scholarly journals HupO, a Novel Regulator Involved in Thiosulfate-Responsive Control of HupSL [NiFe]-Hydrogenase Synthesis in Thiocapsa roseopersicina

2016 ◽  
Vol 82 (7) ◽  
pp. 2039-2049 ◽  
Author(s):  
Ildikó K. Nagy ◽  
Kornél L. Kovács ◽  
Gábor Rákhely ◽  
Gergely Maróti
Keyword(s):  
Transcriptional Repression ◽  
Rhodobacter Capsulatus ◽  
Sulfur Metabolism ◽  
Enzyme Synthesis ◽  
Regulatory Control ◽  
Bacterial Cells ◽  
Purple Sulfur Bacterium ◽  
Content Type ◽  
Thiocapsa Roseopersicina

ABSTRACT[NiFe]-hydrogenases are regulated by various factors to fulfill their physiological functions in bacterial cells. The photosynthetic purple sulfur bacteriumThiocapsa roseopersicinaharbors four functional [NiFe]-hydrogenases: HynSL, HupSL, Hox1, and Hox2. Most of these hydrogenases are functionally linked to sulfur metabolism, and thiosulfate has a central role in this organism. The membrane-associated Hup hydrogenases have been shown to play a role in energy conservation through hydrogen recycling. The expression of Hup-type hydrogenases is regulated by H2inRhodobacter capsulatusandCupriavidus necator; however, it has been shown that the corresponding hydrogen-sensing system is nonfunctional inT. roseopersicinaand that thiosulfate is a regulating factor ofhupexpression. Here, we describe the discovery and analysis of mutants of a putative regulator (HupO) of the Hup hydrogenase inT. roseopersicina. HupO appears to mediate the transcriptional repression of Hup enzyme synthesis under low-thiosulfate conditions. We also demonstrate that the presence of the Hox1 hydrogenase strongly influences Hup enzyme synthesis in thathupexpression was decreased significantly in thehox1mutant. This reduction in Hup synthesis could be reversed by mutation ofhupO, which resulted in strongly elevatedhupexpression, as well as Hup protein levels, and concomitantin vivohydrogen uptake activity in thehox1mutant. However, this regulatory control was observed only at low thiosulfate concentrations. Additionally, weak hydrogen-dependenthupexpression was shown in thehupOmutant strain lacking the Hox1 hydrogenase. HupO-mediated Hup regulation therefore appears to link thiosulfate metabolism and the hydrogenase network inT. roseopersicina.

scholarly journals Assessment of Minocycline and Polymyxin B Combination against Acinetobacter baumannii

2015 ◽  
Vol 59 (5) ◽  
pp. 2720-2725 ◽  
Author(s):  
Dana R. Bowers ◽  
Henry Cao ◽  
Jian Zhou ◽  
Kimberly R. Ledesma ◽  
Dongxu Sun ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Utility ◽  
Efflux Pump ◽  
Polymyxin B ◽  
Intracellular Concentration ◽  
Bacterial Cells ◽  
Efflux Pump Inhibitor ◽  
Content Type

ABSTRACTAntimicrobial resistance amongAcinetobacter baumanniiis increasing worldwide, often necessitating combination therapy. The clinical utility of using minocycline with polymyxin B is not well established. In this study, we investigated the activity of minocycline and polymyxin B against 1 laboratory isolate and 3 clinical isolates ofA. baumannii. Minocycline susceptibility testing was performed with and without an efflux pump inhibitor, phenylalanine-arginine β-naphthylamide (PAβN). The intracellular minocycline concentration was determined with and without polymyxin B (0.5 μg/ml). Time-kill studies were performed over 24 h using approximately 106CFU/ml of each strain with clinically relevant minocycline concentrations (2 μg/ml and 8 μg/ml), with and without polymyxin B (0.5 μg/ml). Thein vivoefficacy of the combination was assessed in a neutropenic murine pneumonia model. Infected animals were administered minocycline (50 mg/kg), polymyxin B (10 mg/kg), or both to achieve clinically equivalent exposures in humans. A reduction in the minocycline MIC (≥4×) was observed in the presence of PAβN. The intracellular concentration andin vitrobactericidal effect of minocycline were both enhanced by polymyxin B. With 2 minocycline-susceptible strains, the bacterial burden in lung tissue at 24 h was considerably reduced by the combination compared to monotherapy with minocycline or polymyxin B. In addition, the combination prolonged survival of animals infected with a minocycline-susceptible strain. Polymyxin B increased the intracellular concentration of minocycline in bacterial cells and enhanced the bactericidal activity of minocycline, presumably due to efflux pump disruption. The clinical utility of this combination should be further investigated.

