|
|
|
|
|
|
Characterization of the 101-Kilobase-Pair Megaplasmid pKB1, Isolated from the Rubber-Degrading Bacterium Gordonia westfalica Kb1. Daniel Bröker, 2004.The complete sequence of the circular 101,016-bp megaplasmid pKB1 from the cis-1,4-polyisoprene-degrading bacterium Gordonia westfalica Kb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined . Plasmid pKB1 harbors 105 open reading frames . The predicted products of 46 of these are significantly related to proteins of known function . Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element . Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome . The origin of replication (oriV) of pKB1 was identified and used for construction of Escherichia coli-Gordonia shuttle vectors, which was also suitable for several other Gordonia species and related genera . The metabolic region included the heavy-metal resistance gene cadA, encoding a P-type ATPase . Expression of cadA in E . coli mediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host . When G . westfalica strain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance . Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1 . Molecular Characterization of Benzimidazole Resistance in Helicobacter pylori. Scott D. Mills, 2004.A family of benzimidazole derivatives (BI) was shown to possess potent and selective activity against Helicobacter pylori, although the precise cellular target of the BIs is unknown . Spontaneous H . pylori mutants were isolated as resistant to a representative BI (compound A) . Genomic DNA was isolated from a BI-resistant mutant, transformed into a BI-sensitive strain, and found to be sufficient to confer BI resistance . The resistance determinant was localized to a 17-kb clone after screening a lambda-based genomic library constructed from the BI-resistant strain . Upon sequencing and mapping onto the H . pylori strain J99 genome, the 17-kb clone was shown to contain the entire nuo operon (NADH:ubiquinone oxidoreductase) . Further subcloning and DNA sequencing revealed that a single point mutation in nuoD was responsible for BI resistance . The mutation resulted in a G398S amino acid change at the C terminus of NuoD . Thirty-three additional spontaneous BI-resistant mutants were characterized . Sequencing of nuoD from 32 isolated mutants revealed three classes of missense mutation resulting in amino acid changes in NuoD: G398S, F404S, and V407M . One BI-resistant isolate did not have a mutation in nuoD . Instead, a T27A amino acid change was identified in NuoB . MIC testing of the wild-type H . pylori strain and four classes of nuo mutants revealed that all NuoD mutant classes were hypersensitive to rotenone, a known inhibitor of complex I (NADH:ubiquinone oxidoreductase) suggested to bind to NuoD . Further, a nuoD knockout verified that it is essential in H . pylori and may be the target of the BI compounds . An Archaeal Chromosomal Autonomously Replicating Sequence Element from an Extreme Halophile, Halobacterium sp . Strain NRC-1. Brian R. Berquist, 2003.We report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophile Halobacterium strain NRC-1 . The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid . Deletion analysis showed that sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation . Sequences located proximal to the two other chromosomally carried haloarchaeal orc genes (orc6 and orc8) are not able to promote efficient autonomous replication . Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp . The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted . Additionally, the inverted repeat structure near orc7 homologs in the genomic sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved . Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication . We discuss the finding of a functional haloarchaeal origin in relation to the large number of orc1-cdc6 homologs identified in the genomes of all haloarchaea to date . A Few-Polyhedra Mutant and Wild-Type Nucleopolyhedrovirus Remain as a Stable Polymorphism during Serial Coinfection in Trichoplusia ni. James C. Bull, 2003.Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture . Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication . Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype . A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo . However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs . We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene . We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae . The levels of both strains remained stable during successive rounds of infection . We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium . Directed Evolution of Thermus Maltogenic Amylase toward Enhanced Thermal Resistance. Young-Wan Kim, 2003.The thermostability of maltogenic amylase from Thermus sp . strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling . Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations . The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L . The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C . The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min . Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level . The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes . The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions . The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds . M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
