Genomic Analysis of Natural Rough Brucella melitensis Rev.1 Vaccine Strains: Identification and Characterization of Mutations in Key Genes Associated with Bacterial LPS Biosynthesis and Virulence
Abstract
:1. Introduction
2. Results and Discussion
2.1. Genomic SNPs
2.2. Characterization of the Specific SNPs of the Natural Rough B. melitensis Rev.1 71036 Vaccine Strain
2.2.1. Genes Involved in LPS Biosynthesis
Phosphomannomutase (BMEII0899)
ABC Transporter Permease (BMEI1416)
2.2.2. Genes Involved in Survival within Environmental Stress Conditions
Glutathione S-Transferase Family Protein (BMEII0256)
NAD(P) Transhydrogenase Subunit Alpha (BMEII0323)
Magnesium Transporter (BMEII0418)
MFS Efflux Pump (BMEI0267)
2.3. Characterization of the Specific SNPs in Genes Involved in LPS Biosynthesis of the Natural Rough B. melitensis Rev.1 44457 Vaccine Strain
2.3.1. Genes Involved in LPS Biosynthesis
Phosphomannomutase (BMEII0899)
GDP-Mannose Dehydratase (BMEI1413)
2.4. In Vitro Acidic and Oxidative Stress Survival of the Natural Rough B. melitensis Rev.1 71036 and 44457 Vaccine Strains
2.5. Confirmation of the Absence of Surface O-PS in the Natural Rough B. melitensis Rev.1 71036 and 44457 Vaccine Strains
3. Materials and Methods
3.1. Strain Information, Extraction of the Genomic DNA, and Sequencing
3.2. SNP Detection
3.3. Validation by Sanger Sequencing
3.4. Structural Analysis
3.4.1. Motif Pattern and Domain Identification
3.4.2. 3D Protein Structure Analysis
3.4.3. Multiple Sequence Alignment
3.4.4. Conservation Analysis
3.4.5. Acid and Oxidative Stress Assays
3.4.6. Whole Cell Immunoassay for the Detection of Surface Brucella OPS
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Von Bargen, K.; Gorvel, J.-P.; Salcedo, S.P. Internal affairs: Investigating the Brucella intracellular lifestyle. FEMS Microbiol. Rev. 2012, 36, 533–562. [Google Scholar] [CrossRef] [Green Version]
- Haag, A.F.; Myka, K.K.; Arnold, M.F.F.; Caro-Hernández, P.; Ferguson, G.P. Importance of lipopolysaccharide and cyclic β-1,2-glucans in Brucella-mammalian infections. Int. J. Microbiol. 2010, 2010, 124509. [Google Scholar] [CrossRef] [Green Version]
- Adone, R.; Muscillo, M.; La Rosa, G.; Francia, M.; Tarantino, M. Antigenic, immunologic and genetic characterization of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18. PLoS ONE 2011, 6, e24073. [Google Scholar] [CrossRef] [Green Version]
- Mancilla, M. Smooth to Rough Dissociation in Brucella: The Missing Link to Virulence. Front. Cell. Infect. Microbiol. 2015, 5, 98. [Google Scholar] [CrossRef] [PubMed]
- Cardoso, P.G.; Macedo, G.C.; Azevedo, V.; Oliveira, S.C. Brucella spp noncanonical LPS: Structure, biosynthesis, and interaction with host immune system. Microb. Cell Fact. 2006, 5, 13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Adone, R.; Francia, M.; Ciuchini, F. Evaluation of Brucella melitensis B115 as rough-phenotype vaccine against B. melitensis and B. ovis infections. Vaccine 2008, 26, 4913–4917. [Google Scholar] [CrossRef]
- Monreal, D.; Grilló, M.J.; González, D.; Marín, C.M.; De Miguel, M.J.; López-Goñi, I.; Blasco, J.M.; Cloeckaert, A.; Moriyón, I. Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model. Infect. Immun. 2003, 71, 3261–3271. [Google Scholar] [CrossRef] [Green Version]
- Billard, E.; Dornand, J.; Gross, A. Interaction of Brucella suis and Brucella abortus rough strains with human dendritic cells. Infect. Immun. 2007, 75, 5916–5923. [Google Scholar] [CrossRef] [Green Version]
- Lapaque, N.; Moriyon, I.; Moreno, E.; Gorvel, J.P. Brucella lipopolysaccharide acts as a virulence factor. Curr. Opin. Microbiol. 2005, 8, 60–66. [Google Scholar] [CrossRef]
- Kianmehr, Z.; Ardestani, S.K.; Soleimanjahi, H.; Fotouhi, F.; Alamian, S.; Ahmadian, S. Comparison of biological and immunological characterization of lipopolysaccharides from Brucella abortus RB51 and S19. Jundishapur J. Microbiol. 2015, 8. [Google Scholar] [CrossRef] [Green Version]
