Identification of protein components and quantitative immunoassay for SEC2 in staphylococcin injection.

PubMed

Ding, Ding; Huang, Peng; Sun, Hongying; Xue, Qiao; Pan, Yingqiu; Chen, Shuqing

2009-08-15

In China, staphylococcin injection has been commonly used in combined cancer therapy to enhance the systemic immune response and reduce the toxicities associated with chemotherapy or radiation therapy in the last decade. It is claimed that the main effective component is staphylococcal enterotoxin C2 (SEC2). However, no standard method based on the concentration of SEC2 has been established for quality control of the injection products. In this study, a sensitive and reliable biotin-streptavidin-ELISA (BS-ELISA) method was established for detection and quantification of SEC2. In addition, 1-D SDS-PAGE coupled with nano-LC-MS/MS was performed to identify the protein components in the injection products from one manufacturing company. The results of the BS-ELISA showed that SEC2 only accounted for less than 0.1% of the total protein in the injection products, and the nano-LC-MS/MS results showed that fifty-five proteins of Staphylococcus aureus were confidently identified in the injection solution. Seventeen out of these proteins, including SEC2, were well-known virulence factors. In addition, eighteen proteins of other Gram-positive bacteria were also confidently identified. Thus, the results indicated that SEC2 is of very low concentration in the injection products and the process of the injection preparation should be improved for health and safety consideration.

Functional diversification of Arabidopsis SEC1-related SM proteins in cytokinetic and secretory membrane fusion.

PubMed

Karnahl, Matthias; Park, Misoon; Krause, Cornelia; Hiller, Ulrike; Mayer, Ulrike; Stierhof, York-Dieter; Jürgens, Gerd

2018-06-12

Sec1/Munc18 (SM) proteins contribute to membrane fusion by interacting with Qa-SNAREs or nascent trans -SNARE complexes. Gymnosperms and the basal angiosperm Amborella have only a single SEC1 gene related to the KEULE gene in Arabidopsis However, the genomes of most angiosperms including Arabidopsis encode three SEC1-related SM proteins of which only KEULE has been functionally characterized as interacting with the cytokinesis-specific Qa-SNARE KNOLLE during cell-plate formation. Here we analyze the closest paralog of KEULE named SEC1B. In contrast to the cytokinesis defects of keule mutants, sec1b mutants are homozygous viable. However, the keule sec1b double mutant was nearly gametophytically lethal, displaying collapsed pollen grains, which suggests substantial overlap between SEC1B and KEULE functions in secretion-dependent growth. SEC1B had a strong preference for interaction with the evolutionarily ancient Qa-SNARE SYP132 involved in secretion and cytokinesis, whereas KEULE interacted with both KNOLLE and SYP132. This differential interaction with Qa-SNAREs is likely conferred by domains 1 and 2a of the two SM proteins. Comparative analysis of all four possible combinations of the relevant SEC1 Qa-SNARE double mutants revealed that in cytokinesis, the interaction of SEC1B with KNOLLE plays no role, whereas the interaction of KEULE with KNOLLE is prevalent and functionally as important as the interactions of both SEC1B and KEU with SYP132 together. Our results suggest that functional diversification of the two SEC1-related SM proteins during angiosperm evolution resulted in enhanced interaction of SEC1B with Qa-SNARE SYP132, and thus a predominant role of SEC1B in secretion.

Surfing the Sec61 channel: bidirectional protein translocation across the ER membrane.

PubMed

Römisch, K

1999-12-01

Misfolded secretory and transmembrane proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded. Degradation is primarily mediated by cytosolic proteasomes and thus requires retrograde transport out of the ER back to the cytosol. The available evidence suggests that the protein-conducting channel formed by the Sec61 complex is responsible for both forward and retrograde transport of proteins across the ER membrane. For transmembrane proteins, retrograde transport can be viewed as a reversal of integration of membrane proteins into the ER membrane. Retrograde transport of soluble proteins through the Sec61 channel after signal-peptide cleavage, however, must be mechanistically distinct from signal-peptide-mediated import into the ER through the same channel.

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation

PubMed Central

Lakkaraju, Asvin K. K.; Thankappan, Ratheeshkumar; Mary, Camille; Garrison, Jennifer L.; Taunton, Jack; Strub, Katharina

2012-01-01

Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120–160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway. PMID:22648169

Transposon-disruption of a maize nuclear gene, tha1, encoding a chloroplast SecA homologue: in vivo role of cp-SecA in thylakoid protein targeting.

PubMed

Voelker, R; Mendel-Hartvig, J; Barkan, A

1997-02-01

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced > or = 40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-SecA function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.

In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes

PubMed Central

Ohta, Naoki; Kato, Yasuhiko; Watanabe, Hajime; Mori, Hirotada; Matsuura, Tomoaki

2016-01-01

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach. PMID:27808179

Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum

PubMed Central

1985-01-01

SEC53, a gene that is required for completion of assembly of proteins in the endoplasmic reticulum in yeast, has been cloned, sequenced, and the product localized by cell fractionation. Complementation of a sec53 mutation is achieved with unique plasmids from genomic or cDNA expression banks. These inserts contain the authentic gene, a cloned copy of which integrates at the sec53 locus. An open reading frame in the insert predicts a 29-kD protein with no significant hydrophobic character. This prediction is confirmed by detection of a 28-kD protein overproduced in cells that carry SEC53 on a multicopy plasmid. To follow Sec53p more directly, a LacZ-SEC53 gene fusion has been constructed which allows the isolation of a hybrid protein for use in production of antibody. With such an antibody, quantitative immune decoration has shown that the sec53-6 mutation decreases the level of Sec53p at 37 degrees C, while levels comparable to wild-type are seen at 24 degrees C. An eightfold overproduction of Sec53p accompanies transformation of cells with a multicopy plasmid containing SEC53. Cell fractionation, performed with conditions that preserve the lumenal content of the endoplasmic reticulum (ER), shows Sec53p highly enriched in the cytosol fraction. We suggest that Sec53p acts indirectly to facilitate assembly in the ER, possibly by interacting with a stable ER component, or by providing a small molecule, other than an oligosaccharide precursor, necessary for the assembly event. PMID:3905826

Fluorophore Absorption Size Exclusion Chromatography (FA-SEC): An Alternative Method for High-Throughput Detergent Screening of Membrane Proteins.

PubMed

Lin, Sung-Yao; Sun, Xing-Han; Hsiao, Yu-Hsuan; Chang, Shao-En; Li, Guan-Syun; Hu, Nien-Jen

2016-01-01

Membrane proteins play key roles in many fundamental functions in cells including ATP synthesis, ion and molecule transporter, cell signalling and enzymatic reactions, accounting for ~30% genes of whole genomes. However, the hydrophobic nature of membrane proteins frequently hampers the progress of structure determination. Detergent screening is the critical step in obtaining stable detergent-solubilized membrane proteins and well-diffracting protein crystals. Fluorescence Detection Size Exclusion Chromatography (FSEC) has been developed to monitor the extraction efficiency and monodispersity of membrane proteins in detergent micelles. By tracing the FSEC profiles of GFP-fused membrane proteins, this method significantly enhances the throughput of detergent screening. However, current methods to acquire FSEC profiles require either an in-line fluorescence detector with the SEC equipment or an off-line spectrofluorometer microplate reader. Here, we introduce an alternative method detecting the absorption of GFP (FA-SEC) at 485 nm, thus making this methodology possible on conventional SEC equipment through the in-line absorbance spectrometer. The results demonstrate that absorption is in great correlation with fluorescence of GFP. The comparably weaker absorption signal can be improved by using a longer path-length flow cell. The FA-SEC profiles were congruent with the ones plotted by FSEC, suggesting FA-SEC could be a comparable and economical setup for detergent screening of membrane proteins.

High-Resolution Genetics Identifies the Lipid Transfer Protein Sec14p as Target for Antifungal Ergolines

PubMed Central

Cotesta, Simona; Perruccio, Francesca; Knapp, Britta; Fu, Yue; Studer, Christian; Pries, Verena; Riedl, Ralph; Helliwell, Stephen B.; Petrovic, Katarina T.; Movva, N. Rao; Sanglard, Dominique; Tao, Jianshi; Hoepfner, Dominic

2016-01-01

Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point. PMID:27855158

Structurally detailed coarse-grained model for Sec-facilitated co-translational protein translocation and membrane integration

PubMed Central

Miller, Thomas F.

2017-01-01

We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 μs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency. PMID:28328943

A "push and slide" mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase.

PubMed

Bauer, Benedikt W; Shemesh, Tom; Chen, Yu; Rapoport, Tom A

2014-06-05

In bacteria, most secretory proteins are translocated across the plasma membrane by the interplay of the SecA ATPase and the SecY channel. How SecA moves a broad range of polypeptide substrates is only poorly understood. Here we show that SecA moves polypeptides through the SecY channel by a "push and slide" mechanism. In its ATP-bound state, SecA interacts through a two-helix finger with a subset of amino acids in a substrate, pushing them into the channel. A polypeptide can also passively slide back and forth when SecA is in the predominant ADP-bound state or when SecA encounters a poorly interacting amino acid in its ATP-bound state. SecA performs multiple rounds of ATP hydrolysis before dissociating from SecY. The proposed push and slide mechanism is supported by a mathematical model and explains how SecA allows translocation of a wide range of polypeptides. This mechanism may also apply to hexameric polypeptide-translocating ATPases. Copyright © 2014 Elsevier Inc. All rights reserved.

The Small GTPase MoSec4 Is Involved in Vegetative Development and Pathogenicity by Regulating the Extracellular Protein Secretion in Magnaporthe oryzae

PubMed Central

Zheng, Huakun; Chen, Simiao; Chen, Xiaofeng; Liu, Shuyan; Dang, Xie; Yang, Chengdong; Giraldo, Martha C.; Oliveira-Garcia, Ely; Zhou, Jie; Wang, Zonghua; Valent, Barbara

2016-01-01

The Rab GTPase proteins play important roles in the membrane trafficking, and consequently protein secretion and development of eukaryotic organisms. However, little is known about the function of Rab GTPases in Magnaporthe oryzae. To further explore the function of Rab GTPases, we deleted the ortholog of the yeast Sec4p protein in M. oryzae, namely MoSEC4. The ΔMosec4 mutant is defective in polarized growth and conidiation, and it displays decreased appressorium turgor pressure and attenuated pathogenicity. Notably, the biotrophic invasive hyphae produced in rice cells are more bulbous and compressed in the ΔMosec4 mutant. Further studies showed that deletion of the MoSEC4 gene resulted in decreased secretion of extracellular enzymes and mislocalization of the cytoplasmic effector PWL2-mCherry-NLS. In accordance with a role in secretion, the GFP-MoSec4 fusion protein mainly accumulates at tips of growing vegetative hyphae. Our results suggest that the MoSec4 protein plays important roles in the secretion of extracellular proteins and consequently hyphal development and pathogenicity in the rice blast fungus. PMID:27729922

Identifying Bacterial Immune Evasion Proteins Using Phage Display.

PubMed

Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

2017-01-01

Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

Demonstration of a Specific Escherichia coli SecY–Signal Peptide Interaction†

PubMed Central

Wang, Ligong; Miller, Alexander; Rusch, Sharyn L.; Kendall, Debra A.

2011-01-01

Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide–SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA. PMID:15476412

Bacterial copper storage proteins.

PubMed

Dennison, Christopher; David, Sholto; Lee, Jaeick

2018-03-30

Copper is essential for most organisms as a cofactor for key enzymes involved in fundamental processes such as respiration and photosynthesis. However, copper also has toxic effects in cells, which is why eukaryotes and prokaryotes have evolved mechanisms for safe copper handling. A new family of bacterial proteins uses a Cys-rich four-helix bundle to safely store large quantities of Cu(I). The work leading to the discovery of these proteins, their properties and physiological functions, and how their presence potentially impacts the current views of bacterial copper handling and use are discussed in this review. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Aggregation factor analysis for protein formulation by a systematic approach using FTIR, SEC and design of experiments techniques.

PubMed

Feng, Yan Wen; Ooishi, Ayako; Honda, Shinya

2012-01-05

A simple systematic approach using Fourier transform infrared (FTIR) spectroscopy, size exclusion chromatography (SEC) and design of experiments (DOE) techniques was applied to the analysis of aggregation factors for protein formulations in stress and accelerated testings. FTIR and SEC were used to evaluate protein conformational and storage stabilities, respectively. DOE was used to determine the suitable formulation and to analyze both the main effect of single factors and the interaction effect of combined factors on aggregation. Our results indicated that (i) analysis at a low protein concentration is not always applicable to high concentration formulations; (ii) an investigation of interaction effects of combined factors as well as main effects of single factors is effective for improving conformational stability of proteins; (iii) with the exception of pH, the results of stress testing with regard to aggregation factors would be available for suitable formulation instead of performing time-consuming accelerated testing; (iv) a suitable pH condition should not be determined in stress testing but in accelerated testing, because of inconsistent effects of pH on conformational and storage stabilities. In summary, we propose a three-step strategy, using FTIR, SEC and DOE techniques, to effectively analyze the aggregation factors and perform a rapid screening for suitable conditions of protein formulation. Copyright © 2011 Elsevier B.V. All rights reserved.

In Candida albicans hyphae, Sec2p is physically associated with SEC2 mRNA on secretory vesicles.

PubMed

Caballero-Lima, David; Hautbergue, Guillaume M; Wilson, Stuart A; Sudbery, Peter E

2014-11-01

Candida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans-Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA.

PubMed

Randall, Linda L; Henzl, Michael T

2010-06-01

Protein export mediated by the general secretory Sec system in Escherichia coli proceeds by a dynamic transfer of a precursor polypeptide from the chaperone SecB to the SecA ATPase motor of the translocon and subsequently into and through the channel of the membrane-embedded SecYEG heterotrimer. The complex between SecA and SecB is stabilized by several separate sites of contact. Here we have demonstrated directly an interaction between the N-terminal residues 2 through 11 of SecA and the C-terminal 13 residues of SecB by isothermal titration calorimetry and analytical sedimentation velocity centrifugation. We discuss the unusual binding properties of SecA and SecB in context of a model for transfer of the precursor along the pathway of export.

SecG is required for antibiotic activities of Pseudomonas sp. YL23 against Erwinia amylovora and Dickeya chrysanthemi.

PubMed

Liu, Youzhou; Baird, Sonya M; Qiao, Junqing; Du, Yan; Lu, Shi-En

2015-05-01

Strain YL23 was isolated from soybean root tips and identified to be Pseudomonas sp. This strain showed broad-spectrum antibacterial activity against bacterial pathogens that are economically important in agriculture. To characterize the genes dedicated to antibacterial activities against microbial phytopathogens, a Tn5-mutation library of YL23 was constructed. Plate bioassays revealed that the mutant YL23-93 lost its antibacterial activities against Erwinia amylovora and Dickeya chrysanthemi as compared with its wild type strain. Genetic and sequencing analyses localized the transposon in a homolog of the secG gene in the mutant YL23-93. Constitutive expression plasmid pUCP26-secG was constructed and electroporated into the mutant YL23-93. Introduction of the plasmid pUCP26-secG restored antibacterial activities of the mutant YL23-93 to E. amylovora and D. chrysanthemi. As expected, empty plasmid pUCP26 could not complement the phenotype of the antibacterial activity in the mutant. Thus the secG gene, belonging to the Sec protein translocation system, is required for antibacterial activity of strain YL23 against E. amylovora and D. chrysanthemi. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Roles of the conserved cytoplasmic region and non-conserved carboxy-terminal region of SecE in Escherichia coli protein translocase.

PubMed

Kontinen, V P; Yamanaka, M; Nishiyama, K; Tokuda, H

1996-06-01

SecE, an essential membrane component of the Escherichia coli protein translocase, consists of 127 amino acid residues. Only a part of the second putative cytoplasmic region comprising some 13 residues is essential for the SecE function as long as the proper topological arrangement is retained. The Trp84 and Pro85 residues of this region are conserved in all eubacterial SecE homologues. The conservation of positively charged residues corresponding to Arg80 and Lys81 is also substantial. We deleted or replaced these residues to assess their roles in the SecE function. Deletion of the Arg80-Lys81 dipeptide did not abolish the SecE function whereas that of Trp84 or Pro85 caused a loss of the function. Strikingly, however, replacement of Pro85 with either Gly, Ser, or Ala, and that of Trp84 with Lys did not abolish the SecE function. These results indicate that the strong conservation of these residues does not reflect their obligatory requirement for the SecE function. A chimeric SecE possessing the cytoplasmic region of the E. coli SecE and the following region of the Bacillus subtilis SecE was able to form the translocation machinery together with SecA, SecY, and SecG. Although a Leu to Arg mutation at position 108 has been thought to cause a loss of signal recognition fidelity and thereby suppress a signal sequence defect, the same mutation at position 111 caused a complete loss of the function. The levels of SecY and SecG in the secEcsE501 mutant, which expresses SecE at a decreased level and is sensitive to low temperature, increased upon the expression of functional SecE derivatives, irrespective of the site of mutation, suggesting that the levels of SecY and SecG are co-operatively determined by the level of functional, but not non-functional, SecE. Based on these results, the SecE function in the translocase is discussed.

Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells.

PubMed

Gans, Jonathan; Osborne, Jonathan; Cheng, Juliet; Djapgne, Louise; Oglesby-Sherrouse, Amanda G

2018-01-01

Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

Pancreatic SEC23B deficiency is sufficient to explain the perinatal lethality of germline SEC23B deficiency in mice

PubMed Central

Khoriaty, Rami; Everett, Lesley; Chase, Jennifer; Zhu, Guojing; Hoenerhoff, Mark; McKnight, Brooke; Vasievich, Matthew P.; Zhang, Bin; Tomberg, Kärt; Williams, John; Maillard, Ivan; Ginsburg, David

2016-01-01

In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes. PMID:27297878

Regulation of the protein-conducting channel by a bound ribosome

PubMed Central

Gumbart, James; Trabuco, Leonardo G.; Schreiner, Eduard; Villa, Elizabeth; Schulten, Klaus

2009-01-01

Summary During protein synthesis, it is often necessary for the ribosome to form a complex with a membrane-bound channel, the SecY/Sec61 complex, in order to translocate nascent proteins across a cellular membrane. Structural data on the ribosome-channel complex are currently limited to low-resolution cryo-electron microscopy maps, including one showing a bacterial ribosome bound to a monomeric SecY complex. Using that map along with available atomic-level models of the ribosome and SecY, we have determined, through molecular dynamics flexible fitting (MDFF), an atomic-resolution model of the ribosome-channel complex. We characterized computationally the sites of ribosome-SecY interaction within the complex and determined the effect of ribosome binding on the SecY channel. We also constructed a model of a ribosome in complex with a SecY dimer by adding a second copy of SecY to the MDFF-derived model. The study involved 2.7-million-atom simulations over altogether nearly 50 ns. PMID:19913480

Novel Allylic Oxidation of α-Cedrene to sec-Cedrenol by a Rhodococcus Strain

PubMed Central

Takigawa, Hirofumi; Kubota, Hiromi; Sonohara, Hiroshi; Okuda, Mitsuyoshi; Tanaka, Shigeyoshi; Fujikura, Yoshiaki; Ito, Susumu

1993-01-01

A bacterial strain, designated KSM-7358, that can use α-cedrene for growth was isolated. The strain was identified as a member of the genus Rhodococcus and catalyzed the novel allylic oxidation of α-cedrene regiospecifically to produce (R)-10-hydroxycedrene (sec-cedrenol) with a very high yield. α-Curcumene was also produced as a possible metabolite of sec-cedrenol. A possible pathway for the microbial conversion of α-cedrene to sec-cedrenol and α-curcumene is proposed. PMID:16348930

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant.

PubMed

Ruíz, Teresa; De la Rosa, José M; Domínguez, Angel; Rodríguez, Luis

2003-05-01

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.

Pleiotropic Alterations in Lipid Metabolism in Yeast sac1 Mutants: Relationship to “Bypass Sec14p” and Inositol Auxotrophy

PubMed Central

Rivas, Marcos P.; Kearns, Brian G.; Xie, Zhigang; Guo, Shuling; Sekar, M. Chandra; Hosaka, Kohei; Kagiwada, Satoshi; York, John D.; Bankaitis, Vytas A.

1999-01-01

SacIp dysfunction results in bypass of the requirement for phosphatidylinositol transfer protein (Sec14p) function in yeast Golgi processes. This effect is accompanied by alterations in inositol phospholipid metabolism and inositol auxotrophy. Elucidation of how sac1 mutants effect “bypass Sec14p” will provide insights into Sec14p function in vivo. We now report that, in addition to a dramatic accumulation of phosphatidylinositol-4-phosphate, sac1 mutants also exhibit a specific acceleration of phosphatidylcholine biosynthesis via the CDP-choline pathway. This phosphatidylcholine metabolic phenotype is sensitive to the two physiological challenges that abolish bypass Sec14p in sac1 strains; i.e. phospholipase D inactivation and expression of bacterial diacylglycerol (DAG) kinase. Moreover, we demonstrate that accumulation of phosphatidylinositol-4-phosphate in sac1 mutants is insufficient to effect bypass Sec14p. These data support a model in which phospholipase D activity contributes to generation of DAG that, in turn, effects bypass Sec14p. A significant fate for this DAG is consumption by the CDP-choline pathway. Finally, we determine that CDP-choline pathway activity contributes to the inositol auxotrophy of sac1 strains in a novel manner that does not involve obvious defects in transcriptional expression of the INO1 gene. PMID:10397762

Bacterial Origin of a Mitochondrial Outer Membrane Protein Translocase

PubMed Central

Harsman, Anke; Niemann, Moritz; Pusnik, Mascha; Schmidt, Oliver; Burmann, Björn M.; Hiller, Sebastian; Meisinger, Chris; Schneider, André; Wagner, Richard

2012-01-01

Mitochondria are of bacterial ancestry and have to import most of their proteins from the cytosol. This process is mediated by Tom40, an essential protein that forms the protein-translocating pore in the outer mitochondrial membrane. Tom40 is conserved in virtually all eukaryotes, but its evolutionary origin is unclear because bacterial orthologues have not been identified so far. Recently, it was shown that the parasitic protozoon Trypanosoma brucei lacks a conventional Tom40 and instead employs the archaic translocase of the outer mitochondrial membrane (ATOM), a protein that shows similarities to both eukaryotic Tom40 and bacterial protein translocases of the Omp85 family. Here we present electrophysiological single channel data showing that ATOM forms a hydrophilic pore of large conductance and high open probability. Moreover, ATOM channels exhibit a preference for the passage of cationic molecules consistent with the idea that it may translocate unfolded proteins targeted by positively charged N-terminal presequences. This is further supported by the fact that the addition of a presequence peptide induces transient pore closure. An in-depth comparison of these single channel properties with those of other protein translocases reveals that ATOM closely resembles bacterial-type protein export channels rather than eukaryotic Tom40. Our results support the idea that ATOM represents an evolutionary intermediate between a bacterial Omp85-like protein export machinery and the conventional Tom40 that is found in mitochondria of other eukaryotes. PMID:22778261

Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development.

PubMed

Townley, Anna K; Feng, Yi; Schmidt, Katy; Carter, Deborah A; Porter, Robert; Verkade, Paul; Stephens, David J

2008-09-15

The COPII coat assembles on endoplasmic reticulum membranes to coordinate the collection of secretory cargo with the formation of transport vesicles. During COPII assembly, Sar1 deforms the membrane and recruits the Sec23-Sec24 complex (Sec23/24), which is the primary cargo-binding adaptor for the system, and Sec13-Sec31 (Sec13/31), which provides a structural outer layer for vesicle formation. Here we show that Sec13 depletion results in concomitant loss of Sec31 and juxtanuclear clustering of pre-budding complexes containing Sec23/24 and cargo. Electron microscopy reveals the presence of curved coated profiles on distended endoplasmic reticulum, indicating that Sec13/31 is not required for the generation or maintenance of the curvature. Surprisingly, export of tsO45-G-YFP, a marker of secretory cargo, is unaffected by Sec13/31 depletion; by contrast, secretion of collagen from primary fibroblasts is strongly inhibited. Suppression of Sec13 expression in zebrafish causes defects in proteoglycan deposition and skeletal abnormalities that are grossly similar to the craniofacial abnormalities of crusher mutant zebrafish and patients with cranio-lenticulo-sutural dysplasia. We conclude that efficient coupling of the inner (Sec23/24) and outer (Sec13/31) layers of the COPII coat is required to drive the export of collagen from the endoplasmic reticulum, and that highly efficient COPII assembly is essential for normal craniofacial development during embryogenesis.

Ancestral and derived protein import pathways in the mitochondrion of Reclinomonas americana.

PubMed

Tong, Janette; Dolezal, Pavel; Selkrig, Joel; Crawford, Simon; Simpson, Alastair G B; Noinaj, Nicholas; Buchanan, Susan K; Gabriel, Kipros; Lithgow, Trevor

2011-05-01

The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or "translocase in the inner mitochondrial membrane complex." Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution.

The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.

PubMed

Zehner, Matthias; Marschall, Andrea L; Bos, Erik; Schloetel, Jan-Gero; Kreer, Christoph; Fehrenschild, Dagmar; Limmer, Andreas; Ossendorp, Ferry; Lang, Thorsten; Koster, Abraham J; Dübel, Stefan; Burgdorf, Sven

2015-05-19

The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER. Copyright © 2015 Elsevier Inc. All rights reserved.

Bacterial ice crystal controlling proteins.

PubMed

Lorv, Janet S H; Rose, David R; Glick, Bernard R

2014-01-01

Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

PubMed

Hovingh, Elise S; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion

PubMed Central

Hovingh, Elise S.; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed. PMID:28066340

Unraveling novel broad-spectrum antibacterial targets in food and waterborne pathogens using comparative genomics and protein interaction network analysis.

PubMed

Jadhav, Ankush; Shanmugham, Buvaneswari; Rajendiran, Anjana; Pan, Archana

2014-10-01

Food and waterborne diseases are a growing concern in terms of human morbidity and mortality worldwide, even in the 21st century, emphasizing the need for new therapeutic interventions for these diseases. The current study aims at prioritizing broad-spectrum antibacterial targets, present in multiple food and waterborne bacterial pathogens, through a comparative genomics strategy coupled with a protein interaction network analysis. The pathways unique and common to all the pathogens under study (viz., methane metabolism, d-alanine metabolism, peptidoglycan biosynthesis, bacterial secretion system, two-component system, C5-branched dibasic acid metabolism), identified by comparative metabolic pathway analysis, were considered for the analysis. The proteins/enzymes involved in these pathways were prioritized following host non-homology analysis, essentiality analysis, gut flora non-homology analysis and protein interaction network analysis. The analyses revealed a set of promising broad-spectrum antibacterial targets, present in multiple food and waterborne pathogens, which are essential for bacterial survival, non-homologous to host and gut flora, and functionally important in the metabolic network. The identified broad-spectrum candidates, namely, integral membrane protein/virulence factor (MviN), preprotein translocase subunits SecB and SecG, carbon storage regulator (CsrA), and nitrogen regulatory protein P-II 1 (GlnB), contributed by the peptidoglycan pathway, bacterial secretion systems and two-component systems, were also found to be present in a wide range of other disease-causing bacteria. Cytoplasmic proteins SecG, CsrA and GlnB were considered as drug targets, while membrane proteins MviN and SecB were classified as vaccine targets. The identified broad-spectrum targets can aid in the design and development of antibacterial agents not only against food and waterborne pathogens but also against other pathogens. Copyright © 2014 Elsevier B.V. All rights

Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus

PubMed Central

Tsukazaki, Tomoya; Mori, Hiroyuki; Fukai, Shuya; Numata, Tomoyuki; Perederina, Anna; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo; Vassylyev, Dmitry G.; Nureki, Osamu; Ito, Koreaki

2006-01-01

Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 Å upon X-ray irradiation, revealing that they belonged to space group P43212. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 Å resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery. PMID:16582489

The super elongation complex (SEC) and MLL in development and disease

PubMed Central

Smith, Edwin; Lin, Chengqi; Shilatifard, Ali

2011-01-01

Transcriptional regulation at the level of elongation is vital for the control of gene expression and metazoan development. The mixed lineage leukemia (MLL) protein and its Drosophila homolog, Trithorax, which exist within COMPASS (complex of proteins associated with Set1)-like complexes, are master regulators of development. They are required for proper homeotic gene expression, in part through methylation of histone H3 on Lys 4. In humans, the MLL gene is involved in a large number of chromosomal translocations that create chimeric proteins, fusing the N terminus of MLL to several proteins that share little sequence similarity. Several frequent translocation partners of MLL were found recently to coexist in a super elongation complex (SEC) that includes known transcription elongation factors such as eleven-nineteen lysine-rich leukemia (ELL) and P-TEFb. Importantly, the SEC is required for HOX gene expression in leukemic cells, suggesting that chromosomal translocations involving MLL could lead to the overexpression of HOX and other genes through the involvement of the SEC. Here, we review the normal developmental roles of MLL and the SEC, and how MLL fusion proteins can mediate leukemogenesis. PMID:21460034

Bacterial Ice Crystal Controlling Proteins

PubMed Central

Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

2014-01-01

Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane.

PubMed Central

He, S Y; Schoedel, C; Chatterjee, A K; Collmer, A

1991-01-01

The plant pathogenic enterobacterium Erwinia chrysanthemi EC16 secretes several extracellular, plant cell wall-degrading enzymes, including pectate lyase isozyme PelE. Secretion kinetics of 35S-labeled PelE indicated that the precursor of PelE was rapidly processed by the removal of the amino-terminal signal peptide and that the resulting mature PelE remained cell bound for less than 60 s before being secreted to the bacterial medium. PelE-PhoA (alkaline phosphatase) hybrid proteins generated in vivo by TnphoA insertions were mostly localized in the periplasm of E. chrysanthemi, and one hybrid protein was observed to be associated with the outer membrane of E. chrysanthemi in an out gene-dependent manner. A gene fusion resulting in the substitution of the beta-lactamase signal peptide for the first six amino acids of the PelE signal peptide did not prevent processing or secretion of PelE in E. chrysanthemi. When pelE was overexpressed, mature PelE protein accumulated in the periplasm rather than the cytoplasm in cells of E. chrysanthemi and Escherichia coli MC4100 (pCPP2006), which harbors a functional cluster of E. chrysanthemi out genes. Removal of the signal peptide from pre-PelE was SecA dependent in E. coli MM52 even in the presence of the out gene cluster. These data indicate that the extracellular secretion of pectic enzymes by E. chrysanthemi is an extension of the Sec-dependent pathway for general export of proteins across the bacterial inner membrane. Images PMID:1829728

Dynamic distribution of the SecA and SecY translocase subunits and septal localization of the HtrA surface chaperone/protease during Streptococcus pneumoniae D39 cell division.

