Crucial roles of intracellular cyclic di-GMP in impacting the genes important for extracellular electron transfer by Geobacter metallireducens

Abstract

To investigate the roles of intracellular c-di-GMP in bacterial extracellular electron transfer (EET), three Geobacter metallireducens strains with high (Gme-H), intermediate (Gme-C), and low (Gme-L) intracellular levels of c-di-GMP were constructed via the synthetic biology approach. Compared to Gme-C, Gme-H showed similar Fe(III) reduction rates, formed thicker biofilms on conductive and nonconductive surfaces, and produced more electricity, but showed delayed ability for electricity production. Gme-L formed thinner biofilms on nonconductive surfaces and reduced Fe(III)-citrate faster, but showed slower reduction of ferrihydrite in comparison to Gme-C. Although it produced electricity much faster, Gme-L produced less electricity and formed slightly less amounts of biofilms on anodes, as compared to Gme-C. The mRNA levels of multiple genes encoding c-type cytochromes (c-Cyts) and extracellular pilin protein PilA-N were differentially regulated in Gme-L or Gme-H in comparison to that in Gme-C. Expressions of the genes for PilA-N and extracellular c-Cyt Gmet2896 were increased by high c-di-GMP. Low c-di-GMP increased the gene expressions of the porin-cytochromes in the outer membrane. Further investigation also identified new c-di-GMP-regulated genes directly involved in the EET of G. metallireducens, such as those for the c-Cyts of extracellular Gmet0601, the periplasmic Gmet1703 and Gmet1809 on the cytoplasmic membrane, as deletions of these genes impaired bacterial reductions of extracellular ferrihydrite and anode. Thus, intracellular c-di-GMP impacted multiple genes of G. metallireducens whose protein products might transfer electrons from the cytoplasmic membrane, through the periplasm, across the outer membrane to and in the extracellular environment.IMPORTANCEBis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is ubiquitous in bacterial cells where it regulates a variety of bacterial processes, which range from biofilm formation, bacterial virulence to cell cycle progression. However, its role in regulating bacterial extracellular electron transfer is much less characterized. This investigation shows the crucial roles of intracellular c-di-GMP in impacting the extracellular electron transfer of the Gram-negative bacterium Geobacter metallireducens. The gene expressions of the multiheme c-type cytochromes in the bacterial cytoplasmic membrane, periplasm, outer membrane, and extracellular environment, as well as the gene expression of extracellular pilin protein PilA-N, are all impacted by c-di-GMP. Although how it impacts the expression of these genes is currently unclear, c-di-GMP affects the entire extracellular electron transfer process of G. metallireducens from the cytoplasmic membrane, through the periplasm and across the outer membrane to and in the extracellular environment.

Keywords: Fe(III)-reduction; Geobacter metallireducens; biofilms; cyclic di-GMP; extracellular electron transfer; microbial fuel cells.

Fig 1

Intracellular levels of c-di-GMP (A) and cGAMP (B) in G. metallireducens (Gme) strains grown under Fe(III)-citrate respiring conditions. All results are reported as mean and standard deviation (n = 3). *, 0.01 < P < 0.05; **, 0.001 < P < 0.01, ***P < 0.001. Gme-L, strain with the plasmid containing a c-di-GMP-degrading gene; Gme-C, strain with the empty vector; Gme-H, strain with the plasmid containing a c-di-GMP-synthesizing gene.

Fig 2

Characterization of the G. metallireducens strains with different intracellular levels of c-di-GMP. (A) Biofilm formation. Shown are the absorbance of crystal violet (OD₅₇₀) extracted from stained biofilms on non-conductive surfaces after 60 hours of growth. All results are reported as mean and standard deviation (n = 4). (B) Fe(III)-citrate reduction. (C) Ferrihydrite reduction. (D) Electricity production. (E) Maximum current density. (F) Amounts of proteins detected in the biofilms on anodes during maximum electricity production. All results are reported as mean and standard deviation (n = 3). For points with no error bar, the error was smaller than the size of the symbol. *0.01 < P < .05; **0.001 < P < .01, ***P < 0.001. (G–I) CLSM images of biofilms on anodes for the bacterial strains Gme-L (G), Gme-C (H), and Gme-H (I).

Fig 3

Transcriptomic and immunoblot analyses of the biofilms of G. metallireducens strains with different intracellular levels of c-di-GMP on anodes during maximum electricity production. (A) Mean-difference (MD) plots for the comparison of differentially expressed genes between Gme-L and Gme-C. (B) MD plots for the comparison of differentially expressed genes between Gme-H and Gme-C. (C) The heatmaps of differentially expressed genes in Gme-L and Gme-H. (D) PilA-N, Gmet2896, Gmet0825, Gmet0913, and DnaK proteins were detected in the biofilms on anodes with their respective antibodies. The migration positions of standard proteins (Stds) in kilodaltons (kDa) are shown on the right. (E) The relative abundance of PilA-N, Gmet2896, Gmet0825, Gmet0913, and DnaK proteins in the biofilms on anodes. NS, P > 0.05; *0.01 < P < 0.05; **0.001 < P < 0.01, ***P < 0.001.

Fig 4

Characterizations of the newly identified genes regulated by intracellular c-di-GMP in G. metallireducens. (A) Fe(III)-citrate reduction. (B) Ferrihydrite reduction. (C) Complementation. Shown are the results on the 12th day after the reduction of ferrihydrite. EV, empty vector; C, complement; NC, no cell control. The amount of 100% Fe(II) produced was 35.48 ± 0.45 mM (n = 3). (D) Electricity production. (E) Maximum current density. (F) Amounts of proteins detected in the biofilms on anodes during maximum electricity production. All results are reported as mean and standard deviation (n = 3). For points with no error bar, the error was smaller than the size of the symbol. Asterisks indicate significance levels of difference between mutant and wild-type strains. NS, P > 0.05; *0.01 < P < .05; **0.001 < P < .01, ***P < 0.001. (G–J) Biofilms on anodes of wild type (WT), ΔGmet0601 (Δ1), ΔGmet1703 (Δ2), and ΔGmet1809 (Δ3) after 30 hours of growth.