Subject(s)
Certification , Education, Medical, Continuing , Specialty Boards , Communicable Diseases , Medicine , Specialization , United StatesABSTRACT
Water-shock treatment of group A Streptococcus pyogenes released a mixture of nucleotide-like substances and small amounts of protein. The amount of protein was much less than found with osmotic shock of Gram-negative bacteria. In group A S. pyogenes the osmolytes released exhibited as much as a 6-fold change in respect to different growth phases. Osmolyte release was dependent on the stabilization agent used and independent of cellular metabolic activity. The released osmolytes were found to be required for optimal intracellular iodophilic polysaccharide (IPS) storage. Stabilization of washing solutions, and IPS storage medium with metabolically inert non-ionic organic compounds prevented osmolyte loss and enhanced IPS storage. Polyvinyl pyrrolidone and polyethyleneglycol (MW greater than 1000) exhibited the same protective effects as found with calf serum. Smaller non-ionic organic compounds provided similar protective action but the bacteria were more susceptible to osmotic stress.
Subject(s)
Polysaccharides, Bacterial/metabolism , Streptococcus pyogenes/metabolism , Maltose/metabolism , Molecular Weight , Osmotic Pressure , Polyethylene Glycols/pharmacology , Streptococcus pyogenes/drug effectsABSTRACT
Calf serum, pooled human plasma, polyvinylpyrollidones (PVP) and polyethylene glycols (PEG) were required as medium components by many cultures of group A.S. pyogenes for intracellular polysaccharide accumulation. These supplements produced a stabilizing effect, probably at the cell surface, and were not associated with nutrition during intracellular iodophilic polysaccharide (IPS) storage. Medium stabilization and the presence of maltose or maltose yielding polysaccharides was found to be a property unique to the group A streptococci. Cariogenic streptococci and Streptococcus agalactive did not require medium stabilization for IPS storage although addition of PVP or PEG was stimulatory. Different degrees of IPS storage in group A.S. pyogenes was found to be strain related and probably dependent on the size and degree of branching of the storage polymer.
Subject(s)
Polysaccharides, Bacterial/metabolism , Streptococcus/metabolism , Culture Media , Polyethylene Glycols/pharmacology , Povidone/pharmacology , Streptococcus/drug effects , Streptococcus agalactiae/drug effects , Streptococcus agalactiae/metabolism , Streptococcus mutans/drug effects , Streptococcus mutans/metabolism , Streptococcus pyogenes/drug effects , Streptococcus pyogenes/metabolismABSTRACT
A method is presented for the rapid, isocratic separation of purine nucleotides using strong anion-exchange high-performance liquid chromatography at ambient temperature. The last peak of interest guanosine 5'-triphosphate (GTP) is eluted within 30 min and immediate reinjection is possible. All adenine and guanine nucleotides can be assayed with a single injection without the use of a gradient for elution. The procedure is particularly useful for the assay of NTP:AMP phosphotransferase reactions and/or the determination of changes in size of cellular purine nucleotide pools and computation of energy charges. An Altex Ultrasil AX prepacked column was used, and virtually identical results were obtained under similar conditions with the Whatman Partisil-10 SAX column. The eluting solution was 200 mM potassium phosphate, pH 6.85.
Subject(s)
Purine Nucleotides/analysis , Adenine Nucleotides/metabolism , Aerobiosis , Anaerobiosis , Animals , Anion Exchange Resins , Cells, Cultured , Chromatography, High Pressure Liquid/methods , Energy Metabolism , Glucose/metabolism , Myocardium/metabolism , RatsABSTRACT
Inhibitors of glycoprotein synthesis in eucaryotic cells also inhibited iodophilic polysaccharide (IPS) storage in group A streptococcus pyogenes. Addition of bacitracin or amphomycin, known inhibitors of polyisoprenol phosphate metabolism or lipid-linked oligosaccharide synthesis, indicated that a key intermediate must be synthesized before IPS storage could be detected. Based on inhibitor action and energy requirements the intermediate was most likely an undecaprenol pyrophosphoryl maltosaccharide. Biosynthesis of the maltosaccharide had an ATP requirement as shown by arsenate action but IPS synthesis via amylomaltase was energy independent if the lipid-linked saccharides were preformed. Maltosaccharide acceptor site blockade with 2-deoxy-d-glucose immediately inhibited IPS storage, which demonstrated the need of acceptor glucose residues for transglycosylation activity of amylomaltase. Tunicamycin failed to inhibit IPS synthesis although it was added in lethal concentrations.
Subject(s)
Anti-Bacterial Agents/pharmacology , Arsenates/pharmacology , Arsenic/pharmacology , Deoxy Sugars/pharmacology , Deoxyglucose/pharmacology , Polysaccharides, Bacterial/metabolism , Streptococcus pyogenes/drug effects , Bacitracin/pharmacology , Lipopeptides , Oligopeptides/pharmacology , Polysaccharides, Bacterial/biosynthesis , Streptococcus pyogenes/metabolism , Tunicamycin/pharmacologyABSTRACT
Optimal conditions for intracellular polysaccharide (IPS) storage by non-proliferating suspensions of group A Streptococcus pyogenes are described. Concentrations of yeast extract and acid-hydrolysed casein which did not support measureable growth prevented bacterial death during storage. For optimal storage, high concentrations of maltose were required to antagonize the inhibitory effect of medium glucose released by intracellular amylomaltase activity. Stabilization of cell washing solutions with 0.5 M sucrose and the minimal storage medium with 8.0% w/v polyethylene glycol MW 6,000 was required for optimal culture viability and IPS storage. The stabilization effect prevented loss of small purine or pyrimidine-containing compounds required for IPS storage as indicated by culture water-shock treatment.