PROKARYOTES
crossm Complete Genome Sequence of the Yogurt Isolate Lactobacillus delbrueckii subsp. bulgaricus ACA-DC 87 Voula Alexandraki,a Maria Kazou,a Bruno Pot,b Effie Tsakalidou,a Konstantinos Papadimitrioua Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, Greecea; Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Vrije Universiteit Brussel, Brussels, Belgiumb
Lactobacillus delbrueckii subsp. bulgaricus is widely used in the production of yogurt and cheese. In this study, we present the complete genome sequence of L. delbrueckii subsp. bulgaricus ACA-DC 87 isolated from traditional Greek yogurt. Whole-genome analysis may reveal desirable technological traits of the strain for dairy fermentations.
ABSTRACT
L
actobacillus delbrueckii subsp. bulgaricus is among the most important starters employed in dairy fermentations and is used mainly in the production of yogurt in protocooperation with Streptococcus thermophilus (1). L. delbrueckii subsp. bulgaricus belongs to the acidophilus complex, a group of lactobacilli which further includes Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus johnsonii, and Lactobacillus gasseri (2). Here, we report the genome sequencing and the genomic features of L. delbrueckii subsp. bulgaricus ACA-DC 87, which was isolated from traditional Greek yogurt (3, 4). The genome was sequenced at the Beijing Genomics Institute (BGI Co., Ltd., Hong Kong). A total of 17,362,540 paired-end reads (500-bp, 2,000-bp, and 6,000-bp libraries) were generated using the Illumina HiSeq 2000 platform with ⬎100-fold sequence coverage. After filtering, the reads were assembled with SOAPdenovo version 1.05, resulting in one circular chromosome (5). The accuracy of the assembly was evaluated through whole-genome alignment of the ACA-DC 87 genome against a reference genome, namely, that of L. delbrueckii subsp. bulgaricus ATCC 11842 (6), using ProgressiveMauve (7). The ACA-DC 87 genome sequence was analyzed using 3 gene prediction programs, i.e., Prodigal (8), MetaGeneAnnotator (9), and FGENESB (10). Additionally, RAST version 2.0 was used both in genome annotation and prediction of rRNA and tRNA genes (11). The GenePRIMP pipeline was also employed for the detection of annotation anomalies, including putative pseudogenes (12). All the results obtained were manually curated. Further bioinformatics analysis focused on the identification of genomic islands (GIs) with IslandViewer 4 (13), clustered regularly interspaced short palindromic repeats (CRISPRs) with CRISPRFinder (14), and Pfam domain-containing proteins with Pfam database (15), while the WebMGA server was used for clusters of orthologous groups (COG) functional annotation (16). The chromosome of ACA-DC 87 comprises 1,856,003 bp with a G⫹C content of 49.8%. A total of 1,993 genes were identified in the genome sequence, including 1,644 protein-coding genes and 229 potential pseudogenes. The genome also contains 93 tRNA genes and 9 complete rRNA operons. Twelve genomic islands (GIs) with a total of 196 genes were predicted in the genome of ACA-DC 87. These genes may have been acquired through horizontal gene transfer and could be related to technological features. Several of these genes encode CRISPR-associated proteins, subunits of Volume 5 Issue 34 e00868-17
Received 11 July 2017 Accepted 12 July 2017 Published 24 August 2017 Citation Alexandraki V, Kazou M, Pot B, Tsakalidou E, Papadimitriou K. 2017. Complete genome sequence of the yogurt isolate Lactobacillus delbrueckii subsp. bulgaricus ACA-DC 87. Genome Announc 5:e00868-17. https://doi.org/10.1128/genomeA.00868-17. Copyright © 2017 Alexandraki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Konstantinos Papadimitriou, [email protected].
genomea.asm.org 1
Alexandraki et al.
