| genetic analysis of a tetracycline resistance element from clostridium difficile and its conjugal transfer to and from bacillus subtilis. | a tetracycline resistance (tcr) determinant from clostridium difficile strain 630 was cloned into the escherichia coli plasmid vector puc13. the resulting plasmid pppm20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp saci-hindiii fragment wholly within the tcr gene was identified. dot-blot hybridization studies with the 1.1 kbp fragment showed that the tcr gene belonged to hybridization class m. tcr could be transferred between c. difficile strains and to bacillus subtilis at a frequ ... | 1990 | 2172445 |
| transfer of macrolide-lincosamide-streptogramin b (mls) resistance in clostridium difficile is linked to a gene homologous with toxin a and is mediated by a conjugative transposon, tn5398. | an mls resistance gene designated ermbz, from a toxigenic clostridium difficile strain (630) could be transferred between c. difficile strains, and to and from bacillus subtilis. the intergeneric transfer occurred in the absence of any detectable plasmid dna and the element responsible for gene transfer entered the recipient's chromosome, behaviour which is characteristic of a conjugative transposon. the element was designated tn5398 and was found in six c. difficile strains. tn5398 could be tra ... | 1995 | 7759394 |
| acd, a peptidoglycan hydrolase of clostridium difficile with n-acetylglucosaminidase activity. | a gene encoding a putative peptidoglycan hydrolase was identified by sequence similarity searching in the clostridium difficile 630 genome sequence, and the corresponding protein, named acd (autolysin of c. difficile) was expressed in escherichia coli. the deduced amino acid sequence of acd shows a modular structure with two main domains: an n-terminal domain exhibiting repeated sequences and a c-terminal catalytic domain. the c-terminal domain exhibits sequence similarity with the glucosaminida ... | 2005 | 16000724 |
| semiquantitative analysis of clinical heat stress in clostridium difficile strain 630 using a gelc/ms workflow with empai quantitation. | clostridium difficile is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. here, we used gelc/ms and the exponentially modified protein abundance index (empai) calculation to determine proteomic changes in response to a clinically relevant heat stress. reproducibility between both biological and technical replicates was good, and a 37°c proteome of 224 proteins was complemented by a 41°c ... | 2014 | 24586458 |
| a quantitative proteomic analysis of the heat stress response in <i>clostridium difficile</i> strain 630. | <i>clostridium difficile</i> is a serious nosocomial pathogen whose prevalence worldwide is increasing. post genomic technologies can now be deployed in order to develop understanding of the evolution and diversity of this important human pathogen, yet little is known about the adaptive ability of <i>c. difficile</i>. we used itraq labelling and 2d-lc-ms/ms driven proteomics to investigate the response of <i>c. difficile</i> 630 to a mild, but clinically relevant heat stress. a statistically val ... | 2011 | 21786815 |
| reannotation of the genome sequence of clostridium difficile strain 630. | a regular update of genome annotations is a prerequisite step to help maintain the accuracy and relevance of the information they contain. five years after the first publication of the complete genome sequence of clostridium difficile strain 630, we manually reannotated each of the coding sequences (cdss), using a high-level annotation platform. the functions of more than 500 genes annotated previously with putative functions were reannotated based on updated sequence similarities to proteins wh ... | 2011 | 21349987 |
| proteomic analysis of the insoluble subproteome of clostridium difficile strain 630. | clostridium difficile, a gram-positive spore-forming anaerobe, causes infections in humans ranging from mild diarrhoeal to potentially life-threatening pseudomembranous colitis. the availability of genomic information for a range of c. difficile strains affords researchers the opportunity to better understand not only the evolution of these organisms but also their basic physiology and biochemistry. we used proteomics to characterize the insoluble subproteome of c. difficile strain 630. gel-base ... | 2010 | 20868380 |
| proteomic and genomic characterization of highly infectious clostridium difficile 630 spores. | clostridium difficile, a major cause of antibiotic-associated diarrhea, produces highly resistant spores that contaminate hospital environments and facilitate efficient disease transmission. we purified c. difficile spores using a novel method and show that they exhibit significant resistance to harsh physical or chemical treatments and are also highly infectious, with <7 environmental spores per cm(2) reproducibly establishing a persistent infection in exposed mice. mass spectrometric analysis ... | 2009 | 19542279 |
| mutations in fusa associated with posttherapy fusidic acid resistance in clostridium difficile. | in silico, we identified fusa (2,067 bp) in clostridium difficile 630. sequencing of fusa in posttherapy fusidic acid-resistant c. difficile isolates from 12 patients with c. difficile-associated diarrhea (cdad) identified fusa mutations, one or two nonsynonymous substitutions, or in one case a deletion of one codon associated with resistance. five of these mutations have previously been described in fusa of fusidic acid-resistant staphylococcus aureus, but seven were novel fusa mutations. fusid ... | 2007 | 17307985 |
| typing and subtyping of clostridium difficile isolates by using multiple-locus variable-number tandem-repeat analysis. | using the genomic sequence of clostridium difficile strain 630, we developed multiple-locus variable-number tandem-repeat analysis (mlva) with automated fragment analysis and multicolored capillary electrophoresis as a typing method for c. difficile. all reference strains, representing 31 serogroups, 25 toxinotypes, and 7 known subtypes of pcr ribotype 001, could be discriminated from each other. application of mlva to 28 isolates from 7 outbreaks due to the emerging hypervirulent pcr ribotype 0 ... | 2007 | 17166961 |
