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Faculty of Science, Medicine and Health - Papers: part A

Escherichia

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Single-Molecule Specific Mislocalization Of Red Fluorescent Proteins In Live Escherichia Coli, Harshad Ghodke, Victor E. A Caldas, Christiaan M. Punter, Antoine M. Van Oijen, Andrew Robinson Jan 2016

Single-Molecule Specific Mislocalization Of Red Fluorescent Proteins In Live Escherichia Coli, Harshad Ghodke, Victor E. A Caldas, Christiaan M. Punter, Antoine M. Van Oijen, Andrew Robinson

Faculty of Science, Medicine and Health - Papers: part A

Tagging of individual proteins with genetically encoded fluorescent proteins (FPs) has been used extensively to study localization and interactions in live cells. Recent developments in single-molecule localization microscopy have enabled the dynamic visualization of individual tagged proteins inside living cells. However, tagging proteins with FPs is not without problems: formation of insoluble aggregates and inhibition of native functions of the protein are well-known issues. Previously reported artifacts manifest themselves at all expression levels of the FP-tagged proteins, making the design of control experiments relatively straightforward. Here, we describe a previously uncharacterized mislocalization artifact of Entacmaea quadricolor red fluorescent protein variants ...


Escherichia Coli Single-Stranded Dna-Binding Protein: Nanoesi-Ms Studies Of Salt-Modulated Subunit Exchange And Dna Binding Transactions, Claire E. Mason, Slobodan Jergic, Allen Lo, Yao Wang, Nicholas E. Dixon, Jennifer L. Beck Jan 2013

Escherichia Coli Single-Stranded Dna-Binding Protein: Nanoesi-Ms Studies Of Salt-Modulated Subunit Exchange And Dna Binding Transactions, Claire E. Mason, Slobodan Jergic, Allen Lo, Yao Wang, Nicholas E. Dixon, Jennifer L. Beck

Faculty of Science, Medicine and Health - Papers: part A

Single-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in ...


Structure Of The Theta Subunit Of Escherichia Coli Dna Polymerase Iii In Complex With The Epsilon Subunit, Max A Keniry, Ah-Young Park, Elisabeth A. Owen, Samir M. Hamdan, Guido Pintacuda, Gottfried Otting, Nicholas E. Dixon Jan 2006

Structure Of The Theta Subunit Of Escherichia Coli Dna Polymerase Iii In Complex With The Epsilon Subunit, Max A Keniry, Ah-Young Park, Elisabeth A. Owen, Samir M. Hamdan, Guido Pintacuda, Gottfried Otting, Nicholas E. Dixon

Faculty of Science, Medicine and Health - Papers: part A

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the α, ε, and θ subunits. The θ subunit is the smallest and least understood subunit. The three-dimensional structure of θ in a complex with the unlabeled N-terminal domain of the ε subunit, ε186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of ε186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of θ ...


Bovine Feces From Animals With Gastrointestinal Infections Are A Source Of Serologically Diverse Atypical Enteropathogenic Escherichia Coli And Shiga Toxin-Producing E. Coli Strains That Commonly Possess Intimin, Michael A Hornitzky, Kim Mercieca, Karl A Bettelheim, Steven P. Djordjevic Jan 2005

Bovine Feces From Animals With Gastrointestinal Infections Are A Source Of Serologically Diverse Atypical Enteropathogenic Escherichia Coli And Shiga Toxin-Producing E. Coli Strains That Commonly Possess Intimin, Michael A Hornitzky, Kim Mercieca, Karl A Bettelheim, Steven P. Djordjevic

Faculty of Science, Medicine and Health - Papers: part A

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) cells were isolated from 191 fecal samples from cattle with gastrointestinal infections (diagnostic samples) collected in New South Wales, Australia. By using a multiplex PCR, E. coli cells possessing combinations of stx1, stx2, eae, and ehxA were detected by a combination of direct culture and enrichment in E. coli (EC) (modified) broth followed by plating on vancomycin-cefixime-cefsulodin blood (BVCC) agar for the presence of enterohemolytic colonies and on sorbitol MacConkey agar for the presence of non-sorbitol-fermenting colonies. The high prevalence of the intimin gene eae was a feature ...


Replication Termination In Escherichia Coli: Structure And Anti-Helicase Activity Of The Tus-Ter Complex, Cameron Neylon, Andrew V. Kralicek, Thomas M. Hill, Nicholas E. Dixon Jan 2005

Replication Termination In Escherichia Coli: Structure And Anti-Helicase Activity Of The Tus-Ter Complex, Cameron Neylon, Andrew V. Kralicek, Thomas M. Hill, Nicholas E. Dixon

Faculty of Science, Medicine and Health - Papers: part A

The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while ...


Serotypes And Virulence Gene Profiles Of Shiga Toxin-Producing Escherichia Coli Strains Isolated From Feces Of Pasture-Fed And Lot-Fed Sheep, Steven Djordjevic, Vidiya Ramachandran, Karl A Bettelheim, Barbara A. Vanselow, Peter Holst, Graham Bailey, Michael A. Hornitsky Jan 2004

Serotypes And Virulence Gene Profiles Of Shiga Toxin-Producing Escherichia Coli Strains Isolated From Feces Of Pasture-Fed And Lot-Fed Sheep, Steven Djordjevic, Vidiya Ramachandran, Karl A Bettelheim, Barbara A. Vanselow, Peter Holst, Graham Bailey, Michael A. Hornitsky

Faculty of Science, Medicine and Health - Papers: part A

Shiga toxin-producing Escherichia coli (STEC) strains possessing genes for enterohemolysin (ehxA) and/or intimin (eae), referred to here as complex STEC (cSTEC), are more commonly recovered from the feces of humans with hemolytic uremic syndrome and hemorrhagic colitis than STEC strains that do not possess these accessory virulence genes. Ruminants, particularly cattle and sheep, are recognized reservoirs of STEC populations that may contaminate foods destined for human consumption. We isolated cSTEC strains from the feces of longitudinally sampled pasture-fed sheep, lot-fed sheep maintained on diets comprising various combinations of silage and grain, and sheep simultaneously grazing pastures with cattle to ...


