Journal articles on the topic 'Nutrient PBP (periplasmic binding protein)'
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1
Meyer, Thibault, Armelle Vigouroux, Magali Aumont-Nicaise, Gilles Comte, Ludovic Vial, Céline Lavire, and Solange Moréra. "The plant defense signal galactinol is specifically used as a nutrient by the bacterial pathogen Agrobacterium fabrum." Journal of Biological Chemistry 293, no. 21 (March 30, 2018): 7930–41. http://dx.doi.org/10.1074/jbc.ra118.001856.
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The bacterial plant pathogen Agrobacterium fabrum uses periplasmic-binding proteins (PBPs) along with ABC transporters to import a wide variety of plant molecules as nutrients. Nonetheless, how A. fabrum acquires plant metabolites is incompletely understood. Using genetic approaches and affinity measurements, we identified here the PBP MelB and its transporter as being responsible for the uptake of the raffinose family of oligosaccharides (RFO), which are the most widespread d-galactose–containing oligosaccharides in higher plants. We also found that the RFO precursor galactinol, recently described as a plant defense molecule, is imported into Agrobacterium via MelB with nanomolar range affinity. Structural analyses and binding mode comparisons of the X-ray structures of MelB in complex with raffinose, stachyose, galactinol, galactose, and melibiose (a raffinose degradation product) revealed how MelB recognizes the nonreducing end galactose common to all these ligands and that MelB has a strong preference for a two-unit sugar ligand. Of note, MelB conferred a competitive advantage to A. fabrum in colonizing the rhizosphere of tomato plants. Our integrative work highlights the structural and functional characteristics of melibiose and galactinol assimilation by A. fabrum, leading to a competitive advantage for these bacteria in the rhizosphere. We propose that the PBP MelB, which is highly conserved among both symbionts and pathogens from Rhizobiace family, is a major trait in these bacteria required for early steps of plant colonization.
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WANG, Chia-Chang, Derk E. SCHULTZ, and Robert A. NICHOLAS. "Localization of a putative second membrane association site in penicillin-binding protein 1B of Escherichia coli." Biochemical Journal 316, no. 1 (May 15, 1996): 149–56. http://dx.doi.org/10.1042/bj3160149.
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We have shown previously that the periplasmic domain of penicillin-binding protein 1B (PBP 1Bper; residues 90–844) from Escherichia coli is insoluble in the absence of detergents, and can be reconstituted into liposomes [Nicholas, Lamson and Schultz (1993) J. Biol. Chem. 268, 5632–5641]. These data suggested that native PBP 1B contains a membrane association site in addition to its N-terminal transmembrane anchor. We have studied the membrane topology of PBP 1B in greater detail by assessing detergent binding and solubility in the absence of detergents for PBP 1Bper and a set of proteolytic fragments of PBP 1B. PBP 1Bper was shown by three independent methods to bind to detergent micelles, which strongly suggests that the periplasmic domain interacts with the hydrophobic milieu of membrane bilayers. Digestion with high weight ratios of thrombin of purified PBP 1B containing an engineered thrombin cleavage site on the periplasmic side of the transmembrane anchor generated four fragments in addition to PBP 1Bper that varied in size from 71 to 48 kDa. In contrast to PBP 1Bper, all fragments of 67 kDa and smaller were eluted from a gel-filtration column in the absence of detergents and did not bind to detergent micelles. The N-terminal sequences of the four fragments were determined, allowing the cleavage sites to be located in the primary sequence of PBP 1B. These data localize the membrane association site of PBP 1B to a region comprising the first 163 amino acids of the periplasmic domain, which falls within the putative transglycosylase domain. Lipid modification does not appear to be the mechanism by which PBP 1Bper associates with membranes.
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Pegos, Vanessa R., Louis Hey, Jacob LaMirande, Rachel Pfeffer, Rosalie Lipsh, Moshe Amitay, Daniel Gonzalez, and Mikael Elias. "Phosphate-binding protein fromPolaromonasJS666: purification, characterization, crystallization and sulfur SAD phasing." Acta Crystallographica Section F Structural Biology Communications 73, no. 6 (May 25, 2017): 342–46. http://dx.doi.org/10.1107/s2053230x17007373.
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Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs. Here, the PBP fromPolaromonasJS666 is focused on. Interestingly, this PBP is predicted to harbor different phosphate-binding residues to currently known PBPs. Here, it is shown that the PBP fromPolaromonasJS666 is capable of binding phosphate, with a maximal binding activity at pH 8. Its structure is expected to reveal its binding-cleft configuration as well as its phosphate-binding mode. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.35 Å resolution of the PBP fromPolaromonasJS666 are reported.
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Nakamura, Nozomi, Yoichi Naoe, Akihiro Doi, Yoshitsugu Shiro, and Hiroshi Sugimoto. "Conformational change of periplasmic heme-binding protein in ABC transporter." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1496. http://dx.doi.org/10.1107/s2053273314085039.
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Iron is one of the essential elements for all living organisms. Pathogenic bacteria acquire heme from the host proteins as an iron source. Gram-negative opportunistic pathogen, Burkholderia cenocepacia have ATP-binding cassette (ABC) transporter BhuUV-T complex to permeate heme through inner membrane. BhuT, periplasmic binding protein (PBP), bind and deliver heme(s) to inner membrane transporter BhuUV complex. BhuUV is 2:2 complex of the transmembrane permease subunit and cytoplasmic ATP-binding subunit which couple ATP hydrolysis to solute translocation. The molecular level mechanism of heme recognition and dissociation by PBP and heme transport by transporter complex are not fully understood. Here we describe the crystal structures of the heme-free and two types of heme-bound state of BhuT. These crystals were obtained in different crystallization conditions. Crystals diffracted to high resolution at SPring-8. BhuT is composed of two globular domains linked by a long a-helix. The transport ligand heme is bound between the two domains. A detailed structural comparison of the conformation of the domain and residues involved in the heme binding will be presented.
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Kim, Min-Jeong, Fu-Shi Quan, Hyun-Hee Kong, Jong-Hyun Kim, and Eun-Kyung Moon. "Specific Detection of Acanthamoeba species using Polyclonal Peptide Antibody Targeting the Periplasmic Binding Protein of A. castellanii." Korean Journal of Parasitology 60, no. 2 (April 20, 2022): 143–47. http://dx.doi.org/10.3347/kjp.2022.60.2.143.
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Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.
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Kim, Min-Jeong, A.-Jeong Ham, A.-Young Park, Hae-Jin Sohn, Ho-Joon Shin, Fu-Shi Quan, Hyun-Hee Kong, and Eun-Kyung Moon. "Detection of Acanthamoeba from Acanthamoeba Keratitis Mouse Model Using Acanthamoeba-Specific Antibodies." Microorganisms 10, no. 9 (August 25, 2022): 1711. http://dx.doi.org/10.3390/microorganisms10091711.
