Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strain and Media
2.2. DNA Extraction and Genome Sequencing
2.3. Genome Analysis
2.3.1. Genome Annotation
2.3.2. Pan-Genome and Phylogenetic Analysis
2.3.3. Prophages Prediction
2.3.4. Genomic Islands Prediction
2.3.5. Genome Mining for Secondary Metabolite Biosynthetic Gene Clusters
2.4. Methods of Genome Streamlining
2.5. Resequencing
2.6. Phenotype Testing
2.6.1. Determination of Growth Profiles
2.6.2. Determination of Nisin Titre
2.7. Statistical Analysis
3. Results and Discussion
3.1. General Features of the Genome
3.2. Phylogeny and Core-Pan Genome
3.3. Megaplasmids of L. lactis N8
3.4. Prophage-Related Fragments of L. lactis N8
3.5. Genomic Islands of L. lactis N8
3.6. Other Characteristics of L. lactis N8
3.6.1. Cell-Surface Protein
3.6.2. PKS/NRPS
3.6.3. Bacteriocin Gene Cluster
3.7. Construction of Streamlined Genome Chassis of L. lactis N8
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strains or Plasmids | Relevant Descriptions | Reference |
---|---|---|
Strains | ||
E. coli DH5α | Cloning host; F-φ80lacZ∆M15endA1 recA1 endA1 hsdR17 (rK-mK+) supE44 thi-1 gyrA 96 relA1 ∆(lacZYA-argF)U169 deoR λ- | [22] |
E. coli DH5α-up-down (L1) | Cmr, Emr, E. coli DH5α derivative containing the whole plasmid pNZ5319∆L1 | [12] |
E. coli DH5α-up-down (L2) | Cmr, Emr, E. coli DH5α derivative containing the whole plasmid pNZ5319∆L2 | This study |
E. coli DH5α-up-down (L3) | Cmr, Emr, E. coli DH5α derivative containing the whole plasmid pNZ5319∆L3 | This study |
E. coli DH5α-up-down (L4) | Cmr, Emr, E. coli DH5α derivative containing the whole plasmid pNZ5319∆L4 | This study |
E. coli DH5α-up-down (L5) | Cmr, Emr, E. coli DH5α derivative containing the whole plasmid pNZ5319∆L5 | This study |
E. coli DH5α-up-down (L6) | Cmr, Emr, E. coli DH5α derivative containing the whole plasmid pNZ5319∆L6 | This study |
E. coli DH5α-up-down (L7) | Cmr, Emr, E. coli DH5α derivative containing the whole plasmid pNZ5319∆L7 | This study |
E. coli DH5α-up-down (L8) | Cmr, Emr, E. coli DH5α derivative containing the whole plasmid pNZ5319∆L8 | This study |
Micrococcus luteus NCIB 8166 | Indicator strains for Nisin agar gel diffusion assay | [23] |
L. lactis N8 | Wild-type (WT) Nisin Z producer | [24] |
L. lactis N8-1 | The first DNA region L1 deletion in L. lactis N8 | [12] |
L. lactis N8-2 | The L2 deletion in L. lactis N8-1 | This study |
L. lactis N8-3 | The L3 deletion in L. lactis N8-2 | This study |
L. lactis N8-4 | The L4 deletion in L. lactis N8-3 | This study |
L. lactis N8-5 | The L5 deletion in L. lactis N8-4 | This study |
L. lactis N8-6 | The L6 deletion in L. lactis N8-5 | This study |
L. lactis N8-7 | The L7 deletion in L. lactis N8-6 | This study |
L. lactis N8-8 | The L8 deletion in L. lactis N8-7 | This study |
