Isolation of Bordetella avium and Novel Bordetella Strain from Patients with Respiratory Disease

Bordetella avium is thought to be strictly an avian pathogen. However, 16S rRNA gene sequencing identified 2 isolates from 2 humans with respiratory disease as B. avium and a novel B. avium–like strain. Thus, B. avium and B. avium–like organisms are rare opportunistic human pathogens

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 agent and must germinate to allow for vegetative cell growth and toxin production. While spore germination in Bacillus is well understood, little is known about C. difficile germination and outgrowth. Here we use genome-wide transcriptional analysis to elucidate the temporal gene expression patterns in C. difficile 630 endospore germination. We have optimized methods for large scale production and purification of spores. The germination characteristics of purified spores have been characterized and RNA extraction protocols have been optimized. Gene expression was highly dynamic during germination and outgrowth, and was found to involve a large number of genes. Using this genome-wide, microarray approach we have identified 511 genes that are significantly up- or down-regulated during C. difficile germination (p≤0.01). A number of functional groups of genes appeared to be co-regulated. These included transport, protein synthesis and secretion, motility and chemotaxis as well as cell wall biogenesis. These data give insight into how C. difficile re-establishes its metabolism, re-builds the basic structures of the vegetative cell and resumes growth

Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

Background \ud Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. \ud \ud Methods \ud In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. \ud \ud Conclusion \ud This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies

How the other half lives: CRISPR-Cas's influence on bacteriophages

CRISPR-Cas is a genetic adaptive immune system unique to prokaryotic cells used to combat phage and plasmid threats. The host cell adapts by incorporating DNA sequences from invading phages or plasmids into its CRISPR locus as spacers. These spacers are expressed as mobile surveillance RNAs that direct CRISPR-associated (Cas) proteins to protect against subsequent attack by the same phages or plasmids. The threat from mobile genetic elements inevitably shapes the CRISPR loci of archaea and bacteria, and simultaneously the CRISPR-Cas immune system drives evolution of these invaders. Here we highlight our recent work, as well as that of others, that seeks to understand phage mechanisms of CRISPR-Cas evasion and conditions for population coexistence of phages with CRISPR-protected prokaryotes.Comment: 24 pages, 8 figure

Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements

<p>Abstract</p> <p>Background</p> <p><it>Clostridium botulinum </it>strains can be divided into four physiological groups that are sufficiently diverged to be considered as separate species. Here we present the first complete genome of a <it>C. botulinum </it>strain from physiological group III, causing animal botulism. We also compare the sequence to three new draft genomes from the same physiological group.</p> <p>Results</p> <p>The 2.77 Mb chromosome was highly conserved between the isolates and also closely related to that of <it>C. novyi</it>. However, the sequence was very different from the human <it>C. botulinum </it>group genomes. Replication-directed translocations were rare and conservation of synteny was high. The largest difference between <it>C. botulinum </it>group III isolates occurred within their surprisingly large plasmidomes and in the pattern of mobile elements insertions. Five plasmids, constituting 13.5% of the total genetic material, were present in the completed genome. Interestingly, the set of plasmids differed compared to other isolates. The largest plasmid, the botulinum-neurotoxin carrying prophage, was conserved at a level similar to that of the chromosome while the medium-sized plasmids seemed to be undergoing faster genetic drift. These plasmids also contained more mobile elements than other replicons. Several toxins and resistance genes were identified, many of which were located on the plasmids.</p> <p>Conclusions</p> <p>The completion of the genome of <it>C. botulinum </it>group III has revealed it to be a genome with dual identity. It belongs to the pathogenic species <it>C. botulinum</it>, but as a genotypic species it should also include <it>C. novyi </it>and <it>C. haemolyticum</it>. The genotypic species share a conserved chromosomal core that can be transformed into various pathogenic variants by modulation of the highly plastic plasmidome.</p

Expression, purification, crystallization and preliminary crystallographic analysis of a putative Clostridium difficile surface protein Cwp19

Cwp19 is a putatively surface-located protein from Clostridium difficile. A recombinant N-terminal protein (residues 27–401) lacking the signal peptide and the C-terminal cell-wall-binding repeats (PFam04122) was crystallized using the sitting-drop vapour-diffusion method and diffracted to 2 Å resolution

