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1
Wu, Si, Roslyn N. Brown, Samuel H. Payne, Da Meng, Rui Zhao, Nikola Tolić, Li Cao, et al. "Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the Bacterium Novosphingobium aromaticivorans." International Journal of Proteomics 2013 (March 10, 2013): 1–10. http://dx.doi.org/10.1155/2013/279590.
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The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans. Our top-down analysis provided the confident identification of 55 proteins in the periplasm and characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm.
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Kurokawa, Yoichi, Hideki Yanagi, and Takashi Yura. "Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli." Applied and Environmental Microbiology 66, no. 9 (September 1, 2000): 3960–65. http://dx.doi.org/10.1128/aem.66.9.3960-3965.2000.
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ABSTRACT Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm ofEscherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli.
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Rambow-Larsen, Amy A., and Alison A. Weiss. "Temporal Expression of Pertussis Toxin and Ptl Secretion Proteins by Bordetella pertussis." Journal of Bacteriology 186, no. 1 (January 1, 2004): 43–50. http://dx.doi.org/10.1128/jb.186.1.43-50.2004.
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ABSTRACT Pertussis toxin is an AB5 toxin comprised of protein subunits S1 through S5. The individual subunits are secreted by a Sec-dependent mechanism into the periplasm, where the toxin is assembled. The Ptl type IV secretion system mediates secretion of assembled toxin past the outer membrane. In this study, we examined the time course of protein expression, toxin assembly, and secretion as a function of the bacterial growth cycle. Logarithmic growth was observed after a 1-h lag phase. Secreted toxin was first observed at 3 h. Secretion continued throughout the logarithmic growth phase and decreased as the culture entered the stationary phase after about 24 h. On a per cell basis, toxin secretion occurred at a constant rate of 3 molecules/min/cell from 2 to 18 h. More of toxin subunits S1, S2, and S3 were produced than were secreted, resulting in periplasmic accumulation. Periplasmic S1, S2, and S3 were found to be soluble in the periplasm, as well as membrane associated. About one-half of the periplasmic S1, S2 and S3 subunits were incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and the level increased between 2 and 24 h from 30 to 1,000 molecules per cell; however, the initial level of PtlF, 30 molecules per cell, supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests that translation rates exceed the rate of secretion and that secretion, not toxin and Ptl complex assembly, is rate limiting.
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Felício, Ana P., Eliandre de Oliveira, M. A. Odena, Oswaldo Garcia Jr., Maria C. Bertolini, Lúcio F. C. Ferraz, Laura M. M. Ottoboni, and Maria T. M. Novo. "A Preliminary Differential Proteomic Analysis of the Periplasmic Fraction in Acidithiobacillus Ferrooxidans Planktonic and Attached Cells Exposed to Chalcopyrite." Advanced Materials Research 71-73 (May 2009): 179–82. http://dx.doi.org/10.4028/www.scientific.net/amr.71-73.179.
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The Acidithiobacillus ferrooxidans periplasmic space is known to have proteins involved in the respiratory chains. There are no reports about the expression of the periplasmic proteins in A. ferrooxidans cells attached to chalcopyrite. In this preliminary work, it was compared the periplasmic protein profiles of A. ferrooxidans planktonic and attached cells after exposure to chalcopyrite for 2 hours. The bacterial response to chalcopyrite was investigated by a proteomic approach (two- dimensional gel electrophoresis and mass spectrometry). Four proteins differentially expressed between planktonic and attached cells after exposure to chalcopyrite were identified. Two of these proteins, repressed in chalcopyrite- attached cells, were both identified as superoxide dismutase, whereas the single strand binding protein (SSB) and the PspA/IM30 protein were induced. These results showed that A. ferrooxidans chalcopyrite- attached and planktonic cells show differential expression of the periplasmic proteins and that a proteomic approach can provide a valuable tool to detect proteins related to the A. ferrooxidans response to attachment to the mineral substrates.
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CastilloKeller, Maria, and Rajeev Misra. "Protease-Deficient DegP Suppresses Lethal Effects of a Mutant OmpC Protein by Its Capture." Journal of Bacteriology 185, no. 1 (January 1, 2003): 148–54. http://dx.doi.org/10.1128/jb.185.1.148-154.2003.
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ABSTRACT The expression of assembly-defective outer membrane proteins can confer lethality if they are not degraded by envelope proteases. We report here that the expression of a mutant OmpC protein, OmpC2Cys, which forms disulfide bonds in the periplasm due to the presence of two non-native cysteine residues, is lethal in cells lacking the major periplasmic protease, DegP. This lethality is not observed in dsbA strains that have diminished ability to form periplasmic disulfide bonds. Our data show that this OmpC2Cys-mediated lethality in a degP::Kmr dsbA + background can be reversed by a DegP variant, DegPS210A, that is devoid of its proteolytic activity but retains its reported chaperone activity. However, DegPS210A does not reverse the lethal effect of OmpC2Cys by correcting its assembly but rather by capturing misfolded mutant OmpC polypeptides and thus removing them from the assembly pathway. Displacement of OmpC2Cys by DegPS210A also alleviates the negative effect that the mutant OmpC protein has on wild-type OmpF.
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Zhou, Xiaofeng, Xiufang Hu, Jinyun Li, and Nian Wang. "A Novel Periplasmic Protein, VrpA, Contributes to Efficient Protein Secretion by the Type III Secretion System in Xanthomonas spp." Molecular Plant-Microbe Interactions® 28, no. 2 (February 2015): 143–53. http://dx.doi.org/10.1094/mpmi-10-14-0309-r.
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Efficient secretion of type III effector proteins from the bacterial cytoplasm to host cell cytosol via a type III secretion system (T3SS) is crucial for virulence of plant-pathogenic bacterium. Our previous study revealed a conserved hypothetical protein, virulence-related periplasm protein A (VrpA), which was identified as a critical virulence factor for Xanthomonas citri subsp. citri. In this study, we demonstrate that mutation of vrpA compromises X. citri subsp. citri virulence and hypersensitive response induction. This deficiency is also observed in the X. campestris pv. campestris strain, suggesting a functional conservation of VrpA in Xanthomonas spp. Our study indicates that VrpA is required for efficient protein secretion via T3SS, which is supported by multiple lines of evidence. A CyaA reporter assay shows that VrpA is involved in type III effector secretion; quantitative reverse-transcription polymerase chain reaction analysis suggests that the vrpA mutant fails to activate citrus-canker-susceptible gene CsLOB1, which is transcriptionally activated by transcription activator-like effector PthA4; in vitro secretion study reveals that VrpA plays an important role in secretion of T3SS pilus, translocon, and effector proteins. Our data also indicate that VrpA in X. citri subsp. citri localizes to bacterial periplasmic space and the periplasmic localization is required for full function of VrpA and X. citri subsp. citri virulence. Protein–protein interaction studies show that VrpA physically interacts with periplasmic T3SS components HrcJ and HrcC. However, the mutation of VrpA does not affect T3SS gene expression. Additionally, VrpA is involved in X. citri subsp. citri tolerance of oxidative stress. Our data contribute to the mechanical understanding of an important periplasmic protein VrpA in Xanthomonas spp.
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van der Wal, Fimme J., G. Koningstein, C. M. ten Hagen, Bauke Oudega, and Joen Luirink. "Optimization of Bacteriocin Release Protein (BRP)-Mediated Protein Release by Escherichia coli: Random Mutagenesis of the pCloDF13-Derived BRP Gene To Uncouple Lethality and Quasi-Lysis from Protein Release." Applied and Environmental Microbiology 64, no. 2 (February 1, 1998): 392–98. http://dx.doi.org/10.1128/aem.64.2.392-398.1998.
