123 research outputs found
Determination of the molecular basis for coprogen import by Gram-negative bacteria
No full textIn order to survive in mixed microbial communities, some species of fungi secrete coprogens, siderophores that facilitate capture of the scarce nutrient iron. The TonB-dependent transporter FhuE is integrated in the outer membrane of Gram-negative bacteria and has been reported to scavenge these fungally produced coprogens. In this work, an Escherichia coli strain was engineered that is dependent solely on FhuE for its access to siderophore-sequestered iron. Using this tool, it is shown that while FhuE is highly active in the import of coprogens, it has some level of promiscuity, acting as a low-affinity transporter for related siderophores. The crystal structure of FhuE in complex with coprogen was determined, providing a structural basis to explain this selective promiscuity. The structural data, in combination with functional analysis, presented in this work show that FhuE has evolved to specifically engage with planar siderophores. A potential evolutionary driver, and a critical consequence of this selectivity, is that it allows FhuE to exclude antibiotics that mimic nonplanar hydroxamate siderophores: these toxic molecules could otherwise cross the outer membrane barrier through a Trojan horse mechanism.</p
Discovering colicin and lectin-like bacteriocins for the creation of disease resistant transgenic plants
No full textThe colicin and lectin-like bacteriocins are a broad class of antimicrobial proteins produced by Gram-negative bacteria. They are generally narrow spectrum, killing or inhibiting the growth of closely related bacteria. Numerous Gram-negative bacteria that are important pathogens of both animals and plants produce and are susceptible to these bacteriocins. As such, these proteins represent an attractive alternative to traditional small molecule antibiotics for controlling bacterial infection.
Very little is known about bacteriocins produced by Gram-negative plant pathogens and so the aim of this work was to discover novel bacteriocins active against globally important plant pathogens from the genera Pectobacterium and Pseudomonas. The bacteriocins discovered in this study were then structurally and functionally characterised and assessed for their ability to impart disease resistance when expressed in a model transgenic system.
This study presents the discovery and characterisation of the bacteriocins syringacin M, syringacin L1 and pyocin L1 from the genus Pseudomonas, As well as the discovery and characterisation of the unusual ferredoxin containing pectocins from the genus Pectobacterium. Also presented is the discovery of a novel virulence related ferredoxin/iron-uptake system in Pectobacterium, which is parasitised by the pectocins for cell entry. Additionally, the transgenic expression of the bacteriocin putidacin L1 in both Arabidopsis thaliana and Nicotiana benthamiana was shown to provide these plants with resistance to infection by strains of the plant pathogen P. syringae
The crystal structure of the TonB-dependent transporter YncD reveals a positively charged substrate-binding site
No full textThe outer membrane of Gram-negative bacteria is highly impermeable to hydrophilic molecules of larger than 600Da, protecting these bacteria from toxins present in the environment. In order to transport nutrients across this impermeable membrane, Gram-negative bacteria utilize a diverse family of outer-membrane proteins called TonB-dependent transporters. The majority of the members of this family transport iron-containing substrates. However, it is becoming increasingly clear that TonB-dependent transporters target chemically diverse substrates. In this work, the structure and phylogenetic distribution of the TonB-dependent transporter YncD are investigated. It is shown that while YncD is present in some enteropathogens, including Escherichia coli and Salmonella spp., it is also widespread in Gammaproteobacteria and Betaproteobacteria of environmental origin. The structure of YncD was determined, showing that despite a distant evolutionary relationship, it shares structural features with the ferric citrate transporter FecA, including a compact positively charged substrate-binding site. Despite these shared features, it is shown that YncD does not contribute to the growth of E. coli in pure culture under iron-limiting conditions or with ferric citrate as an iron source. Previous studies of transcriptional regulation in E. coli show that YncD is not induced under iron-limiting conditions and is unresponsive to the ferric uptake regulator (Fur). These observations, combined with the data presented here, suggest that YncD is not responsible for the transport of an iron-containing substrate.</p
