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Assembly of an Oxalate Decarboxylase Produced under K Control into the Bacillus subtilis Spore Coat. Teresa Costa, 2004.Over 30 polypeptides are synthesized at various times during sporulation in Bacillus subtilis, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat . The coat consists of three main layers,an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-densestriated outer layer . The product of the B . subtilis oxdD genewas previously shown to have oxalate decarboxylase activitywhen it was produced in Escherichia coli and to be a spore constituent.In this study, we found that OxdD specifically associates withthe spore coat structure, and in this paper we describe regulationof its synthesis and assembly . We found that transcription ofoxdD is induced during sporulation as a monocistronic unit underthe control of sigmaK and is negatively regulated by GerE . We alsofound that localization of a functional OxdD-green fluorescentprotein [GFP] at the surface of the developing spore dependson the SafA morphogenetic protein, which localizes at the interfacebetween the spore cortex and coat layers . OxdD-GFP localizesaround the developing spore in a cotE mutant, which does notassemble the spore outer coat layer, but it does not persistin spores produced by the mutant . Together, the data suggestthat OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy alleleof oxdD resulted in production of spores with increased levelsof OxdD that were able to degrade oxalate but were sensitiveto lysozyme. Flexibility in the Receptor-Binding Domain of the Enzymatic Colicin E9 Is Required for Toxicity against Escherichia coli Cells. Christopher N. Penfold, 2004.The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood . It has been suggested that unfolding of the coiled-coil BtuB receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation . We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain . After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9 . On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9 . The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry . The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins . The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB . Rapid Real-Time PCR Genotyping of Mutations Associated with Sulfadoxine-Pyrimethamine Resistance in Plasmodium falciparum. Alisa P. Alker, 2004.The resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) is an emerging public health threat . Resistance to these drugs is associated with point mutations in the genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) . We describe here an assay using real-time PCR and sequence-specific probes that detects these mutations . Using DNA from plasmids, cultured strains, and clinical samples, real-time PCR could distinguish four DHPS polymorphisms (codons 437, 540, 581, and 613) and three DHFR polymorphisms (codons 51, 59, and 108) . This assay is rapid and sensitive, with a detection limit of 10 copies in most cases . This assay is amenable to large-scale studies of drug resistance . Definition of the Mycobacterial SOS Box and Use To Identify LexA-Regulated Genes in Mycobacterium tuberculosis. Elaine O. Davis, 2002.The bases of the mycobacterial SOS box important for LexA binding were determined by replacing each base with every other and examining the effect on the induction of a reporter gene following DNA damage . This analysis revealed that the SOS box was longer than originally thought by 2 bp in each half of the palindromic site . A search of the Mycobacterium tuberculosis genome sequence with the new consensus, TCGAAC(N)4GTTCGA, identified 4 sites which were perfect matches and 12 sites with a single mismatch which were predicted to bind LexA . Genes which could potentially be regulated by these SOS boxes were ascertained from their positions relative to the sites . Examination of expression data for these genes following DNA damage identified 12 new genes which are most likely regulated by LexA as well as the known M . tuberculosis DNA damage-inducible genes recA, lexA, and ruvC . Of these 12 genes, only 2 have a predicted function: dnaE2, a component of DNA polymerase III, and linB, which is similar to 1,3,4,6-tetrachloro-1,4-cylcohexadiene hydrolase . Curiously, of the remaining 10 genes predicted to be LexA regulated, 7 are members of the M . tuberculosis 13E12 repeat family, which has some of the characteristics of mobile elements . Common Extracellular Sensory Domains in Transmembrane Receptors for Diverse Signal Transduction Pathways in Bacteria and Archaea. Igor B. Zhulin, 2003.Transmembrane receptors in microorganisms, such as sensory histidine kinases and methyl-accepting chemotaxis proteins, are molecular devices for monitoring environmental changes . We report here that sensory domain sharing is widespread among different classes of transmembrane receptors . We have identified two novel conserved extracellular sensory domains, named CHASE2 and CHASE3, that are found in at least four classes of transmembrane receptors: histidine kinases, adenylate cyclases, predicted diguanylate cyclases, and either serine/threonine protein kinases (CHASE2) or methyl-accepting chemotaxis proteins (CHASE3) . Three other extracellular sensory domains were