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crossm Complete Genome Sequences of Lactococcus lactis subsp. lactis bv. diacetylactis FM03 and Leuconostoc mesenteroides FM06 Isolated from Cheese Oscar van Mastrigt, Tjakko Abee, Eddy J. Smid Laboratory of Food Microbiology, Wageningen University & Research, Wageningen Campus, Wageningen, The Netherlands
ABSTRACT Here, the genome sequences of Lactococcus lactis subsp. lactis bv. diacetylactis FM03 and Leuconostoc mesenteroides FM06, both isolated from cheese, are presented. FM03 and FM06 contain 7 and 3 plasmids, respectively, that carry genes encoding functions important for growth and survival in dairy fermentations.
L
euconostoc mesenteroides and Lactococcus lactis subsp. lactis bv. diacetylactis are two species of lactic acid bacteria present in starter cultures for cheese production. Compared to L. lactis subsp. cremoris strains, L. mesenteroides and L. lactis subsp. lactis strains show better survival during cheese ripening and thus provide an important contribution to the flavor development (1). Moreover, these two species of lactic acid bacteria also have the ability to utilize citrate, enhancing the production of the buttery aromas acetoin and diacetyl (2). In L. lactis subsp. lactis, the gene for citrate transport is located on a plasmid (3), showing the importance of plasmids for lactococcal performance in dairy applications. Another trait found regularly encoded on plasmids is the resistance to bacteriophages (4). Lactococcus lactis subsp. lactis bv. diacetylactis FM03 and Leuconostoc mesenteroides FM06 were isolated from 10-week-old Samsø cheese. Their genomes were sequenced using an Illumina HiSeq 2500 and a PacBio RS instrument. Respectively, 6.4 and 4.3 million quality-filtered paired-end Illumina sequence reads of 100 bp were de novo assembled into contig sequences using CLC Genomics Workbench version 7.0.4 (CLC bio, Aarhus, Denmark). The contigs were linked and placed into scaffolds based on alignment of the PacBio reads (total, 167 Mbp and 493 Mbp, respectively). This resulted in 8 and 4 scaffolds for FM03 and FM06, respectively. Three out of 12 scaffolds were already circular. The remaining scaffolds were closed with PCR, followed by Sanger sequencing. The complete sequence of L. lactis FM03 was annotated using RAST (5) and manually curated. The sequence of L. mesenteroides FM06 was annotated with the NCBI Prokaryotic Genome Annotation Pipeline (6). The genome sequence of L. lactis FM03 contains a chromosome of 2.43 Mbp, with a G⫹C content of 35.3%, and 7 plasmids with sizes of 3.4, 4.2, 7.5, 8.3, 12.0, 15.2, and 30.3 kbp and G⫹C contents of 33.8, 35.6, 33.6, 34.8, 33.5, 34.1, and 35.2%, respectively. The plasmids contained genes involved in citrate utilization (citQRP), resistance to bacteriophages (five different specificity subunits of a type I restriction-modification system), and resistance to several stresses. In most plasmids, transposons were also identified. The genome sequence of Leuconostoc mesenteroides FM06 contains a chromosomal sequence of 1.89 Mbp, with a G⫹C content of 35.3%, and 3 plasmid sequences with sizes of 8.8, 15.3, and 31.1 kbp and G⫹C contents of 35.2, 39.2, and 36.0%, respectively. Volume 5 Issue 28 e00633-17
Received 17 May 2017 Accepted 18 May 2017 Published 13 July 2017 Citation van Mastrigt O, Abee T, Smid EJ. 2017. Complete genome sequences of Lactococcus lactis subsp. lactis bv. diacetylactis FM03 and Leuconostoc mesenteroides FM06 isolated from cheese. Genome Announc 5:e00633-17. https://doi.org/10.1128/genomeA.00633-17. Copyright © 2017 van Mastrigt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Eddy J. Smid, [email protected].
genomea.asm.org 1
van Mastrigt et al.
