1,721,162 research outputs found
Further strategies for signature-tagged mutagenesis and the application of oligonucleotide microarrays for the quantification of DNA-tagged strains
No full textThe original signature-tagged mutagenesis (STM) technique has certain technical limitations, including high variation in signal intensities after hybridization, owing to the variability of the tag sequences, necessitating further rounds of screening. This chapter describes the basic STM approach to identify potential virulence determinants in Yersinia pseudotuberculosis and to monitor the survival rate of Helicobacter pylori mutants under different environmental conditions. In principle, this technology could be more widely applied to measure the relative abundance of tagged microbial strains in any complex environment. This chapter reasoned that the detection of DNA tagged strains could be improved by using (1) the DNA sequence tags designed to have similar hybridization properties, (2) two tags for each mutant, and (3) sequences corresponding to both strands of the tags arrayed on an affymetrix “bar coding” microarray, containing immobilized oligonucleotide tag sequences
Development and application of the active surveillance of pathogens microarray to monitor bacterial gene flux.
Get PDFBACKGROUND: Human and animal health is constantly under threat by emerging pathogens that have recently acquired genetic determinants that enhance their survival, transmissibility and virulence. We describe the construction and development of an Active Surveillance of Pathogens (ASP) oligonucleotide microarray, designed to 'actively survey' the genome of a given bacterial pathogen for virulence-associated genes.
RESULTS: The microarray consists of 4958 reporters from 151 bacterial species and include genes for the identification of individual bacterial species as well as mobile genetic elements (transposons, plasmid and phage), virulence genes and antibiotic resistance genes. The ASP microarray was validated with nineteen bacterial pathogens species, including Francisella tularensis, Clostridium difficile, Staphylococcus aureus, Enterococcus faecium and Stenotrophomonas maltophilia. The ASP microarray identified these bacteria, and provided information on potential antibiotic resistance (eg sufamethoxazole resistance and sulfonamide resistance) and virulence determinants including genes likely to be acquired by horizontal gene transfer (e.g. an alpha-haemolysin).
CONCLUSION: The ASP microarray has potential in the clinic as a diagnostic tool, as a research tool for both known and emerging pathogens, and as an early warning system for pathogenic bacteria that have been recently modified either naturally or deliberately
Lateral gene transfer between prokaryotes and multicellular eukaryotes: ongoing and significant?
Get PDFAbstract The expansion of genome sequencing projects has produced accumulating evidence for lateral transfer of genes between prokaryotic and eukaryotic genomes. However, it remains controversial whether these genes are of functional importance in their recipient host. Nikoh and Nakabachi, in a recent paper in BMC Biology, take a first step and show that two genes of bacterial origin are highly expressed in the pea aphid Acyrthosiphon pisum. Active gene expression of transferred genes is supported by three other recent studies. Future studies should reveal whether functional proteins are produced and whether and how these are targeted to the appropriate compartment. We argue that the transfer of genes between host and symbiont may occasionally be of great evolutionary importance, particularly in the evolution of the symbiotic interaction itself.</p
Separated at birth? Microarray analysis of two strikingly similar Yersinia species
Get PDFThis is the final version of the article. Available from Wiley via the DOI in this record.We acknowledge financial support for this project from
DSTL and The Wellcome Trust. We also acknowledge
BµG@S (the Bacterial Microarray Group at St George’s)
and The Wellcome Trust for funding their work; Keith Vass
from the University of Glasgow; and Mike Prentice from
St. Bartholomew’s Hospital for his advice and expertise in
all things Yersinia
System-level Strategies for Studying the Metabolism of Mycobacterium tuberculosis
Get PDFBrian D. Robertson and Brendan W. Wre
Investigating novel vaccine candidates for Clostridium difficile
Get PDFClostridium difficile infection (CDI) is the most common cause of nosocomial diarrhoea worldwide. Current treatment options have varied levels of success and there is no licensed vaccine. Most vaccines investigated to date use the C. difficile toxins, as these induce a protective immune response. However, this is only against the symptoms of CDI, therefore there is a need for antigens capable of preventing colonisation and transmission. The results of a C. difficile specific pan-protein array, screened against blood samples from CDI patients and healthy controls, was analysed and a shortlist of proteins identified where the IgG antibody response was significantly higher in the control group. Three of these were expressed and purified in E. coli, and tested against patient samples in an ELISA. For two of the three proteins, there was a higher IgG response in the healthy control group (albeit not significant) confirming the array results. Inactivation of three immunogenic proteins from the array in C. difficile R20291 revealed the putative permease CDR20291_0342, was not involved in efflux of the antimicrobials tested. Inactivation of the putative pilin protein, CDR20291_3343, did not influence surface motility, but motility was almost abolished in a flagella mutant. The putative cobalt transporter CDR20291_0330, was proposed to contribute to a putative vitamin B12 synthesis pathway, which is required for ethanolamine utilisation. Neither R20291Δ0330 or R20291 utilised ethanolamine in the conditions tested but it was found that inactivation of one component of the two-component regulatory system in C. difficile 630Δerm abolished ethanolamine utilisation. Finally, mechanisms of synthesising C. difficile glycoconjugates using bioconjugation were investigated, using a carrier protein and portion of the R20291 flagella glycan. A short acceptor peptide was designed and found to be glycosylated at all sites with a glycan from Campylobacter jejuni but it was not possible to detect glycosylation with the flagella glycan from C. difficile
Investigating the role of N-linked glycosylation system in bacteria
Get PDFGlycosylation is the most abundant protein post-translational modification found in nature. The attachment of glycans to proteins has been shown to play a central role in modulating protein folding, stability, and signalling. In recent years, it has become evident that glycosylation systems are found in all domains of life. Advances in genomics and mass spectrometry have revealed several types of glycosylation systems in bacteria. However, how and why bacterial proteins are modified remains poorly defined. The aim of this study is to a) investigate the role of general N-linked glycosylation in a major food poisoning bacterium; Campylobacter jejuni b) functionally analyse other N-linked glycosylation systems in deep-sea vent bacteria c) Interrogate the role of other forms of N-linked glycosylation systems in bacteria. This study evaluates the differential protein expression in glycosylation deficient C. jejuni compared to its wildtype counterpart. Thus, enriching our understanding of the role of general N-linked glycans and the pleotropic effects caused by knocking out this post-translational modification. Isobaric labelling mass spectrometry results indicate that protein quality control machineries are more abundant in glycosylation deficient C. jejuni. Also, the major multidrug efflux pump; CmeABC and nitrate reduction assembly; NapAB were shown to be impaired in N-linked
glycosylation null C. jejuni. Furthermore, examination of the role of Nlinked glycans in stabilising CmeABC indicated that N-linked glycans modulate protein folding, reduce protein unfolding rate and enhance protein-protein interaction. Computational and functional analysis confirmed the presence and the activity of the oligosaccharyltransferase PglB, of N-linked glycosylation system from deep-sea vent bacteria. Investigating other forms of N-linked glycosylation in the pig pathogen bacterium; Actinobacillus pleuropneumoniae, showed its importance in cell adhesion and pathogenesis. This study provides deeper insights into the roles of N-glycans at the molecular level and enriches our understanding
of microbial glycoproteome
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