Identification and Characterization of the Major Porin of Desulfovibrio vulgaris Hildenborough

ABSTRACT Due in large part to their ability to facilitate the diffusion of a diverse range of solutes across the outer membrane (OM) of Gram-negative bacteria, the porins represent one of the most prominent and important bacterial membrane protein superfamilies. Notably, for the Gram-negative bacterium Desulfovibrio vulgaris Hildenborough, a model organism for studies of sulfate-reducing bacteria, no genes for porins have been identified or proposed in its annotated genome. Results from initial biochemical studies suggested that the product of the DVU0799 gene, which is one of the most abundant proteins of the D. vulgaris Hildenborough OM and purified as a homotrimeric complex, was a strong porin candidate. To investigate this possibility, this protein was further characterized biochemically and biophysically. Structural analyses via electron microscopy of negatively stained protein identified trimeric particles with stain-filled depressions and structural modeling suggested a β-barrel structure for the monomer, motifs common among the known porins. Functional studies were performed in which crude OM preparations or purified DVU0799 was reconstituted into proteoliposomes and the proteoliposomes were examined for permeability against a series of test solutes. The results obtained establish DVU0799 to be a pore-forming protein with permeability properties similar to those observed for classical bacterial porins, such as those of Escherichia coli. Taken together, these findings identify this highly abundant OM protein to be the major porin of D. vulgaris Hildenborough. Classification of DVU0799 in this model organism expands the database of functionally characterized porins and may also extend the range over which sequence analysis strategies can be used to identify porins in other bacterial genomes. IMPORTANCE Porins are membrane proteins that form transmembrane pores for the passive transport of small molecules across the outer membranes of Gram-negative bacteria. The present study identified and characterized the major porin of the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, observing its preference for anionic sugars over neutral ones. Its predicted architecture appears to be novel for a classical porin, as its core β-barrel structure is of a type typically found in solute-specific channels. Broader use of the methods employed here, such as assays for channel permeability and electron microscopy of purified samples, is expected to help expand the database of confirmed porin sequences and improve the range over which sequence analysis-based strategies can be used to identify porins in other Gram-negative bacteria. Functional characterization of these critical gatekeeping proteins from divergent Desulfovibrio species should offer an improved understanding of the physiological features that determine their habitat range and supporting activities.

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β-Aminopeptidases: Insight into Enzymes without a Known Natural Substrate

Applied and Environmental Microbiology ◽

10.1128/aem.00318-19 ◽

2019 ◽

Vol 85 (15) ◽

Author(s):

Marietta John-White ◽

James Gardiner ◽

Priscilla Johanesen ◽

Dena Lyras ◽

Geoffrey Dumsday

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Gram Negative Bacteria ◽

Diverse Range ◽

Α Subunit ◽

Gram Negative ◽

Content Type ◽

Proteolytic Resistance ◽

Insight Into ◽

Unique Capability

ABSTRACT β-Aminopeptidases have the unique capability to hydrolyze N-terminal β-amino acids, with varied preferences for the nature of β-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of β-peptides and to kinetically resolve β-peptides and amides for the production of enantiopure β-amino acids. To date, six β-aminopeptidases have been discovered and functionally characterized, five from Gram-negative bacteria and one from a fungus, Aspergillus. Here we report on the purification and characterization of an additional four β-aminopeptidases, one from a Gram-positive bacterium, Mycolicibacterium smegmatis (BapAMs), one from a yeast, Yarrowia lipolytica (BapAYlip), and two from Gram-negative bacteria isolated from activated sludge identified as Burkholderia spp. (BapABcA5 and BapABcC1). The genes encoding β-aminopeptidases were cloned, expressed in Escherichia coli, and purified. The β-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated α and β, appeared to be tightly associated, as the active enzyme was recovered after immobilized-metal affinity chromatography (IMAC) purification, even though only the α-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations β-homo-Gly (βhGly) and β3-homo-Leu (β3hLeu) with high activities. These enzymes displayed higher activity with H-βhGly-p-nitroanilide (H-βhGly-pNA) than previously characterized enzymes from other microorganisms. These data indicate that the new β-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce β-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the β-aminopeptidases may play a role in some cellular functions. IMPORTANCE β-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic β-peptides as a sole carbon source. Six β-aminopeptidases have been previously characterized with preferences for different β-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic β-peptides but also the synthesis of short β-peptides. Identification of other β-aminopeptidases adds to this toolbox of enzymes with differing β-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized in the sustainable manufacture of β-amino acid derivatives and β-peptides for use in biomedical and biomaterial applications. This is important, because β-amino acids and β-peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to α-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.