The plasmids contained genes involved in metal transport, lactose utilization, and resistance to bacteriophages. Interestingly, genes for lactose utilization (lactose phosphotransferase system [PTS] encoded by lacS [7] and -galactosidase encoded by lacLM [8]) were carried by two different plasmids. Further investigations into the genomes of these lactic acid bacteria may provide more insight into the role of plasmids in growth and survival in dairy fermentations. Accession number(s). The genome sequences of Lactococcus lactis FM03 and Leuconostoc mesenteroides FM06 have been deposited at GenBank under the accession numbers CP020604 to CP020611 and CP020731 to CP020734, respectively. ACKNOWLEDGMENT This work was financially supported by Arla Foods, Aarhus, Denmark.
REFERENCES 1. Erkus O, de Jager VC, Spus M, van Alen-Boerrigter IJ, van Rijswijck IM, Hazelwood L, Janssen PW, van Hijum SA, Kleerebezem M, Smid EJ. 2013. Multifactorial diversity sustains microbial community stability. ISME J 7:2126 –2136. https://doi.org/10.1038/ismej.2013.108. 2. Starrenburg MJ, Hugenholtz J. 1991. Citrate fermentation by Lactococcus and Leuconostoc spp. Appl Environ Microbiol 57:3535–3540. 3. Kempler GM, Mckay LL. 1979. Characterization of plasmid deoxyribonucleic acid in Streptococcus lactis subsp. diacetylactis— evidence for plasmid-linked citrate utilization. Appl Environ Microbiol 37:316 –323. 4. Allison GE, Klaenhammer TR. 1998. Phage resistance mechanisms in lactic acid bacteria. Int Dairy J 8:207–226. https://doi.org/10.1016/S0958 -6946(98)00043-0. 5. 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,
Volume 5 Issue 28 e00633-17
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. 6. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI Prokaryotic Genome Annotation Pipeline. Nucleic Acids Res 44: 6614 – 6624. https://doi.org/10.1093/nar/gkw569. 7. Vaughan EE, David S, de Vos WM. 1996. The lactose transporter in Leuconostoc lactis is a new member of the LacS subfamily of galactosidepentose-hexuronide translocators. Appl Environ Microbiol 62:1574 –1582. 8. David S, Stevens H, van Riel M, Simons G, de Vos WM. 1992. Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes. J Bacteriol 174:4475– 4481. https://doi.org/10.1128/jb.174.13.4475-4481.1992.
genomea.asm.org 2
crossm Complete Genome Sequences of Lactococcus lactis subsp. lactis bv. diacetylactis FM03 and Leuconostoc mesenteroides FM06 Isolated from Cheese Oscar van Mastrigt, Tjakko Abee, Eddy J. Smid Laboratory of Food Microbiology, Wageningen University & Research, Wageningen Campus, Wageningen, The Netherlands
ABSTRACT Here, the genome sequences of Lactococcus lactis subsp. lactis bv. diacetylactis FM03 and Leuconostoc mesenteroides FM06, both isolated from cheese, are presented. FM03 and FM06 contain 7 and 3 plasmids, respectively, that carry genes encoding functions important for growth and survival in dairy fermentations.
L
euconostoc mesenteroides and Lactococcus lactis subsp. lactis bv. diacetylactis are two species of lactic acid bacteria present in starter cultures for cheese production. Compared to L. lactis subsp. cremoris strains, L. mesenteroides and L. lactis subsp. lactis strains show better survival during cheese ripening and thus provide an important contribution to the flavor development (1). Moreover, these two species of lactic acid bacteria also have the ability to utilize citrate, enhancing the production of the buttery aromas acetoin and diacetyl (2). In L. lactis subsp. lactis, the gene for citrate transport is located on a plasmid (3), showing the importance of plasmids for lactococcal performance in dairy applications. Another trait found regularly encoded on plasmids is the resistance to bacteriophages (4). Lactococcus lactis subsp. lactis bv. diacetylactis FM03 and Leuconostoc mesenteroides FM06 were isolated from 10-week-old Samsø cheese. Their genomes were sequenced using an Illumina HiSeq 2500 and a PacBio RS instrument. Respectively, 6.4 and 4.3 million quality-filtered paired-end Illumina sequence reads of 100 bp were de novo assembled into contig sequences using CLC Genomics Workbench