Abstract
Detailed differentiation, classification, and phylogenetic analysis of the order Lactobacillales are performed using molecular techniques that involve the comparison of whole genomes, multilocus sequence analysis, DNA–DNA hybridisation, and 16S rRNA sequencing. Despite the wide application of the latter two techniques, issues associated with them are extensively discussed. Although complete genomic analyses are the most appropriate for phylogenetic studies, they are time-consuming and require high levels of expertise. Many phylogenetic/identification markers have been proposed for enterococci, lactobacilli, streptococci, and lactobacilli. However, none have been established for vagococci and some genera within the order Lactobacillales. The objective of the study was to find novel alternative housekeeping genes for classification, typing, and phylogenetic analysis of selected genera within the order Lactobacillales. We designed primers flanking variable regions of the infB (504 nt) and rpsB (333 nt) genes and amplified and sequenced them in 56 strains of different genera within the order Lactobacillales. Statistical analysis and characteristics of the gene regions suggested that they could be used for taxonomic purposes. Phylogenetic analyses, including assessment of (in)congruence between individual phylogenetic trees indicated the possibility of using the concatenation of the two genes as an alternative tool for the evaluation of phylogeny compared with the 16S rRNA gene representing the standard phylogenetic marker of prokaryotes. Moreover, infB, rpsB regions and their concatenate were phylogenetically consistent with two widely applied alternative genetic markers in taxonomy of particular Lactobacillales genera encoding the 60 kDa chaperonin protein (GroEL-hsp60) and phenylalanyl-tRNA synthetase, alpha subunit (pheS).
Similar content being viewed by others
References
Calmin G, Lefort F, Belbahri L (2008) Multi-loci sequence typing (MLST) for two lacto-acid bacteria (LAB) species: Pediococcus parvulus and P. damnosus. Mol Biotechnol 40:170–179
Carr FJ, Chill D, Maida N (2002) The lactic acid bacteria: a literature survey. Crit Rev Microbiol 28:281–370
Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552
Chao SH, Sasamoto M, Kudo Y, Fujimoto J, Tsai YC, Watanabe K (2010) Lactobacillus odoratitofui sp. nov., isolated from stinky tofu brine. Int J Syst Evol Microbiol 60:2903–2907
Claesson MJ, van Sinderen D, O’Toole PW (2008) Lactobacillus phylogenomics-towards a reclassification of the genus. Int J Syst Evol Microbiol 58:2945–2954
Collins MD (2009) Genus Vagococcus. In: de Vos P, Garrity GM, Jones D, Krieg NR, Ludwig W (eds) Bergey’s manual of systematic bacteriology (The Firmicutes), 2nd edn, vol 3. Springer, New York, pp 616–618
Dan T, Liu W, Sun Z, Lv Q, Xu H, Song Y, Zhang H (2014) A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia. BMC Microbiol 14:150
Dobson CM, Deneer H, Lee S, Hemmingsen S, Glaze S, Ziola B (2002) Phylogenetic analysis of the genus Pediococcus, including Pediococcus claussenii sp. nov., a novel lactic acid bacterium isolated from beer. Int J Syst Evol Microbiol 52:2003–2010
Ehrmann MA, Müller MR, Vogel RF (2003) Molecular analysis of sourdough reveals Lactobacillus mindensis sp. nov. Int J Syst Evol Microbiol 53:7–13
Glaeser SP, Kämpfer P (2015) Multilocus sequence analysis (MLSA) in prokaryotic taxonomy. Syst Appl Microbiol 38:237–245
Goh SH, Facklam RR, Chang M, Hill JE, Tyrrell GJ, Burns EC, Chan D, He C, Rahim T, Shaw C, Hemmingsen SM (2000) Identification of Enterococcus species and phenotypically similar Lactococcus and Vagococcus species by reverse checkerboard hybridization to chaperonin 60 gene sequences. J Clin Microbiol 38:3953–3959
Haakensen M, Dobson CM, Hill JE, Ziola B (2009) Reclassification of Pediococcus dextrinicus (Coster and White 1964) back 1978 (approved lists 1980) as Lactobacillus dextrinicus comb. nov., and emended description of the genus Lactobacillus. Int J Syst Evol Microbiol 59:615–621
