Transcriptomic and proteomic characterization of the Fur modulon in the metal-reducing bacterium Shewanella oneidensis

Abstract

The availability of the complete genome sequence for Shewanella oneidensis MR-1 has permitted a comprehensive characterization of the ferric uptake regulator (Fur) modulon in this dissimilatory metal-reducing bacterium. We have employed targeted gene mutagenesis, DNA microarrays, proteomic analysis using liquid chromatography-mass spectrometry, and computational motif discovery tools to define the S. oneidensis Fur regulon. Using this integrated approach, we identified nine probable operons (containing 24 genes) and 15 individual open reading frames (ORFs), either with unknown functions or encoding products annotated as transport or binding proteins, that are predicted to be direct targets of Fur-mediated repression. This study suggested, for the first time, possible roles for four operons and eight ORFs with unknown functions in iron metabolism or iron transport-related functions. Proteomic analysis clearly identified a number of transporters, binding proteins, and receptors related to iron uptake that were up-regulated in response to a fur deletion and verified the expression of nine genes originally annotated as pseudogenes. Comparison of the transcriptome and proteome data revealed strong correlation for genes shown to be undergoing large changes at the transcript level. A number of genes encoding components of the electron transport system were also differentially expressed in a fur deletion mutant. The gene omcA (SO1779), which encodes a decaheme cytochrome c, exhibited significant decreases in both mRNA and protein abundance in the fur mutant and possessed a strong candidate Fur-binding site in its upstream region, thus suggesting that omcA may be a direct target of Fur activation.

FIG. 1.

Functional distribution of differentially expressed genes in the FUR2 strain. Each bar represents the number of ORFs showing significant changes in mRNA abundance in response to a fur deletion mutation under aerobic and anaerobic growth conditions. The ORFs are grouped in their corresponding functional and homology classes according to the TIGR annotation (http://www.tigr.org/tigr-scripts/CMR2/gene_attribute_results_org_or_role.dbi): (A) amino acid biosynthesis; (B) biosynthesis of cofactors, prosthetic groups, and carriers; (C) cell envelope; (D) cellular processes; (E) central intermediary metabolism; (F) DNA metabolism; (G) energy metabolism; (H) fatty acid and phospholipid metabolism; (I) hypothetical proteins; (J) other categories; (K) protein fate; (L) protein synthesis; (M) purines, pyrimidines, nucleosides, and nucleotides; (N) regulatory functions; (O) signal transduction; (P) transcription; (Q) transport and binding proteins; and (R) unknown function.

FIG. 2.

Comparison of gene expression measurements by microarray hybridization and real-time quantitative RT-PCR. The changes in gene expression in a fur deletion mutant grown under both aerobic and anaerobic respiratory conditions were log transformed (in base 10). The real-time RT-PCR log₁₀ ratio values were plotted against the microarray data log₁₀ values. Comparison of the two methods indicated a high level of concordance (r² = 0.86).

FIG. 3.

Identification of a predicted consensus Fur-binding motif in S. oneidensis MR-1 using computational methods. A sequence logo representation (57) of a palindromic-motif model was derived based on only those sites listed in Table 5 that are directly upstream of genes exhibiting differential expression in response to a fur deletion mutation. The error bars indicate standard deviations of the sequence conservation.