scholarly journals Shewanella oneidensis MR-1 Uses Overlapping Pathways for Iron Reduction at a Distance and by Direct Contact under Conditions Relevant for Biofilms

2005 ◽  
Vol 71 (8) ◽  
pp. 4414-4426 ◽  
Author(s):  
Douglas P. Lies ◽  
Maria E. Hernandez ◽  
Andreas Kappler ◽  
Randall E. Mielke ◽  
Jeffrey A. Gralnick ◽  
...  
Keyword(s):  
Direct Contact ◽  
Iron Reduction ◽  
Shewanella Oneidensis ◽  
Live Cells ◽  
Wild Type ◽  
Electron Shuttling ◽  
Electron Shuttles ◽  
Diffusion Limited ◽  
Culture Supernatants ◽  
Maximal Reduction

ABSTRACT We developed a new method to measure iron reduction at a distance based on depositing Fe(III) (hydr)oxide within nanoporous glass beads. In this “Fe-bead” system, Shewanella oneidensis reduces at least 86.5% of the iron in the absence of direct contact. Biofilm formation accompanies Fe-bead reduction and is observable both macro- and microscopically. Fe-bead reduction is catalyzed by live cells adapted to anaerobic conditions, and maximal reduction rates require sustained protein synthesis. The amount of reactive ferric iron in the Fe-bead system is available in excess such that the rate of Fe-bead reduction is directly proportional to cell density; i.e., it is diffusion limited. Addition of either lysates prepared from anaerobic cells or exogenous electron shuttles stimulates Fe-bead reduction by S. oneidensis, but iron chelators or additional Fe(II) do not. Neither dissolved Fe(III) nor electron shuttling activity was detected in culture supernatants, implying that the mediator is retained within the biofilm matrix. Strains with mutations in omcB or mtrB show about 50% of the wild-type levels of reduction, while a cymA mutant shows less than 20% of the wild-type levels of reduction and a menF mutant shows insignificant reduction. The Fe-bead reduction defect of the menF mutant can be restored by addition of menaquinone, but menaquinone itself cannot stimulate Fe-bead reduction. Because the menF gene encodes the first committed step of menaquinone biosynthesis, no intermediates of the menaquinone biosynthetic pathway are used as diffusible mediators by this organism to promote iron reduction at a distance. CymA and menaquinone are required for both direct and indirect mineral reduction, whereas MtrB and OmcB contribute to but are not absolutely required for iron reduction at a distance.

scholarly journals Flavin Electron Shuttles Dominate Extracellular Electron Transfer by Shewanella oneidensis

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Nicholas J. Kotloski ◽  
Jeffrey A. Gralnick
Keyword(s):  
Electron Transfer ◽  
Direct Electron Transfer ◽  
Shewanella Oneidensis ◽  
Phenotypic Characterization ◽  
Extracellular Electron Transfer ◽  
Wild Type ◽  
Content Type ◽  
Electron Shuttling ◽  
Electron Shuttles ◽  
Set Up

ABSTRACT Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δbfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. IMPORTANCE Extracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.

scholarly journals The Mtr Respiratory Pathway Is Essential for Reducing Flavins and Electrodes in Shewanella oneidensis

2009 ◽  
Vol 192 (2) ◽  
pp. 467-474 ◽  
Author(s):  
Dan Coursolle ◽  
Daniel B. Baron ◽  
Daniel R. Bond ◽  
Jeffrey A. Gralnick
Keyword(s):  
Shewanella Oneidensis ◽  
Flavin Mononucleotide ◽  
Wild Type ◽  
Respiratory Pathway ◽  
Biologically Relevant ◽  
Electron Shuttles ◽  
Substrate Reduction ◽  
Oxidized Iron ◽  
Associated Proteins ◽  
Current Production

ABSTRACT The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.

scholarly journals GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

2009 ◽  
Vol 134 (6) ◽  
pp. 489-521 ◽  
Author(s):  
Fraser J. Moss ◽  
P.I. Imoukhuede ◽  
Kimberly Scott ◽  
Jia Hu ◽  
Joanna L. Jankowsky ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Fluorescent Proteins ◽  
Resonance Energy ◽  
High Order ◽  
Live Cells ◽  
Gaba Uptake ◽  
Cell Periphery ◽  
Wild Type ◽  
Gaba Transporter ◽  
Amplitude Component

