{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:06:01Z","timestamp":1760148361735,"version":"build-2065373602"},"reference-count":41,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2023,4,24]],"date-time":"2023-04-24T00:00:00Z","timestamp":1682294400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key R&amp;D Program of China","award":["2022YFC2304003","82172317","82102412","SKLPB2208"],"award-info":[{"award-number":["2022YFC2304003","82172317","82102412","SKLPB2208"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["2022YFC2304003","82172317","82102412","SKLPB2208"],"award-info":[{"award-number":["2022YFC2304003","82172317","82102412","SKLPB2208"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Foundation of State Key Laboratory of Pathogens and Biosecurity","award":["2022YFC2304003","82172317","82102412","SKLPB2208"],"award-info":[{"award-number":["2022YFC2304003","82172317","82102412","SKLPB2208"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Bacillus anthracis is a Gram-positive bacterium that causes the zoonotic disease anthrax. Here, we studied the characteristic phenotype and virulence attenuation of the putative No. II vaccine strain, PNO2, which was reportedly introduced from the Pasteur Institute in 1934. Characterization of the strain showed that, compared with the control strain, A16Q1, the attenuated PNO2 (PNO2D1) was phospholipase-positive, with impaired protein hydrolysis and significantly reduced sporulation. Additionally, PNO2D1 significantly extended the survival times of anthrax-challenged mice. An evolutionary tree analysis revealed that PNO2D1 was not a Pasteur strain but was more closely related to a Tsiankovskii strain. A database comparison revealed a seven-base insertion mutation in the nprR gene. Although it did not block nprR transcription, the insertion mutation resulted in the premature termination of protein translation. nprR deletion of A16Q1 resulted in a nonproteolytic phenotype that could not sporulate. The database comparison revealed that the abs gene is also prone to mutation, and the abs promoter activity was much lower in PNO2D1 than in A16Q1. Low abs expression may be an important reason for the decreased virulence of PNO2D1.<\/jats:p>","DOI":"10.3390\/biology12050645","type":"journal-article","created":{"date-parts":[[2023,4,25]],"date-time":"2023-04-25T02:06:35Z","timestamp":1682388395000},"page":"645","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Genome Sequence and Phenotypic Analysis of a Protein Lysis-Negative, Attenuated Anthrax Vaccine Strain"],"prefix":"10.3390","volume":"12","author":[{"given":"Lu","family":"Yuan","sequence":"first","affiliation":[{"name":"College of Food Science and Technology, Shanghai Ocean University, 999 Hucheng Huan Road, Nanhui New City, Shanghai 201306, China"},{"name":"State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7077-1992","authenticated-orcid":false,"given":"Dongshu","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"},{"name":"Laboratory of Advanced Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jie","family":"Chen","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Shanghai Ocean University, 999 Hucheng Huan Road, Nanhui New City, Shanghai 201306, China"},{"name":"State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yufei","family":"Lyu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"},{"name":"Laboratory of Advanced Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Erling","family":"Feng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yan","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Shanghai Ocean University, 999 Hucheng Huan Road, Nanhui New City, Shanghai 201306, China"},{"name":"State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiankai","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"},{"name":"Laboratory of Advanced Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8476-4320","authenticated-orcid":false,"given":"Hengliang","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Shanghai Ocean University, 999 Hucheng Huan Road, Nanhui New City, Shanghai 201306, China"},{"name":"State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1146\/annurev.micro.55.1.647","article-title":"Anthrax","volume":"55","author":"Mock","year":"2001","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1128\/iai.39.1.371-376.1983","article-title":"Evidence for plasmid-mediated toxin production in Bacillus anthracis","volume":"39","author":"Mikesell","year":"1983","journal-title":"Infect. Immun."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1128\/iai.49.2.291-297.1985","article-title":"Demonstration of a capsule plasmid in Bacillus anthracis","volume":"49","author":"Green","year":"1985","journal-title":"Infect. Immun."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1128\/IAI.74.1.469-480.2006","article-title":"Roles of Macrophages and Neutrophils in the Early Host Response to Bacillus anthracis Spores in a Mouse Model of Infection","volume":"74","author":"Cote","year":"2006","journal-title":"Infect. Immun."