Secretion of flavins by Shewanella species and their role in extracellular electron transfer

Abstract

Fe(III)-respiring bacteria such as Shewanella species play an important role in the global cycle of iron, manganese, and trace metals and are useful for many biotechnological applications, including microbial fuel cells and the bioremediation of waters and sediments contaminated with organics, metals, and radionuclides. Several alternative electron transfer pathways have been postulated for the reduction of insoluble extracellular subsurface minerals, such as Fe(III) oxides, by Shewanella species. One such potential mechanism involves the secretion of an electron shuttle. Here we identify for the first time flavin mononucleotide (FMN) and riboflavin as the extracellular electron shuttles produced by a range of Shewanella species. FMN secretion was strongly correlated with growth and exceeded riboflavin secretion, which was not exclusively growth associated but was maximal in the stationary phase of batch cultures. Flavin adenine dinucleotide was the predominant intracellular flavin but was not released by live cells. The flavin yields were similar under both aerobic and anaerobic conditions, with total flavin concentrations of 2.9 and 2.1 micromol per gram of cellular protein, respectively, after 24 h and were similar under dissimilatory Fe(III)-reducing conditions and when fumarate was supplied as the sole electron acceptor. The flavins were shown to act as electron shuttles and to promote anoxic growth coupled to the accelerated reduction of poorly crystalline Fe(III) oxides. The implications of flavin secretion by Shewanella cells living at redox boundaries, where these mineral phases can be significant electron acceptors for growth, are discussed.

FIG. 1.

Production of intracellular (A) and extracellular (B) flavins in Shewanella oneidensis MR-1 cultures grown anaerobically and aerobically for 24 h in minimal medium containing 50 mM lactate and 100 mM fumarate. Data are averages for triplicate cultures.

FIG. 2.

Production of extracellular FMN (squares), riboflavin (Rib) (diamonds), and FAD (triangles) during anaerobic growth (circles) of Shewanella oneidensis MR-1 in a defined minimal medium containing 50 mM lactate and 100 mM fumarate. Data are averages for triplicate cultures.

FIG. 3.

Extracellular flavin yields in cultures of Shewanella and Pseudomonas species after 49 h of aerobic growth in minimal medium containing 50 mM lactate and 100 mM fumarate. Data are averages for triplicate cultures. S.sp., Shewanella sp.; S.b., S. baltica; S.o., S. oneidensis; S.f., S. frigidimarina; P.p., P. putida; P.s., P. stutzeri; P.a., P. aeruginosa; E.c., E. coli; RF, riboflavin.

FIG. 4.

Fe(III) reduction rates of Shewanella oneidensis MR-1 cells grown with fumarate (black) or Fe(III)-citrate (gray), with poorly soluble Fe(III) oxide and Fe(III)-citrate as substrates and in the absence of redox mediators (RM) or with 10 μM of the redox mediator FAD, FMN, riboflavin (Rib), or AQDS. Data are averages for triplicate cultures.

FIG. 5.

Reduction of poorly soluble Fe(III) oxide (PSFO; closed symbols) and soluble Fe(III)-citrate (sF; open symbols) by cells of Shewanella strain Hac334. The concentration of riboflavin added to the assay mixtures ranged from 0 to 100 μM (R0 to R100). Data are averages for triplicate cultures.