- Poester, F.P.; Samartino, L.E.; Santos, R.L. Pathogenesis and pathobiology of brucellosis in livestock. Rev. Sci. Tech. 2013, 32, 105–115. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Avila-Calderón, E.D.; Lopez-Merino, A.; Sriranganathan, N.; Boyle, S.M.; Contreras-Rodríguez, A. A history of the development of Brucella vaccines. BioMed Res. Int. 2013, 2013, 743509. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Herzberg, M.; Elberg, S. Immunization against Brucella infection. I. Isolation and characterization of a streptomycin-dependent mutant. J. Bacteriol. 1953, 66, 585–599. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Banai, M. Control of small ruminant brucellosis by use of Brucella melitensis Rev.1 vaccine: Laboratory aspects and field observations. Vet. Microbiol. 2002, 90, 497–519. [Google Scholar] [CrossRef]
- González, D.; Grilló, M.-J.; De Miguel, M.-J.; Ali, T.; Arce-Gorvel, V.; Delrue, R.-M.; Conde-Alvarez, R.; Muñoz, P.; López-Goñi, I.; Iriarte, M.; et al. Brucellosis vaccines: Assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export. PLoS ONE 2008, 3, e2760. [Google Scholar] [CrossRef] [PubMed]
- Moriyón, I.; Grilló, M.J.; Monreal, D.; González, D.; Marín, C.; López-Goñi, I.; Mainar-Jaime, R.C.; Moreno, E.; Blasco, J.M. Rough vaccines in animal brucellosis: Structural and genetic basis and present status. Vet. Res. 2004, 35, 1–38. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Salmon-Divon, M.; Kornspan, D. Transcriptomic analysis of smooth versus rough Brucella melitensis Rev.1 vaccine strains reveals insights into virulence attenuation. Int. J. Med. Microbiol. 2020, 310, 151363. [Google Scholar] [CrossRef]
- Chuang, G.Y.; Mehra-Chaudhary, R.; Ngan, C.H.; Zerbe, B.S.; Kozakov, D.; Vajda, S.; Beamer, L.J. Domain motion and interdomain hot spots in a multidomain enzyme. Protein Sci. 2010, 19, 1662–1672. [Google Scholar] [CrossRef] [Green Version]
- Mitchell, A.L.; Attwood, T.K.; Babbitt, P.C.; Blum, M.; Bork, P.; Bridge, A.; Brown, S.D.; Chang, H.Y.; El-Gebali, S.; Fraser, M.I.; et al. InterPro in 2019: Improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res. 2019, 47, D351–D360. [Google Scholar] [CrossRef] [Green Version]
- Godfroid, F.; Cloeckaert, A.; Taminiau, B.; Danese, I.; Tibor, A.; De Bolle, X.; Mertens, P.; Letesson, J.J. Genetic organisation of the lipopolysaccharide O-antigen biosynthesis region of Brucella melitensis 16M (wbk). Res. Microbiol. 2000, 151, 655–668. [Google Scholar] [CrossRef]
- Anderson, T.D.; Cheville, N.F. Ultrastructural morphometric analysis of Brucella abortus-infected trophoblasts in experimental placentitis. Bacterial replication occurs in rough endoplasmic reticulum. Am. J. Pathol. 1986, 124, 226–237. [Google Scholar] [PubMed]
- Celli, J.; de Chastellier, C.; Franchini, D.-M.; Pizarro-Cerda, J.; Moreno, E.; Gorvel, J.-P. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J. Exp. Med. 2003, 198, 545–556. [Google Scholar] [CrossRef]
- Salcedo, S.P.; Chevrier, N.; Lacerda, T.L.S.; Ben Amara, A.; Gerart, S.; Gorvel, V.A.; de Chastellier, C.; Blasco, J.M.; Mege, J.-L.; Gorvel, J.-P. Pathogenic brucellae replicate in human trophoblasts. J. Infect. Dis. 2013, 207, 1075–1083. [Google Scholar] [CrossRef]
- Teixeira-Gomes, A.P.; Cloeckaert, A.; Zygmunt, M.S. Characterization of heat, oxidative, and acid stress responses in Brucella melitensis. Infect. Immun. 2000, 68, 2954–2961. [Google Scholar] [CrossRef] [Green Version]
- Hayes, J.D.; McLellan, L.I. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic. Res. 1999, 31, 273–300. [Google Scholar] [CrossRef] [PubMed]
