Abstract
The probiotic efficacy and fermentative ability of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus), a widely used probiotic, is majorly affected by its acid tolerance. Here, we conducted whole-genome sequencing of the high acid-tolerant L. bulgaricus LJJ stored in the laboratory. Compared with the whole genome of low acid-tolerant strain L. bulgaricus ATCC11842, the results show that 16 candidate acid-tolerant genes may be involved in the regulation of the acid tolerance of L. bulgaricus LJJ. Association analysis of candidate acid-tolerant genes and acid-tolerant traits of different L. bulgaricus strains revealed that the three genes dapA, dapH, and lysC are the main reasons for the strong acid tolerance of L. bulgaricus LJJ. The results of real-time quantitative PCR (RT-qPCR) supported this conclusion. KEGG pathway analysis showed that these three acid-tolerant genes are involved in the synthesis of lysine; the synthesis of lysine may confer L. bulgaricus LJJ strong acid tolerance. This study successfully revealed the acid tolerance mechanism of L. bulgaricus LJJ and provides a theoretical basis for the subsequent selection of strains with high acid tolerance for improved probiotic functions.
Key points
• Three genes are identified as acid-tolerant genes, respectively, lysC, dapA, and dapH.
• LysC and dapA are the major key genes in the synthesis of lysine.
• The synthesis of lysine may confer L. bulgaricus LJJ strong acid tolerance.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
L. bulgaricus type strain ATCC11842 (NCBI accession number: NC_008054) was purchased from American Type Culture Collection. L. bulgaricus LJJ (NCBI accession number: CP049052) is patently stored in China General Microbiological Culture Collection Center (CGMCC No. 2687). The datasets supporting the results of this article are included within the article and its supplementary files. Requests to access any strains shown in this manuscript should be directed to Xiaoyang Pang (pangxiaoyang@163.com) and Jiaping Lv (lvjiapingcaas@126.com).
References
Anisimova EA, Yarullina DR, Ilinskaya ON (2017) Antagonistic activity of lactobacilli isolated from natural ecotopes. Microbiology 86(6):708–713. https://doi.org/10.1134/s0026261717060054
Azcarate-Peril MA, Altermann E, Hoover-Fitzula RL, Cano RJ, Klaenhammer TR (2004) Identification and inactivation of genetic loci involved with Lactobacillus acidophilus acid tolerance. Appl Environ Microbiol 70(9):5315–5322. https://doi.org/10.1128/aem.70.9.5315-5322.2004
Azcarate-Peril MA, McAuliffe O, Altermann E, Lick S, Russell WM, Klaenhammer TR (2005) Microarray analysis of a two-component regulatory system involved in acid resistance and proteolytic activity in Lactobacillus acidophilus. Appl Environ Microb 71(10):5794–5804. https://doi.org/10.1128/aem.71.10.5794-5804.2005
Benkert P, Biasini M, Schwede T (2011) Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27(3):343–350. https://doi.org/10.1093/bioinformatics/btq662
Bogdanov I, Popkhristov P, Marinov I (1962) Anticancer effect of antibioticum bulgaricum on sarcoma 180 and on the select form of Ehrlich carcinoma. Abstract VIII. Int Ann C
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST plus : architecture and applications. Bmc Bioinformatics 10:421. https://doi.org/10.1186/1471-2105-10-421
Chen X, Sun Z, Meng H, Zhang H (2009) The acid tolerance association with expression of H+-ATPase in Lactobacillus casei. Int J Dairy Technol 62(2):272–276. https://doi.org/10.1111/j.1471-0307.2009.00461.x
Cotter PD, Hill C (2003) Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiol Mol Biol Rev 67(3):429. https://doi.org/10.1128/mmbr.37.3.429-453.2003
Cox MM, Goodman MF, Kreuzer KN, Sherratt DJ, Sandler SJ, Marians KJ (2000) The importance of repairing stalled replication forks. Nature 404(6773):37–41
Darling ACE, Mau B, Blattner FR, Perna NT (2004) Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res 14(7):1394–1403. https://doi.org/10.1101/gr.2289704
De Angelis M, Gobbetti M (2004) Environmental stress responses in Lactobacillus: a review. Proteomics 4(1):106–122. https://doi.org/10.1002/pmic.200300497