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Genome Sequence of Enterococcus faecium Strain ICIS 96 Demonstrating Intermicrobial Antagonism Associated with Bacteriocin Production

Genome Announcements ◽

10.1128/genomea.00126-18 ◽

2018 ◽

Vol 6 (10) ◽

Cited By ~ 2

Author(s):

Tatiana M. Pashkova ◽

Alexey S. Vasilchenko ◽

Yuriy A. Khlopko ◽

Elena E. Kochkina ◽

Olga L. Kartashova ◽

...

Keyword(s):

Antimicrobial Activity ◽

Genome Sequence ◽

Enterococcus Faecium ◽

Antagonistic Activity ◽

Bacteriocin Production ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Faecium Strain

ABSTRACT We report here the complete genome sequence of Enterococcus faecium strain ICIS 96, which was isolated from the feces of a horse. Bacteriological characterization of strain ICIS 96 revealed the absence of pathogenicity factors, while its spectrum of antagonistic activity was found to be broad, having activities associated with both Gram-positive and Gram-negative bacteria. Analysis of the E. faecium ICIS 96 genome revealed five genes associated with antimicrobial activity (enterocin [ent] A, ent B, lactobin A/cerein 7b, and ent L50 A/B). No genes that correlate with human pathogenicity were identified.

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NDM-5 and OXA-181 Beta-Lactamases, a Significant Threat Continues To Spread in the Americas

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00454-17 ◽

2017 ◽

Vol 61 (7) ◽

Cited By ~ 30

Author(s):

Laura J. Rojas ◽

Andrea M. Hujer ◽

Susan D. Rudin ◽

Meredith S. Wright ◽

T. Nicholas Domitrovic ◽

...

Keyword(s):

Abdominal Surgery ◽

Hot Spot ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Beta Lactamases ◽

Carbapenem Resistant ◽

Multiple Copies ◽

Life Threatening

ABSTRACT Among Gram-negative bacteria, carbapenem-resistant infections pose a serious and life-threatening challenge. Here, the CRACKLE network reports a sentinel detection and characterization of a carbapenem-resistant Klebsiella pneumoniae ST147 isolate harboring bla NDM-5 and bla OXA-181 from a young man who underwent abdominal surgery in India. bla NDM-5 was located on an IncFII plasmid of ≈90 kb, whereas bla OXA-181 was chromosomally encoded. Resistome and genome analysis demonstrated multiple copies of the transposable element IS26 and a “hot-spot region” in the IncFII plasmid.

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Al2O3 Nanopowders, a Suitable Compound for Active Control of Biofouling

Journal of Nano Research ◽

10.4028/www.scientific.net/jnanor.32.71 ◽

2015 ◽

Vol 32 ◽

pp. 71-80

Author(s):

Mohsen Mehregan ◽

Hossein Soltaninejad ◽

Behnaz Toluei Nia ◽

Hadi Zare-Zardini ◽

Masoud Zare-Shehneh ◽

...

