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No description available.

No description available.

Campylobacter jejuni is a gram-negative thermotolerant microaerobic pathogen that causes human gastroenteritis worldwide. The production of extracellular polymeric substances to create a biofilm is a mechanism by which C. jejuni can protect itself from unfavourable environments, and is a contributory factor to the survival and transmission of the organism to farms animals and into the human food chain. Bacteriophages are natural predators of bacteria that have the potential for use as targeted biocontrol agents with the advantage that they can penetrate and affect bacteria embedded in biofilms. The effects of virulent Campylobacter-specific bacteriophages CP8 and CP30 on C. jejuni biofilms formed on glass by strains NCTC 11168, PT14 and HPC5 at 37 °C under microaerobic conditions were investigated. Independent bacteriophage treatment led to 1 to 3 Log10 CFU/cm2 reductions in the viable count 24 h postinfection compared with control levels. In contrast, bacteriophage applied under these conditions effected a reduction of less than 1 Log10 CFU/ml in planktonic cells. Resistance to bacteriophage in bacteria surviving bacteriphage treatment of C. jejuni NCTC 11168 biofilms was >80%, whereas bacteriophage resistance was not found in similarly recovered C. jejuni PT14 cells. Concomitant dispersal of the biofilm matrix by bacteriophage was demonstrated by crystal violet staining and transmission electron microscopy (TEM). The resistant survivors of bacteriophage treatment of biofilms formed by HPC5 and PT14 remained closely associated with the phage but not NCTC 11168. Analysis of the DNA contents of these isolates by PFGE and Southern transfer confirmed the presence of phage genomic DNA (approximately 140 kb) leading to the conclusion that these strains represent examples of the carrier state life cycle (CSLC) reported for other bacterial species. TEMs of CSLC cultures demonstrated the association of bacteriophage particles with Campylobacter cells that were devoid of flagella. Physiological studies of the CSLC strains showed the bacteria were non-motile but able to grow at a similar rate to parental cultures until reaching the phage proliferation threshold (7 Log10 CFU/ml) when growth rate declined and the phage titre increased. Of further note the CSLC strains had a greater capacity to survive atmospheric oxygen under nutrient limited conditions. CSLC phages exhibited differences in host binding, efficiency of plating and host range. Transcriptome analyses of CSLC strains harvested from microaerobic cultures at early exponential phase prior to phage proliferation were performed using DNA microarrays to demonstrate changes in host gene expression as compared with parental cultures. Notably genes involved in metabolism and the modification of macromolecules were up-regulated and specific flagella biosynthesis functions down-regulated in the CSLC strains.

N-linked protein glycosylation involves the transfer of a glycan onto an Asparagine residue (N) of a polypeptide chain. It is common in Eukaryotes and has recently been observed in Prokaryotes, most notably in Campylobacter jejuni. The C. jejuni N-linked glycosylation system is encoded on a single pgl gene locus that also functions when expressed in Escherichia coli. The key enzyme involved in N-linked protein glycosylation is encoded by the pglB gene and transfers lipid-linked glycan onto N residues of glycoproteins in the periplasm. It is clear from accumulating genome sequence data that pglB orthologues are present in all Campylobacter species and in related species such as Wolinella succinogenes, Desulfovibrio vulgaris and Desulfovibrio desulfuricans. Most Helicobacter species, including Helicobacter pylori, lack the pglB gene but three related Helicobacter species Helicobacter pullorum, Helicobacter canadensis and Helicobacter winghamensis have two distinct pglB genes. These and other orthologues of C. jejuni pgl genes are located not within a single locus but rather at five distinct loci. One of the two pglB genes, termed pglB1, is required for in vitro N-glycosylation of peptides (Jervis et al., 2010). In this thesis I present data on the role of further pgl gene orthologues and previously uncharacterized genes in H. pullorum N-glycosylation. Furthermore I have also identified a number of H. pullorum glycoproteins and provide data comparing N-glycosylation processes in C. jejuni and H. pullorum. These data expand our preliminary observations on the first Helicobacter N-linked glycosylation system, and provide important information on the similarities and differences between the well characterised C. jejuni system and these more recently identified pathways.

