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Legionella pneumophila utilizes the Dot/Icm type IVB secretion system to deliver hundreds of effector proteins inside eukaryotic cells to ensure intracellular replication. Our understanding of the molecular functions of the largest pathogenic arsenal known to the bacterial world remains incomplete. By leveraging advancements in 3D protein structure prediction, we provide a comprehensive structural analysis of 368 L. pneumophila effectors, representing a global atlas of predicted functional domains summarized in a database ( https://pathogens3d.org/legionella-pneumophila ). Our analysis identified 157 types of diverse functional domains in 287 effectors, including 159 effectors with no prior functional annotations. Furthermore, we identified 35 cryptic domains in 30 effector models that have no similarity with experimentally structurally characterized proteins, thus, hinting at novel functionalities. Using this analysis, we demonstrate the activity of thirteen functional domains, including three cryptic domains, predicted in L. pneumophila effectors to cause growth defects in the Saccharomyces cerevisiae model system. This illustrates an emerging strategy of exploring synergies between predictions and targeted experimental approaches in elucidating novel effector activities involved in infection., Competing Interests: Disclosure and competing interests statement The authors declare no competing interests., (© 2024. The Author(s).)

To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense-directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, the putative active site in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vacuole that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated, but rather, have significant rotational flexibility. Phylogenetic analysis supports that the disulfide bond cysteines are also absent in pangolin-SARS and closely related viruses, consistent with pangolins being the presumed intermediate host in the emergence of SARS-CoV-2. The absence of these cysteines does not impact viral replication or protein translation., Competing Interests: CONFLICTS OF INTEREST K.J.F.S. has a significant financial interest in Situ Biosciences, a contract research organization that conducts research unrelated to this work. T.Y.T. and M.O. are listed as inventors on a patent application filed by the Gladstone Institutes that covers the use of pGLUE to generate SARS-CoV-2 infectious clones and replicons. All other authors declare no conflicts of interest.

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work, we functionally and structurally characterize the Pca regulon regulatory protein (PcaR) transcription factor belonging to the IclR transcription factor family. Here, we present in vitro functional analysis of the ligand profile of PcaR and the construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this transcription factor. This work contributes key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors, in addition to providing more insights into the IclR family of regulators., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Transcription factor (TF)-based biosensors are useful synthetic biology tools for applications in a variety of areas of biotechnology. A major challenge of biosensor circuits is the limited repertoire of identified and well-characterized TFs for applications of interest, in addition to the challenge of optimizing selected biosensors. In this work, we implement the IclR family repressor TF TtgV from Pseudomonas putida DOT-T1E as an indole-derivative biosensor in Escherichia coli . We optimize the genetic circuit utilizing different components, providing insights into biosensor design and expanding on previous studies investigating this TF. We discover novel physiologically relevant ligands of TtgV, such as skatole. The broad specificity of TtgV makes it a useful target for directed evolution and protein engineering toward desired specificity. TtgV, as an indole-derivative biosensor, is a promising genetic component for the detection of compounds with biological activities relevant to health and the gut microbiome.

Extracytoplasmic Ni(II)-binding proteins (NiBPs) are molecular shuttles involved in cellular nickel uptake. Here, we determined the crystal structure of apo CcNikZ-II at 2.38 Å, which revealed a Ni(II)-binding site comprised of the double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Mutagenesis of the site identified Glu60 and His511 as critical for high affinity Ni(II)-binding. Phylogenetic analysis showed 15 protein clusters with two groups containing the HH-prong. Metal-binding assays with 11 purified NiBPs containing this feature yielded higher Ni(II)-binding affinities. Replacement of the wild type v-loop with those from other NiBPs improved the affinity by up to an order of magnitude. This work provides molecular insights into the determinants for Ni(II) affinity and paves way for NiBP engineering., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Transcription factor (TF)-based biosensors that connect small-molecule sensing with readouts such as fluorescence have proven to be useful synthetic biology tools for applications in biotechnology. However, the development of specific TF-based biosensors is hindered by the limited repertoire of TFs specific for molecules of interest since current construction methods rely on a limited set of characterized TFs. In this study, we present an approach for engineering the specificity of TFs through a computation-based workflow using molecular docking that enables targeted alteration of TF ligand specificity. Using this method, we engineer the LysR family BenM TF to alter its specificity from its cognate ligand cis , cis -muconic acid to adipic acid through a single amino acid substitution identified by our computational workflow. When implemented in a cell-free system, the engineered biosensor shows higher ligand sensitivity, expanding the potential applications of this circuit. We further investigate ligand binding through molecular dynamics to analyze the substitution, elucidating the impact of modulating a single amino acid position on the mechanism of BenM ligand binding. This study represents the first application of biomolecular modeling methods for altering BenM specificity and for gaining insights into how mutations influence the structural dynamics of BenM. Such methods can potentially be applied to other TFs to alter specificity and analyze the dynamics responsible for these changes, highlighting the applicability of computational tools for informing experiments. In addition, our developed adipic acid biosensor can be applied for the identification and engineering of enzymes to produce adipic acid., Competing Interests: The authors declare no conflict of interest., (© 2024 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