Keyword(s):

Electron Microscopy ◽

Antibacterial Activity ◽

Blood Cells ◽

Gram Negative Bacteria ◽

Bacterial Strains ◽

X Ray Diffraction ◽

Gram Positive ◽

Gram Negative ◽

X Ray

The formation of biofilm (Biofouling) in different surface is the great concern in types of fields, especially in medical and health system as well as in membrane technology. The present study deals with the synthesis and characterization of Al2O3 nanopowders with antibacterial activity which can be a potentially utilized material for biocompatible implants. Nanostructure was synthesized based on sol-gel method and then, crystallite size, and microstructural and morphological characterization of nanostructure were determined by X-ray diffraction, electron-microscopic techniques - scanning electron microscopy (SEM) and transmission electron microscopy (TEM). According to X-ray diffraction, the value of particle size for Al2O3 nanopowders is 20.85 nm. In the following, the antibacterial activity of Al2O3 nanoparticles was assessed on three gram positive and three gram negative bacteria by radial diffusion assay and measurement of minimum inhibitory concentration (MIC). The toxicity of Al2O3 nanopowders on blood cells was also assessed. The results showed that this nanostructure has potent antibacterial activity against gram positive and gram negative bacteria. The synthesized Al2O3 nanopowders showed the antimicrobial activity against antibiotic resistant bacterium, Staphylococcus aureus. Significant antibacterial activity of this nanostructure was seen to have a greatest effect on Bacillus cereus with the MIC value of 9.2 μg/ml; while, among bacterial strains, Salmonellatyphimurium was investigated to be the most resistant one with the MIC of 35.6 μg/ml. Al2O3 nanopowders showed no toxicity on blood cells. according to acquired data in this study, Al2O3 nanopowders may be a good material for inhibition of biofilm formation.

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Attention Seeker: Production, Modification, and Release of Inflammatory Peptidoglycan Fragments in Neisseria Species

Journal of Bacteriology ◽

10.1128/jb.00354-17 ◽

2017 ◽

Vol 199 (20) ◽

Cited By ~ 6

Author(s):

Jia Mun Chan ◽

Joseph P. Dillard

Keyword(s):

Model Organism ◽

Degradation Process ◽

Host Tissue ◽

Gram Negative Bacteria ◽

Biological Processes ◽

Gram Negative ◽

Content Type ◽

Bacterial Physiology ◽

Neisseria Species ◽

Cellular Integrity

ABSTRACT Maintenance of the structural macromolecule peptidoglycan (PG), which involves regulated cycles of PG synthesis and PG degradation, is pivotal for cellular integrity and survival. PG fragments generated from the degradation process are usually efficiently recycled by Gram-negative bacteria. However, Neisseria gonorrhoeae and a limited number of Gram-negative bacteria release PG fragments in amounts sufficient to induce host tissue inflammation and damage during an infection. Due to limited redundancy in PG-modifying machineries and genetic tractability, N. gonorrhoeae serves as a great model organism for the study of biological processes related to PG. This review summarizes the generation, modification, and release of inflammatory PG molecules by N. gonorrhoeae and related species and discusses these findings in the context of understanding bacterial physiology and pathogenesis.

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Predatory bacteria as living antibiotics – where are we now?

Microbiology ◽

10.1099/mic.0.001025 ◽

2021 ◽

Vol 167 (1) ◽

Cited By ~ 1

Author(s):

Robert J. Atterbury ◽

Jess Tyson

Keyword(s):

Antimicrobial Resistance ◽

Global Health ◽

Type Species ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Biologically Relevant ◽

Link Type ◽

Predatory Bacteria

Antimicrobial resistance (AMR) is a global health and economic crisis. With too few antibiotics in development to meet current and anticipated needs, there is a critical need for new therapies to treat Gram-negative infections. One potential approach is the use of living predatory bacteria, such as Bdellovibrio bacteriovorus (small Gram-negative bacteria that naturally invade and kill Gram-negative pathogens of humans, animals and plants). Moving toward the use of Bdellovibrio as a ‘living antibiotic’ demands the investigation and characterization of these bacterial predators in biologically relevant systems. We review the fundamental science supporting the feasibility of predatory bacteria as alternatives to antibiotics.