- Vuilleumier, S. Bacterial glutathione S-transferases: What are they good for? J. Bacteriol. 1997, 179, 1431–1441. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Allocati, N.; Favaloro, B.; Masulli, M.; Alexeyev, M.F.; Di Ilio, C. Proteus mirabilis glutathione S-transferase B1-1 is involved in protective mechanisms against oxidative and chemical stresses. Biochem. J. 2003, 373, 305–311. [Google Scholar] [CrossRef] [Green Version]
- Jackson, J.B. Proton translocation by transhydrogenase. FEBS Lett. 2003, 545, 18–24. [Google Scholar] [CrossRef] [Green Version]
- Yan, J.; Ralston, M.M.; Meng, X.; Bongiovanni, K.D.; Jones, A.L.; Benndorf, R.; Nelin, L.D.; Joshua Frazier, W.; Rogers, L.K.; Smith, C.V.; et al. Glutathione reductase is essential for host defense against bacterial infection. Free Radic. Biol. Med. 2013, 61, 320–332. [Google Scholar] [CrossRef] [Green Version]
- Hattori, M.; Iwase, N.; Furuya, N.; Tanaka, Y.; Tsukazaki, T.; Ishitani, R.; Maguire, M.E.; Ito, K.; Maturana, A.; Nureki, O. Mg 2-dependent gating of bacterial MgtE channel underlies Mg 2 homeostasis. EMBO J. 2009, 28, 3602–3612. [Google Scholar] [CrossRef] [Green Version]
- Groisman, E.A.; Hollands, K.; Kriner, M.A.; Lee, E.J.; Park, S.Y.; Pontes, M.H. Bacterial Mg2+ homeostasis, transport, and virulence. Annu. Rev. Genet. 2013, 47, 625–646. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Romani, A.M.; Scarpa, A. Regulation of cellular magnesium. Front. Biosci. 2000, 5, D720–D734. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- García Véscovi, E.; Soncini, F.C.; Groisman, E.A. Mg2+ as an extracellular signal: Environmental regulation of Salmonella virulence. Cell 1996, 84, 165–174. [Google Scholar] [CrossRef] [Green Version]
- Smith, R.L.; Maguire, M.E. Microbial magnesium transport: Unusual transporters searching for identity. Mol. Microbiol. 1998, 28, 217–226. [Google Scholar] [CrossRef] [PubMed]
- Grabenstein, J.P.; Marceau, M.; Pujol, C.; Simonet, M.; Bliska, J.B. The response regulator PhoP of Yersinia pseudotuberculosis is important for replication in macrophages and for virulence. Infect. Immun. 2004, 72, 4973–4984. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Oyston, P.C.F.; Dorrell, N.; Williams, K.; Li, S.R.; Green, M.; Titball, R.W.; Wren, B.W. The response regulator PhoP is important for survival under conditions of macrophage-induced stress and virulence in Yersinia pestis. Infect. Immun. 2000, 68, 3419–3425. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ford, D.C.; Joshua, G.W.P.; Wren, B.W.; Oyston, P.C.F. The importance of the magnesium transporter MgtB for virulence of Yersinia pseudotuberculosis and Yersinia pestis. Microbiology 2014, 160, 2710–2717. [Google Scholar] [CrossRef] [PubMed]
- Alix, E.; Blanc-Potard, A.B. MgtC: A key player in intramacrophage survival. Trends Microbiol. 2007, 15, 252–256. [Google Scholar] [CrossRef]
- Lavigne, J.P.; O’Callaghan, D.; Blanc-Potard, A.B. Requirement of MgtC for Brucella suis intramacrophage growth: A potential mechanism shared by Salmonella enterica and Mycobacterium tuberculosis for adaptation to a low-Mg2+ environment. Infect. Immun. 2005, 73, 3160–3163. [Google Scholar] [CrossRef] [Green Version]
- Huang, Y.; Lemieux, M.J.; Song, J.; Auer, M.; Wang, D.N. Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science 2003, 301, 616–620. [Google Scholar] [CrossRef] [Green Version]
- Zhao, Y.; Heng, J.; Zhao, Y.; Liu, M.; Liu, Y.; Fan, J.; Wang, X.; Zhang, X.C. Substrate-bound structure of the E. coli multidrug resistance transporter MdfA. Cell Res. 2015, 25, 1060–1073. [Google Scholar] [CrossRef]
- Piddock, L.J.V. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin. Microbiol. Rev. 2006, 19, 382–402. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pasqua, M.; Grossi, M.; Scinicariello, S.; Aussel, L.; Barras, F.; Colonna, B.; Prosseda, G. The MFS efflux pump EmrKY contributes to the survival of Shigella within macrophages. Sci. Rep. 2019, 9, 2906. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Quillin, S.J.; Schwartz, K.T.; Leber, J.H. The novel Listeria monocytogenes bile sensor BrtA controls expression of the cholic acid efflux pump MdrT. Mol. Microbiol. 2011, 81, 129–142. [Google Scholar] [CrossRef]