The survival of Legionella spp. as intracellular pathogens relies on the combined action of protein effectors delivered inside their eukaryotic hosts by the Dot/Icm ( d efective in o rganelle t rafficking/ i ntra c ellular m ultiplication) type IVb secretion system. The specific repertoire of effector arsenals varies dramatically across over 60 known species of this genera with Legionella pneumophila responsible for most cases of Legionnaires' disease in humans encoding over 360 Dot/Icm effectors. However, a small subset of "core" effectors appears to be conserved across all Legionella species raising an intriguing question of their role in these bacteria's pathogenic strategy, which for most of these effectors remains unknown. L. pneumophila Lpg0103 effector, also known as VipF, represents one of the core effector families that features a tandem of Gcn5-related N-acetyltransferase (GNAT) domains. Here, we present the crystal structure of the Lha0223, the VipF representative from Legionella hackeliae in complex with acetyl-coenzyme A determined to 1.75 Å resolution. Our structural analysis suggested that this effector family shares a common fold with the two GNAT domains forming a deep groove occupied by residues conserved across VipF homologs. Further analysis suggested that only the C-terminal GNAT domain of VipF effectors retains the active site composition compatible with catalysis, whereas the N-terminal GNAT domain binds the ligand in a non-catalytical mode. We confirmed this by in vitro enzymatic assays which revealed VipF activity not only against generic small molecule substrates, such as chloramphenicol, but also against poly-L-lysine and histone-derived peptides. We identified the human eukaryotic translation initiation factor 3 (eIF3) complex co-precipitating with Lpg0103 and demonstrated the direct interaction between the several representatives of the VipF family, including Lpg0103 and Lha0223 with the K subunit of eIF3. According to our data, these interactions involve primarily the C-terminal tail of eIF3-K containing two lysine residues that are acetylated by VipF. VipF catalytic activity results in the suppression of eukaryotic protein translation in vitro , revealing the potential function of VipF "core" effectors in Legionella 's pathogenic strategy.IMPORTANCEBy translocating effectors inside the eukaryotic host cell, bacteria can modulate host cellular processes in their favor. Legionella species, which includes the pneumonia-causing Legionella pneumophila, encode a widely diverse set of effectors with only a small subset that is conserved across this genus. Here, we demonstrate that one of these conserved effector families, represented by L. pneumophila VipF (Lpg0103), is a tandem Gcn5-related N-acetyltransferase interacting with the K subunit of human eukaryotic initiation factor 3 complex. VipF catalyzes the acetylation of lysine residues on the C-terminal tail of the K subunit, resulting in the suppression of eukaryotic translation initiation factor 3-mediated protein translation in vitro . These new data provide the first insight into the molecular function of this pathogenic factor family common across Legionellae ., Competing Interests: The authors declare no conflict of interest.

The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria. Among the most prevalent are aminoglycoside acetyltransferases (AACs) that inactivate the antibiotics through acetyl coenzyme A-mediated modification. Most AACs are members of the GCN5 superfamily of acyltransferases which lack conserved active site residues that participate in catalysis. ApmA is the first reported AAC belonging to the left-handed β-helix superfamily. These enzymes are characterized by an essential active site histidine that acts as an active site base. Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other detoxifying left-handed β-helix superfamily enzymes and canonical GCN5 AACs. We find that the active site histidine plays different functions depending on the acetyl-accepting aminoglycoside substrate. This flexibility in the mechanism of a single enzyme underscores the plasticity of antibiotic resistance elements to co-opt protein catalysts in the evolution of drug detoxification., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Importance: Toxin-antitoxin (TA) systems are parasitic genetic elements found in almost all bacterial genomes. They are exchanged horizontally between cells and are typically poorly conserved across closely related strains and species. Here, we report the characterization of a tripartite TA system in the bacterial pathogen Legionella pneumophila that is highly conserved across Legionella species genomes. This system (denoted HipBST Lp ) is a distant homolog of the recently discovered split-HipA system in Escherichia coli (HipBST Ec ). We present bioinformatic, molecular, and structural analyses of the divergence between these two systems and the functionality of this newly described TA system family. Furthermore, we provide evidence to refute previous claims that the toxin in this system (HipT Lp ) possesses bifunctionality as an L. pneumophila virulence protein. Overall, this work expands our understanding of the split-HipA system architecture and illustrates the potential for undiscovered biology in these abundant genetic elements., Competing Interests: The authors declare no conflict of interest.

Cultivated meat production requires an efficient, robust and highly optimized serum-free cell culture media for the needed upscaling of muscle cell expansion. Existing formulations of serum-free media are complex, expensive and have not been optimized for muscle cells. Thus, we undertook this work to develop a simple and robust serum-free media for the proliferation of bovine satellite cells (SCs) through Design of Experiment (DOE) and Response Surface Methodology (RSM) using precise and high-throughput image-based cytometry. Proliferative attributes were investigated with transcriptomics and long-term performance was validated using multiple live assays. Here we formulated a media based on three highly optimized components; FGF2 (2 ng/mL), fetuin (600 µg/mL) and BSA (75 µg/mL) which together with an insulin-transferrin-selenium (1x) supplement, sustained the proliferation of bovine SCs, porcine SCs and murine C2C12 muscle cells. Remarkably, cells cultured in our media named Tri-basal 2.0+ performed better than cell cultured in 10% FBS, with respect to proliferation. Hence, the optimized Tri-basal 2.0+ enhanced serum-free cell attachment and long-term proliferation, providing an alternative solution to the use of FBS in the production of cultivated meat., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The sulfonamides (sulfas) are the oldest class of antibacterial drugs and inhibit the bacterial dihydropteroate synthase (DHPS, encoded by folP), through chemical mimicry of its co-substrate p-aminobenzoic acid (pABA). Resistance to sulfa drugs is mediated either by mutations in folP or acquisition of sul genes, which code for sulfa-insensitive, divergent DHPS enzymes. While the molecular basis of resistance through folP mutations is well understood, the mechanisms mediating sul-based resistance have not been investigated in detail. Here, we determine crystal structures of the most common Sul enzyme types (Sul1, Sul2 and Sul3) in multiple ligand-bound states, revealing a substantial reorganization of their pABA-interaction region relative to the corresponding region of DHPS. We use biochemical and biophysical assays, mutational analysis, and in trans complementation of E. coli ΔfolP to show that a Phe-Gly sequence enables the Sul enzymes to discriminate against sulfas while retaining pABA binding and is necessary for broad resistance to sulfonamides. Experimental evolution of E. coli results in a strain harboring a sulfa-resistant DHPS variant that carries a Phe-Gly insertion in its active site, recapitulating this molecular mechanism. We also show that Sul enzymes possess increased active site conformational dynamics relative to DHPS, which could contribute to substrate discrimination. Our results reveal the molecular foundation for Sul-mediated drug resistance and facilitate the potential development of new sulfas less prone to resistance., (© 2023. The Author(s).)

