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Effects of Overexpression of Nutrient Receptors on Germination of Spores of Bacillus subtilis. Rosa-Martha Cabrera-Martinez, 2003.The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants . However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K+ (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons . Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK . Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations . However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein . Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA . These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination . We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination . Cloning and Heterologous Expression of a ß-D-Mannosidase (EC 3.2.1.25)-Encoding Gene from Thermobifida fusca TM51. Emese Béki, 2003.Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, ß-xylosidases, endomannanases, and ß-mannosidases, when grown on cellulose or hemicellulose as carbon sources . ß-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms . An expression library of T . fusca, prepared in Streptomyces lividans TK24, was screened for ß-mannosidase activity to clone genes coding for mannosidases . One positive clone was identified, and a ß-mannosidase-encoding gene (manB) was isolated . Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes . The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87 . S . lividans was used as heterologous expression host for the putative ß-mannosidase gene of T . fusca . The purified gene product obtained from the culture filtrate of S . lividans was then subjected to more-detailed biochemical analysis . Temperature and pH optima of the recombinant enzyme were 53°C and 7.17, respectively . Substrate specificity tests revealed that the enzyme exerts only ß-D-mannosidase activity . Its kinetic parameters, determined on para-nitrophenyl ß-D-mannopyranoside (pNP-ßM) substrate were as follows: Km = 180 µM and Vmax = 5.96 µmol min-1 mg-1; the inhibition constant for mannose was Ki = 5.5 mM . Glucono-lacton had no effect on the enzyme activity . A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-  M and pNP-ßM; under these conditions mannosyl groups were transferred by the enzyme from pNP-ßM to pNP-M resulting in the synthesis of small amounts (1 to 2%) of disaccharides .
An In Vitro Study of the Probiotic Potential of a Bile-Salt-Hydrolyzing Lactobacillus fermentum Strain, and Determination of Its Cholesterol-Lowering Properties. Dora I. A. Pereira, 2003.This study evaluated the use of a bile-salt-hydrolyzing Lactobacillus fermentum strain as a probiotic with potential hypocholesterolemic properties . The effect of L . fermentum on representative microbial populations and overall metabolic activity of the human intestinal microbiota was investigated using a three-stage continuous culture system . Also, the use of galactooligosaccharides as a prebiotic to enhance growth and/or activity of the Lactobacillus strain was evaluated . Administration of L . fermentum resulted in a decrease in the overall bifidobacterial population (ca . 1 log unit) . In the in vitro system, no significant changes were observed in the total bacterial, Lactobacillus, Bacteroides, and clostridial populations through L . fermentum supplementation . Acetate production decreased by 9 to 27%, while the propionate and butyrate concentrations increased considerably (50 to 90% and 52 to 157%, respectively) . A general, although lesser, increase in the production of lactate was observed with the administration of the L . fermentum strain . Supplementation of the prebiotic to the culture medium did not cause statistically significant changes in either the numbers or the activity of the microbiota, although an increase in the butyrate production was seen (29 to 39%) . Results from this in vitro study suggest that L . fermentum KC5b is a candidate probiotic which may affect cholesterol metabolism . The short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation, are probably the major mechanism involved in the purported cholesterol-lowering properties of this strain .
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