scholarly journals Biochemical Characterization of Individual Components of the Allochromatium vinosum DsrMKJOP Transmembrane Complex Aids Understanding of Complex Function In Vivo

2010 ◽  
Vol 192 (24) ◽  
pp. 6369-6377 ◽  
Author(s):  
Fabian Grein ◽  
Inês A. C. Pereira ◽  
Christiane Dahl
Keyword(s):  
Biochemical Characterization ◽  
Sulfur Metabolism ◽  
Complex Function ◽  
Redox Potentials ◽  
Allochromatium Vinosum ◽  
Purple Sulfur Bacterium ◽  
Membrane Anchoring ◽  
Integral Proteins ◽  
First Time

ABSTRACT The DsrMKJOP transmembrane complex has a most important function in dissimilatory sulfur metabolism and consists of cytoplasmic, periplasmic, and membrane integral proteins carrying FeS centers and b- and c-type cytochromes as cofactors. In this study, the complex was isolated from the purple sulfur bacterium Allochromatium vinosum and individual components were characterized as recombinant proteins. The two integral membrane proteins DsrM and DsrP were successfully produced in Escherichia coli C43(DE3) and C41(DE3), respectively. DsrM was identified as a diheme cytochrome b, and the two hemes were found to be in low-spin state. Their midpoint redox potentials were determined to be +60 and +110 mV. Although no hemes were predicted for DsrP, it was also clearly identified as a b-type cytochrome. To the best of our knowledge, this is the first time that heme binding has been experimentally proven for a member of the NrfD protein family. Both cytochromes were partly reduced after addition of a menaquinol analogue, suggesting interaction with quinones in vivo. DsrO and DsrK were both experimentally proven to be FeS-containing proteins. In addition, DsrK was shown to be membrane associated, and we propose a monotopic membrane anchoring for this protein. Coelution assays provide support for the proposed interaction of DsrK with the soluble cytoplasmic protein DsrC, which might be its substrate. A model for the function of DsrMKJOP in the purple sulfur bacterium A. vinosum is presented.

scholarly journals Cyanobacterial-Type, Heteropentameric, NAD+-Reducing NiFe Hydrogenase in the Purple Sulfur Photosynthetic Bacterium Thiocapsa roseopersicina

2004 ◽  
Vol 70 (2) ◽  
pp. 722-728 ◽  
Author(s):  
Gábor Rákhely ◽  
Ákos T. Kovács ◽  
Gergely Maróti ◽  
Barna D. Fodor ◽  
Gyula Csanádi ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Photosynthetic Bacterium ◽  
Hydrogenase Activity ◽  
Structural Genes ◽  
Purple Sulfur Bacterium ◽  
Thiocapsa Roseopersicina ◽  
Reverse Transcription Pcr ◽  
Single Transcript

ABSTRACT Structural genes coding for two membrane-associated NiFe hydrogenases in the phototrophic purple sulfur bacterium Thiocapsa roseopersicina (hupSL and hynSL) have recently been isolated and characterized. Deletion of both hydrogenase structural genes did not eliminate hydrogenase activity in the cells, and considerable hydrogenase activity was detected in the soluble fraction. The enzyme responsible for this activity was partially purified, and the gene cluster coding for a cytoplasmic, NAD+-reducing NiFe hydrogenase was identified and sequenced. The deduced gene products exhibited the highest similarity to the corresponding subunits of the cyanobacterial bidirectional soluble hydrogenases (HoxEFUYH). The five genes were localized on a single transcript according to reverse transcription-PCR experiments. A σ54-type promoter preceded the gene cluster, suggesting that there was inducible expression of the operon. The Hox hydrogenase was proven to function as a truly bidirectional hydrogenase; it produced H2 under nitrogenase-repressed conditions, and it recycled the hydrogen produced by the nitrogenase in cells fixing N2. In-frame deletion of the hoxE gene eliminated hydrogen evolution derived from the Hox enzyme in vivo, although it had no effect on the hydrogenase activity in vitro. This suggests that HoxE has a hydrogenase-related role; it likely participates in the electron transfer processes. This is the first example of the presence of a cyanobacterial-type, NAD+-reducing hydrogenase in a phototrophic bacterium that is not a cyanobacterium. The potential physiological implications are discussed.