PubMed

Tsui, Ho-Ching Tiffany; Keen, Susan K; Sham, Lok-To; Wayne, Kyle J; Winkler, Malcolm E

2011-01-01

The Sec translocase pathway is the major route for protein transport across and into the cytoplasmic membrane of bacteria. Previous studies reported that the SecA translocase ATP-binding subunit and the cell surface HtrA protease/chaperone formed a single microdomain, termed "ExPortal," in some species of ellipsoidal (ovococcus) Gram-positive bacteria, including Streptococcus pyogenes. To investigate the generality of microdomain formation, we determined the distribution of SecA and SecY by immunofluorescent microscopy in Streptococcus pneumoniae (pneumococcus), which is an ovococcus species evolutionarily distant from S. pyogenes. In the majority (≥ 75%) of exponentially growing cells, S. pneumoniae SecA (SecA (Spn)) and SecY (Spn) located dynamically in cells at different stages of division. In early divisional cells, both Sec subunits concentrated at equators, which are future sites of constriction. Further along in division, SecA(Spn) and SecY(Spn) remained localized at mid-cell septa. In late divisional cells, both Sec subunits were hemispherically distributed in the regions between septa and the future equators of dividing cells. In contrast, the HtrA (Spn) homologue localized to the equators and septa of most (> 90%) dividing cells, whereas the SrtA(Spn) sortase located over the surface of cells in no discernable pattern. This dynamic pattern of Sec distribution was not perturbed by the absence of flotillin family proteins, but was largely absent in most cells in early stationary phase and in cls mutants lacking cardiolipin synthase. These results do not support the existence of an ExPortal microdomain in S. pneumoniae. Instead, the localization of the pneumococcal Sec translocase depends on the stage of cell division and anionic phospholipid content. Two patterns of Sec translocase distribution, an ExPortal microdomain in certain ovococcus-shaped species like Streptococcus pyogenes and a spiral pattern in rod-shaped species like Bacillus subtilis, have

Haloarchaeal Protein Translocation via the Twin Arginine Translocation Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pohlschroder Mechthild

2009-02-03

Protein transport across hydrophobic membranes that partition cellular compartments is essential in all cells. The twin arginine translocation (Tat) pathway transports proteins across the prokaryotic cytoplasmic membranes. Distinct from the universally conserved Sec pathway, which secretes unfolded proteins, the Tat machinery is unique in that it secretes proteins in a folded conformation, making it an attractive pathway for the transport and secretion of heterologously expressed proteins that are Sec-incompatible. During the past 7 years, the DOE-supported project has focused on the characterization of the diversity of bacterial and archaeal Tat substrates as well as on the characterization of the Tatmore » pathway of a model archaeon, Haloferax volcanii, a member of the haloarchaea. We have demonstrated that H. volcanii uses this pathway to transport most of its secretome.« less

Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

PubMed

Suda, Yasuyuki; Tachikawa, Hiroyuki; Inoue, Ichiro; Kurita, Tomokazu; Saito, Chieko; Kurokawa, Kazuo; Nakano, Akihiko; Irie, Kenji

2018-02-01

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

PubMed Central

Ishii, Eiji; Chiba, Shinobu; Hashimoto, Narimasa; Kojima, Seiji; Homma, Michio; Ito, Koreaki; Akiyama, Yoshinori; Mori, Hiroyuki

2015-01-01

SecDF interacts with the SecYEG translocon in bacteria and enhances protein export in a proton-motive-force-dependent manner. Vibrio alginolyticus, a marine-estuarine bacterium, contains two SecDF paralogs, V.SecDF1 and V.SecDF2. Here, we show that the export-enhancing function of V.SecDF1 requires Na+ instead of H+, whereas V.SecDF2 is Na+-independent, presumably requiring H+. In accord with the cation-preference difference, V.SecDF2 was only expressed under limited Na+ concentrations whereas V.SecDF1 was constitutive. However, it is not the decreased concentration of Na+ per se that the bacterium senses to up-regulate the V.SecDF2 expression, because marked up-regulation of the V.SecDF2 synthesis was observed irrespective of Na+ concentrations under certain genetic/physiological conditions: (i) when the secDF1VA gene was deleted and (ii) whenever the Sec export machinery was inhibited. VemP (Vibrio export monitoring polypeptide), a secretory polypeptide encoded by the upstream ORF of secDF2VA, plays the primary role in this regulation by undergoing regulated translational elongation arrest, which leads to unfolding of the Shine–Dalgarno sequence for translation of secDF2VA. Genetic analysis of V. alginolyticus established that the VemP-mediated regulation of SecDF2 is essential for the survival of this marine bacterium in low-salinity environments. These results reveal that a class of marine bacteria exploits nascent-chain ribosome interactions to optimize their protein export pathways to propagate efficiently under different ionic environments that they face in their life cycles. PMID:26392525

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

PubMed Central

Branco, Luis M; Matschiner, Alex; Fair, Joseph N; Goba, Augustine; Sampey, Darryl B; Ferro, Philip J; Cashman, Kathleen A; Schoepp, Randal J; Tesh, Robert B; Bausch, Daniel G; Garry, Robert F; Guttieri, Mary C

2008-01-01

Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA). Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination. PMID:18538016

N-Glycosylation of Campylobacter jejuni Surface Proteins Promotes Bacterial Fitness

PubMed Central

Nothaft, Harald; Zheng, Jing

2013-01-01

Campylobacter jejuni is the etiologic agent of human bacterial gastroenteritis worldwide. In contrast, despite heavy colonization, C. jejuni maintains a commensal mode of existence in chickens. The consumption of contaminated chicken products is thought to be the principal mode of C. jejuni transmission to the human population. C. jejuni harbors a system for N-linked protein glycosylation that has been well characterized and modifies more than 60 periplasmic and membrane-bound proteins. However, the precise role of this modification in the biology of C. jejuni remains unexplored. We hypothesized that the N-glycans protect C. jejuni surface proteins from the action of gut proteases. The C. jejuni pglB mutant, deficient in the expression of the oligosaccharyltransferase, exhibited reduced growth in medium supplemented with chicken cecal contents (CCC) compared with that of wild-type (WT) cells. Inactivation of the cecal proteases by heat treatment or with protease inhibitors completely restored bacterial viability and partially rescued bacterial growth. Physiological concentrations of trypsin, but not chymotrypsin, also reduced C. jejuni pglB mutant CFU. Live or dead staining indicated that CCC preferentially influenced C. jejuni growth as opposed to bacterial viability. We identified multiple chicken cecal proteases by mass fingerprinting. The use of protease inhibitors that target specific classes indicated that both metalloproteases and serine proteases were involved in the attenuated growth of the oligosaccharyltransferase mutant. In conclusion, protein N-linked glycosylation of surface proteins may enhance C. jejuni fitness by protecting bacterial proteins from cleavage due to gut proteases. PMID:23460522

Computational Analysis of Uncharacterized Proteins of Environmental Bacterial Genome

NASA Astrophysics Data System (ADS)

Coxe, K. J.; Kumar, M.

2017-12-01

Betaproteobacteria strain CB is a gram-negative bacterium in the phylum Proteobacteria and are found naturally in soil and water. In this complex environment, bacteria play a key role in efficiently eliminating the organic material and other pollutants from wastewater. To investigate the process of pollutant removal from wastewater using bacteria, it is important to characterize the proteins encoded by the bacterial genome. Our study combines a number of bioinformatics tools to predict the function of unassigned proteins in the bacterial genome. The genome of Betaproteobacteria strain CB contains 2,112 proteins in which function of 508 proteins are unknown, termed as uncharacterized proteins (UPs). The localization of the UPs with in the cell was determined and the structure of 38 UPs was accurately predicted. These UPs were predicted to belong to various classes of proteins such as enzymes, transporters, binding proteins, signal peptides, transmembrane proteins and other proteins. The outcome of this work will help better understand wastewater treatment mechanism.

Interactome of E. piscicida and grouper liver proteins reveals strategies of bacterial infection and host immune response.

PubMed

Li, Hui; Zhu, Qing-Feng; Peng, Xuan-Xian; Peng, Bo

2017-01-03

The occurrence of infectious diseases is related to heterogeneous protein interactions between a host and a microbe. Therefore, elucidating the host-pathogen interplay is essential. We previously revealed the protein interactome between Edwardsiella piscicida and fish gill cells, and the present study identified the protein interactome between E. piscicida and E. drummondhayi liver cells. E. drummondhayi liver cells and bacterial pull-down approaches were used to identify E. piscicida outer membrane proteins that bind to liver cells and fish liver cell proteins that interact with bacterial cells, respectively. Eight bacterial proteins and 11 fish proteins were characterized. Heterogeneous protein-protein interactions between these bacterial cells and fish liver cells were investigated through far-Western blotting and co-immunoprecipitation. A network was constructed based on 42 heterogeneous protein-protein interactions between seven bacterial proteins and 10 fish proteins. A comparison of the new interactome with the previously reported interactome showed that four bacterial proteins overlapped, whereas all of the identified fish proteins were new, suggesting a difference between bacterial tricks for evading host immunity and the host strategy for combating bacterial infection. Furthermore, these bacterial proteins were found to regulate the expression of host innate immune-related proteins. These findings indicate that the interactome contributes to bacterial infection and host immunity.

Sec16 in conventional and unconventional exocytosis: Working at the interface of membrane traffic and secretory autophagy?

PubMed

Tang, Bor Luen

2017-12-01

Sec16 is classically perceived to be a scaffolding protein localized to the transitional endoplasmic reticulum (tER) or the ER exit sites (ERES), and has a conserved function in facilitating coat protein II (COPII) complex-mediated ER exit. Recent findings have, however, pointed toward a role for Sec16 in unconventional exocytosis of certain membrane proteins, such as the Cystic fibrosis transmembrane conductance regulator (CFTR) in mammalian cells, and possibly also α-integrin in certain contexts of Drosophila development. In this regard, Sec16 interacts with components of a recently deciphered pathway of stress-induced unconventional exocytosis, which is dependent on the tether protein Golgi reassembly stacking proteins (GRASPs) and the autophagy pathway. Intriguingly, Sec16 also appears to be post-translationally modified by autophagy-related signaling processes. Sec16 is known to be phosphorylated by the atypical extracellular signal regulated kinase 7 (Erk7) upon serum and amino acid starvation, both represent conditions that trigger autophagy. Recent work has also shown that Sec16 is phosphorylated, and thus regulated by the prominent autophagy-initiating Unc-51-like autophagy activating kinase 1 (Ulk1), as well as another autophagy modulator Leucine-rich repeat kinase 2 (Lrrk2). The picture emerging from Sec16's network of physical and functional interactors allows the speculation that Sec16 is situated (and may in yet undefined ways function) at the interface between COPII-mediated exocytosis of conventional vesicular traffic and the GRASP/autophagy-dependent mode of unconventional exocytosis. © 2017 Wiley Periodicals, Inc.

Comparison of the structural basis for thermal stability between archaeal and bacterial proteins.

PubMed

Ding, Yanrui; Cai, Yujie; Han, Yonggang; Zhao, Bingqiang

2012-01-01

In this study, the structural basis for thermal stability in archaeal and bacterial proteins was investigated. There were many common factors that confer resistance to high temperature in both archaeal and bacterial proteins. These factors include increases in the Lys content, the bends and blanks of secondary structure, the Glu content of salt bridge; decreases in the number of main-side chain hydrogen bond and exposed surface area, and changes in the bends and blanks of amino acids. Certainly, the utilization of charged amino acids to form salt bridges is a primary factor. In both heat-resistant archaeal and bacterial proteins, most Glu and Asp participate in the formation of salt bridges. Other factors may influence either archaeal or bacterial protein thermostability, which includes the more frequent occurrence of shorter 3(10)-helices and increased hydrophobicity in heat-resistant archaeal proteins. However, there were increases in average helix length, the Glu content in salt bridges, temperature factors and decreases in the number of main-side chain hydrogen bonds, uncharged-uncharged hydrogen bonds, hydrophobicity, and buried and exposed polar surface area in heat-resistant bacterial proteins. Evidently, there are few similarities and many disparities between the heat-resistant mechanisms of archaeal and bacterial proteins.

Selective Photoaffinity Labeling Identifies the Signal Peptide Binding Domain on SecA

PubMed Central

Musial-Siwek, Monika; Rusch, Sharyn L.; Kendall, Debra A.

2007-01-01

SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the Signal Peptide Binding Groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53-residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acids 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA. PMID:17084862

Learning about protein solubility from bacterial inclusion bodies

PubMed Central

Martínez-Alonso, Mónica; González-Montalbán, Nuria; García-Fruitós, Elena; Villaverde, Antonio

2009-01-01

The progressive solving of the conformation of aggregated proteins and the conceptual understanding of the biology of inclusion bodies in recombinant bacteria is providing exciting insights on protein folding and quality. Interestingly, newest data also show an unexpected functional and structural complexity of soluble recombinant protein species and picture the whole bacterial cell factory scenario as more intricate than formerly believed. PMID:19133126

Sec66-Dependent Regulation of Yeast Spindle-Pole Body Duplication Through Pom152

PubMed Central

Katta, Santharam S.; Chen, Jingjing; Gardner, Jennifer M.; Friederichs, Jennifer M.; Smith, Sarah E.; Gogol, Madelaine; Unruh, Jay R.; Slaughter, Brian D.; Jaspersen, Sue L.

2015-01-01

In closed mitotic systems such as Saccharomyces cerevisiae, the nuclear envelope (NE) does not break down during mitosis, so microtubule-organizing centers such as the spindle-pole body (SPB) must be inserted into the NE to facilitate bipolar spindle formation and chromosome segregation. The mechanism of SPB insertion has been linked to NE insertion of nuclear pore complexes (NPCs) through a series of genetic and physical interactions between NPCs and SPB components. To identify new genes involved in SPB duplication and NE insertion, we carried out genome-wide screens for suppressors of deletion alleles of SPB components, including Mps3 and Mps2. In addition to the nucleoporins POM152 and POM34, we found that elimination of SEC66/SEC71/KAR7 suppressed lethality of cells lacking MPS2 or MPS3. Sec66 is a nonessential subunit of the Sec63 complex that functions together with the Sec61 complex in import of proteins into the endoplasmic reticulum (ER). Cells lacking Sec66 have reduced levels of Pom152 protein but not Pom34 or Ndc1, a shared component of the NPC and SPB. The fact that Sec66 but not other subunits of the ER translocon bypass deletion mutants in SPB genes suggests a specific role for Sec66 in the control of Pom152 levels. Based on the observation that sec66∆ does not affect the distribution of Ndc1 on the NE or Ndc1 binding to the SPB, we propose that Sec66-mediated regulation of Pom152 plays an NPC-independent role in the control of SPB duplication. PMID:26510791

Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization

PubMed Central

Johansen, Jesper; Alfaro, Gabriel; Beh, Christopher T.

2016-01-01

Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. PMID:27526190

Rehosting of Bacterial Chaperones for High-Quality Protein Production▿

PubMed Central

Martínez-Alonso, Mónica; Toledo-Rubio, Verónica; Noad, Rob; Unzueta, Ugutz; Ferrer-Miralles, Neus; Roy, Polly; Villaverde, Antonio

2009-01-01

Coproduction of DnaK/DnaJ in Escherichia coli enhances solubility but promotes proteolytic degradation of their substrates, minimizing the yield of unstable polypeptides. Higher eukaryotes have orthologs of DnaK/DnaJ but lack the linked bacterial proteolytic system. By coexpression of DnaK and DnaJ in insect cells with inherently misfolding-prone recombinant proteins, we demonstrate simultaneous improvement of soluble protein yield and quality and proteolytic stability. Thus, undesired side effects of bacterial folding modulators can be avoided by appropriate rehosting in heterologous cell expression systems. PMID:19820142

Bacterial collagen-like proteins that form triple-helical structures

PubMed Central

Yu, Zhuoxin; An, Bo; Ramshaw, John A.M.; Brodsky, Barbara

2014-01-01

A large number of collagen-like proteins have been identified in bacteria during the past ten years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in E. coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35–39 °C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions. PMID:24434612

C-reactive Protein Versus Neutrophil/lymphocyte Ratio in Differentiating Bacterial and Non-bacterial Pneumonia in Children.

PubMed

Gauchan, E; Adhikari, S

2016-09-01

Pneumonia is a leading cause of childhood mortality in a low resource country. Simple laboratory markers can help differentiate between bacterial and non-bacterial pneumonias for appropriate management. In children aged one to 60 months with features of lower respiratory infection, C-reactive protein (CRP) and neutrophil-lymphocyte ratio (NLR) were used to differentiate between bacterial and non-bacterial pneumonias. The cutoff values for detecting bacterial pneumonias were evaluated by statistical tools. Bacterial pneumonia was diagnosed in 285 (43.6%) children out of 654 studied. At a cut-off value of 36 mg/L CRP was predictive of bacterial pneumonias with sensitivity and specificity of 61.8% and 91.3% respectively while the sensitivity and specificity for predicting bacterial pneumonia using NLR was 45.6% and 64% respectively with 1.28 used as a cut-off. Our study shows that CRP is superior to NLR in differentiating bacterial from non-bacterial pneumonias in children.

Polarity and charge of the periplasmic loop determine the YidC and sec translocase requirement for the M13 procoat lep protein.

PubMed

Soman, Raunak; Yuan, Jijun; Kuhn, Andreas; Dalbey, Ross E

2014-01-10

During membrane biogenesis, the M13 procoat protein is inserted into the lipid bilayer in a strictly YidC-dependent manner with both the hydrophobic signal sequence and the membrane anchor sequence promoting translocation of the periplasmic loop via a hairpin mechanism. Here, we find that the translocase requirements can be altered for PClep in a predictable manner by changing the polarity and charge of the peptide region that is translocated across the membrane. When the polarity of the translocated peptide region is lowered and the charged residues in this region are removed, translocation of this loop region occurs largely by a YidC- and Sec-independent mechanism. When the polarity is increased to that of the wild-type procoat protein, the YidC insertase is essential for translocation. Further increasing the polarity, by adding charged residues, switches the insertion pathway to a YidC/Sec mechanism. Conversely, we find that increasing the hydrophobicity of the transmembrane segments of PClep can decrease the translocase requirement for translocation of the peptide chain. This study provides a framework to understand why the YidC and Sec machineries exist in parallel and demonstrates that the YidC insertase has a limited capacity to translocate a peptide chain on its own.

Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2

PubMed Central

Takahashi, Takeshi; Kojima, Kyosuke; Zhang, Wei; Sasaki, Kanae; Ito, Masaru; Suzuki, Hironori; Kawasaki, Masato; Wakatsuki, Soichi; Takahara, Terunao; Shibata, Hideki; Maki, Masatoshi

2015-01-01

ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca2+-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe85, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr180, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined. PMID:25667979

Exocyst sec5 regulates exocytosis of newcomer insulin granules underlying biphasic insulin secretion.

PubMed

Xie, Li; Zhu, Dan; Kang, Youhou; Liang, Tao; He, Yu; Gaisano, Herbert Y

2013-01-01

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca(2+) channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.

The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

PubMed Central

Das, Debanu; Finn, Robert D; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Sri Krishna, S; Kumar, Abhinav; Lam, Winnie W; Marciano, David; Miller, Mitchell D; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Yeh, Andrew; Zhou, Jiadong; Hodgson, Keith O; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2010-01-01

Sufu (Suppressor of Fused), a two-domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu-like proteins have previously been identified based on sequence similarity to the N-terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu-like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu-like protein. The structure revealed a striking similarity to the N-terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ∼15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu-like proteins that are present in ∼200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam. PMID:20836087

Factors affecting the rate of breakdown of bacterial protein in rumen fluid.

PubMed

Wallace, R J; McPherson, C A

1987-09-01

1. The cellular proteins of Butyrivibrio fibrisolvens, Lactobacillus casei, Megasphaera elsdenii, Selenomonas ruminantium and Streptococcus bovis were labelled by growth in the presence of L-[14C]leucine, and the breakdown of labelled protein was measured in incubations of these bacteria with rumen fluid to which unlabelled 5 mM-L-leucine was added. The rate of protein breakdown was estimated from the rate of release of radioactivity into acid-soluble material. 2. Protein breakdown occurred at different rates in different species. The mean rates for B. fibrisolvens, L. casei, M. elsdenii, Sel. ruminantium and Str. bovis were 28.6, 18.1, 17.7, 10.5 and 5.3%/h respectively in samples of strained rumen fluid (SRF) with different protozoal populations. Rates of 3%/h or less were found in SRF from ciliate-free sheep or in faunated SRF from which protozoa had been removed by centrifugation. Further removal of mixed rumen bacteria had little effect. Suspensions of washed protozoa degraded bacterial protein at rates which were of the same order as those found in SRF. 3. The rate of breakdown of bacterial protein in different samples of SRF tended to increase as the numbers of small entodiniomorphid protozoa increased. The numbers of larger entodiniomorphs and holotrichs had no obvious influence on this rate. 4. Autoclaved and u.v.-treated bacteria were generally no different from live bacteria in their susceptibility to breakdown in SRF from faunated sheep, indicating that endogenous protein turnover was not a significant cause of bacterial protein catabolism. 5. The rate of bacterial protein breakdown was unrelated to the proteolytic activity of SRF. 6. It was concluded that predation by small protozoa is by far the most important cause of bacterial protein turnover in the rumen, with autolysis, other lytic factors and endogenous proteolysis being of minor importance.

The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?

PubMed

Rizo, Josep; Südhof, Thomas C

2012-01-01

Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions*

PubMed Central

Canova, Marc J.; Molle, Virginie

2014-01-01

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection. PMID:24554701

Bacterial serine/threonine protein kinases in host-pathogen interactions.

PubMed

Canova, Marc J; Molle, Virginie

2014-04-04

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.

The effect of temperature and bacterial growth phase on protein extraction by means of electroporation.

PubMed

Haberl-Meglič, Saša; Levičnik, Eva; Luengo, Elisa; Raso, Javier; Miklavčič, Damijan

2016-12-01

Different chemical and physical methods are used for extraction of proteins from bacteria, which are used in variety of fields. But on a large scale, many methods have severe drawbacks. Recently, extraction by means of electroporation showed a great potential to quickly obtain proteins from bacteria. Since many parameters are affecting the yield of extracted proteins, our aim was to investigate the effect of temperature and bacterial growth phase on the yield of extracted proteins. At the same time bacterial viability was tested. Our results showed that the temperature has a great effect on protein extraction, the best temperature post treatment being 4°C. No effect on bacterial viability was observed for all temperatures tested. Also bacterial growth phase did not affect the yield of extracted proteins or bacterial viability. Nevertheless, further experiments may need to be performed to confirm this observation, since only one incubation temperature (4°C) and one incubation time before and after electroporation (0.5 and 1h) were tested for bacterial growth phase. Based on our results we conclude that temperature is a key element for bacterial membrane to stay in a permeabilized state, so more proteins flow out of bacteria into surrounding media. Copyright © 2016 Elsevier B.V. All rights reserved.

Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

PubMed Central

Murphy, Patrick J. M.

2014-01-01

Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in

Semi-automated hydrophobic interaction chromatography column scouting used in the two-step purification of recombinant green fluorescent protein.

PubMed

Stone, Orrin J; Biette, Kelly M; Murphy, Patrick J M

2014-01-01

Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth.

PubMed

Bloch, Daria; Pleskot, Roman; Pejchar, Přemysl; Potocký, Martin; Trpkošová, Pavlína; Cwiklik, Lukasz; Vukašinović, Nemanja; Sternberg, Hasana; Yalovsky, Shaul; Žárský, Viktor

2016-10-01

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP 2 ) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP 2 However, the interaction with PIP 2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes. © 2016 American Society of Plant Biologists. All Rights Reserved.

Bacterial-like PPP protein phosphatases: novel sequence alterations in pathogenic eukaryotes and peculiar features of bacterial sequence similarity.

PubMed

Kerk, David; Uhrig, R Glen; Moorhead, Greg B

2013-01-01

Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, "bacterial-like" enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the "eukaryotic-like" phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research.

Sec13 safeguards the integrity of the endoplasmic reticulum and organogenesis of the digestive system in zebrafish.

PubMed

Niu, Xubo; Gao, Chuan; Jan Lo, Li; Luo, Yue; Meng, Chunmei; Hong, Jian; Hong, Wanjin; Peng, Jinrong

2012-07-15

The Sec13-Sec31 heterotetramer serves as the outer coat in the COPII complex, which mediates protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus. Although it has been studied in depth in yeast and cultured cells, the role of COPII in organogenesis in a multicellular organism has not. We report here that a zebrafish sec13(sq198) mutant, which exhibits a phenotype of hypoplastic digestive organs, has a mutation in the sec13 gene. The mutant gene encodes a carboxyl-terminus-truncated Sec13 that loses its affinity to Sec31a, which leads to disintegration of the ER structure in various differentiated cells in sec13(sq198), including chondrocytes, intestinal epithelial cells and hepatocytes. Disruption of the ER structure activates an unfolded protein response that eventually causes the cells to undergo cell-cycle arrest and cell apoptosis, which arrest the growth of developing digestive organs in the mutant. Our data provide the first direct genetic evidence that COPII function is essential for the organogenesis of the digestive system. Copyright © 2012 Elsevier Inc. All rights reserved.

'Drugs from bugs': bacterial effector proteins as promising biological (immune-) therapeutics.

PubMed

Rüter, Christian; Hardwidge, Philip R

2014-02-01

Immune system malfunctions cause many of the most severe human diseases. The immune system has evolved primarily to control bacterial, viral, fungal, and parasitic infections. In turn, over millions of years of coevolution, microbial pathogens have evolved various mechanisms to control and modulate the host immune system for their own benefit and survival. For example, many bacterial pathogens use virulence proteins to modulate and exploit target cell mechanisms. Our understanding of these bacterial strategies opens novel possibilities to exploit 'microbial knowledge' to control excessive immune reactions. Gaining access to strategies of microbial pathogens could lead to potentially huge benefits for the therapy of inflammatory diseases. Most work on bacterial pathogen effector proteins has the long-term aim of neutralizing the infectious capabilities of the pathogen. However, attenuated pathogens and microbial products have been used for over a century with overwhelming success in the form of vaccines to induce specific immune responses that protect against the respective infectious diseases. In this review, we focus on bacterial effector and virulence proteins capable of modulating and suppressing distinct signaling pathways with potentially desirable immune-modulating effects for treating unrelated inflammatory diseases. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Mechanism of conformational coupling in SecA: Key role of hydrogen-bonding networks and water interactions.

PubMed

Milenkovic, Stefan; Bondar, Ana-Nicoleta

2016-02-01

SecA uses the energy yielded by the binding and hydrolysis of adenosine triphosphate (ATP) to push secretory pre-proteins across the plasma membrane in bacteria. Hydrolysis of ATP occurs at the nucleotide-binding site, which contains the conserved carboxylate groups of the DEAD-box helicases. Although crystal structures provide valuable snapshots of SecA along its reaction cycle, the mechanism that ensures conformational coupling between the nucleotide-binding site and the other domains of SecA remains unclear. The observation that SecA contains numerous hydrogen-bonding groups raises important questions about the role of hydrogen-bonding networks and hydrogen-bond dynamics in long-distance conformational couplings. To address these questions, we explored the molecular dynamics of SecA from three different organisms, with and without bound nucleotide, in water. By computing two-dimensional hydrogen-bonding maps we identify networks of hydrogen bonds that connect the nucleotide-binding site to remote regions of the protein, and sites in the protein that respond to specific perturbations. We find that the nucleotide-binding site of ADP-bound SecA has a preferred geometry whereby the first two carboxylates of the DEAD motif bridge via hydrogen-bonding water. Simulations of a mutant with perturbed ATP hydrolysis highlight the water-bridged geometry as a key structural element of the reaction path. Copyright © 2015. Published by Elsevier B.V.

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth1[OPEN

PubMed Central

Bloch, Daria; Pleskot, Roman; Vukašinović, Nemanja

2016-01-01

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2. However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes. PMID:27516531

Ubiquitin-specific protease 8 deubiquitinates Sec31A and decreases large COPII carriers and collagen IV secretion.

PubMed

Kawaguchi, Kohei; Endo, Akinori; Fukushima, Toshiaki; Madoka, Yuka; Tanaka, Toshiaki; Komada, Masayuki

2018-05-15

Nascent cargo proteins in the endoplasmic reticulum are transported to the Golgi by COPII carriers. Typical COPII vesicles are 60-70 nm in diameter, and much larger macromolecules, such as procollagen, are transported by atypical large COPII carriers in mammalian cells. The formation of large COPII carriers is enhanced by Cul3 ubiquitin ligase, which mono-ubiquitinates Sec31A, a COPII coat protein. However, the deubiquitinating enzyme for Sec31A was unclear. Here, we show that the deubiquitinating enzyme USP8 interacts with and deubiquitinates Sec31A. The interaction was mediated by the adaptor protein STAM1. USP8 overexpression inhibited the formation of large COPII carriers. By contrast, USP8 knockdown caused the accumulation of COPII coat proteins around the cis-Golgi, promoted the intracellular trafficking of procollagen IV from the endoplasmic reticulum to the Golgi, and increased collagen IV secretion. We concluded that USP8 deubiquitinates Sec31A and inhibits the formation of large COPII carriers, thereby suppressing collagen IV secretion. Copyright © 2018 Elsevier Inc. All rights reserved.

A screen for over-secretion of proteins by yeast based on a dual component cellular phosphatase and immuno-chromogenic stain for exported bacterial alkaline phosphatase reporter

PubMed Central

2013-01-01

Background To isolate over-secretors, we subjected to saturation mutagenesis, a strain of P.pastoris exporting E. coli alkaline phosphatase (EAP) fused to the secretory domain of the yeast α factor pheromone through cellular PHO1/KEX2 secretory processing signals as the α-sec-EAP reporter protein. Direct chromogenic staining for α-sec-EAP activity is non-specific as its NBT/BCIP substrate cross-reacts with cellular phosphatases which can be inhibited with Levulinic acid. However, the parental E(P) strain only exports detectable levels of α-sec-EAP at 69 hours and not within the 36 hour period post-seeding required for effective screening with the consequent absence of a reference for secretion. We substituted the endogenous cellular phosphatase activity as a comparative reference for secretion rate and levels as well as for colony alignment while elevating specificity and sensitivity of detection of the exported protein with other innovative modifications of the immuno-chromogenic staining application for screening protein export mutants. Results Raising the specificity and utility of staining for α-sec-EAP activity required 5 modifications including some to published methods. These included, exploitation of endogenous phosphatase activity, reduction of the cell/protein burden, establishment of the direct relation between concentrations of transcriptional inducer and exported membrane immobilized protein and concentrations of protein exported into growth media, amplification of immuno-specificity and sensitivity of detection of α-sec-EAP reporter enzyme signal and restriction of staining to optimal concentrations of antisera and time periods. The resultant immuno-chromogenic screen allows for the detection of early secretion and as little as 1.3 fold over-secretion of α-sec-EAP reporter protein by E(M) mutants in the presence of 10 fold -216 fold higher concentrations of HSA. Conclusions The modified immuno-chromogenic screen is sensitive, specific and has

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport

PubMed Central

Helm, Jared R.; Bentley, Marvin; Thorsen, Kevin D.; Wang, Ting; Foltz, Lauren; Oorschot, Viola; Klumperman, Judith; Hay, Jesse C.

2014-01-01

Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery. PMID:25006245

Two-ligand priming mechanism for potentiated phosphoinositide synthesis is an evolutionarily conserved feature of Sec14-like phosphatidylinositol and phosphatidylcholine exchange proteins.

PubMed

Huang, Jin; Ghosh, Ratna; Tripathi, Ashutosh; Lönnfors, Max; Somerharju, Pentti; Bankaitis, Vytas A

2016-07-15

Lipid signaling, particularly phosphoinositide signaling, plays a key role in regulating the extreme polarized membrane growth that drives root hair development in plants. The Arabidopsis AtSFH1 gene encodes a two-domain protein with an amino-terminal Sec14-like phosphatidylinositol transfer protein (PITP) domain linked to a carboxy-terminal nodulin domain. AtSfh1 is critical for promoting the spatially highly organized phosphatidylinositol-4,5-bisphosphate signaling program required for establishment and maintenance of polarized root hair growth. Here we demonstrate that, like the yeast Sec14, the AtSfh1 PITP domain requires both its phosphatidylinositol (PtdIns)- and phosphatidylcholine (PtdCho)-binding properties to stimulate PtdIns-4-phosphate [PtdIns(4)P] synthesis. Moreover, we show that both phospholipid-binding activities are essential for AtSfh1 activity in supporting polarized root hair growth. Finally, we report genetic and biochemical evidence that the two-ligand mechanism for potentiation of PtdIns 4-OH kinase activity is a broadly conserved feature of plant Sec14-nodulin proteins, and that this strategy appeared only late in plant evolution. Taken together, the data indicate that the PtdIns/PtdCho-exchange mechanism for stimulated PtdIns(4)P synthesis either arose independently during evolution in yeast and in higher plants, or a suitable genetic module was introduced to higher plants from a fungal source and subsequently exploited by them. © 2016 Huang, Ghosh, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective

PubMed Central

Chagnot, Caroline; Zorgani, Mohamed A.; Astruc, Thierry; Desvaux, Mickaël

2013-01-01

Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates) is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative), monoderm (archetypal Gram-positive) and diderm-mycolate (archetypal acid-fast) bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors) involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field. PMID:24133488

Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

PubMed Central

Summer, Elizabeth J.; Cline, Kenneth

1999-01-01

Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O2-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and ΔpH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane ΔpH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and ΔpH pathways. PMID:9952453

Identification and characterization of a novel protective antigen, Sec_205 of Streptococcus equi ssp. Zooepidemicus.

PubMed

Liang, Huihuang; Tang, Bin; Zhao, Pengpeng; Deng, Mingyong; Yan, Lili; Zhai, Pan; Wei, Zigong

2018-02-01

Streptococcus equi ssp. zooepidemicus (SEZ) is an important pathogen of swine streptococcal diseases and can infect a wide range of animals as well as human beings. The absence of effective vaccine confounds the control of SEZ infection. Sec_205, a novel protein identified in the previous study, was inducibly over-expressed in Escherichia coli in the present study. The purified recombinant protein could elicit a significant humoral antibody response and provide efficient protection against lethal challenge of SEZ C55138 in mouse model. The protection against SEZ infection was mediated by specific antibodies to Sec_205 to some extent and was identified by the passive protection assay. The Sec_205 was an in vivo-induced antigen confirmed by the real-time PCR and could adhere to the Hep-2 cells by the inhibition assay. These suggest that Sec_205 may play a vital role in pathogenicity and serve as a new vaccine candidate against SEZ infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

Measurement of the incorporation rates of four amino acids into proteins for estimating bacterial production.

