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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The effect of variations in Neisseria meningitidis pili on bacterial interactions with three epithelial cell lines as well as human umbilical vein endothelial cells was studied using a panel of seven strains expressing Class I or Class II pili. Comparison of adherence of piliated and pilus-deficient variants of each strain to epithelial cells suggested that Class I pili may mediate bacterial adherence with all three epithelial cell lines. In contrast, Class II pili of the strains used did not increase bacterial adherence to Hep-2 larynx carcinoma cells, although an increase in adherence to Chang conjunctival and A549 lung carcinoma epithelial cells was observed in the Class II pili-expressing strains. In addition to these inter-class functional variations, differences in adherence to epithelial cells were also observed among Class I and Class II strains. Functionally different pilin variants of one Class I strain, MC58, were obtained by single colony isolation. One piliated variant was identified which had concurrently lost the ability to adhere to both Chang and Hep-2 cells (‘non-adherent’ phenotype; adherence of 〈 2 bacteria per cell). In addition, several adherent pilin variants were isolated from non-adherent Pil- and Pil- bacteria by selection on Chang cells (adherence of 10–25 bacteria per cell). In contrast to epithelial cells, al) variant pili, whether of Class I or Class II, adhered to endothelial cells in substantially larger numbers (〈50 bacteria per cell) and therefore implied the existence of distinct mechanisms in pilus-facilitated interactions of N. meningitidis with endothelial and epithelial cells.
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co-expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil- backswitcher isolated from the hyper-adherent variant was PilC+ but was non-adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC-expressing variant. The hyper-adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper-adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Neisseria meningitidis pili are filamentous protein structures that are essential adhesins in capsulate bacteria. Pili of adhesion variants of meningococcal strain C311 contain glycosyl residues on pilin (PilE), their major structural subunit. Despite the presence of three potential N-linked glycosylation sites, none appears to be occupied in these pilins. Instead, a novel O-linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1-4-linked digalactose moiety covalently linked to a 2,4-diacetamido-2,4,6-trideoxyhexose sugar which is directly attached to pilin. Pilins derived from galactose epimerase (galE) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. Both parental (#3) pilins and those derived from a hyper-adherent variant (#16) contained identical sugar substitutions in this region of pilin, and galE mutants of #3 were similar to the parental phenotype in their adherence to host cells. These studies have confirmed our previous observations that meningococcal pili are glycosylated and provided the first structural evidence for the presence of covalently linked carbohydrate on pili. In addition, they have revealed a completely novel protein/saccharide linkage.
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Adherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (〈150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non-piliated and failed to adhere to endothelial cells (〈1 bacterium/ cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent Mr and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper-adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably tower compared with their apparent Mr values on SDS-PAGE. Hyper-adherent pilins shared unique changes in sequence including substitution of Asn-113 for Asp-113 and changes from Asn-Asp-Thr-Asp to Thr-Asp-Ala-Lys at residues 127-130 in mature pilin. Asn residues 113 and 127 of‘parental’pilin both form part of the typical eukaryotic N-glycosylation motif Asn-X-Ser/Thr and could potentially be glycosylated post-translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS-PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn-60-Asn-61-Thr-62 in an adherent variant was replaced with Asp-60-Asn-61-Ser-62 in a non-adherent variant. Fully adherent backswitchers obtained from the non-adherent variant always regained Asn-60 but retained Ser-62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N-linked glycosylation sites on the pilin subunit.
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  • 5
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Group
    Nature 338 (1989), S. 622-623 
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] BACTERIAL pathogens frequently exhibit marked variation in their surface components, a phenomenon that assists the sequential colonization of tissues during the course of infections and the avoidance of host immune responses. The underlying molecular mechanisms that drive phase variation, in which ...
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  • 6
    ISSN: 1617-4623
    Keywords: Restriction-modification system ; Neisseria gonorrhoeae ; Nucleotide sequence of R.-M. genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The NgoPII restriction endonuclease, which recognizes the sequence 5′-GG↓CC-3′, differs from its isoschizomer HaeIII in being sensitive to methylation at the external cytosine residue. The entire nucleotide sequence of a cloned 3.3 kb segment of Neisseria gonorrhoeae strain P9 chromosomal DNA which harbours the NgoPII restriction-modification system has been determined. This data, coupled with sub-cloning experiments, indicates that the restriction endonuclease (R.NgoII) and modification (M.NgoII) genes are transcribed from separate promoters but are arranged in tandem, with the R.NgoPII gene being located on the 5′ side of the M.NgoPII gene. Unlike all previously reported restriction systems the 3′ end of the endonuclease open reading frame overlaps the 5′ end of the methylase open reading frame by 8 codons. This overlap may have implications for the regulation of the NgoPII restriction-modification system.
