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.

FIG. 1.

(A) The reporter plasmid pCS26-Pac. The salient features of this pZS derivative (28) include low copy number, strong transcriptional terminators, unique promoter cloning sites, and luxCDABE of Photorhabdus luminescens (33). pCS26-Pac was developed by introducing PacI sites adjacent to the pSC101 origin of replication to facilitate transfer of the reporter cassette to other vectors. (B) The mean luminescence activity in counts per second was measured in triplicate for all strains grown in tryptone broth. The growth curves were within 5%, and no significant differences in growth were observed between these strains and the parent strain (data not shown). RP437/pCGNot21 (open square) contains the vector lacking the lux reporter, RP437/pCS26 (closed triangle) contains a promoterless vector, and RP437/pCS16 (closed diamond) contains the flgB promoter in pCS26. RP437/pCS16 luminescence is plotted on the secondary y axis.

FIG. 2.

Scatter plot analysis of duplicate screens under low-iron conditions. Luminescence readings were taken at 6 and 20 h (inset). The lines demarcate the threefold differences in expression level. The linear trend for the duplicate data points indicates that plate-to-plate variability is minimal. Points falling outside the threefold range (3% of data points) can most likely be attributed to growth effects arising from variation in inoculum. Promoter clones with expression levels below or equal to background were not included in the data set.

FIG. 3.

Scatter plot analysis of library screens under low- versus high-iron conditions. Luminescence readings were taken at 6 and 20 h (inset). The colored data points represent promoter clones exhibiting >3-fold differences in expression between the two conditions; red represents promoters active under low-iron conditions, and green represents promoters active under high-iron conditions. Promoter clones with expression levels below or equal to background were not included in the data set.

FIG. 4.

Hierarchical clustering of iron-responsive expression data. Expression levels above the mean center for each gene are indicated by red, below the mean center by green, and around the mean center in black. Clones are clustered on the y axis and the time course is shown on the x axis. (A) Temporal expression data for promoter clones in supplemented M9 minimal (M9) or LB (LB) media containing 200 μM FeCl₃ (Fe) or 200 μM 2,2′-dipyridyl (Dp). Luminescence was measured at 2, 4, 6, 8, and 24 h. Clustering illustrates differential regulation of promoter sets in response to various medium conditions. (B) +Fur, library strains containing pEX18Tc. −Fur, library strains containing fepA-pEX18Tc. Each data set is shown as a time course through 14, 16, and 18 h of the growth curve in high-iron medium (400 μM FeCl₃). Highlighted clusters refer to promoters with increased expression (class A) or decreased expression (class B) when Fur availability is reduced. The remaining promoters (class C) appear to be regulated independently of Fur (not highlighted).