PubMed

Servais, P

1995-03-01

In aquatic ecosystems, [(3)H]thymidine incorporation into bacterial DNA and [(3)H]leucine incorporation into proteins are usually used to estimate bacterial production. The incorporation rates of four amino acids (leucine, tyrosine, lysine, alanine) into proteins of bacteria were measured in parallel on natural freshwater samples from the basin of the river Meuse (Belgium). Comparison of the incorporation into proteins and into the total macromolecular fraction showed that these different amino acids were incorporated at more than 90% into proteins. From incorporation measurements at four subsaturated concentrations (range, 2-77 nm), the maximum incorporation rates were determined. Strong correlations (r > 0.91 for all the calculated correlations) were found between the maximum incorporation rates of the different tested amino acids over a range of two orders of magnitude of bacterial activity. Bacterial production estimates were calculated using theoretical and experimental conversion factors. The productions calculated from the incorporation rates of the four amino acids were in good concordance, especially when the experimental conversion factors were used (slope range, 0.91-1.11, and r > 0.91). This study suggests that the incorporation of various amino acids into proteins can be used to estimate bacterial production.

Functional Analysis of Developmentally Regulated Genes chs7 and sec22 in the Ascomycete Sordaria macrospora.

PubMed

Traeger, Stefanie; Nowrousian, Minou

2015-04-14

During sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the generation and dispersal of spores. In previous studies, we identified genes with evolutionary conserved expression patterns during fruiting body formation in several fungal species. Here, we present the functional analysis of two developmentally up-regulated genes, chs7 and sec22, in the ascomycete Sordaria macrospora. The genes encode a class VII (division III) chitin synthase and a soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) protein, respectively. Deletion mutants of chs7 had normal vegetative growth and were fully fertile but showed sensitivity toward cell wall stress. Deletion of sec22 resulted in a reduced number of ascospores and in defects in ascospore pigmentation and germination, whereas vegetative growth was normal in the mutant. A SEC22-EGFP fusion construct under control of the native sec22 promoter and terminator regions was expressed during different stages of sexual development. Expression of several development-related genes was deregulated in the sec22 mutant, including three genes involved in melanin biosynthesis. Our data indicate that chs7 is dispensable for fruiting body formation in S. macrospora, whereas sec22 is required for ascospore maturation and germination and thus involved in late stages of sexual development. Copyright © 2015 Traeger and Nowrousian.

Functional Analysis of Developmentally Regulated Genes chs7 and sec22 in the Ascomycete Sordaria macrospora

PubMed Central

Traeger, Stefanie; Nowrousian, Minou

2015-01-01

During sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the generation and dispersal of spores. In previous studies, we identified genes with evolutionary conserved expression patterns during fruiting body formation in several fungal species. Here, we present the functional analysis of two developmentally up-regulated genes, chs7 and sec22, in the ascomycete Sordaria macrospora. The genes encode a class VII (division III) chitin synthase and a soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) protein, respectively. Deletion mutants of chs7 had normal vegetative growth and were fully fertile but showed sensitivity toward cell wall stress. Deletion of sec22 resulted in a reduced number of ascospores and in defects in ascospore pigmentation and germination, whereas vegetative growth was normal in the mutant. A SEC22-EGFP fusion construct under control of the native sec22 promoter and terminator regions was expressed during different stages of sexual development. Expression of several development-related genes was deregulated in the sec22 mutant, including three genes involved in melanin biosynthesis. Our data indicate that chs7 is dispensable for fruiting body formation in S. macrospora, whereas sec22 is required for ascospore maturation and germination and thus involved in late stages of sexual development. PMID:25873638

Trigger Factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli

PubMed Central

Ullers, Ronald S.; Ang, Debbie; Schwager, Françoise; Georgopoulos, Costa; Genevaux, Pierre

2007-01-01

Polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytoplasmic, integral membrane, or exported proteins. In Escherichia coli, the chaperones SecB, Trigger Factor (TF), and DnaK are key players in this process. Here, we report that, as with dnaK or dnaJ mutants, a secB null strain exhibits a strong cold-sensitive (Cs) phenotype. Through suppressor analyses, we found that inactivating mutations in the tig gene encoding TF fully relieve both the Cs phenotype and protein aggregation observed in the absence of SecB. This antagonistic effect of TF depends on its ribosome-binding and chaperone activities but unrelated to its peptidyl-prolyl cis/trans isomerase (PPIase) activity. Furthermore, in contrast to the previously known synergistic action of TF and DnaK/DnaJ above 30°C, a tig null mutation partially suppresses the Cs phenotype exhibited by a compromised DnaK/DnaJ chaperone machine. The antagonistic role of TF is further exemplified by the fact that the secB dnaJ double mutant is viable only in the absence of TF. Finally, we show that, in the absence of TF, more SecA and ribosomes are associated with the inner membrane, suggesting that the presence of TF directly or indirectly interferes with the process of cotranslational protein targeting to the Sec translocon. PMID:17360615

Serum lipopolysaccharide-binding protein prediction of severe bacterial infection in cirrhotic patients with ascites.

PubMed

Albillos, Agustín; de-la-Hera, Antonio; Alvarez-Mon, Melchor

2004-05-15

Serum lipopolysaccharide-binding protein is increased in a subset of non-infected ascitic cirrhotic patients, a finding previously related to bacterial passage from the gut to the circulation without overt infection. We prospectively analysed the risk factors associated with a first episode of severe bacterial infection in 84 ascitic cirrhotics, followed up for a median of 46 weeks. The cumulative probability of such infection in patients with raised and normal lipopolysaccharide-binding protein was 32.4% and 8.0% (p=0.004), respectively. Increased lipopolysaccharide-binding protein was the only factor independently associated with severe bacterial infection in a multivariate analysis (relative risk 4.49, 95% CI 1.42-14.1). Monitoring of serum lipopolysaccharide-binding protein could, therefore, help to target cirrhotic patients with ascites for antibiotic prophylaxis.

SURFACE INACTIVATION OF BACTERIAL VIRUSES AND OF PROTEINS

PubMed Central

Adams, Mark H.

1948-01-01

1. The seven bacterial viruses of the T group active against E. coli, are rapidly inactivated at gas-liquid interfaces. 2. The kinetics of this inactivation whether brought about by shaking or by bubbling with nitrogen are those of a first order reaction. 3. This inactivation may be prevented by the addition of enough protein to maintain the gas-liquid interface in a saturated condition. 4. The analogy between this phenomenon and the surface denaturation of proteins is pointed out and discussed. PMID:18917025

Sec34 is implicated in traffic from the endoplasmic reticulum to the Golgi and exists in a complex with GTC-90 and ldlBp.

PubMed

Loh, Eva; Hong, Wanjin

2002-06-14

Sec34p/Grd20p has been implicated in endoplasmic reticulum (ER)-to-Golgi transport and/or post-Golgi trafficking events and exists in a protein complex consisting of at least eight subunits in yeast. Although the mammalian counterpart (Sec34) of Sec34p has been molecularly identified, its role and interacting partners remain undefined. In this study, we have prepared antibodies specifically against the recombinant N-terminal fragment of Sec34 that recognize a polypeptide of about 93 kDa and label the Golgi apparatus. In a well-characterized semi-intact cell assay that reconstitutes transport of the envelope glycoprotein (VSVG) of vesicular stomatitis virus from the ER to the Golgi apparatus, anti-Sec34 antibodies inhibited the transport in a dose-dependent manner. The inhibition by anti-Sec34 antibodies could be neutralized by a noninhibitory amount of the antigen. Large-scale immunoprecipitation of rat liver cytosol with immobilized anti-Sec34 antibodies has co-immunoprecipitated GTC-90 and ldlBp, two peripheral Golgi proteins previously shown to exist in separate protein complexes. Two mammalian homologues (Dor1 and Cod1) of the yeast Sec34 complex were similarly recovered in the Sec34 immunoprecipitates. When expressed in transfected cells, epitope-tagged ldlCp and Cod2 were co-immunoprecipitated with anti-Sec34 antibodies with efficiencies comparable to that observed for tagged ldlBp, Dor1, and Cod1. Direct interactions of Sec34 with ldlBp and ldlCp were further demonstrated in vitro. These results suggest that Sec34, GTC-90, and ldlBp/ldlCp are part of the same protein complex(es) that regulates diverse aspects of Golgi function, including transport from the ER to the Golgi apparatus.

[Structure and function of the bacterial flagellar type III protein export system in Salmonella
].

PubMed

Minamino, Tohru

2015-01-01

The bacterial flagellum is a filamentous organelle that propels the bacterial cell body in liquid media. For construction of the bacterial flagellum beyond the cytoplasmic membrane, flagellar component proteins are transported by its specific protein export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. Flagellar substrate-specific chaperones bind to their cognate substrates in the cytoplasm and escort the substrates to the docking platform of the export gate. The export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane as the energy sources to drive protein export and coordinates protein export with assembly by ordered export of substrates to parallel with their order of assembly. In this review, we summarize our current understanding of the structure and function of the flagellar protein export system in Salmonella enterica serovar Typhimurium.

Plant immunity: a lesson from pathogenic bacterial effector proteins.

PubMed

Cui, Haitao; Xiang, Tingting; Zhou, Jian-Min

2009-10-01

Phytopathogenic bacteria inject an array of effector proteins into host cells to alter host physiology and assist the infection process. Some of these effectors can also trigger disease resistance as a result of recognition in the plant cell by cytoplasmic immune receptors. In addition to effector-triggered immunity, plants immunity can be triggered upon the detection of Pathogen/Microbe-Associated Molecular Patterns by surface-localized immune receptors. Recent progress indicates that many bacterial effector proteins use a variety of biochemical properties to directly attack key components of PAMP-triggered immunity and effector-triggered immunity, providing new insights into the molecular basis of plant innate immunity. Emerging evidence indicate that the evolution of disease resistance in plants is intimately linked to the mechanism by which bacterial effectors promote parasitism. This review focuses on how these studies have conceptually advanced our understanding of plant-pathogen interactions.

Tat transport of a Sec passenger leads to both completely translocated as well as membrane-arrested passenger proteins.

PubMed

Dittmar, Julia; Schlesier, René; Klösgen, Ralf Bernd

2014-02-01

We have studied the membrane transport of the chimeric precursor protein 16/33, which is composed of the Tat(1)-specific transport signal of OEC16 and the Sec passenger protein OEC33, both subunits of the oxygen-evolving system associated with photosystem II. Protein transport experiments performed with isolated pea thylakoids show that the 16/33 chimera is transported in a strictly Tat-dependent manner into the thylakoid vesicles yielding mature OEC33 (mOEC33) in two different topologies. One fraction accumulates in the thylakoid lumen and is thus resistant to externally added protease. A second fraction is arrested during transport in an N-in/C-out topology within the membrane. Chase experiments demonstrate that this membrane-arrested mOEC33 moiety does not represent a translocation intermediate but instead an alternative end product of the transport process. Transport arrest of mOEC33, which is embedded in the membrane with a mildly hydrophobic protein segment, requires more than 26 additional and predominantly hydrophilic residues C-terminal of the membrane-embedded segment. Furthermore, it is stimulated by mutations which potentially affect the conformation of mOEC33 suggesting that at least partial folding of the passenger protein is required for complete membrane translocation. Copyright © 2013 Elsevier B.V. All rights reserved.

Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

NASA Astrophysics Data System (ADS)

Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

1999-04-01

Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

Analysis of the Isolated SecA DEAD Motor Suggests a Mechanism for Chemical-Mechanical Coupling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nithianantham, Stanley; Shilton, Brian H

The preprotein cross-linking domain and C-terminal domains of Escherichia coli SecA were removed to create a minimal DEAD motor, SecA-DM. SecA-DM hydrolyzes ATP and has the same affinity for ADP as full-length SecA. The crystal structure of SecA-DM in complex with ADP was solved and shows the DEAD motor in a closed conformation. Comparison with the structure of the E. coli DEAD motor in an open conformation (Protein Data Bank ID 2FSI) indicates main-chain conformational changes in two critical sequences corresponding to Motif III and Motif V of the DEAD helicase family. The structures that the Motif III and Motifmore » V sequences adopt in the DEAD motor open conformation are incompatible with the closed conformation. Therefore, when the DEAD motor makes the transition from open to closed, Motif III and Motif V are forced to change their conformations, which likely functions to regulate passage through the transition state for ATP hydrolysis. The transition state for ATP hydrolysis for the SecA DEAD motor was modeled based on the conformation of the Vasa helicase in complex with adenylyl imidodiphosphate and RNA (Protein Data Bank ID 2DB3). A mechanism for chemical-mechanical coupling emerges, where passage through the transition state for ATP hydrolysis is hindered by the conformational changes required in Motif III and Motif V, and may be promoted by binding interactions with the preprotein substrate and/or other translocase domains and subunits.« less

Analysis of the Isolated SecA DEAD Motor Suggests a Mechanism for Chemical-Mechanical Coupling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nithianantham, Stanley; Shilton, Brian H

2011-09-28

The preprotein cross-linking domain and C-terminal domains of Escherichia coli SecA were removed to create a minimal DEAD motor, SecA-DM. SecA-DM hydrolyzes ATP and has the same affinity for ADP as full-length SecA. The crystal structure of SecA-DM in complex with ADP was solved and shows the DEAD motor in a closed conformation. Comparison with the structure of the E. coli DEAD motor in an open conformation (Protein Data Bank ID 2FSI) indicates main-chain conformational changes in two critical sequences corresponding to Motif III and Motif V of the DEAD helicase family. The structures that the Motif III and Motifmore » V sequences adopt in the DEAD motor open conformation are incompatible with the closed conformation. Therefore, when the DEAD motor makes the transition from open to closed, Motif III and Motif V are forced to change their conformations, which likely functions to regulate passage through the transition state for ATP hydrolysis. The transition state for ATP hydrolysis for the SecA DEAD motor was modeled based on the conformation of the Vasa helicase in complex with adenylyl imidodiphosphate and RNA (Protein Data Bank ID 2DB3). A mechanism for chemical-mechanical coupling emerges, where passage through the transition state for ATP hydrolysis is hindered by the conformational changes required in Motif III and Motif V, and may be promoted by binding interactions with the preprotein substrate and/or other translocase domains and subunits.« less

Bacterial adhesion to protein-coated surfaces: An AFM and QCM-D study

NASA Astrophysics Data System (ADS)

Strauss, Joshua; Liu, Yatao; Camesano, Terri A.

2009-09-01

Bacterial adhesion to biomaterials, mineral surfaces, or other industrial surfaces is strongly controlled by the way bacteria interact with protein layers or organic matter and other biomolecules that coat the materials. Despite this knowledge, many studies of bacterial adhesion are performed under clean conditions, instead of in the presence of proteins or organic molecules. We chose fetal bovine serum (FBS) as a model protein, and prepared FBS films on quartz crystals. The thickness of the FBS layer was characterized using atomic force microscopy (AFM) imaging under liquid and quartz crystal microbalance with dissipation (QCM-D). Next, we characterized how the model biomaterial surface would interact with the nocosomial pathogen Staphylococcus epidermidis. An AFM probe was coated with S. epidermidis cells and used to probe a gold slide that had been coated with FBS or another protein, fibronectin (FN). These experiments show that AFM and QCM-D can be used in complementary ways to study the complex interactions between bacteria, proteins, and surfaces.

Epithelial organization and cyst lumen expansion require efficient Sec13-Sec31-driven secretion.

PubMed

Townley, Anna K; Schmidt, Katy; Hodgson, Lorna; Stephens, David J

2012-02-01

Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13-Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture.

Phytophthora infestans RXLR Effector AVR1 Interacts with Exocyst Component Sec5 to Manipulate Plant Immunity.

PubMed

Du, Yu; Mpina, Mohamed H; Birch, Paul R J; Bouwmeester, Klaas; Govers, Francine

2015-11-01

Phytophthora infestans secretes numerous RXLR effectors that modulate host defense and thereby pave the way for successful invasion. Here, we show that the RXLR effector AVR1 is a virulence factor that promotes colonization and suppresses callose deposition, a hallmark of basal defense. To identify host targets of AVR1, we performed yeast two-hybrid screens and selected Sec5 as a candidate. Sec5 is a subunit of the exocyst, a protein complex that is involved in vesicle trafficking. AVR1-like (A-L), a close homolog of AVR1, also acts as a virulence factor, but unlike AVR1, A-L does not suppress CRINKLER2 (CRN2)-induced cell death or interact with Sec5. Compared with AVR1, A-L is shorter and lacks the carboxyl-terminal tail, the T-region that is crucial for CRN2-induced cell death suppression and Sec5 interaction. In planta analyses revealed that AVR1 and Sec5 are in close proximity, and coimmunoprecipitation confirmed the interaction. Sec5 is required for secretion of the pathogenesis-related protein PR-1 and callose deposition and also plays a role in CRN2-induced cell death. Our findings show that P. infestans manipulates an exocyst subunit and thereby potentially disturbs vesicle trafficking, a cellular process that is important for basal defense. This is a novel strategy that oomycete pathogens exploit to modulate host defense. © 2015 American Society of Plant Biologists. All Rights Reserved.

Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

PubMed

Brewer, Matthew T; Agbedanu, Prince N; Zamanian, Mostafa; Day, Tim A; Carlson, Steve A

2013-11-01

Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

Behind the lines–actions of bacterial type III effector proteins in plant cells

PubMed Central

Büttner, Daniela

2016-01-01

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

Protein aggregation as bacterial inclusion bodies is reversible.

PubMed

Carrió, M M; Villaverde, A

2001-01-26

Inclusion bodies are refractile, intracellular protein aggregates usually observed in bacteria upon targeted gene overexpression. Since their occurrence has a major economical impact in protein production bio-processes, in vitro refolding strategies are under continuous exploration. In this work, we prove spontaneous in vivo release of both beta-galactosidase and P22 tailspike polypeptides from inclusion bodies resulting in their almost complete disintegration and in the concomitant appearance of soluble, properly folded native proteins with full biological activity. Since, in particular, the tailspike protein exhibits an unusually slow and complex folding pathway involving deep interdigitation of beta-sheet structures, its in vivo refolding indicates that bacterial inclusion body proteins are not collapsed into an irreversible unfolded state. Then, inclusion bodies can be observed as transient deposits of folding-prone polypeptides, resulting from an unbalanced equilibrium between in vivo protein precipitation and refolding that can be actively displaced by arresting protein synthesis. The observation that the formation of big inclusion bodies is reversible in vivo can be also relevant in the context of amyloid diseases, in which deposition of important amounts of aggregated protein initiates the pathogenic process.

Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

PubMed Central

Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

1995-01-01

The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins).

PubMed

Pratt, R F

2008-07-01

The DD-peptidase enzymes (penicillin-binding proteins) catalyze the final transpeptidation reaction of bacterial cell wall (peptidoglycan) biosynthesis. Although there is now much structural information available about these enzymes, studies of their activity as enzymes lag. It is now established that representatives of two low-molecular-mass classes of DD-peptidases recognize elements of peptidoglycan structure and rapidly react with substrates and inhibitors incorporating these elements. No members of other DD-peptidase classes, including the high-molecular-mass enzymes, essential for bacterial growth, appear to interact strongly with any particular elements of peptidoglycan structure. Rational design of inhibitors for these enzymes is therefore challenging.

The crystal structures of EAP domains from Staphylococcus aureus reveal an unexpected homology to bacterial superantigens.

PubMed

Geisbrecht, Brian V; Hamaoka, Brent Y; Perman, Benjamin; Zemla, Adam; Leahy, Daniel J

2005-04-29

The Eap (extracellular adherence protein) of Staphylococcus aureus functions as a secreted virulence factor by mediating interactions between the bacterial cell surface and several extracellular host proteins. Eap proteins from different Staphylococcal strains consist of four to six tandem repeats of a structurally uncharacterized domain (EAP domain). We have determined the three-dimensional structures of three different EAP domains to 1.8, 2.2, and 1.35 A resolution, respectively. These structures reveal a core fold that is comprised of an alpha-helix lying diagonally across a five-stranded, mixed beta-sheet. Comparison of EAP domains with known structures reveals an unexpected homology with the C-terminal domain of bacterial superantigens. Examination of the structure of the superantigen SEC2 bound to the beta-chain of a T-cell receptor suggests a possible ligand-binding site within the EAP domain (Fields, B. A., Malchiodi, E. L., Li, H., Ysern, X., Stauffacher, C. V., Schlievert, P. M., Karjalainen, K., and Mariuzza, R. (1996) Nature 384, 188-192). These results provide the first structural characterization of EAP domains, relate EAP domains to a large class of bacterial toxins, and will guide the design of future experiments to analyze EAP domain structure/function relationships.

Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meining, Winfried, E-mail: wim@csb.ki.se; Scheuring, Johannes; Fischer, Markus

2006-06-01

SecA ATPase from E. faecalis has been cloned, overexpressed, purified and crystallized. Crystals belong to space group C2 and diffract to 2.4 Å resolution. The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 Å resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 Å,more » α = γ = 90.0, β = 119.1°. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.« less

Identification and characterization of a selenoprotein family containing a diselenide bond in a redox motif

PubMed Central

Shchedrina, Valentina A.; Novoselov, Sergey V.; Malinouski, Mikalai Yu.; Gladyshev, Vadim N.

2007-01-01

Selenocysteine (Sec, U) insertion into proteins is directed by translational recoding of specific UGA codons located upstream of a stem-loop structure known as Sec insertion sequence (SECIS) element. Selenoproteins with known functions are oxidoreductases containing a single redox-active Sec in their active sites. In this work, we identified a family of selenoproteins, designated SelL, containing two Sec separated by two other residues to form a UxxU motif. SelL proteins show an unusual occurrence, being present in diverse aquatic organisms, including fish, invertebrates, and marine bacteria. Both eukaryotic and bacterial SelL genes use single SECIS elements for insertion of two Sec. In eukaryotes, the SECIS is located in the 3′ UTR, whereas the bacterial SelL SECIS is within a coding region and positioned at a distance that supports the insertion of either of the two Sec or both of these residues. SelL proteins possess a thioredoxin-like fold wherein the UxxU motif corresponds to the catalytic CxxC motif in thioredoxins, suggesting a redox function of SelL proteins. Distantly related SelL-like proteins were also identified in a variety of organisms that had either one or both Sec replaced with Cys. Danio rerio SelL, transiently expressed in mammalian cells, incorporated two Sec and localized to the cytosol. In these cells, it occurred in an oxidized form and was not reducible by DTT. In a bacterial expression system, we directly demonstrated the formation of a diselenide bond between the two Sec, establishing it as the first diselenide bond found in a natural protein. PMID:17715293

Epithelial organization and cyst lumen expansion require efficient Sec13–Sec31-driven secretion

PubMed Central

Townley, Anna K.; Schmidt, Katy; Hodgson, Lorna; Stephens, David J.

2012-01-01

Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13–Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture. PMID:22331354

Bacterial flagellar capping proteins adopt diverse oligomeric states

DOE Office of Scientific and Technical Information (OSTI.GOV)

Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A.

2016-09-24

Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD fromPseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find thatPseudomonasFliD exhibits unexpected structural similarity to other flagellar proteins atmore » the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies.« less

SEC14 is a specific requirement for secretion of phospholipase B1 and pathogenicity of Cryptococcus neoformans

PubMed Central

Chayakulkeeree, Methee; Johnston, Simon Andrew; Oei, Johanes Bijosono; Lev, Sophie; Williamson, Peter Richard; Wilson, Christabel Frewen; Zuo, Xiaoming; Leal, Ana Lusia; Vainstein, Marilene Henning; Meyer, Wieland; Sorrell, Tania Christine; May, Robin Charles; Djordjevic, Julianne Teresa

2011-01-01

Summary Secreted phospholipase B1 (CnPlb1) is essential for dissemination of Cryptococcus neoformans to the central nervous system (CNS) yet essential components of its secretion machinery remain to be elucidated. Using gene deletion analysis we demonstrate that CnPlb1 secretion is dependent on the CnSEC14 product, CnSec14-1p. CnSec14-1p is a homologue of the phosphatidylinositol transfer protein (PITP) ScSec14p, which is essential for secretion and viability in Saccharomyces cerevisiae. In contrast to CnPlb1, neither laccase 1 (Lac1)-induced melanization within the cell wall nor capsule induction were negatively impacted in CnSEC14-1 deletion mutants (CnΔsec14-1 and CnΔsec14-1CnΔsfh5). Similar to the CnPLB1 deletion mutant (CnΔplb1), CnΔsec14-1 was hypo-virulent in mice and did not disseminate to the CNS by day 14 post infection. Furthermore, macrophage expulsion of live CnΔsec14-1 and CnΔplb1 (vomocytosis) was reduced. Individual deletion of CnSEC14-2, a closely-related CnSEC14-1 homologue, and CnSFH5, a distantly-related SEC fourteen-like homologue, did not abrogate CnPlb1 secretion or virulence. However, reconstitution of CnΔsec14-1 with CnSEC14-1 or CnSEC14-2 restored both phenotypes, consistent with functional genetic redundancy. We conclude that CnPlb1 secretion is SEC14-dependent and that C. neoformans preferentially exports virulence determinants to the cell periphery via distinct pathways. We also demonstrate that CnPlb1 secretion is essential for vomocytosis. PMID:21453402

46 CFR Sec. 3 - Specifications.

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2011-10-01

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Self-organization and positioning of bacterial protein clusters

NASA Astrophysics Data System (ADS)

Murray, Seán M.; Sourjik, Victor

2017-10-01

Many cellular processes require proteins to be precisely positioned within the cell. In some cases this can be attributed to passive mechanisms such as recruitment by other proteins in the cell or by exploiting the curvature of the membrane. However, in bacteria, active self-positioning is likely to play a role in multiple processes, including the positioning of the future site of cell division and cytoplasmic protein clusters. How can such dynamic clusters be formed and positioned? Here, we present a model for the self-organization and positioning of dynamic protein clusters into regularly repeating patterns based on a phase-locked Turing pattern. A single peak in the concentration is always positioned at the midpoint of the model cell, and two peaks are positioned at the midpoint of each half. Furthermore, domain growth results in peak splitting and pattern doubling. We argue that the model may explain the regular positioning of the highly conserved structural maintenance of chromosomes complexes on the bacterial nucleoid and that it provides an attractive mechanism for the self-positioning of dynamic protein clusters in other systems.

Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins

PubMed Central

1991-01-01

The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase. The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins. In contrast, the middle third of SIS1 is not similar to dnaJ proteins. This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD. The SIS1 gene is essential. Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus. About one- fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase. We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1. PMID:1714460

Molecular dynamics simulations on interaction between bacterial proteins: Implication on pathogenic activities.

PubMed

Mondal, Manas; Chakrabarti, Jaydeb; Ghosh, Mahua

2018-03-01

We perform molecular dynamics simulation studies on interaction between bacterial proteins: an outer-membrane protein STY3179 and a yfdX protein STY3178 of Salmonella Typhi. STY3179 has been found to be involved in bacterial adhesion and invasion. STY3178 is recently biophysically characterized. It is a soluble protein having antibiotic binding and chaperon activity capabilities. These two proteins co-occur and are from neighboring gene in Salmonella Typhi-occurrence of homologs of both STY3178 and STY3179 are identified in many Gram-negative bacteria. We show using homology modeling, docking followed by molecular dynamics simulation that they can form a stable complex. STY3178 belongs to aqueous phase, while the beta barrel portion of STY3179 remains buried in DPPC bilayer with extra-cellular loops exposed to water. To understand the molecular basis of interaction between STY3178 and STY3179, we compute the conformational thermodynamics which indicate that these two proteins interact through polar and acidic residues belonging to their interfacial region. Conformational thermodynamics results further reveal instability of certain residues in extra-cellular loops of STY3179 upon complexation with STY3178 which is an indication for binding with host cell protein laminin. © 2017 Wiley Periodicals, Inc.

An allosteric Sec61 inhibitor traps nascent transmembrane helices at the lateral gate

PubMed Central

MacKinnon, Andrew L; Paavilainen, Ville O; Sharma, Ajay; Hegde, Ramanujan S; Taunton, Jack

2014-01-01

Membrane protein biogenesis requires the coordinated movement of hydrophobic transmembrane domains (TMD) from the cytosolic vestibule of the Sec61 channel into the lipid bilayer. Molecular insight into TMD integration has been hampered by the difficulty of characterizing intermediates during this intrinsically dynamic process. In this study, we show that cotransin, a substrate-selective Sec61 inhibitor, traps nascent TMDs in the cytosolic vestibule, permitting detailed interrogation of an early pre-integration intermediate. Site-specific crosslinking revealed the pre-integrated TMD docked to Sec61 near the cytosolic tip of the lateral gate. Escape from cotransin-arrest depends not only on cotransin concentration, but also on the biophysical properties of the TMD. Genetic selection of cotransin-resistant cancer cells uncovered multiple mutations clustered near the lumenal plug of Sec61α, thus revealing cotransin’s likely site of action. Our results suggest that TMD/lateral gate interactions facilitate TMD transfer into the membrane, a process that is allosterically modulated by cotransin binding to the plug. DOI: http://dx.doi.org/10.7554/eLife.01483.001 PMID:24497544

Multi-location gram-positive and gram-negative bacterial protein subcellular localization using gene ontology and multi-label classifier ensemble.

PubMed

Wang, Xiao; Zhang, Jun; Li, Guo-Zheng

2015-01-01

It has become a very important and full of challenge task to predict bacterial protein subcellular locations using computational methods. Although there exist a lot of prediction methods for bacterial proteins, the majority of these methods can only deal with single-location proteins. But unfortunately many multi-location proteins are located in the bacterial cells. Moreover, multi-location proteins have special biological functions capable of helping the development of new drugs. So it is necessary to develop new computational methods for accurately predicting subcellular locations of multi-location bacterial proteins. In this article, two efficient multi-label predictors, Gpos-ECC-mPLoc and Gneg-ECC-mPLoc, are developed to predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. The two multi-label predictors construct the GO vectors by using the GO terms of homologous proteins of query proteins and then adopt a powerful multi-label ensemble classifier to make the final multi-label prediction. The two multi-label predictors have the following advantages: (1) they improve the prediction performance of multi-label proteins by taking the correlations among different labels into account; (2) they ensemble multiple CC classifiers and further generate better prediction results by ensemble learning; and (3) they construct the GO vectors by using the frequency of occurrences of GO terms in the typical homologous set instead of using 0/1 values. Experimental results show that Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently improve prediction accuracy of subcellular localization of multi-location gram-positive and gram-negative bacterial proteins respectively. The online web servers for Gpos-ECC-mPLoc and Gneg-ECC-mPLoc predictors are freely accessible

MINS2: revisiting the molecular code for transmembrane-helix recognition by the Sec61 translocon.

PubMed

Park, Yungki; Helms, Volkhard

2008-08-15

To be fully functional, membrane proteins should not only fold, but also get inserted into the membrane, which is mediated by the Sec61 translocon. Recent experimental studies have attempted to elucidate how the Sec61 translocon accomplishes this delicate task by measuring the translocon-mediated membrane insertion free energies of 357 systematically designed peptides. On the basis of this data set, we have developed MINS2, a novel sequence-based computational method for predicting the membrane insertion free energies of protein sequences. A benchmark analysis of MINS2 shows that MINS2 signi.cantly outperforms previously proposed methods. Importantly, the application of MINS2 to known membrane protein structures shows that a better prediction of membrane insertion free energies does not lead to a better prediction of transmembrane segments of polytopic membrane proteins. A web server for MINS2 is publicly available at http://service.bioinformatik.uni-saarland.de/mins. Supplementary data are available at Bioinformatics online.

46 CFR Sec. 9 - Communications.

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2011-10-01

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46 CFR Sec. 9 - Communications.

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Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

PubMed Central

Matsunaga, James; Barocchi, Michele A.; Croda, Julio; Young, Tracy A.; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A.; Reis, Mitermayer G.; Riley, Lee W.; Haake, David A.; Ko, Albert I.

2005-01-01

Summary Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.

PubMed

Matsunaga, James; Barocchi, Michele A; Croda, Julio; Young, Tracy A; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A; Reis, Mitermayer G; Riley, Lee W; Haake, David A; Ko, Albert I

2003-08-01

Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis.

