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Vertical Transmission of Endobacteria in the Arbuscular Mycorrhizal Fungus Gigaspora margarita through Generation of Vegetative Spores. V. Bianciotto, 2004.Arbuscular mycorrhizal (AM) fungi living in symbiotic association with the roots of vascular plants have also been shown to host endocellular rod-shaped bacteria . Based on their ribosomal sequences, these endobacteria have recently been identified as a new taxon, Candidatus Glomeribacter gigasporarum . In order to investigate the cytoplasmic stability of the endobacteria in their fungal host and their transmission during AM fungal reproduction (asexual), a system based on transformed carrot roots and single-spore inocula of Gigaspora margarita was used . Under these in vitro sterile conditions, with no risk of horizontal contamination, the propagation of endobacteria could be monitored, and it was shown, by using primers designed for both 16S and 23S ribosomal DNAs, to occur through several vegetative spore generations (SG0 to SG4) . A method of confocal microscopy for quantifying the density of endobacteria in spore cytoplasm was designed and applied; endobacteria were consistently found in all of the spore generations, although their number rapidly decreased from SG0 to SG4 . The study demonstrates that a vertical transmission of endobacteria takes place through the fungal vegetative generations (sporulation) of an AM fungus, indicating that active bacterial proliferation occurs in the coenocytic mycelium of the fungus, and suggests that these bacteria are obligate endocellular components of their AM fungal host . Involvement of an Inducible Fructose Phosphotransferase Operon in Streptococcus gordonii Biofilm Formation. C. Y. Loo, 2003.Oral streptococci, such as Streptococcus gordonii, are the predominant early colonizers that initiate biofilm formation on tooth surfaces . Investigation of an S . gordonii::Tn917-lac biofilm-defective mutant isolated by using an in vitro biofilm formation assay showed that the transposon insertion is near the 3' end of an open reading frame (ORF) encoding a protein homologous to Streptococcus mutans FruK . Three genes, fruR, fruK, and fruI, were predicted to encode polypeptides that are part of the fructose phosphotransferase system (PTS) in S . gordonii . These proteins, FruR, FruK, and FruI, are homologous to proteins encoded by the inducible fruRKI operon of S . mutans . In S . mutans, FruR is a transcriptional repressor, FruK is a fructose-1-phosphate kinase, and FruI is the fructose-specific enzyme II (fructose permease) of the phosphoenolpyruvate-dependent sugar PTS . Reverse transcription-PCR confirmed that fruR, fruK, and fruI are cotranscribed as an operon in S . gordonii, and the transposon insertion in S . gordonii fruK::Tn917-lac resulted in a nonpolar mutation . Nonpolar inactivation of either fruK or fruI generated by allelic replacement resulted in a biofilm-defective phenotype, whereas a nonpolar mutant with an inactivated fruR gene retained the ability to form a biofilm . Expression of fruK, as measured by the ß-galactosidase activity of the fruK::Tn917-lac mutant, was observed to be growth phase dependent and was enhanced when the mutant was grown in media with high levels of fructose, sucrose, xylitol, and human serum, indicating that the fructose PTS operon was fructose and xylitol inducible, similar to the S . mutans fructose PTS . The induction by fructose was inhibited by the presence of glucose, indicating that glucose is able to catabolite repress fruK expression . Nonpolar inactivation of the fruR gene in the fruK::Tn917-lac mutant resulted in a greater increase in ß-galactosidase activity when the organism was grown in media supplemented with fructose, confirming that fruR is a transcriptional repressor of the fructose PTS operon . These results suggest that the regulation of fructose transport and metabolism in S . gordonii is intricately tied to carbon catabolite control and the ability to form biofilms . Carbon catabolite control, which modulates carbon flux in response to environmental nutritional levels, appears to be important in the regulation of bacterial biofilms . Involvement of the adc Operon and Manganese Homeostasis in Streptococcus gordonii Biofilm Formation. C. Y. Loo, 2003.Pioneer oral bacteria, including Streptococcus gordonii, initiate the formation of oral biofilms on tooth surfaces, which requires differential expression of genes that recognize unique environmental cues . An S . gordonii::Tn917-lac biofilm-defective mutant was isolated by using an in vitro biofilm formation assay . Subsequent inverse PCR and sequence analyses identified the transposon insertion to be near the 3' end of an open reading frame (ORF) encoding a protein homologous to a Streptococcus pneumoniae repressor, AdcR . The S . gordonii adc operon, consisting of the four ORFs adcR, adcC, adcB, and adcA, is homologous to the adc operon of S . pneumoniae, which plays a role in zinc and/or manganese transport and genetic competence in S . pneumoniae . AdcR is a metal-dependent repressor protein containing a putative metal-binding site, AdcC contains a consensus-binding site for ATP, AdcB is a hydrophobic protein with seven hydrophobic membrane-spanning regions, and AdcA is a lipoprotein permease with a putative metal-binding site . The three proteins (AdcC through -A) are similar to those of the binding-lipoprotein-dependent transport system of gram-positive bacteria . Reverse transcriptase PCR confirmed that adcRCBA are cotranscribed as an operon in S . gordonii and that the transposon insertion in S . gordonii adcR::Tn917-lac had resulted in a polar mutation . Expression of adcR, measured by the ß-galactosidase activity of the adcR::Tn917-lac mutant, was growth phase dependent and increased when the mutant was grown in media with high levels of manganese (>1 mM) and to a lesser extent in media with zinc, indicating that AdcR may be a regulator at high levels of extracellular manganese . A nonpolar inactivation of adcR generated by allelic replacement resulted in a biofilm- and competence-defective phenotype . The biofilm-defective phenotype observed suggests that AdcR is an active repressor when synthesized and acts at a distant site(s) on the chromosome . Thus, the adc operon is involved in manganese acquisition in S . gordonii and manganese homeostasis and appears to modulate sessile growth in this bacterium . Engineering Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Optimal Ring Expansion Activity toward Penicillin G. Chia-Li Wei, 2003.The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application . A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the kcat/Km ratio compared to the wild-type enzyme . Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased kcat/Km values . When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the kcat/Km ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G . Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction .
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