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Identification of rocA, a Positive Regulator of covR Expression in the Group A Streptococcus. Indranil Biswas, 2003.In the group A streptococcus (GAS; Streptococcus pyogenes), a two-component system known as CovRS (or CsrRS) regulates about 15% of the genes, including several important virulence factors like the hyaluronic acid capsule . Most of these genes, including covR itself, are negatively regulated by CovR . We have isolated two independent ISS1 insertions in an open reading frame (ORF) that increases CovR expression as measured by a Pcov-gusA reporter fusion in single copy in the GAS chromosome . This ORF, named rocA for "regulator of Cov," activates covR transcription about threefold . As expected, a rocA mutant is mucoid and produces more transcript from the has promoter since this promoter is repressed by CovR . This effect is dependent on the presence of a wild-type covR gene . In contrast to its activation of Pcov, RocA negatively regulates its own expression . This autoregulation is not dependent on the presence of the covR gene . All the phenotypes of the rocA mutant were complemented by the presence of the rocA gene on a plasmid . The rocA gene is present in strains of all nine M serotypes of GAS tested and is absent from strains representing 11 other groups of streptococci and related bacteria, including strains of the closely related group C and G streptococci . It seems likely that rocA plays an important role in the pathogenesis of GAS since it affects expression of the global regulator CovR . Production of L-Ascorbic Acid by Metabolically Engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii. Michael Sauer, 2004.Yeasts do not possess an endogenous biochemical pathway for the synthesis of vitamin C . However, incubated with L-galactose, L-galactono-1,4-lactone, or L-gulono-1,4-lactone intermediates from the plant or animal pathway leading to L-ascorbic acid, Saccharomyces cerevisiae and Zygosaccharomyces bailii cells accumulate the vitamin intracellularly . Overexpression of the S . cerevisiae enzymes D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase enhances this ability significantly . In fact, the respective recombinant yeast strains even gain the capability to accumulate the vitamin in the culture medium . An even better result is obtainable by expression of the plant enzyme L-galactose dehydrogenase from Arabidopsis thaliana . Budding yeast cells overexpressing the endogenous D-arabinono-1,4-lactone oxidase as well as L-galactose dehydrogenase are capable of producing about 100 mg of L-ascorbic acid liter–1, converting 40% (wt/vol) of the starting compound L-galactose . Mapping the MinE Site Involved in Interaction with the MinD Division Site Selection Protein of Escherichia coli. Lu-Yan Ma, 2003.Interactions between the MinD and MinE proteins are required for proper placement of the Escherichia coli division septum . The site within MinE that is required for interaction with MinD was mapped by studying the effects of site-directed minE mutations on MinD-MinE interactions in yeast two-hybrid and three-hybrid experiments . This confirmed that the MinE N-terminal domain is responsible for the interaction of MinE with MinD . Mutations that interfered with the interaction defined an extended surface on one face of the  -helical region of the MinE N-terminal domain, consistent with the idea that the MinE-MinD interaction involves formation of a coiled-coil structure by interaction with a complementary helical surface within MinD .
Characterization of a Lactococcus lactis Strain That Secretes a Major Epitope of Bovine Beta-Lactoglobulin and Evaluation of Its Immunogenicity in Mice. Jean-Marc Chatel, 2003.Bovine ß-lactoglobulin (Blg) is one of the major cow's milk allergens . Peptide 41-60 of Blg (Blg41-60) was described as a murine T-cell determinant and a murine, rat, and human immunoglobulin E (IgE) epitope . The aim of this study was the expression of Blg41-60 as a fusion protein in the food-grade bacterium Lactococcus lactis and the characterization of its immunogenicity in mice . We constructed a recombinant strain of L . lactis capable of inducible production and secretion of Blg41-60::Nuc, a fusion protein between Blg41-60 and the mature part of the staphylococcal nuclease (Nuc) . The highest production yield of Blg41-60::Nuc (32.5 mg/liter) was reached 4 h after induction . At this time, up to 75% of Blg41-60::Nuc was secreted . When monoclonal antibodies specific for Blg41-60 were used, purified Blg41-60::Nuc and synthetic Blg41-60 exhibited very similar immunoreactivities . Subcutaneous coadministration of purified Blg41-60::Nuc and killed nonrecombinant L . lactis resulted in the induction of specific anti-Blg41-60 IgG2a and IgG1 . The IgG1/IgG2a ratio and the lack of specific IgE suggest a Th1-type immune response, i.e., a nonallergic response . Similar administrations of the killed Blg41-60::Nuc-producing L . lactis strain did not elicit a specific immune response, whereas a transitory mucosal IgA-specific immune response was induced in mice after oral administration of the live Blg41-60::Nuc-producing L . lactis strain .
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