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ABSTRACT Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli periplasm into the culture medium. However, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-lysis) and inhibition of growth on broth agar plates (lethality). To optimize BRP-mediated protein release, the pCloDF13 BRP gene was subjected to random mutagenesis by using PCR techniques. Mutated BRPs with a strongly reduced capacity to cause growth inhibition on broth agar plates were selected, analyzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid cultures. A subset of these BRP derivatives did not cause quasi-lysis and had only a small effect on growth but still functioned in the release of the periplasmic protein β-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture medium. These BRP derivatives can be more efficiently used for extracellular production of proteins by E. coli than can the original BRP.
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Bernhard, Michael, Bärbel Friedrich, and Roman A. Siddiqui. "Ralstonia eutropha TF93 Is Blocked in Tat-Mediated Protein Export." Journal of Bacteriology 182, no. 3 (February 1, 2000): 581–88. http://dx.doi.org/10.1128/jb.182.3.581-588.2000.
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ABSTRACT Ralstonia eutropha (formerly Alcaligenes eutrophus) TF93 is pleiotropically affected in the translocation of redox enzymes synthesized with an N-terminal signal peptide bearing a twin arginine (S/T-R-R-X-F-L-K) motif. Immunoblot analyses showed that the catalytic subunits of the membrane-bound [NiFe] hydrogenase (MBH) and the molybdenum cofactor-binding periplasmic nitrate reductase (Nap) are mislocalized to the cytoplasm and to the inner membrane, respectively. Moreover, physiological studies showed that the copper-containing nitrous oxide reductase (NosZ) was also not translocated to the periplasm in strain TF93. The cellular localization of enzymes exported by the general secretion system was unaffected. The translocation-arrested MBH and Nap proteins were enzymatically active, suggesting that twin-arginine signal peptide-dependent redox enzymes may have their cofactors inserted prior to transmembrane export. The periplasmic destination of MBH, Nap, and NosZ was restored by heterologous expression of Azotobacter chroococcum tatAmobilized into TF93. tatA encodes a bacterial Hcf106-like protein, a component of a novel protein transport system that has been characterized in thylakoids and shown to translocate folded proteins across the membrane.
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Brandon, Lauren D., and Marcia B. Goldberg. "Periplasmic Transit and Disulfide Bond Formation of the Autotransported Shigella Protein IcsA." Journal of Bacteriology 183, no. 3 (February 1, 2001): 951–58. http://dx.doi.org/10.1128/jb.183.3.951-958.2001.
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ABSTRACT The Shigella outer membrane protein IcsA belongs to the family of type V secreted (autotransported) virulence factors. Members of this family mediate their own translocation across the bacterial outer membrane: the carboxy-terminal β domain forms a β barrel channel in the outer membrane through which the amino-terminal α domain passes. IcsA, which is localized at one pole of the bacterium, mediates actin assembly by Shigella, which is essential for bacterial intracellular movement and intercellular dissemination. Here, we characterize the transit of IcsA across the periplasm during its secretion. We show that an insertion in the dsbB gene, whose gene product mediates disulfide bond formation of many periplasmic intermediates, does not affect the surface expression or unipolar targeting of IcsA. However, IcsA forms one disulfide bond in the periplasm in a DsbA/DsbB-dependent fashion. Furthermore, cellular fractionation studies reveal that IcsA has a transient soluble periplasmic intermediate. Our data also suggest that IcsA is folded in a proteinase K-resistant state in the periplasm. From these data, we propose a novel model for the secretion of IcsA that may be applicable to other autotransported proteins.
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Sroga, Grazyna E., and Jonathan S. Dordick. "Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression." Protein Engineering, Design and Selection 14, no. 11 (November 2001): 929–37. http://dx.doi.org/10.1093/protein/14.11.929.
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Kern, Renée, Abderrahim Malki, Jad Abdallah, Jihen Tagourti, and Gilbert Richarme. "Escherichia coli HdeB Is an Acid Stress Chaperone." Journal of Bacteriology 189, no. 2 (November 3, 2006): 603–10. http://dx.doi.org/10.1128/jb.01522-06.
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ABSTRACT We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.
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Scribano, Marzano, Mortera, Sarshar, Vernocchi, Zagaglia, Putignani, Palamara, and Ambrosi. "Insights into the Periplasmic Proteins of Acinetobacter baumannii AB5075 and the Impact of Imipenem Exposure: A Proteomic Approach." International Journal of Molecular Sciences 20, no. 14 (July 13, 2019): 3451. http://dx.doi.org/10.3390/ijms20143451.
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Carbapenem-resistant Acinetobacter baumannii strains cause life-threatening infections due to the lack of therapeutic options. Although the main mechanisms underlying antibiotic-resistance have been extensively studied, the general response to maintain bacterial viability under antibiotic exposure deserves to be fully investigated. Since the periplasmic space contains several proteins with crucial cellular functions, besides carbapenemases, we decided to study the periplasmic proteome of the multidrug-resistant (MDR) A. baumannii AB5075 strain, grown in the absence and presence of imipenem (IMP). Through the proteomic approach, 65 unique periplasmic proteins common in both growth conditions were identified: eight proteins involved in protein fate, response to oxidative stress, energy metabolism, antibiotic-resistance, were differentially expressed. Among them, ABUW_1746 and ABUW_2363 gene products presented the tetratricopeptide repeat motif, mediating protein-protein interactions. The expression switch of these proteins might determine specific protein interactions to better adapt to changing environmental conditions. ABUW_2868, encoding a heat shock protein likely involved in protection against oxidative stress, was upregulated in IMP-exposed bacteria. Accordingly, the addition of periplasmic proteins from A. baumannii cultured with IMP increased bacterial viability in an antioxidant activity assay. Overall, this study provides the first insights about the composition of the periplasmic proteins of a MDR A. baumannii strain, its biological response to IMP and suggests possible new targets to develop alternative antibiotic drugs.
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Barth, S., M. Huhn, B. Matthey, A. Klimka, E. A. Galinski, and A. Engert. "Compatible-Solute-Supported Periplasmic Expression of Functional Recombinant Proteins under Stress Conditions." Applied and Environmental Microbiology 66, no. 4 (April 1, 2000): 1572–79. http://dx.doi.org/10.1128/aem.66.4.1572-1579.2000.
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ABSTRACT The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coliunder osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.
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Beck, Nancy A., Eric S. Krukonis, and Victor J. DiRita. "TcpH Influences Virulence Gene Expression in Vibrio cholerae by Inhibiting Degradation of the Transcription Activator TcpP." Journal of Bacteriology 186, no. 24 (December 15, 2004): 8309–16. http://dx.doi.org/10.1128/jb.186.24.8309-8316.2004.