Hammes WP, Hertel Ch (2009) Genus Lactobacillus. In: de Vos P, Garrity GM, Jones D, Krieg NR, Ludwig W (eds) Bergey’s manual of systematic bacteriology (The Firmicutes), 2nd edn, vol 3. Springer, New York, pp 465–511
Kadri Z, Amar M, Ouadghiri M, Cnockaert M, Aerts M, El Farricha O, Vandamme P (2014) Streptococcus moroccensis sp. nov. and Streptococcus rifensis sp. nov., isolated from raw camel milk. Int J Syst Evol Microbiol 64:2480–2485
Killer J, Dubná S, Sedláček I, Švec P (2014a) Lactobacillus apis sp. nov., from the stomach of honeybees (Apis mellifera), having an in vitro inhibitory effect on the causative agents of American and European foulbrood. Int J Syst Evol Microbiol 64:152–157
Killer J, Votavová A, Valterová I, Vlková E, Rada V, Hroncová Z (2014b) Lactobacillus bombi sp. nov., from the digestive tract of laboratory-reared bumblebee queens (Bombus terrestris). Int J Syst Evol Microbiol 64:2611–2617
Killer J, Švec P, Sedláček I, Cernohlávková J, Benada O, Hroncová Z, Havlík J, Vlková E, Rada V, Kopecny J, Kofronová O (2014c) Vagococcus entomophilus sp. nov., from the digestive tract of a wasp (Vespula vulgaris). Int J Syst Evol Microbiol 64:731–737
Killer J, Mekadim C, Pechar R, Bunešová V, Mrázek J, Vlková E (2018) Gene encoding the CTP synthetase as an appropriate molecular tool for identification and phylogenetic study of the family Bifidobacteriaceae. MicrobiologyOpen. https://doi.org/10.1002/mbo3.579
Lan Y, Rosen G, Hershberg R (2016) Marker genes that are less conserved in their sequences are useful for predicting genome-wide similarity levels between closely related prokaryotic strains. Microbiome 4(1):18
Lang JM, Darling AE, Eisen JA (2013) Phylogeny of bacterial and archaeal genomes using conserved genes: supertrees and supermatrices. PLoS One 8:e62510
Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452
Ludwig W, Schleifer KH, Whitman WB (2009) Order II. Lactobacillales ord. nov. In: de Vos P, Garrity GM, Jones D, Krieg NR, Ludwig W (eds) Bergey’s manual of systematic bacteriology (The Firmicutes), 2nd edn, vol 3. Springer, New York, p 464
Makarova KS, Koonin EV (2007) Evolutionary genomics of lactic acid bacteria. J Bacteriol 189:1199–1208
Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, Pavlov A, Pavlova N, Karamychev V, Polouchine N, Shakhova V, Grigoriev I, Lou Y, Rohksar D, Lucas S, Huang K, Goodstein DM, Hawkins T, Plengvidhya V, Welker D, Hughes J, Goh Y, Benson A, Baldwin K, Lee JH, Díaz-Muñiz I, Dosti B, Smeianov V, Wechter W, Barabote R, Lorca G, Altermann E, Barrangou R, Ganesan B, Xie Y, Rawsthorne H, Tamir D, Parker C, Breidt F, Broadbent J, Hutkins R, O’Sullivan D, Steele J, Unlu G, Saier M, Klaenhammer T, Richardson P, Kozyavkin S, Weimer B, Mills D (2006) Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci USA 103:15611–15616
Mao Y, Chen M, Horvath P (2015) Lactobacillus herbarum sp. nov., a species related to Lactobacillus plantarum. Int J Syst Evol Microbiol 65:4682–4688
Martin DP, Murrell B, Khoosal A, Muhire B (2007) Detecting and analyzing genetic recombination using RDP4. Methods Mol Biol 1525:433–460
Martinez-Murcia AJ, Monera A, Saavedra MJ, Oncina R, Lopez-Alvarez M, Lara E, Figueras MJ (2011) Multilocus phylogenetic analysis of the genus Aeromonas. Syst Appl Microbiol 34:189–199
Mayo B, van Sinderen D, Ventura M (2008) Genome analysis of food grade lactic acid-producing bacteria: from basics to applications. Curr Genom 9:169–183
Naser SM, Thompson FL, Hoste B, Gevers D, Dawyndt P, Vancanneyt M, Swings J (2005a) Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151:2141–2150
Naser SM, Thompson FL, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerck I, Thompson CC, Vancanneyt M, Swings J (2005b) Phylogeny and identification of Enterococci by atpA gene sequence analysis. J Clin Microbiol 43:2224–2230
Naser SM, Dawyndt P, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerck I, Vancanneyt M, Swings J (2007) Identification of lactobacilli by pheS and rpoA gene sequence analyses. Int J Syst Evol Microbiol 57:2777–2789