shared by at least two different classes of transmembrane receptors: histidine kinases and either diguanylate cyclases, adenylate cyclases, or phosphodiesterases . These observations suggest that microorganisms use similar conserved domains to sense similar environmental signals and transmit this information via different signal transduction pathways to different regulatory circuits: transcriptional regulation (histidine kinases), chemotaxis (methyl-accepting proteins), catabolite repression (adenylate cyclases), and modulation of enzyme activity (diguanylate cyclases and phosphodiesterases) . The variety of signaling pathways using the CHASE-type domains indicates that these domains sense some critically important extracellular signals . Null Mutations in a Nudix Gene, ygdP, Implicate an Alarmone Response in a Novel Suppression of Hybrid Jamming. Nicholas J. Hand, 2003.Induction of the toxic LamB-LacZ protein fusion, Hyb42-1, leads to a lethal generalized protein export defect . The prlF1 suppressor causes hyperactivation of the cytoplasmic Lon protease and relieves the inducer sensitivity of Hyb42-1 . Since prlF1 does not cause a detectable change in the stability or level of the hybrid protein, we conducted a suppressor screen, seeking factors genetically downstream of lon with prlF1-like phenotypes . Two independent insertions in the ygdP open reading frame relieve the toxicity of the fusion protein and share two additional properties with prlF1: cold sensitivity and the ability to suppress the temperature sensitivity of a degP null mutation . Despite these similarities, ygdP does not appear to act in the same genetic pathway as prlF1 and lon, suggesting a fundamental link between the phenotypes . We speculate that the common properties of the suppressors relate to secretion defects . The ygdP gene (also known as nudH) has been shown to encode a Nudix protein that acts as a dinucleotide oligophosphate (alarmone) hydrolase . Our results suggest that loss of ygdP function leads to the induction of an alarmone-mediated response that affects secretion . Using an epitope-tagged ygdP construct, we present evidence that this response is sensitive to secretion-related stress and is regulated by differential proteolysis of YgdP in a self-limiting manner . Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para-Hydroxylating Enzyme. Ayelet Fishman, 2004.Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods . Whereas toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 hydroxylates monosubstituted benzenes at the para position and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates at the ortho position, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported previously to hydroxylate toluene at the meta position, producing primarily m-cresol (R . H . Olsen, J . J . Kukor, and B . Kaphammer, J . Bacteriol . 176:3749-3756, 1994) . Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the para position . TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 ± 0.33 nmol/min/mg of protein with an apparent Km value of 250 µM and produced 90% p-cresol and 10% m-cresol . This product mixture was successively transformed to 4-methylcatechol . T4MO, in comparison, produces 97% p-cresol and 3% m-cresol . Pseudomonas aeruginosa PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO . TG1/pBS(Kan)T3MO produced 66% p-nitrophenol and 34% m-nitrophenol from nitrobenzene and 100% p-methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO . Sequencing of the tbu locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors . 1H nuclear magnetic resonance analysis confirmed the structural identity of p-cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO . Enhanced Killing of Acanthamoeba Cysts with a Plant Peroxidase-Hydrogen Peroxide-Halide Antimicrobial System. Reanne Hughes, 2003.The activity of H2O2 against the resistant cyst stage of the pathogenic free-living amoeba Acanthamoeba was enhanced by the addition of KI and either horseradish peroxidase or soybean peroxidase or, to a lesser degree, lactoperoxidase . This resulted in an increase in the cysticidal activity of 3% (wt/vol) H2O2, and there was >3-log killing in 2 h, compared with the 6 h required for comparable results with the peroxide solution alone (P < 0.05) . With 2% H2O2, enhancement was observed at all time points (P < 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone . The activity of sublethal 1% H2O2 was enhanced to give 3-log killing after 8 h of exposure (P < 0.05) . No enhancement was obtained when KCl or catalase was used as a substitute in the reaction mixtures . The H2O2 was not neutralized in the enhanced system during the experiments . However, in the presence of a platinum disk used to neutralize H2O2 in contact lens care systems, the enhanced 2% H2O2 system gave 2.8-log killing after 6 h or total cyst killing by 8 h, and total neutralization of the H2O2 occurred by 4 h . In contrast, 2% H2O2 alone resulted in <0.8-log killing of cysts in the presence of the platinum disk due to rapid (<1 h) neutralization of the peroxide . Our observations could result in significant improvement in the efficacy of H2O2 contact lens disinfection systems against Acanthamoeba cysts and prevention of acanthamoeba keratitis .
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