Evaluation of indigenous potent mushroom growth promoting bacteria (MGPB) on Agaricus bisporus production.

Mushrooms such as Agaricus bisporus, are cultivated for food worldwide. Fruit body initiation in Agaricus bisporus is a phase change from the vegetative to the reproductive stage which depends on the presence of a casing layer with particular physical, chemical and microbiological properties. The phase change is achieved practically by environmental manipulation and the presence of naturally occurring bacteria such as Pseuodomonas putida. In this study, 274 individual bacterial isolates were collected by screening the casing layer of 14 edible mushroom farms. The isolates were analysed with respect to biochemical properties, organic and inorganic phosphate solubilization, production of siderophore and growth in the presence of volatile compound of 1-octen-3-ol. It was found that approximately 97% of the strains were able to grow in the presence of 1-octen-3-ol and 36% were able to solubilize phosphorus. Among the isolates, 23 strains were selected as potent mushroom growth promoting bacteria (MGPB) for inoculation of the casing layer. Field experiments using these strains showed various promoting effects on production of mushroom. Finally, 2 strains (strains Bt4 and Ps7) showing the highest increase in A. bisporus production, were characterized as Pseuodomonas putida by molecular methods and identified as the best suited growth promoting inoculants for application in production farms for increasing the mushroom yield.

The influence of riboflavin and nicotinic acid on Shigella sonnei colony conversion.

BACKGROUND AND OBJECTIVES: Shigella, causative of bacillary dysentery, has two colony forms. The loss of large virulence plasmid from virulent Shigella sonnei form I, during cell storage and subculturing, lead to avirulent form II. Environmental factors, e.g. culture media composition, could affect the conversion of the bacterial forms. MATERIALS AND METHODS: In this study, some components, i.e., B-complex vitamins, nicotinic acid and riboflavin, were added to the bacterial culture medium and their influence on colony conversion were examined. RESULTS: The findings revealed that colony conversion is temperature independent and growth on the SS agar did not stabilize the bacterium in form I. Also, the findings showed that colonies on the minimal media supplemented with nicotinic acid and riboflavin, were stable in form I. In addition, according to the findings, the active OxyR has potential binding sites upstream of two genes involved in the replication of large virulence plasmid and expression of O-polysaccharide, i.e., repB and wbgT, respectively. CONCLUSION: Based on the findings of the present study, it is possible that nicotinic acid and riboflavin activate the transcriptional regulatory protein OxyR via dropping off the intracellular reducing power and in this way stabilize the colonies in form I.

A simplified purification procedure for recombinant human granulocyte macrophage-colony stimulating factor from periplasmic space of Escherichia coli.

Cytoplasmic expression is commonly used for production of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) which most often comes with inclusion body formation. We expressed rhGM-CSF in periplasmic space of Escherichia coli and optimized its extraction by osmotic shock and purification by anion exchange chromatography. Our works show that MgCl2 at 2 mM in osmotic shock buffer improves extraction of the protein and reduces contamination with other proteins. To achieve a simplified purification procedure for rhGM-CSF, efforts were focused on the adjustment of pH of the buffers and application of proper concentration of salt. Following to measurement of the pI of 5.4 for rhGM-CSF by isoelectric focusing, the pH of dialysis buffer and buffers used in anion exchange chromatography were adjusted to 6.5 for optimal binding of the protein to the column and removal of proteins with higher pIs during washing of the column. In addition, it was found that appliance of NaCl at a concentration of 20 mM in dialysis and column washing buffers prior to elution with elution buffer containing 120 mM NaCl significantly improves purification of the protein. Starting with specific amount of total proteins obtained by osmotic shock, it was possible to recover 95% of which following to purification with a purification yield of 72% for rhGM-CSF along with appropriate biological activity.

Epitope analysis of the CS3 fimbrial subunit of human enterotoxigenic Escherichia coli and the construction of novel CS3::ST and CS3::LT-B immunogens.

Enterotoxigenic E. coli (ETEC) are the major cause of traveler's diarrhoea and the CS3 fimbriae/fibrillae are expressed by most strains bearing the colonization factor CFA/II. The cstAH gene cluster determining CS3 biosynthesis has been previously cloned and sequenced and it has been shown that cstH encodes the major fimbrial subunit and cstA-G encode an assembly cassette. In the work described here we have sought to define the surface exposed domains on CS3 and to manipulate them so that CS3 can be used as a means of expressing foreign antigenic determinants on the bacterial surface. Using a panel of 21 monoclonal antibodies, which we have used in western blotting, immunofluorescence microscopy and colony blotting, together with computer predictions, we have identified three domains within CstH. Two of these sites were permissive for insertion and we have introduced, in-frame, either an epitope from the B subunit of LT (heat labile toxin) or the entire coding sequence of mature ST (heat stable toxin) to construct hybrid proteins. These proteins could be assembled into hybrid fimbriae which could be recognized by antibodies to both CS3 and the foreign epitope as shown by immunofluorescence microscopy and colony blotting. The immunogenicity of the constructs has been evaluated following both oral and intraperitoneal immunization of mice with the attenuated Salmonella typhimurium strain G30 harbouring the hybrid cst operons. Although plasmid stability is currently a problem, these experiments showed that antibodies to both the carrier and the foreign epitope were generated.