Proteolytic Maturation of the Outer Membrane c-Type Cytochrome OmcZ by a Subtilisin-Like Serine Protease Is Essential for Optimal Current Production by Geobacter sulfurreducens

Abstract

An outer membrane c-type cytochrome (OmcZ) in Geobacter sulfurreducens is essential for optimal current production in microbial fuel cells. OmcZ exists in two forms, small and large, designated OmcZ_S and OmcZ_L, respectively. However, it is still not known how these two structures are formed. A mutant with a disruption of the GSU2075 gene encoding a subtilisin-like serine protease (designated ozpA for the OmcZprotease), which is located downstream of omcZ, produced low currents at a level similar to that of the omcZ-deficient mutant strain. Biochemical analyses revealed that the ozpA mutant accumulated OmcZ_L and did not produce OmcZ_S, which is thought to be a mature form that is essential for the extracellular electron transfer to the electrode. A heterologous expression system cell lysate from an Escherichia coli strain producing OzpA cleaved OmcZ_L and generated OmcZ_S as the proteolytic product. Among the culture supernatant, loosely bound outer surface, and intracellular protein fractions from wild-type G. sulfurreducens, only the culture supernatant protein fraction showed OmcZ_L cleavage activity, indicating that the mature form of OmcZ, OmcZ_S, can be produced outside the cells. These results indicate that OzpA is an essential protease for current production via the maturation of OmcZ, and OmcZ_S is the key to the extracellular electron transfer to electrodes. This proteolytic maturation of OmcZ is a unique regulation among known c-type cytochromes in G. sulfurreducens. IMPORTANCE Microbial fuel cells are a promising technology for energy generation from various waste types. However, the molecular mechanisms of microbial extracellular electron transfer to the electrode need to be elucidated. G. sulfurreducens is a common key player in electricity generation in mixed-culture microbial fuel cell systems and a model microorganism for the study of extracellular electron transfer. Outer membrane c-type cytochrome OmcZ is essential for an optimal current production by G. sulfurreducens. OmcZ proteolytic cleavage occurs during maturation, but the underlying mechanism is unknown. This study identifies a subtilisin-like protease, OzpA, which plays a role in cleaving OmcZ and generating the mature form of OmcZ (OmcZ_S). OzpA is essential for current production and, thus, the proteolytic maturation of OmcZ. This is a novel regulation of the c-type cytochrome for G. sulfurreducens extracellular electron transfer. This study also provides new insights into the design strategy and development of microbial extracellular electron transfer for an efficient energy conversion from chemical energy to electricity.

Keywords: Geobacter sulfurreducens; c-type cytochrome; electroactive microorganism; extracellular electron transfer; subtilisin.

FIG 1

Profiles of c-type cytochromes in the ozpA-deficient mutant. (A) Heme-stained gel image of whole-cell lysates of wild-type and ozpA-deficient mutant G. sulfurreducens strains and of LBOP fractions of wild-type and ozpA-deficient mutant strains. (B) Western blot using anti-OmcZ antibody for LBOP fractions from wild-type, omcZ-deficient mutant, and ozpA-deficient mutant G. sulfurreducens strains.

FIG 2

Current production of wild-type (black), ozpA-deficient mutant (red), ozpA-complemented (blue), and omcZ-deficient mutant (green) strains. Current production of only ozpA-deficient and omcZ-deficient mutant strains for the comparison are shown in the inset. Representation of triplicates is shown for ozpA-deficient mutant and ozpA-complemented strains.

FIG 3

Proteolytic cleavage of OmcZ by culture supernatant, loosely bound cell surface protein, and intracellular fraction protein from wild-type G. sulfurreducens. Heme-stained gel of LBOP of ozpA-deficient mutant treated with PBS buffer (negative control), culture supernatant, LBOP, and intracellular fraction protein from wild-type G. sulfurreducens.

FIG 4

In vitro proteolytic cleavage of OmcZ by OzpA. Heme-564-stained gel (A) and Western blot using anti-OmcZ antibody (B) of LBOP fraction, LBOP fraction treated with a crude extract of E. coli lacking OzpA, and LBOP fraction treated with a crude extract of E. coli expressing OzpA.