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Microbiology Society logo Microbiology

Volume 171, Issue 11

Identification and functional analysis of two oligopeptide transporters supporting the growth of strain Shirota in milk Open Access

Graphical Abstract

Graphical abstract

strain Shirota (LcS) employs two distinct oligopeptide transporters, Opp and Dpp, to facilitate nitrogen acquisition during growth in milk. These transporters exhibit differential substrate specificity, enabling the uptake of oligopeptides of varying lengths derived from milk proteins.

Abstract

Oligopeptide transporters are important proteins in several lactic acid bacteria (LAB) that facilitate the transport of oligopeptides, the primary nitrogen source for growth in milk. Although the proteolytic mechanisms are well understood in some LAB species, limited research has been conducted on the peptide transport systems of (formerly ) strain Shirota (LcS), particularly its oligopeptide transporters. This study investigated the nitrogen uptake mechanism of LcS, a probiotic lactic acid bacterium, by generating gene knockout (KO) strains of two oligopeptide transporters, Opp and Dpp. Consequently, the disruption of these genes eliminated the ability of the bacterium to grow in milk, identifying Opp and Dpp as the primary oligopeptide transporters in LcS. Growth in a leucine-free chemically defined medium with a Leu-containing peptide as the sole nitrogen source indicated that Opp and Dpp transport peptides of 4–8 and 3–7 residues, respectively. To our knowledge, this study provides the first experimental evidence of oligopeptide transporters in capable of transporting peptides up to eight residues long. Analysis of KO strains targeting OppA or DppA to identify other oligopeptide-binding proteins (OBPs) within each oligopeptide transporter operon that may influence substrate specificity revealed that OppA is the only OBPs in Opp. However, DppA and DppA, encoded at chromosomal locations distant from the Dpp operon, may function as subunits constituting Dpp and DppA. These findings enhance our understanding of nitrogen source utilization in lactobacilli and might inform future strategies to optimize nitrogen sources for LcS and improve culture technology for LcS-based products.

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Keyword(s): lactic acid bacteria , Lacticaseibacillus paracasei strain Shirota , oligopeptide transporters and oligopeptide-binding proteins
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2025-11-20
2025-12-16