Lst1p and Sec24p Cooperate in Sorting of the Plasma Membrane Atpase into Copii Vesicles in Saccharomyces cerevisiae

PubMed Central

Shimoni, Yuval; Kurihara, Tatsuo; Ravazzola, Mariella; Amherdt, Mylène; Orci, Lelio; Schekman, Randy

2000-01-01

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of α-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles. PMID:11086000

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

PubMed

Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

2017-05-01

Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

46 CFR Sec. 2 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Definitions. Sec. 2 Section 2 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS FEDERAL PORT CONTROLLERS Sec. 2... department of the United States) in time of emergency to meet the needs of the national defense and maintain...

46 CFR Sec. 12 - Audit.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Audit. Sec. 12 Section 12 Shipping MARITIME... TRANSACTIONS UNDER AGENCY AGREEMENTS Reports and Audit Sec. 12 Audit. (a) The owner will audit as currently as possible subsequent to audit by the agent, all documents relating to the activities, maintenance and...

46 CFR Sec. 5 - Accounting.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records. [16 FR 2885...

Hierarchical protein export mechanism of the bacterial flagellar type III protein export apparatus.

PubMed

Minamino, Tohru

2018-06-01

The bacterial flagellum is supramolecular motility machinery consisting of the basal body, the hook and the filament. Flagellar proteins are translocated across the cytoplasmic membrane via a type III protein export apparatus, diffuse down the central channel of the growing structure and assemble at the distal end. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. The completion of hook assembly is the most important morphological checkpoint of the sequential flagellar assembly process. When the hook reaches its mature length of about 55 nm in Salmonella enterica, the type III protein export apparatus switches export specificity from proteins required for the structure and assembly of the hook to those responsible for filament assembly, thereby terminating hook assembly and initiating filament assembly. Three flagellar proteins, namely FliK, FlhB and FlhA, are responsible for this substrate specificity switching. Upon completion of the switching event, interactions among FlhA, the cytoplasmic ATPase complex and flagellar type III export chaperones establish the assembly order of the filament at the hook tip. Here, we describe our current understanding of a hierarchical protein export mechanism used in flagellar type III protein export.

Phytophthora infestans RXLR Effector AVR1 Interacts with Exocyst Component Sec5 to Manipulate Plant Immunity1[OPEN

PubMed Central

Du, Yu; Mpina, Mohamed H.; Birch, Paul R.J.; Bouwmeester, Klaas; Govers, Francine

2015-01-01

Phytophthora infestans secretes numerous RXLR effectors that modulate host defense and thereby pave the way for successful invasion. Here, we show that the RXLR effector AVR1 is a virulence factor that promotes colonization and suppresses callose deposition, a hallmark of basal defense. To identify host targets of AVR1, we performed yeast two-hybrid screens and selected Sec5 as a candidate. Sec5 is a subunit of the exocyst, a protein complex that is involved in vesicle trafficking. AVR1-like (A-L), a close homolog of AVR1, also acts as a virulence factor, but unlike AVR1, A-L does not suppress CRINKLER2 (CRN2)-induced cell death or interact with Sec5. Compared with AVR1, A-L is shorter and lacks the carboxyl-terminal tail, the T-region that is crucial for CRN2-induced cell death suppression and Sec5 interaction. In planta analyses revealed that AVR1 and Sec5 are in close proximity, and coimmunoprecipitation confirmed the interaction. Sec5 is required for secretion of the pathogenesis-related protein PR-1 and callose deposition and also plays a role in CRN2-induced cell death. Our findings show that P. infestans manipulates an exocyst subunit and thereby potentially disturbs vesicle trafficking, a cellular process that is important for basal defense. This is a novel strategy that oomycete pathogens exploit to modulate host defense. PMID:26336092

Hydrophobically stabilized open state for the lateral gate of the Sec translocon

PubMed Central

Zhang, Bin; Miller, Thomas F.

2010-01-01

The Sec translocon is a central component of cellular pathways for protein translocation and membrane integration. Using both atomistic and coarse-grained molecular simulations, we investigate the conformational landscape of the translocon and explore the role of peptide substrates in the regulation of the translocation and integration pathways. Inclusion of a hydrophobic peptide substrate in the translocon stabilizes the opening of the lateral gate for membrane integration, whereas a hydrophilic peptide substrate favors the closed lateral gate conformation. The relative orientation of the plug moiety and a peptide substrate within the translocon channel is similarly dependent on whether the substrate is hydrophobic or hydrophilic in character, and the energetics of the translocon lateral gate opening in the presence of a peptide substrate is governed by the energetics of the peptide interface with the membrane. Implications of these results for the regulation of Sec-mediated pathways for protein translocation vs. membrane integration are discussed. PMID:20203009

Mammalian TRAPPIII Complex positively modulates the recruitment of Sec13/31 onto COPII vesicles

PubMed Central

Zhao, Shan; Li, Chun Man; Luo, Xiao Min; Siu, Gavin Ka Yu; Gan, Wen Jia; Zhang, Lin; Wu, William K. K.; Chan, Hsiao Chang; Yu, Sidney

2017-01-01

The Transport protein particle (TRAPP) complex is a tethering factor for COPII vesicle. Of three forms of TRAPP (TRAPPI, II and III) complexes identified so far, TRAPPIII has been largely considered to play a role in autophagy. While depletion of TRAPPIII specific subunits caused defects in the early secretory pathway and TRAPPIII might interact with components of the COPII vesicle coat, its exact role remains to be determined. In this study, we studied the function of TRAPPIII in early secretory pathway using a TRAPPIII-specific subunit, TRAPPC12, as starting point. We found that TRAPPC12 was localized to the ER exit sites and ERGIC. In cells deleted with TRAPPC12, ERGIC and to a lesser extent, the Golgi became dispersed. ER-to-Golgi transport was also delayed. TRAPPC12, but not TRAPPC8, bound to Sec13/Sec31A tetramer but each Sec protein alone could not interact with TRAPPC12. TRAPPIII positively modulated the assembly of COPII outer layer during COPII vesicle formation. These results identified a novel function of TRAPPIII as a positive modulator of the outer layer of the COPII coat. PMID:28240221

SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

PubMed Central

Bruno, Joanne; Chaudhary, Natasha; Iaea, David

2016-01-01

RAB10 is a regulator of insulin-stimulated translocation of the GLUT4 glucose transporter to the plasma membrane (PM) of adipocytes, which is essential for whole-body glucose homeostasis. We establish SEC16A as a novel RAB10 effector in this process. Colocalization of SEC16A with RAB10 is augmented by insulin stimulation, and SEC16A knockdown attenuates insulin-induced GLUT4 translocation, phenocopying RAB10 knockdown. We show that SEC16A and RAB10 promote insulin-stimulated mobilization of GLUT4 from a perinuclear recycling endosome/TGN compartment. We propose RAB10–SEC16A functions to accelerate formation of the vesicles that ferry GLUT4 to the PM during insulin stimulation. Because GLUT4 continually cycles between the PM and intracellular compartments, the maintenance of elevated cell-surface GLUT4 in the presence of insulin requires accelerated biogenesis of the specialized GLUT4 transport vesicles. The function of SEC16A in GLUT4 trafficking is independent of its previously characterized activity in ER exit site formation and therefore independent of canonical COPII-coated vesicle function. However, our data support a role for SEC23A, but not the other COPII components SEC13, SEC23B, and SEC31, in the insulin stimulation of GLUT4 trafficking, suggesting that vesicles derived from subcomplexes of COPII coat proteins have a role in the specialized trafficking of GLUT4. PMID:27354378

Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs

PubMed Central

Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

2016-01-01

In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4+ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals’ endocrine system. PMID:27775083

Protein export through the bacterial flagellar type III export pathway.

PubMed

Minamino, Tohru

2014-08-01

For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. © 2013 Elsevier B.V. All rights reserved.

Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

PubMed

Yang, Huiying; Ke, Yuehua; Wang, Jian; Tan, Yafang; Myeni, Sebenzile K; Li, Dong; Shi, Qinghai; Yan, Yanfeng; Chen, Hui; Guo, Zhaobiao; Yuan, Yanzhi; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

2011-11-01

A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP.

Screening host proteins required for bacterial adherence after H9N2 virus infection.

PubMed

Ma, Li-Li; Sun, Zhen-Hong; Xu, Yu-Lin; Wang, Shu-Juan; Wang, Hui-Ning; Zhang, Hao; Hu, Li-Ping; Sun, Xiao-Mei; Zhu, Lin; Shang, Hong-Qi; Zhu, Rui-Liang; Wei, Kai

2018-01-01

H9N2 subtype low pathogenic avian influenza virus (LPAIV) is distributed worldwide and causes great economic losses in the poultry industry, especially when complicated with other bacterial infections. Tissue damages caused by virus infection provide an opportunity for bacteria invasion, but this mechanism is not sufficient for low pathogenic strains. Moreover, although H9N2 virus infection was demonstrated to promote bacterial infection in several studies, its mechanism remained unclear. In this study, infection experiments in vivo and in vitro demonstrated that the adhesion of Escherichia coli (E. coli) to host cells significantly increased after H9N2 virus infection, and this increase was not caused by pathological damages. Subsequently, we constructed a late chicken embryo infection model and used proteomics techniques to analyze the expression of proteins associated with bacterial adhesion after H9N2 virus infection. A total of 279 significantly differential expressed proteins were detected through isobaric tags for relative and absolute quantitation (iTRAQ) coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis. The results of Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed proteins were enriched in host innate immunity; cell proliferation, differentiation, and apoptosis; and pathogenicity-related signaling pathways. Finally, we screened out several proteins, such as TGF-β1, integrins, cortactin, E-cadherin, vinculin, and fibromodulin, which were probably associated with bacterial adhesion. The study analyzed the mechanism of secondary bacterial infection induced by H9N2 virus infection from a novel perspective, which provided theoretical and data support for investigating the synergistic infection mechanism between the H9N2 virus and bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Extractable Bacterial Surface Proteins in Probiotic–Host Interaction

PubMed Central

do Carmo, Fillipe L. R.; Rabah, Houem; De Oliveira Carvalho, Rodrigo D.; Gaucher, Floriane; Cordeiro, Barbara F.; da Silva, Sara H.; Le Loir, Yves; Azevedo, Vasco; Jan, Gwénaël

2018-01-01

Some Gram-positive bacteria, including probiotic ones, are covered with an external proteinaceous layer called a surface-layer. Described as a paracrystalline layer and formed by the self-assembly of a surface-layer-protein (Slp), this optional structure is peculiar. The surface layer per se is conserved and encountered in many prokaryotes. However, the sequence of the corresponding Slp protein is highly variable among bacterial species, or even among strains of the same species. Other proteins, including surface layer associated proteins (SLAPs), and other non-covalently surface-bound proteins may also be extracted with this surface structure. They can be involved a various functions. In probiotic Gram-positives, they were shown by different authors and experimental approaches to play a role in key interactions with the host. Depending on the species, and sometime on the strain, they can be involved in stress tolerance, in survival within the host digestive tract, in adhesion to host cells or mucus, or in the modulation of intestinal inflammation. Future trends include the valorization of their properties in the formation of nanoparticles, coating and encapsulation, and in the development of new vaccines. PMID:29670603

Identification of the interactome between fish plasma proteins and Edwardsiella tarda reveals tissue-specific strategies against bacterial infection.

PubMed

Li, Hui; Huang, Xiaoyan; Zeng, Zaohai; Peng, Xuan-Xian; Peng, Bo

2016-09-01

Elucidating the complex pathogen-host interaction is essential for a comprehensive understanding of how these remarkable agents invade their hosts and how the hosts defend against these invaders. During the infection, pathogens interact intensively with host to enable their survival, which can be revealed through their interactome. Edwardsiella tarda is a Gram-negative bacterial pathogen causing huge economic loss in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. E. tarda is an ideal model for host-pathogen investigation as it infects fish in three distinct steps: entering the host, circulating through the blood and establishing infection. We adopted a previous established proteomic approach that inactivated E. tarda cells and covalent crosslink fish plasma proteins were used to capture plasma proteins and bacterial outer membrane proteins, respectively. By the combinatorial use of proteomic and biochemical approaches, six plasma proteins and seven outer membrane proteins (OMPs) were identified. Interactions among these proteins were validated with protein-array, far-Western blotting and co-immunoprecipitation. At last, seventeen plasma protein-bacteria protein-protein interaction were confirmed to be involved in the interaction network, forming a complex interactome. Compared to our previous results, different host proteins were detected, whereas some of the bacterial proteins were similar, which indicates that hosts adopt tissue-specific strategies to cope with the same pathogen during infection. Thus, our results provide a robust demonstration of both bacterial initiators and host receptors or interacting proteins to further explore infection and anti-infective mechanisms between hosts and microbes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Toward an SEC-FERC memorandum of understanding. [SEC-FERC (Securities and Exchange Commission-Federal Energy Regulatory Commission)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moeller, J.W.

1994-01-01

The Securities and Exchange Commission (SEC) is under fire. For the past year, Congress has criticized its administration of the Public Utility Holding Company Act of 1935 (PUHCA). The criticism might persuade Congress to transfer the administration of PUHCA from the SEC to the Federal Energy Regulatory Commission (FERC). Section I of this article briefly describes the events of the past year and the implications of transfer of PUHCA from the SEC to the FERC. Section II of this article discusses the background relationship of PUHCA, under which the SEC regulates the securities transactions of public utilities, to the Federalmore » Power Act, under which the FERC regulates the rates of public utilities. Section III discusses the case involving the Ohio Power Company, which resulted in a November 1990 decision of the Supreme Court, that recently has highlighted the conflict in jurisdiction between the SEC and the FERC relative to the regulation of public utilities. Section IV discusses the immediate responses to that conflict--a proposed amendment to a FERC regulation and S. 544--and the reasons that neither response is an ideal solution. Section V proposes an alternative to those immediate responses--the Memorandum of Understanding. An Appendix is provided which proposes a draft Memorandum of Understanding between the SEC and the FERC.« less

Production enhancement of the extracellular lipase LipA in Bacillus subtilis: Effects of expression system and Sec pathway components.

PubMed

Ma, Ran Jing; Wang, Yan Hong; Liu, Lu; Bai, Lei Lei; Ban, Rui

2018-02-01

Lipases are among the most versatile biocatalysts, and are used in a range of industrially relevant bioconversion reactions. However, the production of LipA in recombinant Bacillus subtilis is still limited, due to unresolved issues surrounding the regulation of the expression and secretion systems. In this study, the gene encoding LipA from B. subtilis 168 was expressed in BNA under the control of the P 43 and the P AE promoter. The extracellular lipase activity of the resulting strains BNACL and BNAAL was 7.8 U ml -1 and 12.6 U ml -1 , respectively. To further enhance the expression of LipA, pHP13L was constructed by inserting the P AE -lip into the shuttle vector pHP13, which produced an extracellular lipase activity of 180.5 U ml -1 of BNA/pHP13L. The strain BNAY8 described in Supplement data which lacks eight extracellular proteins was constructed and the deletion a few of the much weaker secreting proteins had no significant effect on the secretion of LipA. Moreover, the four Sec pathway components, secA-prfB, secDF, secYEG, prsA, were individually overexpressed in BNA. The overexpression of secDF and prsA enhanced the production of LipA by 28% and 49%, respectively. Furthermore, the co-overexpression of secDF with prsA improved the extracellular amount of LipA by 59% over that of BNA/pHP13L, reaching 287.8 U ml -1 . It can therefore be said that both regulatory elements and secretion pathway had an impact on the production of secreted LipA. Their optimization and modification is a useful strategy to improve the homologous overproduction of other extracellular proteins in B. subtilis. Copyright © 2017. Published by Elsevier Inc.

Serum amyloid A is a retinol binding protein that transports retinol during bacterial infection

PubMed Central

Derebe, Mehabaw G; Zlatkov, Clare M; Gattu, Sureka; Ruhn, Kelly A; Vaishnava, Shipra; Diehl, Gretchen E; MacMillan, John B; Williams, Noelle S; Hooper, Lora V

2014-01-01

Retinol plays a vital role in the immune response to infection, yet proteins that mediate retinol transport during infection have not been identified. Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization, but their exact functions remain unclear. Here we show that mouse and human SAAs are retinol binding proteins. Mouse and human SAAs bound retinol with nanomolar affinity, were associated with retinol in vivo, and limited the bacterial burden in tissues after acute infection. We determined the crystal structure of mouse SAA3 at a resolution of 2 Å, finding that it forms a tetramer with a hydrophobic binding pocket that can accommodate retinol. Our results thus identify SAAs as a family of microbe-inducible retinol binding proteins, reveal a unique protein architecture involved in retinol binding, and suggest how retinol is circulated during infection. DOI: http://dx.doi.org/10.7554/eLife.03206.001 PMID:25073702

STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

PubMed Central

Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

2009-01-01

Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane[OPEN

PubMed Central

Karnik, Rucha; Zhang, Ben; Waghmare, Sakharam; Aderhold, Christin; Grefen, Christopher; Blatt, Michael R.

2015-01-01

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners. PMID:25747882

Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens.

PubMed

Li, Jie; Lin, Shixian; Wang, Jie; Jia, Shang; Yang, Maiyun; Hao, Ziyang; Zhang, Xiaoyu; Chen, Peng R

2013-05-15

Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.

PubMed

Ittig, Simon J; Schmutz, Christoph; Kasper, Christoph A; Amstutz, Marlise; Schmidt, Alexander; Sauteur, Loïc; Vigano, M Alessandra; Low, Shyan Huey; Affolter, Markus; Cornelis, Guy R; Nigg, Erich A; Arrieumerlou, Cécile

2015-11-23

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network. © 2015 Ittig et al.

Protein deposition and its effect on bacterial adhesion to contact lenses.

PubMed

Omali, Negar Babaei; Zhu, Hua; Zhao, Zhenjun; Willcox, Mark D P

2013-06-01

Bacterial adhesion to contact lenses is believed to be the initial step for the development of several adverse reactions that occur during lens wear such as microbial keratitis. This study examined the effect of combinations of proteins on the adhesion of bacteria to contact lenses. Unworn balafilcon A and senofilcon A lenses were soaked in commercially available pure protein mixtures to achieve the same amount of various proteins as found ex vivo. These lenses were then exposed to Pseudomonas aeruginosa and Staphylococcus aureus. Following incubation, the numbers of P. aeruginosa or S. aureus that adhered to the lenses were measured. The possible effect of proteins on bacterial growth was investigated by incubating bacteria in medium containing protein. Although there was a significant (p < 0.003) increase in the total or viable counts of one strain of S. aureus (031) on balafilcon A lenses soaked in the lysozyme/lactoferrin combination, the protein adhered to lenses did not alter the adhesion of any other strains of P. aeruginosa or S. aureus (p > 0.05). Growth of S. aureus 031 (p < 0.0001) but not of P. aeruginosa 6294 was stimulated by addition of lysozyme/lactoferrin combination (2.8/0.5 mg/mL). Addition of lipocalin did not affect the growth of any strains tested (p > 0.05). Adsorption of amounts of lysozyme and lactoferrin or lipocalin equivalent to those extracted from worn contact lenses did not affect the adhesion of most strains of S. aureus or P. aeruginosa to lens surfaces.

Clinical Prognosis in Neonatal Bacterial Meningitis: The Role of Cerebrospinal Fluid Protein.

PubMed

Tan, Jintong; Kan, Juan; Qiu, Gang; Zhao, Dongying; Ren, Fang; Luo, Zhongcheng; Zhang, Yongjun

2015-01-01

Neonates are at high risk of meningitis and of resulting neurologic complications. Early recognition of neonates at risk of poor prognosis would be helpful in providing timely management. From January 2008 to June 2014, we enrolled 232 term neonates with bacterial meningitis admitted to 3 neonatology departments in Shanghai, China. The clinical status on the day of discharge from these hospitals or at a postnatal age of 2.5 to 3 months was evaluated using the Glasgow Outcome Scale (GOS). Patients were classified into two outcome groups: good (167 cases, 72.0%, GOS = 5) or poor (65 cases, 28.0%, GOS = 1-4). Neonates with good outcome had less frequent apnea, drowsiness, poor feeding, bulging fontanelle, irritability and more severe jaundice compared to neonates with poor outcome. The good outcome group also had less pneumonia than the poor outcome group. Besides, there were statistically significant differences in hemoglobin, mean platelet volume, platelet distribution width, C-reaction protein, procalcitonin, cerebrospinal fluid (CSF) glucose and CSF protein. Multivariate logistic regression analyses suggested that poor feeding, pneumonia and CSF protein were the predictors of poor outcome. CSF protein content was significantly higher in patients with poor outcome. The best cut-offs for predicting poor outcome were 1,880 mg/L in CSF protein concentration (sensitivity 70.8%, specificity 86.2%). After 2 weeks of treatment, CSF protein remained higher in the poor outcome group. High CSF protein concentration may prognosticate poor outcome in neonates with bacterial meningitis.

What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira

PubMed Central

Fouts, Derrick E.; Matthias, Michael A.; Adhikarla, Haritha; Adler, Ben; Amorim-Santos, Luciane; Berg, Douglas E.; Bulach, Dieter; Buschiazzo, Alejandro; Chang, Yung-Fu; Galloway, Renee L.; Haake, David A.; Haft, Daniel H.; Hartskeerl, Rudy; Ko, Albert I.; Levett, Paul N.; Matsunaga, James; Mechaly, Ariel E.; Monk, Jonathan M.; Nascimento, Ana L. T.; Nelson, Karen E.; Palsson, Bernhard; Peacock, Sharon J.; Picardeau, Mathieu; Ricaldi, Jessica N.; Thaipandungpanit, Janjira; Wunder, Elsio A.; Yang, X. Frank; Zhang, Jun-Jie; Vinetz, Joseph M.

2016-01-01

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade’s refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic

What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira.

PubMed

Fouts, Derrick E; Matthias, Michael A; Adhikarla, Haritha; Adler, Ben; Amorim-Santos, Luciane; Berg, Douglas E; Bulach, Dieter; Buschiazzo, Alejandro; Chang, Yung-Fu; Galloway, Renee L; Haake, David A; Haft, Daniel H; Hartskeerl, Rudy; Ko, Albert I; Levett, Paul N; Matsunaga, James; Mechaly, Ariel E; Monk, Jonathan M; Nascimento, Ana L T; Nelson, Karen E; Palsson, Bernhard; Peacock, Sharon J; Picardeau, Mathieu; Ricaldi, Jessica N; Thaipandungpanit, Janjira; Wunder, Elsio A; Yang, X Frank; Zhang, Jun-Jie; Vinetz, Joseph M

2016-02-01

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade's refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic

46 CFR Sec. 16 - Liquidated damages.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 8 2012-10-01 2012-10-01 false Liquidated damages. Sec. 16 Section 16 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 16...

46 CFR Sec. 16 - Liquidated damages.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 8 2013-10-01 2013-10-01 false Liquidated damages. Sec. 16 Section 16 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 16...

46 CFR Sec. 16 - Liquidated damages.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 8 2011-10-01 2011-10-01 false Liquidated damages. Sec. 16 Section 16 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 16...

46 CFR Sec. 16 - Liquidated damages.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Liquidated damages. Sec. 16 Section 16 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 16...

46 CFR Sec. 16 - Liquidated damages.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 8 2014-10-01 2014-10-01 false Liquidated damages. Sec. 16 Section 16 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 16...

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2.

PubMed

Bubenik, Jodi L; Miniard, Angela C; Driscoll, Donna M

2014-01-01

Selenium, a micronutrient, is primarily incorporated into human physiology as selenocysteine (Sec). The 25 Sec-containing proteins in humans are known as selenoproteins. Their synthesis depends on the translational recoding of the UGA stop codon to allow Sec insertion. This requires a stem-loop structure in the 3' untranslated region of eukaryotic mRNAs known as the Selenocysteine Insertion Sequence (SECIS). The SECIS is recognized by SECIS-binding protein 2 (SBP2) and this RNA:protein interaction is essential for UGA recoding to occur. Genetic mutations cause SBP2 deficiency in humans, resulting in a broad set of symptoms due to differential effects on individual selenoproteins. Progress on understanding the different phenotypes requires developing robust tools to investigate SBP2 structure and function. In this study we demonstrate that SBP2 protein produced by in vitro translation discriminates among SECIS elements in a competitive UGA recoding assay and has a much higher specific activity than bacterially expressed protein. We also show that a purified recombinant protein encompassing amino acids 517-777 of SBP2 binds to SECIS elements with high affinity and selectivity. The affinity of the SBP2:SECIS interaction correlated with the ability of a SECIS to compete for UGA recoding activity in vitro. The identification of a 250 amino acid sequence that mediates specific, selective SECIS-binding will facilitate future structural studies of the SBP2:SECIS complex. Finally, we identify an evolutionarily conserved core cysteine signature in SBP2 sequences from the vertebrate lineage. Mutation of multiple, but not single, cysteines impaired SECIS-binding but did not affect protein localization in cells.

Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

PubMed Central

Simone, Domenico; Bay, Denice C.; Leach, Thorin; Turner, Raymond J.

2013-01-01

Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial

Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

PubMed

Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

2016-08-12

Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb*

PubMed Central

Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

2016-01-01

Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

Monitoring substrate enables real-time regulation of a protein localization pathway.

PubMed

Ito, Koreaki; Mori, Hiroyuki; Chiba, Shinobu

2018-06-01

Protein localization machinery supports cell survival and physiology, suggesting the potential importance of its expression regulation. Here, we summarize a remarkable scheme of regulation, which allows real-time feedback regulation of the machinery expression. A class of regulatory nascent polypeptides, called monitoring substrates, undergoes force-sensitive translation arrest. The resulting ribosome stalling on the mRNA then affects mRNA folding to expose the ribosome-binding site of the downstream target gene and upregulate its translation. The target gene encodes a component of the localization machinery, whose physical action against the monitoring substrate leads to arrest cancellation. Thus, this scheme of feedback loop allows the cell to adjust the amount of the machinery to correlate inversely with the effectiveness of the process at a given moment. The system appears to have emerged late in evolution, in which a narrow range of organisms selected a distinct monitoring substrate-machinery combination. Currently, regulatory systems of SecM-SecA, VemP-SecDF2 and MifM-YidC2 are known to occur in different bacterial species.

Selenocysteine in proteins-properties and biotechnological use.

PubMed

Johansson, Linda; Gafvelin, Guro; Arnér, Elias S J

2005-10-30

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.

46 CFR Sec. 3 - Standby agreements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 8 2014-10-01 2014-10-01 false Standby agreements. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS FEDERAL PORT CONTROLLERS Sec. 3 Standby agreements. The Director, NSA, may negotiate the standard form of service agreement, specified in...

46 CFR Sec. 3 - Standby agreements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 8 2013-10-01 2013-10-01 false Standby agreements. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS FEDERAL PORT CONTROLLERS Sec. 3 Standby agreements. The Director, NSA, may negotiate the standard form of service agreement, specified in...

Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection

PubMed Central

Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C.; Rajala, Rahul; Ismail, Ashrafali M.; Seto, Donald; Dyer, David W.

2018-01-01

presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota. PMID:29925671

Object-adapted trapping and shape-tracking to probe a bacterial protein chain motor

NASA Astrophysics Data System (ADS)

Roth, Julian; Koch, Matthias; Rohrbach, Alexander

2015-03-01

The helical bacterium Spiroplasma is a motile plant and anthropod pathogen which swims by propagating pairs of kinks along its cell body. As a well suited model system for bacterial locomotion, understanding the cell's molecular motor is of vital interest also regarding the combat of bacterial diseases. The extensive deformations related to these kinks are caused by a contractile cytoskeletal protein ribbon representing a linear motor in contrast to common rotary motors as, e.g., flagella. We present new insights into the working of this motor through experiments with object-adapted optical traps and shape-tracking techniques. We use the given laser irradiation from the optical trap to hinder bacterial energy (ATP) production through the production of O2 radicals. The results are compared with experiments performed under the influence of an O2-Scavenger and ATP inhibitors, respectively. Our results show clear dependences of the kinking properties on the ATP concentration inside the bacterium. The experiments are supported by a theoretical model which we developed to describe the switching of the ribbon's protein subunits.

46 CFR Sec. 2 - Bank account.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 8 2011-10-01 2011-10-01 false Bank account. Sec. 2 Section 2 Shipping MARITIME... TRANSACTIONS UNDER AGENCY AGREEMENTS Accounts Sec. 2 Bank account. A separate joint bank account will be... the depository or depositories, the owner will issue an order for the establishment of the joint bank...

46 CFR Sec. 2 - Bank account.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Bank account. Sec. 2 Section 2 Shipping MARITIME... TRANSACTIONS UNDER AGENCY AGREEMENTS Accounts Sec. 2 Bank account. A separate joint bank account will be... the depository or depositories, the owner will issue an order for the establishment of the joint bank...

Procalcitonin and C-reactive protein as markers of bacterial infection in critically ill children at onset of systemic inflammatory response syndrome.

PubMed

Simon, Liliana; Saint-Louis, Patrick; Amre, Devendra K; Lacroix, Jacques; Gauvin, France

2008-07-01

To compare the accuracy of procalcitonin and C-reactive protein as diagnostic markers of bacterial infection in critically ill children at the onset of systemic inflammatory response syndrome (SIRS). Prospective cohort study. Tertiary care, university-affiliated pediatric intensive care unit (PICU). Consecutive patients with SIRS. From June to December 2002, all PICU patients were screened daily to include cases of SIRS. At inclusion (onset of SIRS), procalcitonin and C-reactive protein levels as well as an array of cultures were obtained. Diagnosis of bacterial infection was made a posteriori by an adjudicating process (consensus of experts unaware of the results of procalcitonin and C-reactive protein). Baseline and daily data on severity of illness, organ dysfunction, and outcome were collected. Sixty-four patients were included in the study and were a posteriori divided into the following groups: bacterial SIRS (n = 25) and nonbacterial SIRS (n = 39). Procalcitonin levels were significantly higher in patients with bacterial infection compared with patients without bacterial infection (p = .01). The area under the receiver operating characteristic curve for procalcitonin was greater than that for C-reactive protein (0.71 vs. 0.65, respectively). A positive procalcitonin level (>or=2.5 ng/mL), when added to bedside clinical judgment, increased the likelihood of bacterial infection from 39% to 92%, while a negative C-reactive protein level (<40 mg/L) decreased the probability of bacterial infection from 39% to 2%. Procalcitonin is better than C-reactive protein for differentiating bacterial from nonbacterial SIRS in critically ill children, although the accuracy of both tests is moderate. Diagnostic accuracy could be enhanced by combining these tests with bedside clinical judgment.

Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons.

PubMed

Gennaris, Alexandra; Ezraty, Benjamin; Henry, Camille; Agrebi, Rym; Vergnes, Alexandra; Oheix, Emmanuel; Bos, Julia; Leverrier, Pauline; Espinosa, Leon; Szewczyk, Joanna; Vertommen, Didier; Iranzo, Olga; Collet, Jean-François; Barras, Frédéric

2015-12-17

The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts. Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum.

Comparison of exhalation time methods (6 sec vs. 10 sec) of a hand-held exhaled nitric oxide analyzer.

PubMed

Ito, Yasunori; Adachi, Yuichi; Itazawa, Toshiko; Okabe, Yoshie; Adachi, Yoko S; Katsumuma, Toshio; Miyawaki, Toshio

2010-10-01

Standard exhalation time for measuring fractional exhaled nitric oxide (FeNO) is 10 sec, but this is not easy for younger children. We aimed to investigate the agreement between FeNO values during 10-sec (FeNO-10) and 6-sec (FeNO-6) exhalation and the feasibility of measuring FeNO-6, using a hand-held analyzer, NIOX-MINO®. FeNO values measured during 10- and 6-sec (random order) were compared. Success rates of the two different time modes were also evaluated. In 119 asthmatic children (median age 8 years [range 4-15]) who had been already accustomed to NIOX-MINO®, median FeNO-10 (29 ppb [IQR 15.2-42.0]) and FeNO-6 (27 ppb [IQR 16.0-43.5]) did not differ significantly (P = 0.90), and there was a good correlation between both values (r = 0.984, P < 0.001). Mean difference (FeNO-10-FeNO-6) was -0.151 ppb (95% CI: -0.95 to 0.65, limits of agreement: -8.8 to 8.5). In 46 asthmatic children (median age 7 years [range 4-15]) who had never used any FeNO analyzers, all the children aged 8 years and more (n = 21) succeeded in measuring FeNO on both time modes, whereas for children aged younger than 8 years (n = 25) success rates of the 10- and 6-sec mode were 60.0% and 92.0%, respectively. In conclusion, we showed good agreement between FeNO-10 and FeNO-6, and the 6-sec mode of NIOX-MINO® is more feasible than 10-sec mode for measuring FeNO in younger children. © 2010 Wiley-Liss, Inc.

