Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens

Abstract

A 9.6 kDa periplasmic c -type cytochrome, designated PpcA, was purified from the Fe(III)-reducing bacterium Geobacter sulfurreducens and characterized. The purified protein is basic (pI 9.5), contains three haems and has an N-terminal amino acid sequence closely related to those of the previously described trihaem c (7) cytochromes of Geobacter metallireducens and Desulfuromonas acetoxidans. The gene encoding PpcA was identified from the G. sulfurreducens genome using the N-terminal sequence, and encodes a protein of 71 amino acids (molecular mass 9.58 kDa) with 49% identity to the c (7) cytochrome of D. acetoxidans. In order to determine the physiological role of PpcA, a knockout mutant was prepared with a single-step recombination method. Acetate-dependent Fe(III) reduction was significantly inhibited in both growing cultures and cell suspensions of the mutant. When ppcA was expressed in trans, the full capacity for Fe(III) reduction with acetate was restored. The transfer of electrons from acetate to anthraquinone 2,6-disulphonate (AQDS; a humic acid analogue) and to U(VI) was also compromised in the mutant, but acetate-dependent reduction of fumarate was not altered. The rates of reduction of Fe(III), AQDS, U(VI) and fumarate were also the same in the wild type and ppcA mutant when hydrogen was supplied as the electron donor. When taken together with previous studies on other electron transport proteins in G. sulfurreducens, these results suggest that PpcA serves as an intermediary electron carrier from acetate to terminal Fe(III) reductases in the outer membrane, and is also involved in the transfer of electrons from acetate to U(VI) and humics.

Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens

“…Previous studies have shown that the periplasmic c-type cytochrome protein PpcA (GSU0612) is required for efficient reduction of soluble Fe(III) citrate (Lloyd et al, 2003). This study showed that the ppcA gene was downregulated 6.5-fold during growth on Fe(III) oxide compared with Fe(III) citrate (Table 1) and that the PpcAdeficient strain grew as well as the wild-type on Fe(III) oxide (Table 5).…”

“…It has been shown that both MacA and PpcA are involved in the reduction of soluble Fe(III) Kim & Lovley, 2008;Lloyd et al, 2003) and U(VI) (Lloyd et al, 2003;Shelobolina et al, 2007); however, deletion of either of these genes did not affect Mn(IV) reduction-dependent growth (Table 5). Expression of ppcD was fivefold higher on Mn(IV) oxide (Table 4), and in proteomic studies, PpcD was more abundant in Fe(III) oxide-grown than in Fe(III) citrate-grown cells (Ding et al, 2008).…”

Microtoming coupled to microarray analysis to evaluate the spatial metabolic status of Geobacter sulfurreducens biofilms

“…2.05-2.85-fold) decreased (Supplementary Table 4). These included two putative outer-surface c-type cytochromes, OmcX and OmcQ, putative cytochromes encoded by GSU0593 and GSU2743, as well as the periplasmic cytochrome PpcA (Lloyd et al, 2003). However, there seemed to be no significant difference in the expression levels of the vast majority of the ca.…”

Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov.

“…electron transfer in Geobacter species Lloyd et al, 2003). Thus, due to its distinct physiological differences from Geobacter species, a separate genus designation for Pelobacter propionicus may be warranted.…”