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Although the prevalence of Acanthamoeba keratitis (AK) is rare, its incidence in contact lens wearers has increased. Acanthamoeba infections can lead to the loss of vision if the diagnosis and treatment are delayed. In this study, we investigated the diagnostic potential of two antibodies raised against the adenylyl cyclase-associated protein (ACAP) and periplasmic binding protein (PBP) of A. castellanii in the AK mouse model. The specificity of ACAP and PBP antibodies to Acanthamoeba was confirmed by immunocytochemistry. AK mouse models were produced by corneal infections with A. castellanii trophozoites for 7 days and 21 days. Enzyme-linked immunosorbent assay results revealed that both ACAP and PBP antibodies successfully detected Acanthamoeba antigens in the tears and eyeball lysates of the AK mouse model. The detection levels of Acanthamoeba antigens were similar at both infection time points. Anti-Acanthamoeba IgG, IgA, and IgM antibodies were evaluated from the sera of the AK mouse model. Notably, IgM and IgA antibody responses were highest and lowest at both time points, respectively. Our findings revealed that both ACAP and PBP antibodies could detect Acanthamoeba antigens in the tears and eyeball lysates of the AK mouse model. These results provide important information for understanding Acanthamoeba infections and developing a new diagnostic tool for AK.
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Kumar, Vijay, Samantha L. Viviani, Jeeda Ismail, Shreya Agarwal, Robert A. Bonomo, and Focco van den Akker. "Structural analysis of the boronic acid β-lactamase inhibitor vaborbactam binding to Pseudomonas aeruginosa penicillin-binding protein 3." PLOS ONE 16, no. 10 (October 15, 2021): e0258359. http://dx.doi.org/10.1371/journal.pone.0258359.
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Antimicrobial resistance (AMR) mediated by β-lactamases is the major and leading cause of resistance to penicillins and cephalosporins among Gram-negative bacteria. β-Lactamases, periplasmic enzymes that are widely distributed in the bacterial world, protect penicillin-binding proteins (PBPs), the major cell wall synthesizing enzymes, from inactivation by β-lactam antibiotics. Developing novel PBP inhibitors with a non-β-lactam scaffold could potentially evade this resistance mechanism. Based on the structural similarities between the evolutionary related serine β-lactamases and PBPs, we investigated whether the potent β-lactamase inhibitor, vaborbactam, could also form an acyl-enzyme complex with Pseudomonas aeruginosa PBP3. We found that this cyclic boronate, vaborbactam, inhibited PBP3 (IC50 of 262 μM), and its binding to PBP3 increased the protein thermal stability by about 2°C. Crystallographic analysis of the PBP3:vaborbactam complex reveals that vaborbactam forms a covalent bond with the catalytic S294. The amide moiety of vaborbactam hydrogen bonds with N351 and the backbone oxygen of T487. The carboxyl group of vaborbactam hydrogen bonds with T487, S485, and S349. The thiophene ring and cyclic boronate ring of vaborbactam form hydrophobic interactions, including with V333 and Y503. The active site of the vaborbactam-bound PBP3 harbors the often observed ligand-induced formation of the aromatic wall and hydrophobic bridge, yet the residues involved in this wall and bridge display much higher temperature factors compared to PBP3 structures bound to high-affinity β-lactams. These insights could form the basis for developing more potent novel cyclic boronate-based PBP inhibitors to inhibit these targets and overcome β-lactamases-mediated resistance mechanisms.
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MAHAPATRA, Sebabrata, Sanjib BHAKTA, Jasimuddin AHAMED, and Joyoti BASU. "Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae." Biochemical Journal 350, no. 1 (August 9, 2000): 75–80. http://dx.doi.org/10.1042/bj3500075.
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Mycobacterium leprae has two high-molecular-mass multimodular penicillin-binding proteins (PBPs) of class A, termed PBP1 and PBP1* [Lepage, Dubois, Ghosh, Joris, Mahapatra, Kundu, Basu, Chakrabarti, Cole, Nguyen-Disteche and Ghuysen (1997) J. Bacteriol. 179, 4627–4630]. PBP1-Xaa–β-lactamase fusions generated periplasmic β-lactamase activity when Xaa (the amino acid of PBP1 at the fusion junction) was residue 314, 363, 407, 450 or 480. Truncation of the N-terminal part of the protein up to residue Leu-147 generated a penicillin-binding polypeptide which could still associate with the plasma membrane, whereas [∆M1–R314]PBP1 (PBP1 lacking residues Met-1 to Arg-314) failed to associate with the membrane, suggesting that the region between residues Leu-147 and Arg-314 harbours an additional plasma membrane association site for PBP1. Truncation of the C-terminus up to 42 residues downstream of the KTG (Lys-Thr-Gly) motif also generated a polypeptide that retained penicillin-binding activity. [∆M1–R314]PBP1 could be extracted from inclusion bodies and refolded under appropriate conditions to give a form capable of binding penicillin with the same efficiency as full-length PBP1. This is, to the best of our knowledge, the first report of a soluble derivative of a penicillin-resistant high-molecular-mass PBP of class A that is capable of binding penicillin. A chimaeric PBP in which the penicillin-binding (PB) module of PBP1 was fused at its N-terminal end with the non-penicillin-binding (n-PB) module of PBP1* retained pencillin-binding activity similar to that of PBP1, corroborating the finding that the n-PB module of PBP1 is dispensable for its penicillin-binding activity.
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de Sousa Borges, Anabela, Jeanine de Keyzer, Arnold J. M. Driessen, and Dirk-Jan Scheffers. "The Escherichia coli Membrane Protein Insertase YidC Assists in the Biogenesis of Penicillin Binding Proteins." Journal of Bacteriology 197, no. 8 (February 9, 2015): 1444–50. http://dx.doi.org/10.1128/jb.02556-14.
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ABSTRACTMembrane proteins need to be properly inserted and folded in the membrane in order to perform a range of activities that are essential for the survival of bacteria. The Sec translocon and the YidC insertase are responsible for the insertion of the majority of proteins into the cytoplasmic membrane. YidC can act in combination with the Sec translocon in the insertion and folding of membrane proteins. However, YidC also functions as an insertase independently of the Sec translocon for so-called YidC-only substrates. In addition, YidC can act as a foldase and promote the proper assembly of membrane protein complexes. Here, we investigate the effect ofEscherichia coliYidC depletion on the assembly of penicillin binding proteins (PBPs), which are involved in cell wall synthesis. YidC depletion does not affect the total amount of the specific cell division PBP3 (FtsI) in the membrane, but the amount of active PBP3, as assessed by substrate binding, is reduced 2-fold. A similar reduction in the amount of active PBP2 was observed, while the levels of active PBP1A/1B and PBP5 were essentially similar. PBP1B and PBP3 disappeared from higher-Mwbands upon YidC depletion, indicating that YidC might play a role in PBP complex formation. Taken together, our results suggest that the foldase activity of YidC can extend to the periplasmic domains of membrane proteins.IMPORTANCEThis study addresses the role of the membrane protein insertase YidC in the biogenesis of penicillin binding proteins (PBPs). PBPs are proteins containing one transmembrane segment and a large periplasmic or extracellular domain, which are involved in peptidoglycan synthesis. We observe that in the absence of YidC, two critical PBPs are not correctly folded even though the total amount of protein in the membrane is not affected. Our findings extend the function of YidC as a foldase for membrane protein (complexes) to periplasmic domains of membrane proteins.