Human homologues of the bacterial heat-shock protein DnaJ are preferentially expressed in neurons.

PubMed Central

Cheetham, M E; Brion, J P; Anderton, B H

1992-01-01

The bacterial heat-shock protein DnaJ has been implicated in protein folding and protein complex dissociation. The DnaJ protein interacts with the prokaryotic analogue of Hsp70, DnaK, and accelerates the rate of ATP hydrolysis by DnaK. Several yeast homologues of DnaJ, with different proposed subcellular localizations and functions, have recently been isolated and are the only eukaryotic forms of DnaJ so far described. We have isolated cDNAs corresponding to two alternatively spliced transcripts of a novel human gene, HSJ1, which show sequence similarity to the bacterial DnaJ protein and the yeast homologues. The cDNA clones were isolated from a human brain-frontal-cortex expression library screened with a polyclonal antiserum raised to paired-helical-filament (PHF) proteins isolated from extracts of the brains of patients suffering from Alzheimer's disease. The similarity between the predicted human protein sequences and the bacterial and yeast proteins is highest at the N-termini, this region also shows a limited similarity to viral T-antigens and is a possible common motif involved in the interaction with DnaK/Hsp70. Northern-blot analysis has shown that human brain contains higher levels of mRNA for the DnaJ homologue than other tissues examined, and hybridization studies with riboprobes in situ show a restricted pattern of expression of the mRNA within the brain, with neuronal layers giving the strongest signal. These findings suggest that the DnaJ-DnaK (Hsp70) interaction is general to eukaryotes and, indeed, to higher organisms. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1599432

Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

PubMed Central

Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.

2013-01-01

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423

Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.

PubMed

Kirkwood, Kathryn J; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I

2013-12-01

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles*

PubMed Central

Haurat, M. Florencia; Aduse-Opoku, Joseph; Rangarajan, Minnie; Dorobantu, Loredana; Gray, Murray R.; Curtis, Michael A.; Feldman, Mario F.

2011-01-01

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions. PMID:21056982

Phylogenetic and Complementation Analysis of a Single-Stranded DNA Binding Protein Family from Lactococcal Phages Indicates a Non-Bacterial Origin

PubMed Central

Mariadassou, Mahendra; Bardowski, Jacek K.; Bidnenko, Elena

2011-01-01

Background The single-stranded-nucleic acid binding (SSB) protein superfamily includes proteins encoded by different organisms from Bacteria and their phages to Eukaryotes. SSB proteins share common structural characteristics and have been suggested to descend from an ancestor polypeptide. However, as other proteins involved in DNA replication, bacterial SSB proteins are clearly different from those found in Archaea and Eukaryotes. It was proposed that the corresponding genes in the phage genomes were transferred from the bacterial hosts. Recently new SSB proteins encoded by the virulent lactococcal bacteriophages (Orf14bIL67-like proteins) have been identified and characterized structurally and biochemically. Methodology/Principal Findings This study focused on the determination of phylogenetic relationships between Orf14bIL67-like proteins and other SSBs. We have performed a large scale phylogenetic analysis and pairwise sequence comparisons of SSB proteins from different phyla. The results show that, in remarkable contrast to other phage SSBs, the Orf14bIL67–like proteins form a distinct, self-contained and well supported phylogenetic group connected to the archaeal SSBs. Functional studies demonstrated that, despite the structural and amino acid sequence differences from bacterial SSBs, Orf14bIL67 protein complements the conditional lethal ssb-1 mutation of Escherichia coli. Conclusions/Significance Here we identified for the first time a group of phages encoded SSBs which are clearly distinct from their bacterial counterparts. All methods supported the recognition of these phage proteins as a new family within the SSB superfamily. Our findings suggest that unlike other phages, the virulent lactococcal phages carry ssb genes that were not acquired from their hosts, but transferred from an archaeal genome. This represents a unique example of a horizontal gene transfer between Archaea and bacterial phages. PMID:22073223

Coupling MALDI-TOF mass spectrometry protein and specialized metabolite analyses to rapidly discriminate bacterial function

PubMed Central

Clark, Chase M.; Costa, Maria S.

2018-01-01

For decades, researchers have lacked the ability to rapidly correlate microbial identity with bacterial metabolism. Since specialized metabolites are critical to bacterial function and survival in the environment, we designed a data acquisition and bioinformatics technique (IDBac) that utilizes in situ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze protein and specialized metabolite spectra recorded from single bacterial colonies picked from agar plates. We demonstrated the power of our approach by discriminating between two Bacillus subtilis strains in <30 min solely on the basis of their differential ability to produce cyclic peptide antibiotics surfactin and plipastatin, caused by a single frameshift mutation. Next, we used IDBac to detect subtle intraspecies differences in the production of metal scavenging acyl-desferrioxamines in a group of eight freshwater Micromonospora isolates that share >99% sequence similarity in the 16S rRNA gene. Finally, we used IDBac to simultaneously extract protein and specialized metabolite MS profiles from unidentified Lake Michigan sponge-associated bacteria isolated from an agar plate. In just 3 h, we created hierarchical protein MS groupings of 11 environmental isolates (10 MS replicates each, for a total of 110 spectra) that accurately mirrored phylogenetic groupings. We further distinguished isolates within these groupings, which share nearly identical 16S rRNA gene sequence identity, based on interspecies and intraspecies differences in specialized metabolite production. IDBac is an attempt to couple in situ MS analyses of protein content and specialized metabolite production to allow for facile discrimination of closely related bacterial colonies. PMID:29686101

Selective dye-labeling of newly synthesized proteins in bacterial cells.

PubMed

Beatty, Kimberly E; Xie, Fang; Wang, Qian; Tirrell, David A

2005-10-19

We describe fluorescence labeling of newly synthesized proteins in Escherichia coli cells by means of Cu(I)-catalyzed cycloaddition between alkynyl amino acid side chains and the fluorogenic dye 3-azido-7-hydroxycoumarin. The method involves co-translational labeling of proteins by the non-natural amino acids homopropargylglycine (Hpg) or ethynylphenylalanine (Eth) followed by treatment with the dye. As a demonstration, the model protein barstar was expressed and treated overnight with Cu(I) and 3-azido-7-hydroxycoumarin. Examination of treated cells by confocal microscopy revealed that strong fluorescence enhancement was observed only for alkynyl-barstar treated with Cu(I) and the reactive dye. The cellular fluorescence was punctate, and gel electrophoresis confirmed that labeled barstar was localized in inclusion bodies. Other proteins showed little fluorescence. Examination of treated cells by fluorimetry demonstrated that cultures supplemented with Eth or Hpg showed an 8- to 14-fold enhancement in fluorescence intensity after labeling. Addition of a protein synthesis inhibitor reduced the emission intensity to levels slightly above background, confirming selective labeling of newly synthesized proteins in the bacterial cell.

14 CFR Sec. 1-5 - Records.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Records. Sec. 1-5 Section 1-5 Aeronautics... Provisions Sec. 1-5 Records. (a) The general books of account and all books, records, and memoranda which... Accounts. Registers, or other appropriate records, shall be maintained of the history and nature of each...

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria*

PubMed Central

Wang, Yan; Wang, Rui; Jin, Feng; Liu, Yang; Yu, Jiayu; Fu, Xinmiao; Chang, Zengyi

2016-01-01

β-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent β-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving β-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for β-barrel OMP biogenesis. PMID:27298319

Sec61α is required for dorsal closure during Drosophila embryogenesis through its regulation of Dpp signaling

PubMed Central

Wang, Xiaochen; Ward, Robert E.

2010-01-01

During dorsal closure in Drosophila, signaling events in the dorsalmost row of epidermal cells (DME cells) direct the migration of lateral epidermal sheets towards the dorsal midline where they fuse to enclose the embryo. A Jun amino-terminal kinase (JNK) cascade in the DME cells induces the expression of Decapentaplegic (Dpp). Dpp signaling then regulates the cytoskeleton in the DME cells and amnioserosa to affect the cell shape changes necessary to complete dorsal closure. We identified a mutation in Sec61α that specifically perturbs dorsal closure. Sec61α encodes the main subunit of the translocon complex for co-translational import of proteins into the ER. JNK signaling is normal in Sec61α mutant embryos, but Dpp signaling is attenuated and the DME cells fail to maintain an actinomyosin cable as epithelial migration fails. Consistent with this model, dorsal closure is rescued in Sec61α mutant embryos by an activated form of the Dpp receptor Thick veins. PMID:20112345

Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae

PubMed Central

1992-01-01

We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus. PMID:1730771

UGT-29 protein expression and localization during bacterial infection in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Wong, Rui-Rui; Lee, Song-Hua; Nathan, Sheila

2014-09-01

The nematode Caenorhabditis elegans is routinely used as an animal model to delineate complex molecular mechanisms involved in the host response to pathogen infection. Following up on an earlier study on host-pathogen interaction, we constructed a ugt-29::GFP transcriptional fusion transgenic worm strain to examine UGT-29 protein expression and localization upon bacterial infection. UGT-29 orthologs can be found in higher organisms including humans and is proposed as a member of the UDP-Glucoronosyl Transferase family of proteins which are involved in phase II detoxification of compounds detrimental to the host organism. Under uninfected conditions, UGT-29::GFP fusion protein was highly expressed in the C. elegans anterior pharynx and intestine, two major organs involved in detoxification. We further evaluated the localization of the enzyme in worms infected with the bacterial pathogen, Burkholderia pseudomallei. The infected ugt-29::GFP transgenic strain exhibited increased fluorescence in the pharynx and intestine with pronounced fluorescence also extending to body wall muscle. This transcriptional fusion GFP transgenic worm is a convenient and direct tool to provide information on UGT detoxification enzyme gene expression and could be a useful tool for a number of diverse applications.

Fission Yeast Sec3 and Exo70 Are Transported on Actin Cables and Localize the Exocyst Complex to Cell Poles

PubMed Central

Martin, Sophie G.

2012-01-01

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk. PMID:22768263

Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc1 complex

NASA Astrophysics Data System (ADS)

Martin, Daniel R.; Matyushov, Dmitry V.

2015-04-01

Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc1 bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ˜0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins.

Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons

PubMed Central

Henry, Camille; Agrebi, Rym; Vergnes, Alexandra; Oheix, Emmanuel; Bos, Julia; Leverrier, Pauline; Espinosa, Leon; Szewczyk, Joanna; Vertommen, Didier; Iranzo, Olga; Collet, Jean-François; Barras, Frédéric

2015-01-01

The reactive species of oxygen (ROS) and chlorine (RCS) damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine (Met) is converted to methionine sulfoxide (Met-O), which can cause a loss of biological activity. To rescue proteins with Met-O residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts 1-3. Here, we report the identification of an enzymatic system, MsrPQ, repairing Met-O containing proteins in the bacterial cell envelope, a compartment particularly exposed to the ROS and RCS generated by the host defense mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a heme-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid (HOCl), a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from Met oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both R- and S- diastereoisomers of Met-O, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting Met residues from oxidation should prompt search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum (ER). PMID:26641313

Fuel of the Bacterial Flagellar Type III Protein Export Apparatus.

PubMed

Minamino, Tohru; Kinoshita, Miki; Namba, Keiichi

2017-01-01

The flagellar type III export apparatus utilizes ATP and proton motive force (PMF) across the cytoplasmic membrane as the energy sources and transports flagellar component proteins from the cytoplasm to the distal growing end of the growing structure to construct the bacterial flagellum beyond the cellular membranes. The flagellar type III export apparatus coordinates flagellar protein export with assembly by ordered export of substrates to parallel with their order of the assembly. The export apparatus is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. Since the ATPase complex is dispensable for flagellar protein export, PMF is the primary fuel for protein unfolding and translocation. Interestingly, the export gate complex can also use sodium motive force across the cytoplasmic membrane in addition to PMF when the ATPase complex does not work properly. Here, we describe experimental protocols, which have allowed us to identify the export substrate class and the primary fuel of the flagellar type III protein export apparatus in Salmonella enterica serovar Typhimurium.

Structural analysis of a set of proteins resulting from a bacterial genomics project.

PubMed

Badger, J; Sauder, J M; Adams, J M; Antonysamy, S; Bain, K; Bergseid, M G; Buchanan, S G; Buchanan, M D; Batiyenko, Y; Christopher, J A; Emtage, S; Eroshkina, A; Feil, I; Furlong, E B; Gajiwala, K S; Gao, X; He, D; Hendle, J; Huber, A; Hoda, K; Kearins, P; Kissinger, C; Laubert, B; Lewis, H A; Lin, J; Loomis, K; Lorimer, D; Louie, G; Maletic, M; Marsh, C D; Miller, I; Molinari, J; Muller-Dieckmann, H J; Newman, J M; Noland, B W; Pagarigan, B; Park, F; Peat, T S; Post, K W; Radojicic, S; Ramos, A; Romero, R; Rutter, M E; Sanderson, W E; Schwinn, K D; Tresser, J; Winhoven, J; Wright, T A; Wu, L; Xu, J; Harris, T J R

2005-09-01

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB. Copyright 2005 Wiley-Liss, Inc.

46 CFR Sec. 4 - Funding of operations.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 8 2014-10-01 2014-10-01 false Funding of operations. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURAL RULES FOR FINANCIAL TRANSACTIONS UNDER AGENCY AGREEMENTS Funding of Operations Sec. 4 Funding of operations. Cash...

46 CFR Sec. 4 - Funding of operations.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 8 2013-10-01 2013-10-01 false Funding of operations. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURAL RULES FOR FINANCIAL TRANSACTIONS UNDER AGENCY AGREEMENTS Funding of Operations Sec. 4 Funding of operations. Cash...

46 CFR Sec. 4 - Funding of operations.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 8 2011-10-01 2011-10-01 false Funding of operations. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURAL RULES FOR FINANCIAL TRANSACTIONS UNDER AGENCY AGREEMENTS Funding of Operations Sec. 4 Funding of operations. Cash...

Selective expression of a sec1/munc18 member in sea urchin eggs and embryos.

PubMed

Leguia, Mariana; Wessel, Gary M

2004-10-01

Regulated secretion is mediated by SNAREs (soluble NSF attachment receptors) and their regulators and effectors, which include the SM (sec1/munc18) family of proteins. Homologs of the SNAREs have been identified in sea urchins, associated with cortical granule exocytosis at fertilization, with membranes of the cleavage furrow, and in secretory cells later in development. To contribute to the understanding of regulated secretion in sea urchins we have cloned the single SM protein homolog from two species of sea urchin, Lytechinus variegatus and Strongylocentrotus purpuratus. In oocytes and eggs, we find that it localizes to the plasma membrane and the cortical region of the egg, consistent with a role in one of the steps leading to cortical granule exocytosis. The protein is also expressed throughout development, enriched in membranes of the cleavage furrow in early embryos, and in cells of the gut in advanced embryos. Furthermore, we find that sec1/munc18 co-localizes with its cognate binding partner syntaxin. Finally, our biochemical analysis shows that the protein associates with rab3 in high molecular weight complexes, suggesting that the exocytotic machinery functions as a multi-protein subunit to mediate regulated secretion in sea urchins. These results will be instrumental in the future to functionally test the SNARE regulators associated with multiple membrane fusion events.

46 CFR Sec. 3 - Accounting for revenues.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Accounting for revenues. Sec. 3 Section 3 Shipping... FINANCIAL TRANSACTIONS UNDER AGENCY AGREEMENTS Accounting for Revenues Sec. 3 Accounting for revenues. (a... shipper, consignee, weight or measurement, freight rate and basis (whether the freight rate applies on...

36 CFR 67.1 - Sec. 48(g) and Sec. 170(h) of the Internal Revenue Code of 1986.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 36 Parks, Forests, and Public Property 1 2010-07-01 2010-07-01 false Sec. 48(g) and Sec. 170(h) of the Internal Revenue Code of 1986. 67.1 Section 67.1 Parks, Forests, and Public Property NATIONAL PARK..., Alaska 99503: Alaska Mid-Atlantic Regional Office, National Park Service, U.S. Customs House, Second...

An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems.

PubMed

Cheng, Hao-Wen; Chen, Kuan-Chun; Raja, Joseph A J; Li, Jian-Xian; Yeh, Shyi-Dong

2013-04-15

NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, we expressed different green fluorescent protein (GFP)-fused deletions of NSscon in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids, "(109)KFTMHNQIF(117)", denoted as "nss", retain the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag's possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and non-tagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. Copyright © 2013 Elsevier B.V. All rights reserved.

Impact of fluorescent protein fusions on the bacterial flagellar motor.

PubMed

Heo, M; Nord, A L; Chamousset, D; van Rijn, E; Beaumont, H J E; Pedaci, F

2017-10-03

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation- new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.

Sss1p Is Required to Complete Protein Translocon Activation*

PubMed Central

Wilkinson, Barrie M.; Brownsword, Judith K.; Mousley, Carl J.; Stirling, Colin J.

2010-01-01

Protein translocation across the endoplasmic reticulum membrane occurs at the Sec61 translocon. This has two essential subunits, the channel-forming multispanning membrane protein Sec61p/Sec61α and the tail-anchored Sss1p/Sec61γ, which has been proposed to “clamp” the channel. We have analyzed the function of Sss1p using a series of domain mutants and found that both the cytosolic and transmembrane clamp domains of Sss1p are essential for protein translocation. Our data reveal that the cytosolic domain is required for Sec61p interaction but that the transmembrane clamp domain is required to complete activation of the translocon after precursor targeting to Sec61p. PMID:20709746

46 CFR Sec. 2 - Stand-by agreements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 8 2013-10-01 2013-10-01 false Stand-by agreements. Sec. 2 Section 2 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS OPERATING CONTRACT Sec. 2 Stand-by agreements. The Director NSA, Maritime Administration, in advance of an emergency, may negotiate...

46 CFR Sec. 2 - Stand-by agreements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 8 2014-10-01 2014-10-01 false Stand-by agreements. Sec. 2 Section 2 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS OPERATING CONTRACT Sec. 2 Stand-by agreements. The Director NSA, Maritime Administration, in advance of an emergency, may negotiate...

Four Proteins Encoded in the gspB-secY2A2 Operon of Streptococcus gordonii Mediate the Intracellular Glycosylation of the Platelet-Binding Protein GspB

PubMed Central

Takamatsu, Daisuke; Bensing, Barbara A.; Sullam, Paul M.

2004-01-01

Platelet binding by Streptococcus gordonii strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the secY2A2 locus immediately downstream of gspB. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and Orf4 by investigating the expression and glycosylation of a series of glutathione S-transferase-GspB fusion proteins in M99 and in gly, nss, gtf, and orf4 mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the gtf and orf4 mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in Escherichia coli, glycosylation of this protein was observed only in the presence of both gtf and orf4. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB. PMID:15489421

Anticancer Activity of Bacterial Proteins and Peptides.

PubMed

Karpiński, Tomasz M; Adamczak, Artur

2018-04-30

Despite much progress in the diagnosis and treatment of cancer, tumour diseases constitute one of the main reasons of deaths worldwide. The side effects of chemotherapy and drug resistance of some cancer types belong to the significant current therapeutic problems. Hence, searching for new anticancer substances and medicines are very important. Among them, bacterial proteins and peptides are a promising group of bioactive compounds and potential anticancer drugs. Some of them, including anticancer antibiotics (actinomycin D, bleomycin, doxorubicin, mitomycin C) and diphtheria toxin, are already used in the cancer treatment, while other substances are in clinical trials (e.g., p28, arginine deiminase ADI) or tested in in vitro research. This review shows the current literature data regarding the anticancer activity of proteins and peptides originated from bacteria: antibiotics, bacteriocins, enzymes, nonribosomal peptides (NRPs), toxins and others such as azurin, p28, Entap and Pep27anal2. The special attention was paid to the still poorly understood active substances obtained from the marine sediment bacteria. In total, 37 chemical compounds or groups of compounds with antitumor properties have been described in the present article.

46 CFR Sec. 4 - Method of payment.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 8 2011-10-01 2011-10-01 false Method of payment. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE TO BE FOLLOWED BY GENERAL... Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank account...

14 CFR Sec. 1-6 - Accounting entities.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Accounting entities. Sec. 1-6 Section 1-6... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 1-6 Accounting entities. (a) Separate accounting records shall be maintained for each air...

46 CFR Sec. 7 - Job order numbering.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 8 2011-10-01 2011-10-01 false Job order numbering. Sec. 7 Section 7 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 7 Job order numbering. (a) The NSA-LUMPSUMREP Contract number shall be inserted in every job order and supplemental job...

46 CFR Sec. 7 - Job order numbering.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Job order numbering. Sec. 7 Section 7 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 7 Job order numbering. (a) The NSA-LUMPSUMREP Contract number shall be inserted in every job order and supplemental job...

46 CFR Sec. 7 - Job order numbering.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 8 2013-10-01 2013-10-01 false Job order numbering. Sec. 7 Section 7 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 7 Job order numbering. (a) The NSA-LUMPSUMREP Contract number shall be inserted in every job order and supplemental job...

46 CFR Sec. 7 - Job order numbering.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 8 2014-10-01 2014-10-01 false Job order numbering. Sec. 7 Section 7 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 7 Job order numbering. (a) The NSA-LUMPSUMREP Contract number shall be inserted in every job order and supplemental job...

46 CFR Sec. 7 - Job order numbering.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 8 2012-10-01 2012-10-01 false Job order numbering. Sec. 7 Section 7 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 7 Job order numbering. (a) The NSA-LUMPSUMREP Contract number shall be inserted in every job order and supplemental job...

14 CFR Sec. 2-4 - Accounting period.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Accounting period. Sec. 2-4 Section 2-4... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 2-4 Accounting period. (a) The accounting year of each air carrier subject to this Uniform...

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria.

PubMed

Wang, Yan; Wang, Rui; Jin, Feng; Liu, Yang; Yu, Jiayu; Fu, Xinmiao; Chang, Zengyi

2016-08-05

β-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent β-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving β-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for β-barrel OMP biogenesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Factor H-IgG Chimeric Proteins as a Therapeutic Approach against the Gram-Positive Bacterial Pathogen Streptococcus pyogenes.

PubMed

Blom, Anna M; Magda, Michal; Kohl, Lisa; Shaughnessy, Jutamas; Lambris, John D; Ram, Sanjay; Ermert, David

2017-12-01

Bacteria can cause life-threatening infections, such as pneumonia, meningitis, or sepsis. Antibiotic therapy is a mainstay of treatment, although antimicrobial resistance has drastically increased over the years. Unfortunately, safe and effective vaccines against most pathogens have not yet been approved, and thus developing alternative treatments is important. We analyzed the efficiency of factor H (FH)6-7/Fc, a novel antibacterial immunotherapeutic protein against the Gram-positive bacterium Streptococcus pyogenes This protein is composed of two domains of complement inhibitor human FH (FH complement control protein modules 6 and 7) that bind to S. pyogenes , linked to the Fc region of IgG (FH6-7/Fc). FH6-7/Fc has previously been shown to enhance complement-dependent killing of, and facilitate bacterial clearance in, animal models of the Gram-negative pathogens Haemophilus influenzae and Neisseria meningitidis We hypothesized that activation of complement by FH6-7/Fc on the surface of Gram-positive bacteria such as S. pyogenes will enable professional phagocytes to eliminate the pathogen. We found that FH6-7/Fc alleviated S. pyogenes- induced sepsis in a transgenic mouse model expressing human FH ( S. pyogenes binds FH in a human-specific manner). Furthermore, FH6-7/Fc, which binds to protein H and selected M proteins, displaced FH from the bacterial surface, enhanced alternative pathway activation, and reduced bacterial blood burden by opsonophagocytosis in a C3-dependent manner in an ex vivo human whole-blood model. In conclusion, FH-Fc chimeric proteins could serve as adjunctive treatments against multidrug-resistant bacterial infections. Copyright © 2017 by The American Association of Immunologists, Inc.

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

PubMed

Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

2017-01-01

There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for

Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent.

PubMed

Upadhyay, Vaibhav; Singh, Anupam; Jha, Divya; Singh, Akansha; Panda, Amulya K

2016-06-08

Formation of inclusion bodies poses a major hurdle in recovery of bioactive recombinant protein from Escherichia coli. Urea and guanidine hydrochloride have routinely been used to solubilize inclusion body proteins, but many times result in poor recovery of bioactive protein. High pH buffers, detergents and organic solvents like n-propanol have been successfully used as mild solubilization agents for high throughput recovery of bioactive protein from bacterial inclusion bodies. These mild solubilization agents preserve native-like secondary structures of proteins in inclusion body aggregates and result in improved recovery of bioactive protein as compared to conventional solubilization agents. Here we demonstrate solubilization of human growth hormone inclusion body aggregates using 30% trifluoroethanol in presence of 3 M urea and its refolding into bioactive form. Human growth hormone was expressed in E. coli M15 (pREP) cells in the form of inclusion bodies. Different concentrations of trifluoroethanol with or without addition of low concentration (3 M) of urea were used for solubilization of inclusion body aggregates. Thirty percent trifluoroethanol in combination with 3 M urea was found to be suitable for efficient solubilization of human growth hormone inclusion bodies. Solubilized protein was refolded by dilution and purified by anion exchange and size exclusion chromatography. Purified protein was analyzed for secondary and tertiary structure using different spectroscopic tools and was found to be bioactive by cell proliferation assay. To understand the mechanism of action of trifluoroethanol, secondary and tertiary structure of human growth hormone in trifluoroethanol was compared to that in presence of other denaturants like urea and guanidine hydrochloride. Trifluoroethanol was found to be stabilizing the secondary structure and destabilizing the tertiary structure of protein. Finally, it was observed that trifluoroethanol can be used to solubilize

14 CFR Sec. 1-6 - Accounting entities.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Accounting entities. Sec. 1-6 Section 1-6... Provisions Sec. 1-6 Accounting entities. (a) Separate accounting records shall be maintained for each air transport entity for which separate reports to the BTS are required to be made by sections 21(g) and for...

Sequential Super-Resolution Imaging of Bacterial Regulatory Proteins: The Nucleoid and the Cell Membrane in Single, Fixed E. coli Cells.

PubMed

Spahn, Christoph; Glaesmann, Mathilda; Gao, Yunfeng; Foo, Yong Hwee; Lampe, Marko; Kenney, Linda J; Heilemann, Mike

2017-01-01

Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.

Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

PubMed

Hu, Yaozhong; Romão, Ema; Vertommen, Didier; Vincke, Cécile; Morales-Yánez, Francisco; Gutiérrez, Carlos; Liu, Changxiao; Muyldermans, Serge

2017-09-01

The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated. Copyright © 2017 Elsevier Inc. All rights reserved.

Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

PubMed Central

2010-01-01

Background Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this

DBSecSys: A Database of Burkholderia mallei Secretion Systems

DTIC Science & Technology

2014-07-16

toxins and the lipases, as well as non-proteinaceous substrates, e.g., cyclic β- glucans and polysaccharides. 2* - Represents a Sec/Tat-dependent system...divided into three types: 1) the archetypal bacterial proteins exported into the periplasm via the Sec system; 2) trimeric proteins with a single beta ...barrel domain; and 3) pairs of proteins in which one partner carries the beta barrel domain and the other partner is the secreted protein. 6

CoSEC: Connecting Living With a Star Research

NASA Astrophysics Data System (ADS)

Hurlburt, N.; Freeland, S.; Bose, P.; Zimdars, A.; Slater, G.

2006-12-01

The Collaborative Sun-Earth Connector (CoSEC) provide the means for heliophysics researchers to compose the data sources and processing services published by their peers into processing workflows that reliably generate publication-worthy data. It includes: composition of computational and data services into easy-to- read workflows with data quality and version traceability; straightforward translation of existing services into workflow components, and advertisement of those components to other members of the CoSEC community; annotation of published services with functional attributes to enable discovery of capabilities required by particular workflows and identify peer subgroups in the CoSEC community; and annotation of published services with nonfunctional attributes to enable selection on the basis of quality of service (QoS). We present an overview and demonstration of the CoSEC system, discuss applications, the lessons learned and future developments.

Bacterial membrane proteomics.

PubMed

Poetsch, Ansgar; Wolters, Dirk

2008-10-01

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

[Identification of proteins interacting with the circadian clock protein PER1 in tumors using bacterial two-hybrid system technique].

PubMed

Zhang, Yu; Yao, Youlin; Jiang, Siyuan; Lu, Yilu; Liu, Yunqiang; Tao, Dachang; Zhang, Sizhong; Ma, Yongxin

2015-04-01

To identify protein-protein interaction partners of PER1 (period circadian protein homolog 1), key component of the molecular oscillation system of the circadian rhythm in tumors using bacterial two-hybrid system technique. Human cervical carcinoma cell Hela library was adopted. Recombinant bait plasmid pBT-PER1 and pTRG cDNA plasmid library were cotransformed into the two-hybrid system reporter strain cultured in a special selective medium. Target clones were screened. After isolating the positive clones, the target clones were sequenced and analyzed. Fourteen protein coding genes were identified, 4 of which were found to contain whole coding regions of genes, which included optic atrophy 3 protein (OPA3) associated with mitochondrial dynamics and homo sapiens cutA divalent cation tolerance homolog of E. coli (CUTA) associated with copper metabolism. There were also cellular events related proteins and proteins which are involved in biochemical reaction and signal transduction-related proteins. Identification of potential interacting proteins with PER1 in tumors may provide us new insights into the functions of the circadian clock protein PER1 during tumorigenesis.

Comprehensive assessment and performance improvement of effector protein predictors for bacterial secretion systems III, IV and VI.

PubMed

An, Yi; Wang, Jiawei; Li, Chen; Leier, André; Marquez-Lago, Tatiana; Wilksch, Jonathan; Zhang, Yang; Webb, Geoffrey I; Song, Jiangning; Lithgow, Trevor

2018-01-01

Bacterial effector proteins secreted by various protein secretion systems play crucial roles in host-pathogen interactions. In this context, computational tools capable of accurately predicting effector proteins of the various types of bacterial secretion systems are highly desirable. Existing computational approaches use different machine learning (ML) techniques and heterogeneous features derived from protein sequences and/or structural information. These predictors differ not only in terms of the used ML methods but also with respect to the used curated data sets, the features selection and their prediction performance. Here, we provide a comprehensive survey and benchmarking of currently available tools for the prediction of effector proteins of bacterial types III, IV and VI secretion systems (T3SS, T4SS and T6SS, respectively). We review core algorithms, feature selection techniques, tool availability and applicability and evaluate the prediction performance based on carefully curated independent test data sets. In an effort to improve predictive performance, we constructed three ensemble models based on ML algorithms by integrating the output of all individual predictors reviewed. Our benchmarks demonstrate that these ensemble models outperform all the reviewed tools for the prediction of effector proteins of T3SS and T4SS. The webserver of the proposed ensemble methods for T3SS and T4SS effector protein prediction is freely available at http://tbooster.erc.monash.edu/index.jsp. We anticipate that this survey will serve as a useful guide for interested users and that the new ensemble predictors will stimulate research into host-pathogen relationships and inspiration for the development of new bioinformatics tools for predicting effector proteins of T3SS, T4SS and T6SS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

46 CFR Sec. 15 - Subcontracts.

Code of Federal Regulations, 2012 CFR

2012-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 15 Subcontracts. Under Article 29 of the NSA-LUMPSUMREP Contract, the Contractor is authorized to subcontract...

46 CFR Sec. 15 - Subcontracts.

Code of Federal Regulations, 2013 CFR

2013-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 15 Subcontracts. Under Article 29 of the NSA-LUMPSUMREP Contract, the Contractor is authorized to subcontract...

46 CFR Sec. 15 - Subcontracts.

Code of Federal Regulations, 2014 CFR

2014-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 15 Subcontracts. Under Article 29 of the NSA-LUMPSUMREP Contract, the Contractor is authorized to subcontract...

46 CFR Sec. 15 - Subcontracts.

Code of Federal Regulations, 2011 CFR

2011-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 15 Subcontracts. Under Article 29 of the NSA-LUMPSUMREP Contract, the Contractor is authorized to subcontract...

46 CFR Sec. 15 - Subcontracts.

Code of Federal Regulations, 2010 CFR

2010-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 15 Subcontracts. Under Article 29 of the NSA-LUMPSUMREP Contract, the Contractor is authorized to subcontract...

Protein expression of preferred human codon-optimized Gaussia luciferase genes with an artificial open-reading frame in mammalian and bacterial cells.

