Project description:Purpose: Bacillus anthracis produces three regulators, AtxA, AcpA and AcpB, which control virulence gene transcription and belong to an emerging class of regulators termed ‘PCVRs’ (Phosphoenolpyruvate-dependent phosphotransferase regulation Domain-Containing Virulence Regulators). AtxA, named for its control of toxin gene expression, is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. Reports of PCVR activity suggest overlapping function. We used RNA-Seq to assess the regulons of the paralogous regulators in strains constructed to express individual PCVRs at native levels. Methods: Cultures were incubated in toxin-inducing conditions, and expression of each PCVR in the atxAacpAacpB-null background strain was induced with an IPTG concentration that yielded native protein levels for each PCVR. RNA was extracted when cultures reached late-exponential phase using saturated acid phenol extraction, followed by RNA precipitation from the aqueous phase. Creation of libraries for NGS analysis used total RNA (1.0 μg). Samples were treated with Ribo-Zero (Epicentre) to remove ribosomal RNA prior to fragmentation using divalent cations and heat. Libraries were created using an Illumina TruSeq sample preparation kit following the protocol as recommended by the manufacturer. NGS sequencing was performed as a paired-end 50 base sequence using an Illumina HiSeq 1500 following the protocol recommended by the manufacturer. Results: Plasmid and chromosome-borne genes were PCVR controlled, with AtxA, AcpA and AcpB having a ≥4-fold effect on transcript levels of 145, 130 and 49 genes respectively. Several genes were coregulated by two or three PCVRs. Of the 203 transcripts associated with pXO1, 15 were altered at least fourfold: 13 by AtxA and 2 by AcpA. No pXO1-derived transcripts were affected fourfold or greater by AcpB. Read maps revealed that many of the regulated transcripts from pXO1 were associated with the 35 kb region that lies within a larger 44.8 kb pathogenicity island (PAI) (Thorne, 1993). We detected transcripts for 110 genes on pXO2, of which 21 were regulated fourfold or greater by at least one of the three PCVRs. The highly regulated genes clustered within a 35.5 kb region of the plasmid, which includes the capsule biosynthetic operon capBCADE followed by the weakly co-transcribed acpB gene. A significantly smaller proportion of PCVR-regulated genes mapped to the chromosome. Of the 5593 transcripts, 198 were altered fourfold or greater by one or more PCVRs. Clustering of PCVR-regulated chromosome genes was not apparent. Among the most highly regulated chromosomal genes were many genes associated with branched chain amino acid (BCAA) synthesis and uptake. Of the 17 genes implicated in BCAA biosynthesis, expression of 13 of these genes is repressed by AtxA and AcpB. In this work, we have shown that the three PRD-containing virulence regulators of B. anthracis exhibit overlapping and divergent target gene specificities. Conclusions: In this work, we show the relative activities of the PCVRs for specific and co-regulated genes. The results reveal the vast effects of the PCVRs on B. anthracis gene expression and indicate a high degree of functional similarity among the regulators. Our investigations provide insight into control of B. anthracis gene expression and expand our knowledge of structure and function of this emerging class of virulence gene regulators.

Project description:The goal of this project was to screen soil samples for bacteria that may harbor B. anthracis virulence-associated genes (VAGs). There is currently no information about the prevalence of these types of organisms in the environment. Due to increased environmental monitoring of select agents by programs such as BioWatch and biodetection systems in place at the United States Post Offices and Department of State locations, it has become critical that we not only better understand the natural range of B. anthracis but also how widespread B. anthracis virulence genes are in environmental communities. Naturally occurring isolates containing the B. anthracis virulence genes could generate false-positive results in tests that detect the anthrax toxins, capsule or their associated genes. Understanding the true diversity and pathogenic potential of Bacillus spp. and particularly the B. cereus group is crucial not only in terms of understanding data from environmental monitoring but also diagnosing patients with clinical presentations similar to anthrax in the future. Severe and fatal disease caused by strains similar to B. anthracis could unnecessarily initiate emergency responses if anthrax was incorrectly suspected. Conversely, these strains may be used as bioterror agents requiring science-based responses; presently our limited understanding of these organisms does not permit data-driven decision making. We have investigated 700 aerobic sporoform soil isolates obtained from two areas in the Southwest of the US. Soil samples from the first site had been taken from public access land approximately 50 meters across from the work site of a fatal pneumonia case in a welding factory. This took place in year 2003 when B. cereus was isolated from a metal worker. The second site was targeted because of a recent case involving a deceased mule suspected to have died of a B. anthracis infection. Soil samples were initially analyzed at the CDC. Isolates were obtained by heating the soil at 65 degrees Celcius for 30 minutes followed by plating on agar media. All isolates were screened by PCR for the presence of B. anthracis genomic traits such as toxin genes (cya, lef and pag) as well as chromosomal markers. All isolates were also tested for their hemolytic activity as well as phage sensitivity.

Project description:The goal of this project was to screen soil samples for bacteria that may harbor B. anthracis virulence-associated genes (VAGs). There is currently no information about the prevalence of these types of organisms in the environment. Due to increased environmental monitoring of select agents by programs such as BioWatch and biodetection systems in place at the United States Post Offices and Department of State locations, it has become critical that we not only better understand the natural range of B. anthracis but also how widespread B. anthracis virulence genes are in environmental communities. Naturally occurring isolates containing the B. anthracis virulence genes could generate false-positive results in tests that detect the anthrax toxins, capsule or their associated genes. Understanding the true diversity and pathogenic potential of Bacillus spp. and particularly the B. cereus group is crucial not only in terms of understanding data from environmental monitoring but also diagnosing patients with clinical presentations similar to anthrax in the future. Severe and fatal disease caused by strains similar to B. anthracis could unnecessarily initiate emergency responses if anthrax was incorrectly suspected. Conversely, these strains may be used as bioterror agents requiring science-based responses; presently our limited understanding of these organisms does not permit data-driven decision making. We have investigated 700 aerobic sporoform soil isolates obtained from two areas in the Southwest of the US. Soil samples from the first site had been taken from public access land approximately 50 meters across from the work site of a fatal pneumonia case in a welding factory. This took place in year 2003 when B. cereus was isolated from a metal worker. The second site was targeted because of a recent case involving a deceased mule suspected to have died of a B. anthracis infection. Soil samples were initially analyzed at the CDC. Isolates were obtained by heating the soil at 65 degrees Celcius for 30 minutes followed by plating on agar media. All isolates were screened by PCR for the presence of B. anthracis genomic traits such as toxin genes (cya, lef and pag) as well as chromosomal markers. All isolates were also tested for their hemolytic activity as well as phage sensitivity. Eighty-four query strains were investigated in this study, with each query strain hybridized against the reference strain, Sterne. Two dye-swap experiments were performed with seventeen strains, for a total of four hybridizations per query strain. The other strains have a single dye experiment, for a total of two hybridizations per query strain. Each 70mer oligo spotted on the B. cereus species microarray is spotted once. Positive controls on the array consist of oligos designed from the sequenced reference genome, Sterne, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.

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Direct regulons of the master virulence regulator AtxA of Bacillus anthracis

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