Supplementary Table 1 Bignell et al from Secondary metabolite arsenal of an opportunistic pathogenic fungus

Analysis A. fumigatus 293 genom

Local duplications in β-lactamase PenA.

<p>A. Map of the peptides subjected to duplication and pairs of small nucleotide sequences that apparently mediated the underlying DNA duplication (formation of TRs). The entire omega loop is displayed at the top with the amino acids denoted using the one-letter codes and positions numbered according to Ambler <i>et al.</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004640#pgen.1004640-Ambler1" target="_blank">[47]</a>. Direct repeats or same-strand complementary sequences (SCSs) associated with each DNA template are denoted by pink or blue boxes, respectively, and matching positions between the pair of SCSs are denoted with red dots. Green horizontal bars underneath denote the ends of the DNA templates. B. SCSs associated with the TRs in β-lactamase genes from clinical isolates. The SCSs identified in two β-lactamase genes with TR mutations, SHV-16 gene from <i>K. pneumoniae</i> and <i>ampC</i> from <i>Enterobacter cloacae</i> GC1, are shown in boxes. Below of each of the genes are their parental alleles, the SHV-1 gene and <i>ampC</i> in <i>E. cloacae</i> strain P99, respectively. The paired upstream and downstream elements are denoted in blue boxes.</p

Reversion assays with strains carrying different <i>penA</i> alleles.

<p>A. Reversion assays. The strains with the <i>penA</i> mutations TR11, TR10, Cys69Tyr, and Asp179Asn were tested for their ability to undergo reversion. Serial dilutions of cells were spotted on selection plates containing amoxicillin (AMX), and spots of 10⁶ or 10⁵ cells from which revertant colonies arose (from the TR mutants) are shown. B. Reversion frequencies. Each measurement was plotted, and the mean value is denoted by a red line.</p

The Tandem Repeats Enabling Reversible Switching between the Two Phases of β-Lactamase Substrate Spectrum

<div><p>Expansion or shrinkage of existing tandem repeats (TRs) associated with various biological processes has been actively studied in both prokaryotic and eukaryotic genomes, while their origin and biological implications remain mostly unknown. Here we describe various duplications (<i>de novo</i> TRs) that occurred in the coding region of a β-lactamase gene, where a conserved structure called the omega loop is encoded. These duplications that occurred under selection using ceftazidime conferred substrate spectrum extension to include the antibiotic. Under selective pressure with one of the original substrates (amoxicillin), a high level of reversion occurred in the mutant β-lactamase genes completing a cycle back to the original substrate spectrum. The <i>de novo</i> TRs coupled with reversion makes a genetic toggling mechanism enabling reversible switching between the two phases of the substrate spectrum of β-lactamases. This toggle exemplifies the effective adaptation of <i>de novo</i> TRs for enhanced bacterial survival. We found pairs of direct repeats that mediated the DNA duplication (TR formation). In addition, we found different duos of sequences that mediated the DNA duplication. These novel elements—that we named SCSs (same-strand complementary sequences)—were also found associated with β-lactamase TR mutations from clinical isolates. Both direct repeats and SCSs had a high correlation with TRs in diverse bacterial genomes throughout the major phylogenetic lineages, suggesting that they comprise a fundamental mechanism shaping the bacterial evolution.</p></div

Mutation study with an SCS element.

<p>A. Two G to T substitution mutations introduced into a region overlapping seven SCS elements. Positions subjected to substitution mutations are shown in red. B. TR formation patterns compared between the wild type and the mutated <i>penA</i>. C. TRs formed in the mutated <i>penA</i> gene. M-TRs 1 to 8 that were newly formed in the mutated gene and TR10′, a version of TR10 in the wild type gene (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004640#pgen-1004640-g001" target="_blank">Figure 1A</a>), are shown. Common SCSs for M-TRs 3 to 8 are denoted with red arrows.</p

Proposed models.