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Brambilla, Luciano, Jorgelina Morán-Barrio, and Alejandro M. Viale. "Low-Molecular-Mass Penicillin Binding Protein 6b (DacD) Is Required for Efficient GOB-18 Metallo-β-Lactamase Biogenesis in Salmonella enterica and Escherichia coli." Antimicrobial Agents and Chemotherapy 58, no. 1 (October 21, 2013): 205–11. http://dx.doi.org/10.1128/aac.01224-13.
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ABSTRACTMetallo-β-lactamases (MBLs) are Zn2+-containing secretory enzymes of clinical relevance, whose final folding and metal ion assembly steps in Gram-negative bacteria occur after secretion of the apo form to the periplasmic space. In the search of periplasmic factors assisting MBL biogenesis, we found thatdacDnull (ΔdacD) mutants ofSalmonella entericaandEscherichia coliexpressing the pre-GOB-18 MBL gene from plasmids showed significantly reduced resistance to cefotaxime and concomitant lower accumulation of GOB-18 in the periplasm. This reduced accumulation of GOB-18 resulted from increased accessibility to proteolytic attack in the periplasm, suggesting that the lack of DacD negatively affects the stability of secreted apo MBL forms. Moreover, ΔdacDmutants ofS. entericaandE. colishowed an altered ability to develop biofilm growth. DacD is a widely distributed low-molecular-mass (LMM) penicillin binding protein (PBP6b) endowed with lowdd-carboxypeptidase activity whose functions are still obscure. Our results indicate roles for DacD in assisting biogenesis of particular secretory macromolecules in Gram-negative bacteria and represent to our knowledge the first reported phenotypes for bacterial mutants lacking this LMM PBP.
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Elitas, Meltem. "On-Chip Isoniazid Exposure of Mycobacterium smegmatis Penicillin-Binding Protein (PBP) Mutant Using Time-Lapse Fluorescent Microscopy." Micromachines 9, no. 11 (October 31, 2018): 561. http://dx.doi.org/10.3390/mi9110561.
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Antibiotic resistance has been one of the biggest threats to global health. Despite the available prevention and control strategies and efforts in developing new antibiotics, the need remains for effective approaches against antibiotic resistance. Efficient strategies to cope with antimicrobial resistance require a quantitative and deeper understanding of microbial behavior, which can be obtained using different techniques to provide the missing pieces of the current antibiotic-resistance puzzle. Microfluidic-microscopy techniques are among the most promising methods that contribute modernization of traditional assays in microbiology. They provide monitoring and manipulation of cells at micro-scale volumes. Here, we combined population-level, culture-based assays with single-cell resolution, microfluidic-microscopy systems to investigate isoniazid response of Mycobacterium smegmatis penicillin-binding protein (PBP) mutant. This mutant exhibited normal growth in plain medium and sensitivity to stress responses when treated with thermal stress (45 °C), detergent stress (0.1% sodium dodecyl sulfate), acid stress (pH 4.5), and nutrient starvation (1XPBS). The impact of msm0031 transposon insertion on drug-mediated killing was determined for isoniazid (INH, 50 µg/mL), rifampicin (RIF, 200 µg/mL), ethionamide (ETH, 200 µg/mL), and ethambutol (EMB, 5 µg/mL). The PBP mutant demonstrated remarkable isoniazid-killing phenotype in batch culture. Therefore, we hypothesized that single-cell analysis will show increased lysis kinetics and fewer intact cells after drug treatment. However, the single-cell analysis data showed that upon isoniazid exposure, the percentage of the intact PBP mutant cells was 24%, while the percentage of the intact wild-type cells was 4.6%. The PBP mutant cells exhibited decreased cell-lysis profile. Therefore, the traditional culture-based assays were not sufficient to provide insights about the subpopulation of viable but non-culture cells. Consequently, we need more adequate tools to be able to comprehend and fight the antibiotic resistance of bacteria.
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Marty, Loïc, Armelle Vigouroux, Magali Aumont-Nicaise, Franck Pelissier, Thibault Meyer, Céline Lavire, Yves Dessaux, and Solange Moréra. "Structural basis for two efficient modes of agropinic acid opine import into the bacterial pathogen Agrobacterium tumefaciens." Biochemical Journal 476, no. 1 (January 15, 2019): 165–78. http://dx.doi.org/10.1042/bcj20180861.
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AbstractAgrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. These opines serve as nutrients and are imported into bacteria via periplasmic-binding proteins (PBPs) in association with ABC transporters. Structural and affinity data on agropine and agropinic acid opines bound to PBPs are currently lacking. Here, we investigated the molecular basis of AgtB and AgaA, proposed as the specific PBP for agropine and agropinic acid import, respectively. Using genetic approaches and affinity measurements, we identified AgtB and its transporter as responsible for agropine uptake in agropine-assimilating agrobacteria. Nonetheless, we showed that AgtB binds agropinic acid with a higher affinity than agropine, and we structurally characterized the agropinic acid-binding mode through three crystal structures at 1.4, 1.74 and 1.9 Å resolution. In the crystallization time course, obtaining a crystal structure of AgtB with agropine was unsuccessful due to the spontaneous lactamization of agropine into agropinic acid. AgaA binds agropinic acid only with a similar affinity in nanomolar range as AgtB. The structure of AgaA bound to agropinic acid at 1.65 Å resolution defines a different agropinic acid-binding signature. Our work highlights the structural and functional characteristics of two efficient agropinic acid assimilation pathways, of which one is also involved in agropine assimilation.
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BHAKTA, Sanjib, and Joyoti BASU. "Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1∗ and its soluble derivative from Mycobacterium tuberculosis." Biochemical Journal 361, no. 3 (January 25, 2002): 635–39. http://dx.doi.org/10.1042/bj3610635.
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The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacteriumtuberculosis, has been named penicillin-binding protein 1∗ (PBP1∗), by analogy to the previously characterized PBP1∗ of M. leprae. This gene has been overexpressed in Escherichia coli. His6-tagged PBP1∗ localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1∗ binds benzylpenicillin and several other β-lactams, notably cefotaxime, with high affinity. Truncation of the N-terminal 64 amino acid residues results in an expressed protein present exclusively in inclusion bodies and unable to associate with the membrane. The C-terminal module encompassing amino acids 272–663 can be extracted from inclusion bodies under denaturing conditions using guanidine/HCl and refolded to give a protein fully competent in penicillin-binding. Deletion of Gly95—Gln143 results in the expression of a protein, which is localized in the cytosol. The soluble derivative of PBP1∗ binds benzylpenicillin with the same efficiency as the full-length protein. This is the first report of a soluble derivative of a class A high-molecular-mass PBP.