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References

  1. Chopin A. Organization and regulation of genes for amino acid biosynthesis in lactic acid bacteria. FEMS Microbiol Rev 1993; 12:21–37 [View Article] [PubMed]
     [Google Scholar]
  2. Morishita T, Fukada T, Shirota M, Yura T. Genetic basis of nutritional requirements in Lactobacillus casei. J Bacteriol 1974; 120:1078–1084 [View Article] [PubMed]
     [Google Scholar]
  3. Savijoki K, Ingmer H, Varmanen P. Proteolytic systems of lactic acid bacteria. Appl Microbiol Biotechnol 2006; 71:394–406 [View Article] [PubMed]
     [Google Scholar]
  4. Kok J, Leenhouts KJ, Haandrikman AJ, Ledeboer AM, Venema G. Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2. Appl Environ Microbiol 1988; 54:231–238 [View Article] [PubMed]
     [Google Scholar]
  5. Doeven MK, Kok J, Poolman B. Specificity and selectivity determinants of peptide transport in Lactococcus lactis and other microorganisms. Mol Microbiol 2005; 57:640–649 [View Article] [PubMed]
     [Google Scholar]
  6. Griffiths MW, Tellez AM. Lactobacillus helveticus: the proteolytic system. Front Microbiol 2013; 4:30 [View Article] [PubMed]
     [Google Scholar]
  7. Zhang H, Xu M, Hu S, Zhao H, Zhang B. The enzyme gene expression of protein utilization and metabolism by Lactobacillus helveticus CICC 22171. Microorganisms 2022; 10:1724 [View Article]
     [Google Scholar]
  8. Rodríguez-Serrano GM, Garcia-Garibay JM, Cruz-Guerrero AE, Gomez-Ruiz L del C, Ayala-Nino A et al. Proteolytic system of Streptococcus thermophilus. J Microbiol Biotechnol 2018; 28:1581–1588 [View Article]
     [Google Scholar]
  9. Tynkkynen S, Buist G, Kunji E, Kok J, Poolman B et al. Genetic and biochemical characterization of the oligopeptide transport system of Lactococcus lactis. J Bacteriol 1993; 175:7523–7532 [View Article] [PubMed]
     [Google Scholar]
  10. Juillard V, Le Bars D, Kunji ER, Konings WN, Gripon JC et al. Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk. Appl Environ Microbiol 1995; 61:3024–3030 [View Article] [PubMed]
     [Google Scholar]
  11. Liu M, Bayjanov JR, Renckens B, Nauta A, Siezen RJ. The proteolytic system of lactic acid bacteria revisited: a genomic comparison. BMC Genom 2010; 11:36 [View Article]
     [Google Scholar]
  12. Monnet V. Bacterial oligopeptide-binding proteins. Cell Mol Life Sci 2003; 60:2100–2114 [View Article] [PubMed]
     [Google Scholar]
  13. Biemans-Oldehinkel E, Doeven MK, Poolman B. ABC transporter architecture and regulatory roles of accessory domains. FEBS Lett 2006; 580:1023–1035 [View Article] [PubMed]
     [Google Scholar]
  14. Detmers FJ, Lanfermeijer FC, Abele R, Jack RW, Tampe R et al. Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of Lactococcus lactis. Proc Natl Acad Sci U S A 2000; 97:12487–12492 [View Article] [PubMed]
     [Google Scholar]
  15. Sanz Y, Toldrá F, Renault P, Poolman B. Specificity of the second binding protein of the peptide ABC-transporter (Dpp) of Lactococcus lactis IL1403. FEMS Microbiol Lett 2003; 227:33–38 [View Article] [PubMed]
     [Google Scholar]
  16. Kunji ER, Smid EJ, Plapp R, Poolman B, Konings WN. Di-tripeptides and oligopeptides are taken up via distinct transport mechanisms in Lactococcus lactis. J Bacteriol 1993; 175:2052–2059 [View Article] [PubMed]
     [Google Scholar]
  17. Garault P, Le Bars D, Besset C, Monnet V. Three oligopeptide-binding proteins are involved in the oligopeptide transport of Streptococcus thermophilus. J Biol Chem 2002; 277:32–39 [View Article] [PubMed]
     [Google Scholar]
  18. Sanz Y, Lanfermeijer FC, Renault P, Bolotin A, Konings WN et al. Genetic and functional characterization of dpp genes encoding a dipeptide transport system in Lactococcus lactis. Arch Microbiol 2001; 175:334–343 [View Article] [PubMed]
     [Google Scholar]
  19. Peltoniemi K, Vesanto E, Palva A. Genetic characterization of an oligopeptide transport system from Lactobacillus delbrueckii subsp. bulgaricus. Arch Microbiol 2002; 177:457–467 [View Article] [PubMed]
     [Google Scholar]
  20. Kok J, van Gijtenbeek LA, de Jong A, van der Meulen SB, Solopova A et al. The evolution of gene regulation research in Lactococcus lactis. FEMS Microbiol Rev 2017; 41:S220–S243 [View Article] [PubMed]
     [Google Scholar]
  21. Iwamoto D, Ishizaki M, Miura T, Sasaki Y. Novel shuttle vector pGMβ1 for conjugative chromosomal manipulation of Lactobacillus delbrueckii subsp. bulgaricus. Biosci Microbiota Food Health 2022; 41:20–29 [View Article] [PubMed]
     [Google Scholar]
  22. Yasuda E, Serata M, Sako T. Suppressive effect on activation of macrophages by Lactobacillus casei strain Shirota genes determining the synthesis of cell wall-associated polysaccharides. Appl Environ Microbiol 2008; 74:4746–4755 [View Article] [PubMed]
     [Google Scholar]
  23. Banjonjit S, Taweechotipatr M, Rungsiyanont S. Effect of probiotic Lactobacillus paracasei on tumor necrosis factor-alpha level in gingival crevicular fluid of patients undergoing impacted third molar removal. J Oral Sci 2022; 64:185–189 [View Article] [PubMed]
     [Google Scholar]
  24. Xie Z, Zhang G, Liu R, Wang Y, Tsapieva AN et al. Heat-Killed Lacticaseibacillus paracasei repairs lipopolysaccharide-induced intestinal epithelial barrier damage via MLCK/MLC pathway activation. Nutrients 2023; 15:1758 [View Article] [PubMed]
     [Google Scholar]
  25. Serata M, Iino T, Yasuda E, Sako T. Roles of thioredoxin and thioredoxin reductase in the resistance to oxidative stress in Lactobacillus casei. Microbiology 2012; 158:953–962 [View Article] [PubMed]
     [Google Scholar]
  26. Serata M, Kiwaki M, Iino T. Functional analysis of a novel hydrogen peroxide resistance gene in Lactobacillus casei strain Shirota. Microbiology 2016; 162:1885–1894 [View Article] [PubMed]
     [Google Scholar]
  27. Alcántara C, Bäuerl C, Revilla-Guarinos A, Pérez-Martínez G, Monedero V et al. Peptide and amino acid metabolism is controlled by an OmpR-family response regulator in Lactobacillus casei. Mol Microbiol 2016; 100:25–41 [View Article] [PubMed]
     [Google Scholar]
  28. Team RDC R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; 2007 http://wwwR-projectorg
  29. Hothorn T, Bretz F, Westfall P. Simultaneous inference in general parametric models. Biom J 2008; 50:346–363 [View Article] [PubMed]
     [Google Scholar]
  30. Wickham H. ggplot2: Elegant Graphics for Data Analysis Springer-Verlag New York; 2016 [View Article]
     [Google Scholar]
  31. Hagting A, Kunji ER, Leenhouts KJ, Poolman B, Konings WN. The di- and tripeptide transport protein of Lactococcus lactis. A new type of bacterial peptide transporter. J Biol Chem 1994; 269:11391–11399 [PubMed]
     [Google Scholar]
  32. Jenkinson HF, Baker RA, Tannock GW. A binding-lipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides. J Bacteriol 1996; 178:68–77 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001624
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Identification and functional analysis of two oligopeptide transporters supporting the growth of Lacticaseibacillus paracasei strain Shirota in milk
Microbiology 171, 001624 (2025); https://doi.org/10.1099/mic.0.001624
/content/journal/micro/10.1099/mic.0.001624
/content/journal/micro/10.1099/mic.0.001624
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