26 research outputs found
Eubacterial SpoVG homologs constitute a new family of site-specific DNA-binding proteins
A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG α-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent β-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins
\u3cem\u3eBorrelia burgdorferi\u3c/em\u3e EbfC Defines a Newly-Identified, Widespread Family of Bacterial DNA-Binding Proteins
The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where ‘n’ can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5′ of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel α-helical ‘tweezer’-like structure
A method for assessing of ship fuel system failures resulting from fuel changeover imposed by environmental requirements
Environmental regulations instigated the technological and procedural revolution in shipping. One of the challenges has been sulfur emission control areas (SECA) and requirement of fuel changeover. Initially, many reports anticipated that new grades of low sulfur fuels might increase various technical problems in ship operation. This research develops a simple and easy to use method of the failure severity and intensity assessment in relation to fuel changeover. The scale of failure rate in the ship’s fuel system was evaluated qualitatively and quantitively, using developed failure frequency indicator and the time between failure. Based on 77 records of fuel system failures collected on seven ships, it has been found that frequency of failures related to SECA fuel changeover is on average nearly three times higher compared to the rest of sailing time. Their severity did not significantly change, but the structure of failures changed considerably. The method and presented results may help in improvement of ship’s systems design and on-board operational procedures
Borrelia burgdorferi RevA Antigen Binds Host Fibronectinâ–¿
Borrelia burgdorferi, the Lyme disease-causing spirochete, can persistently infect its vertebrate hosts for years. B. burgdorferi is often found associated with host connective tissue, where it interacts with components of the extracellular matrix, including fibronectin. Some years ago, a borrelial surface protein, named BBK32, was identified as a fibronectin-binding protein. However, B. burgdorferi BBK32 mutants are still able to bind fibronectin, indicating that the spirochete possesses additional mechanisms for adherence to fibronectin. We now demonstrate that RevA, an unrelated B. burgdorferi outer surface protein, binds mammalian fibronectin in a saturable manner. Site-directed mutagenesis studies identified the amino terminus of the RevA protein as being required for adhesion to fibronectin. RevA bound to the amino-terminal region of fibronectin. RevA binding to fibronectin was not inhibited by salt or heparin, suggesting that adhesin-ligand interactions are primarily nonionic and occur through the non-heparin-binding regions of the fibronectin amino-terminal domains. revA genes are widely distributed among Lyme disease spirochetes, and the present studies determined that all RevA alleles tested bound fibronectin. In addition, RevB, a paralogous protein found in a subset of B. burgdorferi strains, also bound fibronectin. We also confirmed that RevA is produced during mammalian infection but not during colonization of vector ticks and determined that revA transcription is controlled through a mechanism distinct from that of BBK32
Borrelia burgdorferi EbfC, a Novel, Chromosomally Encoded Protein, Binds Specific DNA Sequences Adjacent to erp Loci on the Spirochete's Resident cp32 Prophages
All examined isolates of the Lyme disease spirochete, Borrelia burgdorferi, naturally maintain numerous variants of a prophage family as circular cp32 episomes. Each cp32 carries a locus encoding one or two different Erp outer membrane, surface-exposed lipoproteins. Many of the Erp proteins bind a host complement regulator, factor H, which is hypothesized to protect the spirochete from complement-mediated killing. We now describe the isolation and characterization of a novel, chromosomally encoded protein, EbfC, that binds specific DNA sequences located immediately 5′ of all erp loci. This is one of the first site-specific DNA-binding proteins to be identified in any spirochete. The location of the ebfC gene on the B. burgdorferi chromosome suggests that the cp32 prophages have evolved to use this bacterial host protein for their own benefit and that EbfC probably plays additional roles in the bacterium. A wide range of other bacteria encode homologs of EbfC, none of which have been well characterized, so demonstration that B. burgdorferi EbfC is a site-specific DNA-binding protein has broad implications across the eubacterial kingdom
Transcriptional Regulation of the Borrelia burgdorferi Antigenically Variable VlsE Surface Protein
The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5′ DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator
Synthesis and Structural Characterization of Magnesium Drug Complexes: Efficient Initiators for Forming Polylactide–Drug Conjugates
Five novel magnesium alkoxides supported
by drug chelating agents <i>pridinolum</i> (PriOH = 1,1-diphenyl-3-(1-piperidinyl)-1-propanol)
and <i>venlafaxinum</i> (VenlOH = (<i>RS</i>)-1-[2-dimethylamino-1-(4-methoxyphenyl)-ethyl]Âcyclohexanol)
were successfully synthesized and characterized. Direct reaction of
PriOH and VenlOH with MgBu<sub>2</sub> (1:1) in toluene gives the