Codon choice in genes depends on flanking sequence information—implications for theoretical reverse translation

Algorithms for theoretical reverse translation have direct applications in degenerate PCR. The conventional practice is to create several degenerate primers each of which variably encode the peptide region of interest. In the current work, for each codon we have analyzed the flanking residues in proteins and determined their influence on codon choice. From this, we created a method for theoretical reverse translation that includes information from flanking residues of the protein in question. Our method, named the neighbor correlation method (NCM) and its enhancement, the consensus-NCM (c-NCM) performed significantly better than the conventional codon-usage statistic method (CSM). Using the methods NCM and c-NCM, we were able to increase the average sequence identity from 77% up to 81%. Furthermore, we revealed a significant increase in coverage, at 80% identity, from < 20% (CSM) to > 75% (c-NCM). The algorithms, their applications and implications are discussed herein

Genome Evolution of Wolbachia Strain wPip from the Culex pipiens Group

The obligate intracellular bacterium Wolbachia pipientis strain wPip induces cytoplasmic incompatibility (CI), patterns of crossing sterility, in the Culex pipiens group of mosquitoes. The complete sequence is presented of the 1.48-Mbp genome of wPip which encodes 1386 coding sequences (CDSs), representing the first genome sequence of a B-supergroup Wolbachia. Comparisons were made with the smaller genomes of Wolbachia strains wMel of Drosophila melanogaster, an A-supergroup Wolbachia that is also a CI inducer, and wBm, a mutualist of Brugia malayi nematodes that belongs to the D-supergroup of Wolbachia. Despite extensive gene order rearrangement, a core set of Wolbachia genes shared between the 3 genomes can be identified and contrasts with a flexible gene pool where rapid evolution has taken place. There are much more extensive prophage and ankyrin repeat encoding (ANK) gene components of the wPip genome compared with wMel and wBm, and both are likely to be of considerable importance in wPip biology. Five WO-B–like prophage regions are present and contain some genes that are identical or highly similar in multiple prophage copies, whereas other genes are unique, and it is likely that extensive recombination, duplication, and insertion have occurred between copies. A much larger number of genes encode ankyrin repeat (ANK) proteins in wPip, with 60 present compared with 23 in wMel, many of which are within or close to the prophage regions. It is likely that this pattern is partly a result of expansions in the wPip lineage, due for example to gene duplication, but their presence is in some cases more ancient. The wPip genome underlines the considerable evolutionary flexibility of Wolbachia, providing clear evidence for the rapid evolution of ANK-encoding genes and of prophage regions. This host–Wolbachia system, with its complex patterns of sterility induced between populations, now provides an excellent model for unraveling the molecular systems underlying host reproductive manipulation

Characterization of Clostridium difficile isolates using capillary gel electrophoresis-based PCR ribotyping

We have developed a Clostridium difficile PCR ribotyping method based on capillary gel electrophoresis and have compared it with conventional PCR ribotyping. A total of 146 C. difficile isolates were studied: five isolates were reference strains (PCR ribotypes 001, 014, 017, 027 and 053); 141 were clinical isolates comprising 39 Austrian PCR ribotypes collected in the period 2006–2007 at 25 Austrian healthcare facilities. Capillary gel electrophoresis yielded up to 11 fragments per isolate and 47 ribotype patterns. All but one of the five PCR ribotypes of reference strains were clearly reflected in the chromatograms of capillary-based typing. Capillary gel electrophoresis divided 24 isolates belonging to PCR ribotype type 014 into seven subgroups, whereas subtyping the same isolates using multiple-locus variable-number tandem-repeat analysis yielded three unrelated subgroups, without obvious correlation to sr subgroups. Using a web-based software program (http://webribo.ages.at), we were able to correctly identify these 014 isolates by simply allocating the seven subgroup patterns to one ribotype, i.e. to PCR ribotype 014. We consider capillary gel electrophoresis-based PCR ribotyping to be a way of overcoming the problems associated with inter-laboratory comparisons of typing results, while at the same time substantially diminishing the hands-on time for PCR ribotyping