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ABSTRACT Expression of toxT, the transcription activator of cholera toxin and pilus production in Vibrio cholerae, is the consequence of a complex cascade of regulatory events that culminates in activation of the toxT promoter by TcpP and ToxR, two membrane-localized transcription factors. Both are encoded in operons with genes whose products, TcpH and ToxS, which are also membrane localized, are hypothesized to control their activity. In this study we analyzed the role of TcpH in controlling TcpP function. We show that a mutant of V. cholerae lacking TcpH expressed virtually undetectable levels of TcpP, although tcpP mRNA levels remain unaffected. A time course experiment showed that levels of TcpP, expressed from a plasmid, are dramatically reduced over time without co-overexpression of TcpH. By contrast, deletion of toxS did not affect ToxR protein levels. A fusion protein in which the TcpP periplasmic domain is replaced with that of ToxR remains stable, suggesting that the periplasmic domain of TcpP is the target for degradation of the protein. Placement of the periplasmic domain of TcpP on ToxR, an otherwise stable protein, results in instability, providing further evidence for the hypothesis that the periplasmic domain of TcpP is a target for degradation. Consistent with this interpretation is our finding that derivatives of TcpP lacking a periplasmic domain are more stable in V. cholerae than are derivatives in which the periplasmic domain has been truncated. This work identifies at least one role for the periplasmic domain of TcpP, i.e., to act as a target for a protein degradation pathway that regulates TcpP levels. It also provides a rationale for why the V. cholerae tcpH mutant strain is avirulent. We hypothesize that regulator degradation may be an important mechanism for regulating virulence gene expression in V. cholerae.
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Stewart, F., and P. P. Tuffnell. "Cloning the cDNA for horse growth hormone and expression in Escherichia coli." Journal of Molecular Endocrinology 6, no. 2 (April 1991): 189–96. http://dx.doi.org/10.1677/jme.0.0060189.
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ABSTRACT A 514 bp cDNA transcript coding for 78% of horse (Equus caballus) GH has been cloned and sequenced. The deduced amino acid sequence corresponded precisely to that previously obtained by protein sequencing and, in addition, provided new sequence information for the signal peptide. The missing 3′ fragment of the cDNA was reconstructed using synthetic oligonucleotides and site-specific directed mutagenesis. The complete cDNA sequence was then inserted into an expression vector (PIN-III-lppp−5) which utilizes a bacterial signal peptide to secrete the expressed product into the periplasmic space of Escherichia coli. Western blot analysis of cell lysates and periplasmic fractions prepared from cells harbouring this construct revealed significant quantities of immunoreactive GH and indicated that the bacterial signal peptide was successfully cleaved from the fusion protein on secretion. Recombinant-derived horse GH, recovered by osmotic shock from the periplasm, was active in a heterologous radioimmunoassay and a horse liver radioreceptor assay and resulted in a recovery of 0·5–2 mg GH/l cell culture. An apparent limitation on the secretion rate of horse GH in E. coli, possibly involving a block to translocation across the cytoplasmic membrane, prevented higher levels of expression being obtained.
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Brangulis, Kalvis, Inara Akopjana, Andris Kazaks, and Kaspars Tars. "Crystal structure of the N-terminal domain of the major virulence factor BB0323 from the Lyme disease agent Borrelia burgdorferi." Acta Crystallographica Section D Structural Biology 75, no. 9 (August 22, 2019): 825–30. http://dx.doi.org/10.1107/s2059798319010751.
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Lyme disease is an infection caused by the spirochete Borrelia burgdorferi after it is transmitted to a mammalian organism during a tick blood meal. B. burgdorferi encodes at least 140 lipoproteins located on the outer or inner membrane, thus facing the surroundings or the periplasmic space, respectively. However, most of the predicted lipoproteins are of unknown function, and only a few proteins are known to be essential for the persistence and virulence of the pathogen. One such protein is the periplasmic BB0323, which is indispensable for B. burgdorferi to cause Lyme disease and the function of which is associated with cell fission and outer membrane integrity. After expression and transport to the periplasm, BB0323 is cleaved into C-terminal and N-terminal domains by the periplasmic serine protease BB0104. The resulting N-terminal domain is sufficient to ensure the survival of B. burgdorferi throughout the mouse–tick infection cycle. The crystal structure of the N-terminal domain of BB0323 was determined at 2.35 Å resolution. The overall fold of the protein belongs to the spectrin superfamily, with the characteristic interconnected triple-helical bundles known as spectrin repeats that function as linkers between different cell components in other organisms. Overall, the reported three-dimensional structure of the N-terminal domain of BB0323 not only reveals the molecular details of a protein that is essential for B. burgdorferi membrane integrity, cell fission and infectivity, but also suggests that spectrin repeats in bacteria are not limited to the EzrA proteins.
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Nally, Jarlath E., John F. Timoney, and Brian Stevenson. "Temperature-Regulated Protein Synthesis by Leptospira interrogans." Infection and Immunity 69, no. 1 (January 1, 2001): 400–404. http://dx.doi.org/10.1128/iai.69.1.400-404.2001.
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ABSTRACT Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37°C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37°C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37°C. A 36-kDa protein synthesized at 30 but not at 37°C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection withL. interrogans serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection.
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Delgado, Mónica, Héctor Toledo, and Carlos A. Jerez. "Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans." Applied and Environmental Microbiology 64, no. 7 (July 1, 1998): 2380–85. http://dx.doi.org/10.1128/aem.64.7.2380-2385.1998.
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ABSTRACT We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacteriumLeptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillumchemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. ThelcrI gene showed both ς28-like and ς70-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP fromEscherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed inE. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.
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Rasooli, I., M. Alipour, and S. L. Mousavi Gargari. "Isolation, Cloning, Expression and Immunoactivity of Periplasmic Binding Protein, FepB." International Journal of Infectious Diseases 12 (December 2008): e252. http://dx.doi.org/10.1016/j.ijid.2008.05.682.
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Marczak, Małgorzata, Andrzej Mazur, Jarosław E. Król, Wiesław I. Gruszecki, and Anna Skorupska. "Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: Subcellular Localization and Possible Involvement in Exopolysaccharide Export." Journal of Bacteriology 188, no. 19 (October 1, 2006): 6943–52. http://dx.doi.org/10.1128/jb.00651-06.
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ABSTRACT Surface expression of exopolysaccharides (EPS) in gram-negative bacteria depends on the activity of proteins found in the cytoplasmic membrane, the periplasmic space, and the outer membrane. pssTNOP genes identified in Rhizobium leguminosarum bv. trifolii strain TA1 encode proteins that might be components of the EPS polymerization and secretion system. In this study, we have characterized PssN protein. Employing pssN-phoA and pssN-lacZ gene fusions and in vivo acylation with [3H]palmitate, we demonstrated that PssN is a 43-kDa lipoprotein directed to the periplasm by an N-terminal signal sequence. Membrane detergent fractionation followed by sucrose gradient centrifugation showed that PssN is an outer membrane-associated protein. Indirect immunofluorescence with anti-PssN and fluorescein isothiocyanate-conjugated antibodies and protease digestion of spheroplasts and intact cells of TA1 provided evidence that PssN is oriented towards the periplasmic space. Chemical cross-linking of TA1 and E. coli cells overproducing PssN-His6 protein showed that PssN might exist as a homo-oligomer of at least two monomers. Investigation of the secondary structure of purified PssN-His6 protein by Fourier transform infrared spectroscopy revealed the predominant presence of β-structure; however, α-helices were also detected. Influence of an increased amount of PssN protein on the TA1 phenotype was assessed and correlated with a moderate enhancement of EPS production.
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Haendler, B., A. Becker, C. Noeske-Jungblut, J. Krätzschmar, P. Donner, and W. D. Schleuning. "Expression of active recombinant pallidipin, a novel platelet aggregation inhibitor, in the periplasm of Escherichia coli." Biochemical Journal 307, no. 2 (April 15, 1995): 465–70. http://dx.doi.org/10.1042/bj3070465.