PubMed

Inouye, Satoshi; Suzuki, Takahiro

2016-12-01

The protein expressions of three preferred human codon-optimized Gaussia luciferase genes (pGLuc, EpGLuc, and KpGLuc) were characterized in mammalian and bacterial cells by comparing them with those of wild-type Gaussia luciferase gene (wGLuc) and human codon-optimized Gaussia luciferase gene (hGLuc). Two synthetic genes of EpGLuc and KpGLuc containing the complete preferred human codons have an artificial open-reading frame; however, they had the similar protein expression levels to those of pGLuc and hGLuc in mammalian cells. In bacterial cells, the protein expressions of pGLuc, EpGLuc, and KpGLuc with approximately 65% GC content were the same and showed approximately 60% activities of wGLuc and hGLuc. The artificial open-reading frame in EpGLuc and KpGLuc did not affect the protein expression in mammalian and bacterial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

46 CFR Sec. 13 - Insurance.

Code of Federal Regulations, 2012 CFR

2012-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance. Article 9 of the NSA-LUMPSUMREP Contract sets forth the Contractor's liabilities and obligations with...

46 CFR Sec. 13 - Insurance.

Code of Federal Regulations, 2014 CFR

2014-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance. Article 9 of the NSA-LUMPSUMREP Contract sets forth the Contractor's liabilities and obligations with...

46 CFR Sec. 13 - Insurance.

Code of Federal Regulations, 2011 CFR

2011-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance. Article 9 of the NSA-LUMPSUMREP Contract sets forth the Contractor's liabilities and obligations with...

46 CFR Sec. 13 - Insurance.

Code of Federal Regulations, 2013 CFR

2013-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance. Article 9 of the NSA-LUMPSUMREP Contract sets forth the Contractor's liabilities and obligations with...

46 CFR Sec. 13 - Insurance.

Code of Federal Regulations, 2010 CFR

2010-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance. Article 9 of the NSA-LUMPSUMREP Contract sets forth the Contractor's liabilities and obligations with...

Legionella pneumophila effector WipA, a bacterial PPP protein phosphatase with PTP activity.

PubMed

Jia, Qian; Lin, Yun; Gou, Xuejing; He, Lei; Shen, Dong; Chen, Dongni; Xie, Wei; Lu, Yongjun

2018-04-26

The gram-negative bacterium Legionella pneumophila invades human's lung and causes Legionnaires' disease. To benefit its survival and replication in cellular milieu, L. pneumophila secrets at least 330 effector proteins into host cells. We found that the effector WipA has the protein tyrosine phosphatase (PTP) activity but does not depend on the classical CX5R motif for activity, suggesting that WipA is an unconventional PTP. Meanwhile, the presence of three other highly conserved motifs typically seen in protein serine/threonine phosphatases and the poor inhibition of WipA activity by okadaic acid led us to propose that WipA is a bacterial protein phosphatase. In addition, the determination of the 2.55-Å crystal structure of WipA revealed that WipA resembles cold-active protein tyrosine phosphatase (CAPTPase), and therefore very likely shares the same catalytic mechanism.

Effect of simulated lunar impact on the survival of bacterial spores.

NASA Technical Reports Server (NTRS)

Whitfield, O.; Merek, E. L.; Oyama, V. I.

1973-01-01

In order to test the effect of impact on organisms, the survival of bacterial spores after being propelled at high velocity in Pyrex and plastic beads into crushed basalt was measured. The beads were fired into sterilized canisters by both a conventional powder and a light gas gun. Results indicate that at the minimum (2.4 km/sec) lunar capture velocity, the number of colony forming units (CFUs) decreased by five orders of magnitude, and at 5.5 km/sec, statistically a more probable capture velocity, no CFUs were found. The decrease in CFUs observed with increasing velocity indicates that the spores were most probably killed by the impact.

10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 4 2013-01-01 2013-01-01 false Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to... to Subpart A of Part 710—Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141...

10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 4 2014-01-01 2014-01-01 false Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to... to Subpart A of Part 710—Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141...

10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 4 2012-01-01 2012-01-01 false Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to... to Subpart A of Part 710—Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141...

10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 4 2011-01-01 2011-01-01 false Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to... to Subpart A of Part 710—Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141...

10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 4 2010-01-01 2010-01-01 false Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to... to Subpart A of Part 710—Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141...

Somatic overgrowth associated with homozygous mutations in both MAN1B1 and SEC23A

PubMed Central

Gupta, Swati; Fahiminiya, Somayyeh; Wang, Tracy; Dempsey Nunez, Laura; Rosenblatt, David S.; Gibson, William T.; Gilfix, Brian; Bergeron, John J. M.; Jerome-Majewska, Loydie A.

2016-01-01

Using whole-exome sequencing, we identified homozygous mutations in two unlinked genes, SEC23A c.1200G>C (p.M400I) and MAN1B1 c.1000C>T (p.R334C), associated with congenital birth defects in two patients from a consanguineous family. Patients presented with carbohydrate-deficient transferrin, tall stature, obesity, macrocephaly, and maloccluded teeth. The parents were healthy heterozygous carriers for both mutations and an unaffected sibling with tall stature carried the heterozygous mutation in SEC23A only. Mutations in SEC23A are responsible for craniolenticosultura dysplasia (CLSD). CLSD patients are short, have late-closing fontanels, and have reduced procollagen (pro-COL1A1) secretion because of abnormal pro-COL1A1 retention in the endoplasmic reticulum (ER). The mutation we identified in MAN1B1 was previously associated with reduced MAN1B1 protein and congenital disorders of glycosylation (CDG). CDG patients are also short, are obese, and have abnormal glycan remodeling. Molecular analysis of fibroblasts from the family revealed normal levels of SEC23A in all cells and reduced levels of MAN1B1 in cells with heterozygous or homozygous mutations in SEC23A and MAN1B1. Secretion of pro-COL1A1 was increased in fibroblasts from the siblings and patients, and pro-COL1A1 was retained in Golgi of heterozygous and homozygous mutant cells, although intracellular pro-COL1A1 was increased in patient fibroblasts only. We postulate that increased pro-COL1A1 secretion is responsible for tall stature in these patients and an unaffected sibling, and not previously discovered in patients with mutations in either SEC23A or MAN1B1. The patients in this study share biochemical and cellular characteristics consistent with mutations in MAN1B1 and SEC23A, indicating a digenic disease. PMID:27148587

Modulating bacterial and gut mucosal interactions with engineered biofilm matrix proteins.

PubMed

Duraj-Thatte, Anna M; Praveschotinunt, Pichet; Nash, Trevor R; Ward, Frederick R; Joshi, Neel S

2018-02-22

Extracellular appendages play a significant role in mediating communication between bacteria and their host. Curli fibers are a class of bacterial fimbria that is highly amenable to engineering. We demonstrate the use of engineered curli fibers to rationally program interactions between bacteria and components of the mucosal epithelium. Commensal E. coli strains were engineered to produce recombinant curli fibers fused to the trefoil family of human cytokines. Biofilms formed from these strains bound more mucins than those producing wild-type curli fibers, and modulated mucin rheology as well. When treated with bacteria producing the curli-trefoil fusions mammalian cells behaved identically in terms of their migration behavior as when they were treated with the corresponding soluble trefoil factors. Overall, this demonstrates the potential utility of curli fibers as a scaffold for the display of bioactive domains and an untapped approach to rationally modulating host-microbe interactions using bacterial matrix proteins.

SEC sensor parametric test and evaluation system

NASA Technical Reports Server (NTRS)

1978-01-01

This system provides the necessary automated hardware required to carry out, in conjunction with the existing 70 mm SEC television camera, the sensor evaluation tests which are described in detail. The Parametric Test Set (PTS) was completed and is used in a semiautomatic data acquisition and control mode to test the development of the 70 mm SEC sensor, WX 32193. Data analysis of raw data is performed on the Princeton IBM 360-91 computer.

Motion of single MreB bacterial actin proteins in Caulobacter show treadmilling in vivo

NASA Astrophysics Data System (ADS)

Moerner, W. E.; Kim, Soyeon; Gitai, Zemer; Kinkhabwala, Anika; McAdams, Harley; Shapiro, Lucy

2006-03-01

Ensemble imaging of a bacterial actin homologue, the MreB protein, suggests that the MreB proteins form a dynamic filamentous spiral along the long axis of the cell in Caulobacter crescentus. MreB contracts and expands along the cell axis and plays an important role in cell shape and polarity maintenance, as well as chromosome segregation and translocation of the origin of replication during cell division. In this study we investigated the real-time polymerization of MreB in Caulobacter crescentus using single-molecule fluorescence imaging. With time-lapse imaging, polymerized MreB could be distinguished from cytoplasmic MreB monomers, because single monomeric MreB showed fast motion characteristic of Brownian diffusion, while single polymerized MreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer implies that treadmilling is the predominant mechanism in MreB filament formation. These single-molecule imaging experiments provide the first available information on the velocity of bacterial actin polymerization in a living cell.

Defense Against Cannibalism: The SdpI Family of Bacterial Immunity/Signal Transduction Proteins

PubMed Central

Povolotsky, Tatyana Leonidovna; Orlova, Ekaterina; Tamang, Dorjee G.

2010-01-01

The SdpI family consists of putative bacterial toxin immunity and signal transduction proteins. One member of the family in Bacillus subtilis, SdpI, provides immunity to cells from cannibalism in times of nutrient limitation. SdpI family members are transmembrane proteins with 3, 4, 5, 6, 7, 8, or 12 putative transmembrane α-helical segments (TMSs). These varied topologies appear to be genuine rather than artifacts due to sequencing or annotation errors. The basic and most frequently occurring element of the SdpI family has 6 TMSs. Homologues of all topological types were aligned to determine the homologous TMSs and loop regions, and the positive-inside rule was used to determine sidedness. The two most conserved motifs were identified between TMSs 1 and 2 and TMSs 4 and 5 of the 6 TMS proteins. These showed significant sequence similarity, leading us to suggest that the primordial precursor of these proteins was a 3 TMS–encoding genetic element that underwent intragenic duplication. Various deletional and fusional events, as well as intragenic duplications and inversions, may have yielded SdpI homologues with topologies of varying numbers and positions of TMSs. We propose a specific evolutionary pathway that could have given rise to these distantly related bacterial immunity proteins. We further show that genes encoding SdpI homologues often appear in operons with genes for homologues of SdpR, SdpI’s autorepressor. Our analyses allow us to propose structure–function relationships that may be applicable to most family members. Electronic supplementary material The online version of this article (doi:10.1007/s00232-010-9260-7) contains supplementary material, which is available to authorized users. PMID:20563570

Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family*

PubMed Central

Broussard, Tyler C.; Miller, Darcie J.; Jackson, Pamela; Nourse, Amanda; White, Stephen W.; Rock, Charles O.

2016-01-01

Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

Perturbation of bacterial ice nucleation activity by a grass antifreeze protein.

PubMed

Tomalty, Heather E; Walker, Virginia K

2014-09-26

Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9°C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Effect of ceramide-1-phosphate transfer protein on intestinal bacterial translocation in severe acute pancreatitis.

PubMed

Wang, Jiang; Li, Chang; Jiang, Yingjian; Zheng, Hongmei; Li, Dehui; Liang, Yibo; Deng, Wensheng; Zhang, Dianliang

2017-02-01

The aim of the study was to investigate the effects of ceramide-1-phosphate transfer protein (CPTP) on the intestinal epithelial tight junction proteins in patients with severe acute pancreatitis (SAP). Fifty patients with SAP were classified into two groups according to the presence of bacterial translocation (BT) in the blood. Thirty healthy individuals were included in the control group. The presence of BT was analyzed by polymerase chain reaction. The expression of tight junction proteins and CPTP was determined using immunohistochemistry and western blotting. Bacterial DNA was detected in the peripheral blood of 62.0% of the patients with SAP. The expression of CPTP and tight junction proteins in SAP patients was lower than that in healthy controls. Among the patients with SAP, those positive for BT(+) showed a lower level of CPTP and occluding (OC) and zonula occludens-1 (ZO-1) expression and a higher level of IVA cPLA2 expression than BT(-) patients. Moreover, the expression of CPTP was significantly associated with ZO-1 and showed a negative correlation with expression of IVA cPLA2 in SAP-BT(+) patients. CPTP affects the expression of tight junction proteins and may protects the intestinal epithelial barrier by downregulating the expression of IVA cPLA2. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

46 CFR Sec. 8 - Extra work and changes.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Extra work and changes. Sec. 8 Section 8 Shipping... Sec. 8 Extra work and changes. (a) At any time after the award of an original job order and during the time the work thereunder is being performed, additional or extra work or changes in the work covered by...

46 CFR Sec. 8 - Extra work and changes.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 8 2011-10-01 2011-10-01 false Extra work and changes. Sec. 8 Section 8 Shipping... Sec. 8 Extra work and changes. (a) At any time after the award of an original job order and during the time the work thereunder is being performed, additional or extra work or changes in the work covered by...

Comparative Genomic Analyses of the Bacterial Phosphotransferase System

PubMed Central

Barabote, Ravi D.; Saier, Milton H.

2005-01-01

We report analyses of 202 fully sequenced genomes for homologues of known protein constituents of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). These included 174 bacterial, 19 archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not identified in archaea or eukaryotes, showing that the horizontal transfer of genes encoding PTS proteins has not occurred between the three domains of life. Of the 174 bacterial genomes (136 bacterial species) analyzed, 30 diverse species have no PTS homologues, and 29 species have cytoplasmic PTS phosphoryl transfer protein homologues but lack recognizable PTS permeases. These soluble homologues presumably function in regulation. The remaining 77 species possess all PTS proteins required for the transport and phosphorylation of at least one sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS proteins. These homologues were analyzed for family association, range of protein types, domain organization, and organismal distribution. Different strains of a single bacterial species often possess strikingly different complements of PTS proteins. Types of PTS protein domain fusions were analyzed, showing that certain types of domain fusions are common, while others are rare or prohibited. Select PTS proteins were analyzed from different phylogenetic standpoints, showing that PTS protein phylogeny often differs from organismal phylogeny. The results document the frequent gain and loss of PTS protein-encoding genes and suggest that the lateral transfer of these genes within the bacterial domain has played an important role in bacterial evolution. Our studies provide insight into the development of complex multicomponent enzyme systems and lead to predictions regarding the types of protein-protein interactions that promote efficient PTS-mediated phosphoryl transfer. PMID:16339738

Selective molecular transport through the protein shell of a bacterial microcompartment organelle

DOE PAGES

Chowdhury, Chiranjit; Chun, Sunny; Pang, Allan; ...

2015-02-23

Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. In this paper, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux ofmore » propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Finally and overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.« less

Tuning membrane protein mobility by confinement into nanodomains

NASA Astrophysics Data System (ADS)

Karner, Andreas; Nimmervoll, Benedikt; Plochberger, Birgit; Klotzsch, Enrico; Horner, Andreas; Knyazev, Denis G.; Kuttner, Roland; Winkler, Klemens; Winter, Lukas; Siligan, Christine; Ollinger, Nicole; Pohl, Peter; Preiner, Johannes

2017-03-01

High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.

Meat, dairy and plant proteins alter bacterial composition of rat gut bacteria.

PubMed

Zhu, Yingying; Lin, Xisha; Zhao, Fan; Shi, Xuebin; Li, He; Li, Yingqiu; Zhu, Weiyun; Xu, Xinglian; Li, Chunbao; Lu, Chunbao; Zhou, Guanghong

2015-10-14

Long-term consumption of red meat has been considered a potential risk to gut health, but this is based on clinic investigations, excessive intake of fat, heme and some injurious compounds formed during cooking or additions to processed meat products. Whether intake of red meat protein affects gut bacteria and the health of the host remains unclear. In this work, we compared the composition of gut bacteria in the caecum, by sequencing the V4-V5 region of 16S ribosomal RNA gene, obtained from rats fed with proteins from red meat (beef and pork), white meat (chicken and fish) and other sources (casein and soy). The results showed significant differences in profiles of gut bacteria between the six diet groups. Rats fed with meat proteins had a similar overall structure of caecal bacterial communities separated from those fed non-meat proteins. The beneficial genus Lactobacillus was higher in the white meat than in the red meat or non-meat protein groups. Also, rats fed with meat proteins and casein had significantly lower levels of lipopolysaccharide-binding proteins, suggesting that the intake of meat proteins may maintain a more balanced composition of gut bacteria, thereby reducing the antigen load and inflammatory response in the host.

Meat, dairy and plant proteins alter bacterial composition of rat gut bacteria

PubMed Central

Zhu, Yingying; Lin, Xisha; Zhao, Fan; Shi, Xuebin; Li, He; Li, Yingqiu; Zhu, Weiyun; Xu, Xinglian; Lu, Chunbao; Zhou, Guanghong

2015-01-01

Long-term consumption of red meat has been considered a potential risk to gut health, but this is based on clinic investigations, excessive intake of fat, heme and some injurious compounds formed during cooking or additions to processed meat products. Whether intake of red meat protein affects gut bacteria and the health of the host remains unclear. In this work, we compared the composition of gut bacteria in the caecum, by sequencing the V4-V5 region of 16S ribosomal RNA gene, obtained from rats fed with proteins from red meat (beef and pork), white meat (chicken and fish) and other sources (casein and soy). The results showed significant differences in profiles of gut bacteria between the six diet groups. Rats fed with meat proteins had a similar overall structure of caecal bacterial communities separated from those fed non-meat proteins. The beneficial genus Lactobacillus was higher in the white meat than in the red meat or non-meat protein groups. Also, rats fed with meat proteins and casein had significantly lower levels of lipopolysaccharide-binding proteins, suggesting that the intake of meat proteins may maintain a more balanced composition of gut bacteria, thereby reducing the antigen load and inflammatory response in the host. PMID:26463271

Bacterial Iron–Sulfur Regulatory Proteins As Biological Sensor-Switches

PubMed Central

Crack, Jason C.; Green, Jeffrey; Hutchings, Matthew I.; Thomson, Andrew J.

2012-01-01

Abstract Significance: In recent years, bacterial iron–sulfur cluster proteins that function as regulators of gene transcription have emerged as a major new group. In all cases, the cluster acts as a sensor of the environment and enables the organism to adapt to the prevailing conditions. This can range from mounting a response to oxidative or nitrosative stress to switching between anaerobic and aerobic respiratory pathways. The sensitivity of these ancient cofactors to small molecule reactive oxygen and nitrogen species, in particular, makes them ideally suited to function as sensors. Recent Advances: An important challenge is to obtain mechanistic and structural information about how these regulators function and, in particular, how the chemistry occurring at the cluster drives the subsequent regulatory response. For several regulators, including FNR, SoxR, NsrR, IscR, and Wbl proteins, major advances in understanding have been gained recently and these are reviewed here. Critical Issues: A common theme emerging from these studies is that the sensitivity and specificity of the cluster of each regulatory protein must be exquisitely controlled by the protein environment of the cluster. Future Directions: A major future challenge is to determine, for a range of regulators, the key factors for achieving control of sensitivity/specificity. Such information will lead, eventually, to a system understanding of stress response, which often involves more than one regulator. Antioxid. Redox Signal. 17, 1215–1231. PMID:22239203

Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

PubMed

Merchant, Mark; Kinney, Clint; Sanders, Paige

2009-12-01

Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

46 CFR Sec. 2 - Terms.

Code of Federal Regulations, 2013 CFR

2013-10-01

... PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 2 Terms. The terms employed in this order shall have the same meaning as those contained in NSA Order No. 47. ...

46 CFR Sec. 2 - Terms.

Code of Federal Regulations, 2011 CFR

2011-10-01

... PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 2 Terms. The terms employed in this order shall have the same meaning as those contained in NSA Order No. 47. ...

46 CFR Sec. 2 - Terms.

Code of Federal Regulations, 2012 CFR

2012-10-01

... PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 2 Terms. The terms employed in this order shall have the same meaning as those contained in NSA Order No. 47. ...

46 CFR Sec. 2 - Terms.

Code of Federal Regulations, 2014 CFR

2014-10-01

... PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 2 Terms. The terms employed in this order shall have the same meaning as those contained in NSA Order No. 47. ...

46 CFR Sec. 2 - Terms.

Code of Federal Regulations, 2010 CFR

2010-10-01

... PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 2 Terms. The terms employed in this order shall have the same meaning as those contained in NSA Order No. 47. ...

The participation of outer membrane proteins in the bacterial sensitivity to nanosilver.

PubMed

Kędziora, Anna; Krzyżewska, Eva; Dudek, Bartłomiej; Bugla-Płoskońska, Gabriela

2016-06-13

The presented study is to analyze the participation of outer membrane proteins of Gram- negative bacteria in sensitivity to silver nanomaterials. The mechanism of interaction of silver with the bacterial cell is best described in this group of microorganisms. There are several theories regarding the effectiveness of antimicrobial ions and nanosilver, and at the indicated differences in the way they work. Outer membrane proteins of Gram-negative bacteria are involved in the procurement of silver from the environment and contribute to the development mechanisms of resistance to nanometals. They are measurable parameter in the field of cell phenotypic response to the presence of Gram-negative bacteria in the environment silver nanoforms: its properties, chemical composition, content or times of action. Proteomic methods (including two dimensional electrophoresis and MALDI‑TOF MS) are therefore relevant techniques for determining the susceptibility of bacteria to silver and the changes taking place in the outer membrane under the influence: uptime/exposure and physical and chemical parameters of silver nanomaterials. Many products containing nanosilver is still in the research phase in terms of physico‑chemical characteristics and biological activity, others have been already implemented in many industries. During the very fast nanotechnology developing and introduction to the market products based on the nanosilver the bacterial answer to nanosilver is needed.

Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maruyama, Yukie; Ochiai, Akihito; Mikami, Bunzo

Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit.more » Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.« less

Soy protein films for wound-healing applications: antibiotic release, bacterial inhibition and cellular response.

PubMed

Peles, Zachi; Binderman, Itzhak; Berdicevsky, Israela; Zilberman, Meital

2013-05-01

Use of naturally derived materials is becoming widespread in the biomedical field. Soy protein has advantages over the various types of natural proteins employed for biomedical applications, due to its low price, non-animal origin and relatively long storage time and stability. In the current study, soy protein isolate (SPI) was investigated as a matrix for wound-dressing applications. The antibiotic drug gentamicin was incorporated into the matrix for local controlled release and thus continuous bactericidal effect. Homogeneous high-quality films were cast from aqueous solutions and tested for the effects of gentamicin release on bacterial inhibition. The cytotoxicity and in vitro biocompatibility of these films were also examined. The gentamicin release profiles exhibited a moderate burst effect followed by a decreasing release rate, which was maintained for at least 4 weeks, thus enabling a suitable bacterial inhibition effect. The materials released from the films during an indirect cytotoxicity test were found to be safe, except for a slight inhibitory effect in the presence of high concentrations of glycerol. The biocompatibility test showed confluent cell cultures in close proximity to the SPI films. It is clear that these new antibiotic-eluting SPI films exhibit a high potential for use as wound dressings. Copyright © 2012 John Wiley & Sons, Ltd.

Emerging therapeutic delivery capabilities and challenges utilizing enzyme/protein packaged bacterial vesicles.

PubMed

Alves, Nathan J; Turner, Kendrick B; Medintz, Igor L; Walper, Scott A

2015-07-01

Nanoparticle-based therapeutics are poised to play a critical role in treating disease. These complex multifunctional drug delivery vehicles provide for the passive and active targeted delivery of numerous small molecule, peptide and protein-derived pharmaceuticals. This article will first discuss some of the current state of the art nanoparticle classes (dendrimers, lipid-based, polymeric and inorganic), highlighting benefits/drawbacks associated with their implementation. We will then discuss an emerging class of nanoparticle therapeutics, bacterial outer membrane vesicles, that can provide many of the nanoparticle benefits while simplifying assembly. Through molecular biology techniques; outer membrane vesicle hijacking potentially allows for stringent control over nanoparticle production allowing for targeted protein packaged nanoparticles to be fully synthesized by bacteria.

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

PubMed

Dröge, Melloney J; Boersma, Ykelien L; Braun, Peter G; Buining, Robbert Jan; Julsing, Mattijs K; Selles, Karin G A; van Dijl, Jan Maarten; Quax, Wim J

2006-07-01

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

14 CFR Sec. 19-7 - Passenger origin-destination survey.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Passenger origin-destination survey. Sec... AIR CARRIERS Operating Statistics Classifications Sec. 19-7 Passenger origin-destination survey. (a... carriers) shall participate in a Passenger Origin-Destination (O & D) Survey covering domestic and...

14 CFR Sec. 19-7 - Passenger origin-destination survey.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Passenger origin-destination survey. Sec... AIR CARRIERS Operating Statistics Classifications Sec. 19-7 Passenger origin-destination survey. (a... carriers) shall participate in a Passenger Origin-Destination (O & D) Survey covering domestic and...

The Protein Interactome of Streptococcus pneumoniae and Bacterial Meta-interactomes Improve Function Predictions.

PubMed

Wuchty, S; Rajagopala, S V; Blazie, S M; Parrish, J R; Khuri, S; Finley, R L; Uetz, P

2017-01-01

The functions of roughly a third of all proteins in Streptococcus pneumoniae , a significant human-pathogenic bacterium, are unknown. Using a yeast two-hybrid approach, we have determined more than 2,000 novel protein interactions in this organism. We augmented this network with meta-interactome data that we defined as the pool of all interactions between evolutionarily conserved proteins in other bacteria. We found that such interactions significantly improved our ability to predict a protein's function, allowing us to provide functional predictions for 299 S. pneumoniae proteins with previously unknown functions. IMPORTANCE Identification of protein interactions in bacterial species can help define the individual roles that proteins play in cellular pathways and pathogenesis. Very few protein interactions have been identified for the important human pathogen S. pneumoniae . We used an experimental approach to identify over 2,000 new protein interactions for S. pneumoniae , the most extensive interactome data for this bacterium to date. To predict protein function, we used our interactome data augmented with interactions from other closely related bacteria. The combination of the experimental data and meta-interactome data significantly improved the prediction results, allowing us to assign possible functions to a large number of poorly characterized proteins.

Phosphatidic Acid Sequesters Sec18p from cis-SNARE Complexes to Inhibit Priming.

PubMed

Starr, Matthew L; Hurst, Logan R; Fratti, Rutilio A

2016-10-01

Yeast vacuole fusion requires the activation of cis-SNARE complexes through priming carried out by Sec18p/N-ethylmaleimide sensitive factor and Sec17p/α-SNAP. The association of Sec18p with vacuolar cis-SNAREs is regulated in part by phosphatidic acid (PA) phosphatase production of diacylglycerol (DAG). Inhibition of PA phosphatase activity blocks the transfer of membrane-associated Sec18p to SNAREs. Thus, we hypothesized that Sec18p associates with PA-rich membrane microdomains before transferring to cis-SNARE complexes upon PA phosphatase activity. Here, we examined the direct binding of Sec18p to liposomes containing PA or DAG. We found that Sec18p preferentially bound to liposomes containing PA compared with those containing DAG by approximately fivefold. Additionally, using a specific PA-binding domain blocked Sec18p binding to PA-liposomes and displaced endogenous Sec18p from isolated vacuoles. Moreover, the direct addition of excess PA blocked the priming activity of isolated vacuoles in a manner similar to chemically inhibiting PA phosphatase activity. These data suggest that the conversion of PA to DAG facilitates the recruitment of Sec18p to cis-SNAREs. Purified vacuoles from yeast lacking the PA phosphatase Pah1p showed reduced Sec18p association with cis-SNAREs and complementation with plasmid-encoded PAH1 or recombinant Pah1p restored the interaction. Taken together, this demonstrates that regulating PA concentrations by Pah1p activity controls SNARE priming by Sec18p. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

46 CFR Sec. 5 - Measures to protect ship's payrolls.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 8 2012-10-01 2012-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to...

Bacterial actin MreB assembles in complex with cell shape protein RodZ.

PubMed

van den Ent, Fusinita; Johnson, Christopher M; Persons, Logan; de Boer, Piet; Löwe, Jan

2010-03-17

Bacterial actin homologue MreB is required for cell shape maintenance in most non-spherical bacteria, where it assembles into helical structures just underneath the cytoplasmic membrane. Proper assembly of the actin cytoskeleton requires RodZ, a conserved, bitopic membrane protein that colocalises to MreB and is essential for cell shape determination. Here, we present the first crystal structure of bacterial actin engaged with a natural partner and provide a clear functional significance of the interaction. We show that the cytoplasmic helix-turn-helix motif of Thermotoga maritima RodZ directly interacts with monomeric as well as filamentous MreB and present the crystal structure of the complex. In vitro and in vivo analyses of mutant T. maritima and Escherichia coli RodZ validate the structure and reveal the importance of the MreB-RodZ interaction in the ability of cells to propagate as rods. Furthermore, the results elucidate how the bacterial actin cytoskeleton might be anchored to the membrane to help constrain peptidoglycan synthesis in the periplasm.

The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

PubMed

Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

2014-12-18

Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

OsSNDP1, a Sec14-nodulin domain-containing protein, plays a critical role in root hair elongation in rice.

PubMed

Huang, Jin; Kim, Chul Min; Xuan, Yuan-hu; Park, Soon Ju; Piao, Hai Long; Je, Byoung Il; Liu, Jingmiao; Kim, Tae Ho; Kim, Bo-Kyeong; Han, Chang-Deok

2013-05-01

Rice is cultivated in water-logged paddy lands. Thus, rice root hairs on the epidermal layers are exposed to a different redox status of nitrogen species, organic acids, and metal ions than root hairs growing in drained soil. To identify genes that play an important role in root hair growth, a forward genetics approach was used to screen for short-root-hair mutants. A short-root-hair mutant was identified and isolated by using map-based cloning and sequencing. The mutation arose from a single amino acid substitution of OsSNDP1 (Oryza sativa Sec14-nodulin domain protein), which shows high sequence homology with Arabidopsis COW1/AtSFH1 and encodes a phosphatidylinositol transfer protein (PITP). By performing complementation assays with Atsfh1 mutants, we demonstrated that OsSNDP1 is involved in growth of root hairs. Cryo-scanning electron microscopy was utilized to further characterize the effect of the Ossndp1 mutation on root hair morphology. Aberrant morphogenesis was detected in root hair elongation and maturation zones. Many root hairs were branched and showed irregular shapes due to bulged nodes. Many epidermal cells also produced dome-shaped root hairs, which indicated that root hair elongation ceased at an early stage. These studies showed that PITP-mediated phospholipid signaling and metabolism is critical for root hair elongation in rice.

Growth performance and carcase quality in broiler chickens fed on bacterial protein grown on natural gas.

PubMed

Øverland, M; Schøyen, H F; Skrede, A

2010-10-01

1. The effects of increasing concentrations (0, 40, 80 or 120 g/kg) of bacterial protein meal (BPM) and bacterial protein autolysate (BPA) grown on natural gas on growth performance and carcase quality in broiler chickens were examined. 2. Adding BPM to diets reduced feed intake and improved gain: feed from 0 to 21 d and overall to 35 d, but did not significantly affect weight gain compared to the soybean meal based control diet. 3. Increasing concentrations of BPA significantly reduced growth rate, feed intake, gain: feed, carcase weight and dressing percentage, but significantly increased carcase dry matter, fat and energy content. 4. Adding BPM to diets had no effect on viscosity of diets and jejunal digesta, and minor effects on litter quality, whereas BPA increased the viscosity of diets and jejunal digesta, improved litter quality at 21 d, but decreased litter quality at 32 d. 5. To conclude, broiler chickens performed better on a BPM product with intact proteins than on an autolysate with ruptured cell walls and a high content of free amino acids and low molecular-weight peptides.

STUDIES ON THE BACTERIOPHAGE OF D'HERELLE : IX. EVIDENCE OF HYDROLYSIS OF BACTERIAL PROTEIN DURING LYSIS.

PubMed

Hetler, D M; Bronfenbrenner, J

1928-07-31

1. During the process of lysis by bacteriophage, there is an appreciable increase in the amount of free amino acid present in the culture. 2. The increase of free amino acid is due to hydrolysis of bacterial protein.

17 CFR 200.80b - Appendix B-SEC releases.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 17 Commodity and Securities Exchanges 3 2014-04-01 2014-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION... corporate reorganization releases, and the litigation releases are contained in the SEC Docket, which may be...

17 CFR 200.80b - Appendix B-SEC releases.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 17 Commodity and Securities Exchanges 2 2013-04-01 2013-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION... corporate reorganization releases, and the litigation releases are contained in the SEC Docket, which may be...

17 CFR 200.80b - Appendix B-SEC releases.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION... corporate reorganization releases, and the litigation releases are contained in the SEC Docket, which may be...

