High adaptability of the omega loop underlies the substrate-spectrum-extension evolution of a class A β-lactamase, PenL

The omega loop in β-lactamases plays a pivotal role in substrate recognition and catalysis, and some mutations in this loop affect the adaptability of the enzymes to new antibiotics. Various mutations, including substitutions, deletions, and intragenic duplications resulting in tandem repeats (TRs), have been associated with β-lactamase substrate spectrum extension. TRs are unique among the mutations as they cause severe structural perturbations in the enzymes. We explored the process by which TRs are accommodated in order to test the adaptability of the omega loop. Structures of the mutant enzymes showed that the extra amino acid residues in the omega loop were freed outward from the enzyme, thereby maintaining the overall enzyme integrity. This structural adjustment was accompanied by disruptions of the internal α-helix and hydrogen bonds that originally maintained the conformation of the omega loop and the active site. Consequently, the mutant enzymes had a relaxed binding cavity, allowing for access of new substrates, which regrouped upon substrate binding in an induced-fit manner for subsequent hydrolytic reactions. Together, the data demonstrate that the design of the binding cavity, including the omega loop with its enormous adaptive capacity, is the foundation of the continuous evolution of β-lactamases against new drugs

The Early Stage of Bacterial Genome-Reductive Evolution in the Host

The equine-associated obligate pathogen Burkholderia mallei was developed by reductive evolution involving a substantial portion of the genome from Burkholderia pseudomallei, a free-living opportunistic pathogen. With its short history of divergence (∼3.5 myr), B. mallei provides an excellent resource to study the early steps in bacterial genome reductive evolution in the host. By examining 20 genomes of B. mallei and B. pseudomallei, we found that stepwise massive expansion of IS (insertion sequence) elements ISBma1, ISBma2, and IS407A occurred during the evolution of B. mallei. Each element proliferated through the sites where its target selection preference was met. Then, ISBma1 and ISBma2 contributed to the further spread of IS407A by providing secondary insertion sites. This spread increased genomic deletions and rearrangements, which were predominantly mediated by IS407A. There were also nucleotide-level disruptions in a large number of genes. However, no significant signs of erosion were yet noted in these genes. Intriguingly, all these genomic modifications did not seriously alter the gene expression patterns inherited from B. pseudomallei. This efficient and elaborate genomic transition was enabled largely through the formation of the highly flexible IS-blended genome and the guidance by selective forces in the host. The detailed IS intervention, unveiled for the first time in this study, may represent the key component of a general mechanism for early bacterial evolution in the host

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

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

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

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