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The platelet aggregation inhibitor pallidipin is a protein present in the saliva of the blood-sucking triatomine bug Triatoma pallidipennis. Expression of recombinant pallidipin in the periplasm of Escherichia coli was achieved by placing its coding sequence downstream of the alkaline phosphatase (APase) or trc promoter in frame with bacterial leader peptide DNA sequences derived from APase or from the periplasmic form of cyclophilin (Cph). In each case the DNA sequence of mature pallidipin was merged to the leader peptide coding part, either directly, or while introducing additional amino acids, in order to assess their influence on the activity of the leader peptidase and on the biological activity of the recombinant protein. All tested constructs gave rise to abundant periplasmic expression of pallidipin, which was then purified by a combination of cation- and anion-exchange chromatography followed by size-exclusion gel chromatography. Recombinant pallidipin had the expected molecular mass (approximately 19 kDa) and was correctly processed, as demonstrated by SDS/PAGE and N-terminal amino acid sequencing. The highest expression levels were obtained with the three APase-derived expression plasmids. Platelet aggregation tests revealed that E. coli-derived pallidipin was fully active, with an IC50 of 33-89 nM, comparable with that of the native protein, except when an additional N-terminal lysyl-isoleucyl dipeptide was present, which resulted in an IC50 more than ten times higher.
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Tran, Tram Anh T., Douglas K. Struck, and Ry Young. "Periplasmic Domains Define Holin-Antiholin Interactions in T4 Lysis Inhibition." Journal of Bacteriology 187, no. 19 (October 1, 2005): 6631–40. http://dx.doi.org/10.1128/jb.187.19.6631-6640.2005.
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ABSTRACT Bacteriophage T4 effects host lysis with a holin, T, and an endolysin, E. T and E accumulate in the membrane and cytoplasm, respectively, throughout the period of late gene expression. At an allele-specific time, T triggers to disrupt the membrane, allowing E to enter the periplasm and attack the peptidoglycan. T triggering can be blocked by secondary infections, leading to the state of lysis inhibition (LIN). LIN requires the T4 antiholin, RI, and is sensitive to the addition of energy poisons. T is unusual among holins in having a large C-terminal periplasmic domain. The rI gene encodes a polypeptide of 97 residues, of which 72 are predicted to be a periplasmic domain. Here, we show that the periplasmic domain of RI is necessary and sufficient to block T-mediated lysis. Moreover, when overexpressed, the periplasmic domain of T (TCTD) was found to abolish LIN in T4 infections and to convert wild-type (wt) T4 plaques from small and fuzzy edged to the classic “r” large, sharp-edged plaque morphology. Although RI could be detected in whole cells, attempts to monitor it during subcellular fractionation were unsuccessful, presumably because RI is a highly unstable protein. However, fusing green fluorescence protein (GFP) to the N terminus of RI created a more stable chimera that could be demonstrated to form complexes with wild-type TCTD and also with its LIN-defective T75I variant. These results suggest that the function of the unusual periplasmic domain of T is to transduce environmental information for the real-time control of lysis timing.
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Ge, Yigong, Chunhao Li, Linda Corum, Clive A. Slaughter, and Nyles W. Charon. "Structure and Expression of the FlaA Periplasmic Flagellar Protein of Borrelia burgdorferi." Journal of Bacteriology 180, no. 9 (May 1, 1998): 2418–25. http://dx.doi.org/10.1128/jb.180.9.2418-2425.1998.
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ABSTRACT The spirochete which causes Lyme disease, Borrelia burgdorferi, has many features common to other spirochete species. Outermost is a membrane sheath, and within this sheath are the cell cylinder and periplasmic flagella (PFs). The PFs are subterminally attached to the cell cylinder and overlap in the center of the cell. Most descriptions of the B. burgdorferi flagellar filaments indicate that these organelles consist of only one flagellin protein (FlaB). In contrast, the PFs from other spirochete species are comprised of an outer layer of FlaA and a core of FlaB. We recently found that a flaA homolog was expressed in B. burgdorferi and that it mapped in a fla/che operon. These results led us to analyze the PFs and FlaA of B. burgdorferi in detail. Using Triton X-100 to remove the outer membrane and isolate the PFs, we found that the 38.0-kDa FlaA protein purified with the PFs in association with the 41.0-kDa FlaB protein. On the other hand, purifying the PFs by using Sarkosyl resulted in no FlaA in the isolated PFs. Sarkosyl has been used by others to purifyB. burgdorferi PFs, and our results explain in part their failure to find FlaA. Unlike other spirochetes, B. burgdorferi FlaA was expressed at a lower level than FlaB. In characterizing FlaA, we found that it was posttranslationally modified by glycosylation, and thus it resembles its counterpart fromSerpulina hyodysenteriae. We also tested if FlaA was synthesized in a spontaneously occurring PF mutant of B. burgdorferi (HB19Fla−). Although this mutant still synthesizedflaA message in amounts similar to the wild-type amounts, it failed to synthesize FlaA protein. These results suggest that, in agreement with data found for FlaB and other spirochete flagellar proteins, FlaA is likely to be regulated on the translational level. Western blot analysis using Treponema pallidum anti-FlaA serum indicated that FlaA was antigenically well conserved in several spirochete species. Taken together, the results indicate that both FlaA and FlaB comprise the PFs of B. burgdorferi and that they are regulated differently from flagellin proteins of other bacteria.
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Yohannes, Elizabeth, D. Michael Barnhart, and Joan L. Slonczewski. "pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12." Journal of Bacteriology 186, no. 1 (January 1, 2004): 192–99. http://dx.doi.org/10.1128/jb.186.1.192-199.2004.
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ABSTRACT During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.
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Oloomi, M., and S. Bouzari. "Increasing the efficiency of protein production by periplasmic expression in E.coli." Biochemical Society Transactions 30, no. 5 (October 1, 2002): A130. http://dx.doi.org/10.1042/bst030a130b.
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Boiani, R., F. Andreoni, G. Serafini, I. Bianconi, R. Pierleoni, S. Dominici, F. Gorini, and M. Magnani. "Expression and characterization of the periplasmic cobalamin-binding protein ofPhotobacterium damselaesubsp.piscicida." Journal of Fish Diseases 32, no. 9 (September 2009): 745–53. http://dx.doi.org/10.1111/j.1365-2761.2009.01050.x.
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Dumont, Estelle, Julia Vergalli, Jelena Pajovic, Satya P. Bhamidimarri, Koldo Morante, Jiajun Wang, Dmitrijs Lubriks, et al. "Mechanistic aspects of maltotriose-conjugate translocation to the Gram-negative bacteria cytoplasm." Life Science Alliance 2, no. 1 (December 28, 2018): e201800242. http://dx.doi.org/10.26508/lsa.201800242.
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Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin–fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of theEscherichia colimaltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathwayviathe activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.
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Comerci, Diego J., Guido D. Pollevick, Ana M. Vigliocco, Alberto C. C. Frasch, and Rodolfo A. Ugalde. "Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortusS19." Infection and Immunity 66, no. 8 (August 1, 1998): 3862–66. http://dx.doi.org/10.1128/iai.66.8.3862-3866.1998.
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ABSTRACT A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruziwas used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae.