restriction-modification systems, and proteins implicated in exopolysaccharide biosynthesis. The genome also carries one confirmed CRISPR array with a size of 761 bp, containing 11 spacers. Furthermore, 1,322 proteins with Pfam domains were identified. The COG annotation results revealed that approximately 87% of the protein-coding genes (1,284 proteins) were assigned to at least one functional category. The higher number of proteins were allocated to the category of translation, ribosomal structure and biogenesis (J: 8.5%), followed by the categories of amino acid transport and metabolism (E: 7.8%) and replication, recombination, and repair (L: 7.4%). Further analysis of the ACA-DC 87 genome sequence may decipher the technological potential of the strain toward its application in the production of fermented dairy products. Accession number(s). The genome sequence of L. delbrueckii subsp. bulgaricus ACA-DC 87 was deposited at the European Nucleotide Archive under the accession number LT899687. ACKNOWLEDGMENTS We thank Nikos Kyrpides at the Joint Genome Institute (United States Department of Energy) for analysis of the ACA-DC 87 genome with the GenePRIMP pipeline. The present work was cofinanced by the European Social Fund and the national resources EPEAEK and YPEPTH through the Thales project.
REFERENCES 1. Angelov M, Kostov G, Simova E, Beshkova D, Koprinkova-Hristova P. 2009. Proto-cooperation factors in yogurt starter cultures. Rev Genie Indust 3:4 –12. 2. Zhang ZG, Ye ZQ, Yu L, Shi P. 2011. Phylogenomic reconstruction of lactic acid bacteria: an update. BMC Evol Biol 11:1. https://doi.org/10 .1186/1471-2148-11-1. 3. Tsakalidou E, Manolopoulou E, Kabaraki E, Zoidou E, Pot B, Kersters K, Kalantzopoulos G. 1994. The combined use of whole-cell protein extracts for the identification (SDS-PAGE) and enzyme activity screening of lactic acid bacteria isolated from traditional Greek dairy products. Syst Appl Microbiol 17:444 – 458. https://doi.org/10.1016/S0723-2020(11)80062-7. 4. Tsakalidou E, Zoidou E, Kalantzopoulos G. 1992. SDS-polyacrylamide gel electrophoresis of cell proteins from Lactobacillus delbreuckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus strains isolated from yoghurt and cheese. Milchwissenschaft 47:296 –298. 5. Li R, Li Y, Kristiansen K, Wang J. 2008. SOAP: short oligonucleotide alignment program. Bioinformatics 24:713–714. https://doi.org/10.1093/ bioinformatics/btn025. 6. 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, Bessières 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 103:9274 –9279. https://doi.org/10.1073/pnas .0603024103. 7. Darling AE, Mau B, Perna NT. 2010. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One 5:e11147. https://doi.org/10.1371/journal.pone.0011147. 8. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119. https://doi.org/10.1186/ 1471-2105-11-119. 9. Noguchi H, Taniguchi T, Itoh T. 2008. MetaGeneAnnotator: detecting
Volume 5 Issue 34 e00868-17
10.
11.
12.
13.
14.
15.
16.
species-specific patterns of ribosomal binding site for precise gene prediction in anonymous prokaryotic and phage genomes. DNA Res 15:387–396. https://doi.org/10.1093/dnares/dsn027. Solovyev V, Salamov A. 2011. Automatic annotation of microbial genomes and metagenomic sequences, p 61–78. In Li RW (ed), Metagenomics and its applications in agriculture, biomedicine and environmental studies. Nova Science Publishers, New York, NY. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75. https://doi.org/10.1186/1471-2164-9-75. Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, Kyrpides NC. 2010. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods 7:455– 457. https://doi.org/10 .1038/nmeth.1457. Bertelli C, Laird MR, Williams KP; Simon Fraser University Research Computing Group, Lau BY, Hoad G, Winsor GL, Brinkman FSL. 2 May 2017. IslandViewer 4: expanded prediction of genomic islands for largerscale data sets. Nucleic Acids Res. https://doi.org/10.1093/nar/gkx343. Grissa I, Vergnaud G, Pourcel C. 2007. CRISPRFinder: a Web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52–W57. https://doi.org/10.1093/nar/gkm360. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A. 2016. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279 –D285. https://doi.org/10.1093/nar/ gkv1344. Wu S, Zhu Z, Fu L, Niu B, Li W. 2011. WebMGA: a customizable Web server for fast metagenomic sequence analysis. BMC Genomics 12:444. https://doi.org/10.1186/1471-2164-12-444.