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2237","DOI":"10.1007\/s00018-006-6312-3","article-title":"Anthrax vaccines: Pasteur to the present","volume":"63","author":"Scorpio","year":"2006","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"506","DOI":"10.1080\/21505594.2016.1186325","article-title":"Pasteur revisited: An unexpected finding in Bacillus anthracis vaccine strains","volume":"7","author":"Thorkildson","year":"2016","journal-title":"Virulence"},{"key":"ref_7","first-page":"557","article-title":"Virulence and Immunogenicity in Experimental Animals of Bacillus anthracis Strains Harbouring or Lacking 110 MDa and 60 MDa Plasmids","volume":"132","author":"Uchida","year":"1986","journal-title":"J. Gen. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1128\/iai.52.2.454-458.1986","article-title":"Immunization studies with attenuated strains of Bacillus anthracis","volume":"52","author":"Ivins","year":"1986","journal-title":"Infect. Immun."},{"key":"ref_9","first-page":"33","article-title":"Anthrax Vaccines","volume":"271","author":"Friedlander","year":"2002","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"578","DOI":"10.1080\/21505594.2016.1164366","article-title":"Identification of the pXO1 plasmid in attenuated Bacillus anthracis vaccine strains","volume":"7","author":"Liang","year":"2016","journal-title":"Virulence"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1016\/0882-4010(91)90053-D","article-title":"Plasmid-associated virulence factors of non-toxigenic (pX01\u2212) Bacillus anthracis","volume":"10","author":"Welkos","year":"1991","journal-title":"Microb. Pathog."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1086\/341299","article-title":"Effect of the Lower Molecular Capsule Released from the Cell Surface of Bacillus anthracis on the Pathogenesis of Anthrax","volume":"186","author":"Makino","year":"2002","journal-title":"J. Infect. Dis."},{"key":"ref_13","first-page":"5","article-title":"Construction of Putative S-Layer Protein SLP Deletion Mutant in Bacillus anthracis","volume":"20","author":"Tang","year":"2009","journal-title":"Lett. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1006\/mpat.1993.1037","article-title":"Non-toxigenic derivatives of the Ames strain of Bacillus anthracis are fully virulent for mice: Role of plasmid pX02 and chromosome in strain-dependent virulence","volume":"14","author":"Welkos","year":"1993","journal-title":"Microb. Pathog."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1846","DOI":"10.1016\/S0264-410X(97)00132-1","article-title":"Expression of cereolysine AB genes in Bacillus anthracis vaccine strain ensures protection against experimental hemolytic anthrax infection","volume":"15","author":"Pomerantsev","year":"1997","journal-title":"Vaccine"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.jbiotec.2015.06.408","article-title":"Genome sequence of Bacillus anthracis attenuated vaccine strain A16R used for human in China","volume":"210","author":"Liu","year":"2015","journal-title":"J. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"648","DOI":"10.1046\/j.1365-2672.2000.01005.x","article-title":"Characterization of Bacillus anthracis strains used for vaccination","volume":"88","author":"Cataldi","year":"2000","journal-title":"J. Appl. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1046\/j.1365-2672.2002.01660.x","article-title":"Sequence analysis of the genes encoding for the major virulence factors of Bacillus anthracis vaccine strain \u2018Carbosap\u2019","volume":"93","author":"Adone","year":"2002","journal-title":"J. Appl. Microbiol."},{"key":"ref_19","first-page":"104890","article-title":"Phylogenetics of Bacillus anthracis isolates from Russia and bordering countries","volume":"92","author":"Eremenko","year":"2021","journal-title":"Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Liu, X., Wang, D., Wang, H., Feng, E., Zhu, L., and Wang, H. (2012). Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid Incompatibility. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0029875"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"590851","DOI":"10.3389\/fmicb.2020.590851","article-title":"Integrated Transcriptomic and Proteomic Analyses Reveal the Role of NprR in Bacillus anthracis Extracellular Protease Expression Regulation and Oxidative Stress Responses","volume":"11","author":"Wang","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/j.mimet.2018.05.024","article-title":"pheS* as a counter-selectable marker for marker-free genetic manipulations in Bacillus anthracis","volume":"151","author":"Wang","year":"2018","journal-title":"J. Microbiol. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"536357","DOI":"10.3389\/fmicb.2021.536357","article-title":"Application of CRISPR\/Cas9 System for Plasmid Elimination and Bacterial Killing of Bacillus cereus Group Strains","volume":"12","author":"Wang","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1932","DOI":"10.3389\/fmicb.2019.01932","article-title":"Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR\/Cas9 System","volume":"10","author":"Wang","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5421","DOI":"10.1128\/AEM.01453-16","article-title":"Editing of the Bacillus subtilis genome by the CRISPR-Cas9 system","volume":"82","author":"Altenbuchner","year":"2011","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1111\/j.1365-2958.1994.tb00405.x","article-title":"Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis","volume":"13","author":"Agaisse","year":"1994","journal-title":"Mol. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"126438","DOI":"10.1016\/j.micres.2020.126438","article-title":"Transcription in the acetoin catabolic pathway is regulated by AcoR and CcpA in Bacillus thuringiensis","volume":"235","author":"Peng","year":"2020","journal-title":"Microbiol. Res."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Chen, M., Lyu, Y., Feng, E., Zhu, L., Pan, C., Wang, D., Liu, X., and Wang, H. (2020). SpoVG is Necessary for Sporulation in Bacillus anthracis. Microorganisms, 8.","DOI":"10.3390\/microorganisms8040548"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1093\/infdis\/38.4.341","article-title":"Studies on Anthrax Immunity: I. The Attentuation of Bacillus anthracis by means of Sodium Chloride and other Chemicals","volume":"38","author":"Schilling","year":"1926","journal-title":"J. Infect. Dis."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1276","DOI":"10.1111\/j.1365-2958.2011.07519.x","article-title":"Papillation in Bacillus anthracis colonies: A tool for finding new mutators","volume":"79","author":"Yang","year":"2011","journal-title":"Mol. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"126414","DOI":"10.1016\/j.micres.2020.126414","article-title":"Genotyping and population diversity of Bacillus anthracis in China based on MLVA and canSNP analysis","volume":"233","author":"Wang","year":"2020","journal-title":"Microbiol. Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1038\/nmeth.2474","article-title":"Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data","volume":"10","author":"Chin","year":"2013","journal-title":"Nat. Methods"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"722","DOI":"10.1101\/gr.215087.116","article-title":"Canu: Scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation","volume":"27","author":"Koren","year":"2017","journal-title":"Genome Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"D1020","DOI":"10.1093\/nar\/gkaa1105","article-title":"RefSeq: Expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation","volume":"49","author":"Li","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"524","DOI":"10.1186\/s13059-014-0524-x","article-title":"The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes","volume":"15","author":"Treangen","year":"2014","journal-title":"Genome Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"3022","DOI":"10.1093\/molbev\/msab120","article-title":"MEGA11: Molecular Evolutionary Genetics Analysis Version 11","volume":"38","author":"Tamura","year":"2021","journal-title":"Mol. Biol. Evol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1073","DOI":"10.1136\/bmj.328.7447.1073","article-title":"The logrank test","volume":"328","author":"Bland","year":"2004","journal-title":"BMJ"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1016\/j.mimet.2011.05.006","article-title":"Reference genes for quantitative, reverse-transcription PCR in Bacillus cereus group strains throughout the bacterial life cycle","volume":"86","author":"Reiter","year":"2011","journal-title":"J. Microbiol. Methods"},{"key":"ref_39","unstructured":"Miller, J.H. (1972). Experiments in Molecular Genetics, Cold Spring Harbor Laboratory."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1046\/j.1365-2958.2003.03861.x","article-title":"Bacillus anthracis requires siderophore biosynthesis for growth in macrophages and mouse virulence","volume":"51","author":"Cendrowski","year":"2004","journal-title":"Mol. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e02079-18","DOI":"10.1128\/mBio.02079-18","article-title":"Petrobactin Protects against Oxidative Stress and Enhances Sporulation Efficiency in Bacillus anthracis Sterne","volume":"9","author":"Hagan","year":"2018","journal-title":"mBio"}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/12\/5\/645\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:22:38Z","timestamp":1760124158000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/12\/5\/645"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,4,24]]},"references-count":41,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2023,5]]}},"alternative-id":["biology12050645"],"URL":"https:\/\/doi.org\/10.3390\/biology12050645","relation":{},"ISSN":["2079-7737"],"issn-type":[{"type":"electronic","value":"2079-7737"}],"subject":[],"published":{"date-parts":[[2023,4,24]]}}}