Different outer membrane c‐type cytochromes are involved in direct interspecies electron transfer to Geobacter or Methanosarcina species

Abstract Direct interspecies electron transfer (DIET) may be most important in methanogenic environments, but mechanistic studies of DIET to date have primarily focused on cocultures in which fumarate was the terminal electron acceptor. To better understand DIET with methanogens, the transcriptome of Geobacter metallireducens during DIET‐based growth with G. sulfurreducens reducing fumarate was compared with G. metallireducens grown in coculture with diverse Methanosarcina. The transcriptome of G. metallireducens cocultured with G. sulfurreducens was significantly different from those with Methanosarcina. Furthermore, the transcriptome of G. metallireducens grown with Methanosarcina barkeri, which lacks outer‐surface c‐type cytochromes, differed from those of G. metallireducens cocultured with M. acetivorans or M. subterranea, which have an outer‐surface c‐type cytochrome that serves as an electrical connect for DIET. Differences in G. metallireducens expression patterns for genes involved in extracellular electron transfer were particularly notable. Cocultures with c‐type cytochrome deletion mutant strains, ∆Gmet_0930, ∆Gmet_0557 and ∆Gmet_2896, never became established with G. sulfurreducens but adapted to grow with all three Methanosarcina. Two porin–cytochrome complexes, PccF and PccG, were important for DIET; however, PccG was more important for growth with Methanosarcina. Unlike cocultures with G. sulfurreducens and M. acetivorans, electrically conductive pili were not needed for growth with M. barkeri. Shewanella oneidensis, another electroactive microbe with abundant outer‐surface c‐type cytochromes, did not grow via DIET. The results demonstrate that the presence of outer‐surface c‐type cytochromes does not necessarily confer the capacity for DIET and emphasize the impact of the electron‐accepting partner on the physiology of the electron‐donating DIET partner.

In addition to apparent differences in extracellular electron transfer mechanisms utilized by G. metallireducens for growth via DIET with different electron accepting partners, a number of other genes involved in Geobacter physiology were also differentially expressed.Some of these differences are described here in Supplementary Material.

Differential expression of quinone oxidoreductase complexes
Studies of G. sulfurreducens have shown that putative quinone oxidoreductase complexes composed of proteins that contain a transmembrane diheme b-type cytochrome domain fused or adjacent to a multi-heme c-type cytochrome localized to the inner membrane are differentially expressed in response to changes in the reduction potential of the extracellular electron acceptor (1)(2)(3)(4).These cytochrome bc (Cbc) complexes are highly conserved among Geobacter (5,6) and are also present in the G. metallireducens genome (7).Many of these Cbc complex genes were differentially expressed by G. metallireducens when grown in various co-culture conditions.The gene coding for CbcL (Gmet_0100) was >2.96 times more highly expressed in co-cultures with G. sulfurreducens than it was in any of the Methanosarcina co-cultures (Table 1).In G.
sulfurreducens, CbcL was found to be important for electron transfer to extracellular electron acceptors with low reduction potentials such as insoluble Fe(III) oxide or an electrode poised at a potential of -0.1 V (3).Genes from another operon that codes for a Cbc quinone oxidoreductase complex composed of 5 proteins; 3 periplasmic c-type cytochromes cbcD, cbcA and cbcC (Gmet_2931, Gmet_2928, Gmet_2930), an inner membrane b-type cytochrome cbcB (Gmet_2929), and a membrane protein cbcE (Gmet_2932) were also much more highly expressed in co-cultures with G. sulfurreducens than co-cultures with Methanosarcina (Table 1).Transcriptomic and genetic studies showed that the CbcABCDE complex is important for Fe(III) oxide reduction by G. sulfurreducens (8), however, deletion of cbcA and cbcC in G. metallireducens did not affect co-culture growth with any of the partners (this study).
CbcSTU is another Cbc complex that had high transcript abundance in G. sulfurreducens cells grown with Fe(III) oxide ( 8) that has two homologs in G. metallireducens (Gmet_0325-0327 and Gmet_3518-3520).Genes for both of the CbcSTU complexes were also more highly expressed by G. metallireducens cells grown in co-culture with G. sulfurreducens than in co-culture with Methanosarcina (Table 1), but they were not differentially expressed by G. metallireducens cells grown with Fe(III) oxide compared to Fe(III) citrate (9).
