Genomic Profiling of Iron-Responsive Genes in Salmonella enterica Serovar Typhimurium by High-Throughput Screening of a Random Promoter Library

Abstract

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.

Iron and citrate export by a major facilitator superfamily pump regulates metabolism and stress resistance inSalmonellaTyphimurium

“…To confirm the anticipated patterns of gene expression in the Δfur mutant, expression of bfd, sitA, and bfr were also determined. As expected, expression of the Furrepressed bfd and sitA genes was elevated in the Δfur mutant, whereas expression of bfr was essentially unchanged, which may reflect the low levels of bfr expression observed during the exponential phase of growth (36)(37)(38).…”

“…To determine whether the mdtABCD baeSR operon is induced under conditions of iron stress, expression was examined in an isogenic Δfur mutant (EF394) in which iron uptake systems were constitutively expressed. A previous study to identify iron-responsive genes in S. Typhimurium failed to detect a response of the mdtABCD baeSR operon to iron restriction or supplementation per se (36). Therefore, disrupted iron homeostasis in a Δfur mutant, rather than direct regulation by Fur, may lead to mdtABCD baeSR expression (Fig.…”

Comparative Genetics of the rdar Morphotype inSalmonella

“…purified, sequentially digested with XhoI and BamHI (Invitrogen Canada Inc.), and ligated using T4 DNA ligase (Invitrogen Canada Inc.) into pCS26-Pac (XhoI-BamHI) or pU220 (BamHI-XhoI) reporter vectors containing the lux CDABE operon from Photorhabdus luminescens (7). The RpoS-responsive synthetic promoter::lux fusion sig38-H4 has been previously described (68).…”

“…Promoter::lux plasmids were purified from Salmonella (QIAspin miniprep kit; QIAGEN Inc.), and DNA sequencing was performed by Macrogen (Seoul, South Korea) using primers pZE05 and pZE06 (7). DNA sequences were assembled using Contig Express (Vector NTI 7.0; Informax, Bethesda, MD).…”

Staphylococcus aureus promoter-lux reporters for drug discovery

“…11,12 These assays are extremely sensitive, accurately reflect real-time changes in gene expression and are well suited for monitoring the activity of promoters on an individual or a genome-wide level. [13][14][15][16] We have used an array of promoter-lux clones in S. aureus for the purpose of detecting low concentrations of bioactive compounds produced by actinomycetes. The approach is based on the observation that almost all antibiotics, at subinhibitory concentrations (sub-MIC), alter the transcription of up to 5% of genes in bacteria.…”