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Antimicrobial resistance represents a major global health concern and environmental bacteria are considered a source of resistance genes. Carbapenems are often used as the last antibiotic option to treat multidrug-resistant bacteria. Metallo-β-lactamases (MBLs) are able to render resistance to almost all β-lactam antibiotics, including carbapenems. Unfortunately, there are no inhibitors against MBLs for clinical use. Subclass B2 MBLs are the only enzymes working as strict carbapenemases, under-represented, encoded in chromosome genes and only functional as mono-zinc enzymes. Despite current efforts in MBLs inhibitor development, B2 carbapenemase activity is especially difficult to suppress, even in vitro. In this study we characterized BioF, a novel subclass B2 MBL identified in a new environmental Pseudomonas sp. strain isolated from an on-farm biopurification system (BPS). Although blaBioF is most likely a chromosomal gene, it is found in a genomic island and may represent a step previous to the horizontal transmission of B2 genes. The new B2 MBL is active as a mono-zinc enzyme and is a potent carbapenemase with incipient activity against some cephalosporins. BioF activity is not affected by excess zinc and is only inhibited at high metal chelator concentrations. The discovery and characterization of B2 MBL BioF as a potent carbapenemase in a BPS bacterial isolate emphasizes the importance of exploring antibiotic resistances existing in the environmental microbiota under the influence of human activities before they could emerge clinically.

Antibiotic resistance development has been studied using approaches that range from laboratory experimental evolution, surveillance and epidemiology, to clinical isolate sequencing. However, evolutionary trajectories depend on the environment in which selection takes place, compelling to address evolutionary analyses in antibiotic-treated patients, to embrace the whole inherent environmental complexities as well as their dynamics over time. Herein, we address the complexity of the bacterial adaptive response to changing antibiotic selective pressures by studying the long-term in-patient evolution of a broad diversity of β-lactam resistant Pseudomonas aeruginosa clones. By using mutational and ultra-deep amplicon sequencing, we analyzed multiple generations of a P. aeruginosa hypermutator strain persisting for more than 26 years of chronic infection in the airways of a cystic fibrosis (CF) patient. We identified the accumulation of multiple alterations targeting the chromosomally encoded class C β-lactamase (blaPDC), providing structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrate selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. Surprisingly, “a gain of function” of collateral resistance towards ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the diversity of evolutionary trajectories driven by bacteria in the natural CF environmental setting, towards a multidrug resistant phenotype after years of antibiotic treatment against a formidable pathogen.

Eukaryotic integral membrane proteins (IMPs) are difficult to study due to low functional expression levels. To investigate factors for efficient biogenesis of eukaryotic IMPs in the prokaryotic model organism Escherichia coli, important, e.g., for isotope-labeling for NMR, we selected for E. coli cells expressing high levels of functional G protein-coupled receptors (GPCRs) by FACS. Utilizing an E. coli strain library with all non-essential genes systematically deleted, we unexpectedly discovered upon whole-genome sequencing that the improved phenotype was not conferred by the deleted genes but by various subtle alterations in the “housekeeping” sigma 70 factor (RpoD). When analyzing effects of the rpoD mutations at the transcriptome level we found that toxic effects incurred on wild-type E. coli during receptor expression were diminished by two independent and synergistic effects: a slower but longer-lasting GPCR biosynthesis and an optimized transcriptional pattern, augmenting growth and expression at low temperature, setting the basis for further bacterial strain engineering.

