Genome Sequence and Phenotypic Analysis of a Protein Lysis-Negative, Attenuated Anthrax Vaccine Strain

SIMPLE SUMMARY: The 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 of nprR gene resulted in the nonproteolytic phenotype of PNO2 and attenuated sporulation. The nprR was then found to be required for Bacillus anthracis sporulation. Further, inactivation or low expression of abs genes may be an important cause of vaccine strain virulence attenuation. ABSTRACT: 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.