Simple SummaryThe whole-genome sequencing of a putative No. II vaccine strain PNO2 with specific phenotypes was completed. The PNO2 strain is more like a Tsiankovskii strain than a Pasteur II strain. The inactivation ofnprRgene resulted in the nonproteolytic phenotype of PNO2 and attenuated sporulation. ThenprRwas then found to be required forBacillus anthracissporulation. Further, inactivation or low expression ofabsgenes may be an important cause of vaccine strain virulence attenuation.AbstractBacillus anthracisis 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 thenprRgene. Although it did not blocknprRtranscription, the insertion mutation resulted in the premature termination of protein translation.nprRdeletion of A16Q1 resulted in a nonproteolytic phenotype that could not sporulate. The database comparison revealed that theabsgene is also prone to mutation, and theabspromoter activity was much lower in PNO2D1 than in A16Q1. Lowabsexpression may be an important reason for the decreased virulence of PNO2D1.