Stx1c Is The Most Common Shiga Toxin 1 Subtype Among Shiga Toxin-Producing Escherichia Coli Isolates From Sheep But Not Among Isolates From Cattle, Kim Brett, Vidiya Ramachandran, Michael A Hornitzky, Karl A Bettelheim, Mark J. Walker, Steven P. Djordjevic Jan 2003

Stx1c Is The Most Common Shiga Toxin 1 Subtype Among Shiga Toxin-Producing Escherichia Coli Isolates From Sheep But Not Among Isolates From Cattle, Kim Brett, Vidiya Ramachandran, Michael A Hornitzky, Karl A Bettelheim, Mark J. Walker, Steven P. Djordjevic

Faculty of Science, Medicine and Health - Papers: part A

Unlike Shiga toxin 2 (stx2) genes, most nucleotide sequences of Shiga toxin 1 (stx1) genes from Shiga toxin-producing Escherichia coli (STEC), Shigella dysenteriae, and several bacteriophages (H19B, 933J, and H30) are highly conserved. Consequently, there has been little incentive to investigate variants of stx1 among STEC isolates derived from human or animal sources. However stx1OX3, originally identified in an OX3:H8 isolate from a healthy sheep in Germany, differs from other stx1 subtypes by 43 nucleotides, resulting in changes to 12 amino acid residues, and has been renamed stx1c. In this study we describe ...


Bovine Non-O157 Shiga Toxin 2-Containing Escherichia Coli Isolates Commonly Possess Stx2-Edl933 And/Or Stx2vhb Subtypes, Kim Brett, Michael A Hornitzky, Karl A Bettelheim, Mark J. Walker, Steven Djordjevic Jan 2003

Bovine Non-O157 Shiga Toxin 2-Containing Escherichia Coli Isolates Commonly Possess Stx2-Edl933 And/Or Stx2vhb Subtypes, Kim Brett, Michael A Hornitzky, Karl A Bettelheim, Mark J. Walker, Steven Djordjevic

Faculty of Science, Medicine and Health - Papers: part A

stx2 genes from 138 Shiga toxin-producing Escherichia coli (STEC) isolates, of which 127 were of bovine origin (58 serotypes) and 11 of human origin (one serotype; O113:H21), were subtyped. The bovine STEC isolates from Australian cattle carried ehxA and/or eaeA and predominantly possessed stx2-EDL933 (103 of 127; 81.1%) either in combination with stx2vhb (32 of 127; 25.2%) or on its own (52 of 127; 40.4%). Of 22 (90.9%) bovine isolates of serotype O113:H21, a serotype increasingly recovered from patients with hemolytic uremic syndrome (HUS) or hemorrhagic colitis, 20 contained both ...


Distribution Of Intimin Subtypes Among Escherichia Coli Isolates From Ruminant And Human Sources, Vidiya Ramachandran, Kim Brett, Michael A Hornitzky, Mark Dowton, Karl A Bettelheim, Mark J. Walker, Steven P. Djordjevic Jan 2003

Distribution Of Intimin Subtypes Among Escherichia Coli Isolates From Ruminant And Human Sources, Vidiya Ramachandran, Kim Brett, Michael A Hornitzky, Mark Dowton, Karl A Bettelheim, Mark J. Walker, Steven P. Djordjevic

Faculty of Science, Medicine and Health - Papers: part A

The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (α1, α2, β, γ, κ, ɛ, η, ι, λ, θ, and ζ) and three new intimin genes ...


Flexibility Revealed By The 1.85 Å Crystal Structure Of The Β Sliding-Clamp Subunit Of Escherichia Coli Dna Polymerase Iii, Aaron J. Oakley, Pavel Prosselkov, Gene Wijffels, Jennifer L. Beck, Matthew Cj Wilce, Nicholas E. Dixon Jan 2003

Flexibility Revealed By The 1.85 Å Crystal Structure Of The Β Sliding-Clamp Subunit Of Escherichia Coli Dna Polymerase Iii, Aaron J. Oakley, Pavel Prosselkov, Gene Wijffels, Jennifer L. Beck, Matthew Cj Wilce, Nicholas E. Dixon

Faculty of Science, Medicine and Health - Papers: part A

The subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding clamp that interacts with the (polymerase) subunit to maintain the high processivity of the enzyme. The protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 Å [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 Å at ...


The Common Ovine Shiga Toxin 2-Containing Escherichia Coli Serotypes And Human Isolates Of The Same Serotypes Possess A Stx2d Toxin Type, Vidiya Ramachandran, Michael A. Hornitzky, Karl A. Bettelheim, Mark J. Walker, Steven P. Djordjevic Jan 2001

The Common Ovine Shiga Toxin 2-Containing Escherichia Coli Serotypes And Human Isolates Of The Same Serotypes Possess A Stx2d Toxin Type, Vidiya Ramachandran, Michael A. Hornitzky, Karl A. Bettelheim, Mark J. Walker, Steven P. Djordjevic

Faculty of Science, Medicine and Health - Papers: part A

Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease. In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models. We have characterized the stx2 subtype of 168 Shiga toxin-producing Escherichia coli (STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans. The ovine STEC isolates were of serotypes that have been shown to occur commonly in the gastrointestinal tract of healthy sheep. The major stx2 subtype in the ovine isolates was ...