Strigolactones (SLs) regulate many aspects of plant development, but ambiguities remain about how this hormone is perceived because SL-complexed receptor structures do not exist. We find that when SL binds the Striga receptor, ShHTL5, a series of conformational changes relative to the unbound state occur, but these events are not sufficient for signalling. Ligand-complexed receptors, however, form internal tunnels that posit an explanation for how SL exits its receptor after hydrolysis., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The ability to transition between yeast and filamentous growth states is critical for virulence of the leading human fungal pathogen Candida albicans. Large-scale genetic screens have identified hundreds of genes required for this morphological switch, but the mechanisms by which many of these genes orchestrate this developmental transition remain largely elusive. In this study, we characterized the role of Ent2 in governing morphogenesis in C. albicans. We showed that Ent2 is required for filamentous growth under a wide range of inducing conditions and is also required for virulence in a mouse model of systemic candidiasis. We found that the epsin N-terminal homology (ENTH) domain of Ent2 enables morphogenesis and virulence and does so via a physical interaction with the Cdc42 GTPase-activating protein (GAP) Rga2 and regulation of its localization. Further analyses revealed that overexpression of the Cdc42 effector protein Cla4 can overcome the requirement for the ENTH-Rga2 physical interaction, indicating that Ent2 functions, at least in part, to enable proper activation of the Cdc42-Cla4 signaling pathway in the presence of a filament-inducing cue. Overall, this work characterizes the mechanism by which Ent2 regulates hyphal morphogenesis in C. albicans, unveils the importance of this factor in enabling virulence in an in vivo model of systemic candidiasis and adds to the growing understanding of the genetic control of a key virulence trait. IMPORTANCE Candida albicans is a leading human fungal pathogen that can cause life-threatening infections in immunocompromised individuals, with mortality rates of ~40%. The ability of this organism to grow in both yeast and filamentous forms is critical for the establishment of systemic infection. Genomic screens have identified many genes required for this morphological transition, yet our understanding of the mechanisms that regulate this key virulence trait remains incomplete. In this study, we characterized Ent2 as a core regulator of C. albicans morphogenesis. We show that Ent2 regulates hyphal morphogenesis through an interaction between its ENTH domain and the Cdc42 GAP, Rga2, which signals through the Cdc42-Cla4 signaling pathway. Finally, we show that the Ent2 protein, and specifically its ENTH domain, is required for virulence in a mouse model of systemic candidiasis. Overall, this work identifies Ent2 as a key regulator of filamentation and virulence in C. albicans.

The NleGs are the largest family of type 3 secreted effectors in attaching and effacing (A/E) pathogens, such as enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and Citrobacter rodentium. NleG effectors contain a conserved C-terminal U-box domain acting as a ubiquitin protein ligase and target host proteins via a variable N-terminal portion. The specific roles of these effectors during infection remain uncertain. Here, we demonstrate that the three NleG effectors-NleG1 Cr , NleG7 Cr , and NleG8 Cr -encoded by C. rodentium DBS100 play distinct roles during infection in mice. Using individual nleG Cr knockout strains, we show that NleG7 Cr contributes to bacterial survival during enteric infection while NleG1 Cr promotes the expression of diarrheal symptoms and NleG8 Cr contributes to accelerated lethality in susceptible mice. Furthermore, the NleG8 Cr effector contains a C-terminal PDZ domain binding motif that enables interaction with the host protein GOPC. Both the PDZ domain binding motif and the ability to engage with host ubiquitination machinery via the intact U-box domain proved to be necessary for NleG8 Cr function, contributing to the observed phenotype during infection. We also establish that the PTZ binding motif in the EHEC NleG8 (NleG8 Ec ) effector, which shares 60% identity with NleG8 Cr , is engaged in interactions with human GOPC. The crystal structure of the NleG8 Ec C-terminal peptide in complex with the GOPC PDZ domain, determined to 1.85 Å, revealed a conserved interaction mode similar to that observed between GOPC and eukaryotic PDZ domain binding motifs. Despite these common features, nleG8 Ec does not complement the Δ nleG8 Cr phenotype during infection, revealing functional diversification between these NleG effectors.

Rising concerns about climate change and sustainable energy have attracted efforts towards developing environmentally friendly alternatives to fossil fuels. Biosynthesis of n-butane, a highly desirable petro-chemical, fuel additive and diluent in the oil industry, remains a challenge. In this work, we first engineered enzymes Tes, Car and AD in the termination module to improve the selectivity of n-butane biosynthesis, and ancestral reconstruction and a synthetic RBS significantly improved the AD abundance. Next, we did ribosome binding site (RBS) calculation to identify potential metabolic bottlenecks, and then mitigated the bottleneck with RBS engineering and precursor propionyl-CoA addition. Furthermore, we employed a model-assisted strain design and a nonrepetitive extra-long sgRNA arrays (ELSAs) and quorum sensing assisted CRISPRi to facilitate a dynamic two-stage fermentation. Through systems engineering, n-butane production was increased by 168-fold from 0.04 to 6.74 mg/L. Finally, the maximum n-butane production from acetate was predicted using parsimonious flux balance analysis (pFBA), and we achieved n-butane production from acetate produced by electrocatalytic CO reduction. Our findings pave the way for selectively producing n-butane from renewable carbon source., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

Fungal pathogens are a continuing challenge due to few effective antifungals and a rise in resistance. In previous work, we described the inhibition of Candida albicans virulence following exposure to the 68 amino acid bacteriocin, EntV, secreted by Enterococcus faecalis. Here, to optimize EntV as a potential therapeutic and better understand its antifungal features, an X-ray structure is obtained. The structure consists of six alpha helices enclosing a seventh 16 amino acid helix (α7). The individual helices are tested for antifungal activity using in vitro and nematode infection assays. Interestingly, α7 retains antifungal, but not antibacterial activity and is also effective against Candida auris and Cryptococcus neoformans. Further reduction of α7 to 12 amino acids retains full antifungal activity, and excellent efficacy is observed in rodent models of C. albicans oropharyngeal, systemic, and venous catheter infections. Together, these results showcase EntV-derived peptides as promising candidates for antifungal therapeutic development., (© 2022. The Author(s).)

