Direct involvement of ombB, omaB, and omcB genes in extracellular reduction of Fe(III) by Geobacter sulfurreducens PCA

Abstract

The tandem gene clusters orfR-ombB-omaB-omcB and orfS-ombC-omaC-omcC of the metal-reducing bacterium Geobacter sulfurreducens PCA are responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III)-citrate and ferrihydrite [a poorly crystalline Fe(III) oxide]. Each gene cluster encodes a putative transcriptional factor (OrfR/OrfS), a porin-like outer-membrane protein (OmbB/OmbC), a periplasmic c-type cytochrome (c-Cyt, OmaB/OmaC) and an outer-membrane c-Cyt (OmcB/OmcC). The individual roles of OmbB, OmaB and OmcB in extracellular reduction of Fe(III), however, have remained either uninvestigated or controversial. Here, we showed that replacements of ombB, omaB, omcB, and ombB-omaB with an antibiotic gene in the presence of ombC-omaC-omcC had no impact on reduction of Fe(III)-citrate by G. sulfurreducens PCA. Disruption of ombB, omaB, omcB, and ombB-omaB in the absence of ombC-omaC-omcC, however, severely impaired the bacterial ability to reduce Fe(III)-citrate as well as ferrihydrite. These results unequivocally demonstrate an overlapping role of ombB-omaB-omcB and ombC-omaC-omcC in extracellular Fe(III) reduction by G. sulfurreducens PCA. Involvement of both ombB-omaB-omcB and ombC-omaC-omcC in extracellular Fe(III) reduction reflects the importance of these trans-outer membrane protein complexes in the physiology of this bacterium. Moreover, the kinetics of Fe(III)-citrate and ferrihydrite reduction by these mutants in the absence of ombC-omaC-omcC were nearly identical, which suggests that absence of any protein subunit eliminates function of OmaB/OmbB/OmcB protein complex. Finally, orfS was found to have a negative impact on the extracellular reduction of Fe(III)-citrate and ferrihydrite in G. sulfurreducens PCA probably by serving as a transcriptional repressor.

Keywords: Fe(III) reduction; Geobacter; extracellular electron transfer; porin-cytochrome; trans-outer membrane protein complex.

FIGURE 1

The orfR gene clusters of Geobacter sulfurreducens PCA. The genes encoding transcriptional factors, porin-like outer-membrane proteins, the periplasmic c-type cytochromes and the outer-membrane c-type cytochromes are labeled in black, green, red, and purple, respectively.

FIGURE 2

Characterization of ombB, omaB, and omcB in the presence of ombC-omaC-omcC. (A) Growth on fumarate. (B) Fe(III)-citrate reduction. The curves are labeled in the same way in (A,B), except that no cell control is omitted in (A). The values plotted at each time point are the average OD₆₀₀ (A) and 0.5 N HCl extractable Fe(II) (B) measured for each strain from triplicate assays, respectively, and error bars are standard deviations. For points without error bar, the error was smaller than the symbol.

FIGURE 3

Characterization of ombB, omaB, and omcB in the absence of ombC-omaC-omcC. (A) Growth on fumarate. (B) Fe(III)-citrate reduction at 26 h. (C) Ferrihydrite reduction at 360 h. The values plotted at each time point are the average OD₆₀₀ (A) and 0.5 N HCl extractable Fe(II) (B,C) measured for each strain from triplicate assays, respectively, and error bars are standard deviations. For points without error bar, the error was smaller than the symbol (A).

FIGURE 4

Characterization of orfS. (A) Growth on fumarate. (B) Fe(III)-citrate reduction. (C) Ferrihydrite reduction. The curves are labeled in the same way in (A–C), except that no cell control is omitted in (A). The values plotted at each time point are the average OD₆₀₀ (A) and 0.5 N HCl extractable Fe(II) (B,C) measured for each strain from triplicate assays, respectively, and error bars are standard deviations. For points without error bar, the error was smaller than the symbol.