Crystal structures and biochemical characterization of DNA sliding clamps from three Gram-negative bacterial pathogens

Abstract Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3′-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3′ CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH2 of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.

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Identification and Biochemical Characterization of a Novel Protein Phosphatase 2C-Like Ser/Thr Phosphatase in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00225-18 ◽

2018 ◽

Vol 200 (18) ◽

Cited By ~ 9

Author(s):

Krithika Rajagopalan ◽

Elizabeth Nagle ◽

Jonathan Dworkin

Keyword(s):

Escherichia Coli ◽

Protein Phosphatase ◽

Biochemical Characterization ◽

Regulatory Protein ◽

Negative Bacterium ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Novel Protein

Regulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria, including the model Gram-negative bacteriumEscherichia coli, demonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thr phosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

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First biochemical and crystallographic characterization of a fast-performing ferritin from a marine invertebrate

Biochemical Journal ◽

10.1042/bcj20170681 ◽

2017 ◽

Vol 474 (24) ◽

pp. 4193-4206 ◽

Cited By ~ 8

Author(s):

Evelien De Meulenaere ◽

Jake Brian Bailey ◽

Faik Akif Tezcan ◽

Dimitri Dominique Deheyn

Keyword(s):

Crystal Structures ◽

Reaction Rate ◽

Biochemical Characterization ◽

Marine Invertebrate ◽

Catalytic Performance ◽

Catalytic Sites ◽

Ferroxidase Activity ◽

Biochemical Analyses ◽

And Storage

Ferritin, a multimeric cage-like enzyme, is integral to iron metabolism across all phyla through the sequestration and storage of iron through efficient ferroxidase activity. While ferritin sequences from ∼900 species have been identified, crystal structures from only 50 species have been reported, the majority from bacterial origin. We recently isolated a secreted ferritin from the marine invertebrate Chaetopterus sp. (parchment tube worm), which resides in muddy coastal seafloors. Here, we present the first ferritin from a marine invertebrate to be crystallized and its biochemical characterization. The initial ferroxidase reaction rate of recombinant Chaetopterus ferritin (ChF) is 8-fold faster than that of recombinant human heavy-chain ferritin (HuHF). To our knowledge, this protein exhibits the fastest catalytic performance ever described for a ferritin variant. In addition to the high-velocity ferroxidase activity, ChF is unique in that it is secreted by Chaetopterus in a bioluminescent mucus. Previous work has linked the availability of Fe2+ to this long-lived bioluminescence, suggesting a potential function for the secreted ferritin. Comparative biochemical analyses indicated that both ChF and HuHF showed similar behavior toward changes in pH, temperature, and salt concentration. Comparison of their crystal structures shows no significant differences in the catalytic sites. Notable differences were found in the residues that line both 3-fold and 4-fold pores, potentially leading to increased flexibility, reduced steric hindrance, or a more efficient pathway for Fe2+ transportation to the ferroxidase site. These suggested residues could contribute to the understanding of iron translocation through the ferritin shell to the ferroxidase site.

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FplA FromFusobacterium nucleatumis a Type Vd autotransporter phospholipase with a proposed role in altered host signaling and evasion of autophagy

10.1101/136598 ◽

2017 ◽

Cited By ~ 1

Author(s):

Michael A. Casasanta ◽

Christopher C. Yoo ◽

Hans B. Smith ◽

A. Jane Duncan ◽

Kyla Cochrane ◽

...

