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The Streptococcus gordonii Platelet Binding Protein GspB Undergoes Glycosylation Independently of Export. Barbara A. Bensing, 2004.The binding of bacteria and platelets may play a central role in the pathogenesis of infective endocarditis . Platelet binding by Streptococcus gordonii strain M99 is predominantly mediated by the 286-kDa cell wall-anchored protein GspB . This unusually large protein lacks a typical amino-terminal signal peptide and is translocated from the cytoplasm via a dedicated transport system . A 14-kb segment just downstream of gspB encodes SecA2 and SecY2, two components of the GspB-specific transport system . The downstream segment also encodes several putative glycosyl transferases that may be responsible for the posttranslational modification of GspB . In this study, we compared the abilities of M99 and two GspB- mutant strains to bind various lectins . GspB was found to have affinity for lectins that bind N-acetylglucosamine . We also examined variant forms of GspB that lack a carboxy-terminal cell wall-anchoring domain and thus are free of covalent linkage to cell wall peptidoglycan . Like native GspB, these truncated proteins appear to be heavily glycosylated, as evidenced by migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass >100 kDa in excess of the predicted mass, negligible staining with conventional protein stains, and reactivity with hydrazide following periodate oxidation . Furthermore, analysis of the carbohydrate associated with the GspB variants by high-pH anion-exchange chromatography revealed the presence of  70
to 100 monosaccharide residues per GspB polypeptide (primarily N-acetylglucosamine
and glucose) . Analysis of GspB in protoplasts of secA2 or
secY2 mutant strains, which do not export GspB, indicates that
GspB is glycosylated in the cytoplasm of these strains . The combined
data suggest that the native GspB is a glycoprotein and that it may
be glycosylated prior to export .
Multiple-Antibiotic Resistance of Enterococcus spp . Isolated from Commercial Poultry Production Environments. Joshua R. Hayes, 2004.The potential impact of food animals in the production environment on the bacterial population as a result of antimicrobial drug use for growth enhancement continues to be a cause for concern . Enterococci from 82 farms within a poultry production region on the eastern seaboard were isolated to establish a baseline of susceptibility profiles for a number of antimicrobials used in production as well as clinical environments . Of the 541 isolates recovered, Enterococcus faecalis (53%) and E . faecium (31%) were the predominant species, while multiresistant antimicrobial phenotypes were observed among all species . The prevalence of resistance among isolates of E . faecalis was comparatively higher among lincosamide, macrolide, and tetracycline antimicrobials, while isolates of E . faecium were observed to be more frequently resistant to fluoroquinolones and penicillins . Notably, 63% of the E . faecium isolates were resistant to the streptogramin quinupristin-dalfopristin, while high-level gentamicin resistance was observed only among the E . faecalis population, of which 7% of the isolates were resistant . The primary observations are that enterococci can be frequently isolated from the poultry production environment and can be multiresistant to antimicrobials used in human medicine . The high frequency with which resistant enterococci are isolated from this environment suggests that these organisms might be useful as sentinels to monitor the development of resistance resulting from the usage of antimicrobial agents in animal production . Allosteric Regulation of Bacillus subtilis NAD Kinase by Quinolinic Acid. Silvia Garavaglia, 2003.NADP is essential for biosynthetic pathways, energy, and signal transduction . In living organisms, NADP biosynthesis proceeds through the phosphorylation of NAD with a reaction catalyzed by NAD kinase . We expressed, purified, and characterized Bacillus subtilis NAD kinase . This enzyme represents a new member of the inorganic polyphosphate [poly(P)]/ATP NAD kinase subfamily, as it can use poly(P), ATP, or other nucleoside triphosphates as phosphoryl donors . NAD kinase showed marked positive cooperativity for the substrates ATP and poly(P) and was inhibited by its product, NADP, suggesting that the enzyme plays a major regulatory role in NADP biosynthesis . We discovered that quinolinic acid, a central metabolite in NAD(P) biosynthesis, behaved like a strong allosteric activator for the enzyme . Therefore, we propose that NAD kinase is a key enzyme for both NADP metabolism and quinolinic acid metabolism . Detection of Listeria monocytogenes from a Model Food by Fluorescence Resonance Energy Transfer-Based PCR with an Asymmetric Fluorogenic Probe Set. Kai Koo, 2003.It has been shown that fluorescence resonance energy transfer (FRET)-based PCR, including the TaqMan assay and molecular beacons, has potential for rapid detection of pathogens . In these promising real-time detection assays a single internal oligonucleotide probe labeled on both the 5' (reporter) and 3' (quencher) ends is used for selective generation of fluorescence . In this paper, we describe the use of a previously reported novel probe design for FRET-based PCR detection of Listeria monocytogenes in pure culture and in a model food commodity . In the assay described here an asymmetric probe set is used; this probe set consists of a long 5' fluorescein-labeled reporter probe and a short, complementary 3' DABCYL-labeled quencher oligonucleotide, which are used in a 5' nuclease amplification and detection assay . By using the listeriolysin O (hly) and p60 (iap) genes as amplification targets, the performance of two primer-probe sets in amplification and subsequent detection of target DNA was evaluated . In studies performed with pure cultures of L . monocytogenes, the PCR profiles indicated that the relative change in fluorescence intensity was correlated with both the initial number of cells and the accumulation of specific amplicons for both hly and iap gene fragments . Experiments were also done to determine the applicability of the method to the detection of L . monocytogenes by targeting hly DNA and its short-lived mRNA product in a model food commodity . Twenty-five-milliliter samples of reconstituted nonfat dry milk (NFDM) were seeded with L . monocytogenes and processed to concentrate the bacteria by centrifugation, and this was followed by nucleic acid extraction and amplification with hly-specific primers . Endpoint detection of PCR and reverse transcription-PCR amplicons could be achieved at inoculum levels of 103 and 104 CFU of L . monocytogenes/25 ml of NFDM, respectively . This study demonstrated that this asymmetric FRET-based amplification and detection protocol provides an alternative approach for endpoint detection of nucleic acid amplification products as applied to detection of pathogens in a model food .
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