version 7.0.4 (CLC bio, Aarhus, Denmark). The contigs were linked and placed into scaffolds based on alignment of the PacBio reads (total, 167 Mbp and 493 Mbp, respectively). This resulted in 8 and 4 scaffolds for FM03 and FM06, respectively. Three out of 12 scaffolds were already circular. The remaining scaffolds were closed with PCR, followed by Sanger sequencing. The complete sequence of L. lactis FM03 was annotated using RAST (5) and manually curated. The sequence of L. mesenteroides FM06 was annotated with the NCBI Prokaryotic Genome Annotation Pipeline (6). The genome sequence of L. lactis FM03 contains a chromosome of 2.43 Mbp, with a G⫹C content of 35.3%, and 7 plasmids with sizes of 3.4, 4.2, 7.5, 8.3, 12.0, 15.2, and 30.3 kbp and G⫹C contents of 33.8, 35.6, 33.6, 34.8, 33.5, 34.1, and 35.2%, respectively. The plasmids contained genes involved in citrate utilization (citQRP), resistance to bacteriophages (five different specificity subunits of a type I restriction-modification system), and resistance to several stresses. In most plasmids, transposons were also identified. The genome sequence of Leuconostoc mesenteroides FM06 contains a chromosomal sequence of 1.89 Mbp, with a G⫹C content of 35.3%, and 3 plasmid sequences with sizes of 8.8, 15.3, and 31.1 kbp and G⫹C contents of 35.2, 39.2, and 36.0%, respectively. Volume 5 Issue 28 e00633-17
Received 17 May 2017 Accepted 18 May 2017 Published 13 July 2017 Citation van Mastrigt O, Abee T, Smid EJ. 2017. Complete genome sequences of Lactococcus lactis subsp. lactis bv. diacetylactis FM03 and Leuconostoc mesenteroides FM06 isolated from cheese. Genome Announc 5:e00633-17. https://doi.org/10.1128/genomeA.00633-17. Copyright © 2017 van Mastrigt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Eddy J. Smid, [email protected].
genomea.asm.org 1
van Mastrigt et al.
The plasmids contained genes involved in metal transport, lactose utilization, and resistance to bacteriophages. Interestingly, genes for lactose utilization (lactose phosphotransferase system [PTS] encoded by lacS [7] and -galactosidase encoded by lacLM [8]) were carried by two different plasmids. Further investigations into the genomes of these lactic acid bacteria may provide more insight into the role of plasmids in growth and survival in dairy fermentations. Accession number(s). The genome sequences of Lactococcus lactis FM03 and Leuconostoc mesenteroides FM06 have been deposited at GenBank under the accession numbers CP020604 to CP020611 and CP020731 to CP020734, respectively. ACKNOWLEDGMENT This work was financially supported by Arla Foods, Aarhus, Denmark.
REFERENCES 1. Erkus O, de Jager VC, Spus M, van Alen-Boerrigter IJ, van Rijswijck IM, Hazelwood L, Janssen PW, van Hijum SA, Kleerebezem M, Smid EJ. 2013. Multifactorial diversity sustains microbial community stability. ISME J 7:2126 –2136. https://doi.org/10.1038/ismej.2013.108. 2. Starrenburg MJ, Hugenholtz J. 1991. Citrate fermentation by Lactococcus and Leuconostoc spp. Appl Environ Microbiol 57:3535–3540. 3. Kempler GM, Mckay LL. 1979. Characterization of plasmid deoxyribonucleic acid in Streptococcus lactis subsp. diacetylactis— evidence for plasmid-linked citrate utilization. Appl Environ Microbiol 37:316 –323. 4. Allison GE, Klaenhammer TR. 1998. Phage resistance mechanisms in lactic acid bacteria. Int Dairy J 8:207–226. https://doi.org/10.1016/S0958 -6946(98)00043-0. 5. 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,
Volume 5 Issue 28 e00633-17
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. 6. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI Prokaryotic Genome Annotation Pipeline. Nucleic Acids Res 44: 6614 – 6624. https://doi.org/10.1093/nar/gkw569. 7. Vaughan EE, David S, de Vos WM. 1996. The lactose transporter in Leuconostoc lactis is a new member of the LacS subfamily of galactosidepentose-hexuronide translocators. Appl Environ Microbiol 62:1574 –1582. 8. David S, Stevens H, van Riel M, Simons G, de Vos WM. 1992. Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes. J Bacteriol 174:4475– 4481. https://doi.org/10.1128/jb.174.13.4475-4481.1992.
genomea.asm.org 2