Keyword(s):

Colorectal Cancer ◽

Outer Membrane ◽

Virulence Factors ◽

Biochemical Characterization ◽

Bacterial Virulence ◽

Chemical Probes ◽

Gram Negative ◽

Enzyme Family ◽

Host Signaling

ABSTRACTFusobacterium nucleatumis a pathogenic oral bacterium that is linked to multiple human infections and colorectal cancer. While most Gram-negative pathogens utilize secretion systems for cellular invasion and infection,F. nucleatumlacks Type I, II, III, IV, and VI secretion. By contrast,F. nucleatumstrains are enriched in Type V secreted autotransporters, which are Gram-negative bacterial virulence factors critical for binding and entry into host cells. Here we present the first biochemical characterization of aF. nucleatumType Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby renameFusobacteriumphospholipase autotransporter (FplA). FplA is expressed as a full-length 85 kDa outer membrane embedded protein, or as a truncated phospholipase domain that remains associated with the outer membrane. Using multiple FplA constructs we characterized lipid substrate specificity, potent inhibitors, and chemical probes to detect and track this enzyme family. While the role of FplA is undetermined inF. nucleatumvirulence, homologous phospholipases from intracellular pathogens are critical for vacuole escape, altered host signaling, and intracellular survival. We hypothesize that upon intracellular invasion of the host, FplA could play a role in phagosomal escape, subversion of autophagy, or eicosanoid-mediated inflammatory signaling, as we show that FplA binds with high affinity to host phosphoinositide signaling lipids critical to these processes. Our identification of substrates, inhibitors, and chemical probes for FplA, in combination with anfplAgene deletion strain, encompass a powerful set of tools for the future analysis of FplAin vivo. In addition, these studies will guide the biochemical characterization of additional Type Vd autotransporter phospholipases.IMPORTANCEF. nucleatumis an emerging pathogen that is linked to the pathogenesis of colorectal cancer, yet there is a critical knowledge gap in the mechanisms used by this bacterium to elicit changes in the host for intracellular entry and survival. As phospholipases are critical virulence factors for intracellular bacteria to initiate vacuole lysis, cell-to-cell spread, and evasion of autophagy, we set out to characterize a unique Type Vd secreted phospholipase A1 enzyme fromF. nucleatum. Our results show a potential role for modulating host signaling pathways through cleavage of phosphoinositide dependent signaling lipids. These studies open the door for further characterization of this unique enzyme family in bacterial virulence, host-pathogen interactions, and forF. nucleatum, in colorectal carcinogenesis.

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Mutasynthetic Production and Antimicrobial Characterization of Darobactin Analogs

Microbiology Spectrum ◽

10.1128/spectrum.01535-21 ◽

2021 ◽

Vol 9 (3) ◽

Author(s):

Nils Böhringer ◽

Robert Green ◽

Yang Liu ◽

Ute Mettal ◽

Michael Marner ◽

...

Keyword(s):

Antibiotic Resistance ◽

Heterologous Expression ◽

Bacterial Pathogens ◽

Therapeutic Options ◽

The Novel ◽

Proof Of Concept ◽

Gram Negative ◽

Pharmacokinetic Properties

Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties.

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Characterization of two relacidines belonging to a novel class of circular lipopeptides that act against Gram‐negative bacterial pathogens

Environmental Microbiology ◽

10.1111/1462-2920.15145 ◽

2020 ◽

Vol 22 (12) ◽

pp. 5125-5136 ◽

Cited By ~ 1

Author(s):

Zhibo Li ◽

Parichita Chakraborty ◽

Reinder H. Vries ◽

Chunxu Song ◽

Xinghong Zhao ◽

...

Keyword(s):

Bacterial Pathogens ◽

Gram Negative

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Genetic and Biochemical Characterization of an Acquired Subgroup B3 Metallo-β-Lactamase Gene,blaAIM-1, and Its Unique Genetic Context in Pseudomonas aeruginosa from Australia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05654-11 ◽

2012 ◽

Vol 56 (12) ◽

pp. 6154-6159 ◽

Cited By ~ 46

Author(s):

Dongeun Yong ◽

Mark A. Toleman ◽

Jan Bell ◽

Brett Ritchie ◽

Rachael Pratt ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Southern Hybridization ◽