| the multidrug-resistant human pathogen clostridium difficile has a highly mobile, mosaic genome. | we determined the complete genome sequence of clostridium difficile strain 630, a virulent and multidrug-resistant strain. our analysis indicates that a large proportion (11%) of the genome consists of mobile genetic elements, mainly in the form of conjugative transposons. these mobile elements are putatively responsible for the acquisition by c. difficile of an extensive array of genes involved in antimicrobial resistance, virulence, host interaction and the production of surface structures. th ... | 2006 | 16804543 |
| generation of an erythromycin-sensitive derivative of clostridium difficile strain 630 (630deltaerm) and demonstration that the conjugative transposon tn916deltae enters the genome of this strain at multiple sites. | erythromycin resistance in clostridium difficile strain 630 is conferred by a genetic element termed tn5398 which contains two erm(b) genes: erm1(b) and erm2(b). an erythromycin-sensitive derivative of strain 630 (designated 630deltaerm) was generated by spontaneous mutation after continuous subculture for 30 days. this strain had lost the erm2(b) gene from within tn5398 but retained erm1(b). however, the strain could revert to erythromycin resistance at a frequency of 2.79 x 10(-8), although it ... | 2005 | 15673506 |
| the macrolide-lincosamide-streptogramin b resistance determinant from clostridium difficile 630 contains two erm(b) genes. | the ermb macrolide-lincosamide-streptogramin b (mls) resistance determinant from clostridium difficile 630 contains two copies of an erm(b) gene, separated by a 1.34-kb direct repeat also found in an erm(b) determinant from clostridium perfringens. in addition, both erm(b) genes are flanked by variants of the direct repeat sequence. this genetic arrangement is novel for an ermb mls resistance determinant. | 2000 | 10639372 |
| comparative transcriptional analysis of clinically relevant heat stress response in clostridium difficile strain 630. | clostridium difficile is considered to be one of the most important causes of health care-associated infections worldwide. in order to understand more fully the adaptive response of the organism to stressful conditions, we examined transcriptional changes resulting from a clinically relevant heat stress (41 °c versus 37 °c) in c. difficile strain 630 and identified 341 differentially expressed genes encompassing multiple cellular functional categories. while the transcriptome was relatively resi ... | 2012 | 22860125 |
| proteases and sonication specifically remove the exosporium layer of spores of clostridium difficile strain 630. | clostridium difficile spores are the means through which this anaerobic pathogen may persist in hospital surfaces and in the host. there is a lack of knowledge in the proteins that localize to the surface of c. difficile spores primarily due to the lack of established methods to efficiently separate the outermost layer, the exosporium. in this work, we propose methods to remove the exosporium layer of c. difficile spores through either protease digestion or sonication treatment leaving the spore ... | 2013 | 23384826 |
| transcriptional analysis of temporal gene expression in germinating clostridium difficile 630 endospores. | clostridium difficile is the leading cause of hospital acquired diarrhoea in industrialised countries. under conditions that are not favourable for growth, the pathogen produces metabolically dormant endospores via asymmetric cell division. these are extremely resistant to both chemical and physical stress and provide the mechanism by which c. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants. spores are the primary infective a ... | 2013 | 23691138 |
| solution nmr structure of cd1104b from pathogenic clostridium difficile reveals a distinct α-helical architecture and provides first structural representative of protein domain family pf14203. | a high-quality structure of the 68-residue protein cd1104b from clostridium difficile strain 630 exhibits a distinct all α-helical fold. the structure presented here is the first representative of bacterial protein domain family pf14203 (currently 180 members) of unknown function (duf4319) and reveals that the side-chains of the only two strictly conserved residues (glu 8 and lys 48) form a salt bridge. moreover, these two residues are located in the vicinity of the largest surface cleft which i ... | 2013 | 24048810 |
| structures of a bifunctional cell wall hydrolase cwlt containing a novel bacterial lysozyme and an nlpc/p60 dl-endopeptidase. | tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. orf14 within the conjugation module encodes a bifunctional cell wall hydrolase cwlt that consists of an n-terminal bacterial lysozyme domain (n-acetylmuramidase, blysg) and a c-terminal nlpc/p60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. we determined the crystal structures of cwlt from two pathogens, ... | 2014 | 24051416 |
| structural and biochemical analyses of alanine racemase from the multidrug-resistant clostridium difficile strain 630. | clostridium difficile, a gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients. c. difficile is frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudomembranous colitis. the severity of c. difficile infections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated w ... | 2014 | 25004969 |