Transcriptomic and genetic studies also showed that the CbcWXV complex was important for Fe(III) oxide reduction by G. metallireducens, G. sulfurreducens, and G. uraniireducens (8,9), and genes from this complex were most highly expressed in cocultures with M. barkeri (Table 2).
G. metallireducens cells grown with Type II Methanosarcina had high transcript abundance for genes from a Cbc complex consisting of a b-type cytochrome (cbcP; Gmet_0539), a transmembrane protein (cbcQ; Gmet_0533), an iron-sulfur cluster binding protein (cbcO; Gmet_0538), and three c-type cytochromes (cbcM; Gmet_0536), (cbcN; Gmet_0537) and (cbcR; Gmet_0534) (Table 3).Transcriptomic studies showed that genes from this complex were important for Fe(III) oxide reduction by G. sulfurreducens and G. metallireducens (8,9), however, deletion of cbcR did not impact Fe(III) oxide reduction by G. metallireducens (9) and also did not affect co-culture growth with any of the partners (this study).
The G. metallireducens genome also has a cluster of genes (pcmABCDEFG) that code for another putative quinol-cytochrome bc complex without a homolog in G. sulfurreducens that is found in an operon with genes coding for proteins involved in paracresol degradation (10)(11)(12).The PcmABCDEFG complex contains genes for an inner membrane protein (Gmet_2117), two inner membrane associated b-type cytochromes (pcmC and pcmD; Gmet_2119-2120), two iron sulfur cluster proteins (pcmB, pcmE; Gmet_2118, Gmet_2121), and two periplasmic c-type cytochrome proteins (pcmF and pcmG, Gmet_2122-2123) that were all more highly expressed in co-cultures with M. barkeri (Table 2).
Other genes that were more highly expressed by G. metallireducens grown in coculture with G. sulfurreducens Transcript levels for genes for the flagellar filament protein, FliC, many of the flagellar biosynthesis proteins and the carbon storage regulator protein A (CsrA), which positively regulates flagellar synthesis (13), were most abundant in co-cultures with G. sulfurreducens (Supplementary Table S6).This high expression of G. metallireducens flagellar components during growth with G. sulfurreducens can be explained by the fact that G. sulfurreducens strain PCA does not produce a functional flagellum (14) and motility helps with the formation of aggregates.Flagella, however, have been observed on M. acetivorans (15).Although they are not directly involved in electron transfer, studies have shown that Geobacter flagella are important for growth on extracellular electron acceptors.Motility is needed for Geobacter to access insoluble electron acceptors, such as insoluble Fe(III) oxide, electrodes, or other organisms for participation in DIET.In fact, G. metallireducens strains lacking fliC were not capable of growth when insoluble Fe(III) oxide was provided as an electron acceptor (16) and it has been proposed that Geobacter flagella can serve as biofilm matrix scaffolds that enhance biofilm conductivity on current harvesting anodes (17).Deletion of fliC significantly increased the amount of time needed for establishment of aggregates with all DIET partners (this study).However, once aggregates became established, growth was similar to the wild-type co-cultures.
Consistent with the finding that many more genes coding for c-type cytochromes were more highly expressed by G. metallireducens grown in co-culture with G. sulfurreducens (Figure 2), genes coding for heme biogenesis proteins were also more highly expressed in the GM/GS co-culture (Supplementary Table S3).L-glutamate is a precursor for siroheme biosynthesis (18) and glutamate biosynthesis genes such as NADP dependent glutamate dehydrogenase (gdhA; Gmet_1186) and ferredoxin dependent glutamate synthase (gltS; Gmet_0147) were also much more highly expressed in GM/GS co-cultures than any of the Methanosarcina co-cultures (Supplementary Table S3C).In addition, genes coding for cysteine were more highly expressed in GM/GS co-cultures (Supplementary Table S3D), likely because synthesis of multi-heme cytochrome c holoproteins require high concentrations of cysteine because each heme group is covalently attached to two cysteine residues (19).