Plasmids | ||
pNZ5319 | Cmr, Emr, used as knock-out vector | [25] |
pNZTS-Cre | Emr, cre gene cloned at the EcoRI and HindIII sites (cat gene deletion vector) | [20] |
pNZ5319∆L1 | Cmr, Emr, upstream and downstream homology arm of L1 amplified from L. lactis N8 genome cloned into pNZ5319 | [12] |
pNZ5319∆L2 | Cmr, Emr, upstream and downstream homology arm of L2 amplified from L. lactis N8 genome cloned into pNZ5319 | This study |
pNZ5319∆L3 | Cmr, Emr, upstream and downstream homology arm of L3 amplified from L. lactis N8 genome cloned into pNZ5319 | This study |
pNZ5319∆L4 | Cmr, Emr, upstream and downstream homology arm of L4 amplified from L. lactis N8 genome cloned into pNZ5319 | This study |
pNZ5319∆L5 | Cmr, Emr, upstream and downstream homology arm of L5 amplified from L. lactis N8 genome cloned into pNZ5319 | This study |
pNZ5319∆L6 | Cmr, Emr, upstream and downstream homology arm of L6 amplified from L. lactis N8 genome cloned into pNZ5319 | This study |
pNZ5319∆L7 | Cmr, Emr, upstream and downstream homology arm of L7 amplified from L. lactis N8 genome cloned into pNZ5319 | This study |
pNZ5319∆L8 | Cmr, Emr, upstream and downstream homology arm of L8 amplified from L. lactis N8 genome cloned into pNZ5319 | This study |
Feature | Size/Percentage/Number |
---|---|
Genome size (bp) | 2,421,567 |
G + C content | 35.1% |
rRNAs | 19 |
tRNAs | 64 |
ncRNAs | 4 |
Total genes | 2521 |
Coding sequences (CDs) | 2434 |
Prophages | 7 |
Genomic islands (GIs) | 12 |
Plasmids | 2 |
Organism Name | Strain | BioSample | Number of Plasmids | Total Size of Plasmids | Release Date |
---|---|---|---|---|---|
Lactococcus lactis subsp. lactis | N8 | SAMN15500618 | 2 | 0.151562 | 22 October 2020 |
Lactococcus lactis subsp. lactis | No attributes | SAMN14223931 | 1 | 0.060232 | 10 September 1998 |
Lactococcus lactis subsp. lactis | CBA3619 | SAMN11843663 | 1 | 0.107586 | 16 August 2019 |
Lactococcus lactis subsp. lactis | FDAARGOS_865 | SAMN13450395 | 1 | 0.058335 | 15 December 2020 |
Lactococcus lactis subsp. lactis | FDAARGOS_887 | SAMN13450417 | 4 | 0.180362 | 15 December 2020 |
Lactococcus lactis subsp. lactis | FDAARGOS_866 | SAMN13450396 | 4 | 0.241108 | 15 December 2020 |
Lactococcus lactis subsp. lactis | FDAARGOS_1064 | SAMN16357233 | 4 | 0.112979 | 21 December 2020 |
Lactococcus lactis subsp. lactis | WiKim0098 | SAMN16788728 | 1 | 0.076987 | 18 January 2021 |
Lactococcus lactis subsp. lactis | A12 | SAMEA4005236 | 4 | 0.126727 | 23 July 2016 |
Lactococcus lactis subsp. lactis | 229 | SAMN04955249 | 5 | 0.165685 | 5 April 2017 |
Lactococcus lactis subsp. lactis | 275 | SAMN04955252 | 4 | 0.25801 | 5 April 2017 |
Lactococcus lactis subsp. lactis | UC063 | SAMN04956302 | 5 | 0.149078 | 5 April 2017 |
Lactococcus lactis subsp. lactis | UL8 | SAMN04956402 | 3 | 0.037067 | 10 April 2017 |
