scholarly journals The DyP-type peroxidase DtpA is a Tat-substrate required for GlxA maturation and morphogenesis in Streptomyces

Open Biology ◽  
2016 ◽  
Vol 6 (1) ◽  
pp. 150149 ◽  
Author(s):  
Marloes L. C. Petrus ◽  
Erik Vijgenboom ◽  
Amanda K. Chaplin ◽  
Jonathan A. R. Worrall ◽  
Gilles P. van Wezel ◽  
...  
Keyword(s):  
Active Site ◽  
Deletion Mutants ◽  
Copper Trafficking ◽  
Twin Arginine Translocation ◽  
Trafficking Pathway ◽  
Aerial Growth ◽  
Pellet Formation ◽  
Morphological Defects ◽  
Related Proteins ◽  
Copper Oxidase

The filamentous bacterium Streptomyces lividans depends on the radical copper oxidase GlxA for the formation of reproductive aerial structures and, in liquid environments, for the formation of pellets. Incorporation of copper into the active site is essential for the formation of a cross-linked tyrosyl-cysteine cofactor, which is needed for enzymatic activity. In this study, we show a crucial link between GlxA maturation and a group of copper-related proteins including the chaperone Sco and a novel DyP-type peroxidase hereinafter called DtpA. Under copper-limiting conditions, the sco and dtpA deletion mutants are blocked in aerial growth and pellet formation, similarly to a glxA mutant. Western blot analysis showed that GlxA maturation is perturbed in the sco and dtpA mutants, but both maturation and morphology can by rescued by increasing the bioavailability of copper. DtpA acts as a peroxidase in the presence of GlxA and is a substrate for the twin-arginine translocation (Tat) translocation pathway. In agreement, the maturation status of GlxA is also perturbed in tat mutants, which can be compensated for by the addition of copper, thereby partially restoring their morphological defects. Our data support a model wherein a copper-trafficking pathway and Tat-dependent secretion of DtpA link to the GlxA-dependent morphogenesis pathway.

scholarly journals Manganese Superoxide Dismutase inSaccharomyces cerevisiaeAcquires Its Metal Co-factor through a Pathway Involving the Nramp Metal Transporter, Smf2p

2001 ◽  
Vol 276 (50) ◽  
pp. 47556-47562 ◽  
Author(s):  
Edward E-Ching Luk ◽  
Valeria Cizewski Culotta
Keyword(s):  
Superoxide Dismutase ◽  
Manganese Superoxide Dismutase ◽  
Protein Glycosylation ◽  
Copper Trafficking ◽  
Metal Transporter ◽  
Trafficking Pathway ◽  
Manganese Superoxide ◽  
Steady State Levels ◽  
Copper Zinc ◽  
Intracellular Vesicles

Eukaryotes express both copper/zinc (SOD1)- and manganese (SOD2)-requiring superoxide dismutase enzymes that guard against oxidative damage. Although SOD1 acquires its copper through a specific copper trafficking pathway, nothing is known regarding the intracellular manganese trafficking pathway for SOD2. We demonstrate here that inSaccharomyces cerevisiaecells delivery of manganese to SOD2 in the mitochondria requires the Nramp metal transporter, Smf2p. SOD2 activity is greatly diminished insmf2Δ mutants, even though the mature SOD2 polypeptide accumulates to normal levels in mitochondria. Treatingsmf2Δ cells with manganese supplements corrected the SOD2 defect, as did elevating intracellular manganese through mutations inPMR1.Hence, manganese appears to be inaccessible to mitochondrial SOD2 insmf2mutants. Cells lackingSMF2also exhibited defects in manganese-dependent steps in protein glycosylation and showed an overall decrease in steady-state levels of accumulated manganese. By comparison, mutations in the cell surface Nramp transporter, Smf1p, had very little impact on manganese accumulation and trafficking. Smf2p resides in intracellular vesicles and shows no evidence of plasma membrane localization, even in anend4mutant blocked for endocytosis. We propose a model in which Smf2p-containing vesicles play a central role in manganese trafficking to the mitochondria and other cellular sites as well.

scholarly journals Role of the Twin-Arginine Translocation Pathway in Staphylococcus

2009 ◽  
Vol 191 (19) ◽  
pp. 5921-5929 ◽  
Author(s):  
Lalitha Biswas ◽  
Raja Biswas ◽  
Christiane Nerz ◽  
Knut Ohlsen ◽  
Martin Schlag ◽  
...  
Keyword(s):  
Functional Unit ◽  
Signal Sequence ◽  
Protein A ◽  
Mouse Kidney ◽  
Deletion Mutants ◽  
Twin Arginine Translocation ◽  
High Affinity ◽  
Tat Pathway ◽  
First Time

ABSTRACT In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (ΔtatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of ΔtatAC and Δtat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.

Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli: comparison with functionally related proteins

2001 ◽  
Vol 310 (2) ◽  
pp. 449-470 ◽  
Author(s):  
Nicolas Foloppe ◽  
Johan Sagemark ◽  
Kerstin Nordstrand ◽  
Kurt D. Berndt ◽  
Lennart Nilsson
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Structure Dynamics ◽  
Related Proteins

scholarly journals Proteomics Investigation of the Time Course Responses of RAW264.7 Macrophages to Infections With the Wild-Type and Twin-Arginine Translocation Mutant Strains of Brucella melitensis

2021 ◽  
Vol 11 ◽  
Author(s):  
Xin Yan ◽  
Sen Hu ◽  
Yan Yang ◽  
Da Xu ◽  
Wenxing Liu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Time Course ◽  
Nitrosative Stress ◽  
Brucella Melitensis ◽  
No Production ◽  
Wild Type ◽  
Twin Arginine Translocation ◽  
Raw264.7 Macrophages ◽  
Mutant Strains ◽  
Related Proteins

Brucella, a notorious intracellular pathogen, causes chronic infections in many mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane; protein substrates translocated by Brucella include ABC transporters, oxidoreductases, and cell envelope biosynthesis proteins. Previously, we showed that a Tat mutant of Brucella melitensis M28 exhibits reduced survival within murine macrophages. In this study, we compared the host responses elicited by wild-type M28 and its Tat-mutant strains ex vivo. We utilized label-free quantitative proteomics to assess proteomic changes in RAW264.7 macrophages after infection with M28 and its Tat mutants. A total of 6085 macrophage proteins were identified with high confidence, and 79, 50, and 99 proteins were differentially produced upon infection with the Tat mutant at 4, 24, and 48 hpi, respectively, relative to the wild-type infection. Gene ontology and KEGG enrichment analysis indicated that immune response-related proteins were enriched among the upregulated proteins. Compared to the wild-type M28 infection, the most upregulated proteins upon Tat-mutant infection included the cytosolic nucleic acid signaling pathway-related proteins IFIH1, DHX58, IFI202, IFI204, and ISG15 and the NF-κB signaling pathway-related proteins PTGS2, CD40, and TRAF1, suggesting that the host increases the production of these proteins in response to Tat mutant infection. Upregulation of some proteins was further verified by a parallel reaction monitoring (PRM) assay. ELISA and qRT-PCR assays indicated that Tat mutant infection significantly induced proinflammatory cytokine (TNF-α and IL-6) and nitric oxide (NO) production. Finally, we showed that the Tat mutant displays higher sensitivity to nitrosative stress than the wild type and that treatment with the NO synthase inhibitor L-NMMA significantly increases the intracellular survival of the Tat mutant, indicating that NO production contributes to restricting Tat mutant survival within macrophages. Collectively, this work improves our understanding of host immune responses to Tat mutants and provides insights into the mechanisms underlying the attenuated virulence of Tat mutants.

The small GTPase KlRho5 responds to oxidative stress and affects cytokinesis

2021 ◽  
Vol 134 (18) ◽  
Author(s):  
Marius Musielak ◽  
Carolin C. Sterk ◽  
Felix Schubert ◽  
Christian Meyer ◽  
Achim Paululat ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Dynamics ◽  
Kluyveromyces Lactis ◽  
Neck Region ◽  
Small Gtpase ◽  
Nutrient Sensing ◽  
Deletion Mutants ◽  
Nucleotide Exchange ◽  
Morphological Defects

ABSTRACT Rho5 is the yeast homolog of the human small GTPase Rac1. We characterized the genes encoding Rho5 and the subunits of its dimeric activating guanine-nucleotide-exchange factor (GEF), Dck1 and Lmo1, in the yeast Kluyveromyces lactis. Rapid translocation of the three GFP-tagged components to mitochondria upon oxidative stress and carbon starvation indicate a similar function of KlRho5 in energy metabolism and mitochondrial dynamics as described for its Saccharomyces cerevisiae homolog. Accordingly, Klrho5 deletion mutants are hyper-resistant towards hydrogen peroxide. Moreover, synthetic lethalities of rho5 deletions with key components in nutrient sensing, such as sch9 and gpr1, are not conserved in K. lactis. Instead, Klrho5 deletion mutants display morphological defects with strengthened lateral cell walls and protruding bud scars. The latter result from aberrant cytokinesis, as observed by following the budding process in vivo and by transmission electron microscopy of the bud neck region. This phenotype can be suppressed by KlCDC42G12V, which encodes a hyper-active variant. Data from live-cell fluorescence microscopy support the notion that KlRho5 interferes with the actin moiety of the contractile actomyosin ring, with consequences different from those previously reported for mutants lacking myosin.

scholarly journals Widespread Distribution and Functional Specificity of the Copper Importer CcoA: Distinct Cu Uptake Routes for Bacterial Cytochrome c Oxidases