scholarly journals Shape Changes and Interaction Mechanism of Escherichia coli Cells Treated with Sericin and Use of a Sericin-Based Hydrogel for Wound Healing

2016 ◽  
Vol 82 (15) ◽  
pp. 4663-4672 ◽  
Author(s):  
Rui Xue ◽  
Yalong Liu ◽  
Qingsong Zhang ◽  
Congcong Liang ◽  
Huazhen Qin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Cell Membrane ◽  
Interaction Mechanism ◽  
Bacterial Cells ◽  
Content Type ◽  
E Coli ◽  
Membrane Blebbing ◽  
Sterile Gauze

ABSTRACTTo verify the interaction mechanism between sericin andEscherichia coli, especially the morphological and structural changes in the bacterial cells, the antimicrobial activity of sericin againstE. colias a model for Gram-negative bacteria was investigated. The antibacterial activity of sericin onE. coliand the interaction mechanism were investigated in this study by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with sericin. The changes in morphology and cellular compositions of bacterial cells treated with sericin were observed by an inverted fluorescence microscope, scanning electron microscopy, and transmission electron microscopy. Changes in electrical conductivity, total sugar concentration of the broth for the bacteria, and protein expression of the bacteria were determined to investigate the permeability of the cell membrane. A sericin-based hydrogel was prepared for anin vivostudy of wound dressing. The results showed that the antibacterial activity of the hydrogel increased with the increase in the concentration of sericin from 10 g/liter to 40 g/liter. The introduction of sericin induces membrane blebbing ofE. colicells caused by antibiotic action on the cell membrane. The cytoplasm shrinkage phenomenon was accompanied by blurring of the membrane wall boundaries. WhenE. colicells were treated with sericin, release of intracellular components quickly increased. The electrical conductivity assay indicated that the charged ions are reduced after exposure to sericin so that the integrity of the cell membrane is weakened and metabolism is blocked. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that sericin hinders the expression of bacterial protein. Sericin may damage the integrity of the bacterial cell membrane, thereby eventually inhibiting the growth and reproduction ofE. coli. Compared to sterile gauze, the sericin-based hydrogel promoted fibroblast cell proliferation and accelerated the formation of granulation tissues and neovessels.IMPORTANCEThe specific relationship and interaction mechanism between sericin andE. colicells were investigated and elucidated. The results show that after 12 h of treatment, sericin molecules induce membrane blebbing ofE. colicells, and the bacteria show decreases in liquidity and permeability of biological membrane, resulting in alterations in the conductivity of the culture medium and the integrity of the outer membrane. The subsequentin vivoresults demonstrate that the sericin-poly(N-isopropylacrylamide-N,N′-methylene-bis-acrylamide [NIPAm-MBA]) hydrogel accelerated wound healing compared to that with sterile gauze, which is a beneficial result for future applications in clinical medicine and the textile, food, and coating industries.

scholarly journals A Comparative Quantitative Proteomic Study Identifies New Proteins Relevant for Sulfur Oxidation in the Purple Sulfur Bacterium Allochromatium vinosum

2014 ◽  
Vol 80 (7) ◽  
pp. 2279-2292 ◽  
Author(s):  
Thomas Weissgerber ◽  
Marc Sylvester ◽  
Lena Kröninger ◽  
Christiane Dahl
Keyword(s):  
Sulfur Metabolism ◽  
Sulfur Oxidation ◽  
Hypothetical Protein ◽  
Mrna Levels ◽  
Reduced Sulfur Compounds ◽  
Allochromatium Vinosum ◽  
Purple Sulfur Bacterium ◽  
Content Type ◽  
Protein Levels ◽  
New Genes