The understanding of protein evolution depends on the ability to relate the impact of mutations on molecular traits to organismal fitness. Biological activity and robustness have been regarded as important features in shaping protein evolutionary landscapes. Conformational dynamics, which is essential for protein function, has received little attention in the context of evolutionary analyses. Here we employ NMR spectroscopy, the chief experimental tool to describe protein dynamics at atomic level in solution at room temperature, to study the intrinsic dynamic features of a metallo- Β: -lactamase enzyme and three variants identified during a directed evolution experiment that led to an expanded substrate profile. We show that conformational dynamics in the catalytically relevant microsecond to millisecond timescale is optimized along the favored evolutionary trajectory. In addition, we observe that the effects of mutations on dynamics are epistatic. Mutation Gly262Ser introduces slow dynamics on several residues that surround the active site when introduced in the wild-type enzyme. Mutation Asn70Ser removes the slow dynamics observed for few residues of the wild-type enzyme, but increases the number of residues that undergo slow dynamics when introduced in the Gly262Ser mutant. These effects on dynamics correlate with the epistatic interaction between these two mutations on the bacterial phenotype. These findings indicate that conformational dynamics is an evolvable trait, and that proteins endowed with more dynamic active sites also display a larger potential for promoting evolution. Fil: Gonzalez, Mariano Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Abriata, Luciano Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Tomatis, Pablo Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Protein evolution is crucial for organismal adaptation and fitness. This process takes place by shaping a given 3-dimensional fold for its particular biochemical function within the metabolic requirements and constraints of the environment. The complex interplay between sequence, structure, functionality, and stability that gives rise to a particular phenotype has limited the identification of traits acquired through evolution. This is further complicated by the fact that mutations are pleiotropic, and interactions between mutations are not always understood. Antibiotic resistance mediated by β-lactamases represents an evolutionary paradigm in which organismal fitness depends on the catalytic efficiency of a single enzyme. Based on this, we have dissected the structural and mechanistic features acquired by an optimized metallo-β-lactamase (MβL) obtained by directed evolution. We show that antibiotic resistance mediated by this enzyme is driven by 2 mutations with sign epistasis. One mutation stabilizes a catalytically relevant intermediate by fine tuning the position of 1 metal ion; whereas the other acts by augmenting the protein flexibility. We found that enzyme evolution (and the associated antibiotic resistance) occurred at the expense of the protein stability, revealing that MβLs have not exhausted their stability threshold. Our results demonstrate that flexibility is an essential trait that can be acquired during evolution on stable protein scaffolds. Directed evolution aided by a thorough characterization of the selected proteins can be successfully used to predict future evolutionary events and design inhibitors with an evolutionary perspective.

The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal beta-lactam drug. This compound and the monobactam aztreonam were assayed as substrates of the Metallo-beta-lactamase BcII. None of them was hydrolyzed by the enzyme. While the azetidinone was not able to bind BcII, aztreonam was shown to bind in a nonproductive mode. These results provide an explanation for the unability of Metallo-beta-lactamases to inactive monobactams and give some clues for inhibitor design.

Understanding the driving forces behind protein evolution requires the ability to correlate the molecular impact of mutations with organismal fitness. To address this issue, we employ here metallo--lactamases as a model system, which are Zn(II) dependent enzymes that mediate antibiotic resistance. We present a study of all the possible evolutionary pathways leading to a metallo--lactamase variant optimized by directed evolution. By studying the activity, stability and Zn(II) binding capabilities of all mutants in the preferred evolutionary pathways, we show that this local fitness landscape is strongly conditioned by epistatic interactions arising from the pleiotropic effect of mutations in the different molecular features of the enzyme. Activity and stability assays in purified enzymes do not provide explanatory power. Instead, measurement of these molecular features in an environment resembling the native one provides an accurate description of the observed antibiotic resistance profile. We report that optimization of Zn(II) binding abilities of metallo--lactamases during evolution is more critical than stabilization of the protein to enhance fitness. A global analysis of these parameters allows us to connect genotype with fitness based on quantitative biochemical and biophysical parameters. Fil: Meini, María Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Tomatis, Pablo Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Weinreich, Daniel M.. University Brown; Estados Unidos Fil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Metallo-β-lactamases (MBLs) represent the latest generation of β-lactamases. The structural diversity and broad substrate profile of MBLs allow them to confer resistance to most β-lactam antibiotics. To explore the evolutionary potential of these enzymes, we have subjected the Bacillus cereus MBL (BcII) to a directed evolution scheme, which resulted in an increased hydrolytic efficiency toward cephalexin. A systematic study of the hydrolytic profile, substrate binding, and active-site features of the evolved lactamase reveal that directed evolution has shaped the active site by means of remote mutations to better hydrolyze cephalosporins with small, uncharged C-3 substituents. One of these mutations is found in related enzymes from pathogenic bacteria and is responsible for the increase in that enzyme's hydrolytic profile. The mutations lowered the activation energy of the rate-limiting step rather than improved the affinity of the enzyme toward these substrates. The following conclusions can be made: ( i ) MBLs are able to expand their substrate spectrum without sacrificing their inherent hydrolytic capabilities; ( ii ) directed evolution is able to mimic mutations that occur in nature; ( iii ) the metal-ligand strength is tuned by second-shell mutations, thereby influencing the catalytic efficiency; and ( iv ) changes in the position of the second Zn(II) ion in MBLs affect the substrate positioning in the active site. Overall, these results show that the evolution of enzymatic catalysis can take place by remote mutations controlling reactivity.

