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

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Protocol publication

[…] Whole-genome sequencing (WGS) was performed in the Genome Sequencer FLX (454 Life Sciences/Roche) using FLX or FLX Titanium reagents according to the manufacturer's protocols and instructions []. Signal processing of FLX data was performed on the sequencer itself, whereas signal processing of Titanium data was performed off-rig on a Linux cluster of 10 nodes connected via gigabit ethernet. Each node contained eight 64-bit processing cores running at 2.3 GHz with 8 GB of RAM. De novo or reference-guided assembly of either FLX or Titanium sequences, preliminary annotation of the genome using the DIYA pipeline [] and identification of variations were performed using the above-described computer cluster and Sterne (NC 005945) as reference genome as described []. A variant flagged by GS Reference Mapper was defined as high confidence if it met the following criteria: a) at least three non-duplicated reads showed the same variation; b) at least one read in each of the forward and reverse orientations showed this variation, unless there are at least five reads with quality scores over 20 (or 30 if the difference involves a 5-mer or higher). A more detailed description of differences is available in the Genome Sequencer Data Analysis Software manual. In this study, a high quality variation is defined as a high confidence (as defined by 454/Roche) and high concordance (≥75% reads have the variation) variation. [...] Two plasmids were engineered to facilitate allelic exchange in B. anthracis, as improvements to a published system []. Details of the improvements will be published elsewhere. Briefly, pRP1028 and pSS4332 serve the functions of pBKJ236 and pBKJ223, respectively, and contain many of the same features. In pRP1028, the erythromycin resistance of pBKJ236 was replaced with spectinomycin resistance, and in pSS4332, the tetracycline resistance of pBKJ223 was replaced with kanamycin resistance. Allelic exchange constructs derived from pRP1028 were transferred to B. anthracis, and plasmid integrants were isolated following a temperature shift []. Introduction of pSS4332 into the integrant led to I-SceI-mediated cleavage of the integrated plasmid, stimulating the second crossover event. The presence of the desired changes was confirmed by PCR and sequencing.Specifically, to reconstruct the point mutations found in the spontaneous AP50R mutants, the mutations and approximately 500 bp of flanking homology on each side were PCR-amplified from the B. anthracis strains and cloned into pRP1028. To delete csaB, standard cloning methods were used to engineer a construct with the sequence GTG CGG CCCGGG GGA TCT TAA, representing START+one codon+XmaI site+two codons+STOP, along with approximately 500 bp of flanking homology.To restore the wild-type allele to the csaB deletion strain, a derivative of pRP1028 was engineered with the csaB sequence with the adenosine at position 693 changed to cytosine (creating the XmaI site CCCGGG without changing the amino acid sequence), along with approximately 1kb of flanking homology. The change was confirmed by PCR and sequencing, as well as by digestion of the PCR product with XmaI (Figure ).To identify conserved residues of CsaB, the nucleotide sequence (BAS0840) was aligned with the 163 proteins in the seed alignment of Pfam family PF04230 (polysaccharide pyruvyl transferase) [] using ClustalW []. The resulting alignment was processed with WebLogo [] to calculate the consensus sequence and percent conservation at each position (Figure and Additional file : Figure S1). The histidine at position 270 of BAS0840 was found to be 100% conserved in these proteins. Standard cloning techniques were used to engineer a derivative of pRP1028 in which the histidine codon (CAT) was changed to alanine (GCT) (Figure ), flanked by approximately 850 bp of homology. The presence of the H270A change was confirmed by PCR and sequencing.DNA sequences used to generate ΔBAS3946 and BAS3946 G1024A were synthesized (Gene Oracle, Mountain View, CA). For ΔBAS3946, the sequence included the first 6 codons of the gene, followed by the last 5 codons of the gene and the STOP codon, all flanked by approximately 500 bp of homology. For BAS3946 G1024A, the sequence included the mutation flanked by 500 bp of homology. In each case, DNA was cloned into pRP1028, allelic exchange was performed as described above, and the presence of the desired changes was confirmed by PCR and sequencing. […]

Pipeline specifications

Software tools DIYA, Clustal W, WebLogo
Databases Pfam
Applications WGS analysis, Genome data visualization
Organisms Bacillus anthracis