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Bahia Khalfaoui-Hassani ◽  
Hongjiang Wu ◽  
Crysten E. Blaby-Haas ◽  
Yang Zhang ◽  
Federica Sandri ◽  
...  
Keyword(s):  
Rhodobacter Capsulatus ◽  
Biochemical Characterization ◽  
Genomic Analysis ◽  
Primary Electron ◽  
Major Facilitator Superfamily ◽  
Close Relative ◽  
Comparative Genomic ◽  
Copper Uptake ◽  
Copper Trafficking ◽  
Copper Oxidase

ABSTRACT Cytochrome c oxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (CuB) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the CuB atom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacterium Rhodobacter capsulatus indicated that the copper atom needed for the CuB site of cbb 3-type cytochrome c oxidase (cbb 3-Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis of CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochrome c oxidases. To define the specificity of CcoA activity, we investigated its function in Rhodobacter sphaeroides, a close relative of R. capsulatus that contains both cbb 3- and aa 3-Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production of cbb 3-Cox and not that of aa 3-Cox was affected. We therefore concluded that CcoA is dedicated solely to cbb 3-Cox biogenesis, establishing that distinct copper uptake systems provide the CuB atoms to the catalytic sites of these two similar cytochrome c oxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions. IMPORTANCE The cbb 3- and aa 3-type cytochrome c oxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and cofactor composition, yet they all share a conserved heme-copper binuclear site within their catalytic subunit. In this study, we show that the copper atoms of the catalytic center of two similar cytochrome c oxidases from this superfamily are provided by different copper uptake systems during their biogenesis. This finding illustrates different strategies by which organisms fine-tune the trafficking of copper, which is an essential but toxic micronutrient.

scholarly journals The C-terminus of factor H: monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor H-related proteins

1998 ◽  
Vol 331 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Wolfgang M. PRODINGER ◽  
Jens HELLWAGE ◽  
Martin SPRUTH ◽  
Manfred P. DIERICH ◽  
Peter F. ZIPFEL
Keyword(s):  
Monoclonal Antibodies ◽  
Unique Feature ◽  
Factor H ◽  
Deletion Mutants ◽  
Heparin Binding ◽  
Specific Detection ◽  
Short Consensus Repeats ◽  
C Terminus ◽  
Inhibitory Antibodies ◽  
Related Proteins

We have generated monoclonal antibodies (mAbs) specific for the C-terminus of factor H that can be used as inhibitory antibodies for heparin binding and for the specific detection of factor H and factor H-related proteins (FHRs) in plasma and triacylglycerol-rich lipoproteins. Four distinct mAbs were established: IXF9 (IgG1), VD3 (IgG2a), VIG8 (IgG1) and IIC5 (IgG1). Each reacts specifically with FHR-1 and factor H (and also with FHR-2 in the case of VIG8), but none binds to the related FHR-3 and FHR-4 proteins nor to factor H-like protein 1. By the use of deletion mutants of factor H and by comparing the reactivity with FHR-1 and FHR-2, the binding epitopes of the mAbs were identified and localized to different short consensus repeats (SCRs): mAbs IXF9 and VD3 bind to related or even identical sites within SCR 18 (factor H) and SCR 3 (FHR-1) respectively. mAbs VIG8 and IIC5 bind to different epitopes located within SCRs 19 to 20 of factor H and SCRs 4 to 5 of FHR-1 respectively. Only mAb VIG8 reacts with the corresponding SCRs 3 to 4 of FHR-2. These antibodies are useful for the detection of the corresponding proteins in biological specimens such as fractions of lipoproteins. In addition, mAb VIG8 has the unique feature of inhibiting binding of factor H to heparin. Given the recent identification of a heparin- and a C3b-binding domain within the C-terminus of factor H, these mAbs should provide useful tools for functional analysis and for the precise localization of the domain(s) required for this interaction.

Cytochrome cbb3 oxidase and bacterial microaerobic metabolism

2002 ◽  
Vol 30 (4) ◽  
pp. 653-658 ◽  
Author(s):  
R. S. Pitcher ◽  
T. Brittain ◽  
N. J. Watmugh
Keyword(s):  
Active Site ◽  
Reduction Potential ◽  
Oxygen Affinity ◽  
Pseudomonas Stutzeri ◽  
Spectroscopic Characterization ◽  
High Oxygen Affinity ◽  
Copper Oxidase ◽  
Reduced State

Cytochrome cbb3 oxidase is a member of the haem-copper oxidase superfamily. It is characterized by its high oxygen affinity, while retaining the ability to pump protons. These attributes are central to its proposed role in bacterial microaerobic metabolism. Recent spectroscopic characterization of both the cytochrome cbb3 oxidase complex from Pseudomonas stutzeri and the dihaem ccoP subunit expressed separately in Escherichia coli has revealed the presence of a low-spin His/His co-ordinated c-type cytochrome. The low midpoint reduction potential of this haem (Em < + 100 mV), together with its unexpected ability to bind CO in the reduced state at the expense of the distal histidine ligand, raises questions about the role of the ccoP subunit in the delivery of electrons to the active site.

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