Culturing eukaryotic cells has widespread applications in research and industry, including the emerging field of cell-cultured meat production colloquially referred to as "cellular agriculture". These applications are often restricted by the high cost of growth medium necessary for cell growth. Mitogenic protein growth factors (GFs) are essential components of growth medium and account for upwards of 90% of the total costs. Here, we present a set of expression constructs and a simplified protocol for recombinant production of functionally active GFs, including FGF2, IGF1, PDGF-BB, and TGF-β1 in Escherichia coli . Using this E. coli expression system, we produced soluble GF orthologs from species including bovine, chicken, and salmon. Bioactivity analysis revealed orthologs with improved performance compared to commercially available alternatives. We estimated that the production cost of GFs using our methodology will significantly reduce the cost of cell culture medium, facilitating low-cost protocols tailored for cultured meat production and tissue engineering., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Transcription factor (TF)-based biosensors have been applied in biotechnology for a variety of functions, including protein engineering, dynamic control, environmental detection, and point-of-care diagnostics. Such biosensors are promising analytical tools due to their wide range of detectable ligands and modular nature. However, designing biosensors tailored for applications of interest with the desired performance parameters, including ligand specificity, remains challenging. Biosensors often require significant engineering and tuning to meet desired specificity, sensitivity, dynamic range, and operating range parameters. Another limitation is the orthogonality of biosensors across hosts, given the role of the cellular context. Here, we describe recent advances and examples in the engineering and optimization of TF-based biosensors for plug-and-play small molecule detection. We highlight novel developments in TF discovery and biosensor design, TF specificity engineering, and biosensor tuning, with emphasis on emerging computational methods., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In the human fungal pathogen Candida albicans , ARO1 encodes an essential multi-enzyme that catalyses consecutive steps in the shikimate pathway for biosynthesis of chorismate, a precursor to folate and the aromatic amino acids. We obtained the first molecular image of C. albicans Aro1 that reveals the architecture of all five enzymatic domains and their arrangement in the context of the full-length protein. Aro1 forms a flexible dimer allowing relative autonomy of enzymatic function of the individual domains. Our activity and in cellulo data suggest that only four of Aro1's enzymatic domains are functional and essential for viability of C. albicans , whereas the 3-dehydroquinate dehydratase (DHQase) domain is inactive because of active site substitutions. We further demonstrate that in C. albicans , the type II DHQase Dqd1 can compensate for the inactive DHQase domain of Aro1, suggesting an unrecognized essential role for this enzyme in shikimate biosynthesis. In contrast, in Candida glabrata and Candida parapsilosis , which do not encode a Dqd1 homolog, Aro1 DHQase domains are enzymatically active, highlighting diversity across Candida species., (© 2022 Stogios et al.)

Crop parasites of the Striga genera are a major biological deterrent to food security in Africa and are one of the largest obstacles to poverty alleviation on the continent. Striga seeds germinate by sensing small-molecule hormones, strigolactones (SLs), that emanate from host roots. Although SL receptors (Striga hermonthica HYPOSENSITIVE TO LIGHT [ShHTL]) have been identified, discerning their function has been difficult because these parasites cannot be easily grown under laboratory conditions. Moreover, many Striga species are obligate outcrossers that are not transformable, hence not amenable to genetic analysis. By combining phenotypic screening with ShHTL structural information and hybrid drug discovery methods, we discovered a potent SL perception inhibitor for Striga, dormirazine (DOZ). Structural analysis of this piperazine-based antagonist reveals a novel binding mechanism, distinct from that of known SLs, blocking access of the hormone to its receptor. Furthermore, DOZ reduces the flexibility of protein-protein interaction domains important for receptor signaling to downstream partners. In planta, we show, via temporal additions of DOZ, that SL receptors are required at a specific time during seed conditioning. This conditioning is essential to prime seed germination at the right time; thus, this SL-sensitive stage appears to be critical for adequate receptor signaling. Aside from uncovering a function for ShHTL during seed conditioning, these results suggest that future Ag-Biotech Solutions to Striga infestations will need to carefully time the application of antagonists to exploit receptor availability and outcompete natural SLs, critical elements for successful parasitic plant invasions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an aac(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment., (© 2022. The Author(s).)

The fungus Candida albicans is an opportunistic pathogen that can exploit imbalances in microbiome composition to invade its human host, causing pathologies ranging from vaginal candidiasis to fungal sepsis. Bacteria of the genus Lactobacillus are colonizers of human mucosa and can produce compounds with bioactivity against C. albicans. Here, we show that some Lactobacillus species produce a small molecule under laboratory conditions that blocks the C. albicans yeast-to-filament transition, an important virulence trait. It remains unexplored whether the compound is produced in the context of the human host. Bioassay-guided fractionation of Lactobacillus-conditioned medium linked this activity to 1-acetyl-β-carboline (1-ABC). We use genetic approaches to show that filamentation inhibition by 1-ABC requires Yak1, a DYRK1-family kinase. Additional biochemical characterization of structurally related 1-ethoxycarbonyl-β-carboline confirms that it inhibits Yak1 and blocks C. albicans biofilm formation. Thus, our findings reveal Lactobacillus-produced 1-ABC can prevent the yeast-to-filament transition in C. albicans through inhibition of Yak1., (© 2021. The Author(s).)

Uncovering the basis of small-molecule hormone receptors' evolution is paramount to a complete understanding of how protein structure drives function. In plants, hormone receptors for strigolactones are well suited to evolutionary inquiries because closely related homologs have different ligand preferences. More importantly, because of facile plant transgenic systems, receptors can be swapped and quickly assessed functionally in vivo. Here, we show that only three mutations are required to turn the nonstrigolactone receptor, KAI2, into a receptor that recognizes the plant hormone strigolactone. This modified receptor still retains its native function to perceive KAI2 ligands. Our directed evolution studies indicate that only a few keystone mutations are required to increase receptor promiscuity of KAI2, which may have implications for strigolactone receptor evolution in parasitic plants., Competing Interests: The authors declare no competing interest.