Genes involved in sulfate/sulfur assimilation and transport were among the most highly expressed genes in GM/GS co-cultures.In fact, genes for operons coding for sulfate/thiosulfate transport system proteins (cysPTWA; Gmet_1903-1906) and sulfatesulfur assimilation proteins (cysDN; Gmet_2859-2860) were 42-731 and 4-15 times more highly expressed in GM/GS co-cultures than those with Methanosarcina (Supplementary Table S3E).
Transcript abundance for iron uptake and metabolism genes was also higher in GM/GS co-cultures.For example, the number of transcripts for the gene that codes for the iron transport protein, FeoB (Gmet_2444) was >3 times more highly expressed in GM/GS co-cultures than those with Methanosarcina (Supplementary Table S3F).
Genes involved in synthesis of ferredoxin were more highly expressed in GM/GS co-cultures.The genes for cysteine desulfurase (iscS; Gmet_0872) and its transcriptional regulator (iscR; Gmet_0871) were 10 to 36 times more highly transcribed in GM/GS cocultures (Supplementary Table S3G).In addition, the gene coding for the chaperone involved in maturation of iron-sulfur cluster containing proteins, HscA (Gmet_3532) and numerous genes coding for proteins with iron-sulfur binding domains were more highly expressed in GM/GS co-cultures (Supplementary Table S2).
Transcriptomic analyses revealed that the composition of the G. metallireducens cell membrane was slightly different when it was grown via DIET with G. sulfurreducens compared to growth with any of the Methanosarcina species.G. metallireducens membranes are known to contain hopanoid lipids (20), which are important for bacterial membrane stability and functioning (21).Genes for hopene biosynthesis were more highly expressed in GM/GS co-cultures (Supplementary Table S3H).Studies have shown that the presence of the hopanoid lipid, hopene, lowers membrane ion permeability, membrane fluidity and membrane conductance (20,22) and this might be important for electron transfer to other Geobacter species.However, further investigation into this possibility is required.

Differences between Type I and Type II Methanosarcina
Aside from differences in quinol oxidoreductase complex proteins, many of the electron transport genes more highly expressed in GM/MB co-cultures than in other coculture conditions coded for periplasmic c-type cytochromes (Table 2).These included Gmet_1703 and Gmet_2156, which code for periplasmic 7-and 9-heme cytochromes, a periplasmic tri-heme cytochrome, ppcF (Gmet_0335), and 5 periplasmic di-heme cytochromes, ppcB (Gmet_3166), ccpB (Gmet_1210), ppcE (Gmet_1846), narC (Gmet_0328), and ppcA (Gmet_2902).The only one of these c-type cytochromes that was more highly expressed by G. metallireducens during growth on Fe(III) oxide was Gmet_2156 (9).A ccpB homolog in G. sulfurreducens codes for a cytochrome c peroxidase protein that protects the cell from reactive oxygen species (23), and Ppc cytochromes are periplasmic electron transporters involved in Fe(III) respiration by G. sulfurreducens (24,25).
Genes coding for several c-type cytochrome proteins were also more highly expressed in Type II Methanosarcina compared to M. barkeri (Table 3).These included a periplasmic monoheme cytochrome (Gmet_0232), the periplasmic di-heme cytochrome peroxidase CcpA (Gmet_3091) (23), and Gmet_0679, which codes for a 5-heme cytochrome with unknown localization.Gmet_0679 was highly expressed in Fe(III) oxide grown G. metallireducens cells, but gene deletion studies revealed that it was not essential for Fe(III) oxide reduction (9).Deletion of Gmet_0679 and Gmet_0232 from G. metallireducens also did not impact growth via DIET with any of the electron-accepting partners (this study).
Genes coding for proteins involved in the biosynthesis of menaquinone and ubiquinone (26,27), lipid soluble electron carriers involved in electron shuttling across the inner membrane, were most highly expressed by G. metallireducens grown via DIET with Type II Methanosarcina (Supplementary Table S3I).The gene coding for UbiA, which codes for a 4-hydroxy benzoate octaprenyl transferase thought to be a key enzyme of ubiquinone biosynthesis (26) was more than ~4 times more highly expressed in Type II Methanosarcina co-cultures.