Lactococcus lactis subsp. lactis | 14B4 | SAMN08792430 | 1 | 0.0597 | 1 June 2018 |
Lactococcus lactis subsp. lactis | 184 | SAMN04955247 | 6 | 0.042416 | 11 January 2019 |
Lactococcus lactis subsp. lactis | C10 | SAMN04956267 | 3 | 0.056865 | 11 January 2019 |
Lactococcus lactis subsp. lactis | UC06 | SAMN04956292 | 6 | 0.155687 | 13 January 2019 |
Lactococcus lactis subsp. lactis | UC08 | SAMN04956293 | 5 | 0.166766 | 16 January 2019 |
Lactococcus lactis subsp. lactis | UC77 | SAMN04956303 | 5 | 0.178184 | 3 January 2019 |
Lactococcus lactis subsp. lactis | UC11 | SAMN04956294 | 6 | 0.156382 | 11 September 2019 |
Lactococcus lactis subsp. lactis | G121 | SAMN14943687 | 3 | 0.123307 | 7 September 2020 |
Lactococcus lactis subsp. lactis | 223 | SAMN09847869 | 6 | 0.05587 | 25 February 2021 |
Lactococcus lactis subsp. lactis | WM1 | SAMN09847649 | 5 | 0.180672 | 23 February 2021 |
Lactococcus lactis subsp. lactis | 267 | SAMN09847870 | 5 | 0.164327 | 27 February 2021 |
Lactococcus lactis subsp. lactis | DRC3 | SAMN16604567 | 7 | 0.232822 | 10 April 2021 |
Lactococcus lactis subsp. lactis | Lac3 | SAMN18740314 | 1 | 0.007367 | 13 May 2021 |
Lactococcus lactis subsp. lactis bv. diacetylactis | FM03 | SAMN06061939 | 7 | 0.0809 | 24 May 2017 |
Lactococcus lactis subsp. lactis bv. diacetylactis | SD96 | SAMN12502795 | 10 | 0.219931 | 7 October 2019 |
Lactococcus lactis subsp. lactis bv. diacetylactis | BGBU1-4 | SAMN12627231 | 1 | 0.00633 | 17 November 2019 |
Lactococcus lactis subsp. lactis bv. diacetylactis | S50 | SAMN10167144 | 6 | 0.240412 | 10 September 2020 |
Lactococcus lactis subsp. lactis bv. diacetylactis | No attributes | SAMN14226089 | 1 | 0.018977 | 1 August 2011 |
Lactococcus lactis subsp. lactis bv. diacetylactis | No attributes | SAMN14226088 | 1 | 0.021728 | 1 August 2011 |
Lactococcus lactis subsp. lactis bv. diacetylactis | No attributes | SAMN14226087 | 1 | 0.022166 | 1 August 2011 |
Lactococcus lactis subsp. lactis bv. diacetylactis | No attributes | SAMN14226080 | 1 | 0.053876 | 1 August 2011 |
Lactococcus lactis subsp. lactis | CV56 | SAMN02603398 | 5 | 0.119279 | 1 May 2012 |
Lactococcus lactis subsp. lactis | KF147 | SAMN02603087 | 1 | 0.03751 | 22 December 2009 |
Lactococcus lactis subsp. lactis | KLDS 4.0325 | SAMN02603468 | 6 | 0.171622 | 10 May 2018 |
Lactococcus lactis subsp. lactis | NCDO 2118 | SAMN02471376 | 1 | 0.037571 | 21 January 2015 |
Average | 3.66 | 0.031197 |
Prophage | Start | End | Size (bp) | Att Core Sequence | G+C Content | Status |
---|---|---|---|---|---|---|
LLN8-1 (chromosome) | 1,369,374 | 1,405,566 | 36,193 | TTTAATTTAGAAA | 35.27% | intact |
LLN8-2 (chromosome) | 1,949,930 | 1,988,317 | 38,388 | AACGTAACTAAAAACGTAACTAA | 35.18% | intact |
LLN8-3 (chromosome) | 2,128,037 | 2,162,403 | 34,367 | AACTTATTTTTAT | 34.31% | incomplete |
