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Genomic Profiling of Iron-Responsive Genes in Salmonella enterica Serovar Typhimurium by High-Throughput Screening of a Random Promoter Library. Jaime Bjarnason, 2003.The importance of iron to bacteria is shown by the presence of numerous iron-scavenging and transport systems and by many genes whose expression is tightly regulated by iron availability . We have taken a global approach to gene expression analysis of Salmonella enterica serovar Typhimurium in response to iron by combining efficient, high-throughput methods with sensitive, luminescent reporting of gene expression using a random promoter library . Real-time expression profiles of the library were generated under low- and high-iron conditions to identify iron-regulated promoters, including a number of previously identified genes . Our results indicate that approximately 7% of the genome may be regulated directly or indirectly by iron . Further analysis of these clones using a Fur titration assay revealed three separate classes of genes; two of these classes consist of Fur-regulated genes . A third class was Fur independent and included both negatively and positively iron-responsive genes . These may reflect new iron-dependent regulons . Iron-responsive genes included iron transporters, iron storage and mobility proteins, iron-containing proteins (redox proteins, oxidoreductases, and cytochromes), transcriptional regulators, and the energy transducer tonB . By identifying a wide variety of iron-responsive genes, we extend our understanding of the global effect of iron availability on gene expression in the bacterial cell . Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2'-Deoxyribonucleoside Production. Nobuyuki Horinouchi, 2003.The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4 . This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues . The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli . The DERA of K . pneumoniae was expressed in recombinant E . coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K . pneumoniae cell extract . One of the E . coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde . The E . coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP . Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E . coli cells . The DR5P produced was further transformed to 2'-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase . These results indicated that production of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP .
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