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. 2002 Feb;68(2):881-92.
doi: 10.1128/AEM.68.2.881-892.2002.

Transcriptional and proteomic analysis of a ferric uptake regulator (fur) mutant of Shewanella oneidensis: possible involvement of fur in energy metabolism, transcriptional regulation, and oxidative stress

Dorothea K Thompson  1 , Alexander S Beliaev, Carol S Giometti, Sandra L Tollaksen, Tripti Khare, Douglas P Lies, Kenneth H Nealson, Hanjo Lim, John Yates 3rd, Craig C Brandt, James M Tiedje, Jizhong Zhou
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Transcriptional and proteomic analysis of a ferric uptake regulator (fur) mutant of Shewanella oneidensis: possible involvement of fur in energy metabolism, transcriptional regulation, and oxidative stress

Dorothea K Thompson et al. Appl Environ Microbiol. 2002 Feb.

Abstract

The iron-directed, coordinate regulation of genes depends on the fur (ferric uptake regulator) gene product, which acts as an iron-responsive, transcriptional repressor protein. To investigate the biological function of a fur homolog in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1, a fur knockout strain (FUR1) was generated by suicide plasmid integration into this gene and characterized using phenotype assays, DNA microarrays containing 691 arrayed genes, and two-dimensional polyacrylamide gel electrophoresis. Physiological studies indicated that FUR1 was similar to the wild-type strain when they were compared for anaerobic growth and reduction of various electron acceptors. Transcription profiling, however, revealed that genes with predicted functions in electron transport, energy metabolism, transcriptional regulation, and oxidative stress protection were either repressed (ccoNQ, etrA, cytochrome b and c maturation-encoding genes, qor, yiaY, sodB, rpoH, phoB, and chvI) or induced (yggW, pdhC, prpC, aceE, fdhD, and ppc) in the fur mutant. Disruption of fur also resulted in derepression of genes (hxuC, alcC, fhuA, hemR, irgA, and ompW) putatively involved in iron uptake. This agreed with the finding that the fur mutant produced threefold-higher levels of siderophore than the wild-type strain under conditions of sufficient iron. Analysis of a subset of the FUR1 proteome (i. e. , primarily soluble cytoplasmic and periplasmic proteins) indicated that 11 major protein species reproducibly showed significant (P < 0. 05) differences in abundance relative to the wild type. Protein identification using mass spectrometry indicated that the expression of two of these proteins (SodB and AlcC) correlated with the microarray data. These results suggest a possible regulatory role of S. oneidensis MR-1 Fur in energy metabolism that extends the traditional model of Fur as a negative regulator of iron acquisition systems VSports手机版. .

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Figures

FIG. 1.
FIG. 1.
Amino acid sequence alignment of fur-encoded proteins from P. aeruginosa (GenBank accession no. A40622), V. cholerae (GenBank accession no. A42282), S. oneidensis MR-1 (http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi), and E. coli (GenBank accession no. S07308). Conserved amino acid residues are shaded.
FIG. 2.
FIG. 2.
Analysis of microarray gene expression data. Mean signal intensity ratios of mutant to wild-type mRNA levels were obtained from two to four independent replicate experiments for each gene. Genes with mean fluorescence intensities less than two standard deviations above background in both channels were excluded. Expression ratios were transformed to natural log, such that a 2.7-fold increase (solid line) in transcript levels in the fur mutant equaled 1, whereas a 2.7-fold decrease (dashed line) equaled −1. The relative error of each expression ratio is presented.
FIG. 3.
FIG. 3.
2-D PAGE of whole-cell lysates of S. oneidensis MR-1 (A) and FUR1 (B) grown in LB medium (high concentration of iron) under aerobic conditions. Protein spots showing significant quantitative differences (a P value of at least <0.05) between the two strains are indicated by arrows and numbers. The gel images are oriented with the isoelectric focusing dimension horizontal and the SDS-PAGE dimension vertical. The approximate pI is indicated along the horizontal axis. The approximate positions of the SDS-PAGE molecular mass (MW) standards are presented in kilodaltons along the vertical axis.
FIG. 3.
FIG. 3.
2-D PAGE of whole-cell lysates of S. oneidensis MR-1 (A) and FUR1 (B) grown in LB medium (high concentration of iron) under aerobic conditions. Protein spots showing significant quantitative differences (a P value of at least <0.05) between the two strains are indicated by arrows and numbers. The gel images are oriented with the isoelectric focusing dimension horizontal and the SDS-PAGE dimension vertical. The approximate pI is indicated along the horizontal axis. The approximate positions of the SDS-PAGE molecular mass (MW) standards are presented in kilodaltons along the vertical axis.
FIG. 4.
FIG. 4.
Anaerobic growth of MR-1 and FUR1 strains of S. oneidensis on LB medium supplemented with 20 mM Na lactate and 10 mM fumarate (A) or 8 mM nitrate (B). Cultures pregrown anaerobically overnight on the tested substrates were used to inoculate the medium. Values represent independent means ± standard deviations of OD600 readings obtained for triplicate cultures of each strain assessed in parallel.
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