The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 α-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an α-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The α-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the α-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Apramycin is an aminoglycoside antibiotic with the potential to be developed to combat multidrug-resistant pathogens. Its unique structure evades the clinically widespread mechanisms of aminoglycoside resistance that currently compromise the efficacy of other members in this drug class. Of the aminoglycoside-modifying enzymes that chemically alter these antibiotics, only AAC(3)-IVa has been demonstrated to confer resistance to apramycin through N -acetylation. Knowledge of other modification mechanisms is important to successfully develop apramycin for clinical use. Here, we show that ApmA is structurally unique among the previously described aminoglycoside-modifying enzymes and capable of conferring a high level of resistance to apramycin. In vitro experiments indicated ApmA to be an N -acetyltransferase, but in contrast to AAC(3)-IVa, ApmA has a unique regiospecificity of the acetyl transfer to the N2' position of apramycin. Crystallographic analysis of ApmA conclusively showed that this enzyme is an acetyltransferase from the left-handed β-helix protein superfamily (LβH) with a conserved active site architecture. The success of apramycin will be dependent on consideration of the impact of this potential form of clinical resistance. IMPORTANCE Apramycin is an aminoglycoside antibiotic that has been traditionally used in veterinary medicine. Recently, it has become an attractive candidate to repurpose in the fight against multidrug-resistant pathogens prioritized by the World Health Organization. Its atypical structure circumvents most of the clinically relevant mechanisms of resistance that impact this class of antibiotics. Prior to repurposing apramycin, it is important to understand the resistance mechanisms that could be a liability. Our study characterizes the most recently identified apramycin resistance element, apmA We show ApmA does not belong to the protein families typically associated with aminoglycoside resistance and is responsible for modifying a different site on the molecule. The data presented will be critical in the development of apramycin derivatives that will evade apmA in the event it becomes prevalent in the clinic., (Copyright © 2021 Bordeleau et al.)

New strategies are urgently needed to counter the threat to human health posed by drug-resistant fungi. To explore an as-yet unexploited target space for antifungals, we screened a library of protein kinase inhibitors for the ability to reverse resistance of the most common human fungal pathogen, Candida albicans, to caspofungin, a widely used antifungal. This screen identified multiple 2,3-aryl-pyrazolopyridine scaffold compounds capable of restoring caspofungin sensitivity. Using chemical genomic, biochemical, and structural approaches, we established the target for our most potent compound as Yck2, a casein kinase 1 family member. Combination of this compound with caspofungin eradicated drug-resistant C. albicans infection while sparing co-cultured human cells. In mice, genetic depletion of YCK2 caused an ∼3-log 10 decline in fungal burden in a model of systemic caspofungin-resistant C. albicans infection. Structural insights and our tool compound's profile in culture support targeting the Yck2 kinase function as a broadly active antifungal strategy., Competing Interests: Declaration of Interests L.E.C. and L.W. are co-founders and shareholders in Bright Angel Therapeutics, a platform company for development of novel antifungal therapeutics. L.E.C. is a consultant for Boragen, a small-molecule development company focused on leveraging the unique chemical properties of boron chemistry for crop protection and animal health., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A previous soil metagenomics study recovered a novel cephalosporin resistance determinant, pbp TET A6 , for which the exact resistance mechanism was unclear. This study used a three-dimensional structure-guided mutagenesis approach to demonstrate that PBP TET A6 is likely to be a class A penicillin-binding protein (PBP), and that its ability to confer cephalosporin resistance is directly linked to the functional integrity of its transpeptidase (TP) catalytic core. Screening of a library of PBP TET A6 variants carrying randomly introduced point mutations revealed additional residue modifications that compromised resistance, all of which were proximal to the TP active site except one which was found in a 29-amino-acid-long superstructure (α6-α7 loop) absent in other class A PBP homologues. Based on the site-specific mutagenesis results, it is hypothesized that residue arginine-400 plays an important role in limiting the access of certain cephalosporin compounds to the enzymatic core of the TP domain of PBP TET A6 . Using a combination of adaptive evolution assays and whole-genome sequencing, the potential impact of PBP TET A6 on promoting the development of resistance in the clinically significant opportunistic pathogen Pseudomonas aeruginosa was investigated. Under the selective pressure of serial ceftazidime exposures, the pbp TET A6 -expressing P. aeruginosa population readily evolved by excluding a ~400-kbp chromosomal element to acquire additional resistance against cephalosporins, suggesting that PBP TET A6 has a catalytic effect on facilitating antibiotic-resistance-associated genome adaptation. Overall, the soil environment contains genes conferring resistance to critically important antibiotics by cryptic mechanisms. Understanding what impact anthropogenic activities might have on the abundance and evolution of these genes should be a priority., (Copyright © 2020. Published by Elsevier B.V.)

Vancomycin and related glycopeptides are drugs of last resort for the treatment of severe infections caused by Gram-positive bacteria such as Enterococcus species, Staphylococcus aureus, and Clostridium difficile. Vancomycin was long considered immune to resistance due to its bactericidal activity based on binding to the bacterial cell envelope rather than to a protein target as is the case for most antibiotics. However, two types of complex resistance mechanisms, each comprised of a multi-enzyme pathway, emerged and are now widely disseminated in pathogenic species, thus threatening the clinical efficiency of vancomycin. Vancomycin forms an intricate network of hydrogen bonds with the d-Ala-d-Ala region of Lipid II, interfering with the peptidoglycan layer maturation process. Resistance to vancomycin involves degradation of this natural precursor and its replacement with d-Ala-d-lac or d-Ala-d-Ser alternatives to which vancomycin has low affinity. Through extensive research over 30 years after the initial discovery of vancomycin resistance, remarkable progress has been made in molecular understanding of the enzymatic cascades responsible. Progress has been driven by structural studies of the key components of the resistance mechanisms which provided important molecular understanding such as, for example, the ability of this cascade to discriminate between vancomycin sensitive and resistant peptidoglycan precursors. Important structural insights have been also made into the molecular evolution of vancomycin resistance enzymes. Altogether this molecular data can accelerate inhibitor discovery and optimization efforts to reverse vancomycin resistance. Here, we overview our current understanding of this complex resistance mechanism with a focus on the structural and molecular aspects., (© 2020 The Protein Society.)

