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

P1-2: Action Makes it Clear: Motor Capability Enhances Visual Sensitivity in Distant Space

Distance is a critical factor determining the perceptual quality of visual objects. The closer they are, the better we see. Our visual experience, however, does not entirely depend on physical aspects of objects. In fact, the distinction between far and close distance is quite subjective. We often see distant objects with greater acuity as if they were near us. The present study investigated effects of motor capability on visual analysis in the space distant from the body (extrapersonal space). Since the vision has its critical role in guiding the action, we hypothesized that if a person is able to perform an action by using a tool, visual representations of the extrapersonal space might be altered. Using a keyboard, participants manipulated a ball on the screen at a distance they could not reach by hands. Each participant's contrast threshold was measured before and after the manipulation task. As results, participants who were able to control the ball showed improved visual sensitivity relative to those who passively watched the ball moving (Experiment 1). Furthermore, when participants could move the ball in a certain area of the screen but not in the other, improved visual sensitivity was observed only in the area where motor capability was experienced (Experiment 2A). The effect of motor capability, however, was not significant in the peripersonal space (Experiment 2B). Overall, our results demonstrate that visual analysis of distant space can be improved by motor capability, which was temporarily induced by tool-use. It further suggests the tight link between action and vision

Opine-Based Agrobacterium Competitiveness: Dual Expression Control of the Agrocinopine Catabolism (acc) Operon by Agrocinopines and Phosphate Levels ▿ †

Agrobacterium tumefaciens strain C58 can transform plant cells to produce and secrete the sugar-phosphate conjugate opines agrocinopines A and B. The bacterium then moves in response to the opines and utilizes them as exclusive sources of carbon, energy, and phosphate via the functions encoded by the acc operon. These privileged opine-involved activities contribute to the formation of agrobacterial niches in the environment. We found that the expression of the acc operon is induced by agrocinopines and also by limitation of phosphate. The main promoter is present in front of the first gene, accR, which codes for a repressor. This operon structure enables efficient repression when opine levels are low. The promoter contains two putative operators, one overlapping the −10 sequence and the other in the further upstream from it; two partly overlapped putative pho boxes between the two operators; and two consecutive transcription start sites. DNA fragments containing either of the operators bound purified repressor AccR in the absence of agrocinopines but not in the presence of the opines, demonstrating the on-off switch of the promoter. Induction of the acc operon can occur under low-phosphate conditions in the absence of agrocinopines and further increases when the opines also are present. Such opine-phosphate dual regulatory system of the operon may ensure maximum utilization of agrocinopines when available and thereby increase the chances of agrobacterial survival in the highly competitive environment with limited general food sources

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

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

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