Characterization of transcriptional regulation of Shewanella frigidimarina Fe(III)-induced flavocytochrome c reveals a novel iron-responsive gene regulation system

Abstract

The bacterium Shewanella frigidimarina can grow anaerobically by utilizing Fe(III) as a respiratory electron acceptor. This results in the synthesis of a number of periplasmic c-type cytochromes, which are absent when the organism is grown in the absence of added Fe(III). One cytochrome, IfcA, is synthesized when Fe(III) is present as the sole respiratory electron acceptor or when it is present in combination with oxygen, fumarate, or nitrate. The ifcA gene was thus selected for a study of iron-responsive gene regulation of respiratory proteins in S. frigidimarina. The monocistronic ifcA gene clusters with two other monocistronic genes, ifcO, encoding a putative outer membrane porin, and ifcR, encoding a putative transcriptional regulator of the LysR superfamily. Analysis of transcription of all three genes under a range of growth conditions in the wild type and an ifcR insertion mutant and analysis of a strain that constitutively expresses ifcR revealed that iron regulation is exerted at the level of ifcR transcription. In the presence of Fe(III) IfcR is synthesized and acts positively to regulate expression of ifcO and ifcA. Control of Fe(III) respiration by this novel regulatory system differs markedly from Fur-mediated regulation of iron assimilation, in which Fur serves as an Fe(II)-activated repressor.

FIG. 1.

Heme-linked peroxidase staining of periplasmic fractions of wild-type S. frigidimarina. Aliquots (40 μg) of periplasmic fractions were loaded onto an SDS-10% (wt/vol) PAGE gel. Lane 1, purified sample of IfcA (1 μg); lane 2, periplasm from cells grown anaerobically with fumarate; lane 3, periplasm from cells grown anaerobically with nitrate; lane 4, periplasm from cells grown anaerobically with ferric citrate; lane 5, periplasm from cells grown anaerobically with ferric citrate and fumarate; lane 6, periplasm from cells grown anaerobically with ferric citrate and nitrate; lane 7, periplasm from cells grown anaerobically with manganese; lane 8, periplasm from cells grown microaerobically; lane 9 periplasm from cells grown microaerobically with ferric citrate.

FIG. 2.

Fe(III) reduction by wild-type S. frigidimarina. A color change from black to yellow in the medium is an indication of Fe(III) reduction, as observed when S. frigidimarina cells were grown with ferric citrate or with fumarate and ferric citrate as electron acceptors but not when S. frigidimarina cells were grown with nitrate and ferric citrate as respiratory substrates.

FIG. 3.

Schematic representation of the organization of the ifcR, ifcO, and ifcA genes in S. frigidimarina and S. oneidensis. The arrows indicate directions of transcription. Annotation and preliminary genomic sequence data were provided by The Institute for Genomic Research (http://www.tigr.org). The numbers indicate the levels of amino acid identity and similarity (expressed as percentages) between the corresponding proteins from the two species.

FIG. 4.

Heme-linked peroxidase staining of periplasmic fractions from S. frigidimarina PSD106 (ifcR) and PSD201 (ifcR⁺) after anaerobic growth with ferric citrate. Aliquots (40 μg) of periplasmic fractions were loaded onto an SDS-10% (wt/vol) PAGE gel. Lane 1, pure IfcA protein (1 μg); lane 2, periplasm from S. frigidimarina PSD106 (ifcR); lane 3, periplasm from S. frigidimarina PSD201 (PSD106 complemented with ifcR⁺); lane 4, purified Fcc3 protein (1 μg).

FIG. 5.

Regulation of ifcO and ifcA transcription. (A) Intergenic region of ifcO and ifcA. The arrows indicate the transcription start site of each gene. Putative ribosome-binding sites are indicated by boldface type, and the −35 and −10 RNA polymerase-binding sites of each gene are indicated by boldface type and underlining. (B) Primer extension analysis of ifcA and ifcO performed with total RNA isolated from wild-type cells of S. frigidimarina and cells of ifcR mutant strain PSD106. +O₂, RNA from cells grown microaerobically on LB medium; −O₂ +Fu, RNA from cells grown anaerobically with fumarate as the sole electron acceptor; −O₂+Fe, RNA from cells grown anaerobically with iron as the electron acceptor. Primer extension reactions were performed by using oligonucleotides P2 and P3 (see Materials and Methods), which hybridized with ifcO and ifcA, respectively. Sequencing reactions were performed with the oligonucleotides that were used in the primer extension reactions.

FIG. 6.

Regulation of ifcR expression. (A) Regulatory region of the ifcR gene. The arrow indicates the transcription start site of the ifcR gene. The proposed ribosome-binding site is indicated by boldface type, and the putative −35 and −10 RNA polymerase recognition sequences are indicated by boldface type and underlining. A perfect palindromic sequence is underlined. (B) Primer extension analysis of ifcR performed with total RNA isolated from wild-type cells of S. frigidimarina and cells of ifcR mutant strain PSD106. +O₂, RNA from cells grown microaerobically on LB medium; −O₂+Fu, RNA from cells grown anaerobically with fumarate as the sole electron acceptor; −O₂+Fe, RNA from cells grown anaerobically with iron as the sole electron acceptor. Primer extension reactions were performed by using labeled oligonucleotide P1 (see Materials and Methods). Sequencing reactions were performed with the oligonucleotide that was used in the primer extension reactions. (C) Primer extension analysis of ifcR performed with total RNA isolated from wild-type cells of S. frigidimarina. +Fe, RNA from cells grown anaerobically with iron as the sole electron acceptor; Fe+Fu, RNA from cells grown anaerobically with fumarate and iron as electrons acceptors; Fe+Ni, RNA from cells grown anaerobically with iron and nitrate as electrons acceptors; +O₂, RNA from cells grown microaerobically on LB medium; +O₂ +Fe, RNA from cells grown microaerobically with ferric citrate.

FIG. 7.

Heme-linked peroxidase staining of periplasmic fractions from S. frigidimarina PSR33 (ifcR⁺). (A) Aliquots (40 μg) of periplasmic fractions were loaded onto an SDS-10% PAGE gel. Lane 1, periplasm from wild-type cells grown anaerobically with ferric citrate (positive control); lane 2, periplasm from PSR33 cells grown anaerobically with nitrate and ferric citrate; lane 3, periplasm from PSR33 cells grown anaerobically with nitrate; lane 4, periplasm from PSD106 cells grown anaerobically with nitrate and ferric citrate; lane 5, periplasm from PSD106 cells grown anaerobically with nitrate; lane 6, periplasm from PSR33 cells grown anaerobically with fumarate and ferric citrate; lane 7, periplasm from PSR33 cells grown anaerobically with fumarate. (B) Lane 1, purified IfcA (1 μg); lane 2, periplasm from PSR33 cells grown microaerobically on LB medium with 1 mM IPTG; lane 3, periplasm from PSR33 cells grown microaerobically on LB medium without IPTG; lane 4, periplasm from wild-type cells carrying plasmid pRK415 grown microaerobically on LB medium with 1 mM IPTG; lane 5, purified Fcc3 (1 μg).