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.

Comparative genomics analyses on EPS biosynthesis genes required for floc formation of Zoogloea resiniphila and other activated sludge bacteria

“…The transposon insertion sites were mapped as previously described (Bouhenni et al, 2005). For in-frame deletion mutants, the two-step protocol of selection (single cross-over, antibiotic resistance) and counter-selection (double crossover, sucrose sensitivity) was conducted using the suicide vector pDS3.0 (R6K replicon, sacB, Gm R )-based constructs with a fusion of upstream and downstream sequences of the target gene as previously described (Wan et al, 2004). For genetic complementation analyses, the target genes were PCR amplified and cloned into the pBBR1-MCS5 or pHERD30 vector (Qiu et al, 2008).…”

“…Nitrate reduction was assayed in the modified M1 medium supplemented with 50 mM sodium lactate as the electron donor and carbon source and sodium nitrate as the electron acceptors (Wan et al, 2004). The bacterial cultures were incubated under microoxic conditions (without shaking to limit aeration).…”

The Role of Shewanella oneidensis MR‐1 Outer Surface Structures in Extracellular Electron Transfer

“…BG167, a pilD mutant of S. oneidensis MR-1, was generated using the Mini-himar RB-1 transposon [27]. Chromosomal deletions of gspG, type IV pilus biogenesis, Msh pilus biogenesis, and flagellin genes were generated using the modified suicide plasmid pER2, a derivative of pDS3 [28] using described protocols [29]. DNA fragments for generation of deletions or complementation of mutants were amplified by PCR using Phusion polymerase (Clontech) and primers listed in Table 2.…”

Shewanella oneidensis MR-1 Sensory Box Protein Involved in Aerobic and Anoxic Growth

“…An approximately 2.4-kbp fragment was deleted from SO3389 (2,639 bp) with PCR-based crossover amplification as described previously (1,17,56). The primer pairs used to generate two DNA fragments with 3Ј-staggered ends were as follows: No-5Ј-CGCGAGCTCGTTCGGACCTTCG ATAAATC-3Ј and Ni-5Ј-TGTTTAAACTTAGTGGATGGGGCCCAATCTGT TAACTGCTT-3Ј and Co-5Ј-CGCGAGCTCGCACCTTGACGCAAATGAT C-3Ј and Ci-5Ј-CCCATCCACTAAGTTTAAACAGCGGATGTTGAGGCCTT TTT-3Ј (noncomplementary tag sequences are underlined), and the outside primers were used to amplify a fusion molecule.…”

“…The crossover PCR product was purified and digested by SacI, cloned into treated suicide vector pDS3.0, and electroporated into E. coli S17-1/ pir . The suicide plasmid construct was moved into MR-1 as previously described (56), and the mutated SO3389 gene was verified by PCR (5Ј-GAGTGGCATTCAGCACTAGA-3Ј and 5Ј-CATACGGT CGGCTTCATCAA-3Ј) and sequence determination. The resulting mutant did not possess any of the predicted domains after completion of the in-frame deletion.…”