Από 3 πρότυπα στελέχη Pseudomonas aeruginosa το Smooth στέλεχος PAC-IR και τα Rough στελέχη PAC-557 και PAC-605, που δώρησε ευγενώς στο Εργαστήριο Μικροβιολογίας του Πανεπιστημίου Πατρών η Dr. ΡΜ Meadow του University Co 1 lege, London παρελήφθησαν με ειδική μεθοδολογία ο λιποπολυσακχαρίτης L.P.S, η εξωκυττάρια ουσία (Slime) και οι εξωτερικές μεμβράνες. Έγιναν ηλεκτροφορήσεις με αποδιατακτικούς παράγοντες (SDS-PAGE) και χρώση των πηκτωμάτων για πρωτεΐνες με Coomassie blue, και για L.P.S με AgNÖ3. Οι ηλεκτροφορητικές εικόνες του Slime, του L.P.S και των εξωτερικών μεμβρανών διαφέρουν μεταξύ τους και στα 3 στελέχη. Ανάλυση των ουδετέρων σακχάρων έδειξε ότι και τα 3 Slime περί έχουν με ποσοτικές διαφορές 6 σάκχαρα, τη ραμνόζη, τη φουκόζη, τη ξυλόζη, τη μαννόζη και τη γαλακτόζη. Αντίθετα μαννόζη και γαλακτόζη δεν περιέχονται στο Smooth L.P.S, ενώ οι Rough L.P.Ss περί έχουν μόνο ραμνόζη και γλυκόζη. Ανάλυση του λιπιδικού στοιχείου των 3 Slime και των 3 L.P.Ss έδειξε ότι ποιοτικά περιέχουν 5 ίδια λιπαρά οξέα. Δωδεκανοικό οξύ (~Cii), 20Η-δωδεκανοικό (-Ci?), δεκατετρανικό οξύ (~Ci 4), δεκαεξανικό οξύ (~Cit), δεκαοκτανικό οξύ (-Ci s) και τα ισομερή του. Μετά από χρωματογραφία μοριακής διήθησης στα 3 Slime ο πολυσακχαρίτης που παραλαμβάνεται από την υδατανθρακική κορυφή δεν περιέχει L πρωτεΐνη και είναι μοριακού βάρους 40.000-100.000 daltons. Τα αποτελέσματα αυτά αποδεικνύουν ότι η παραγωγή του Slime είναι κοινή ιδιότητα Smooth και Rough στελεχών Ρ.aeruginosa και ότι το υδατανθρακικό στοιχείο του Slime είναι αυτοτελής ουσία, ελεύθερη πρωτεϊνών, που δεν αποτελείται από τις πλευρικές αλυσίδες του L.P.S. / Lipopolysaccharide (L.P.S), slime and outer membranes were obtained from a smooth, nonmucoid Pseudomonas aeruginosa strain, PAC-IR and its two rough mutants, PAC~ 557 and PAC-605 (Kindly provided by Dr. PM Meadow, University College, London). Electrophoretic analysis on SDS-polyacrylamide gels and staining with silver nitrate and Coomassie blue showed different profiles between L.P.S, Slime and outer membranes in all three strains. Comparative analysis of the saccharide moiety between L.P.S and Slime of each strain by H.P.L.C demonstrated that the saccharide moiety of slime has different composition from that of L.P.S. Six neutral sugars, rhamnose, fucose, xylose, mannose, galactose and glucose were identified in all three slimes though in different amounts. Mannose and galactose were not found in the smooth L.P.S, whereas only rhamnose and glucose were identified in the L.P.Ss of the rough strains. Comparative analysis of the lipid moiety between L.P.S and Slime in all three strains with Gas Chromatography and Mass Spectroscopy indicated that lipid moiety had no quaiitative differences concerning the lipid acids. Five lipid acids were identified in all three slimes and L.P.Ss dodecanoic acid (~Ci2)> 20H~dodecanoic acid (-C2?), tetradecanoic (-C14), exadecanoic (Ci(,), octadecanoic (- Cis) and its isomeric forms. After gel filtration of all three slimes the polysaccharide obtained from the carbohydrate peak fraction was found to be protein free. By gel filtration the mo 1 ecular size of the polysaccharide was estimated to be about 40000-100000 daltons. Whether the polysaccharide moiety is a glycolipid is not clear at the present. This problem is currently under investigation. Our results suggest that slime production is a common property shared by both Smooth and Hough Ρ.aeruginosa strains. The saccharide moiety of slime does not represent the side chains of L.P.S and it is protein free.