| fluoroquinolone resistance does not impose a cost on the fitness of clostridium difficile in vitro. | point mutations conferring resistance to fluoroquinolones were introduced in the gyr genes of the reference strain clostridium difficile 630. only mutants with the substitution thr-82→ile in gyra, which characterizes the hypervirulent epidemic clone iii/027/nap1, were resistant to all fluoroquinolones tested. the absence of a fitness cost in vitro for the most frequent mutations detected in resistant clinical isolates suggests that resistance will be maintained even in the absence of antibiotic ... | 2015 | 25534738 |
| genome resequencing of the virulent and multidrug-resistant reference strain clostridium difficile 630. | we resequenced the complete genome of the virulent and multidrug-resistant pathogen clostridium difficile strain 630. a combination of single-molecule real-time and illumina sequencing technology revealed the presence of an additional rrna gene cluster, additional trnas, and the absence of a transposon in comparison to the published and reannotated genome sequence. | 2015 | 25858846 |
| what's a snp between friends: the influence of single nucleotide polymorphisms on virulence and phenotypes of clostridium difficile strain 630 and derivatives. | clostridium difficile is a major cause of antibiotic induced diarrhea worldwide, responsible for significant annual mortalities and represents a considerable economic burden on healthcare systems. the two main c. difficile virulence factors are toxins a and b. isogenic toxin b mutants of 2 independently isolated erythromycin-sensitive derivatives (630e and 630δerm) of strain 630 were previously shown to exhibit substantively different phenotypes. compared to 630, strain 630e and its progeny grow ... | 2016 | 27652799 |
| insight into the 3d structure and substrate specificity of previously uncharacterized gnat superfamily acetyltransferases from pathogenic bacteria. | members of the gcn5-related n-acetyltransferase (gnat) superfamily catalyze the acetylation of a wide range of small molecule and protein substrates. due to their abundance in all kingdoms of life and diversity of their functions, they are implicated in many aspects of eukaryotic and prokaryotic physiology. although numerous gnats have been identified thus far, many remain structurally and functionally uncharacterized. the elucidation of their structures and functions is critical for broadening ... | 2017 | 27783928 |
| manual curation and reannotation of the genomes of clostridium difficile 630δerm and clostridium difficile 630. | we resequenced the genome of clostridium difficile 630δerm (dsm 28645), a model strain commonly used for the generation of insertion mutants. the genome sequence was obtained by a combination of single-molecule real-time (smrt) and illumina sequencing technology. detailed manual curation and comparison to the previously published genomic sequence revealed sequence differences including inverted regions and the presence of plasmid pcd630. manual curation of our previously deposited genome sequenc ... | 2017 | 28068217 |
| the cwb2 cell wall-anchoring module is revealed by the crystal structures of the clostridium difficile cell wall proteins cwp8 and cwp6. | bacterial cell wall proteins play crucial roles in cell survival, growth, and environmental interactions. in gram-positive bacteria, cell wall proteins include several types that are non-covalently attached via cell wall binding domains. of the two conserved surface-layer (s-layer)-anchoring modules composed of three tandem slh or cwb2 domains, the latter have so far eluded structural insight. the crystal structures of cwp8 and cwp6 reveal multi-domain proteins, each containing an embedded cwb2 ... | 2017 | 28132783 |
| proteome mining for the identification and in-silico characterization of putative drug targets of multi-drug resistant clostridium difficile strain 630. | clostridium difficile is an enteric pathogen that causes approximately 20% to 30% of antibiotic-associated diarrhea. in recent years, there has been a substantial rise in the rate of c. difficile infections as well as the emergence of virulent and antibiotic resistant c. difficile strains. so, there is an urgent need for the identification of therapeutic potential targets and development of new drugs for the treatment and prevention of c. difficile infections. in the current study, we used a hyb ... | 2017 | 28235560 |
| protein composition of the outermost exosporium-like layer of clostridium difficile 630 spores. | clostridium difficile spores are considered the morphotype of infection, transmission and persistence of c. difficile infections. there is a lack of information on the composition of the outermost exosporium layer of c. difficile spores. using recently developed exosporium removal methods combined with ms/ms, we have established a gel-free approach to analyze the proteome of the exosporium of c. difficile spores of strain 630. a total of 184 proteins were found in the exosporium layer of c. diff ... | 2015 | 25849250 |
| structural insight into the clostridium difficile ethanolamine utilisation microcompartment. | bacterial microcompartments form a protective proteinaceous barrier around metabolic enzymes that process unstable or toxic chemical intermediates. the genome of the virulent, multidrug-resistant clostridium difficile 630 strain contains an operon, eut, encoding a bacterial microcompartment with genes for the breakdown of ethanolamine and its utilisation as a source of reduced nitrogen and carbon. the c. difficile eut operon displays regulatory genetic elements and protein encoding regions in co ... | 2012 | 23144756 |
| clostridium difficile has a single sortase, srtb, that can be inhibited by small-molecule inhibitors. | bacterial sortases are transpeptidases that covalently anchor surface proteins to the peptidoglycan of the gram-positive cell wall. sortase protein anchoring is mediated by a conserved cell wall sorting signal on the anchored protein, comprising of a c-terminal recognition sequence containing an "lpxtg-like" motif, followed by a hydrophobic domain and a positively charged tail. | 2014 | 25183427 |