Patwardhan A, Ray S, Roy A (2014) Molecular markers in phylogenetic studies—a review. J Phylogen Evol Biol 2:131
Puertas A, Arahal DR, Ibarburu I, Elizaquível P, Aznar R, Dueñas MT (2014) Lactobacillus sicerae sp. nov., a lactic acid bacterium isolated from Spanish natural cider. Int J Syst Evol Microbiol 64:2949–2955
Rosselló-Móra R, Amann R (2001) The species concept for prokaryotes. FEMS Microbiol Rev 25:39–67
Rosselló-Móra R, Amann R (2015) Past and future species definitions for Bacteria and Archaea. Syst Appl Microbiol 38:209–216
Roux S, Enault F, Bronner G, Debroas D (2011) Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems. FEMS Microbiol Ecol 78:617–628
Salvetti E, Fondi M, Fani R, Torriani S, Felis GE (2013) Evolution of lactic acid bacteria in the order Lactobacillales as depicted by analysis of glycolysis and pentose phosphate pathways. Syst Appl Microbiol 36:291–305
Švec P, Devriese LA (2009) Genus Enterococcus. In: de Vos P, Garrity GM, Jones D, Krieg NR, Ludwig W (edn) Bergey’s manual of systematic bacteriology (The Firmicutes), 2nd edn, vol 3. Springer, New York, pp 594–607
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729
Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikolskaya AN, Rao BS, Smirnov S, Sverdlov AV, Vasudevan S, Wolf YI, Yin JJ, Natale DA (2003) The COG database: an updated version includes eukaryotes. BMC Bioinform 4:41
Tindall BJ, Rosselló-Móra R, Busse HJ, Ludwig W, Kämpfer P (2010) Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 60:249–266
Tomasini N, Lauthier JJ, Llewellyn MS, Diosque P (2013) MLSTest: novel software for multi-locus sequence data analysis in eukaryotic organisms. Infect Genet Evol 20:188–196
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3-new capabilities and interfaces. Nucleic Acids Res 40:e115
Ventura M, Canchaya C, Meylan V, Klaenhammer TR, Zink R (2003) Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification. Appl Environ Microbiol 69:6908–6922
Wang Z, Wu M (2013) A phylum-level bacterial phylogenetic marker database. Mol Biol Evol 30(6):1258–1262
Wu D, Jospin G, Eisen JA (2013) Systematic identification of gene families for use as “markers” for phylogenetic and phylogeny-driven ecological studies of bacteria and archaea and their major subgroups. PLoS One 8(10):e77033
Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017) Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 67:1613–1617
Zhang ZG, Ye ZQ, Yu L, Shi P (2011) Phylogenomic reconstruction of lactic acid bacteria: an update. BMC Evol Biol 11:1
Zheng J, Ruan L, Sun M, Gänzle MA (2015) Genomic view of Lactobacilli and Pediococci demonstrates that phylogeny matches ecology and physiology. Appl Environ Microbiol 81:7233–7243
Acknowledgements
This study was funded by the Project Excellence (no. CZ.02.1.01/0.0/0.0/15_003/0000460), the Czech Health Research Council (Project no. 16-27449A) and the Institution research Project (no. RO 0318) of the Food Research Institute Prague (Ministry of Agriculture of the Czech Republic). This work is dedicated to the memory of our teacher prof. Ing. Vojtěch Rada, C.Sc., whose enthusiasm, support and precious advices contributed so much to this work. We will always be grateful to him for this.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethical statement
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in the study involving animals were in accordance with the ethical standards of the institution or practice at which the study was conducted.
Conflict of interest
The authors have no conflicts of interest.
Additional information
Communicated by Erko Stackebrandt.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Mekadim, C., Killer, J., Mrázek, J. et al. Evaluation of the infB and rpsB gene fragments as genetic markers intended for identification and phylogenetic analysis of particular representatives of the order Lactobacillales. Arch Microbiol 200, 1427–1437 (2018). https://doi.org/10.1007/s00203-018-1554-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00203-018-1554-7