LLN8-4 (chromosome) | 2,323,015 | 2,342,870 | 19,856 | ACGCTTTTTACTACGTTCG | 34.56% | incomplete |
LLN8-5 (plasmid1) | 40,333 | 69,929 | 29,597 | AAAATAAAAAGT | 32.05% | incomplete |
LLN8-6 (plasmid2) | 8658 | 53,552 | 44,895 | TTTCGAACATTT | 36.09% | questionable |
LLN8-7 (plasmid2) | 53,728 | 69,125 | 15,398 | AGGTTCTGTTGCAAAGTT | 35.32% | questionable |
Genomic Island | Start | End | Size (bp) | G+C Content % |
---|---|---|---|---|
GI001 (chromosome) | 583,454 | 626,486 | 43,032 | 30.63 |
GI002 (chromosome) | 1,188,697 | 1,213,410 | 24,713 | 32.54 |
GI003 (chromosome) | 1,431,036 | 1,435,913 | 4877 | 39.57 |
GI004 (chromosome) | 1,560,579 | 1,604,865 | 44,286 | 35.98 |
GI005 (chromosome) | 1,622,309 | 1,629,361 | 7052 | 30.91 |
GI006 (chromosome) | 1,950,028 | 1,955,065 | 5037 | 30.06 |
GI007 (chromosome) | 1,976,159 | 1,989,281 | 13,122 | 33.38 |
GI008 (chromosome) | 2,322,308 | 2,341,772 | 19,464 | 34.55 |
GI009 (plasmid 1) | 56,384 | 60,867 | 4483 | 37.20 |
GI010 (plasmid 1) | 58,996 | 67,736 | 8740 | 33.58 |
GI011 (plasmid 2) | 15,929 | 22,625 | 6696 | 36.81 |
GI012 (plasmid 2) | 55,774 | 62,660 | 6886 | 32.64 |
L. lactis | Removed (bp) | Cumulative (bp) | Deletion (%) | μmax (h−1) | Generation Time (min) |
---|---|---|---|---|---|
N8 | 0 | 0 | 0% | 0.44 ± 0.04 | 45.7 ± 2.7 |
N8-1 | 19,739 | 19,739 | 0.77% | 0.48 ± 0.05 | 41.5 ± 5.0 |
N8-2 | 1638 | 21,377 | 0.83% | 0.49 ± 0.06 | 42.7 ± 4.1 |
N8-3 | 18,628 | 40,005 | 1.55% | 0.50 ± 0.03 | 40.7 ± 5.0 |
N8-4 | 10,659 | 50,664 | 1.97% | 0.47 ± 0.03 | 40.0 ± 5.1 |
N8-5 | 13,502 | 64,166 | 2.49% | 0.44 ± 0.02 | 41.3 ± 4.9 |
N8-6 | 39,203 | 103,369 | 4.02% | 0.53 ± 0.09 | 41.1 ± 3.2 |
N8-7 | 58,594 | 161,963 | 6.29% | 0.52 ± 0.03 | 39.3 ± 5.2 |
N8-8 | 14,465 | 176,428 | 6.86% | 0.50 ± 0.05 | 39.0 ± 4.2 |
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Qiao, W.; Liu, F.; Wan, X.; Qiao, Y.; Li, R.; Wu, Z.; Saris, P.E.J.; Xu, H.; Qiao, M. Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8. Microorganisms 2022, 10, 47. https://doi.org/10.3390/microorganisms10010047
Qiao W, Liu F, Wan X, Qiao Y, Li R, Wu Z, Saris PEJ, Xu H, Qiao M. Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8. Microorganisms. 2022; 10(1):47. https://doi.org/10.3390/microorganisms10010047
Chicago/Turabian StyleQiao, Wanjin, Fulu Liu, Xing Wan, Yu Qiao, Ran Li, Zhenzhou Wu, Per Erik Joakim Saris, Haijin Xu, and Mingqiang Qiao. 2022. "Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8" Microorganisms 10, no. 1: 47. https://doi.org/10.3390/microorganisms10010047
APA StyleQiao, W., Liu, F., Wan, X., Qiao, Y., Li, R., Wu, Z., Saris, P. E. J., Xu, H., & Qiao, M. (2022). Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8. Microorganisms, 10(1), 47. https://doi.org/10.3390/microorganisms10010047