Del Piano M, Morelli L, Strozzi GP, Allesina S, Barba M, Deidda F, Lorenzini P, Ballaré M, Montino F, Orsello M, Sartori M, Garello E, Carmagnola S, Pagliarulo M, Capurso L (2006) Probiotics: from research to consumer. Dig Liver Dis 38(Suppl 2):S248–S255
Dhiman A, Bhatnagar S, Kulshreshtha P, Bhatnagar R (2014) Functional characterization of WalRK: a two-component signal transduction system from Bacillus anthracis. Febs Open Bio 4:65–76. https://doi.org/10.1016/j.fob.2013.12.005
Duwat P, Cesselin B, Sourice S, Gruss A (2000) Lactococcus lactis, a bacterial model for stress responses and survival. Int J Food Microbiol 55(1–3):83–86. https://doi.org/10.1016/s0168-1605(00)00179-3
Gordon D, Macrae J, Wheater DM (1957) A Lactobacillus preparation for use with antibiotics. Lancet 1(MAY4):899–901
Guillouard I, Lim E-M, Van de Guchte M, Grimaldi C, Penaud S, Maguin E (2004) Tolérance et réponse adaptative au stress acide chez Lactobacillus delbrueckii ssp. bulgaricus. Lait 84(1–2):1–6
Hao P, Zheng H, Yu Y, Ding G, Gu W, Chen S, Yu Z, Ren S, Oda M, Konno T, Wang S, Li X, Ji ZS, Zhao G (2011) Complete sequencing and pan-genomic analysis of Lactobacillus delbrueckiisubsp.bulgaricus reveal its genetic basis for industrial yogurt production. PloS one 6(1):e15964. https://doi.org/10.1371/journal.pone.0015964
Higuchi T, Hayashi H, Abe K (1997) Exchange of glutamate and gamma-aminobutyrate in a Lactobacillus strain. J Bacteriol 179(10):3362–3364. https://doi.org/10.1128/jb.179.10.3362-3364.1997
Huang R, Pan M, Wan C, Shah NP, Tao X, Wei H (2016) Physiological and transcriptional responses and cross protection of Lactobacillus plantarum ZDY2013 under acid stress. J Dairy Sci 99(2):1002–1010. https://doi.org/10.3168/jds.2015-9993
Jan G, Leverrier P, Pichereau V, Boyaval P (2001) Changes in protein synthesis and morphology during acid adaptation of Propionibacterium freudenreichii. Appl Environ Microb 67(5):2029–2036. https://doi.org/10.1128/aem.67.5.2029-2036.2001
Kandola S, Teotia US, Kumar R, Mishra AK, Singh A (2016) Investigation of acid tolerance attribute of various Lactobacillus casei group strains. Indian J Anim Res 50(2):190–193. https://doi.org/10.18805/ijar.9539
Kelley DR, Liu B, Delcher AL, Pop M, Salzberg SL (2012) Gene prediction with glimmer for metagenomic sequences augmented by classification and clustering. Nucleic Acids Res 40(1):e9. https://doi.org/10.1093/nar/gkr1067
Knight DJW, Girling KJ (2003) Gut flora in health and disease. Lancet 361(9371):1831. https://doi.org/10.1016/s0140-6736(03)12489-0
Koebmann BJ, Nilsson D, Kuipers OP, Jensen PR (2000) The membrane-bound H+-ATPase complex is essential for growth of Lactococcus lactis. J Bacteriol 182(17):4738–4743. https://doi.org/10.1128/jb.182.17.4738-4743.2000
Kouzarides T (2000) Acetylation: a regulatory modification to rival phosphorylation? EMBO J 19(6):1176–1179. https://doi.org/10.1093/emboj/19.6.1176
Landete JM, Garcia-Haro L, Blasco A, Manzanares P, Berbegal C, Monedero V, Zuniga M (2010) Requirement of the Lactobacillus casei MaeKR two-component system for L-malic acid utilization via a malic enzyme pathway. Appl Environ Microb 76(1):84–95. https://doi.org/10.1128/aem.02145-09
Lee KH, Park JH, Kim TY, Kim HU, Lee SY (2007) Systems metabolic engineering of Escherichia coli for L-threonine production. Mol Syst Biol 3:149. https://doi.org/10.1038/msb4100196
Lim EM, Ehrlich SD, Maguin E (2000) Identification of stress-inducible proteins in Lactobacillus delbrueckii subsp bulgaricus. Electrophoresis 21(12):2557–2561. https://doi.org/10.1002/1522-2683(20000701)21:12<2557::aid-elps2557>3.0.co;2-b
Liu SQ, Pilone GJ (1998) A review: arginine metabolism in wine lactic acid bacteria and its practical significance. J Appl Microbiol 84(3):315–327. https://doi.org/10.1046/j.1365-2672.1998.00350.x
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods (San Diego, Calif) 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Maghnouj A, Cabral TFD, Stalon V, Vander Wauven C (1998) The arcABDC gene cluster, encoding the arginine deiminase pathway of Bacillus licheniformis, and its activation by the arginine repressor ArgR. J Bacteriol 180(24):6468–6475
Ming H, Xu D, Guo Z, Liu Y (2016) Adaptive evolution of Lactobacillus casei under acidic conditions enhances multiple-stress tolerance. Food Sci Technol Res 22(3):331–336. https://doi.org/10.3136/fstr.22.331