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Mosbahi, Khedidja, Marta Wojnowska, Amaya Albalat, and Daniel Walker. "Bacterial iron acquisition mediated by outer membrane translocation and cleavage of a host protein." Proceedings of the National Academy of Sciences 115, no. 26 (June 11, 2018): 6840–45. http://dx.doi.org/10.1073/pnas.1800672115.
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Iron is an essential micronutrient for most bacteria and is obtained from iron-chelating siderophores or directly from iron-containing host proteins. For Gram-negative bacteria, classical iron transport systems consist of an outer membrane receptor, a periplasmic binding protein, and an inner membrane ABC transporter, which work in concert to deliver iron from the cell surface to the cytoplasm. We recently showed thatPectobacteriumspp. are able to acquire iron from ferredoxin, a small and stable 2Fe-2S iron sulfur cluster containing protein and identified the ferredoxin receptor, FusA, a TonB-dependent receptor that binds ferredoxin on the cell surface. The genetic context offusAsuggests an atypical iron acquisition system, lacking a periplasmic binding protein, although the mechanism through which iron is extracted from the captured ferredoxin has remained unknown. Here we show that FusC, an M16 family protease, displays a highly targeted proteolytic activity against plant ferredoxin, and that growth enhancement ofPectobacteriumdue to iron acquisition from ferredoxin is FusC-dependent. The periplasmic location of FusC indicates a mechanism in which ferredoxin is imported into the periplasm via FusA before cleavage by FusC, as confirmed by the uptake and accumulation of ferredoxin in the periplasm in a strain lackingfusC. The existence of homologous uptake systems in a range of pathogenic bacteria suggests that protein uptake for nutrient acquisition may be widespread in bacteria and shows that, similar to their endosymbiotic descendants mitochondria and chloroplasts, bacteria produce dedicated protein import systems.
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Cockerell, Steven R., Alex C. Rutkovsky, Josiah P. Zayner, Rebecca E. Cooper, Lindsay R. Porter, Sam S. Pendergraft, Zach M. Parker, Marcus W. McGinnis, and Ece Karatan. "Vibrio cholerae NspS, a homologue of ABC-type periplasmic solute binding proteins, facilitates transduction of polyamine signals independent of their transport." Microbiology 160, no. 5 (May 1, 2014): 832–43. http://dx.doi.org/10.1099/mic.0.075903-0.
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The polyamines norspermidine and spermidine are among the environmental signals that regulate Vibrio cholerae biofilm formation. The effects of these polyamines are mediated by NspS, a member of the bacterial periplasmic solute binding protein superfamily. Almost all members of this superfamily characterized to date are components of ATP-binding cassette-type transporters involved in nutrient uptake. Consequently, in the current annotation of the V. cholerae genome, NspS has been assigned a function in transport. The objective of this study was to further characterize NspS and investigate its potential role in transport. Our results support a role for NspS in signal transduction in response to norspermidine and spermidine, but not their transport. In addition, we provide evidence that these polyamine signals are processed by c-di-GMP signalling networks in the cell. Furthermore, we present comparative genomics analyses which reveal the presence of NspS-like proteins in a variety of bacteria, suggesting that periplasmic ligand binding proteins may be widely utilized for sensory transduction.
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Zainol, Mohd Khairi, Z. Mohd Zin, R. S. T. Linforth, and D. J. Scott. "Overexpression, extraction, purification and characterisation of DppA from <i>Escherichia coli</i>." Journal of Biochemistry, Microbiology and Biotechnology 2, no. 2 (December 30, 2014): 40–46. http://dx.doi.org/10.54987/jobimb.v2i2.147.
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Components such as protein within the periplasmic space serve some vital purposes: such as buffering against the outside environment, processing of essential nutrients for transport into the cytoplasmic space, and cell wall biosynthesis. Dipeptide binding protein a (DppA) is a member of a family of ABC proteins and is involved in the transportation of potentially beneficial dipeptides as nutrient source through the periplasmic space and into cell. DppA was successfully cloned into expression vectors and over expressed in Escherichia coli, extracted, purified, and characterized. DppA was subjected to biophysical characterization using mass spectrometry. Mass spectrometry (ms) analysis and analytical ultra-centrifugation were used to evaluate the recombinant DppA’s molecular weight. The findings presented in this study highlighted the ability to overexpress, extract and purify the recombinant DppA and reflect the differences techniques to asses its purity.
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Krewulak, Karla D., and Hans J. Vogel. "TonB or not TonB: is that the question?This paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process." Biochemistry and Cell Biology 89, no. 2 (April 2011): 87–97. http://dx.doi.org/10.1139/o10-141.
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Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.
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Moreira, B., S. Boyle-Vavra, B. L. deJonge, and R. S. Daum. "Increased production of penicillin-binding protein 2, increased detection of other penicillin-binding proteins, and decreased coagulase activity associated with glycopeptide resistance in Staphylococcus aureus." Antimicrobial Agents and Chemotherapy 41, no. 8 (August 1997): 1788–93. http://dx.doi.org/10.1128/aac.41.8.1788.
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The mechanism of glycopeptide resistance in the genus Staphylococcus is unknown. Since these antimicrobial compounds act by binding the peptidoglycan precursor terminus, the target of transglycosylase and transpeptidase enzymes, it was hypothesized that resistance might be mediated in Staphylococcus aureus by increased production or activity of these enzymes, commonly called penicillin-binding proteins (PBPs). To evaluate this possibility, glycopeptide-resistant mutants were prepared by passage of several clinical isolates of this species in nutrient broth containing successively increasing concentrations of the glycopeptide vancomycin or teicoplanin. Decreased coagulase activity and increased resistance to lysostaphin were uniformly present in the vancomycin-resistant mutants. Peptidoglycan cross-linking increased in one resistant isolate and decreased in two resistant isolates. The amounts of radioactive penicillin that bound to each PBP in susceptible and resistant strains were compared; PBP2 production was also evaluated by Western blotting. Increased penicillin labeling and production of PBP2 were found in all resistant derivatives selected by either vancomycin or teicoplanin. Moreover, the increase in PBP2 penicillin labeling occurred early in a series of vancomycin-selected derivatives and was strongly correlated (r > 0.9) with the increase in vancomycin and teicoplanin MIC. An increase in penicillin labeling also occurred, variably, in PBP1, PBP3, and/or PBP4. These data demonstrate a strong correlation between resistance to glycopeptides and increased PBP activity and/or production in S. aureus. Such an increase could allow PBPs to better compete with glycopeptides for the peptidoglycan precursor.
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Keegan, Ronan, David G. Waterman, David J. Hopper, Leighton Coates, Graham Taylor, Jingxu Guo, Alun R. Coker, Peter T. Erskine, Steve P. Wood, and Jonathan B. Cooper. "The 1.1 Å resolution structure of a periplasmic phosphate-binding protein fromStenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank." Acta Crystallographica Section D Structural Biology 72, no. 8 (July 27, 2016): 933–43. http://dx.doi.org/10.1107/s2059798316010433.