ABSTRACTIn the present study, we compared the proteome response ofAllochromatium vinosumwhen growing photoautotrophically in the presence of sulfide, thiosulfate, and elemental sulfur with the proteome response when the organism was growing photoheterotrophically on malate. Applying tandem mass tag analysis as well as two-dimensional (2D) PAGE, we detected 1,955 of the 3,302 predicted proteins by identification of at least two peptides (59.2%) and quantified 1,848 of the identified proteins. Altered relative protein amounts (≥1.5-fold) were observed for 385 proteins, corresponding to 20.8% of the quantifiedA. vinosumproteome. A significant number of the proteins exhibiting strongly enhanced relative protein levels in the presence of reduced sulfur compounds are well documented essential players during oxidative sulfur metabolism, e.g., the dissimilatory sulfite reductase DsrAB. Changes in protein levels generally matched those observed for the respective relative mRNA levels in a previous study and allowed identification of new genes/proteins participating in oxidative sulfur metabolism. One gene cluster (hyd; Alvin_2036-Alvin_2040) and one hypothetical protein (Alvin_2107) exhibiting strong responses on both the transcriptome and proteome levels were chosen for gene inactivation and phenotypic analyses of the respective mutant strains, which verified the importance of the so-called Isp hydrogenase supercomplex for efficient oxidation of sulfide and a crucial role of Alvin_2107 for the oxidation of sulfur stored in sulfur globules to sulfite. In addition, we analyzed the sulfur globule proteome and identified a new sulfur globule protein (SgpD; Alvin_2515).

scholarly journals Brucella melitensis Invades Murine Erythrocytes during Infection

2014 ◽  
Vol 82 (9) ◽  
pp. 3927-3938 ◽  
Author(s):  
Marie-Alice Vitry ◽  
Delphine Hanot Mambres ◽  
Michaël Deghelt ◽  
Katrin Hack ◽  
Arnaud Machelart ◽  
...  
Keyword(s):  
Brucella Melitensis ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Bacterial Cells ◽  
Content Type ◽  
Type Iv ◽  
Transmission Electron ◽  
Time Point

ABSTRACTBrucellaspp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed thatBrucella melitensisis able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced followingBrucellavaccination. In contrast toBartonella, the type IV secretion system and flagellar expression are not critically required for the persistence ofBrucellain blood. ImageStream analysis of blood cells showed that following a brief extracellular phase,Brucellais associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated thatB. melitensisis able to invade erythrocytesin vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host's immune response and hamper antibiotic treatment and suggests possibleBrucellatransmission by bloodsucking insects in nature.

scholarly journals Escherichia coliZipA Organizes FtsZ Polymers into Dynamic Ring-Like Protofilament Structures

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Marcin Krupka ◽  
Marta Sobrinos-Sanguino ◽  
Mercedes Jiménez ◽  
Germán Rivas ◽  
William Margolin
Keyword(s):  
Cell Division ◽  
High Surface ◽  
Bacterial Cells ◽  
Content Type ◽  
E Coli ◽  
Membrane Attachment ◽  
Lipid Surface ◽  
Confocal Fluorescence Imaging

ABSTRACTZipA is an essential cell division protein inEscherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfacesin vitro, visible as GTP-dependent chiral vortices several hundred nanometers in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with somein vitrostudies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also usedE. colimutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filamentsin vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill.IMPORTANCEBacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at midcell that forms a wall separating the two daughter cells and allows cells to divide. The key protein in this ring is FtsZ, a homolog of tubulin that forms dynamic polymers. Here, we use electron microscopy and confocal fluorescence imaging to show that one of the proteins required to attach FtsZ polymers to the membrane duringE. colicell division, ZipA, can promote dynamic swirls of FtsZ on a lipid surfacein vitro. Importantly, these swirls are observed only when ZipA is present at low, physiologically relevant surface densities. Although ZipA has been thought to enhance bundling of FtsZ polymers, we find little evidence for bundlingin vitro. In addition, we present several lines ofin vivoevidence indicating that ZipA does not act to directly bundle FtsZ polymers.