genomea.asm.org 2
crossm Complete Genome Sequence of the Yogurt Isolate Lactobacillus delbrueckii subsp. bulgaricus ACA-DC 87 Voula Alexandraki,a Maria Kazou,a Bruno Pot,b Effie Tsakalidou,a Konstantinos Papadimitrioua Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, Greecea; Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Vrije Universiteit Brussel, Brussels, Belgiumb
Lactobacillus delbrueckii subsp. bulgaricus is widely used in the production of yogurt and cheese. In this study, we present the complete genome sequence of L. delbrueckii subsp. bulgaricus ACA-DC 87 isolated from traditional Greek yogurt. Whole-genome analysis may reveal desirable technological traits of the strain for dairy fermentations.
ABSTRACT
L
actobacillus delbrueckii subsp. bulgaricus is among the most important starters employed in dairy fermentations and is used mainly in the production of yogurt in protocooperation with Streptococcus thermophilus (1). L. delbrueckii subsp. bulgaricus belongs to the acidophilus complex, a group of lactobacilli which further includes Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus johnsonii, and Lactobacillus gasseri (2). Here, we report the genome sequencing and the genomic features of L. delbrueckii subsp. bulgaricus ACA-DC 87, which was isolated from traditional Greek yogurt (3, 4). The genome was sequenced at the Beijing Genomics Institute (BGI Co., Ltd., Hong Kong). A total of 17,362,540 paired-end reads (500-bp, 2,000-bp, and 6,000-bp libraries) were generated using the Illumina HiSeq 2000 platform with ⬎100-fold sequence coverage. After filtering, the reads were assembled with SOAPdenovo version 1.05, resulting in one circular chromosome (5). The accuracy of the assembly was evaluated through whole-genome alignment of the ACA-DC 87 genome against a reference genome, namely, that of L. delbrueckii subsp. bulgaricus ATCC 11842 (6), using ProgressiveMauve (7). The ACA-DC 87 genome sequence was analyzed using 3 gene prediction programs, i.e., Prodigal (8), MetaGeneAnnotator (9), and FGENESB (10). Additionally, RAST version 2.0 was used both in genome annotation and prediction of rRNA and tRNA genes (11). The GenePRIMP pipeline was also employed for the detection of annotation anomalies, including putative pseudogenes (12). All the results obtained were manually curated. Further bioinformatics analysis focused on the identification of genomic islands (GIs) with IslandViewer 4 (13), clustered regularly interspaced short palindromic repeats (CRISPRs) with CRISPRFinder (14), and Pfam domain-containing proteins with Pfam database (15), while the WebMGA server was used for clusters of orthologous groups (COG) functional annotation (16). The chromosome of ACA-DC 87 comprises 1,856,003 bp with a G⫹C content of 49.8%. A total of 1,993 genes were identified in the genome sequence, including 1,644 protein-coding genes and 229 potential pseudogenes. The genome also contains 93 tRNA genes and 9 complete rRNA operons. Twelve genomic islands (GIs) with a total of 196 genes were predicted in the genome of ACA-DC 87. These genes may have been acquired through horizontal gene transfer and could be related to technological features. Several of these genes encode CRISPR-associated proteins, subunits of Volume 5 Issue 34 e00868-17
Received 11 July 2017 Accepted 12 July 2017 Published 24 August 2017 Citation Alexandraki V, Kazou M, Pot B, Tsakalidou E, Papadimitriou K. 2017. Complete genome sequence of the yogurt isolate Lactobacillus delbrueckii subsp. bulgaricus ACA-DC 87. Genome Announc 5:e00868-17. https://doi.org/10.1128/genomeA.00868-17. Copyright © 2017 Alexandraki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Konstantinos Papadimitriou, [email protected].
genomea.asm.org 1
Alexandraki et al.