dimeric compounds [MgÂ(μ,η<sup>2</sup>-OPri)<sup><i>n</i></sup>Bu]<sub>2</sub> (<b>1</b>) and [MgÂ(μ,η<sup>2</sup>-VenlO)<sup><i>n</i></sup>Bu]<sub>2</sub> (<b>2</b>), respectively. Furthermore, the crystallization of an equimolar
mixture of <b>1</b> and <b>2</b> in toluene yields heteroleptic
magnesium complex [MgÂ(μ,η<sup>2</sup>-OVenl)Â(η<sup>1</sup>-OPri)]<sub>2</sub> (<b>3</b>). Moreover, reactions
of <b>1</b> and <b>2</b> with 2 molar equivs of the corresponding
drug–ligands give the homoleptic magnesium bis-alkoxides [MgÂ(μ,η<sup>2</sup>-OPri)Â(η<sup>1</sup>-OPri)]<sub>2</sub> (<b>4</b>) and [MgÂ(μ,η<sup>2</sup>-OVenl)Â(η<sup>1</sup>-OVenl)]<sub>2</sub> (<b>5</b>). The treatment of compound <b>1</b> with 2 equivs of VenlOH or <b>2</b> with 2 equivs of PriOH
leads to the formation of <b>3</b>. Complexes <b>1</b>–<b>5</b> were characterized by elemental analysis,
nuclear magnetic resonance, and single crystal X-ray diffraction (for <b>1</b>–<b>4</b>). It was found that complexes <b>1</b>–<b>5</b> are efficient initiators of the ring-opening
polymerization of l-LA, yielding PLA-OPri and PLA-OVenl conjugates,
respectively. Moreover, the ring-opening polymerization of l-LA initiated by <b>3</b> led to the simultaneous generation
of a blend of poly-l-lactide conjugates with end-capped VenlO
and PriO groups
Coordinated Expression of Borrelia burgdorferi Complement Regulator-Acquiring Surface Proteins during the Lyme Disease Spirochete's Mammal-Tick Infection Cycleâ–¿
The Lyme disease spirochete, Borrelia burgdorferi, is largely resistant to being killed by its hosts’ alternative complement activation pathway. One possible resistance mechanism of these bacteria is to coat their surfaces with host complement regulators, such as factor H. Five different B. burgdorferi outer surface proteins having affinities for factor H have been identified: complement regulator-acquiring surface protein 1 (BbCRASP-1), encoded by cspA; BbCRASP-2, encoded by cspZ; and three closely related proteins, BbCRASP-3, -4, and -5, encoded by erpP, erpC, and erpA, respectively. We now present analyses of the recently identified BbCRASP-2 and cspZ expression patterns throughout the B. burgdorferi infectious cycle, plus novel analyses of BbCRASP-1 and erp-encoded BbCRASPs. Our results, combined with data from earlier studies, indicate that BbCRASP-2 is produced primarily during established mammalian infection, while BbCRASP-1 is produced during tick-to-mammal and mammal-to-tick transmission stages but not during established mammalian infection, and Erp-BbCRASPs are produced from the time of transmission from infected ticks into mammals until they are later acquired by other feeding ticks. Transcription of cspZ and synthesis of BbCRASP-2 were severely repressed during cultivation in laboratory medium relative to mRNA levels observed during mammalian infection, and cspZ expression was influenced by culture temperature and pH, observations which will assist identification of the mechanisms employed by B. burgdorferi to control expression of this borrelial infection-associated protein
Borrelia burgdorferi Regulates Expression of Complement Regulator-Acquiring Surface Protein 1 during the Mammal-Tick Infection Cycle
During the natural mammal-tick infection cycle, the Lyme disease spirochete Borrelia burgdorferi comes into contact with components of the alternative complement pathway. B. burgdorferi, like many other human pathogens, has evolved the immune evasion strategy of binding two host-derived fluid-phase regulators of complement, factor H and factor H-like protein 1 (FHL-1). The borrelial complement regulator-acquiring surface protein 1 (CRASP-1) is a surface-exposed lipoprotein that binds both factor H and FHL-1. Analysis of CRASP-1 expression during the mammal-tick infectious cycle indicated that B. burgdorferi expresses this protein during mammalian infection, supporting the hypothesized role for CRASP-1 in immune evasion. However, CRASP-1 synthesis was repressed in bacteria during colonization of vector ticks. Analysis of cultured bacteria indicated that CRASP-1 is differentially expressed in response to changes in pH. Comparisons of CRASP-1 expression patterns with those of other infection-associated B. burgdorferi proteins, including the OspC, OspA, and Erp proteins, indicated that each protein is regulated through a unique mechanism
SpoVG is a highly conserved Eubacteria protein.
<p><b>Illustrated is an alignment of the predicted sequences of SpoVG proteins from 19 different families of 6 different phyla.</b> Alignment was performed using Geneious software, Pfam 200, with 1000 iterations. Identical or homologous amino acids found in the same position of multiple proteins are indicated by same-colored boxes. Note that these analyses grouped the SpoVG protein of the opportunistic oral pathogen <i>Prevotella dentalis</i> with the Gram-positive <i>Bacilli</i> class, although it is currently considered to be a member of the <i>Bacteroides</i>. Consistent with these results, <i>P. dentalis</i> has morphological and biochemical features which differ from other species in the genus <i>Prevotella</i> and class <i>Bacteroides </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066683#pone.0066683-Willems1" target="_blank">[51]</a>. Red arrows indicate residues demonstrated to be involved in SpoVG-DNA interactions. Green asterisks denote conserved residues that were found to be not required for binding DNA. The magenta box indicates residues of SpoVG<i><sub>Bb</sub></i> and SpoVG<i><sub>Sa</sub></i> involved in DNA sequence specificity.</p