Genes for biosynthesis of the coenzymes cobalamin, folate, molybdopterin, and thiamine were more significantly expressed by G. metallireducens in Type II Methanosarcina co-cultures (Supplementary Table S3J-M).Methanosarcina make several methanogenesis pathway enzymes that require such cofactors (28)(29)(30) and studies have shown that cross-feeding of vitamins like cobalamin are common in interspecies symbiotic partnerships (31)(32)(33)(34).It is possible that G. metallireducens makes higher concentrations of these coenzymes in the presence of Type II Methanosarcina because it participates in higher rates of cross-feeding with these methanogens than with the other electron accepting partners.Evidence for increased cross-feeding to Type II Methanosarcina is provided by the finding that expression of btuB (Gmet_1245) which codes for an outer membrane cobalamin transporter (35), was >5 and >23 fold higher in Type II Methanosarcina-GM co-cultures than GM/GS and GM/MB co-cultures (Supplementary Table S2).

Similarities in expression of non-cytochrome genes during DIET
A gene just upstream from Gmet_0930, Gmet_0933, was among the most highly expressed genes in all 4 conditions (Supplementary Table S1).Gmet_0933 codes for a protein that shares many similarities to Bap (biofilm-associated protein) proteins, which are large secreted proteins required for biofilm formation in various bacteria (36,37).
Similar to previously characterized Bap proteins, Gmet_0933 has a number of tandem repeats (24 repeats), contains two cadherin like domains, an EF-hand calcium binding motif, a serine rich region, and 6 immunoglobulin like domains.Gmet_0933 also contains 7 mirror beta grasp (MBG) domains that are associated with bacterial surface proteins (38).Another gene (Gmet_2043) that codes for a large and highly repetitive (15 tandem repeats) outer surface associated protein that is potentially involved in biofilm formation was also highly transcribed by G metallireducens in all 4 co-culture conditions (Supplementary Table S1).It has five SdrD (SD-repeat containing protein D) B-like domains, which are found in cell surface adhesion proteins (39).
The gene coding for OmpJ (Gmet_3254) was the most highly expressed membrane associated protein in all 4 co-cultures (Supplementary Table S1).It is homologous to an outer membrane porin found in G. sulfurreducens that is indirectly required for Fe(III) oxide reduction (40).It serves as a putative porin that helps stabilize the integrity of the periplasmic space necessary for proper folding and functioning of periplasmic and outer membrane electron transport components.Another gene coding for an outer membrane porin protein, OmpA (Gmet_0342) was also among the most highly expressed genes in all 4 conditions.The gene coding for OmpA was also highly expressed by G. sulfurreducens and G. metallireducens cells during growth in the presence of Fe(III) (9,41), and it has been shown to play a role in biofilm formation (42).Almost all of the genes from an operon (Gmet_2022-2033) that codes for proteins involved in production of exopolysaccharides and lipopolysaccharides were also highly expressed by G. metallireducens in all 4 conditions.Lipopolysaccharides (LPS) are important for bacterial adherence to surfaces during biofilm formation in many Gramnegative bacteria (43).Transcriptomic and genetic studies showed that Gmet_2029 which codes for a putative lipopolysaccharide synthesis protein was critical for Fe(III) oxide reduction (9), and it was important for DIET in all 4 co-cultures.In fact, G. metallireducens strains in which Gmet_2029 was deleted were unable to form aggregates with G. sulfurreducens.Establishment of co-culture growth with Methanosarcina species was significantly delayed and once co-cultures were established they continued to grow 2.2 (p=0.002),2.81 (p=0.0003), and 3.4 (p=0.009)times slower in ∆Gmet_2029/MB, ∆Gmet_2029/MA, and ∆Gmet_2029/MS co-cultures than the wild-type co-cultures after 4 transfers (this study).
Supplementary TableS6.Fold differences for genes coding for flagellin and flagellum assembly proteins in G. metallireducens in cocultures grown with G. sulfurreducens (GS) compared to co-cultures grown with the 3 different Methanosarcina species (M.barkeri (MB); M. acetivorans (MA); M. subterranea (MS)).p-valuesareavailable in Supplementary TableS2but all comparisons shown have p-values that are <0.05.NS: no significant difference