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Miller, Kurt W., Robin Schamber, Yanling Chen, and Bibek Ray. "Production of Active Chimeric Pediocin AcH inEscherichia coli in the Absence of Processing and Secretion Genes from the Pediococcus pap Operon." Applied and Environmental Microbiology 64, no. 1 (January 1, 1998): 14–20. http://dx.doi.org/10.1128/aem.64.1.14-20.1998.
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ABSTRACT Minimum requirements have been determined for synthesis and secretion of the Pediococcus antimicrobial peptide, pediocin AcH, in Escherichia coli. The functional mature domain of pediocin AcH (Lys+1 to Cys+44) is targeted into the E. coli sec machinery and secreted to the periplasm in active form when fused in frame to the COOH terminus of the secretory protein maltose-binding protein (MBP). The PapC-PapD specialized secretion machinery is not required for secretion of the MBP-pediocin AcH chimeric protein, indicating that inPediococcus, PapC and PapD probably are required for recognition and processing of the leader peptide rather than for translocation of the mature pediocin AcH domain across the cytoplasmic membrane. The chimeric protein displays bactericidal activity, suggesting that the NH2 terminus of pediocin AcH does not span the phospholipid bilayer in the membrane-interactive form of the molecule. However, the conserved Lys+1-Tyr-Tyr-Gly-Asn-Gly-Val+7-sequence at the NH2 terminus is important because deletion of this sequence abolishes activity. The secreted chimeric protein is released into the culture medium when expressed in a periplasmic leaky E. coli host. The MBP fusion-periplasmic leaky expression system should be generally advantageous for production and screening of the activity of bioactive peptides.
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Lam, Siu Ling, Shane Kirby, and Anthony B. Schryvers. "Foreign signal peptides can constitute a barrier to functional expression of periplasmic proteins in Haemophilus influenzae." Microbiology 149, no. 11 (November 1, 2003): 3155–64. http://dx.doi.org/10.1099/mic.0.26411-0.
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To study the periplasmic branch of iron (ferric ion) uptake systems in Gram-negative bacteria, genetic reconstitution experiments were initiated in Haemophilus influenzae involving exchange of the periplasmic iron-binding protein. The expression of many of the heterologous periplasmic ferric-binding proteins (FbpAs) was quite limited. Transformation experiments with the fbpA gene from Neisseria gonorrhoeae yielded two colony sizes with different phenotypic characteristics. The small colonies contained the intact N. gonorrhoeae fbpA gene and were deficient in utilization of transferrin iron. The large colonies contained hybrid H. influenzae/N. gonorrhoeae fbpA genes, were proficient in transferrin iron utilization and had enhanced levels of expression of FbpA. These hybrid genes included several that encoded the mature N. gonorrhoeae FbpA with the H. influenzae signal peptide. To more fully evaluate the effect of foreign signal peptides, a series of hybrid genes were prepared that exchanged the signal peptides from H. influenzae FbpA, N. gonorrhoeae FbpA and the TEM-1 β-lactamase. The presence of the H. influenzae leader was required for functional expression of FbpAs and was shown to dramatically increase the level of β-lactamase activity.
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Soares, C. R. J., F. I. C. Gomide, E. K. M. Ueda, and P. Bartolini. "Periplasmic expression of human growth hormone via plasmid vectors containing the PL promoter: use of HPLC for product quantification." Protein Engineering Design and Selection 16, no. 12 (December 1, 2003): 1131–38. http://dx.doi.org/10.1093/protein/gzg114.
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Lattemann, Claus T., Jochen Maurer, Elke Gerland, and Thomas F. Meyer. "Autodisplay: Functional Display of Active β-Lactamase on the Surface of Escherichia coli by the AIDA-I Autotransporter." Journal of Bacteriology 182, no. 13 (July 1, 2000): 3726–33. http://dx.doi.org/10.1128/jb.182.13.3726-3733.2000.
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ABSTRACT Members of the protein family of immunoglobulin A1 protease-like autotransporters comprise multidomain precursors consisting of a C-terminal autotransporter domain that promotes the translocation of N-terminally attached passenger domains across the cell envelopes of gram-negative bacteria. Several autotransporter domains have recently been shown to efficiently promote the export of heterologous passenger domains, opening up an effective tool for surface display of heterologous proteins. Here we report on the autotransporter domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I), which was genetically fused to the C terminus of the periplasmic enzyme β-lactamase, leading to efficient expression of the fusion protein in E. coli. The β-lactamase moiety of the fusion protein was presented on the bacterial surface in a stable manner, and the surface-located β-lactamase was shown to be enzymatically active. Enzymatic activity was completely removed by protease treatment, indicating that surface display of β-lactamase was almost quantitative. The periplasmic domain of the outer membrane protein OmpA was not affected by externally added proteases, demonstrating that the outer membranes of E. coli cells expressing the β-lactamase AIDA-I fusion protein remained physiologically intact.
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Bingle, Wade H., Harry D. Kurtz Jr., and John Smit. "An ''all-purpose" cellulase reporter for gene fusion studies and application to the paracrystalline surface (S)-layer protein of Caulobacter crescentus." Canadian Journal of Microbiology 39, no. 1 (January 1, 1993): 70–80. http://dx.doi.org/10.1139/m93-010.
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The secreted endoglucanase (CenA) from the Gram-positive bacterium Cellulomonas fimi and a deletion derivative (ΔCenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of ΔCenA as a reporter molecule in Caulobacter crescentus. Expression of cenA in C. crescentus yielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium. In contrast, expression of ΔcenA yielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in the C. crescentus cytoplasm. Using the putative cytoplasmic and periplasmic forms of ΔCenA as markers, a simple assay for periplasmic ΔCenA hybrids was developed. This assay indicated that ΔCenA activity was largely independent of cellular location. To facilitate the use of ΔCenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating ΔcenA was constructed. This vector was used to investigate factors important to the expression of the gene (rsaA) encoding the paracrystalline surface protein (S-layer) of the bacterium. It was found that altering the 5′ untranslated region of the rsaA mRNA reduced gene expression by 70%. One rsaA:ΔcenA gene fusion resulting from these experiments that incorporated only rsaA translation initiation information was further modified to serve as a general reporter for creating transcriptional gene fusions with other promoters. Gene fusions between alkaline phosphatase (phoA) and either cenA or lacZ were used to supplement information about RsaA secretion derived from rsaA:phoA gene fusions. It was found that linkage of the N-terminal leader peptide of CenA to PhoA yielded 50–100 times more cell-associated PhoA activity in C. crescentus than linkage of the RsaA N terminus. Taken together, these experiments indicated that ΔCenA was useful for tagging proteins localized to the cytoplasm, exported to the periplasm, or secreted from the cell, as well as for monitoring events in the cytoplasm such as examining factors important to the level of gene expression. Further, because ΔCenA was active in all cell compartments, it could be used to estimate the efficiency of hybrid protein export-secretion from enzyme activity measurements alone. In short, ΔCenA possessed many of the attributes of an "all-purpose" reporter.Key words: gene fusions, protein secretion, cellulase, alkaline phosphatase, Caulobacter crescentus.
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Rajabi Memari, Hamid, Ramakrishnan Ramanan, and Arbakariya Ariff. "Comparison of expression systems for the production of human interferon-α2b." Open Life Sciences 5, no. 4 (August 1, 2010): 446–55. http://dx.doi.org/10.2478/s11535-010-0036-y.
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AbstractThe production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 µg/L of culture, while the amount of nuclear expression in the plant was 4.46 µg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform.