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ∼30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

scholarly journals Sulfur-mediated electron shuttling during bacterial iron reduction

Science ◽  
2014 ◽  
Vol 344 (6187) ◽  
pp. 1039-1042 ◽  
Author(s):  
T. M. Flynn ◽  
E. J. O'Loughlin ◽  
B. Mishra ◽  
T. J. DiChristina ◽  
K. M. Kemner
Keyword(s):  
Iron Reduction ◽  
Electron Shuttling

scholarly journals Biophysical basis underlying dynamic Lck activation visualized by ZapLck FRET biosensor

2019 ◽  
Vol 5 (6) ◽  
pp. eaau2001 ◽  
Author(s):  
Rongxue Wan ◽  
Jenny Wu ◽  
Mingxing Ouyang ◽  
Lei Lei ◽  
Jiaming Wei ◽  
...  
Keyword(s):  
T Cells ◽  
Kinase Activity ◽  
Diffusion Rate ◽  
Live Cells ◽  
Wild Type ◽  
Tcr Signaling ◽  
Lck Kinase ◽  
Full Activation

Lck plays crucial roles in TCR signaling. We developed a new and sensitive FRET biosensor (ZapLck) to visualize Lck kinase activity with high spatiotemporal resolutions in live cells. ZapLck revealed that 62% of Lck signal was preactivated in T-cells. In Lck-deficient JCam T-cells, Lck preactivation was abolished, which can be restored to 51% by reconstitution with wild-type Lck (LckWT) but not a putatively inactive mutant LckY394F. LckWT also showed a stronger basal Lck-Lck interaction and a slower diffusion rate than LckY394F. Interestingly, aggregation of TCR receptors by antibodies in JCam cells led to a strong activation of reconstituted LckY394F similar to LckWT. Both activated LckY394F and LckWT diffused more slowly and displayed increased Lck-Lck interaction at a similar level. Therefore, these results suggest that a phosphorylatable Y394 is necessary for the basal-level interaction and preactivation of LckWT, while antibody-induced TCR aggregation can trigger the full activation of LckY394F.

scholarly journals Τhe Nematicidal Potential of Bioactive Streptomyces Strains Isolated from Greek Rhizosphere Soils Tested on Arabidopsis Plants of Varying Susceptibility to Meloidogyne spp.

Plants ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 699
Author(s):  
Christianna Meidani ◽  
Alexandros Savvidis ◽  
Evaggelia Lampropoulou ◽  
Aggeliki Sagia ◽  
Efstathios Katsifas ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Arabidopsis Thaliana ◽  
Meloidogyne Incognita ◽  
Meloidogyne Javanica ◽  
Wild Type ◽  
Biological Cycle ◽  
Second Stage ◽  
Cycle Arrest ◽  
Meloidogyne Spp ◽  
Culture Supernatants

A total of 461 indigenous Streptomycetes strains recovered from various Greek rhizosphere habitats were tested for their bioactivity. All isolates were examined for their ability to suppress the growth of 12 specific target microorganisms. Twenty-six were found to exert antimicrobial activity and were screened for potential nematicidal action. S. monomycini ATHUBA 220, S. colombiensis ATHUBA 438, S. colombiensis ATHUBA 431, and S. youssoufensis ATHUBA 546 were proved to have a nematicidal effect and thus were further sequenced. Batch culture supernatants and solvent extracts were assessed for paralysis on Meloidogyne javanica and Meloidogyne incognita second-stage juveniles (J2). The solvent extracts of S. monomycini ATHUBA 220 and S. colombiensis ATHUBA 438 had the highest paralysis rates, so these Streptomycetes strains were further on tested for nematodes’ biological cycle arrest on two Arabidopsis thaliana plants; the wild type (Col-0) and the katanin mutant fra2, which is susceptible to M. incognita. Interestingly, S. monomycini ATHUBA 220 and S. colombiensis ATHUBA 438 were able to negatively affect the M. incognita biological cycle in Col-0 and fra2 respectively, and increased growth in Col-0 upon M. incognita infection. However, they were ineffective against M. javanica. Fra2 plants were also proved susceptible to M. javanica infestation, with a reduced growth upon treatments with the Streptomyces strains. The nematicidal action and the plant-growth modulating abilities of the selected Streptomycetes strains are discussed.

