Identification of a Diguanylate Cyclase That Facilitates Biofilm Formation on Electrodes by Shewanella oneidensis MR-1

Abstract

In many bacteria, cyclic diguanosine monophosphate (c-di-GMP), synthesized by diguanylate cyclase (DGC), serves as a second messenger involved in the regulation of biofilm formation. Although studies have suggested that c-di-GMP also regulates the formation of electrochemically active biofilms (EABFs) by Shewanella oneidensis MR-1, DGCs involved in this process remained to be identified. Here, we report that the SO_1646 gene, hereafter named dgcS, is upregulated under medium flow conditions in electrochemical flow cells (EFCs), and its product (DgcS) functions as a major DGC in MR-1. In vitro assays demonstrated that purified DgcS catalyzed the synthesis of c-di-GMP from GTP. Comparisons of intracellular c-di-GMP levels in the wild-type strain and a dgcS deletion mutant (ΔdgcS mutant) showed that production of c-di-GMP was markedly reduced in the ΔdgcS mutant when cells were grown in batch cultures and on electrodes in EFCs. Cultivation of the ΔdgcS mutant in EFCs also revealed that the loss of DgcS resulted in impaired biofilm formation and decreased current generation. These findings demonstrate that MR-1 uses DgcS to synthesize c-di-GMP under medium flow conditions, thereby activating biofilm formation on electrodes.IMPORTANCE Bioelectrochemical systems (BESs) have attracted wide attention owing to their utility in sustainable biotechnology processes, such as microbial fuel cells and electrofermentation systems. In BESs, electrochemically active bacteria (EAB) form biofilms on electrode surfaces, thereby serving as effective catalysts for the interconversion between chemical and electric energy. It is therefore important to understand mechanisms for the formation of biofilm by EAB grown on electrodes. Here, we show that a model EAB, S. oneidensis MR-1, expresses DgcS as a major DGC, thereby activating the formation of biofilms on electrodes via c-di-GMP-dependent signal transduction cascades. The findings presented herein provide the molecular basis for improving electrochemical interactions between EAB and electrodes in BESs. The results also offer molecular insights into how Shewanella regulates biofilm formation on solid surfaces in the natural environment.

Keywords: biofilm; cyclic di-GMP; diguanylate cyclase; electrochemically active bacteria; flow cell.

FIG 1

Enzymatic characterization of DgcS. (A) Predicted domain structure of DgcS. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of C-ht-DgcS protein samples. Protein samples (5 μg) were analyzed on 12.5% SDS-polyacrylamide gels. Lanes: C, E. coli BL21(DE3) (pET-C-ht-dgcS) crude extract; M, molecular weight marker; P; purified C-ht-DgcS. (C) Specific DGC activity of E. coli BL21(DE3) (pET-C-ht-dgcS) crude extract and purified C-ht-DgcS. Results are expressed as μmol c-di-GMP produced min⁻¹mg⁻¹ protein. Error bars represent standard deviations calculated from the results of three independent experiments.

FIG 2

Intracellular c-di-GMP contents in S. oneidensis derivatives. Cells were grown under aerobic conditions and harvested at the stationary growth phase. Error bars represent standard deviations calculated from the results of three independent experiments. Asterisks indicate a statistically significant difference (P < 0.05; one-way ANOVA followed by LSD test; ns, not significant).

FIG 3

Time courses of current generation by MR-1_c and the ΔdgcS mutant in EFC. Results and error bars represent the means and standard deviations, respectively, calculated from the results of three independent experiments.

FIG 4

Characterization of the ΔdgcS mutant in EFC. (A) Representative CLSM images of MR-1_c and the ΔdgcS mutant grown in EFC. Vertical (x-z) and horizontal (x-y) images were obtained from EFCs operated for 168 h. Horizontal (x-y) images were obtained at heights of 3 μm and 10 μm from the WE surface (z = 3 μm and 10 μm, respectively). (B) Vertical distribution of AFP signal intensities in CLSM images obtained from EFCs operated for 168 h. (C to G) Protein contents (C), maximum current density normalized to protein contents (D), intracellular c-di-GMP contents (E), polysaccharide contents (F), and ratios of polysaccharide contents to protein contents (G) in WE-associated MR-1_c and ΔdgcS biofilms. Cells were grown in EFCs for 168 h. In all graphs, asterisks indicate a statistically significant difference (P < 0.05; Student's t test; ns, not significant). Error bars represent standard deviations calculated from the results of three independent experiments.