A Resonance Raman Enhancement Mechanism for Axial Vibrational Modes in the Pyridine Adduct of Myoglobin Proximal Cavity Mutant (H93G)

The proximal cavity mutant of myoglobin consists of a mutation of the proximal histidine to glycine (H93G), which permits exogenous ligands to bind to the heme iron. A non-native pyridine ligand can ligate to the heme to yield a five-coordinate adduct, H93G­(Pyr), that cannot be formed freely in solution since the six-coordinate bis-pyridine adduct is more stable than the five-coordinate adduct. We have used resonance Raman spectroscopy in the Soret band region of the heme to study the enhancement of axial vibrations of bound pyridine in the H93G­(Pyr) adduct. The observation that the pyridine ring breathing mode (ν₁) and the symmetric ring stretching (ν₃) modes are enhanced under these conditions is explained by a computational approach that shows that coupling of the π-system of the heme with the p-orbitals of the pyridine is analogous to π-backbonding in diatomic ligand adducts of heme proteins. The result has the broader significance that it suggests that the resonance enhancement of pyridine modes could be an important aspect of Raman scattering of pyridine on conducting surfaces such as those studied in surface enhanced Raman scattering experiments

Lack of association of CDSs in the accessory genome with outbreak clades and statistical test of neutrality of non-synonymous mutations in the core genome.

<p>A) Likelihood of association of individual CDSs within the accessory genome with outbreak clades. Each of the 79 genomic islands and plasmids is indicated by alternating light and dark blue segments consisting of one vertical line per CDS. Individual genomes were assigned to one of four outbreak clades or one of 28 non-outbreak clades. Only eight CDSs within GI21 were significantly associated with all outbreak clades (Fisher exact test, p = 0.032). After a Bonferroni correction for multiple tests, no CDS was significantly associated with outbreak clades. B) Numbers of non-synonymous mutations per CDS in the non-recombinant, non-repetitive core genome as a function of gene length whose mean expectations are indicated by an internal white line. Each gene is represented by a circle, whose size is proportional to the deviation according to a χ² statistic from theoretical expectations of a non-parametric test (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003471#pgen.1003471.s015" target="_blank">Figure S5</a>). Shades of blue indicate different α-thresholds (0.05, 0.01, 0.001) of the confidence intervals of the theoretical expectations, where 0.05 indicate CI 95%. The sole outlier identified in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003471#pgen.1003471.s017" target="_blank">Figure S7B</a> is indicated in red.</p

Reconstruction of MRCA dates and changes in population size.

<p>Analyses were based on analyses of 846 non-recombinant, non-repetitive, non-mobile concatenated core SNPs by the relaxed GMRF model in Beast v1.7.1 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003471#pgen.1003471-Drummond1" target="_blank">[36]</a>. (A) A maximum clade credibility tree. Branch colors indicate substitution rates transformed to a logarithmic scale (Rate Key), except that posterior probabilities of <0.5 for nodes and branches are indicated in black. (B) Bayesian skyride plot showing changes in effective population size of serovar Agona over time (black line) with the extent of the 95% confidence intervals shaded in blue.</p

Insertions (solid lines) and deletions (dashed lines) of IS elements (Tables S7, S8, S9) in mobile elements (red boxes) or the core genome (black ellipses) mapped on a SNP genealogy of 73 Agona genomes.

<p>The SNP genealogy is based on a maximum parsimony tree based on 846 non-recombinant, non-mobile core SNPs (as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003471#pgen-1003471-g001" target="_blank">Figure 1</a>), but drawn in radial fashion (Mega) for convenience. The tips of the branches include strain ID numbers, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003471#pgen-1003471-g001" target="_blank">Figure 1</a> and Dataset S2. Node, clade and branch designations are also according to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003471#pgen-1003471-g001" target="_blank">Figure 1</a> and Dataset S2.</p

Genealogy of 73 Agona genomes based on SNPs and indels <i>versus</i> PFGE patterns.

<p>Left: Maximum parsimony genealogy inferred from 846 non-homoplastic, non-recombinant, non-mobile, non-repetitive core SNPs. The numbers of independent insertions or deletions of mobile elements (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003471#pgen.1003471.s011" target="_blank">Figure S1</a>, Dataset S1) are indicated by symbols at the nodes within the genealogy (Key). Isolates that are related to outbreaks were clustered in clades A–D and their sub-clades (A1, A2, <i>etc.</i>). Centre: Strain designations include a serial number, an abbreviation for Host and the year of isolation (Dataset S2). Right: PFGE patterns and profiles of <i>XbaI</i>-digested DNA for each strain except for the reference genome (25.H.03 [SL483]), which was predicted <i>in silico</i>. All observed PFGE bands were also predicted from genomic analyses except for bands in dashed boxes. The detailed sizes and existence of each band are listed in Dataset S3.</p

Histograms of the frequencies of numbers of differences between pairs of genomes in SNPs, genomic islands, accessory genes, or PFGE bands.

<p>Histograms of the frequencies of numbers of differences between pairs of genomes in SNPs (A), genomic islands (B), accessory genes (C), or PFGE bands (D). The X axes of histograms A and C indicate the maximal number of differences within the indicated ranges in order to ensure that the first column only includes identical pairs (maximum = 0). These frequency distributions correspond to the same data which were used for comparisons in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003471#pgen-1003471-g008" target="_blank">Figure 8</a>, except that those referred to genetic similarity and the data here refer to the converse, which are differences.</p

SNP densities in recombinant and non-recombinant regions of the Agona core genome.

<p>(A) Average frequency of SNPs per Kb from pair-wise, 1 Kb sliding window comparisons between the genome of Agona SL483 <i>versus</i> 12 genomes from serovars Choleraesuis, Dublin, Enteritidis, Gallinarium, Heidelberg, Newport, Paratyphi A, Paratyphi B, Paratyphi C, Schwarzengrund, Typhi, and Typhimurium (hatched boxes). A comparable distribution of mean pair-wise diversity between all 73 Agona genomes is indicated by black boxes. (B). SNP density in recombination segments. (C) Frequency distributions of the length of recombinant segments (x-axis) and number of SNPs per recombinant region (y-axis). Each recombination region is indicated by an open circle with an internal dot. The linear regression of these data indicates that 9.2 SNPs are found per recombinant Kb (<i>R</i>² = 0.97).</p

Features of the <i>S. enterica</i> Agona non-repetitive non-mobile core genome.

<p>Features of the <i>S. enterica</i> Agona non-repetitive non-mobile core genome.</p

Multiple introductions and deletions of mobile elements.

<p>Each introduction or deletion in a node of the genealogy is counted only once, even if that introduced mobile element is present in multiple descendent genomes. The number of distinct sequence variants within each category is indicated by (Number) after Type. Where mobile elements integrated into multiple locations within the genome, each such integration was associated with a distinct sequence variant (Dataset S1), and they represent independent insertions. Additional, unrelated non-IS mobile elements were each introduced on a single occasion (Dataset S1), consisting of 2 ICE/IMEs, 3 bacteriophages, 3 plasmids and 4 other genomic islands, for a total of 95 introductions of non-IS mobile elements. Five other genomic islands were each deleted once, for a total of 22 deletions of non-IS mobile elements. Similarly, five IS elements were introduced on one occasion each (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003471#pgen.1003471.s027" target="_blank">Table S8</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003471#pgen.1003471.s028" target="_blank">S9</a>), for a total of 54 introductions.</p

Number of CDSs <i>versus</i> number of genomes in which they were present.

<p>All CDSs in the pan-genome were used for these calculations, except that repetitive CDSs such as IS elements were each counted as one CDS.</p