Acyl-coenzyme A-dependent N-acetyltransferases (AACs) catalyze the modification of aminoglycosides rendering the bacteria carrying such enzymes resistant to this class of antibiotics. Here we present the crystal structure of AAC(3)-Ia enzyme from Serratia marcescens in complex with coenzyme A determined to 1.8 Å resolution. This enzyme served as an architype for the AAC enzymes targeting the amino group at Position 3 of aminoglycoside main aminocyclitol ring. The structure of this enzyme has been previously determined only in truncated form and was interpreted as distinct from subsequently characterized AACs. The reason for the unusual arrangement of secondary structure elements of AAC(3)-Ia was not further investigated. By determining the full-length structure of AAC(3)-Ia we establish that this enzyme adopts the canonical AAC fold conserved across this family and it does not undergo through significant rearrangement of secondary structure elements upon ligand binding as was proposed previously. In addition, our results suggest that the C-terminal tail in AAC(3)-Ia monomer forms intramolecular hydrogen bonds that contributes to formation of stable dimer, representing the predominant oligomeric state for this enzyme., (© 2019 The Protein Society.)

Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of ( R )-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans , TM1559 from Thermotoga maritima , and DeoC from Escherichia coli The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr 224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys 155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis., (© 2020 Kim et al.)

Bacteria have evolved sophisticated mechanisms to inhibit the growth of competitors 1 . One such mechanism involves type VI secretion systems, which bacteria can use to inject antibacterial toxins directly into neighbouring cells. Many of these toxins target the integrity of the cell envelope, but the full range of growth inhibitory mechanisms remains unknown 2 . Here we identify a type VI secretion effector, Tas1, in the opportunistic pathogen Pseudomonas aeruginosa. The crystal structure of Tas1 shows that it is similar to enzymes that synthesize (p)ppGpp, a broadly conserved signalling molecule in bacteria that modulates cell growth rate, particularly in response to nutritional stress 3 . However, Tas1 does not synthesize (p)ppGpp; instead, it pyrophosphorylates adenosine nucleotides to produce (p)ppApp at rates of nearly 180,000 molecules per minute. Consequently, the delivery of Tas1 into competitor cells drives rapid accumulation of (p)ppApp, depletion of ATP, and widespread dysregulation of essential metabolic pathways, thereby resulting in target cell death. Our findings reveal a previously undescribed mechanism for interbacterial antagonism and demonstrate a physiological role for the metabolite (p)ppApp in bacteria.

Glycoside hydrolase family 74 (GH74) is a historically important family of endo -β-glucanases. On the basis of early reports of detectable activity on cellulose and soluble cellulose derivatives, GH74 was originally considered to be a "cellulase" family, although more recent studies have generally indicated a high specificity toward the ubiquitous plant cell wall matrix glycan xyloglucan. Previous studies have indicated that GH74 xyloglucanases differ in backbone cleavage regiospecificities and can adopt three distinct hydrolytic modes of action: exo , endo -dissociative, and endo -processive. To improve functional predictions within GH74, here we coupled in-depth biochemical characterization of 17 recombinant proteins with structural biology-based investigations in the context of a comprehensive molecular phylogeny, including all previously characterized family members. Elucidation of four new GH74 tertiary structures, as well as one distantly related dual seven-bladed β-propeller protein from a marine bacterium, highlighted key structure-function relationships along protein evolutionary trajectories. We could define five phylogenetic groups, which delineated the mode of action and the regiospecificity of GH74 members. At the extremes, a major group of enzymes diverged to hydrolyze the backbone of xyloglucan nonspecifically with a dissociative mode of action and relaxed backbone regiospecificity. In contrast, a sister group of GH74 enzymes has evolved a large hydrophobic platform comprising 10 subsites, which facilitates processivity. Overall, the findings of our study refine our understanding of catalysis in GH74, providing a framework for future experimentation as well as for bioinformatics predictions of sequences emerging from (meta)genomic studies., (© 2019 Arnal et al.)

Paenibacillus odorifer produces a single multimodular enzyme containing a glycoside hydrolase (GH) family 74 module (AIQ73809). Recombinant production and characterization of the GH74 module ( Po GH74 cat ) revealed a highly specific, processive endo -xyloglucanase that can hydrolyze the polysaccharide backbone at both branched and unbranched positions. X-ray crystal structures obtained for the free enzyme and oligosaccharide complexes evidenced an extensive hydrophobic binding platform - the first in GH74 extending from subsites -4 to +6 - and unique mobile active-site loops. Site-directed mutagenesis revealed that glycine-476 was uniquely responsible for the promiscuous backbone-cleaving activity of Po GH74 cat ; replacement with tyrosine, which is conserved in many GH74 members, resulted in exclusive hydrolysis at unbranched glucose units. Likewise, systematic replacement of the hydrophobic platform residues constituting the positive subsites indicated their relative contributions to the processive mode of action. Specifically, W347 (+3 subsite) and W348 (+5 subsite) are essential for processivity, while W406 (+2 subsite) and Y372 (+6 subsite) are not strictly essential, but aid processivity., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Src homology 2 (SH2) domains play a critical role in signal transduction in mammalian cells by binding to phosphorylated Tyr (pTyr). Apart from a few isolated cases in viruses, no functional SH2 domain has been identified to date in prokaryotes. Here we identify 93 SH2 domains from Legionella that are distinct in sequence and specificity from mammalian SH2 domains. The bacterial SH2 domains are not only capable of binding proteins or peptides in a Tyr phosphorylation-dependent manner, some bind pTyr itself with micromolar affinities, a property not observed for mammalian SH2 domains. The Legionella SH2 domains feature the SH2 fold and a pTyr-binding pocket, but lack a specificity pocket found in a typical mammalian SH2 domain for recognition of sequences flanking the pTyr residue. Our work expands the boundary of phosphotyrosine signalling to prokaryotes, suggesting that some bacterial effector proteins have acquired pTyr-superbinding characteristics to facilitate bacterium-host interactions.