17 CFR 200.80b - Appendix B-SEC releases.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 2 2011-04-01 2011-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION... corporate reorganization releases, and the litigation releases are contained in the SEC Docket, which may be...

17 CFR 200.80b - Appendix B-SEC releases.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 17 Commodity and Securities Exchanges 2 2012-04-01 2012-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION... corporate reorganization releases, and the litigation releases are contained in the SEC Docket, which may be...

14 CFR Sec. 2-5 - Revenue and accounting practices.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Revenue and accounting practices. Sec. 2-5... General Accounting Provisions Sec. 2-5 Revenue and accounting practices. (a) Revenue accounting practices... physically verify the reliability of its passenger revenue accounting practice at least once each accounting...

The Protein Interactome of Streptococcus pneumoniae and Bacterial Meta-interactomes Improve Function Predictions

PubMed Central

Rajagopala, S. V.; Blazie, S. M.; Parrish, J. R.; Khuri, S.; Finley, R. L.

2017-01-01

ABSTRACT The functions of roughly a third of all proteins in Streptococcus pneumoniae, a significant human-pathogenic bacterium, are unknown. Using a yeast two-hybrid approach, we have determined more than 2,000 novel protein interactions in this organism. We augmented this network with meta-interactome data that we defined as the pool of all interactions between evolutionarily conserved proteins in other bacteria. We found that such interactions significantly improved our ability to predict a protein’s function, allowing us to provide functional predictions for 299 S. pneumoniae proteins with previously unknown functions. IMPORTANCE Identification of protein interactions in bacterial species can help define the individual roles that proteins play in cellular pathways and pathogenesis. Very few protein interactions have been identified for the important human pathogen S. pneumoniae. We used an experimental approach to identify over 2,000 new protein interactions for S. pneumoniae, the most extensive interactome data for this bacterium to date. To predict protein function, we used our interactome data augmented with interactions from other closely related bacteria. The combination of the experimental data and meta-interactome data significantly improved the prediction results, allowing us to assign possible functions to a large number of poorly characterized proteins. PMID:28744484

Effects of Trx2p and Sec23p expression on stable production of hepatitis B surface antigen S domain in recombinant Saccharomyces cerevisiae.

PubMed

Park, Young-Kyoung; Jung, Sang-Min; Lim, Hyung-Kwon; Son, Young-Jin; Park, Yong-Cheol; Seo, Jin-Ho

2012-08-31

The S domain of hepatitis B virus surface antigen (sHBsAg) is the primary component for vaccine development against virus infection. For stable expression of sHBsAg in recombinant Saccharomyces cerevisiae, new accessory genes necessary for foreign protein expression were screened by DNA microarray. Among 600 genes of interest, genes coding for an activating protein of ATPase in Hsp90 (Aha1p), S. cerevisiae DnaJ (Scj1p), thioredoxin 2 (Trx2p) and a GTPase-activator specific for Sar1 (Sec23p) as well as Pdi1p were selected in transcriptome analysis, which are known to facilitate disulfide bond formation or induce protein transport in the secretion pathway. Individual and combinatorial expression of SEC23, TRX2 and PDI1 increased total sHBsAg concentration by 1.9-6.5-fold, relative to the control strain expressing sHBsAg only. Additionally, moderate expression of Kex2p protease able to cut off the signal peptide enhanced the portion of the authentic sHBsAg to total sHBsAg. Fed-batch fermentation of the S. cerevisiae 2805 strain coexpressing the sHBsAg, SEC23, PDI1 and KEX2 genes resulted in 70.6mg/L final sHBsAg concentration which was 5.6 times higher than that of the control. Transmission electron microscopic analysis of the yeast cells elucidated the effects of the accessory gene coexpression on the intracellular localization of sHBsAg. Like PDI1, coexpression of both SEC23 and/or TRX2 newly isolated in this study is expected to improve the target protein expression in S. cerevisiae. Copyright © 2012 Elsevier B.V. All rights reserved.

New Type of Antimicrobial Protein Produced by the Plant Pathogen Clavibacter michiganensis subsp. michiganensis

PubMed Central

Liu, Zhanliang; Ma, Ping; Holtsmark, Ingrid; Skaugen, Morten; Eijsink, Vincent G. H.

2013-01-01

It has previously been shown that the tomato pathogen Clavibacter michiganensis subsp. michiganensis secretes a 14-kDa protein, C. michiganensis subsp. michiganensis AMP-I (CmmAMP-I), that inhibits growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato. Using sequences obtained from tryptic fragments, we have identified the gene encoding CmmAMP-I and we have recombinantly produced the protein with an N-terminal intein tag. The gene sequence showed that CmmAMP-I contains a typical N-terminal signal peptide for Sec-dependent secretion. The recombinant protein was highly active, with 50% growth inhibition (IC50) of approximately 10 pmol, but was not toxic to potato leaves or tubers. CmmAMP-I does not resemble any known protein and thus represents a completely new type of bacteriocin. Due to its high antimicrobial activity and its very narrow inhibitory spectrum, CmmAMP-1 may be of interest in combating potato ring rot disease. PMID:23851100

ssaD1, a suppressor of secA51(Ts) that renders growth of Escherichia coli cold sensitive, is an early amber mutation in the transcription factor gene nusB.

PubMed Central

Rajapandi, T.; Oliver, D.

1994-01-01

Complementation analysis of the ssaD1 mutation, isolated as a suppressor of the secA51(Ts) mutation that renders growth of Escherichia coli cold sensitive, was used to show that ssaD corresponds to nusB, a gene known to be important in transcription antitermination. DNA sequence analysis of the ssaD1 allele showed that it creates an amber mutation in the 15th codon of nusB. Analysis of the effect of different levels of NusB protein on secA transcription and translation suggested that NusB plays little or no role in the control of secA expression. Accordingly, mechanisms by which nusB inactivation can lead to suppression of secA51(Ts) and secY24(Ts) mutations without affecting secA expression need to be considered. PMID:8021230

A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts

PubMed Central

Galperin, Michael Y

2005-01-01

Background Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. Results This paper presents results of a comprehensive census of signal transduction proteins – histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases – encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. Conclusion The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set) can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the

A census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial IQ, extroverts and introverts.

PubMed

Galperin, Michael Y

2005-06-14

Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. This paper presents results of a comprehensive census of signal transduction proteins--histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases--encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set) can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the current leader Wolinella succinogenes

46 CFR Sec. 2 - Description of NSA-WORKSMALREP Contract.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 8 2013-10-01 2013-10-01 false Description of NSA-WORKSMALREP Contract. Sec. 2 Section... REPAIRS-NSA-WORKSMALREP Sec. 2 Description of NSA-WORKSMALREP Contract. This is an individual fixed price contract which may be awarded to any firm not holding an NSA-LUMPSUMREP Contract, as a result of formal...

46 CFR Sec. 2 - Description of NSA-WORKSMALREP Contract.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 8 2012-10-01 2012-10-01 false Description of NSA-WORKSMALREP Contract. Sec. 2 Section... REPAIRS-NSA-WORKSMALREP Sec. 2 Description of NSA-WORKSMALREP Contract. This is an individual fixed price contract which may be awarded to any firm not holding an NSA-LUMPSUMREP Contract, as a result of formal...

46 CFR Sec. 2 - Description of NSA-WORKSMALREP Contract.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 8 2014-10-01 2014-10-01 false Description of NSA-WORKSMALREP Contract. Sec. 2 Section... REPAIRS-NSA-WORKSMALREP Sec. 2 Description of NSA-WORKSMALREP Contract. This is an individual fixed price contract which may be awarded to any firm not holding an NSA-LUMPSUMREP Contract, as a result of formal...

46 CFR Sec. 2 - Description of NSA-WORKSMALREP Contract.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 8 2011-10-01 2011-10-01 false Description of NSA-WORKSMALREP Contract. Sec. 2 Section... REPAIRS-NSA-WORKSMALREP Sec. 2 Description of NSA-WORKSMALREP Contract. This is an individual fixed price contract which may be awarded to any firm not holding an NSA-LUMPSUMREP Contract, as a result of formal...

Financial Journalism under Fire: The SEC and Newsroom Ethics.

ERIC Educational Resources Information Center

Spellman, Robert L.

Although noting that the Securities and Exchange Commission (SEC) has been a valuable ally of journalists, this paper suggests that recent efforts of the SEC in prosecuting the case of R. Foster Winans, Jr., a former writer for the "Wall Street Journal," may be unconstitutional. Following an introduction to the First Amendment issues…

Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins.

PubMed

Chaikam, Vijay; Karlson, Dale T

2010-01-01

The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs. [BMB reports 2010; 43(1): 1-8].

De novo generation of infectious prions with bacterially expressed recombinant prion protein.

PubMed

Zhang, Zhihong; Zhang, Yi; Wang, Fei; Wang, Xinhe; Xu, Yuanyuan; Yang, Huaiyi; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

2013-12-01

The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

46 CFR Sec. 5 - Measures to protect ship's payrolls.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5... SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to... paying off the crew should be either the Master, or purser, or some other member of the ship's personnel...

46 CFR Sec. 5 - Measures to protect ship's payrolls.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 8 2011-10-01 2011-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5... SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to... paying off the crew should be either the Master, or purser, or some other member of the ship's personnel...

46 CFR Sec. 5 - Measures to protect ship's payrolls.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 8 2014-10-01 2014-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5... SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to... paying off the crew should be either the Master, or purser, or some other member of the ship's personnel...

46 CFR Sec. 5 - Measures to protect ship's payrolls.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 8 2013-10-01 2013-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5... SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to... paying off the crew should be either the Master, or purser, or some other member of the ship's personnel...

Identification of cyclin B1 and Sec62 as biomarkers for recurrence in patients with HBV-related hepatocellular carcinoma after surgical resection.

PubMed

Weng, Li; Du, Juan; Zhou, Qinghui; Cheng, Binbin; Li, Jun; Zhang, Denghai; Ling, Changquan

2012-06-08

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Frequent tumor recurrence after surgery is related to its poor prognosis. Although gene expression signatures have been associated with outcome, the molecular basis of HCC recurrence is not fully understood, and there is no method to predict recurrence using peripheral blood mononuclear cells (PBMCs), which can be easily obtained for recurrence prediction in the clinical setting. According to the microarray analysis results, we constructed a co-expression network using the k-core algorithm to determine which genes play pivotal roles in the recurrence of HCC associated with the hepatitis B virus (HBV) infection. Furthermore, we evaluated the mRNA and protein expressions in the PBMCs from 80 patients with or without recurrence and 30 healthy subjects. The stability of the signatures was determined in HCC tissues from the same 80 patients. Data analysis included ROC analysis, correlation analysis, log-lank tests, and Cox modeling to identify independent predictors of tumor recurrence. The tumor-associated proteins cyclin B1, Sec62, and Birc3 were highly expressed in a subset of samples of recurrent HCC; cyclin B1, Sec62, and Birc3 positivity was observed in 80%, 65.7%, and 54.2% of the samples, respectively. The Kaplan-Meier analysis revealed that high expression levels of these proteins was associated with significantly reduced recurrence-free survival. Cox proportional hazards model analysis revealed that cyclin B1 (hazard ratio [HR], 4.762; p = 0.002) and Sec62 (HR, 2.674; p = 0.018) were independent predictors of HCC recurrence. These results revealed that cyclin B1 and Sec62 may be candidate biomarkers and potential therapeutic targets for HBV-related HCC recurrence after surgery.

Iron metabolism mutant hbd mice have a deletion in Sec15l1, which has homology to a yeast gene for vesicle docking.

PubMed

White, Robert A; Boydston, Leigh A; Brookshier, Terri R; McNulty, Steven G; Nsumu, Ndona N; Brewer, Brandon P; Blackmore, Krista

2005-12-01

Defects in iron absorption and utilization lead to iron deficiency and anemia. While iron transport by transferrin receptor-mediated endocytosis is well understood, it is not completely clear how iron is transported from the endosome to the mitochondria where heme is synthesized. We undertook a positional cloning project to identify the causative mutation for the hemoglobin-deficit (hbd) mouse mutant, which suffers from a microcytic, hypochromic anemia apparently due to defective iron transport in the endocytosis cycle. As shown by previous studies, reticulocyte iron accumulation in homozygous hbd/hbd mice is deficient despite normal binding of transferrin to its receptor and normal transferrin uptake in the cell. We have identified a strong candidate gene for hbd, Sec15l1, a homologue to yeast SEC15, which encodes a key protein in vesicle docking. The hbd mice have an exon deletion in Sec15l1, which is the first known mutation of a SEC gene homologue in mammals.

46 CFR Sec. 11 - Guarantee obligations.

Code of Federal Regulations, 2013 CFR

2013-10-01

... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP Contract...

46 CFR Sec. 11 - Guarantee obligations.

Code of Federal Regulations, 2012 CFR

2012-10-01

... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP Contract...

46 CFR Sec. 11 - Guarantee obligations.

Code of Federal Regulations, 2014 CFR

2014-10-01

... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP Contract...

46 CFR Sec. 11 - Guarantee obligations.

Code of Federal Regulations, 2011 CFR

2011-10-01

... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP Contract...

Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes

NASA Astrophysics Data System (ADS)

Shen, Hsin-Hui; Leyton, Denisse L.; Shiota, Takuya; Belousoff, Matthew J.; Noinaj, Nicholas; Lu, Jingxiong; Holt, Stephen A.; Tan, Khershing; Selkrig, Joel; Webb, Chaille T.; Buchanan, Susan K.; Martin, Lisandra L.; Lithgow, Trevor

2014-10-01

In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

46 CFR Sec. 10 - Bonds.

Code of Federal Regulations, 2010 CFR

2010-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form 25...

46 CFR Sec. 10 - Bonds.

Code of Federal Regulations, 2011 CFR

2011-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form 25...

46 CFR Sec. 10 - Bonds.

Code of Federal Regulations, 2012 CFR

2012-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form 25...

46 CFR Sec. 10 - Bonds.

Code of Federal Regulations, 2014 CFR

2014-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form 25...

46 CFR Sec. 10 - Bonds.

Code of Federal Regulations, 2013 CFR

2013-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form 25...

Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

PubMed

Nisa-Martínez, Rafael; Laporte, Philippe; Jiménez-Zurdo, José Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolás

2013-01-01

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

PubMed Central

Nisa-Martínez, Rafael; Laporte, Philippe; Jiménez-Zurdo, José Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolás

2013-01-01

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns. PMID:24391881

pLoc-mGneg: Predict subcellular localization of Gram-negative bacterial proteins by deep gene ontology learning via general PseAAC.

PubMed

Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

2017-10-06

Information of the proteins' subcellular localization is crucially important for revealing their biological functions in a cell, the basic unit of life. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to develop computational tools for timely identifying their subcellular locations based on the sequence information alone. The current study is focused on the Gram-negative bacterial proteins. Although considerable efforts have been made in protein subcellular prediction, the problem is far from being solved yet. This is because mounting evidences have indicated that many Gram-negative bacterial proteins exist in two or more location sites. Unfortunately, most existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions important for both basic research and drug design. In this study, by using the multi-label theory, we developed a new predictor called "pLoc-mGneg" for predicting the subcellular localization of Gram-negative bacterial proteins with both single and multiple locations. Rigorous cross-validation on a high quality benchmark dataset indicated that the proposed predictor is remarkably superior to "iLoc-Gneg", the state-of-the-art predictor for the same purpose. For the convenience of most experimental scientists, a user-friendly web-server for the novel predictor has been established at http://www.jci-bioinfo.cn/pLoc-mGneg/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. Copyright © 2017 Elsevier Inc. All rights reserved.

Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

PubMed Central

Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

2016-01-01

Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

75 FR 44781 - Joint CFTC-SEC Advisory Committee on Emerging Regulatory Issues

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-29

...; File No. 265-26] Joint CFTC-SEC Advisory Committee on Emerging Regulatory Issues AGENCY: Commodity... Meeting of Joint CFTC-SEC Advisory Committee on Emerging Regulatory Issues. SUMMARY: The Joint CFTC-SEC Advisory Committee on Emerging Regulatory Issues will hold a public meeting on August 11, 2010, from 9 a.m...

75 FR 66362 - Joint CFTC-SEC Advisory Committee on Emerging Regulatory Issues

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-28

...; File No. 265-26] Joint CFTC-SEC Advisory Committee on Emerging Regulatory Issues AGENCY: Commodity... meeting of Joint CFTC-SEC Advisory Committee on Emerging Regulatory Issues. SUMMARY: The Joint CFTC-SEC Advisory Committee on Emerging Regulatory Issues will hold a public meeting on November 5, 2010, from 9 a.m...

Yeast syntaxins Sso1p and Sso2p belong to a family of related membrane proteins that function in vesicular transport.

PubMed Central

Aalto, M K; Ronne, H; Keränen, S

1993-01-01

The yeast SEC1 gene encodes a hydrophilic protein that functions at the terminal stage in secretion. We have cloned two yeast genes, SSO1 and SSO2, which in high copy number can suppress sec1 mutations and also mutations in several other late acting SEC genes, such as SEC3, SEC5, SEC9 and SEC15. SSO1 and SSO2 encode small proteins with N-terminal hydrophilic domains and C-terminal hydrophobic tails. The two proteins are 72% identical in sequence and together perform an essential function late in secretion. Sso1p and Sso2p show significant sequence similarity to six other proteins. Two of these, Sed5p and Pep12p, are yeast proteins that function in transport from ER to Golgi and from Golgi to the vacuole, respectively. Also related to Sso1p and Sso2p are three mammalian proteins: epimorphin, syntaxin A/HPC-1 and syntaxin B. A nematode cDNA product also belongs to the new protein family. The new protein family is thus present in a wide variety of eukaryotic cells, where its members function at different stages in vesicular transport. Images PMID:8223426

Serum C-reactive protein as a diagnostic biomarker in dogs with bacterial respiratory diseases.

PubMed

Viitanen, S J; Laurila, H P; Lilja-Maula, L I; Melamies, M A; Rantala, M; Rajamäki, M M

2014-01-01

C-reactive protein (CRP) is a major acute-phase protein in dogs. Serum concentrations are low in healthy animals, but increase rapidly after inflammatory stimuli. The aim of the study was to investigate CRP concentrations in various respiratory diseases of dogs and to determine if CRP can be used as a biomarker in the diagnosis of bacterial respiratory diseases. A total of 106 privately owned dogs with respiratory diseases (17 with bacterial tracheobronchitis [BTB], 20 with chronic bronchitis [CB], 20 with eosinophilic bronchopneumopathy [EBP], 12 with canine idiopathic pulmonary fibrosis [CIPF], 15 with cardiogenic pulmonary edema [CPE], and 22 with bacterial pneumonia [BP]) and 72 healthy controls. The study was conducted as a prospective cross-sectional observational study. CRP was measured in serum samples. Diagnosis was confirmed by clinical and laboratory findings, diagnostic imaging, and selected diagnostic methods such as cytological and microbiological analysis of respiratory samples, echocardiography, and histopathology. Dogs with BP had significantly higher CRP concentrations (median, 121 mg/L; interquartile range, 68-178 mg/L) than dogs with BTB (23, 15-38, P = .0003), CB (13, 8-14, P < .0001), EBP (5, 5-15, P < .0001), CIPF (17, 10-20, P < .0001), or CPE (19, 13-32, P < .0001) and healthy controls (14, 8-20, P < .0001). Dogs with BTB had significantly higher CRP concentrations than dogs with CB (P = .001) or EBP (P < .0001) and healthy controls (P = .029). These results indicate that CRP has potential for use as an additional biomarker, especially in the diagnostics of BP. Copyright © 2013 by the American College of Veterinary Internal Medicine.

Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms.

PubMed

Randrianjatovo, I; Girbal-Neuhauser, E; Marcato-Romain, C-E

2015-06-01

Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7 μg to as high as 50 μg per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250 μg ml(-1)) showed little or no sugar interference up to 2000 μg ml(-1), thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.1 ± 3.1, 38.3 ± 7.1 and 0.3 ± 0.1 μg equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms.

Putative bacterial volatile-mediated growth in soybean (Glycine max L. Merrill) and expression of induced proteins under salt stress.

PubMed

Vaishnav, A; Kumari, S; Jain, S; Varma, A; Choudhary, D K

2015-08-01

Plant root-associated rhizobacteria elicit plant immunity referred to as induced systemic tolerance (IST) against multiple abiotic stresses. Among multibacterial determinants involved in IST, the induction of IST and promotion of growth by putative bacterial volatile compounds (VOCs) is reported in the present study. To characterize plant proteins induced by putative bacterial VOCs, proteomic analysis was performed by MALDI-MS/MS after exposure of soybean seedlings to a new strain of plant growth promoting rhizobacteria (PGPR) Pseudomonas simiae strain AU. Furthermore, expression analysis by Western blotting confirmed that the vegetative storage protein (VSP), gamma-glutamyl hydrolase (GGH) and RuBisCo large chain proteins were significantly up-regulated by the exposure to AU strain and played a major role in IST. VSP has preponderant roles in N accumulation and mobilization, acid phosphatase activity and Na(+) homeostasis to sustain plant growth under stress condition. More interestingly, plant exposure to the bacterial strain significantly reduced Na(+) and enhanced K(+) and P content in root of soybean seedlings under salt stress. In addition, high accumulation of proline and chlorophyll content also provided evidence of protection against osmotic stress during the elicitation of IST by bacterial exposure. The present study reported for the first time that Ps. simiae produces a putative volatile blend that can enhance soybean seedling growth and elicit IST against 100 mmol l(-1) NaCl stress condition. The identification of such differentially expressed proteins provide new targets for future studies that will allow assessment of their physiological roles and significance in the response of glycophytes to stresses. Further work should uncover more about the chemical side of VOC compounds and a detailed study about their molecular mechanism responsible for plant growth. © 2015 The Society for Applied Microbiology.

Increased bacterial cell density and recombinant protein yield using a commercial microbial cultivation system.

PubMed

Peck, Grantley R; Bowden, Timothy R; Shiell, Brian J; Michalski, Wojtek P

2014-01-01

EnBase (BioSilta, Finland) is a microbial cultivation system that replicates fed-batch systems through sustained release of glucose by enzymatic degradation of a polymeric substrate. Achievable bacterial cell densities and recombinant capripoxvirus protein expression levels, solubility, and antigenicity using the EnBase system were assessed. BL21-AI Escherichia coli expressing capripoxvirus proteins achieved up to eightfold higher cell densities when grown in EnBase media compared with standard media. Greater yields of capripoxvirus proteins were attained using EnBase media, either through increases in the amount of expressed protein per cell in conjunction with higher cell density or through the increase in cell density alone. Addition of EnBase booster enhanced protein yield for one of the proteins tested but reduced yield for the other. However, the amount of soluble forms of the capripoxvirus proteins tested was not different from that observed from cultures grown under standard conditions. Purified capripoxvirus proteins expressed using EnBase or standard media were assessed for their performance by enzyme-linked immunosorbent assay (ELISA) and were shown to be equally capable of specifically binding capripoxvirus antibodies.

A symbiont-produced protein and bacterial symbiosis in Amoeba proteus.

PubMed

Pak, J W; Jeon, K W

1997-01-01

Gram symbiotic X-bacteria present in the xD strain of Amoeba proteus as required cell components, synthesize and export a large amount of a 29-kDa protein (S29x) into the host's cytoplasm across bacterial and symbiosome membranes. The S29x protein produced by E. coli transformed with the s29x gene is also rapidly secreted into the culture medium. Inside amoebae, S29x enters the host's nucleus as detected by confocal and immunoelectron microscopy, although it is not clear if S29x is selectively accumulated inside the nucleus. The deduced amino-acid sequence of S29x has a stretch of basic amino acids that could act as a nuclear localization signal, but there is no signal peptide at the N-terminus and the transport of S29x is energy independent. The functions of S29x are not known, but in view of its prominent presence inside the amoeba's nucleus, S29x is suspected to be involved in affecting the expression of amoeba's nuclear gene(s).

Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

PubMed Central

Gauss, George H.; Reott, Michael A.; Rocha, Edson R.; Young, Mark J.; Douglas, Trevor

2012-01-01

A factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical infections, is the bacterium's extreme aerotolerance, which allows survival in oxygenated tissues prior to anaerobic abscess formation. We investigated the role of the bacterioferritin-related (bfr) gene in the B. fragilis oxidative stress response. The bfr mRNA levels are increased in stationary phase or in response to O2 or iron. In addition, bfr null mutants exhibit reduced aerotolerance, and the bfr gene product protects DNA from hydroxyl radical cleavage in vitro. Crystallographic studies revealed a protein with a dodecameric structure and greater similarity to an archaeal DNA protection in starved cells (DPS)-like protein than to the 24-subunit bacterioferritins. Similarity to the DPS-like (DPSL) protein extends to the subunit and includes a pair of conserved cysteine residues juxtaposed to a buried dimetal binding site within the four-helix bundle. Compared to archaeal DPSLs, however, this bacterial DPSL protein contains several unique features, including a significantly different conformation in the C-terminal tail that alters the number and location of pores leading to the central cavity and a conserved metal binding site on the interior surface of the dodecamer. Combined, these characteristics confirm this new class of miniferritin in the bacterial domain, delineate the similarities and differences between bacterial DPSL proteins and their archaeal homologs, allow corrected annotations for B. fragilis bfr and other dpsl genes within the bacterial domain, and suggest an evolutionary link within the ferritin superfamily that connects dodecameric DPS to the (bacterio)ferritin 24-mer. PMID:22020642

Public Files of the SEC.

ERIC Educational Resources Information Center

Woodward, Steve

Some 180 different forms are used by the Securities and Exchange Commission (SEC) to cover the broad range of business activities regulated by the agency. This report examines the forms that are of greatest use to those seeking information about business. These forms are grouped in the following categories: general business information,…

78 FR 42526 - Compliance Policy Guide Sec. 690.800 Salmonella

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-16

... withdrawal of ``Compliance Policy Guide Sec. 690.700 Salmonella Contamination of Dry Dog Food.'' The... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2010-D-0378] Compliance Policy Guide Sec. 690.800 Salmonella in Food for Animals; Availability AGENCY: Food and Drug...

S-layer proteins from Lactobacillus sp. inhibit bacterial infection by blockage of DC-SIGN cell receptor.

PubMed

Prado Acosta, Mariano; Ruzal, Sandra M; Cordo, Sandra M

2016-11-01

Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Serum protein electrophoresis: an interesting diagnosis tool to distinguish viral from bacterial community-acquired pneumonia.

PubMed

Davido, B; Badr, C; Lagrange, A; Makhloufi, S; De Truchis, P; Perronne, C; Salomon, J; Dinh, A

2016-06-01

29-69 % of pneumonias are microbiologically documented because it can be considered as an invasive procedure with variable test sensitivity. However, it drastically impacts therapeutic strategy in particular the use of antibiotics. Serum protein electrophoresis (SPEP) is a routine and non-invasive test commonly used to identify serum protein disorders. As virus and bacteria may induce different globulins production, we hypothesize that SPEP can be used as an etiological diagnosis test. Retrospective study conducted from 1/1/13 until 5/1/15 among patient hospitalized for an acute community-acquired pneumonia based on fever, crackles and radiological abnormalities. α/β, α/γ, β/γ globulins and albumin/globulin (A/G) ratio were calculated from SPEP. Data were analyzed in 3 groups: documented viral (DVP) or bacterial pneumonia (DBP) and supposedly bacterial pneumonia (SBP). We used ANOVA statistic test with multiple comparisons using CI95 and ROC curve to compare them. 109 patients included divided into DBP (n = 16), DVP (n = 26) and SBP (n = 67). Mean age was 62 ± 18 year-old with a sex ratio M/F of 1.3. Underlying conditions (e.g. COPD, diabetes) were comparable between groups in multivariate analysis. Means of A/G ratio were 0.80 [0.76-0.84], 0.96 [0.91-1.01], 1.08 [0.99-1.16] respectively for DBP, SBP and DVP (p = 0.0002). A/G ratio cut-off value of 0.845 has a sensitivity of 87.5 % and a specificity of 73.1 %. A/G ratio seems to be an easy diagnostic tool to differentiate bacterial from viral pneumonia. A/G ratio cut-off value below 0.845 seems to be predictable of a bacterial origin and support the use of antibiotics.

Subcutaneous immunization with inactivated bacterial components and purified protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes prevents puerperal metritis in Holstein dairy cows.

PubMed

Machado, Vinícius Silva; Bicalho, Marcela Luccas de Souza; Meira Junior, Enoch Brandão de Souza; Rossi, Rodolfo; Ribeiro, Bruno Leonardo; Lima, Svetlana; Santos, Thiago; Kussler, Arieli; Foditsch, Carla; Ganda, Erika Korzune; Oikonomou, Georgios; Cheong, Soon Hon; Gilbert, Robert Owen; Bicalho, Rodrigo Carvalho

2014-01-01

In this study we evaluate the efficacy of five vaccine formulations containing different combinations of proteins (FimH; leukotoxin, LKT; and pyolysin, PLO) and/or inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes) in preventing postpartum uterine diseases. Inactivated whole cells were produced using two genetically distinct strains of each bacterial species (E. coli, F. necrophorum, and T. pyogenes). FimH and PLO subunits were produced using recombinant protein expression, and LKT was recovered from culturing a wild F. necrophorum strain. Three subcutaneous vaccines were formulated: Vaccine 1 was composed of inactivated bacterial whole cells and proteins; Vaccine 2 was composed of proteins only; and Vaccine 3 was composed of inactivated bacterial whole cells only. Two intravaginal vaccines were formulated: Vaccine 4 was composed of inactivated bacterial whole cells and proteins; and Vaccine 5 was composed of PLO and LKT. To evaluate vaccine efficacy, a randomized clinical trial was conducted at a commercial dairy farm; 371 spring heifers were allocated randomly into one of six different treatments groups: control, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifers assigned to one of the vaccine groups were each vaccinated twice: at 230 and 260 days of pregnancy. When vaccines were evaluated grouped as subcutaneous and intravaginal, the subcutaneous ones were found to significantly reduce the incidence of puerperal metritis. Additionally, subcutaneous vaccination significantly reduced rectal temperature at 6±1 days in milk. Reproduction was improved for cows that received subcutaneous vaccines. In general, vaccination induced a significant increase in serum IgG titers against all antigens, with subcutaneous vaccination again being more effective. In conclusion, subcutaneous vaccination with inactivated bacterial components and/or protein subunits of E. coli, F. necrophorum and T. pyogenes

Purification and Characterization of an Inverting Stereo- and Enantioselective sec-Alkylsulfatase from the Gram-Positive Bacterium Rhodococcus ruber DSM 44541

PubMed Central

Pogorevc, Mateja; Faber, Kurt

2003-01-01

Whole cells of Rhodococcus ruber DSM 44541 were found to hydrolyze (±)-2-octyl sulfate in a stereo- and enantiospecific fashion. When growing on a complex medium, the cells produced two sec-alkylsulfatases and (at least) one prim-alkylsulfatase in the absence of an inducer, such as a sec-alkyl sulfate or a sec-alcohol. From the crude cell-free lysate, two proteins responsible for sulfate ester hydrolysis (designated RS1 and RS2) were separated from each other based on their different hydrophobicities and were subjected to further chromatographic purification. In contrast to sulfatase RS1, enzyme RS2 proved to be reasonably stable and thus could be purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at a molecular mass of 43 kDa. Maximal enzyme activity was observed at 30°C and at pH 7.5. Sulfatase RS2 showed a clear preference for the hydrolysis of linear secondary alkyl sulfates, such as 2-, 3-, or 4-octyl sulfate, with remarkable enantioselectivity (an enantiomeric ratio of up to 21 [23]). Enzymatic hydrolysis of (R)-2-octyl sulfate furnished (S)-2-octanol without racemization, which revealed that the enzymatic hydrolysis proceeded through inversion of the configuration at the stereogenic carbon atom. Screening of a broad palette of potential substrates showed that the enzyme exhibited limited substrate tolerance; while simple linear sec-alkyl sulfates (C7 to C10) were freely accepted, no activity was found with branched and mixed aryl-alkyl sec-sulfates. Due to the fact that prim-sulfates were not accepted, the enzyme was classified as sec-alkylsulfatase (EC 3.1.6.X). PMID:12732552

46 CFR Sec. 12 - Disposition of removed equipment and scrap.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Disposition of removed equipment and scrap. Sec. 12... CONTRACT-NSA-LUMPSUMREP Sec. 12 Disposition of removed equipment and scrap. (a) Article 8 of the NSA-LUMPSUMREP Contract provides that any ship equipment, fuel, lube oil, supplies, stores, furniture, fixtures...