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Autotransporter-Based Antigen Display in Bacterial Ghosts

Applied and Environmental Microbiology ◽

10.1128/aem.02733-14 ◽

2014 ◽

Vol 81 (2) ◽

pp. 726-735 ◽

Cited By ~ 15

Author(s):

Anna Hjelm ◽

Bill Söderström ◽

David Vikström ◽

Wouter S. P. Jong ◽

Joen Luirink ◽

...

Keyword(s):

Electron Microscopy ◽

Flow Cytometry ◽

Fluorescence Microscopy ◽

Surface Display ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Bacterial Ghosts ◽

Cell Envelopes

ABSTRACTBacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage ϕX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered theEscherichia coliautotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using theMycobacterium tuberculosisvaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorateE. coliandSalmonellaghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E inE. coliandSalmonellacan be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.

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Neutrophil pseudoplatelets in subgingival plaque and crevicular fluid samples from periodontal diseased sites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156298 ◽

1989 ◽

Vol 47 ◽

pp. 862-863 ◽

Cited By ~ 1

Author(s):

J Hanker ◽

E.J. Burkes ◽

G. Greco ◽

R. Scruggs ◽

B. Giammara

Keyword(s):

Electron Microscopy ◽

Bone Marrow ◽

Peripheral Blood ◽

Gingival Crevicular Fluid ◽

Light And Electron Microscopy ◽

Subgingival Plaque ◽

Staining Reaction ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Crevicular Fluid

The mature neutrophil with a segmented nucleus (usually having 3 or 4 lobes) is generally considered to be the end-stage cell of the neutrophil series. It is usually found as such in the bone marrow and peripheral blood where it normally is the most abundant leukocyte. Neutrophils, however, must frequently leave the peripheral blood and migrate into areas of infection to combat microorganisms. It is in such areas that neutrophils were first observed to fragment to form platelet-size particles some of which have a nuclear lobe. These neutrophil pseudoplatelets (NPP) can readily be distinguished from true platelets because they stain for neutrophil myeloperoxidase. True platelets are not positive in this staining reaction because their peroxidase Is inhibited by glutaraldehyde. Neutrophil pseudoplatelets, as well as neutrophils budding to form NPP, could frequently be observed in peripheral blood or bone marrow samples of leukemia patients. They are much more prominent, however, in smears of inflammatory exudates that contain gram-negative bacteria and in gingival crevicular fluid samples from periodontal disease sites. In some of these samples macrophages ingesting, or which contained, pseudoplatelets could be observed. The myeloperoxidase in the ingested pseudoplatelets was frequently active. Despite these earlier observations we did not expect to find many NPP in subgingival plaque smears from diseased sites. They were first seen by light microscopy (Figs. 1, 3-5) in smears on coverslips stained with the PATS reaction, a variation of the PAS reaction which deposits silver for light and electron microscopy. After drying replicate PATS-stained coverslips with hexamethyldisilazane, they were sputter coated with gold and then examined by the SEI and BEI modes of scanning electron microscopy (Fig. 2). Unstained replicate coverslips were fixed, and stained for the demonstration of myeloperoxidase in budding neutrophils and NPP. Neutrophils, activated macrophages and spirochetes as well as other gram-negative bacteria were also prominent in the PATS stained samples. In replicate subgingival plaque smears stained with our procedure for granulocyte peroxidases only neutrophils, budding neutrophils or NPP were readily observed (Fig. 6).

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Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

10.26434/chemrxiv.8282204.v1 ◽

2019 ◽

Author(s):

Jiajun Wang ◽

Rémi Terrasse ◽

Jayesh Arun Bafna ◽

Lorraine Benier ◽

Mathias Winterhalter

Keyword(s):

Outer Membrane ◽

Lipid Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Multi Drug Resistance ◽

Gram Negative Bacteria ◽

Membrane Channels ◽

Gram Negative ◽

Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from Escherichia coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (kon) and lower dissociation (koff) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

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