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  • 7
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
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  • 8
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: A peat monolith (15 cm diameter, 35 cm length) from Ellergower Moss (New Galloway, Scotland), kept outdoors and maintained water-saturated, was investigated for the distributions of gases (O2, CO2, CH4), micro-organisms, total archaeal DNA and methanogen DNA. From the water table (at the surface of the Sphagnum), a steep oxycline gave 〈0.25 μM O2 at 2 cm depth (as shown by membrane inlet mass spectrometry and oxygen electrode methods) and 〈10 nM O2 at 6 cm depth (photobacterium gas diffusion probe). Redox potential measurements indicated a steep decline between 6 cm and 13 cm to a value of −90 mV. At the oxic surface of the peat, CO2 measured 0.5 mM and CH4 〈1 μM. Below 7 cm both gases increased to plateaux at 2 mM and 550 μM, respectively; CH4 concentrations also indicated two distinct zones (7 μM to 2.5 cm depth, then to 28 μM at between 3 and 6 cm). Confocal laser scanning microscopy using the fluorophores 5-cyano-2,3-ditolyl tetrazolium chloride or 3,3-dihexyloxacarbocyanine iodide was used to image micro-organisms with redox active electron transport activities or transmembrane electrochemical potentials, respectively. Samples from 1–5 cm depth showed the presence of active aerobic organisms, whereas those from 10 and 20 cm depth were more active anaerobically, and especially so under H2. Archaeal DNA was present throughout the core; strongest hybridisation was below 9 cm. Two methanogen-specific primers, ME1 and ME2 (which amplify a region of the α-subunit of methyl coenzyme M reductase), hybridised with DNA extracted from below 9 cm depth. Here we describe the concerted application of a number of techniques providing direct information on the precise location and activities of microbes involved in the flux of gases from peatlands.
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  • 9
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Homology to IncN, P, Q and W inc regions was investigated amongst 114 Hg2+-resistant or antibiotic-resistant bacteria isolated from lakewater sediments. No hybridisation signals were found with Inc P, Q and W probes, and only one plasmid, pLV1402, hybridised to the IncN probe. PCR primers designed to conserved regions in the replicon of the IncN plasmid pCU1 and the related beta replicon from pGSH500 were used to amplify a 978-bp fragment from pLV1402, with sequence analysis showing a close relationship (99.2% identity) between their replication genes. A 387-bp region from the pLV1402 rep gene was used to re-screen the isolates and identified another related plasmid, pLV1403. A 3.7-kb probe containing the alpha replicon from pGSH500 hybridised to both pLV1402 and pLV1403, suggesting that both are multi-replicon plasmids. The PCR primers and probes described will be useful in future studies of plasmid diversity.
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  • 10
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The application of pig slurry, high in ammonia-nitrogen concentration, to agricultural land is a practice whose effect on soil microbial communities is poorly understood. The autotrophic ammonia-oxidising bacteria are an integral component of the nitrogen cycle in soil, and their activity will be affected by addition of nitrogenous fertilizer. Molecular biological techniques were applied to the direct detection and identification of ammonia-oxidiser populations in cultivated soil plots treated with different amounts of pig slurry. Members of the genus Nitrosospira were shown by 16S rDNA-directed PCR to be present in both unamended and amended soils, regardless of the quantity of pig slurry applied. In contrast, members of the genus Nitrosomonas were detected by the same approach only in those soil plots that had received high loadings of slurry. The fidelity of amplification products was always confirmed by oligonucleotide probing. In addition, we used high stringency PCR and confirmatory gene probing to detect the presence of the ammonia monooxygenase gene (amoA) of Nitrosomonas europaea directly in all amended soil samples, with hybridization signal intensities that increased with the amount of pig slurry applied to plots. Nitrosomonas europaea amoA DNA could not be detected in soil from the untreated plot. These data support the view that nitrosospiras are ubiquitous as important members of nitrifying populations in the environment. The direct detection of nitrosomonad DNA only in amended soils supports the hypothesis that these nitrosomonads become highly competitive under conditions analogous to laboratory enrichment cultures.
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