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Genetic and Biochemical Characterization of a Chromosome-Encoded Carbapenem-Hydrolyzing Ambler Class D ß-Lactamase from Shewanella algae. Claire Héritier, 2004.A chromosome-encoded ß-lactamase gene from Shewanella algae clinical isolate KB-1 was cloned and expressed in Escherichia coli . It encoded the Ambler class D enzyme OXA-55, sharing less than 55% identity with any other oxacillinases . Although conferring a narrow-spectrum ß-lactam resistance phenotype, OXA-55 had carbapenem-hydrolyzing activity that mirrored the reduced susceptibility to imipenem observed in S . algae KB-1 . Very similar oxacillinases were found in other S . algae isolates . Comparative Analysis of Physical Maps of Four Bacillus subtilis (natto) Genomes. Dongru Qiu, 2004.The complete SfiI and I-CeuI physical maps of four Bacillus subtilis (natto) strains, which were previously isolated as natto (fermented soybean) starters, were constructed to elucidate the genome structure . Not only the similarity in genome size and organization but also the microheterogeneity of the gene context was revealed . No large-scale genome rearrangements among the four strains were indicated by mapping of the genes, including 10 rRNA operons (rrn) and relevant genes required for natto production, to the loci corresponding to those of the B . subtilis strain Marburg 168 . However, restriction fragment length polymorphism and the presence or absence of strain-specific DNA sequences, such as the prophages SPß, skin element, and PBSX, as well as the insertion element IS4Bsu1, could be used to identify one of these strains as a Marburg type and the other three strains as natto types . The genome structure and gene heterogeneity were also consistent with the type of indigenous plasmids harbored by the strains . Requirement of a Relatively High Threshold Level of Mg2+ for Cell Growth of a Rhizoplane Bacterium, Sphingomonas yanoikuyae EC-S001. Henny Hoo, 2004.Mg2+ is one of the essential elements for bacterial cell growth . The presence of the magnesium cation (Mg2+) in various concentrations often affects cell growth restoration in plant-associating bacteria . This study attempted to determine whether Mg2+ levels in Sphingomonas yanoikuyae EC-S001 affected cell growth restoration in the host plant and what the threshold level is . S . yanoikuyae EC-S001, isolated from the rhizoplane of spinach seedlings grown from surface-sterilized seeds under aseptic conditions, displayed uniform dispersion and attachment throughout the rhizoplane and phylloplane of the host seedlings . S . yanoikuyae EC-S001 did not grow in potato-dextrose broth medium but grew well in an aqueous extract of spinach leaves . Chemical investigation of the growth factor in the spinach leaf extract led to identification of the active principle as the magnesium cation . A concentration of ca . 0.10 mM Mg2+ or more allowed S . yanoikuyae EC-S001 to grow in potato-dextrose broth medium . Some saprophytic and/or diazotrophic bacteria used in our experiment were found to have diverse threshold levels for their Mg2+ requirements . For example, Burkholderia cepacia EC-K014, originally isolated from the rhizoplane of a Melastoma sp., could grow even in Mg2+-free Hoagland's no . 2 medium with saccharose and glutamine (HSG medium) and requires a trace level of Mg2+ for its growth . In contrast, S . yanoikuyae EC-S001, together with Bacillus subtilis IFO12113, showed the most drastic restoring responses to subsequent addition of 0.98 mM Mg2+ to Mg2+-free HSG medium . Our studies concluded that Mg2+ is more than just the essential trace element needed for cell growth restoration in S . yanoikuyae EC-S001 and that certain nonculturable bacteria may require a higher concentration of Mg2+ or another specific essential element for their growth . Anthranilate Synthase Can Generate Sufficient Phosphoribosyl Amine for Thiamine Synthesis in Salmonella enterica. I. Ramos, 2003.In bacteria, the biosynthetic pathway for the hydroxymethyl pyrimidine moiety of thiamine shares metabolic intermediates with purine biosynthesis . The two pathways branch after the compound aminoimidazole ribotide . Past work has shown that the first common metabolite, phosphoribosyl amine (PRA), can be generated in the absence of the first enzyme in purine biosynthesis, PurF . PurF-independent PRA synthesis is dependent on both strain background and growth conditions . Standard genetic approaches have not identified a gene product singly responsible for PurF-independent PRA formation . This result has led to the hypothesis that multiple enzymes contribute to PRA synthesis, possibly as the result of side products from their dedicated reaction . A mutation that was able to restore PRA synthesis in a purF gnd mutant strain was identified and found to map in the gene coding for the TrpD subunit of the anthranilate synthase (AS)-phosphoribosyl transferase (PRT) complex . Genetic analyses indicated that wild-type AS-PRT was able to generate PRA in vivo and that the P362L mutant of TrpD facilitated this synthesis . In vitro activity assays showed that the mutant AS was able to generate PRA from ammonia and phosphoribosyl pyrophosphate . This work identifies a new reaction catalyzed by AS-PRT and considers it in the context of cellular thiamine synthesis and metabolic flexibility . The SopE  Phage Integrates into the ssrA Gene of Salmonella enterica Serovar Typhimurium A36 and Is Closely Related to the Fels-2 Prophage.Cosima Pelludat, 2003.Salmonella spp . are enteropathogenic gram-negative bacteria that use a large array of virulence factors to colonize the host, manipulate host cells, and resist the host's defense mechanisms . Even closely related Salmonella strains have different repertoires of virulence factors . Bacteriophages contribute substantially to this diversity . There is increasing evidence that the reassortment of virulence factor repertoires by converting phages like the GIFSY phages and SopE may represent an important mechanism in the adaptation of Salmonella spp . to specific hosts and to the emergence of new epidemic strains . Here, we have analyzed in more detail SopE, a P2-like phage from Salmonella enterica serovar Typhimurium DT204 that encodes the virulence factor SopE . We have cloned and characterized the attachment site (att) of SopE and found that its 47-bp core sequence overlaps the 3' terminus of the ssrA gene of serovar Typhimurium . Furthermore, we have demonstrated integration of SopE into the cloned attB site of serovar Typhimurium A36 . Sequence analysis of the plasmid-borne prophage revealed that SopE is closely related to (60 to 100% identity over 80% of the genome) but clearly distinct from the Fels-2 prophage of serovar Typhimurium LT2 and from P2-like phages in the serovar Typhi CT18 genome . Our results demonstrate that there is considerable variation among the P2-like phages present in closely related Salmonella spp .
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