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A Dominant-Negative fur Mutation in Bradyrhizobium japonicum. Heather P. Benson, 2004.In many bacteria, the ferric uptake regulator [Fur] proteinplays a central role in the regulation of iron uptake genes.Because iron figures prominently in the agriculturally importantsymbiosis between soybean and its nitrogen-fixing endosymbiontBradyrhizobium japonicum, we wanted to assess the role of Furin the interaction . We identified a fur mutant by selectingfor manganese resistance . Manganese interacts with the Fur proteinand represses iron uptake genes . In the presence of high levelsof manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive . TheB . japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron . Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild-type cells leads to a fur phenotype . Unlike a B . japonicum fur-null mutant, thestrain carrying the dominant-negative fur mutation is unableto form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis. Molecular Characterization of a Cephamycin-Hydrolyzing and Inhibitor-Resistant Class A ß-Lactamase, GES-4, Possessing a Single G170S Substitution in the  -Loop.Jun-ichi Wachino, 2004.The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type ß-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new ß-lactamase, GES-3 . In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated . This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 µg/ml) and ß-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 ± 1.7 µM) . The GES-4 enzyme had a single G170S substitution in the -loop region compared with the GES-3 sequence . This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of ß-lactamase inhibitors to GES-4 . A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate blaGES-4 genes mediated by two distinct class 1 integrons with similar gene cassette configurations . Moreover, the genetic environments of the blaGES-4 genes found in strain KG502 were almost identical to that of blaGES-3 in strain KG525 . From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage . The blaGES-4 gene found in strain KG502 might well emerge from a point mutation in the blaGES-3 gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems .
Novel Epibiotic Thiothrix Bacterium on a Marine Amphipod. David C. Gillan, 2004.Comparative analysis of the 16S rRNA gene and fluorescent in situ hybridization (FISH) was used to identify epibiotic filamentous bacteria living on the marine amphipod crustacean Urothoe poseidonis . The epibionts belong to the gamma proteobacteria and represent a novel marine phylotype within the genus Thiothrix . FISH and denaturing gradient gel electrophoresis revealed that the Thiothrix filaments are present on the majority of the amphipods examined . Instability of Pathogenicity Islands in Uropathogenic Escherichia coli 536. Barbara Middendorf, 2004.The uropathogenic Escherichia coli strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs) . One main feature of these distinct DNA regions is their instability . We applied the so-called island-probing approach and individually labeled all five PAIs of E . coli 536 with the counterselectable marker sacB to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions . Furthermore, we investigated the boundaries of these PAIs . According to our experiments, PAI II536 and PAI III536 were the most unstable islands followed by PAI I536 and PAI V536, whereas PAI IV536 was stable . In addition, we found that deletion of PAI II536 and PAI III536 was induced by several environmental stimuli . Whereas excision of PAI I536, PAI II536, and PAI V536 was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III536 was deleted either by site-specific recombination or by homologous recombination between two IS100-specific sequences . In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates . Such extrachromosomal derivatives of PAI II536 and PAI III536 were detected by a specific PCR assay . Our data indicate that the genome content of uropathogenic E . coli can be modulated by deletion of PAIs .
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