Olsen EB, Russell JB, Henickkling T (1991) Electrogenic l-malate transport by Lactobacillus plantarum - a basis for energy derivation from malolactic fermentation. J Bacteriol 173(19):6199–6206. https://doi.org/10.1128/jb.173.19.6199-6206.1991
Paradeshi JS, Patil SN, Koli SH, Chaudhari BL (2018) Effect of copper on probiotic properties of Lactobacillus helveticus CD6. Int J Dairy Technol 71:204–212. https://doi.org/10.1111/1471-0307.12384
Reeve BWP, Reid SJ (2016) Glutamate and histidine improve both solvent yields and the acid tolerance response of Clostridium beijerinckii NCP260. J Appl Microbiol 120(5):1271–1281. https://doi.org/10.1111/jam.13067
Remmert M, Biegert A, Hauser A, Soding J (2012) HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods 9(2):173–175. https://doi.org/10.1038/nmeth.1818
Schwenk WS, Donovan TM (2011) A multispecies framework for landscape conservation planning. Conserv Biol 25(5):1010–1021. https://doi.org/10.1111/j.1523-1739.2011.01723.x
Small PLC, Waterman SR (1998) Acid stress, anaerobiosis and gadCB lessons from Lactococcus lactis and Escherichia coli. Trends Microbiol 6(6):214–216. https://doi.org/10.1016/s0966-842x(98)01285-2
van de Guchte M, Penaud S, Grimaldi C, Barbe V, Bryson K, Nicolas P, Robert C, Oztas S, Mangenot S, Couloux A, Loux V, Dervyn R, Bossy R, Bolotin A, Batto JM, Walunas T, Gibrat JF, Bessieres P, Weissenbach J, Ehrlich SD, Maguin E (2006) The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution. Proc Natl Acad Sci U S A 103(24):9274–9279. https://doi.org/10.1073/pnas.0603024103
Wang Q, Zhang Y, Yang C, Xiong H, Lin Y, Yao J, Li H, Xie L, Zhao W, Yao Y, Ning Z-B, Zeng R, Xiong Y, Guan K-L, Zhao S, Zhao G-P (2010) Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux. Science 327(5968):1004–1007. https://doi.org/10.1126/science.1179687
Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46(W1):W296–W303. https://doi.org/10.1093/nar/gky427
Wu C, Zhang J, Wang M, Du G, Chen J (2012) Lactobacillus casei combats acid stress by maintaining cell membrane functionality. J Ind Microbiol Biotechnol 39(7):1031–1039. https://doi.org/10.1007/s10295-012-1104-2
Wu C, Zhang J, Du G, Chen J (2013) Aspartate protects Lactobacillus casei against acid stress. Appl Microbiol Biotechnol 97(9):4083–4093. https://doi.org/10.1007/s00253-012-4647-2
Zhai Z, An H, Wang G, Luo Y, Hao Y (2015) Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid. Sci Rep 5(1):17024. https://doi.org/10.1038/srep17024
Acknowledgments
The authors would like to thank Na Chen for providing some pretty inspiration and Majorbio Co., Ltd. for some experimental service.
Code availability
Gene prediction: Glimmer 3.02 (http://www.cbcb.umd.edu/software/glimmer/)
Genome-wide circle map: Circos v0.64 (http://circos.ca/)
Genome-wide collinearity: Mauve (http://darlinglab.org/mauve/mauve.html)
Primer design: Primer Premier 5, (http://www.premierbiosoft.com/primerdesign/) and Snapgene 3.2.1 (https://www.snapgene.com/)
Visualization of protein structure: Pymol (https://pymol.org/2/)
GraphPad Prism 8 (https://www.graphpad.com/scientific-software/prism/)
Funding
This work was financially supported by National Key R&D Program of China (2017YFC1600903), National Natural Science Foundation of China (Grant No. 31871833), and the Science and Technology Project of Beijing Education Committee (No. KM201812448003).
Author information
Authors and Affiliations
Contributions
X.Y P, J.P L, S.W Z, and J L designed the study. W.X L, L Y, X.Y Pang, and O.J U wrote the original draft. W.X L and L Y performed experiments. W.L N performed a part of bioinformatics analysis. W.X L and L Y generated the figures and tables. All authors contributed with writing, reviewing and editing. All authors read and approved the final version of the manuscript. W.X L and L Y have equal contribution.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Ethics approval
Not applicable.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Li, W., Yang, L., Nan, W. et al. Whole-genome sequencing and genomic-based acid tolerance mechanisms of Lactobacillus delbrueckii subsp. bulgaricus LJJ. Appl Microbiol Biotechnol 104, 7631–7642 (2020). https://doi.org/10.1007/s00253-020-10788-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-020-10788-5