- Bina, X.R.; Provenzano, D.; Nguyen, N.; Bina, J.E. Vibrio cholerae RND family efflux systems are required for antimicrobial resistance, optimal virulence factor production, and colonization of the infant mouse small intestine. Infect. Immun. 2008, 76, 3595–3605. [Google Scholar] [CrossRef] [Green Version]
- Wang-Kan, X.; Blair, J.M.A.; Chirullo, B.; Betts, J.; La Ragione, R.M.; Ivens, A.; Ricci, V.; Opperman, T.J.; Piddock, L.J.V. Lack of AcrB efflux function confers loss of virulence on Salmonella enterica serovar typhimurium. MBio 2017, 8. [Google Scholar] [CrossRef] [Green Version]
- Zygmunt, M.S.; Blasco, J.M.; Letesson, J.J.; Cloeckaert, A.; Moriyn, I. DNA polymorphism analysis of Brucella lipopolysaccharide genes reveals marked differences in O-polysaccharide biosynthetic genes between smooth and rough Brucella species and novel species-specific markers. BMC Microbiol. 2009, 9, 92. [Google Scholar] [CrossRef] [Green Version]
- Allen, C.A.; Adams, L.G.; Ficht, T.A. Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival. Infect. Immun. 1998, 66, 1008–1016. [Google Scholar] [CrossRef] [Green Version]
- Andrews, S.; Krueger, F.; Seconds-Pichon, A.; Biggins, F.; Wingett, S. FastQC 0.10.1. A Quality Control Tool for High Throughput Sequence Data. Babraham Bioinformatics. Available online: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/%0Ahttp://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/ (accessed on 7 December 2020).
- Andrews, S.; Krueger, F.; Segonds-Pichon, A.; Biggins, L.; Krueger, C.; Wingett, S. Trim Galore 0.65. 2012. Available online: http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ (accessed on 7 December 2020).
- Langmead, B.; Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 2012, 9, 357–359. [Google Scholar] [CrossRef] [Green Version]
- Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G.; Abecasis, G.; Durbin, R. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009, 25, 2078–2079. [Google Scholar] [CrossRef] [Green Version]
- Cingolani, P.; Platts, A.; Wang, L.L.; Coon, M.; Nguyen, T.; Wang, L.; Land, S.J.; Lu, X.; Ruden, D.M. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 2012, 6, 80–92. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- DelVecchio, V.G.; Kapatral, V.; Redkar, R.J.; Patra, G.; Mujer, C.; Los, T.; Ivanova, N.; Anderson, I.; Bhattacharyya, A.; Lykidis, A.; et al. The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc. Natl. Acad. Sci. USA 2002, 99, 443–448. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Salmon-Divon, M.; Yeheskel, A.; Kornspan, D. Genomic analysis of the original Elberg Brucella melitensis Rev.1 vaccine strain reveals insights into virulence attenuation. Virulence 2018, 9, 1436–1448. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Salmon-Divon, M.; Banai, M.; Bardenstein, S.; Blum, S.E.; Kornspan, D. Complete genome sequence of the live attenuated vaccine strain Brucella melitensis Rev.1. Genome Announc. 2018, 6. [Google Scholar] [CrossRef] [Green Version]
- Untergasser, A.; Cutcutache, I.; Koressaar, T.; Ye, J.; Faircloth, B.C.; Remm, M.; Rozen, S.G. Primer3-new capabilities and interfaces. Nucleic Acids Res. 2012, 40, e115. [Google Scholar] [CrossRef] [Green Version]
- Kent, W.J.; Sugnet, C.W.; Furey, T.S.; Roskin, K.M.; Pringle, T.H.; Zahler, A.M.; Haussler, A.D. The Human Genome Browser at UCSC. Genome Res. 2002, 12, 996–1006. [Google Scholar] [CrossRef] [Green Version]
- SerialBasics Serial Cloner 2.6. Available online: http://serialbasics.free.fr/Serial_Cloner.html (accessed on 7 December 2020).