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During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) fromAlcaligenessp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement usingMOLREPin which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in theMOLREPpeak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. ABLASTsearch then indicated that the molecule was most probably a phosphate-binding protein fromStenotrophomonas maltophilia(UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of theS. maltophiliaprotein has been refined to anRfactor of 10.15% and anRfreeof 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate anion is bound tightly between the two domains of the protein and interacts with conserved residues and a number of helix dipoles.
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Coutinho, Bruna G., Emily Mevers, Amy L. Schaefer, Dale A. Pelletier, Caroline S. Harwood, Jon Clardy, and E. Peter Greenberg. "A plant-responsive bacterial-signaling system senses an ethanolamine derivative." Proceedings of the National Academy of Sciences 115, no. 39 (September 6, 2018): 9785–90. http://dx.doi.org/10.1073/pnas.1809611115.
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Certain plant-associated Proteobacteria sense their host environment by detecting an unknown plant signal recognized by a member of a LuxR subfamily of transcription factors. This interkingdom communication is important for both mutualistic and pathogenic interactions. The Populus root endophyte Pseudomonas sp. GM79 possesses such a regulator, named PipR. In a previous study we reported that PipR activates an adjacent gene (pipA) coding for a proline iminopeptidase in response to Populus leaf macerates and peptides and that this activation is dependent on a putative ABC-type transporter [Schaefer AL, et al. (2016) mBio 7:e01101-16]. In this study we identify a chemical derived from ethanolamine that induces PipR activity at picomolar concentrations, and we present evidence that this is the active inducer present in plant leaf macerates. First, a screen of more than 750 compounds indicated ethanolamine was a potent inducer for the PipR-sensing system; however, ethanolamine failed to bind to the periplasmic-binding protein (PBP) required for the signal response. This led us to discover that a specific ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), binds to the PBP and serves as a potent PipR-dependent inducer. We also show that a compound, which coelutes with HEHEAA in HPLC and induces pipA gene expression in a PipR-dependent manner, can be found in Populus leaf macerates. This work sheds light on how plant-associated bacteria can sense their environment and on the nature of inducers for a family of plant-responsive LuxR-like transcription factors found in plant-associated bacteria.
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Wang, Mingxing, Qiong Guo, Kongfu Zhu, Bo Fang, Yifan Yang, Maikun Teng, Xu Li, and Yuyong Tao. "Interface switch mediates signal transmission in a two-component system." Proceedings of the National Academy of Sciences 117, no. 48 (November 16, 2020): 30433–40. http://dx.doi.org/10.1073/pnas.1912080117.
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Two-component systems (TCS), which typically consist of a membrane-embedded histidine kinase and a cytoplasmic response regulator, are the dominant signaling proteins for transduction of environmental stimuli into cellular response pathways in prokaryotic cells. HptRSA is a recently identified TCS consisting of the G6P-associated sensor protein (HptA), transmembrane histidine kinase (HptS), and cytoplasmic effector (HptR). HptRSA mediates glucose-6-phosphate (G6P) uptake to supportStaphylococcus aureusgrowth and multiplication within various host cells. How the mechanism by which HptRSA perceives G6P and triggers a downstream response has remained elusive. Here, we solved the HptA structures in apo and G6P-bound states. G6P binding in the cleft between two HptA domains caused a conformational closing movement. The solved structures of HptA in complex with the periplasmic domain of HptS showed that HptA interacts with HptS through both constitutive and switchable interfaces. The G6P-free form of HptA binds to the membrane-distal side of the HptS periplasmic domain (HptSp), resulting in a parallel conformation of the HptSp protomer pair. However, once HptA associates with G6P, its intramolecular domain closure switches the HptA-HptSp contact region into the membrane-proximal domain, which causes rotation and closure of the C termini of each HptSp protomer. Through biochemical and growth assays of HptA and HptS mutant variants, we proposed a distinct mechanism of interface switch-mediated signaling transduction. Our results provide mechanistic insights into bacterial nutrient sensing and expand our understanding of the activation modes by which TCS communicates external signals.
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Lakaye, Bernard, Alain Dubus, Bernard Joris, and Jean-Marie Frère. "Method for Estimation of Low Outer Membrane Permeability to β-Lactam Antibiotics." Antimicrobial Agents and Chemotherapy 46, no. 9 (September 2002): 2901–7. http://dx.doi.org/10.1128/aac.46.9.2901-2907.2002.
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ABSTRACT The outer membrane of gram-negative bacteria plays a major role in β-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with β-lactamases and efflux systems. Up to now, the quantitative estimation of low outer membrane permeability by the method of Zimmermann and Rosselet was difficult because of the secreted and cell surface-associated β-lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of β-lactam penetration into the periplasm. The method is dedicated to measurement of low permeability and is only valid when the diffusion rate through the outer membrane is rate limiting. Cytoplasmic membrane associated PBPs do not interfere since they are acylated after the very sensitive BlaR-CTD. This method was used to measure the permeability of β-lactamase-deficient strains of Enterobacter cloacae and Enterobacter aerogenes to benzylpenicillin, ampicillin, carbenicillin, cefotaxime, aztreonam, and cephacetrile. Except for that of cephacetrile, the permeability coefficients were equal to or below 10−7 cm/s. For cephacetrile, carbenicillin, and benzylpenicillin, the outer membrane of E. cloacae was 20 to 60 times less permeable than that of Escherichia coli, whereas for cefotaxime, aztreonam, and ampicillin it was, respectively, 400, 1,000, and 700 times less permeable. The permeability coefficient for aztreonam is the lowest ever measured (P = 3.2 × 10−9 cm/s). Using these values, the MICs for a β-lactamase-overproducing strain of E. cloacae were successfully predicted, demonstrating the validity of the method.
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Zhou, Daoguo, Wolf-Dietrich Hardt, and Jorge E. Galán. "Salmonella typhimurium Encodes a Putative Iron Transport System within the Centisome 63 Pathogenicity Island." Infection and Immunity 67, no. 4 (April 1, 1999): 1974–81. http://dx.doi.org/10.1128/iai.67.4.1974-1981.1999.
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ABSTRACT Upon entry into the host, Salmonella enterica strains are presumed to encounter an iron-restricted environment. Consequently, these bacteria have evolved a variety of often-redundant high-affinity acquisition systems to obtain iron in this restricted environment. We have identified an iron transport system that is encoded within the centisome 63 pathogenicity island of Salmonella typhimurium. The nucleotide composition of this locus is significantly different from that of the rest of this pathogenicity island, suggesting a different ancestry and a mosaic structure for this region of the S. typhimurium chromosome. This locus, designated sit, consists of four open reading frames which encode polypeptides with extensive homology to the yfe ABC iron transport system of Yersinia pestis, as well as other ABC transporters. The sitA gene encodes a putative periplasmic binding protein, sitB encodes an ATP-binding protein, and sitC and sitD encode two putative permeases (integral membrane proteins). This operon is capable of complementing the growth defect of the enterobactin-deficient Escherichia coli strain SAB11 in iron-restricted minimal medium. Transcription of the sitoperon is repressed under iron-rich growth conditions in afur-dependent manner. Introduction of a sitBCDdeletion into wild-type S. typhimuriumresulted in no apparent growth defect in either nutrient-rich or minimal medium and no measurable virulence phenotype. These results further support the existence of redundant iron uptake systems in S. enterica.