scholarly journals Delivery of antibacterial silver nanoclusters to Pseudomonas aeruginosa using species-specific DNA aptamers

2020 ◽  
Vol 69 (4) ◽  
pp. 640-652 ◽  
Author(s):  
Jennifer Soundy ◽  
Darren Day
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Activity ◽  
Type Species ◽  
Galleria Mellonella ◽  
Host Cells ◽  
Silver Nanoclusters ◽  
Bacterial Cells ◽  
Content Type ◽  
Link Type

Introduction. The use of silver as an antimicrobial therapeutic is limited by its toxicity to host cells compared with that required to kill bacterial pathogens. Aim. To use aptamer targeting of DNA scaffolded silver nanoclusters as an antimicrobial agent for treating Pseudomonas aeruginosa infections. Methodology. Antimicrobial activity was assessed in planktonic cultures and in vivo using an invertebrate model of infection. Results. The aptamer conjugates that we call aptabiotics have potent antimicrobial activity. Targeted silver nanoclusters were more effective at killing P. aeruginosa than the equivalent quantity of untargeted silver nanoclusters. The aptabiotics have an IC50 of 1.3–2.6 µM against planktonically grown bacteria. Propidium iodide staining showed that they rapidly depolarize bacterial cells to kill approximately 50 % of the population within 10 min following treatment. In vivo testing in the Galleria mellonella model of infection prolonged survival from an otherwise lethal infection. Conclusion. Using P. aeruginosa as a model, we show that targeting of DNA-scaffolded silver nanoclusters with an aptamer has effective fast-acting antimicrobial activity in vitro and in an in vivo animal model.

scholarly journals Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

2013 ◽  
Vol 82 (3) ◽  
pp. 1017-1029 ◽  
Author(s):  
Carmen Gil ◽  
Cristina Solano ◽  
Saioa Burgui ◽  
Cristina Latasa ◽  
Begoña García ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Response ◽  
Extracellular Proteins ◽  
Protective Immune Response ◽  
Surrounding Tissue ◽  
Bacterial Cells ◽  
Content Type ◽  
Biofilm Infection ◽  
Biofilm Matrix

ABSTRACTTheStaphylococcus aureusbiofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation ofS. aureusbiofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response againstS. aureusinfections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinicalS. aureusstrains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing ofS. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using anin vivomodel of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine againstS. aureusbiofilm-associated infections.

scholarly journals Pse-T2, an Antimicrobial Peptide with High-Level, Broad-Spectrum Antimicrobial Potency and Skin Biocompatibility against Multidrug-ResistantPseudomonas aeruginosaInfection

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
Hee Kyoung Kang ◽  
Chang Ho Seo ◽  
Tudor Luchian ◽  
Yoonkyung Park
Keyword(s):  
Antimicrobial Peptide ◽  
Membrane Integrity ◽  
Helical Structure ◽  
Multidrug Resistant ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Bacterial Membranes

ABSTRACTPseudin-2, isolated from the frogPseudis paradoxa, exhibits potent antibacterial activity but also cytotoxicity. In an effort to develop clinically applicable antimicrobial peptides (AMPs), we designed pseudin-2 analogs with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. In addition, truncated analogs of pseudin-2 and Lys-substituted peptides were synthesized to produce linear 18-residue amphipathic α-helices, which were further investigated for their mechanism and functions. These truncated analogs exhibited higher antimicrobial activity and lower cytotoxicity than pseudin-2. In particular, Pse-T2 showed marked pore formation, permeabilization of the outer/inner bacterial membranes, and DNA binding. Fluorescence spectroscopy and scanning electron microscopy showed that Pse-T2 kills bacterial cells by disrupting membrane integrity.In vivo, wounds infected with multidrug-resistant (MDR)Pseudomonas aeruginosahealed significantly faster when treated with Pse-T2 than did untreated wounds or wounds treated with ciprofloxacin. Moreover, Pse-T2 facilitated infected-wound closure by reducing inflammation through suppression of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α). These data suggest that the small antimicrobial peptide Pse-T2 could be useful for future development of therapeutic agents effective against MDR bacterial strains.

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