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Stauffer, Lorraine T., and George V. Stauffer. "Antagonistic Roles for GcvA and GcvB in hdeAB Expression in Escherichia coli." ISRN Microbiology 2012 (May 16, 2012): 1–10. http://dx.doi.org/10.5402/2012/697308.
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In E. coli, the periplasmic proteins HdeA and HdeB have chaperone-like functions, suppressing aggregation of periplasmic proteins under acidic conditions. A microarray analysis of RNA isolated from an E. coli wild type and a ΔgcvB strain grown to mid-log phase in Luria-Bertani broth indicated the hdeAB operon, encoding the HdeA and HdeB proteins, is regulated by the sRNA GcvB. We wanted to verify that GcvB and its coregulator Hfq play a role in regulation of the hdeAB operon. In this study, we show that GcvB positively regulates hdeA::lacZ and hdeB::lacZ translational fusions in cells grown in Luria-Bertani broth and in glucose minimal media + glycine. Activation also requires the Hfq protein. Although many sRNAs dependent on Hfq regulate by an antisense mechanism, GcvB regulates hdeAB either directly or indirectly at the level of transcription. GcvA, the activator of gcvB, negatively regulates hdeAB at the level of transcription. Although expression of gcvB is dependent on GcvA, activation of hdeAB by GcvB occurs independently of GcvA’s ability to repress the operon. Cell survival and growth at low pH are consistent with GcvA negatively regulating and GcvB positively regulating the hdeAB operon.
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TURNER, Susan, Eleanor REID, Harry SMITH, and Jeffrey COLE. "A novel cytochrome c peroxidase from Neisseria gonorrhoeae: a lipoprotein from a Gram-negative bacterium." Biochemical Journal 373, no. 3 (August 1, 2003): 865–73. http://dx.doi.org/10.1042/bj20030088.
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A cytochrome c peroxidase (CCP) produced by Neisseria gonorrhoeae has been shown to have novel characteristics by investigating its location, expression and role in Neisseria gonorrhoeae and by expression in Escherichia coli. Analysis of the N-terminus of CCP indicated that it is a lipoprotein with a signal peptide for cleavage by signal peptidase II. Expression of the gonococcal CCP in E. coli revealed that it is first synthesized as a pro-apo-cytochrome that is translocated across the cytoplasmic membrane. The signal peptide is cleaved and haem is attached in the periplasm. The gonococcal CCP was associated with the membrane of both E. coli and N. gonorrhoeae. The expression of a MalE–CCP fusion protein has allowed characterization of CCP in vitro. Evidence is presented that CCP protects gonococci from hydrogen peroxide, presumably in the periplasmic compartment of the cell. The expression of CCP is dependent on the transcription factor FNR, but is repressed by nitrite, indicating that it could be most important in the stationary-phase response. These data support the hypothesis that the gonococcal lipoprotein CCP is anchored to the membrane in the periplasm, where it might be responsible for the reduction of hydrogen peroxide. Other putative CCP lipoproteins have been identified, representing a new subclass of bacterial CCP proteins.
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Justice, Sheryl S., David A. Hunstad, Jill Reiss Harper, Amy R. Duguay, Jerome S. Pinkner, James Bann, Carl Frieden, Thomas J. Silhavy, and Scott J. Hultgren. "Periplasmic Peptidyl Prolyl cis-trans Isomerases Are Not Essential for Viability, but SurA Is Required for Pilus Biogenesis in Escherichia coli." Journal of Bacteriology 187, no. 22 (November 15, 2005): 7680–86. http://dx.doi.org/10.1128/jb.187.22.7680-7686.2005.
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ABSTRACT In Escherichia coli, FkpA, PpiA, PpiD, and SurA are the four known periplasmic cis-trans prolyl isomerases. These isomerases facilitate proper protein folding by increasing the rate of transition of proline residues between the cis and trans states. Genetic inactivation of all four periplasmic isomerases resulted in a viable strain that exhibited a decreased growth rate and increased susceptibility to certain antibiotics. Levels of the outer membrane proteins LamB and OmpA in the quadruple mutant were indistinguishable from those in the surA single mutant. In addition, expression of P and type 1 pili (adhesive organelles produced by uropathogenic strains of E. coli and assembled by the chaperone/usher pathway) were severely diminished in the absence of the four periplasmic isomerases. Maturation of the usher was significantly impaired in the outer membranes of strains devoid of all four periplasmic isomerases, resulting in a defect in pilus assembly. Moreover, this defect in pilus assembly and usher stability could be attributed to the absence of SurA. The data presented here suggest that the four periplasmic isomerases are not essential for growth under laboratory conditions but may have significant roles in survival in environmental and pathogenic niches, as indicated by the effect on pilus production.
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Pezza, Alejandro, Lucas B. Pontel, Carolina López, and Fernando C. Soncini. "Compartment and signal-specific codependence in the transcriptional control of Salmonella periplasmic copper homeostasis." Proceedings of the National Academy of Sciences 113, no. 41 (September 27, 2016): 11573–78. http://dx.doi.org/10.1073/pnas.1603192113.
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Copper homeostasis is essential for bacterial pathogen fitness and infection, and has been the focus of a number of recent studies. In Salmonella, envelope protection against copper overload and macrophage survival depends on CueP, a major copper-binding protein in the periplasm. This protein is also required to deliver the metal ion to the Cu/Zn superoxide dismutase SodCII. The Salmonella-specific CueP-coding gene was originally identified as part of the Cue regulon under the transcriptional control of the cytoplasmic copper sensor CueR, but its expression differs from the rest of CueR-regulated genes. Here we show that cueP expression is controlled by the concerted action of CueR, which detects the presence of copper in the cytoplasm, and by CpxR/CpxA, which monitors envelope stress. Copper-activated CueR is necessary for the appropriate spatial arrangement of the −10 and −35 elements of the cueP promoter, and CpxR is essential to recruit the RNA polymerase. The integration of two ancestral sensory systems—CueR, which provides signal specificity, and CpxR/CpxA, which detects stress in the bacterial envelope—restricts the expression of this periplasmic copper resistance protein solely to cells encountering surplus copper that disturbs envelope homeostasis, emulating the role of the CusR/CusS regulatory system present in other enteric bacteria.
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Suaste-Olmos, Fernando, Clelia Domenzain, José Cruz Mireles-Rodríguez, Sebastian Poggio, Aurora Osorio, Georges Dreyfus, and Laura Camarena. "The Flagellar Protein FliL Is Essential for Swimming in Rhodobacter sphaeroides." Journal of Bacteriology 192, no. 23 (October 1, 2010): 6230–39. http://dx.doi.org/10.1128/jb.00655-10.
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ABSTRACT In this work we characterize the function of the flagellar protein FliL in Rhodobacter sphaeroides. Our results show that FliL is essential for motility in this bacterium and that in its absence flagellar rotation is highly impaired. A green fluorescent protein (GFP)-FliL fusion forms polar and lateral fluorescent foci that show different spatial dynamics. The presence of these foci is dependent on the expression of the flagellar genes controlled by the master regulator FleQ, suggesting that additional components of the flagellar regulon are required for the proper localization of GFP-FliL. Eight independent pseudorevertants were isolated from the fliL mutant strain. In each of these strains a single nucleotide change in motB was identified. The eight mutations affected only three residues located on the periplasmic side of MotB. Swimming of the suppressor mutants was not affected by the presence of the wild-type fliL allele. Pulldown and yeast two-hybrid assays showed that that the periplasmic domain of FliL is able to interact with itself but not with the periplasmic domain of MotB. From these results we propose that FliL could participate in the coupling of MotB with the flagellar rotor in an indirect fashion.