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Goldberg, Joel, Christopher Bethel, Andrea M. Hujer, Steven Marshall, Magdalena A. Taracila, Krisztina M. Papp-Wallce, Vijay Kumar, Focco van den Akker, Mark Plummer, and Robert A. Bonomo. "1256. In Vivo Activity and Structural Characterization of a New Generation γ-Lactam Siderophore Antibiotic Against Multidrug-Resistant Gram-Negative Bacteria and Acinetobacter spp." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S645. http://dx.doi.org/10.1093/ofid/ofaa439.1440.
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Abstract Background Multidrug-resistant (MDR) A. baumannii presents a critical need for innovative antibacterial development. We have identified a new series of γ-lactam (oxopyrazole) antibiotics that target penicillin binding proteins (PBPs) and incorporate a siderophore moiety to facilitate periplasmic uptake. YU253911, an advanced iteration of this class shows potent in vitro activity against clinically relevant Gram-negative organisms including Acinetobacter spp. Methods Minimum inhibitory concentrations (MICs) for YU253911 were determined using broth microdilution against a 198-member panel of clinical isolates of Acinetobacter spp. Resistant strains were further evaluated for susceptibility to YU253911 in combination with sulbactam. The antibiotic’s target protein was evaluated by binding studies with Bocillin™, a fluorescent penicillin analogue, and modeled in the PBP active site. YU253911 was evaluated in vivo in a mouse soft tissue infection model. Results MIC testing for YU253911 revealed an MIC50 of 0.5 μg/mL and an MIC90 of 16 μg/mL, which compared favorably to all tested β-lactam antibiotics including penicillins, cephalosporins, monobactams and carbapenems (MIC50 = 2 to > 16 μg/mL). Combination with sulbactam augmented the activity of the agent. There was no apparent correlation between YU253911-resistance and the presence of specific β-lactamase genes, and incubation with representative β-lactamase proteins (KPC-2, OXA-23, OXA-24, PER-2, PDC-3, NDM-1, VIM-2, and IMP-1) showed negligible hydrolysis of the agent. YU253911 showed promising preclinical pharmacokinetics in mice with a 15 h half-life from intravenous administration and demonstrated a dose-dependent reduction in colony forming units from 50 and 100 mg/kg q6h dosing in a mouse thigh infection model using P. aeruginosa. Conclusion YU253911, a new generation γ-lactam antibiotic effective against MDR A. baumannii demonstrated promising in in vitro potency and favorable pharmacokinetics which correlated with in vivo efficacy. Disclosures Krisztina M. Papp-Wallce, PhD, Entasis (Grant/Research Support)Merck (Grant/Research Support)Venatorx (Grant/Research Support) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)
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Zeth, Kornelius, Vera Kozjak-Pavlovic, Michaela Faulstich, Martin Fraunholz, Robert Hurwitz, Oliver Kepp, and Thomas Rudel. "Structure and function of the PorB porin from disseminating Neisseria gonorrhoeae." Biochemical Journal 449, no. 3 (January 9, 2013): 631–42. http://dx.doi.org/10.1042/bj20121025.
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The outer membrane of Gram-negative bacteria contains a large number of channel-forming proteins, porins, for the uptake of small nutrient molecules. Neisseria gonorrhoeae PorBIA (PorB of serotype A) are associated with disseminating diseases and mediate a rapid bacterial invasion into host cells in a phosphate-sensitive manner. To gain insights into this structure–function relationship we analysed PorBIA by X-ray crystallography in the presence of phosphate and ATP. The structure of PorBIA in the complex solved at a resolution of 3.3 Å (1 Å=0.1 nm) displays a surplus of positive charges inside the channel. ATP ligand-binding in the channel is co-ordinated by the positively charged residues of the channel interior. These residues ligate the aromatic, sugar and pyrophosphate moieties of the ligand. Two phosphate ions were observed in the structure, one of which clamped by two arginine residues (Arg92 and Arg124) localized at the extraplasmic channel exit. A short β-bulge in β2-strand together with the long L3 loop narrow the barrel diameter significantly and further support substrate specificity through hydrogen bond interactions. Interestingly the structure also comprised a small peptide as a remnant of a periplasmic protein which physically links porin molecules to the peptidoglycan network. To test the importance of Arg92 on bacterial invasion the residue was mutated. In vivo assays of bacteria carrying a R92S mutation confirmed the importance of this residue for host-cell invasion. Furthermore systematic sequence and structure comparisons of PorBIA from Neisseriaceae indicated Arg92 to be unique in disseminating N. gonorrhoeae thereby possibly distinguishing invasion-promoting porins from other neisserial porins.
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Shukla, Shantanu, Dean A. Myles, and Matthew J. Cuneo. "Mapping periplasmic binding protein oligosaccharide recognition with neutron crystallography." Scientific Reports 12, no. 1 (October 21, 2022). http://dx.doi.org/10.1038/s41598-022-20542-8.
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AbstractNumerous studies have shown how periplasmic binding proteins (PBPs) bind substrates with exquisite specificity, even distinguishing between sugar epimers and anomers, or structurally similar ions. Yet, marked substrate promiscuity is also a feature encoded in some PBPs. Except for three sub-Ångström crystal structures, there are no reports of hydrogen atom positions in the remaining (> 1000) PBP structures. The previous X-ray crystal structure of the maltodextrin periplasmic-binding protein from Thermotoga maritima (tmMBP) complexed with oligosaccharide showed a large network of interconnected water molecules stretching from one end of the substrate binding pocket to the other. These water molecules are positioned to form multiple hydrogen bonds, as well as forming interactions between the protein and substrate. Here we present the neutron crystal structure of tmMBP to a resolution of 2.1 Å. This is the first neutron crystal structure from the PBP superfamily and here we unambiguously identify the nature and orientation of the hydrogen bonding and water-mediated interactions involved in stabilizing a tetrasaccharide in the binding site. More broadly, these results demonstrate the conserved intricate mechanisms that underlie substrate-specificity and affinity in PBPs.
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Chu, Byron C. H., and Hans J. Vogel. "A structural and functional analysis of type III periplasmic and substrate binding proteins: their role in bacterial siderophore and heme transport." Biological Chemistry 392, no. 1-2 (February 1, 2011). http://dx.doi.org/10.1515/bc.2011.012.
