protein Eng 1997; 10:479–486 [View Article]
     [Google Scholar]
  45. Balan A, de Souza CS, Moutran A, Ferreira RCC, Franco CS et al. Purification and in vitro characterization of the maltose-binding protein of the plant pathogen Xanthomonas citri. Protein Expr Purif 2005; 43:103–110 [View Article] [PubMed]
     [Google Scholar]
  46. Szmelcman S, Schwartz M, Silhavy TJ, Boos W. Maltose transport in Escherichia coli K12. Eur J Biochem 1976; 65:13–19 [View Article]
     [Google Scholar]
  47. Oldham ML, Chen S, Chen J. Structural basis for substrate specificity in the Escherichia coli maltose transport system. Proc Natl Acad Sci USA 2013; 110:18132–18137 [View Article] [PubMed]
     [Google Scholar]
  48. Jeckelmann JM, Erni B. Transporters of glucose and other carbohydrates in bacteria. Pflugers Arch 2020; 472:1129–1153 [View Article] [PubMed]
     [Google Scholar]
  49. SUMAWONG V, GREGOIRE AT, JOHNSON WD, RAKOFF AE. Identification of carbohydrates in the vaginal fluid of normal females. Fertil Steril 1962; 13:270–280 [View Article] [PubMed]
     [Google Scholar]
  50. Quiocho FA, Spurlino JC, Rodseth LE. Extensive features of tight oligosaccharide binding revealed in high-resolution structures of the maltodextrin transport/chemosensory receptor. Structure 1997; 5:997–1015 [View Article]
     [Google Scholar]
  51. Quiocho FA. Atomic structures of periplasmic binding proteins and the high-affinity active transport systems in bacteria. Philos Trans R Soc Lond B Biol Sci 1990; 326:341–351 [View Article] [PubMed]
     [Google Scholar]
  52. Telmer PG, Shilton BH. Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants. J Biol Chem 2003; 278:34555–34567 [View Article] [PubMed]
     [Google Scholar]
  53. Keiler KC, Sauer RT. Identification of active site residues of the Tsp protease. J Biol Chem 1995; 270:28864–28868 [View Article] [PubMed]
     [Google Scholar]
  54. Hoffmann SV, Fano M, Weert M. Circular dichroism spectroscopy for structural characterization of proteins. In Müllertz A, Perrie Y, Rades T. eds Analytical Techniques in the Pharmaceutical Sciences New York, NY: Springer; 2016 pp 223–251 [View Article]
     [Google Scholar]
  55. Connelly PR, Aldape RA, Bruzzese FJ, Chambers SP, Fitzgibbon MJ et al. Enthalpy of hydrogen bond formation in a protein-ligand binding reaction. Proc Natl Acad Sci USA 1994; 91:1964–1968 [View Article] [PubMed]
     [Google Scholar]
  56. Desantis F, Miotto M, Di Rienzo L, Milanetti E, Ruocco G. Spatial organization of hydrophobic and charged residues affects protein thermal stability and binding affinity. Sci Rep 2022; 12:12087 [View Article] [PubMed]
     [Google Scholar]
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Ligand-binding properties of substrate binding proteins of a maltose uptake system in Gardnerella swidsinskii
Microbiology 172, 001685 (2026); https://doi.org/10.1099/mic.0.001685
/content/journal/micro/10.1099/mic.0.001685
/content/journal/micro/10.1099/mic.0.001685
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