The phosphoglucomutase (pgm) gene codes for a key enzyme required for the formation of UDP-glucose and ADP-glucose, the sugar donors for the biosynthesis of glucose containing polysaccharides. A Mesorhizobium loti pgm null mutant obtained in this study contains an altered form of lipopolysaccharide (LPS), lacks exopolysaccharide (EPS), β cyclic glucan, and glycogen and is unable to nodulate Lotus tenuis. The nonnodulating phenotype of the pgm mutant was not due to the absence of glycogen, since a glycogen synthase (glgA) null mutant effectively nodulates this legume. In M. loti, pgm is part of the glycogen metabolism gene cluster formed by GlgP (glycogen phosphorylase), glgB (glycogen branching), glgC (ADP-glucose pyrophos-phorylase), glgA, pgm, and glgX (glycogen debranching). The genes are transcribed as a single transcript from glgP to at least pgm under the control of a strong promoter (promoter I) upstream of glgP. An alternative promoter (promoter II), mapping in a 154-bp DNA fragment spanning 85 bp upstream of the glgA start codon and the first 69 bp of the glgA coding region, controls the expression of glgA and pgm, independently of the rest of the upstream genes. Primer extension experiments showed that transcription starts 19 bp upstream of the glgA start codon.

A dataset of TEM lactamase variants with different substrate and inhibition profiles was compiled and analyzed. Trends show that loops are the main evolvable regions in these enzymes, gradually accumulating mutations to generate increasingly complex functions. Notably, many mutations present in evolved enzymes are also found in simpler variants, probably originating functional promiscuity. Following a function-stability tradeoff, the increase in functional complexity driven by accumulation of mutations fosters the incorporation of other stability-restoring substitutions, although our analysis suggests they might not be as “global” as generally accepted and seem instead specific to different networks of protein sites. Finally, we show how this dataset can be used to model functional changes in TEMs based on the physicochemical properties of the amino acids.

A number of multiresistant bacterial pathogens inactivate antibiotics by producing Zn(II)-dependent β-lactamases. We show that metal uptake leading to an active dinuclear enzyme in the periplasmic space of Gram-negative bacteria is ensured by a cysteine residue, an unusual metal ligand in oxidizing environments. Kinetic, structural and affinity data show that such Zn(II)-cysteine interaction is an adaptive trait that tunes the metal binding affinity, thus enabling antibiotic resistance at restrictive Zn(II) concentrations.

Metallo-beta-lactamases (MbetaLs) are zinc enzymes able to hydrolyze almost all beta-lactam antibiotics, rendering them inactive, at the same time endowing bacteria high levels of resistance. The design of inhibitors active against all classes of MbetaLs has been hampered by their structural diversity and by the heterogeneity in metal content in enzymes from different sources. BcII is the metallo-beta-lactamase from Bacillus cereus, which is found in both the mononuclear and dinuclear forms. Despite extensive studies, there is still controversy about the nature of the active BcII species. Here we have designed two mutant enzymes in which each one of the metal binding sites was selectively removed. Both mutants were almost inactive, despite preserving most of the structural features of each metal site. These results reveal that neither site isolated in the MbetaL scaffold is sufficient to render a fully active enzyme. This suggests that only the dinuclear species is active or that the mononuclear variants can be active only if aided by other residues that would be metal ligands in the dinuclear species.

The plant kingdom produces an overwhelming number of structurally diverse specialised metabolites. One of the less studied groups of specialised metabolites are flavonoid dimers known as biflavonoids, which consist of two identical or non-identical flavonoid units. They exhibit higher bioactivity than their monomeric subunits and are considered potential drugs in the pharmaceutical industry and potential antioxidants and antimicrobials in the food industry. However, it remains unclear why some plant species produce dimeric structures of flavonoids, under what conditions, and what role biflavonoids play in plant growth and development. In our project, we address such limitations by studying biflavonoids in Ginkgo biloba L., a so-called "living fossil" and a plant widely used in traditional medicine. It is one of the most characteristic plants. It possesses a number of fascinating characteristics, including a large genome, outstanding resistance/tolerance to abiotic and biotic stresses, and dioecious reproduction, making it an ideal model species for biological studies. Its great tolerance to adverse environmental conditions is demonstrated by the fact that Ginko trees growing within 1-2 kilometres of Hiroshima since the atomic bomb explosion in 1945 were among the few living things in the area that survived the blast and were soon healthy again. This poster will provide an overview of the structure and biological activity of biflavonoids and explain why G. biloba is an ideal model for studying biflavonoids. We will also present the preliminary results of the recently launched project "Biflavonoids role in plants: Ginkgo biloba L. as a model system", funded by the Croatian Science Foundation (HRZZ- UIP-2019-04-1018).