Plazomicin is a next-generation, semisynthetic aminoglycoside antibiotic currently under development for the treatment of infections due to multidrug-resistant Enterobacteriaceae. The compound was designed by chemical modification of the natural product sisomicin to provide protection from common aminoglycoside modifying enzymes that chemically alter these drugs via N-acetylation, O-adenylylation, or O-phosphorylation. In this study, plazomicin was profiled against a panel of isogenic strains of Escherichia coli individually expressing twenty-one aminoglycoside resistance enzymes. Plazomicin retained antibacterial activity against 15 of the 17 modifying enzyme-expressing strains tested. Expression of only two of the modifying enzymes, aac(2')-Ia and aph(2″)-IVa, decreased plazomicin potency. On the other hand, expression of 16S rRNA ribosomal methyltransferases results in a complete lack of plazomicin potency. In vitro enzymatic assessment confirmed that AAC(2')-Ia and APH(2'')-IVa (aminoglycoside acetyltransferase, AAC; aminoglycoside phosphotransferase, APH) were able to utilize plazomicin as a substrate. AAC(2')-Ia and APH(2'')-IVa are limited in their distribution to Providencia stuartii and Enterococci, respectively. These data demonstrate that plazomicin is not modified by a broad spectrum of common aminoglycoside modifying enzymes including those commonly found in Enterobacteriaceae. However, plazomicin is inactive in the presence of 16S rRNA ribosomal methyltransferases, which should be monitored in future surveillance programs.

Lipid A phosphoethanolamine (PEtN) transferases render bacteria resistant to the last resort antibiotic colistin. The recent discoveries of pathogenic bacteria harboring plasmid-borne PEtN transferase ( mcr) genes have illustrated the serious potential for wide dissemination of these resistance elements. The origin of mcr-1 is traced to Moraxella species co-occupying environmental niches with Enterobacteriaceae. Here, we describe the crystal structure of the catalytic domain of the chromosomally encoded colistin resistance PEtN transferase, ICR Mc (for intrinsic colistin resistance) of Moraxella catarrhalis. The ICR Mc structure in complex with PEtN reveals key molecular details including specific residues involved in catalysis and PEtN binding. It also demonstrates that ICR Mc catalytic domain dimerization is required for substrate binding. Our structure-guided phylogenetic analysis provides sequence signatures defining potentially colistin-active representatives in this enzyme family. Combined, these results advance the molecular and mechanistic understanding of PEtN transferases and illuminate their origins.

Background: Acetylated 4- O -(methyl)glucuronoxylan (GX) is the main hemicellulose in deciduous hardwood, and comprises a β-(1→4)-linked xylopyranosyl (Xyl p ) backbone substituted by both acetyl groups and α-(1→2)-linked 4- O -methylglucopyranosyluronic acid (MeGlc p A). Whereas enzymes that target singly acetylated Xyl p or doubly 2,3- O -acetyl-Xyl p have been well characterized, those targeting (2- O -MeGlc p A)3- O -acetyl-Xyl p structures in glucuronoxylan have remained elusive., Results: An unclassified carbohydrate esterase (FjoAcXE) was identified as a protein of unknown function from a polysaccharide utilization locus (PUL) otherwise comprising carbohydrate-active enzyme families known to target xylan. FjoAcXE was shown to efficiently release acetyl groups from internal (2- O -MeGlc p A)3- O -acetyl-Xyl p structures, an activity that has been sought after but lacking in known carbohydrate esterases. FjoAcXE action boosted the activity of α-glucuronidases from families GH67 and GH115 by five and nine times, respectively. Moreover, FjoAcXE activity was not only restricted to GX, but also deacetylated (3- O -Ara f )2- O -acetyl-Xyl p of feruloylated xylooligomers, confirming the broad substrate range of this new carbohydrate esterase., Conclusion: This study reports the discovery and characterization of the novel carbohydrate esterase, FjoAcXE. In addition to cleaving singly acetylated Xyl p , and doubly 2,3- O -acetyl-Xyl p , FjoAcXE efficiently cleaves internal 3- O -acetyl-Xyl p linkages in (2- O -MeGlc p A)3- O -acetyl-Xyl p residues along with densely substituted and branched xylooligomers; activities that until now were missing from the arsenal of enzymes required for xylan conversion.

Host colonization by Gram-negative pathogens often involves delivery of bacterial proteins called "effectors" into the host cell. The pneumonia-causing pathogen Legionella pneumophila delivers more than 330 effectors into the host cell via its type IVB Dot/Icm secretion system. The collective functions of these proteins are the establishment of a replicative niche from which Legionella can recruit cellular materials to grow while evading lysosomal fusion inhibiting its growth. Using a combination of structural, biochemical, and in vivo approaches, we show that one of these translocated effector proteins, Ceg4, is a phosphotyrosine phosphatase harboring a haloacid dehalogenase-hydrolase domain. Ceg4 could dephosphorylate a broad range of phosphotyrosine-containing peptides in vitro and attenuated activation of MAPK-controlled pathways in both yeast and human cells. Our findings indicate that L. pneumophila 's infectious program includes manipulation of phosphorylation cascades in key host pathways. The structural and functional features of the Ceg4 effector unraveled here provide first insight into its function as a phosphotyrosine phosphatase, paving the way to further studies into L. pneumophila pathogenicity.

Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.