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Tabatabai, Louisa B., and Glynn H. Frank. "Conservation of Expression and N-Terminal Sequences of the Pasteurella haemolytica 31-Kilodalton andPasteurella trehalosi 29-Kilodalton Periplasmic Iron-Regulated Proteins." Clinical Diagnostic Laboratory Immunology 6, no. 4 (July 1, 1999): 617–20. http://dx.doi.org/10.1128/cdli.6.4.617-620.1999.
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ABSTRACT This study examined the conservation of expression of a 31-kDa iron-regulated protein by serotypes of Pasteurella haemolytica and Pasteurella trehalosi associated with pasteurellosis of cattle and sheep. A polyclonal antibody prepared against the purified 31-kDa periplasmic iron-regulated protein fromP. haemolytica serotype A1 showed that all P. haemolytica serotypes expressed similar 31-kDa proteins with identical N-terminal sequences, whereas P. trehalosiserotypes expressed immunologically different 29-kDa proteins with a different N-terminal sequence. Antibody to the 31-kDa iron-regulated protein was a useful tool to distinguish similarities and differences of the iron-regulated proteins of P. haemolytica andP. trehalosi.
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Nazifi, Narges, Mojtaba Tahmoorespur, Mohammad Hadi Sekhavati, Alireza Haghparast, and Mohammad Ali Behroozikhah. "Assessment of Signal Peptides to Optimize Interleukin 2 (IL-2) Folding and Expression." Current Proteomics 16, no. 3 (February 18, 2019): 188–98. http://dx.doi.org/10.2174/1570164615666181024113612.
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Background:Using a bacterial expression system such as Escherichia coli (E. coli) is very common for protein expression because of its simplicity, low cost and high efficiency.Objective:In order to express proteins that contain di-sulfide bands, an oxidative space such as the periplasmic environment of the bacteria is required. Therefore, a leader sequence which named Signal Peptide (SP) is needed to direct recombinant protein to fold in periplasmic space. Interleukin-2 (IL-2) is a prominent cytokine which known as growth factor for T-cells and typically produced by a variety of immune cells that stimulate and regulate inflammatory and immune responses.Methods:This study was designed to predict the best signal peptides to express IL-2 in E. coli. To predict the best signal peptides for the expression of IL-2 in Gram-negative bacteria (E. coli), forty-five sequences of SPs were extracted from data base. Some most important details such as n, h and c regions of signal peptides and their probability were studied through the signalP software. </P><P> Afterwards, physico–chemical features of SPs were analyzed by Portparam and Solpro tools. Finally, secretion-pathway and sub-cellular localization sites were evaluated by PRED-TAT and ProtcompB softwares.Results:At the end of the in-silico analyzes, it was determined that ccmH, PelB, traU, yohN, lolA, yhcN are the most reliable SPs, respectively, with highest score and best performing to express the IL-2 protein in E. coli.
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Schicklberger, Marcus, Clemens Bücking, Bjoern Schuetz, Heinrich Heide, and Johannes Gescher. "Involvement of theShewanella oneidensisDecaheme Cytochrome MtrA in the Periplasmic Stability of the β-Barrel Protein MtrB." Applied and Environmental Microbiology 77, no. 4 (December 17, 2010): 1520–23. http://dx.doi.org/10.1128/aem.01201-10.
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ABSTRACTTheShewanella oneidensisouter membrane β-barrel protein MtrB is part of a membrane-spanning protein complex (MtrABC) which is necessary for dissimilatory iron reduction. Quantitative PCR, heterologous gene expression, and mutant studies indicated that MtrA is required for periplasmic stability of MtrB. DegP depletion compensated for this MtrA dependence.
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Pilonieta, M. Carolina, Kimberly D. Erickson, Robert K. Ernst, and Corrella S. Detweiler. "A Protein Important for Antimicrobial Peptide Resistance, YdeI/OmdA, Is in the Periplasm and Interacts with OmpD/NmpC." Journal of Bacteriology 191, no. 23 (September 18, 2009): 7243–52. http://dx.doi.org/10.1128/jb.00688-09.
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ABSTRACT Antimicrobial peptides (AMPs) kill or prevent the growth of microbes. AMPs are made by virtually all single and multicellular organisms and are encountered by bacteria in diverse environments, including within a host. Bacteria use sensor-kinase systems to respond to AMPs or damage caused by AMPs. Salmonella enterica deploys at least three different sensor-kinase systems to modify gene expression in the presence of AMPs: PhoP-PhoQ, PmrA-PmrB, and RcsB-RcsC-RcsD. The ydeI gene is regulated by the RcsB-RcsC-RcsD pathway and encodes a 14-kDa predicted oligosaccharide/oligonucleotide binding-fold (OB-fold) protein important for polymyxin B resistance in broth and also for virulence in mice. We report here that ydeI is additionally regulated by the PhoP-PhoQ and PmrA-PmrB sensor-kinase systems, which confer resistance to cationic AMPs by modifying lipopolysaccharide (LPS). ydeI, however, is not important for known LPS modifications. Two independent biochemical methods found that YdeI copurifies with OmpD/NmpC, a member of the trimeric β-barrel outer membrane general porin family. Genetic analysis indicates that ompD contributes to polymyxin B resistance, and both ydeI and ompD are important for resistance to cathelicidin antimicrobial peptide, a mouse AMP produced by multiple cell types and expressed in the gut. YdeI localizes to the periplasm, where it could interact with OmpD. A second predicted periplasmic OB-fold protein, YgiW, and OmpF, another general porin, also contribute to polymyxin B resistance. Collectively, the data suggest that periplasmic OB-fold proteins can interact with porins to increase bacterial resistance to AMPs.
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Bonnard, N., A. Tresierra-Ayala, E. J. Bedmar, and M. J. Delgado. "Molybdate-dependent expression of the periplasmic nitrate reductase in Bradyrhizobium japonicum." Biochemical Society Transactions 33, no. 1 (February 1, 2005): 127–29. http://dx.doi.org/10.1042/bst0330127.
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The napEDABC genes of Bradyrhizobium japonicum encode the periplasmic nitrate reductase, an Mo-containing enzyme which catalyses the reduction of nitrate to nitrite when oxygen concentrations are limiting. In this bacterium, another set of genes, modABC, code for a high affinity ABC-type Mo transport system. A B. japonicum modA mutant has been obtained that is not capable of growing anaerobically with nitrate and lacks nitrate reductase activity. Under nitrate respiring conditions, when Mo concentrations are limiting, the B. japonicum modA mutant lacked both the 90 kDa protein corresponding to the NapA component of the periplasmic nitrate reductase, and the membrane-bound 25 kDa c-type cytochrome NapC. Regulatory studies using a napE–lacZ fusion indicated that napE expression was highly reduced in the modA mutant background when the cells were incubated anaerobically with nitrate under Mo-deficient conditions.
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Dietz, H., D. Pfeifle, and B. Wiedemann. "Location of N-acetylmuramyl-L-alanyl-D-glutamylmesodiaminopimelic acid, presumed signal molecule for beta-lactamase induction, in the bacterial cell." Antimicrobial Agents and Chemotherapy 40, no. 9 (September 1996): 2173–77. http://dx.doi.org/10.1128/aac.40.9.2173.
