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In preliminary experiments, the absence of anthrose in our genetic Sterne knockout was found to decrease pagA expression in BHI broth containing glucose (Fig 5A and B). More...

In preliminary experiments, the absence of anthrose in our genetic Sterne knockout was found to decrease pagA expression in BHI broth containing glucose (Fig 5A and B). This contrasted with the large spike in pagA expression when the Sterne antC mutant is grown in protein rich HIB media which does not contain sugars. When exogenous anthrose was added during growth in BHI broth, expression from PpagA increased in both WT (purple line in Fig 5A) and antC Sterne (purple line in Fig 5B). Many bacteria respond to levels of metabolic intermediates and modulate gene expression accordingly. We hypothesized intracellular or extracellular anthrose levels could impact the vegetative cells through modification of gene expression during vegetative growth. Wildtype B. anthracis Sterne was grown in the presence and absence of exogenously added pure anthrose to avoid any possibility of confounding data in a deletion mutant. Bacteria were harvested at two timepoints post-addition of anthrose, 30 min and 2 h, and compared to diluent only treated cultures (Fig 5C). After 30 min treatment, there were 6 genes significantly upregulated more than two-fold while 17 genes were downregulated (Fig 5D). These genes were mainly involved in generating metabolic intermediates such as pyruvate and trehalose. The most upregulated gene was a putative membrane protein (log2FC=10.98) followed by a gene encoding a GerPF homologue at a log2FC of 10.98. Mutation of GerPF family proteins have been linked to a super-dormant spore phenotype[10]. The gene experiencing the highest downregulation was a putative lipoprotein (log2FC = -20.02). No genes located on pXO1 were detected at 30 min. A subset of differentially transcribed genes from the 30 min timepoint are listed in Fig 5F. After 2 h of anthrose treatment, 52 genes experienced significant up regulation, 18 of which were hypothetical proteins (Fig 5E). Forty-five genes experienced significant down regulation across replicates, 13 of which were hypothetical proteins. Overall design: B. anthracis Sterne strain 34F2 was grown overnight in BHI broth at 37C and standardized to an OD600 of 0.5 and divided into 6 tubes: triplicate for pure anthrose (Sigma) as treatment at a final concentration of 10 ug/ml, and triplicate for a mock of water added. Samples were collected in triplicate at 30 min and triplicate at 120 min after introduction of either liquid. Samples at each timepoint were immediately processed through the Zymo Direct-zol RNA Miniprep Plus (Zymo Resrearch; Irvine, CA, USA) kit including the optional bead beating steps as well as the on column DNAse treatment. Resulting RNA was quantified on a NanoDrop 2000 (Thermo Fisher Scientific; Waltham, MA, USA) and saved at –80°C. To remove contaminating rRNAs ~1.6ug of each sample was treated with Terminator™ 5′-Phosphate-Dependent Exonuclease (Lucigen; Middleton, WI, USA) and cleaned up using the Ambion MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific; Waltham, MA, USA) while removing tRNAs and sRNAs. Less...