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Longitudinal Study of Campylobacter jejuni Bacteriophages and Their Hosts from Broiler Chickens. P. L. Connerton, 2004.A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages . Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn . Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3 . The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content . All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE) . PFGE and multilocus sequence typing indicated that this C . jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C . jejuni types insensitive to the resident bacteriophages . All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct . These results are significant because this is the first study of an environmental population of C . jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens . The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens . In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance . Thermoadaptation of  -Galactosidase AgaB1 in Thermus thermophilus.Olafur Fridjonsson, 2002.The evolutionary potential of a thermostable -galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria . For this purpose, hybrid
-galactosidase genes of agaA and agaB from Bacillus stearothermophilus KVE39, designated agaA1 and agaB1, were cloned into an autonomously replicating Thermus vector and introduced into Thermus thermophilus OF1053GD ( agaT) by transformation . This selector strain is unable to metabolize melibiose (-galactoside) without recombinant
-galactosidases, because the native
-galactosidase gene, agaT, has been deleted . Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded agaA1 gene but unable when harboring a plasmid-encoded agaB1 gene . With incubation of the agaB1 plasmid-harboring strain under selective pressure at a restrictive temperature (67°C) in a minimal melibiose medium, spontaneous mutants as well as N-methyl-N'-nitro-N-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated . The mutant
-galactosidase genes were amplified by PCR, cloned in Escherichia coli, and sequenced . A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant agaB1 genes . The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability . To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell .
Salmonella enterica Serovar Typhi Strains from Which SPI7, a 134-Kilobase Island with Genes for Vi Exopolysaccharide and Other Functions, Has Been Deleted. Satheesh Nair, 2004.Salmonella enterica serovar Typhi has a 134-kb island of DNA identified as salmonella pathogenicity island 7 (SPI7), inserted between pheU and 'pheU (truncated), two genes for tRNAPhe . SPI7 has genes for Vi exopolysaccharide, for type IVB pili, for putative conjugal transfer, and for sopE bacteriophage . Pulsed-field gel electrophoresis following digestion with the endonuclease I-CeuI, using DNA from a set of 120 wild-type strains of serovar Typhi assembled from several sources, identified eight strains in which the I-CeuI G fragment, which contains SPI7, had a large deletion . In addition, agglutination tests with Vi antiserum and phage typing with Vi phages show that all eight strains are Vi negative . We therefore tested these strains for deletion of SPI7 by multiplex PCR, by microarray analysis, and by sequencing of PCR amplicons . Data show that seven of the eight strains are precise deletions of SPI7: a primer pair flanking SPI7 results in a PCR amplicon containing a single pheU gene; microarrays show that all SPI7 genes are deleted . Two of the strains produce amplicons which have A derived from pheU at bp 27, while five have C derived from 'pheU at this position; thus, the position of the crossover which results in the deletion can be inferred . The deletion in the eighth strain, TYT1669, removes 175 kb with junction points in genes STY4465 and STY4664; the left junction of SPI7 and adjacent genes, as well as part of SPI7 including the viaB operon for Vi exopolysaccharide, was removed, while the right junction of SPI7 was retained . We propose that these deletions occurred during storage following isolation .
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