46 CFR Sec. 5 - Disbursements at principal office of agent.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Disbursements at principal office of agent. Sec. 5 Section 5 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURAL RULES FOR FINANCIAL TRANSACTIONS UNDER AGENCY AGREEMENTS Disbursements Sec. 5 Disbursements at principal office of agent. All expenses...

46 CFR Sec. 5 - Accounting.

Code of Federal Regulations, 2013 CFR

2013-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... compensation paid under sections 3(a), 3(b), 3(c), and 3(d) of NSA Order No. 47. Note: Invoices and account...

46 CFR Sec. 5 - Accounting.

Code of Federal Regulations, 2012 CFR

2012-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... compensation paid under sections 3(a), 3(b), 3(c), and 3(d) of NSA Order No. 47. Note: Invoices and account...

46 CFR Sec. 5 - Accounting.

Code of Federal Regulations, 2011 CFR

2011-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... compensation paid under sections 3(a), 3(b), 3(c), and 3(d) of NSA Order No. 47. Note: Invoices and account...

46 CFR Sec. 5 - Accounting.

Code of Federal Regulations, 2014 CFR

2014-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... compensation paid under sections 3(a), 3(b), 3(c), and 3(d) of NSA Order No. 47. Note: Invoices and account...

Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Economou, Nicoleta J.; Zentner, Isaac J.; Lazo, Edwin

2013-04-01

Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex.more » The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance.« less

Subcellular localization for Gram positive and Gram negative bacterial proteins using linear interpolation smoothing model.

PubMed

Saini, Harsh; Raicar, Gaurav; Dehzangi, Abdollah; Lal, Sunil; Sharma, Alok

2015-12-07

Protein subcellular localization is an important topic in proteomics since it is related to a protein׳s overall function, helps in the understanding of metabolic pathways, and in drug design and discovery. In this paper, a basic approximation technique from natural language processing called the linear interpolation smoothing model is applied for predicting protein subcellular localizations. The proposed approach extracts features from syntactical information in protein sequences to build probabilistic profiles using dependency models, which are used in linear interpolation to determine how likely is a sequence to belong to a particular subcellular location. This technique builds a statistical model based on maximum likelihood. It is able to deal effectively with high dimensionality that hinders other traditional classifiers such as Support Vector Machines or k-Nearest Neighbours without sacrificing performance. This approach has been evaluated by predicting subcellular localizations of Gram positive and Gram negative bacterial proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bacterial Cell Mechanics.

PubMed

Auer, George K; Weibel, Douglas B

2017-07-25

Cellular mechanical properties play an integral role in bacterial survival and adaptation. Historically, the bacterial cell wall and, in particular, the layer of polymeric material called the peptidoglycan were the elements to which cell mechanics could be primarily attributed. Disrupting the biochemical machinery that assembles the peptidoglycan (e.g., using the β-lactam family of antibiotics) alters the structure of this material, leads to mechanical defects, and results in cell lysis. Decades after the discovery of peptidoglycan-synthesizing enzymes, the mechanisms that underlie their positioning and regulation are still not entirely understood. In addition, recent evidence suggests a diverse group of other biochemical elements influence bacterial cell mechanics, may be regulated by new cellular mechanisms, and may be triggered in different environmental contexts to enable cell adaptation and survival. This review summarizes the contributions that different biomolecular components of the cell wall (e.g., lipopolysaccharides, wall and lipoteichoic acids, lipid bilayers, peptidoglycan, and proteins) make to Gram-negative and Gram-positive bacterial cell mechanics. We discuss the contribution of individual proteins and macromolecular complexes in cell mechanics and the tools that make it possible to quantitatively decipher the biochemical machinery that contributes to bacterial cell mechanics. Advances in this area may provide insight into new biology and influence the development of antibacterial chemotherapies.

Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology.

PubMed

Gatti-Lafranconi, Pietro; Natalello, Antonino; Ami, Diletta; Doglia, Silvia Maria; Lotti, Marina

2011-07-01

Cells have evolved complex and overlapping mechanisms to protect their proteins from aggregation. However, several reasons can cause the failure of such defences, among them mutations, stress conditions and high rates of protein synthesis, all common consequences of heterologous protein production. As a result, in the bacterial cytoplasm several recombinant proteins aggregate as insoluble inclusion bodies. The recent discovery that aggregated proteins can retain native-like conformation and biological activity has opened the way for a dramatic change in the means by which intracellular aggregation is approached and exploited. This paper summarizes recent studies towards the direct use of inclusion bodies in biotechnology and for the detection of bottlenecks in the folding pathways of specific proteins. We also review the major biophysical methods available for revealing fine structural details of aggregated proteins and which information can be obtained through these techniques. © 2011 The Authors Journal compilation © 2011 FEBS.

The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane.

PubMed

Gunasinghe, Sachith D; Shiota, Takuya; Stubenrauch, Christopher J; Schulze, Keith E; Webb, Chaille T; Fulcher, Alex J; Dunstan, Rhys A; Hay, Iain D; Naderer, Thomas; Whelan, Donna R; Bell, Toby D M; Elgass, Kirstin D; Strugnell, Richard A; Lithgow, Trevor

2018-05-29

The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

14 CFR Sec. 2-2 - Basis of allocation between entities.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Basis of allocation between entities. Sec... AIR CARRIERS General Accounting Provisions Sec. 2-2 Basis of allocation between entities. (a) The... the air carrier, as well as each transport entity and organizational division of the air carrier for...

Chirality Switching by Martensitic Transformation in Protein Cylindrical Crystals: Application to Bacterial Flagella

NASA Astrophysics Data System (ADS)

Komai, Ricardo Kiyohiro

Martensitic transformations provide unique engineering properties that, when designed properly, become important parts of new technology. Martensitic transformations have been studied for many years in traditional alloys (iron, steel, titanium, etc.), however there is still much to be learned in regards to these transformations in biological materials. Olson and Hartman showed in 1982 that these transformations are also observed in bacterial flagella and T4 bacteriophage viral sheaths, allowing for propulsion of bacteria in a fluid environment and, for the virus, is responsible for the infection mechanism. This work demonstrates, using the bacterial flagella as an example, that these transformations can be modelled using thermodynamic methods that are also used to model the transformations in alloys. This thesis work attempts to explain the transformations that occur in bacterial flagella, which are capable of small strain, highly reversible martensitic transformations. The first stress/temperature phase diagrams of these flagella were created by adding the mechanical energy of the transformation of the flagella to limited chemical thermodynamics information of the transformation. Mechanical energy is critical to the transformation process because the bacterial body applies a torque to the radius of the flagella. Finally, work has begun and will be completed in regards to understanding the kinetics of the transformation of the flagella. The motion of the transformation interface can be predicted by using a Landau-Ginzburg model. The crystallography of the transformation in bacterial flagella is also being computed to determine the invariant lines of transformation that occur within this cylindrical crystal. This work has shown that it is possible to treat proteins in a similar manner that alloys are treated when using thermodynamic modelling. Much can be learned from translating what is known regarding phase transformations in hard material systems to soft, organic

Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon

PubMed Central

Kuhn, Patrick; Draycheva, Albena; Vogt, Andreas; Petriman, Narcis-Adrian; Sturm, Lukas; Drepper, Friedel; Warscheid, Bettina; Wintermeyer, Wolfgang

2015-01-01

Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY–SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP–RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY–FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the translocon is guided by the interaction between SRP and translocon-bound FtsY in a quaternary targeting complex. PMID:26459600

A simple model for DNA bridging proteins and bacterial or human genomes: bridging-induced attraction and genome compaction

NASA Astrophysics Data System (ADS)

Johnson, J.; Brackley, C. A.; Cook, P. R.; Marenduzzo, D.

2015-02-01

We present computer simulations of the phase behaviour of an ensemble of proteins interacting with a polymer, mimicking non-specific binding to a piece of bacterial DNA or eukaryotic chromatin. The proteins can simultaneously bind to the polymer in two or more places to create protein bridges. Despite the lack of any explicit interaction between the proteins or between DNA segments, our simulations confirm previous results showing that when the protein-polymer interaction is sufficiently strong, the proteins come together to form clusters. Furthermore, a sufficiently large concentration of bridging proteins leads to the compaction of the swollen polymer into a globular phase. Here we characterise both the formation of protein clusters and the polymer collapse as a function of protein concentration, protein-polymer affinity and fibre flexibility.

GOLGI TRANSPORT 1B Regulates Protein Export from the Endoplasmic Reticulum in Rice Endosperm Cells[OPEN

PubMed Central

Liu, Feng; Wang, Yunlong; Liu, Xi; Wang, Di; Zhu, Xiaopin; Jing, Ruonan; Wu, Mingming; Hao, Yuanyuan; Jiang, Ling; Wang, Chunming

2016-01-01

Coat protein complex II (COPII) mediates the first step of anterograde transport of newly synthesized proteins from the endoplasmic reticulum (ER) to other endomembrane compartments in eukaryotes. A group of evolutionarily conserved proteins (Sar1, Sec23, Sec24, Sec13, and Sec31) constitutes the basic COPII coat machinery; however, the details of how the COPII coat assembly is regulated remain unclear. Here, we report a protein transport mutant of rice (Oryza sativa), named glutelin precursor accumulation4 (gpa4), which accumulates 57-kD glutelin precursors and forms two types of ER-derived abnormal structures. GPA4 encodes the evolutionarily conserved membrane protein GOT1B (also known as GLUP2), homologous to the Saccharomyces cerevisiae GOT1p. The rice GOT1B protein colocalizes with Arabidopsis thaliana Sar1b at Golgi-associated ER exit sites (ERESs) when they are coexpressed in Nicotiana benthamiana. Moreover, GOT1B physically interacts with rice Sec23, and both proteins are present in the same complex(es) with rice Sar1b. The distribution of rice Sar1 in the endomembrane system, its association with rice Sec23c, and the ERES organization pattern are significantly altered in the gpa4 mutant. Taken together, our results suggest that GOT1B plays an important role in mediating COPII vesicle formation at ERESs, thus facilitating anterograde transport of secretory proteins in plant cells. PMID:27803308

Protein Crystals and their Growth

NASA Technical Reports Server (NTRS)

Chernov, A. A.

2004-01-01

Recent results on binding between protein molecules in crystal lattice, crystal-solution surface energy, elastic properties and strength and spontaneous crystal cracking are reviewed and discussed in the first half of this paper (Sea 2-4). In the second par&, some basic approaches to solubility of proteins are followed by overview on crystal nucleation and growth (Sec 5). It is argued that variability of mixing in batch crystallization may be a source for scattering of crystal number ultimately appearing in the batch. Frequency at which new molecules join crystal lattice is measured by kinetic coefficient and related to the observable crystal growth rate. Numerical criteria to discriminate diffusion and kinetic limited growth are discussed on this basis in Sec 7. In Sec 8, creation of defects is discussed with the emphasis on the role of impurities and convection on macromolecular crystal I;erfection.

Assembling the bacterial segrosome.

PubMed

Hayes, Finbarr; Barillà, Daniela

2006-05-01

Genome segregation in prokaryotes is a highly ordered process that integrates with DNA replication, cytokinesis and other fundamental facets of the bacterial cell cycle. The segrosome is the nucleoprotein complex that mediates DNA segregation in bacteria, its assembly and organization is best understood for plasmid partition. The recent elucidation of structures of the ParB plasmid segregation protein bound to centromeric DNA, and of the tertiary structures of other segregation proteins, are key milestones in the path to deciphering the molecular basis of bacterial DNA segregation.

Isolation of cell-free bacterial inclusion bodies.

PubMed

Rodríguez-Carmona, Escarlata; Cano-Garrido, Olivia; Seras-Franzoso, Joaquin; Villaverde, Antonio; García-Fruitós, Elena

2010-09-17

Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10⁻¹ cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

secHsp70 as a tool to approach amyloid-β42 and other extracellular amyloids.

PubMed

De Mena, Lorena; Chhangani, Deepak; Fernandez-Funez, Pedro; Rincon-Limas, Diego E

2017-07-03

Self-association of amyloidogenic proteins is the main pathological trigger in a wide variety of neurodegenerative disorders. These aggregates are deposited inside or outside the cell due to hereditary mutations, environmental exposures or even normal aging. Cumulative evidence indicates that the heat shock chaperone Hsp70 possesses robust neuroprotection against various intracellular amyloids in Drosophila and mouse models. However, its protective role against extracellular amyloids was largely unknown as its presence outside the cells is very limited. Our recent manuscript in PNAS revealed that an engineered form of secreted Hsp70 (secHsp70) is highly protective against toxicity induced by extracellular deposition of the amyloid-β42 (Aβ42) peptide. In this Extra View article, we extend our analysis to other members of the heat shock protein family. We created PhiC31-based transgenic lines for human Hsp27, Hsp40, Hsp60 and Hsp70 and compared their activities in parallel against extracellular Aβ42. Strikingly, only secreted Hsp70 exhibits robust protection against Aβ42-triggered toxicity in the extracellular milieu. These observations indicate that the ability of secHsp70 to suppress Aβ42 insults is quite unique and suggest that targeted secretion of Hsp70 may represent a new therapeutic approach against Aβ42 and other extracellular amyloids. The potential applications of this engineered chaperone are discussed.

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

PubMed

Goldenzweig, Adi; Goldsmith, Moshe; Hill, Shannon E; Gertman, Or; Laurino, Paola; Ashani, Yacov; Dym, Orly; Unger, Tamar; Albeck, Shira; Prilusky, Jaime; Lieberman, Raquel L; Aharoni, Amir; Silman, Israel; Sussman, Joel L; Tawfik, Dan S; Fleishman, Sarel J

2016-07-21

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris.

PubMed

Luong, Truc Thanh; Reardon-Robinson, Melissa E; Siegel, Sara D; Ton-That, Hung

2017-05-15

Posttranslocational protein folding in the Gram-positive biofilm-forming actinobacterium Actinomyces oris is mediated by a membrane-bound thiol-disulfide oxidoreductase named MdbA, which catalyzes oxidative folding of nascent polypeptides transported by the Sec translocon. Reoxidation of MdbA involves a bacterial v itamin K ep o xide r eductase (VKOR)-like protein that contains four cysteine residues, C93/C101 and C175/C178, with the latter forming a canonical CXXC thioredoxin-like motif; however, the mechanism of VKOR-mediated reoxidation of MdbA is not known. We present here a topological view of the A. oris membrane-spanning protein VKOR with these four exoplasmic cysteine residues that participate in MdbA reoxidation. Like deletion of the VKOR gene, alanine replacement of individual cysteine residues abrogated polymicrobial interactions and biofilm formation, concomitant with the failure to form adhesive pili on the bacterial surface. Intriguingly, the mutation of the cysteine at position 101 to alanine (C101A mutation) resulted in a high-molecular-weight complex that was positive for MdbA and VKOR by immunoblotting and was absent in other alanine substitution mutants and the C93A C101A double mutation and after treatment with the reducing agent β-mercaptoethanol. Consistent with this observation, affinity purification followed by immunoblotting confirmed this MdbA-VKOR complex in the C101A mutant. Furthermore, ectopic expression of the Mycobacterium tuberculosis VKOR analog in the A. oris VKOR deletion (ΔVKOR) mutant rescued its defects, in contrast to the expression of M. tuberculosis VKOR variants known to be nonfunctional in the disulfide relay that mediates reoxidation of the disulfide bond-forming catalyst DsbA in Escherichia coli Altogether, the results support a model of a disulfide relay, from its start with the pair C93/C101 to the C175-X-X-C178 motif, that is required for MdbA reoxidation and appears to be conserved in members of the class

Structure and function of Helicobacter pylori CagA, the first-identified bacterial protein involved in human cancer

PubMed Central

HATAKEYAMA, Masanori

2017-01-01

Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor of gastric cancer. The cagA gene-encoded CagA protein is delivered into gastric epithelial cells via bacterial type IV secretion, where it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. Delivered CagA then acts as a non-physiological scaffold/hub protein by interacting with multiple host signaling molecules, most notably the pro-oncogenic phosphatase SHP2 and the polarity-regulating kinase PAR1/MARK, in both tyrosine phosphorylation-dependent and -independent manners. CagA-mediated manipulation of intracellular signaling promotes neoplastic transformation of gastric epithelial cells. Transgenic expression of CagA in experimental animals has confirmed the oncogenic potential of the bacterial protein. Structural polymorphism of CagA influences its scaffold function, which may underlie the geographic difference in the incidence of gastric cancer. Since CagA is no longer required for the maintenance of established gastric cancer cells, studying the role of CagA during neoplastic transformation will provide an excellent opportunity to understand molecular processes underlying “Hit-and-Run” carcinogenesis. PMID:28413197

Effects of a salivary stimulant, slaframine, on ruminal fermentation, bacterial protein synthesis and digestion in frequently fed steers.

PubMed

Froetschel, M A; Amos, H E; Evans, J J; Croom, W J; Hagler, W M

1989-03-01

Slaframine (SF), a parasympathomimetic salivary stimulant, was administered i.m. (10, 15 or 20 micrograms SF/kg BW) to ruminally and abomasally fistulated steers at 12-h intervals for 18-d periods in a latin square-designed experiment. Steers were fed semicontinuously (12 times daily) a 40:60 roughage:concentrate diet at twice their net energy requirement for maintenance. Ruminal digestion coefficients for DM, ADF and starch were 10 to 16% lower and linearly related in an inverse manner to the level of SF administered (P less than .05). Postruminal digestion of DM, ADF and starch increased as much as 46.7, 9.5 and 44.0%, respectively, in a fashion linearly related (P less than .05) to the level of SF administered. Total tract digestion of DM and ADF were not affected by SF; however, total tract starch digestion was increased as much as 5% and was related linearly (P less than .05) to SF treatment. With SF administration, as much as 13% more bacterial protein exited the rumen, resulting in a 16.5% linear improvement (P less than .1) in the efficiency of ruminal bacterial protein production per 100 g of OM fermented. Ruminal concentrations of VFA, ammonia and pH were not affected by SF. These results demonstrate a positive relationship between salivation and ruminal bacterial protein synthesis and suggest that feed utilization by ruminants may be improved by pharmacological stimulation of salivary secretions.

The Yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins.

PubMed

Siggers, Keri A; Lesser, Cammie F

2008-07-17

Microbial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors.

Phylogenetic and Protein Sequence Analysis of Bacterial Chemoreceptors.

PubMed

Ortega, Davi R; Zhulin, Igor B

2018-01-01

Identifying chemoreceptors in sequenced bacterial genomes, revealing their domain architecture, inferring their evolutionary relationships, and comparing them to chemoreceptors of known function become important steps in genome annotation and chemotaxis research. Here, we describe bioinformatics procedures that enable such analyses, using two closely related bacterial genomes as examples.

A Bacteriophage Capsid Protein Is an Inhibitor of a Conserved Transcription Terminator of Various Bacterial Pathogens.

PubMed

Ghosh, Gairika; Reddy, Jayavardhana; Sambhare, Susmit; Sen, Ranjan

2018-01-01

Rho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibits Escherichia coli Rho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, including Mycobacterium smegmatis , Mycobacterium bovis , Mycobacterium tuberculosis , Xanthomonas campestris , Xanthomonas oryzae , Corynebacterium glutamicum , Vibrio cholerae , Salmonella enterica , and Pseudomonas syringae The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from the E. coli transcription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins. In vivo pulldown assays revealed direct binding of Psu with many of these Rho proteins. In vivo expression of psu induced killing of M. smegmatis , M. bovis , X. campestris , and E. coli expressing S. enterica Rho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the "universal" inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions. IMPORTANCE Bacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of the E. coli transcription

Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1

PubMed Central

Belov, George A.; Kovtunovych, Gennadiy; Jackson, Catherine L.; Ehrenfeld, Ellie

2010-01-01

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the Arf GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to BFA, an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. PMID:20497182

Studies on Bacterial Proteins Corona Interaction with Saponin Imprinted ZnO Nanohoneycombs and Their Toxic Responses.

PubMed

Sharma, Deepali; Ashaduzzaman, Md; Golabi, Mohsen; Shriwastav, Amritanshu; Bisetty, Krishna; Tiwari, Ashutosh

2015-11-04

Molecular imprinting generates robust, efficient, and highly mesoporous surfaces for biointeractions. Mechanistic interfacial interaction between the surface of core substrate and protein corona is crucial to understand the substantial microbial toxic responses at a nanoscale. In this study, we have focused on the mechanistic interactions between synthesized saponin imprinted zinc oxide nanohoneycombs (SIZnO NHs), average size 80-125 nm, surface area 20.27 m(2)/g, average pore density 0.23 pore/nm and number-average pore size 3.74 nm and proteins corona of bacteria. The produced SIZnO NHs as potential antifungal and antibacterial agents have been studied on Sclerotium rolfsii (S. rolfsii), Pythium debarynum (P. debarynum) and Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), respectively. SIZnO NHs exhibited the highest antibacterial (∼50%) and antifungal (∼40%) activity against Gram-negative bacteria (E. coli) and fungus (P. debarynum), respectively at concentration of 0.1 mol. Scanning electron spectroscopy (SEM) observation showed that the ZnO NHs ruptured the cell wall of bacteria and internalized into the cell. The molecular docking studies were carried out using binding proteins present in the gram negative bacteria (lipopolysaccharide and lipocalin Blc) and gram positive bacteria (Staphylococcal Protein A, SpA). It was envisaged that the proteins present in the bacterial cell wall were found to interact and adsorb on the surface of SIZnO NHs thereby blocking the active sites of the proteins used for cell wall synthesis. The binding affinity and interaction energies were higher in the case of binding proteins present in gram negative bacteria as compared to that of gram positive bacteria. In addition, a kinetic mathematical model (KMM) was developed in MATLAB to predict the internalization in the bacterial cellular uptake of the ZnO NHs for better understanding of their controlled toxicity. The results obtained from KMM exhibited a good

46 CFR Sec. 20 - Reports of awards.

Code of Federal Regulations, 2011 CFR

2011-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 20 Reports... submitted by the General Agents pursuant to section 3(d) of NSA Order 34 (SRM-3, Revised). ...

46 CFR Sec. 20 - Reports of awards.

Code of Federal Regulations, 2014 CFR

2014-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 20 Reports... submitted by the General Agents pursuant to section 3(d) of NSA Order 34 (SRM-3, Revised). ...

46 CFR Sec. 20 - Reports of awards.

Code of Federal Regulations, 2012 CFR

2012-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 20 Reports... submitted by the General Agents pursuant to section 3(d) of NSA Order 34 (SRM-3, Revised). ...

46 CFR Sec. 20 - Reports of awards.

Code of Federal Regulations, 2010 CFR

2010-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 20 Reports... submitted by the General Agents pursuant to section 3(d) of NSA Order 34 (SRM-3, Revised). ...

46 CFR Sec. 20 - Reports of awards.

Code of Federal Regulations, 2013 CFR

2013-10-01

... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 20 Reports... submitted by the General Agents pursuant to section 3(d) of NSA Order 34 (SRM-3, Revised). ...

Connecting the dots between bacterial biofilms and ice cream.

PubMed

Stanley-Wall, Nicola R; MacPhee, Cait E

2015-12-18

Emerging research is revealing a diverse array of interfacially-active proteins that are involved in varied biological process from foaming horse sweat to bacterial raincoat formation. We describe an interdisciplinary approach to study the molecular and biophysical mechanisms controlling the activity of an unusual bacterial protein called BslA. This protein is needed for biofilm formation and forms a protective layer or raincoat over the bacterial community, but also has a multitude of potential applications in multiphase formulations. Here we document our journey from fundamental research to an examination of the applications for this surface-active protein in ice cream.

Connecting the dots between bacterial biofilms and ice cream

NASA Astrophysics Data System (ADS)

Stanley-Wall, Nicola R.; MacPhee, Cait E.

2015-12-01

Emerging research is revealing a diverse array of interfacially-active proteins that are involved in varied biological process from foaming horse sweat to bacterial raincoat formation. We describe an interdisciplinary approach to study the molecular and biophysical mechanisms controlling the activity of an unusual bacterial protein called BslA. This protein is needed for biofilm formation and forms a protective layer or raincoat over the bacterial community, but also has a multitude of potential applications in multiphase formulations. Here we document our journey from fundamental research to an examination of the applications for this surface-active protein in ice cream.

Expression of an Atriplex nummularia gene encoding a protein homologous to the bacterial molecular chaperone DnaJ.

PubMed Central

Zhu, J K; Shi, J; Bressan, R A; Hasegawa, P M

1993-01-01

DnaJ is a 36-kD heat shock protein that functions together with Dnak (Hsp70) as a molecular chaperone in Escherichia coli. We have obtained a cDNA clone from the higher plant Atriplex nummularia that encodes a 46.6-kD polypeptide (ANJ1) with an overall 35.2% amino acid sequence identity with the E. coli DnaJ. ANJ1 has 43.4% overall sequence identity with the Saccharomyces cerevisiae cytoplasmic DnaJ homolog YDJ1/MAS5. Complementation of the yeast mas5 mutation indicated that ANJ1 is a functional homolog of YDJ1/MAS5. The presence of other DnaJ homologs in A. nummularia was demonstrated by the detection of proteins that are antigenically related to the yeast mitochondrial DnaJ homolog SCJ1 and the yeast DnaJ-related protein Sec63. Expression of the ANJ1 gene was compared with that of an A. nummularia Hsp70 gene. Expression of both ANJ1 and Hsp70 transcripts was coordinately induced by heat shock. However, noncoordinate accumulation of ANJ1 and Hsp70 mRNAs occurred during the cell growth cycle and in response to NaCl stress. PMID:8467224

Expression of an Atriplex nummularia gene encoding a protein homologous to the bacterial molecular chaperone DnaJ.

PubMed

Zhu, J K; Shi, J; Bressan, R A; Hasegawa, P M

1993-03-01

DnaJ is a 36-kD heat shock protein that functions together with Dnak (Hsp70) as a molecular chaperone in Escherichia coli. We have obtained a cDNA clone from the higher plant Atriplex nummularia that encodes a 46.6-kD polypeptide (ANJ1) with an overall 35.2% amino acid sequence identity with the E. coli DnaJ. ANJ1 has 43.4% overall sequence identity with the Saccharomyces cerevisiae cytoplasmic DnaJ homolog YDJ1/MAS5. Complementation of the yeast mas5 mutation indicated that ANJ1 is a functional homolog of YDJ1/MAS5. The presence of other DnaJ homologs in A. nummularia was demonstrated by the detection of proteins that are antigenically related to the yeast mitochondrial DnaJ homolog SCJ1 and the yeast DnaJ-related protein Sec63. Expression of the ANJ1 gene was compared with that of an A. nummularia Hsp70 gene. Expression of both ANJ1 and Hsp70 transcripts was coordinately induced by heat shock. However, noncoordinate accumulation of ANJ1 and Hsp70 mRNAs occurred during the cell growth cycle and in response to NaCl stress.

14 CFR Sec. 1-5 - Records.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Records. Sec. 1-5 Section 1-5 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC... information as will render certain the identification of all facts essential to a verification of the nature...

46 CFR Sec. 6 - Awarding of work.

Code of Federal Regulations, 2012 CFR

2012-10-01

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46 CFR Sec. 6 - Awarding of work.

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2014-10-01

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46 CFR Sec. 6 - Awarding of work.

Code of Federal Regulations, 2013 CFR

2013-10-01

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EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation.

PubMed

Pearson, Jaclyn S; Giogha, Cristina; Mühlen, Sabrina; Nachbur, Ueli; Pham, Chi L L; Zhang, Ying; Hildebrand, Joanne M; Oates, Clare V; Lung, Tania Wong Fok; Ingle, Danielle; Dagley, Laura F; Bankovacki, Aleksandra; Petrie, Emma J; Schroeder, Gunnar N; Crepin, Valerie F; Frankel, Gad; Masters, Seth L; Vince, James; Murphy, James M; Sunde, Margaret; Webb, Andrew I; Silke, John; Hartland, Elizabeth L

2017-01-13

Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3), TIR-domain-containing adapter-inducing interferon-β (TRIF) and Z-DNA-binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI) that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling 1-3 . RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis 4,5 . Here, we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM-containing proteins RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This requires a previously unrecognized tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaves within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL leads to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of tumour necrosis factor (TNF), lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C))-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibits TNF-induced phosphorylation and plasma membrane localization of mixed lineage kinase domain-like pseudokinase (MLKL). In vivo, EspL cysteine protease activity contributes to persistent colonization of mice by the EPEC-like mouse pathogen Citrobacter rodentium. The activity of EspL defines a family of T3SS cysteine protease effectors found in a range of bacteria and reveals a mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.

DNA-binding by Haemophilus influenzae and Escherichia coli YbaB, members of a widely-distributed bacterial protein family.

PubMed

Cooley, Anne E; Riley, Sean P; Kral, Keith; Miller, M Clarke; DeMoll, Edward; Fried, Michael G; Stevenson, Brian

2009-07-13

Genes orthologous to the ybaB loci of Escherichia coli and Haemophilus influenzae are widely distributed among eubacteria. Several years ago, the three-dimensional structures of the YbaB orthologs of both E. coli and H. influenzae were determined, revealing a novel "tweezer"-like structure. However, a function for YbaB had remained elusive, with an early study of the H. influenzae ortholog failing to detect DNA-binding activity. Our group recently determined that the Borrelia burgdorferi YbaB ortholog, EbfC, is a DNA-binding protein. To reconcile those results, we assessed the abilities of both the H. influenzae and E. coli YbaB proteins to bind DNA to which B. burgdorferi EbfC can bind. Both the H. influenzae and the E. coli YbaB proteins bound to tested DNAs. DNA-binding was not well competed with poly-dI-dC, indicating some sequence preferences for those two proteins. Analyses of binding characteristics determined that both YbaB orthologs bind as homodimers. Different DNA sequence preferences were observed between H. influenzae YbaB, E. coli YbaB and B. burgdorferi EbfC, consistent with amino acid differences in the putative DNA-binding domains of these proteins. Three distinct members of the YbaB/EbfC bacterial protein family have now been demonstrated to bind DNA. Members of this protein family are encoded by a broad range of bacteria, including many pathogenic species, and results of our studies suggest that all such proteins have DNA-binding activities. The functions of YbaB/EbfC family members in each bacterial species are as-yet unknown, but given the ubiquity of these DNA-binding proteins among Eubacteria, further investigations are warranted.

46 CFR Sec. 4 - Method of payment.

Code of Federal Regulations, 2010 CFR

2010-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank account...

46 CFR Sec. 4 - Method of payment.

Code of Federal Regulations, 2014 CFR

2014-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank account...

46 CFR Sec. 4 - Method of payment.

Code of Federal Regulations, 2013 CFR

2013-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank account...

46 CFR Sec. 4 - Method of payment.

Code of Federal Regulations, 2012 CFR

2012-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank account...

Structural differences in the bacterial flagellar motor among bacterial species.

PubMed

Terashima, Hiroyuki; Kawamoto, Akihiro; Morimoto, Yusuke V; Imada, Katsumi; Minamino, Tohru

2017-01-01

The bacterial flagellum is a supramolecular motility machine consisting of the basal body as a rotary motor, the hook as a universal joint, and the filament as a helical propeller. Intact structures of the bacterial flagella have been observed for different bacterial species by electron cryotomography and subtomogram averaging. The core structures of the basal body consisting of the C ring, the MS ring, the rod and the protein export apparatus, and their organization are well conserved, but novel and divergent structures have also been visualized to surround the conserved structure of the basal body. This suggests that the flagellar motors have adapted to function in various environments where bacteria live and survive. In this review, we will summarize our current findings on the divergent structures of the bacterial flagellar motor.

46 CFR Sec. 3 - Master's requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Master's requirements. Sec. 3 Section 3 Shipping... Master's requirements. The Master shall: (a) Receive and receipt for the quantities of slop chest items... therefor, at the unit price, specified by the Slop Chest Statement furnished the Master by the General...

46 CFR Sec. 3 - Master's requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 8 2011-10-01 2011-10-01 false Master's requirements. Sec. 3 Section 3 Shipping... Master's requirements. The Master shall: (a) Receive and receipt for the quantities of slop chest items... therefor, at the unit price, specified by the Slop Chest Statement furnished the Master by the General...

46 CFR Sec. 3 - Master's requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 8 2012-10-01 2012-10-01 false Master's requirements. Sec. 3 Section 3 Shipping... Master's requirements. The Master shall: (a) Receive and receipt for the quantities of slop chest items... therefor, at the unit price, specified by the Slop Chest Statement furnished the Master by the General...

46 CFR Sec. 3 - Master's requirements.

Code of Federal Regulations, 2014 CFR