Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing

Background The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents not only for epidemiological surveillance of the intentional outbreak but also for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. It is clear from the 2001 cases that survival may be markedly improved by administration of antimicrobial therapy during the early symptomatic phase of the illness; i.e., within 3 days of appearance of symptoms. Microbiological detection methods are feasible only for organisms that can be cultured in vitro and cannot detect all genetic modifications with the exception of antibiotic resistance. Currently available immuno or nucleic acid-based rapid detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic resistant or virulence enhanced Bacillus anthracis, to advise on therapeutic treatments. Methodology/Principal Findings We show here that the Roche 454-based pyrosequencing can generate whole genome draft sequences of deep and broad enough coverage of a bacterial genome in less than 24 hours. Furthermore, using the unfinished draft sequences, we demonstrate that unbiased identification of known as well as heretofore-unreported genetic modifications that include indels and single nucleotide polymorphisms conferring antibiotic and phage resistances is feasible within the next 12 hours. Conclusions/Significance Second generation sequencing technologies have paved the way for sequence-based rapid identification of both known and previously undocumented genetic modifications in cultured, conventional and newly emerging biothreat agents. Our findings have significant implications in the context of whole genome sequencing-based routine clinical diagnostics as well as epidemiological surveillance of natural disease outbreaks caused by bacterial and viral agents

Whole genome sequencing of phage resistant Bacillus anthracis mutants reveals an essential role for cell surface anchoring protein CsaB in phage AP50c adsorption

BACKGROUND: Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50(R)) exhibit a mucoid colony phenotype and secrete an extracellular matrix. METHODS: Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection. RESULTS: Using whole genome sequence data, we mapped the relevant mutations in six AP50(R) strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains. CONCLUSIONS: Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1)

Whole genome sequencing of phage resistant Bacillus anthracis mutants reveals an essential role for cell surface anchoring protein CsaB in phage AP50c adsorption

Abstract Background Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix. Methods Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection. Results Using whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains. Conclusions Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1)

Genomic Comparison of <em>Escherichia coli</em> O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding Phage stx2

<div><p>In May of 2011, an enteroaggregative <em>Escherichia coli</em> O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical <em>E. coli</em> O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the <em>mer/tet</em> antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of <em>Yersinia pestis</em>. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing <em>E. coli</em> O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.</p> </div

The chromosomal architecture of 2009 and 2011 strains is similar. A)

<p>Alignment of the chromosomes of the 2011 (2011C–3493) and 2009 outbreak genomes (2009EL–2050, 2009EL–2071) in MAUVE. Large regions of divergence are shown as white gaps. <b>B)</b> Locations of SNPs and small insertion/deletions in the 2009 and 2011 genomes. Most of the apparent differences between TY2482 and 2011EL–3493 in this map are due to sequence errors in the TY2482 sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048228#pone.0048228-Grad1" target="_blank">[65]</a>; see Table S4. <b>C)</b> Location of insertion sequences (IS) and IS-like elements.</p

Analysis of prophage content of O104:H4 strains.

<p><b>A)</b> Location of prophages in the genomes of the EAggEc strains analyzed in this study. Linear maps of the genomes and the location of prophages as boxes are shown. All genome sequences have the same starting position as described in methods. The prophage locations are drawn to scale. Phages are color-coded according to similarity; for example the red box indicates the stx2a phages. The exact genomic locations of the prophages in their respective genomes are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048228#pone-0048228-t006" target="_blank">Table 6</a>. <b>B)</b> Architecture of individual prophages. Phage proteins are colored according to their predicted functions. The <i>stx2ab</i> genes are boxed in red; the island of pyrimidine biosynthesis genes identified as a part of this prophage by Phage_Finder is indicated by the blue box. In all cases the <i>int</i> genes are positioned on the left, regardless of the orientation of the prophage within the chromosome.</p

List of loci disrupted, deleted, or replaced by IS elements.

<p>List of loci disrupted, deleted, or replaced by IS elements.</p

A unique repertoire of genomic islands is present in each strain. A)

<p>Identification of chromosomal Regions of Divergence for each strain. Pair-wise BLASTp analysis of annotated protein sequences in RAST was used to determine reciprocal best-hits in each strain. Each closed, complete strain was used as a query against the other strains. RDs were defined as regions exhibiting 4 or more adjacent genes that were absent (red) or significantly divergent (<99% identical at the protein level) (yellow) in the target strains. ^*Annotation differences initially showed an RD3 locus but subsequent BLASTn alignments did not confirm. <b>B)</b> The Tet/Mer locus of the 2011 and 2009 outbreak strains. Genes encoding resistance are shown in red, transport/efflux functions are indicated in blue, regulatory functions in green, and transposon functions in dark red. Hypothetical proteins and proteins of unknown function are indicated in yellow. <b>C)</b> Loss of ascorbate/lyxose-metabolism genes from 2009EL-2071 due to insertion of an IS1B element.</p

Comparison of plasmid sequences to databases.

<p>Comparison of plasmid sequences to databases.</p