restriction-modification systems, and proteins implicated in exopolysaccharide biosynthesis. The genome also carries one confirmed CRISPR array with a size of 761 bp, containing 11 spacers. Furthermore, 1,322 proteins with Pfam domains were identified. The COG annotation results revealed that approximately 87% of the protein-coding genes (1,284 proteins) were assigned to at least one functional category. The higher number of proteins were allocated to the category of translation, ribosomal structure and biogenesis (J: 8.5%), followed by the categories of amino acid transport and metabolism (E: 7.8%) and replication, recombination, and repair (L: 7.4%). Further analysis of the ACA-DC 87 genome sequence may decipher the technological potential of the strain toward its application in the production of fermented dairy products. Accession number(s). The genome sequence of L. delbrueckii subsp. bulgaricus ACA-DC 87 was deposited at the European Nucleotide Archive under the accession number LT899687. ACKNOWLEDGMENTS We thank Nikos Kyrpides at the Joint Genome Institute (United States Department of Energy) for analysis of the ACA-DC 87 genome with the GenePRIMP pipeline. The present work was cofinanced by the European Social Fund and the national resources EPEAEK and YPEPTH through the Thales project.
REFERENCES 1. Angelov M, Kostov G, Simova E, Beshkova D, Koprinkova-Hristova P. 2009. Proto-cooperation factors in yogurt starter cultures. Rev Genie Indust 3:4 –12. 2. Zhang ZG, Ye ZQ, Yu L, Shi P. 2011. Phylogenomic reconstruction of lactic acid bacteria: an update. BMC Evol Biol 11:1. https://doi.org/10 .1186/1471-2148-11-1. 3. Tsakalidou E, Manolopoulou E, Kabaraki E, Zoidou E, Pot B, Kersters K, Kalantzopoulos G. 1994. The combined use of whole-cell protein extracts for the identification (SDS-PAGE) and enzyme activity screening of lactic acid bacteria isolated from traditional Greek dairy products. Syst Appl Microbiol 17:444 – 458. https://doi.org/10.1016/S0723-2020(11)80062-7. 4. Tsakalidou E, Zoidou E, Kalantzopoulos G. 1992. SDS-polyacrylamide gel electrophoresis of cell proteins from Lactobacillus delbreuckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus strains isolated from yoghurt and cheese. Milchwissenschaft 47:296 –298. 5. Li R, Li Y, Kristiansen K, Wang J. 2008. SOAP: short oligonucleotide alignment program. Bioinformatics 24:713–714. https://doi.org/10.1093/ bioinformatics/btn025. 6. 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, Bessières 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 103:9274 –9279. https://doi.org/10.1073/pnas .0603024103. 7. Darling AE, Mau B, Perna NT. 2010. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One 5:e11147. https://doi.org/10.1371/journal.pone.0011147. 8. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119. https://doi.org/10.1186/ 1471-2105-11-119. 9. Noguchi H, Taniguchi T, Itoh T. 2008. MetaGeneAnnotator: detecting
Volume 5 Issue 34 e00868-17
10.
11.
12.
13.
14.
15.
16.
species-specific patterns of ribosomal binding site for precise gene prediction in anonymous prokaryotic and phage genomes. DNA Res 15:387–396. https://doi.org/10.1093/dnares/dsn027. Solovyev V, Salamov A. 2011. Automatic annotation of microbial genomes and metagenomic sequences, p 61–78. In Li RW (ed), Metagenomics and its applications in agriculture, biomedicine and environmental studies. Nova Science Publishers, New York, NY. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75. https://doi.org/10.1186/1471-2164-9-75. Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, Kyrpides NC. 2010. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods 7:455– 457. https://doi.org/10 .1038/nmeth.1457. Bertelli C, Laird MR, Williams KP; Simon Fraser University Research Computing Group, Lau BY, Hoad G, Winsor GL, Brinkman FSL. 2 May 2017. IslandViewer 4: expanded prediction of genomic islands for largerscale data sets. Nucleic Acids Res. https://doi.org/10.1093/nar/gkx343. Grissa I, Vergnaud G, Pourcel C. 2007. CRISPRFinder: a Web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52–W57. https://doi.org/10.1093/nar/gkm360. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A. 2016. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279 –D285. https://doi.org/10.1093/nar/ gkv1344. Wu S, Zhu Z, Fu L, Niu B, Li W. 2011. WebMGA: a customizable Web server for fast metagenomic sequence analysis. BMC Genomics 12:444. https://doi.org/10.1186/1471-2164-12-444.
genomea.asm.org 2