The structure of the bacterial iron-catecholate transporter Fiu suggests that it imports substrates via a two-step mechanism
No full textThe ferric iron uptake (Fiu) transporter from Escherichia coli functions in the transport of iron-catecholate complexes across the bacterial outer membrane, providing the bacterium with iron, which is essential for growth. Recently it has become clear that Fiu also represents a liability for E. coli because its activity allows import of antimicrobial compounds that mimic catecholate. This inadvertent import suggests the potential utility of antimicrobial catechol siderophore mimetics in managing bacterial infections. However, to fully exploit these compounds, a detailed understanding of the mechanism of transport through Fiu and related transporters is required. To address this question, we determined the crystal structure of Fiu at 2.1-2.9 Å and analyzed its function in E. coli Through analysis of the Fiuo crystal structure, in combination with in silico docking and mutagenesis, we provide insight into how Fiu and related transporters bind catecholate in a surface-exposed cavity. Moreover, through determination of the structure of Fiu in multiple crystal states, we revealed the presence of a large, selectively gated cavity in the interior of this transporter. This chamber is large enough to accommodate the Fiu substrate and may allow import of substrates via a two-step mechanism. This would avoid channel formation through the transporter and inadvertent import of toxic molecules. As Fiu and its homologs are the targets of substrate-mimicking antibiotics, these results may assist in the development of these compounds.</p
Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in <i>Pectobacterium spp</i>
No full textIn order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium <i>Pectobacterium carotovorum</i> carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of <i>Pectobacterium carotovorum</i> and <i>Pectobacterium atrosepticum</i> with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that <i>Pectobacterium spp.</i> carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of <i>Pectobacterium carotovorum</i> and <i>atrosepticum</i> that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells
The modular nature of the β-barrel assembly machinery, illustrated inBorrelia burgdorferi
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New host records of chewing lice (Mallophaga) on birds in Florida 3
No full textHere, I report some new county and Florida host records as well as two new U.S. host records for avian lice. County records for avian lice are less significant than the host records themselves. Considering that most avian lice are host specific, the county records should resemble the area the host would most likely be encountered. The main source of material for this publication has been the Marathon Wildlife Rehabilitation Center in Marathon, Monroe County, Florida, with Kelly Grinter as the official curator and keeper. The center takes in about 90 different species of birds per year. The following is a list of the avian lice species, hosts, and localities with collection dates. All of the following specimens were collected by the author, Kelly Grinter, or Leslie Straub. Representatives of all records below are deposited in the Florida State Collection of Arthropods and/or the author’s collection
Understanding the catalytic and gas-sensing properties of metal oxide nanostructures with advanced surface science techniques
No full textTitle: Understanding the catalytic and gassensing properties of metal oxide nanostructures with advanced surface science techniques Author: Lesia Piliai Department: Department of Surface and Plasma Science Supervisor: doc. Mgr. Michael Vorochta, Ph.D., Department of Surface and Plasma Science Abstract: This thesis presents a comprehensive investigation of the catalytic and gassensing properties of CeO2, FeOx, ZnO and WO3 nanostructures. Using a combination of NAPXPS and other surface sensitive techniques, the research has elucidated the morphological and electronic structures of these metal oxides, thereby improving the understanding of their performance in catalytic and gas sensing applications. For CO oxidation, model Au/CeO2, Pt/CeO2 and Fe/CeO2 catalysts have been studied under simulated reaction conditions, focusing on the metal/oxide and gas/oxide interfaces. The results obtained shed light on the nature of surface active sites, charge distribution, redox properties and morphology of the catalyst during CO oxidation. In addition, stable FeOx epitaxial films on ceria have been successfully prepared. In the field of gassensing, 1D ZnO and WO3 nanostructures have been investigated for the detection of CO gas and ethanol vapor. By studying the gassolid interaction and correlating it with the..
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