scholarly journals Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

2010 ◽  
Vol 192 (18) ◽  
pp. 4651-4659 ◽  
Author(s):  
Wendy D. Smith ◽  
Jonathan A. Pointon ◽  
Emily Abbot ◽  
Hae Joo Kang ◽  
Edward N. Baker ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Human Keratinocytes ◽  
Mass Spectrometry Analysis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Liquid Chromatography Tandem Mass ◽  
Protein Adhesion ◽  
Culture Supernatants

ABSTRACT Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.

scholarly journals N- and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1229-1236 ◽  
Author(s):  
MR Fibi ◽  
P Hermentin ◽  
JU Pauly ◽  
L Lauffer ◽  
G Zettlmeissl
Keyword(s):  
Transient Expression ◽  
Cell Lines ◽  
Recombinant Human Erythropoietin ◽  
Biologically Active ◽  
Wild Type ◽  
Expression Levels ◽  
Human Erythropoietin ◽  
Culture Supernatants ◽  
Secretion Rates

Single-site glycomuteins of recombinant human erythropoietin (rhuEpo) were constructed and transiently and stably expressed in BHK-21 cells. The transient expression levels varied among muteins, being highest for mutein rhuEpoGln24 followed by wild-type rhuEpo (rhuEpowt). All other glycomuteins, including rhuEpoGln38, rhuEpoGln83, rhuEpoThr126, and rhuEpoGly126, were secreted at lower levels than rhuEpowt. Muteins expressed in stable cell lines showed similar differences in expression levels. Also each mutein could be affinity-purified from culture supernatants, and was biologically active in vivo. Based on secretion rates from BHK-21 cells, the most potent erythropoietin was rhuEpoGln24. This mutein is also considered to have biologic activities that are superior to rhuEpowt.

Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants

2009 ◽  
Vol 191 (7) ◽  
pp. 571-581 ◽  
Author(s):  
M. Victoria Delpino ◽  
Diego J. Comerci ◽  
Mary Ann Wagner ◽  
Michel Eschenbrenner ◽  
Cesar V. Mujer ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Wild Type ◽  
Culture Supernatants

scholarly journals Lipopolysaccharide biosynthesis-related genes are required for colony pigmentation of Porphyromonas gingivalis

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1282-1293 ◽  
Author(s):  
Keiko Sato ◽  
Nobuo Kido ◽  
Yukitaka Murakami ◽  
Charles I. Hoover ◽  
Koji Nakayama ◽  
...  
Keyword(s):  
Cell Surface ◽  
Porphyromonas Gingivalis ◽  
Dependent Manner ◽  
Wild Type ◽  
Gene Encoding ◽  
Lipopolysaccharide Biosynthesis ◽  
Ladder Pattern ◽  
Surface Polysaccharides ◽  
Culture Supernatants ◽  
Dose Dependent

The periodontopathic bacterium Porphyromonas gingivalis forms pigmented colonies when incubated on blood agar plates as a result of accumulation of μ-oxo haem dimer on the cell surface. Gingipain–adhesin complexes are responsible for production of μ-oxo haem dimer from haemoglobin. Non-pigmented mutants (Tn6-5, Tn7-1, Tn7-3 and Tn10-4) were isolated from P. gingivalis by Tn4351 transposon mutagenesis [Hoover & Yoshimura (1994), FEMS Microbiol Lett 124, 43–48]. In this study, we found that the Tn6-5, Tn7-1 and Tn7-3 mutants carried Tn4351 DNA in a gene homologous to the ugdA gene encoding UDP-glucose 6-dehydrogenase, a gene encoding a putative group 1 family glycosyltransferase and a gene homologous to the rfa gene encoding ADP heptose-LPS heptosyltransferase, respectively. The Tn10-4 mutant carried Tn4351 DNA at the same position as that for Tn7-1. Gingipain activities associated with cells of the Tn7-3 mutant (rfa) were very weak, whereas gingipain activities were detected in the culture supernatants. Immunoblot and mass spectrometry analyses also revealed that gingipains, including their precursor forms, were present in the culture supernatants. A lipopolysaccharide (LPS) fraction of the rfa deletion mutant did not show the ladder pattern that was usually seen for the LPS of the wild-type P. gingivalis. A recombinant chimera gingipain was able to bind to an LPS fraction of the wild-type P. gingivalis in a dose-dependent manner. These results suggest that the rfa gene product is associated with biosynthesis of LPS and/or cell-surface polysaccharides that can function as an anchorage for gingipain–adhesin complexes.

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