|
|
|
|
|
|
A Phosphoprotein from the Archaeon Sulfolobus solfataricus with Protein-Serine/Threonine Kinase Activity. Brian H. Lower, 2004.Sulfolobus solfataricus contains a membrane-associated protein kinase activity that displays a strong preference for threonine as the phospho-acceptor amino acid residue . When a partially purified detergent extract of the membrane fraction from the archaeon S . solfataricus that had been enriched for this activity was incubated with [  -32P]ATP,
radiolabeled phosphate was incorporated into roughly a dozen
polypeptides, several of which contained phosphothreonine . One of the
phosphothreonine-containing proteins was identified by mass peptide
profiling as the product of open reading frame [ORF] sso0469.
Inspection of the DNA-derived amino acid sequence of the predicted
protein product of ORF sso0469 revealed the presence of
sequence characteristics faintly reminiscent of the "eukaryotic"
protein kinase superfamily . ORF sso0469 therefore was cloned,
and its polypeptide product was expressed in Escherichia coli .
The recombinant protein formed insoluble aggregates that could be
dispersed using urea or detergents . The solubilized polypeptide
phosphorylated several exogenous proteins in vitro, including casein,
myelin basic protein, and bovine serum albumin . Mutagenic alteration
of amino acids predicted to be essential for catalytic activity
abolished or severely reduced catalytic activity . Phosphorylation of
exogenous substrates took place on serine and, occasionally,
threonine . This new archaeal protein kinase displayed no catalytic
activity when GTP was substituted for ATP as the phospho-donor
substrate, while Mn2+ was the preferred cofactor .
Clade-Specific Flucytosine Resistance Is Due to a Single Nucleotide Change in the FUR1 Gene of Candida albicans. Andrew R. Dodgson, 2004.Population studies have indicated that natural resistance to flucytosine (5FC) in Candida albicans is limited to one of the five major clades, clade I . In addition, while 73% of clade I isolates are less susceptible to 5FC (MIC  0.5 µg/ml), only 2% of non-clade I isolates are less susceptible . In order to determine the genetic basis for this clade-specific resistance, we sequenced two genes involved in the metabolism of 5FC that had previously been linked to resistance (cytosine deaminase and uracil phosphoribosyltransferase), in 48 isolates representative of all clades . Our results demonstrate that a single nucleotide change from cytosine to thymine at position 301 in the uracil phosphoribosyltransferase gene (FUR1) of C . albicans is responsible for 5FC resistance . The mutant allele was found only in group I isolates . The 5FC MICs for strains without copies of the mutant allele were almost exclusively
 0.25 µg/ml, those for strains with one copy of the mutant allele were
0.5 µg/ml, and those for strains with two copies of the mutant allele were
16 µg/ml . Thus, the two alleles were codominant . The presence of this allele is responsible for clade I-specific resistance to 5FC within the C . albicans population and thus by inference is likely to be the major underlying 5FC resistance mechanism in C . albicans . This represents the first description of the genetic mutation responsible for 5FC resistance .
Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds. Barbara Morasch, 2004.Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria . Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation . The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors (  ) of –1.5 and –3.9 , which is in the same order of magnitude as data provided before for anaerobic toluene degradation . Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms . In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic
(intrinsic) were calculated . A comparison of
intrinsic at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific
elucidated that both varied on average to the same extent . Despite variations during the degradation of different substrates, the range of
found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average
if no fractionation factor is available for single compounds .
FepA with Globular Domain Deletions Lacks Activity. Hema L. Vakharia, 2002.TonB-gated transporters have ß-barrels containing an amino-terminal globular domain that occludes the interior of the barrel . Mutations in the globular domain prevent transport of ligands across the outer membrane . Surprisingly, FepA with deletions of the globular domain (amino acids 3 to 150 and 17 to 150) was previously reported to retain significant sensitivity to colicins B and D and to use ferric enterochelin, all in a TonB-dependent fashion . To further understand TonB interaction with the ß-barrel, in the present study, proteins with deletions of amino acids 1 to 152, 7 to 152, 20 to 152, and 17 to 150 in fepA were constructed and expressed in a  fepA strain . In contrast to previous studies of fepA globular domain deletions, constructs in this study did not retain sensitivity to colicin B and conferred only marginal sensitivity to colicin D . Consistent with these observations, they failed to bind colicin B and detectably cross-link to TonB in vivo . To address this discrepancy, constructs were tested in other strains, one of which (RWB18-60) did support activity of the FepA globular domain deletion proteins constructed in this study . The characteristics of that strain, as well as the strain in which the
FhuA globular domain mutants were seen to be active, suggests the hypothesis that interprotein complementation by two individually nonfunctional proteins restores TonB-dependent activity .
Modularity and Specialization in Superfamily 1 and 2 Helicases. Martin R. Singleton, 2002. Characterization of Benzoyl Coenzyme A Biosynthesis Genes in the Enterocin-Producing Bacterium "Streptomyces maritimus". Longkuan Xiang, 2003.The novel benzoyl coenzyme A (benzoyl-CoA) biosynthesis pathway in "Streptomyces maritimus" was investigated through a series of target-directed mutations . Genes involved in benzoyl-CoA formation were disrupted through single-crossover homologous recombination, and the resulting mutants were analyzed for their ability to biosynthesize the benzoyl-CoA-primed polyketide antibiotic enterocin . Inactivation of the unique phenylalanine ammonia-lyase-encoding gene encP was previously shown to be absolutely required for benzoyl-CoA formation in "S . maritimus" . The fatty acid ß-oxidation-related genes encH, -I, and -J, on the other hand, are necessary but not required . In each case, the yield of benzoyl-CoA-primed enterocin dropped below wild-type levels . We attribute the reduced benzoyl-CoA formation in these specific mutants to functional substitution and cross-talk between the products of genes encH, -I, and -J and the enzyme homologues of primary metabolism . Disruption of the benzoate-CoA ligase encN gene did not perturb enterocin production, however, demonstrating that encN is extraneous and that benzoic acid is not a pathway intermediate . EncN rather serves as a substitute pathway for utilizing exogenous benzoic acid . These experiments provide further support that benzoyl-CoA is formed in a novel bacterial pathway that resembles the eukaryotic assembly of benzoyl-CoA from phenylalanine via a ß-oxidative path . Synthesis of Imidazol-2-yl Amino Acids by Using Cells from Alkane-Oxidizing Bacteria. Annett Mikolasch, 2003.Sixty-one strains of alkane-oxidizing bacteria were tested for their ability to oxidize N-(2-hexylamino-4-phenylimidazol-1-yl)-acetamide to imidazol-2-yl amino acids applicable for pharmaceutical purposes . After growth with n-alkane, 15 strains formed different imidazol-2-yl amino acids identified by chemical structure analysis (mass and nuclear magnetic resonance spectrometry) . High yields of imidazol-2-yl amino acids were produced by the strains Gordonia rubropertincta SBUG 105, Gordonia terrae SBUG 253, Nocardia asteroides SBUG 175, Rhodococcus erythropolis SBUG 251, and Rhodococcus erythropolis SBUG 254 . Biotransformation occurred via oxidation of the alkyl side chain and produced 1-acetylamino-4-phenylimidazol-2-yl-6-aminohexanoic acid and the butanoic acid derivative . In addition, the acetylamino group of these products and of the substrate was transformed to an amino group . The product pattern as well as the transformation pathway of N-(2-hexylamino-4-phenylimidazol-1-yl)-acetamide differed in the various strains used .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