The production of antibiotics by microbes in the environment and their use in medicine and agriculture select for existing and emerging resistance. To address this inevitability, prudent development of antibiotic drugs requires careful consideration of resistance evolution. Here, we identify the molecular basis for expanded substrate specificity in MphI, a macrolide kinase (Mph) that does not confer resistance to erythromycin, in contrast to other known Mphs. Using a combination of phylogenetics, drug-resistance phenotypes, and in vitro enzyme assays, we find that MphI and MphK phosphorylate erythromycin poorly resulting in an antibiotic-sensitive phenotype. Using likelihood reconstruction of ancestral sequences and site-saturation combinatorial mutagenesis, supported by Mph crystal structures, we determine that two non-obvious mutations in combination expand the substrate range. This approach should be applicable for studying the functional evolution of any antibiotic resistance enzyme and for evaluating the evolvability of resistance enzymes to new generations of antibiotic scaffolds.

Aminoglycoside N-acetyltransferases (AACs) confer resistance against the clinical use of aminoglycoside antibiotics. The origin of AACs can be traced to environmental microbial species representing a vast reservoir for new and emerging resistance enzymes, which are currently undercharacterized. Here, we performed detailed structural characterization and functional analyses of four metagenomic AAC (meta-AACs) enzymes recently identified in a survey of agricultural and grassland soil microbiomes ( Forsberg et al. Nature 2014 , 509 , 612 ). These enzymes are new members of the Gcn5-Related-N-Acetyltransferase superfamily and confer resistance to the aminoglycosides gentamicin C, sisomicin, and tobramycin. Moreover, the meta-AAC0020 enzyme demonstrated activity comparable with an AAC(3)-I enzyme that serves as a model AAC enzyme identified in a clinical bacterial isolate. The crystal structure of meta-AAC0020 in complex with sisomicin confirmed an unexpected AAC(6') regiospecificity of this enzyme and revealed a drug binding mechanism distinct from previously characterized AAC(6') enzymes. Together, our data highlights the presence of highly active antibiotic-modifying enzymes in the environmental microbiome and reveals unexpected diversity in substrate specificity. These observations of additional AAC enzymes must be considered in the search for novel aminoglycosides less prone to resistance.

Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6') subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6')-Ig and AAC(6')-Ih from Acinetobacter spp. The crystal structure of AAC(6')-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6')-Ib enzyme. Enzymatic analysis indicated that AAC(6')-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6') enzymes. Structure- and function-informed phylogenetic analysis of AAC(6') enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6') functionality originating through convergent evolution from diverse Gcn5-related-N-acetyltransferase (GNAT) ancestral enzymes, with AAC(6')-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties.

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds.

Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

Unlabelled: Sinorhizobium meliloti forms N2-fixing root nodules on alfalfa, and as a free-living bacterium, it can grow on a very broad range of substrates, including l-proline and several related compounds, such as proline betaine, trans-4-hydroxy-l-proline (trans-4-l-Hyp), and cis-4-hydroxy-d-proline (cis-4-d-Hyp). Fourteen hyp genes are induced upon growth of S. meliloti on trans-4-l-Hyp, and of those, hypMNPQ encodes an ABC-type trans-4-l-Hyp transporter and hypRE encodes an epimerase that converts trans-4-l-Hyp to cis-4-d-Hyp in the bacterial cytoplasm. Here, we present evidence that the HypO, HypD, and HypH proteins catalyze the remaining steps in which cis-4-d-Hyp is converted to α-ketoglutarate. The HypO protein functions as a d-amino acid dehydrogenase, converting cis-4-d-Hyp to Δ(1)-pyrroline-4-hydroxy-2-carboxylate, which is deaminated by HypD to α-ketoglutarate semialdehyde and then converted to α-ketoglutarate by HypH. The crystal structure of HypD revealed it to be a member of the N-acetylneuraminate lyase subfamily of the (α/β)8 protein family and is consistent with the known enzymatic mechanism for other members of the group. It was also shown that S. meliloti can catabolize d-proline as both a carbon and a nitrogen source, that d-proline can complement l-proline auxotrophy, and that the catabolism of d-proline is dependent on the hyp cluster. Transport of d-proline involves the HypMNPQ transporter, following which d-proline is converted to Δ(1)-pyrroline-2-carboxylate (P2C) largely via HypO. The P2C is converted to l-proline through the NADPH-dependent reduction of P2C by the previously uncharacterized HypS protein. Thus, overall, we have now completed detailed genetic and/or biochemical characterization of 9 of the 14 hyp genes., Importance: Hydroxyproline is abundant in proteins in animal and plant tissues and serves as a carbon and a nitrogen source for bacteria in diverse environments, including the rhizosphere, compost, and the mammalian gut. While the main biochemical features of bacterial hydroxyproline catabolism were elucidated in the 1960s, the genetic and molecular details have only recently been determined. Elucidating the genetics of hydroxyproline catabolism will aid in the annotation of these genes in other genomes and metagenomic libraries. This will facilitate an improved understanding of the importance of this pathway and may assist in determining the prevalence of hydroxyproline in a particular environment., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

CouR, a MarR-type transcriptional repressor, regulates the cou genes, encoding p-hydroxycinnamate catabolism in the soil bacterium Rhodococcus jostii RHA1. The CouR dimer bound two molecules of the catabolite p-coumaroyl-CoA (Kd = 11 ± 1 μM). The presence of p-coumaroyl-CoA, but neither p-coumarate nor CoASH, abrogated CouR's binding to its operator DNA in vitro. The crystal structures of ligand-free CouR and its p-coumaroyl-CoA-bound form showed no significant conformational differences, in contrast to other MarR regulators. The CouR-p-coumaroyl-CoA structure revealed two ligand molecules bound to the CouR dimer with their phenolic moieties occupying equivalent hydrophobic pockets in each protomer and their CoA moieties adopting non-equivalent positions to mask the regulator's predicted DNA-binding surface. More specifically, the CoA phosphates formed salt bridges with predicted DNA-binding residues Arg36 and Arg38, changing the overall charge of the DNA-binding surface. The substitution of either arginine with alanine completely abrogated the ability of CouR to bind DNA. By contrast, the R36A/R38A double variant retained a relatively high affinity for p-coumaroyl-CoA (Kd = 89 ± 6 μM). Together, our data point to a novel mechanism of action in which the ligand abrogates the repressor's ability to bind DNA by steric occlusion of key DNA-binding residues and charge repulsion of the DNA backbone., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)