- Di Tommaso, P.; Moretti, S.; Xenarios, I.; Orobitg, M.; Montanyola, A.; Chang, J.M.; Taly, J.F.; Notredame, C. T-Coffee: A web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 2011, 39, W13. [Google Scholar] [CrossRef]
- Albà, M. Making alignments prettier. Genome Biol. 2000, 1, reports2047. [Google Scholar] [CrossRef]
- Sigrist, C.J.A.; de Castro, E.; Cerutti, L.; Cuche, B.A.; Hulo, N.; Bridge, A.; Bougueleret, L.; Xenarios, I. New and continuing developments at PROSITE. Nucleic Acids Res. 2013, 41, D344–D347. [Google Scholar] [CrossRef] [Green Version]
- El-Gebali, S.; Mistry, J.; Bateman, A.; Eddy, S.R.; Luciani, A.; Potter, S.C.; Qureshi, M.; Richardson, L.J.; Salazar, G.A.; Smart, A.; et al. The Pfam protein families database in 2019. Nucleic Acids Res. 2019, 47, D427–D432. [Google Scholar] [CrossRef]
- Waterhouse, A.; Bertoni, M.; Bienert, S.; Studer, G.; Tauriello, G.; Gumienny, R.; Heer, F.T.; De Beer, T.A.P.; Rempfer, C.; Bordoli, L.; et al. SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Res. 2018, 46, W296–W303. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zimmermann, L.; Stephens, A.; Nam, S.Z.; Rau, D.; Kübler, J.; Lozajic, M.; Gabler, F.; Söding, J.; Lupas, A.N.; Alva, V. A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J. Mol. Biol. 2018, 430, 2237–2243. [Google Scholar] [CrossRef] [PubMed]
- Webb, B.; Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinform. 2016, 54, 5.6.1–5.6.37. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
- Katoh, K.; Rozewicki, J.; Yamada, K.D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 2018, 20, 1160–1166. [Google Scholar] [CrossRef] [Green Version]
- Ashkenazy, H.; Abadi, S.; Martz, E.; Chay, O.; Mayrose, I.; Pupko, T.; Ben-Tal, N. ConSurf 2016: An improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 2016, 44, W344–W350. [Google Scholar] [CrossRef] [Green Version]
- Hans, R.; Yadav, P.K.; Sharma, P.K.; Boopathi, M.; Thavaselvam, D. Development and validation of immunoassay for whole cell detection of Brucella abortus and Brucella melitensis. Sci. Rep. 2020, 10, 8543. [Google Scholar] [CrossRef]
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Kornspan, D.; Lubkovskaia, R.; Mathur, S.; Yeheskel, A.; Salmon-Divon, M. Genomic Analysis of Natural Rough Brucella melitensis Rev.1 Vaccine Strains: Identification and Characterization of Mutations in Key Genes Associated with Bacterial LPS Biosynthesis and Virulence. Int. J. Mol. Sci. 2020, 21, 9341. https://doi.org/10.3390/ijms21249341
Kornspan D, Lubkovskaia R, Mathur S, Yeheskel A, Salmon-Divon M. Genomic Analysis of Natural Rough Brucella melitensis Rev.1 Vaccine Strains: Identification and Characterization of Mutations in Key Genes Associated with Bacterial LPS Biosynthesis and Virulence. International Journal of Molecular Sciences. 2020; 21(24):9341. https://doi.org/10.3390/ijms21249341
Chicago/Turabian StyleKornspan, David, Regina Lubkovskaia, Shubham Mathur, Adva Yeheskel, and Mali Salmon-Divon. 2020. "Genomic Analysis of Natural Rough Brucella melitensis Rev.1 Vaccine Strains: Identification and Characterization of Mutations in Key Genes Associated with Bacterial LPS Biosynthesis and Virulence" International Journal of Molecular Sciences 21, no. 24: 9341. https://doi.org/10.3390/ijms21249341
APA StyleKornspan, D., Lubkovskaia, R., Mathur, S., Yeheskel, A., & Salmon-Divon, M. (2020). Genomic Analysis of Natural Rough Brucella melitensis Rev.1 Vaccine Strains: Identification and Characterization of Mutations in Key Genes Associated with Bacterial LPS Biosynthesis and Virulence. International Journal of Molecular Sciences, 21(24), 9341. https://doi.org/10.3390/ijms21249341