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<p>Bacillus anthracis is an endemic soil bacterium that exhibits two different lifestyles. In the soil environment, B. anthracis undergoes a cycle of saprophytic growth, sporulation, and germination. In mammalian hosts, the pathogenic lifestyle of B. anthracis is spore germination followed by vegetative cell replication, but cells do not sporulate. During infection, and in specific culture conditions, transcription of the structural genes for the anthrax toxin proteins and the biosynthetic operon for capsule synthesis is positively controlled by the regulatory protein AtxA. A critical role for the atxA gene in B. anthracis virulence has been established. Here we report an inverse relationship between toxin production and sporulation that is linked to AtxA levels. During culture in conditions favoring sporulation, B. anthracis produces little to no AtxA. When B. anthracis is cultured in conditions favoring toxin gene expression, AtxA is expressed at relatively high levels and sporulation rate and efficiency are reduced. We found that a mutation within the atxA promoter region resulting in AtxA over-expression leads to a marked sporulation defect. The sporulation phenotype of the mutant is dependent upon pXO2-0075, an atxA-regulated open reading frame located on virulence plasmid pXO2. The predicted amino acid sequence of the pXO2-0075 protein has similarity to the sensor domain of sporulation sensor histidine kinases. It was shown previously that pXO2-0075 overexpression suppresses sporulation. We have designated pXO2-0075 “skiA” for “sporulation kinase inhibitor.” Our results indicate that in addition to serving as a positive regulator of virulence gene expression, AtxA modulates B. anthracis development.</p

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<p>Bacillus anthracis is an endemic soil bacterium that exhibits two different lifestyles. In the soil environment, B. anthracis undergoes a cycle of saprophytic growth, sporulation, and germination. In mammalian hosts, the pathogenic lifestyle of B. anthracis is spore germination followed by vegetative cell replication, but cells do not sporulate. During infection, and in specific culture conditions, transcription of the structural genes for the anthrax toxin proteins and the biosynthetic operon for capsule synthesis is positively controlled by the regulatory protein AtxA. A critical role for the atxA gene in B. anthracis virulence has been established. Here we report an inverse relationship between toxin production and sporulation that is linked to AtxA levels. During culture in conditions favoring sporulation, B. anthracis produces little to no AtxA. When B. anthracis is cultured in conditions favoring toxin gene expression, AtxA is expressed at relatively high levels and sporulation rate and efficiency are reduced. We found that a mutation within the atxA promoter region resulting in AtxA over-expression leads to a marked sporulation defect. The sporulation phenotype of the mutant is dependent upon pXO2-0075, an atxA-regulated open reading frame located on virulence plasmid pXO2. The predicted amino acid sequence of the pXO2-0075 protein has similarity to the sensor domain of sporulation sensor histidine kinases. It was shown previously that pXO2-0075 overexpression suppresses sporulation. We have designated pXO2-0075 “skiA” for “sporulation kinase inhibitor.” Our results indicate that in addition to serving as a positive regulator of virulence gene expression, AtxA modulates B. anthracis development.</p

<i>B. anthracis</i> gene products identified by IVIAT.

<p><i>B. anthracis</i> gene products identified by IVIAT.</p

mRNA expression profiles during in vitro and in vivo growth of IVIAT-identified and paralog/similar <i>B. anthracis</i> genes.

<p>Quantitative RT-PCR was performed on RNA recovered from in vitro grown <i>B. anthracis</i> (Ames strain cells grown in BHI and air; late-log phase) and compared to RNA recovered from mice infected with vegetative Ames strain <i>B. anthracis</i> via intravenous injection. RNA from mice was isolated from spleens 18 hr post-infection. Transcript numbers on the Y-axis are normalized against 16S rRNA. Displayed are gene profiles of <i>B. anthracis</i> genes up-regulated in vivo compared to in vitro. In bar graphs, expression levels represent mean values; error bars represent standard deviations (SD). In summary table, expression given as mRNA transcript copies (× 10⁻⁵) per copy of 16S rRNA.</p

<i>Bacillus</i> strains used in this study.

<p><i>Bacillus</i> strains used in this study.</p

<i>B. anthracis</i> species-specific autolysis effects following addition of exogenous putative autolysins N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446: (A) reduction in A₅₉₅ optical density, (B) reduction in colony forming units, and (C, D) morphological changes of <i>B. cereus</i> (C) or <i>B. anthracis</i> (D).

<p><i>B. anthracis</i> (Sterne strain), <i>B. cereus</i> (ATCC 14579), <i>B. subtilis</i> (168), and <i>E. coli</i> (DH5α) vegetative cells were resuspended in 20 mM sodium phosphate buffer containing BA0485 and BA2446 (final concentration 2 uM) or buffer alone (BA0485, pH 7.0; BA2446, pH 8.7). (A) A₅₉₅ readings were recorded every 2 min. and reported as a percentage of starting A₅₉₅. Buffer-only controls were not different for cultures containing <i>B. cereus, B. subtilis,</i> or <i>E. coli</i>. (B) Dilutions of cells were plated prior to addition of BA0485 and BA2446 proteins and then again following 20 min of incubation with buffer alone or buffer containing exogenous BA0485 or BA2446, and CFU are reported as percentage of the starting CFU. (C, D) BA0485, BA2446, or buffer alone were added to cell suspensions of <i>B. anthracis</i>, <i>B. cereus, B. subtilis,</i> and <i>E. coli</i>, and incubated for 20 min. Bacterial morphological changes were only evident following protein addition to <i>B. anthracis</i> (D); representative <i>B. cereus</i> samples are shown (C). Gram stains were performed and all images were captured at 1000×.</p