Biochemical Characterization ◽

Gene Bank ◽

Clinical Isolates ◽

Gram Negative ◽

Content Type ◽

Lactamase Gene ◽

Genetic Context

ABSTRACTThree clinicalPseudomonas aeruginosaisolates (WCH2677, WCH2813, and WCH2837) isolated from the Women's and Children's Hospital, Adelaide, Australia, produced a metallo-β-lactamase (MBL)-positive Etest result. All isolates were PCR negative for known MBL genes. A gene bank was created, and an MBL gene, designatedblaAIM-1, was cloned and fully characterized. The encoded enzyme, AIM-1, is a group B3 MBL that has the highest level of identity to THIN-B and L1. It is chromosomal and flanked by two copies (one intact and one truncated) of an ISCRelement, ISCR15. Southern hybridization studies indicated the movement of both ISCR15andblaAIM-1within the three different clinical isolates. AIM-1 hydrolyzes most β-lactams, with the exception of aztreonam and, to a lesser extent, ceftazidime; however, it possesses significantly higherkcatvalues for cefepime and carbapenems than most other MBLs. AIM-1 was the first mobile group B3 enzyme detected and signals further problems for already beleaguered antimicrobial regimes to treat seriousP. aeruginosaand other Gram-negative infections.

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X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798317013353 ◽

2017 ◽

Vol 73 (10) ◽

pp. 822-828 ◽

Cited By ~ 8

Author(s):

Harry B. Gristick ◽

Haoqing Wang ◽

Pamela J. Bjorkman

Keyword(s):

Crystal Structures ◽

Biochemical Characterization ◽

Complex Type ◽

Cryo Electron Microscopy ◽

High Mannose ◽

Complex Glycans ◽

Anti Hiv ◽

Hiv 1 ◽

Trimeric Envelope

The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy.

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Cultural and Biochemical Characterization of Actinobacillus and Actinobacillus-Like Species from Ram Lambs with Epididymitis

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063878900100402 ◽

1989 ◽

Vol 1 (4) ◽

pp. 288-294 ◽

Cited By ~ 5

Author(s):

Charles M. Scanlan ◽

Mark C. Healey ◽

Anthony R. Torres ◽

Alice V. Johnston

Keyword(s):

Biochemical Characterization ◽

Culture Collection ◽

Carbohydrate Utilization ◽

Gram Negative ◽

As Species ◽

Atcc Strain ◽

Yellow Color ◽

Group A ◽

Group B

Cellular, colonial, cultural, and biochemical characteristics of 25 field strains of gram-negative pleomorphic bacilli from rams with epididymitis were compared with Actinobacillus actinomycetemcomitans American Type Culture Collection (ATCC) strain 29522 and Actinobacillus seminis ATCC strain 15768. Three field strains were identified as A. actinomycetemcomitans, 15 as A. seminis, and 2 as Haemophilus agni; however, 5 strains (3 in group A and 2 in group B) were not identified as species in the genera Actinobacillus, Haemophilus, or Pasteurella based on the taxonomic criteria in Bergey's manual of systematic bacteriology. The 5 Actinobacillus-like organisms in groups A and B were predominantly gram-negative coccobacilli and exhibited less pleomorphism than the 2 Actinobacillus species. The colonial morphologies of groups A and B were similar to the 2 Actinobacillus species but were smaller in diameter and had a pale yellow color. Groups A and B, like the actinobacilli, were facultative anaerobic and capnophilic, did not grow on MacConkey agar, and were catalase-positive and oxidase-positive. Group A reduced nitrate but group B did not. The A. seminis strains utilized ornithine, and group A utilized arginine; but group B did not utilize either omithine or arginine. All strains failed to utilize lysine or tryptophane. All strains produced acid but no gas from glucose, and the utilization of other carbohydrates varied markedly both between and within the 5 groups of bacteria. Based on carbohydrate utilization profiles, the 15 field strains of A. seminis represented 13 biotypes, but with a commercial semi-quantitative enzyme system only 1 biotype was identified. All the A. actinomycetemcomitans, A. seminis, and group B strains produced moderate to high levels of alkaline phosphatase and acid phosphatase, but the group A strains were negative. Groups A and B were negative for beta-glucuronidase, but all the A. actinomycetemcomitans and A. seminis strains produced this enzyme.

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