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AbstractInEscherichia colithe Fhu, Fep and Fec transport systems are involved in the uptake of chelated ferric iron-siderophore complexes, whereas in pathogenic strains heme can also be used as an iron source. An essential step in these pathways is the movement of the ferric-siderophore complex or heme from the outer membrane transporter across the periplasm to the cognate cytoplasmic membrane ATP-dependent transporter. This is accomplished in each case by a dedicated periplasmic binding protein (PBP). Ferric-siderophore binding PBPs belong to the PBP protein superfamily and adopt a bilobal type III structural fold in which the two independently folded amino and carboxy terminal domains are linked together by a single long α-helix of approximately 20 amino acids. Recent structural studies reveal how the PBPs of the Fhu, Fep, Fec and Chu systems are able to bind their corresponding ligands. These complex structures will be discussed and placed in the context of our current understanding of the entire type III family of Gram-negative periplasmic binding proteins and related Gram-positive substrate binding proteins.
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Bonneau, Anne, Béatrice Roche, and Isabelle J. Schalk. "Iron acquisition in Pseudomonas aeruginosa by the siderophore pyoverdine: an intricate interacting network including periplasmic and membrane proteins." Scientific Reports 10, no. 1 (January 10, 2020). http://dx.doi.org/10.1038/s41598-019-56913-x.
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AbstractPyoverdine (PVDI) has been reported to act both as a siderophore for scavenging iron (a key nutrient) and a signaling molecule for the expression of virulence factors. This compound is itself part of a core set of virulence factors produced by Pseudomonas aeruginosa during infections. Once secreted into the bacterial environment and having scavenged ferric iron, PVDI-Fe3+ is taken back into the P. aeruginosa periplasm via the outer membrane transporters FpvAI and FpvB. Iron release from PVDI in the bacterial periplasm involves numerous proteins encoded by the fpvGHJKCDEF genes and a mechanism of iron reduction. Here, we investigated the global interacting network between these various proteins using systematic bacterial two-hybrid screening. We deciphered a network of five interacting proteins composed of two inner-membrane proteins, FpvG (iron reductase) and FpvH (unknown function), and three periplasmic proteins, FpvJ (unknown function), FpvF (periplasmic PVDI-binding protein), and FpvC (iron periplasmic-binding protein). This interacting network strongly suggests the existence of a large protein machinery composed of these five proteins, all playing a role in iron acquisition by PVDI. Furthermore, we discovered an interaction between the periplasmic siderophore binding protein FpvF and the PvdRT-OpmQ efflux pump, also suggesting a role for FpvF in apo-PVDI recycling and secretion after iron delivery. These results highlight a multi-protein complex that drives iron release from PVDI in the periplasm of P. aeruginosa.
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Oswald, Christine, Sander H. J. Smits, Marina Höing, Erhard Bremer, and Lutz Schmitt. "Structural analysis of the choline-binding protein ChoX in a semi-closed and ligand-free conformation." Biological Chemistry 390, no. 11 (November 1, 2009). http://dx.doi.org/10.1515/bc.2009.113.
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Abstract The periplasmic ligand-binding protein ChoX is part of the ABC transport system ChoVWX that imports choline as a nutrient into the soil bacterium Sinorhizobium meliloti. We have recently reported the crystal structures of ChoX in complex with its ligands choline and acetylcholine and the structure of a fully closed but substrate-free state of ChoX. This latter structure revealed an architecture of the ligand-binding site that is superimposable to the closed, ligand-bound form of ChoX. We report here the crystal structure of ChoX in an unusual, ligand-free conformation that represents a semi-closed form of ChoX. The analysis revealed a subdomain movement in the N-lobe of ChoX. Comparison with the two well-characterized substrate binding proteins, MBP and HisJ, suggests the presence of a similar subdomain in these proteins.
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Herrou, Julien, Jonathan W. Willett, Daniel M. Czyż, Gyorgy Babnigg, Youngchang Kim, and Sean Crosson. "Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria." Journal of Bacteriology 199, no. 5 (December 19, 2016). http://dx.doi.org/10.1128/jb.00746-16.
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ABSTRACT Brucella abortus σE1 is an EcfG family sigma factor that regulates the transcription of dozens of genes in response to diverse stress conditions and is required for maintenance of chronic infection in a mouse model. A putative ATP-binding cassette transporter operon, bab1_0223-bab1_0226, is among the most highly activated gene sets in the σE1 regulon. The proteins encoded by the operon resemble quaternary ammonium-compatible solute importers but are most similar in sequence to the broadly conserved YehZYXW system, which remains largely uncharacterized. Transcription of yehZYXW is activated by the general stress sigma factor σS in Enterobacteriaceae, which suggests a functional role for this transport system in bacterial stress response across the classes Alphaproteobacteria and Gammaproteobacteria. We present evidence that B. abortus YehZYXW does not function as an importer of known compatible solutes under physiological conditions and does not contribute to the virulence defect of a σE1-null strain. The sole in vitro phenotype associated with genetic disruption of this putative transport system is reduced growth in the presence of high Li+ ion concentrations. A crystal structure of B. abortus YehZ revealed a class II periplasmic binding protein fold with significant structural homology to Archaeoglobus fulgidus ProX, which binds glycine betaine. However, the structure of the YehZ ligand-binding pocket is incompatible with high-affinity binding to glycine betaine. This is consistent with weak measured binding of YehZ to glycine betaine and related compatible solutes. We conclude that YehZYXW is a conserved, stress-regulated transport system that is phylogenetically and functionally distinct from quaternary ammonium-compatible solute importers. IMPORTANCE Brucella abortus σE1 regulates transcription in response to stressors encountered in its mammalian host and is necessary for maintenance of chronic infection in a mouse model. The functions of the majority of genes regulated by σE1 remain undefined. We present a functional/structural analysis of a conserved putative membrane transport system (YehZYXW) whose expression is strongly activated by σE1. Though annotated as a quaternary ammonium osmolyte uptake system, experimental physiological studies and measured ligand-binding properties of the periplasmic binding protein (PBP), YehZ, are inconsistent with this function. A crystal structure of B. abortus YehZ provides molecular insight into differences between bona fide quaternary ammonium osmolyte importers and YehZ-related proteins, which form a distinct phylogenetic and functional group of PBPs.
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Huang, Yi-Wei, Yu Wang, Yun Lin, Chin Lin, Yi-Tsung Lin, Cheng-Chih Hsu, and Tsuey-Ching Yang. "Impacts of Penicillin Binding Protein 2 Inactivation on β-Lactamase Expression and Muropeptide Profile in Stenotrophomonas maltophilia." mSystems 2, no. 4 (August 29, 2017). http://dx.doi.org/10.1128/msystems.00077-17.