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Using a chromatographic method for the isolation and detection of periplasmic and cytoplasmic muropeptides avoiding radioactive labeling, we found that in the ampD-negative strain JRG582 the anhydromuropeptide N-acetylmuramyl-L-alanyl-D-glutamylmesodiaminopimelic acid (anhMurNAc tripeptide) accumulates not only in the cytoplasm but also in the periplasm. Simultaneously JRG582 carrying the Enterobacter cloacae genes ampC and ampR, which are necessary for the induction of beta-lactamase expression, overproduces beta-lactamase. We confirmed that the transmembrane protein AmpG transports a precursor muropeptide into the cytoplasm and that the formation of the anhMurNAc tripeptide takes place in the cytoplasm. anhMurNAc tripeptide can then be secreted into the periplasm. Therefore, the amount of anhMurNAc tripeptide in the cytoplasm is reduced not only by AmpD but also by transport out of the cell.
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Smith, Roxanne P., Andrew E. Whitten, Jason J. Paxman, Charlene M. Kahler, Martin J. Scanlon, and Begoña Heras. "Production, biophysical characterization and initial crystallization studies of the N- and C-terminal domains of DsbD, an essential enzyme inNeisseria meningitidis." Acta Crystallographica Section F Structural Biology Communications 74, no. 1 (January 1, 2018): 31–38. http://dx.doi.org/10.1107/s2053230x17017800.
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The membrane protein DsbD is a reductase that acts as an electron hub, translocating reducing equivalents from cytoplasmic thioredoxin to a number of periplasmic substrates involved in oxidative protein folding, cytochromecmaturation and oxidative stress defence. DsbD is a multi-domain protein consisting of a transmembrane domain (t-DsbD) flanked by two periplasmic domains (n-DsbD and c-DsbD). Previous studies have shown that DsbD is required for the survival of the obligate human pathogenNeisseria meningitidis. To help understand the structural and functional aspects ofN. meningitidisDsbD, the two periplasmic domains which are required for electron transfer are being studied. Here, the expression, purification and biophysical properties of n-NmDsbD and c-NmDsbD are described. The crystallization and crystallographic analysis of n-NmDsbD and c-NmDsbD are also described in both redox states, which differ only in the presence or absence of a disulfide bond but which crystallized in completely different conditions. Crystals of n-NmDsbDOx, n-NmDsbDRed, c-NmDsbDOxand c-NmDsbDReddiffracted to 2.3, 1.6, 2.3 and 1.7 Å resolution and belonged to space groupsP213,P321,P41andP1211, respectively.
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Harris, Chris R., and Thomas J. Silhavy. "Mapping an Interface of SecY (PrlA) and SecE (PrlG) by Using Synthetic Phenotypes and In Vivo Cross-Linking." Journal of Bacteriology 181, no. 11 (June 1, 1999): 3438–44. http://dx.doi.org/10.1128/jb.181.11.3438-3444.1999.
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ABSTRACT SecY and SecE are integral cytoplasmic membrane proteins that form an essential part of the protein translocation machinery inEscherichia coli. Sites of direct contact between these two proteins have been suggested by the allele-specific synthetic phenotypes exhibited by pairwise combinations of prlA andprlG signal sequence suppressor mutations in these genes. We have introduced cysteine residues within the first periplasmic loop of SecY and the second periplasmic loop of SecE, at a specific pair of positions identified by this genetic interaction. The expression of the cysteine mutant pair results in a dominant lethal phenotype that requires the presence of DsbA, which catalyzes the formation of disulfide bonds. A reducible SecY-SecE complex is also observed, demonstrating that these amino acids must be sufficiently proximal to form a disulfide bond. The use of cysteine-scanning mutagenesis enabled a second contact site to be discovered. Together, these two points of contact allow the modeling of a limited region of quaternary structure, establishing the first characterized site of interaction between these two proteins. This study proves that actual points of protein-protein contact can be identified by using synthetic phenotypes.
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Guzman, Luz-Maria, James J. Barondess, and Jon Beckwith. "FtsL, an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia coli." Journal of Bacteriology 174, no. 23 (December 1992): 7717–28. http://dx.doi.org/10.1128/jb.174.23.7717.1992.
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We have identified a gene involved in bacterial cell division, located immediately upstream of the ftsI gene in the min 2 region of the Escherichia coli chromosome. This gene, which we named ftsL , was detected through characterization of Tn phoA insertions in a plasmid containing this chromosomal region. Tn phoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the ftsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segment, and a periplasmic carboxy terminus. The ftsL gene is essential for cell growth and division. A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis. Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall. The FtsL protein is estimated to be present at about 20 to 40 copies per cell. The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization.
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Yuan, Liu, Meng Guo-Quan, Zhou Jian-Ping, Zhang Teng, Feng Juan, and Ren Zheng-long. "Expression, purification and identification of gibberellin-induced cysteine-rich protein ofGymanadenia conopsea." Chinese Journal of Agricultural Biotechnology 6, no. 3 (December 2009): 271–76. http://dx.doi.org/10.1017/s1479236209990155.
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AbstractPrimers bearing restriction enzyme sites forEcoR I andHind III were designed according to the known partial cDNA sequence for gibberellin-induced cysteine-rich protein and were then used to amplify the full-length open reading frame (ORF) and signal peptide-truncated fragment of thegcgasagene. Two fragments with lengths 319 and 238 bp were obtained and were further cloned into plasmid pET-32(a). Following transformation intoEscherichia coliBL21(DE3), the fusion proteins were observed to appear at ~26.0 and 25.2 kDa after induction from 1 mmol/l isopropyl-beta-D-thiogalactopyronoside (IPTG). The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) of an ultra-thin section revealed that the presence of signal peptide gave rise to the formation of an inclusion body located in the periplasmic space; however, the absence of signal peptide greatly enhanced the solubility of the target protein. The expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.
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Stewart, Valley, Peggy J. Bledsoe, and Stanly B. Williams. "Dual Overlapping Promoters Control napF (Periplasmic Nitrate Reductase) Operon Expression in Escherichia coli K-12." Journal of Bacteriology 185, no. 19 (October 1, 2003): 5862–70. http://dx.doi.org/10.1128/jb.185.19.5862-5870.2003.
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ABSTRACT Escherichia coli elaborates a flexible respiratory metabolism, involving differential synthesis of isoenzymes for many oxidation and reduction reactions. Periplasmic nitrate reductase, encoded by the napFDAGHBC operon, functions with concentrations of nitrate that are too low to support respiration by membrane-bound nitrate reductase. The napF operon control region exhibits unusual organization of DNA binding sites for the transcription regulators Fnr and NarP, which activate transcription in response to anaerobiosis and nitrate, respectively. Previous studies have shown that the napF operon control region directs synthesis of two transcripts whose 5′ ends differ by about 3 nucleotides. We constructed mutant control regions in which either of the two promoter −10 regions is inactivated. Results indicate that the downstream promoter (P1) was responsible for Fnr- and NarP-regulated napF operon expression, whereas transcription from the upstream promoter (P2) was activated only weakly by the Fnr protein and was inhibited by phospho-NarP and -NarL proteins. The physiological function of promoter P2 is unknown. These results establish the unconventional napF operon control region architecture, in which the major promoter P1 is activated by the Fnr protein bound to a site centered at −64.5 with respect to the transcription initiation site, working in conjunction with the phospho-NarP protein bound to a site centered at −44.5.