<p>A. A model for the DNA duplication-reversion cycle serving as a genetic toggling mechanism for the β-lactamase substrate spectrum. Same-strand complementary sequences (SCSs)- or direct repeat-mediated DNA duplication can be selected for in a β-lactamase gene by exposure to ceftazidime (CAZ). The resulting tandem repeat (TR) in the omega loop encodes a duplicated peptide, which results in an alteration in the active site cavity extending the substrate spectrum to include CAZ (substrate spectrum B). The β-lactamase gene with a TR mutation can be reverted to the wild type by exposure to an original antibiotic, amoxicillin (AMX) (substrate spectrum A). The reversion of the TR does not require direct repeats or SCSs but is likely mediated by the replication slippage mechanism, involving general cellular pathway. B. Models for DNA secondary structures. A model is shown for mispaired direct repeats (DRs) that can occur during DNA replication, leading to DNA duplication of the bordered DNA template. TRs 1 to 3 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004640#pgen-1004640-g001" target="_blank">Fig. 1A</a>) and M-TRs 1 to 2 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004640#pgen-1004640-g002" target="_blank">Fig. 2C</a>) may have resulted from this mechanism. Two models of DNA structures that may form through non-canonical base pairings between SCS elements. Such DNA structures may predispose to DNA duplication via an unknown mechanism. Lastly, a model is shown for a DNA structure formed by strand slippage during replication, which leads to reversion.</p

SCSs and direct repeats contents in the omega loop compared with the rest of the <i>penA</i> gene and the genome of <i>B. thailandensis</i>.

<p>The region encoding the omega loop has high contents for SCSs and direct repeats (DR).</p

The MIC levels of the TR mutants.

<p>The MIC levels for ceftazidime (CAZ) and ampicillin (AMP) are shown in the bar graph. WT is the wild type <i>B. thailandensis</i> E264; TRs 1 to 11 are, E264 (<i>penA</i>-TRs 1 to 11); TR10-1 or TR11-1 are, E264 (Δ<i>penA</i>-TR10 or TR11); TR10-2 or TR11-2 are, E264 (Δ<i>penA</i>-TR10 or TR11, complemented with a plasmid carrying the undisrupted gene).</p

Genome-level correlation analysis between TRs and SCSs or direct repeats.

<p>A. TRs-direct repeats and TRs-SCSs correlations in <i>Burkholderia</i>. A high Pearson correlation coefficient of 0.99 was measured in both analyses, suggesting that the formation of TRs are largely attributable to both direct repeats and SCSs in <i>Burkholderia</i>. B. SCSs-TRs correlations of two genera representing mid and low levels (<i>Streptococcus</i> and <i>Shewanella</i>, respectively). The entire set of the data is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004640#pgen.1004640.s003" target="_blank">Figure S3</a>. C. The SCSs-TRs correlation profile in the bacterial phylogenetic tree. Bacterial genera are color-coded based on the levels of the Pearson correlation coefficients.</p

Twelve Positions in a β-Lactamase That Can Expand Its Substrate Spectrum with a Single Amino Acid Substitution

<div><p>The continuous evolution of β-lactamases resulting in bacterial resistance to β-lactam antibiotics is a major concern in public health, and yet the underlying molecular basis or the pattern of such evolution is largely unknown. We investigated the mechanics of the substrate fspectrum expansion of the class A β-lactamase using PenA of <em>Burkholderia thailandensis</em> as a model. By analyzing 516 mutated enzymes that acquired the ceftazidime-hydrolyzing activity, we found twelve positions with single amino acid substitutions (altogether twenty-nine different substitutions), co-localized at the active-site pocket area. The ceftazidime MIC (minimum inhibitory concentration) levels and the relative frequency in the occurrence of substitutions did not correlate well with each other, and the latter appeared be largely influenced by the intrinsic mutational biases present in bacteria. Simulation studies suggested that all substitutions caused a congruent effect, expanding the space in a conserved structure called the omega loop, which in turn increased flexibility at the active site. A second phase of selection, in which the mutants were placed under increased antibiotic pressure, did not result in a second mutation in the coding region, but a mutation that increased gene expression arose in the promoter. This result suggests that the twelve amino acid positions and their specific substitutions in PenA may represent a comprehensive repertoire of the enzyme’s adaptability to a new substrate. These mapped substitutions represent a comprehensive set of general mechanical paths to substrate spectrum expansion in class A β-lactamases that all share a functional evolutionary mechanism using common conserved residues.</p> </div