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ABSTRACT Inducible expression of chromosomally encoded β-lactamase(s) is a key mechanism for β-lactam resistance in Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The muropeptides produced during the peptidoglycan recycling pathway act as activator ligands for β-lactamase(s) induction. The muropeptides 1,6-anhydromuramyl pentapeptide and 1,6-anhydromuramyl tripeptide are the known activator ligands for ampC β-lactamase expression in E. cloacae. Here, we dissected the type of muropepetides for L1/L2 β-lactamase expression in an mrdA deletion mutant of S. maltophilia. Distinct from the findings with the ampC system, 1,6-anhydromuramyl tetrapeptide is the candidate for ΔmrdA-mediated β-lactamase expression in S. maltophilia. Our work extends the understanding of β-lactamase induction and provides valuable information for combating the occurrence of β-lactam resistance. Penicillin binding proteins (PBPs) are involved in peptidoglycan synthesis, and their inactivation is linked to β-lactamase expression in ampR–β-lactamase module–harboring Gram-negative bacteria. There are seven annotated PBP genes, namely, mrcA, mrcB, pbpC, mrdA, ftsI, dacB, and dacC, in the Stenotrophomonas maltophilia genome, and these genes encode PBP1a, PBP1b, PBP1c, PBP2, PBP3, PBP4, and PBP6, respectively. In addition, S. maltophilia harbors two β-lactamase genes, L1 and L2, whose expression is induced via β-lactam challenge. The impact of PBP inactivation on L1/L2 expression was assessed in this study. Inactivation of mrdA resulted in increased L1/L2 expression in the absence of β-lactam challenge, and the underlying mechanism was further elucidated. The roles of ampNG, ampD I (the homologue of Escherichia coli ampD), nagZ, ampR, and creBC in L1/L2 expression mediated by a ΔmrdA mutant strain were assessed via mutant construction and β-lactamase activity determinations. Furthermore, the strain ΔmrdA-mediated change in the muropeptide profile was assessed using liquid chromatography mass spectrometry (LC-MS). The mutant ΔmrdA-mediated L1/L2 expression relied on functional AmpNG, AmpR, and NagZ, was restricted by AmpDI, and was less related to the CreBC two-component system. Inactivation of mrdA significantly increased the levels of total and periplasmic N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamyl-meso-diamnopimelic acid-d-alanine (GlcNAc-anhMurNAc tetrapeptide, or M4N), supporting that the critical activator ligands for mutant strain ΔmrdA-mediated L1/L2 expression are anhMurNAc tetrapeptides. IMPORTANCE Inducible expression of chromosomally encoded β-lactamase(s) is a key mechanism for β-lactam resistance in Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The muropeptides produced during the peptidoglycan recycling pathway act as activator ligands for β-lactamase(s) induction. The muropeptides 1,6-anhydromuramyl pentapeptide and 1,6-anhydromuramyl tripeptide are the known activator ligands for ampC β-lactamase expression in E. cloacae. Here, we dissected the type of muropepetides for L1/L2 β-lactamase expression in an mrdA deletion mutant of S. maltophilia. Distinct from the findings with the ampC system, 1,6-anhydromuramyl tetrapeptide is the candidate for ΔmrdA-mediated β-lactamase expression in S. maltophilia. Our work extends the understanding of β-lactamase induction and provides valuable information for combating the occurrence of β-lactam resistance.
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Pazos, Manuel, Katharina Peters, Adrien Boes, Yalda Safaei, Calem Kenward, Nathanael A. Caveney, Cedric Laguri, et al. "SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli." mBio 11, no. 6 (November 3, 2020). http://dx.doi.org/10.1128/mbio.02796-20.
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ABSTRACT Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum. IMPORTANCE Escherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division.
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Kamini, Dasvit Shetty, Vikas D. Trivedi, Madhushri Varunjikar, and Prashant S. Phale. "Compartmentalization of the Carbaryl Degradation Pathway: Molecular Characterization of Inducible Periplasmic Carbaryl Hydrolase from Pseudomonas spp." Applied and Environmental Microbiology 84, no. 2 (October 27, 2017). http://dx.doi.org/10.1128/aem.02115-17.
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ABSTRACTPseudomonassp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis ofmcbA, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm ofEscherichia coli. The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis ofE. coliexpressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparentKmandVmaxin the range of 98 to 111 μM and 69 to 73 μmol · min−1· mg−1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm ofPseudomonasspp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.IMPORTANCEProteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm ofPseudomonasspp. Predicted transmembrane domain and signal peptide sequences ofPseudomonaswere found to be functional inEscherichia coliand to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.
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Lundgren, Benjamin R., Joseph M. Shoytush, Ryan A. Scheel, Safreen Sain, Zaara Sarwar, and Christopher T. Nomura. "Utilization of L-glutamate as a preferred or sole nutrient in Pseudomonas aeruginosa PAO1 depends on genes encoding for the enhancer-binding protein AauR, the sigma factor RpoN and the transporter complex AatJQMP." BMC Microbiology 21, no. 1 (March 15, 2021). http://dx.doi.org/10.1186/s12866-021-02145-x.
Full textAbstract:
Abstract Background Glutamate and aspartate are preferred nutrients for a variety of microorganisms. In the case for many Pseudomonas spp., utilization of these amino acids is believed to be dependent on a transporter complex comprised of a periplasmic-solute binding protein (AatJ), two permease domains (AatQM) and an ATP-binding component (AatP). Notably, expression of this transporter complex is hypothesized to be regulated at the transcriptional level by the enhancer-binding protein AauR and the alternative sigma factor RpoN. The purpose of the current study was to determine the biological significance of the putative aatJ-aatQMP operon and its regulatory aauR and rpoN genes in the utilization of L-glutamate, L-glutamine, L-aspartate and L-asparagine in Pseudomonas aeruginosa PAO1. Results Deletion of the aatJ-aatQMP, aauR or rpoN genes did not affect the growth of P. aeruginosa PAO1 on L-glutamate, L-glutamine, L-aspartate and L-asparagine equally. Instead, only growth on L-glutamate as the sole carbon source was abolished with the deletion of any one of these genes. Interestingly, growth of the aauR mutant on L-glutamate was readily restored via plasmid-based expression of the aatQMP genes, suggesting that it is the function of AatQMP (and not AatJ) that is limiting in the absence of the aauR gene. Subsequent analysis of beta-galactosidase reporters revealed that both aatJ and aatQ were induced in response to L-glutamate, L-glutamine, L-aspartate or L-asparagine in a manner dependent on the aauR and rpoN genes. In addition, both aatJ and aatQ were expressed at reduced levels in the absence of the inducing-amino acids and the regulatory aauR and rpoN genes. The expression of the aatJ-aatQMP genes is, therefore, multifaceted. Lastly, the expression levels of aatJ were significantly higher (> 5 fold) than that of aatQ under all tested conditions. Conclusions The primary function of AauR in P. aeruginosa PAO1 is to activate expression of the aatJ-aatQMP genes in response to exogenous acidic amino acids and their amide derivatives. Importantly, it is the AauR-RpoN mediated induction of the aatQMP genes that is the pivotal factor enabling P. aeruginosa PAO1 to effectively utilize or consume L-glutamate as a sole or preferred nutrient.