Analysis of a Ms mazei ∆fpoO mutant

Subunit FpoO of the Fpo complex in Ms. mazei is a small, hydrophilic 15 kDa protein encoded by the last gene in the fpo operon (Figure 17). FpoO can also be found in the close relatives Ms. acetivorans and Ms. barkeri and other members of the Methanosarcinales like Methanohalobium evestigatum, Methanolobus tindarius or Methanococcoides burtonii.

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However, FpoO does not have a counterpart in the well-studied Nuo or the Fqo complex. Furthermore, protein blast analysis revealed no homology to any other protein characterized so far. Therefore, the function of FpoO in the Fpo complex is unclear. One [2Fe2S] cluster is predicted with Prosite, which is in accordance with the fact that 1.5 nmol of non-heme iron and 1.6 nmol of acid labile sulfur per nmol protein were detected in a purified, recombinant FpoO protein (Hofmann, 2003). This points to a possible involvement in electron transport processes. The Fpo complex is distinguished from homologous protein complexes by the fact that it transfers electrons to methanophenazine (MP), which is a membrane-integral cofactor specific for methanogens. Therefore, FpoO might be involved in electron transfer from F420H2

to MP.

As a first approach to evaluate the role of FpoO in the metabolism of Ms. mazei, growth experiments were performed with a mutant lacking fpoO (mutant constructed by Cornelia Welte, Radboud Universiteit Nijmegen, The Netherlands). In this mutant, the gene encoding FpoO is replaced by a puromycin resistance cassette (Figure 18).

Figure 18: Structure of the fpo operon in the Ms. mazei ∆fpoO mutant. The gene encoding fpoO was

substituted by a puromycin resistance cassette (pac).

It has to be mentioned here, that in Ms. barkeri the Fpo complex was demonstrated to play a minor role in F420H2 oxidation. Mutants lacking the fpo operon did not show a phenotype

when they were cultivated with methanol as a substrate. It was found that instead of the Fpo complex, the F420-reducing hydrogenase is used to produce H2 from F420H2. H2 is re-oxidized

by the Vho hydrogenase and electrons are channeled into the respiratory chain (Kulkarni et al., 2009). However, in Ms. mazei the Fpo complex is much more important. It was demonstrated that in mutants lacking the whole Fpo complex or subunit FpoF, F420H2

oxidation was severely impaired. In growth experiments with TMA as a substrate, the wild type had a doubling time of 7.7 h and mutants missing the whole complex or subunit FpoF showed a prolonged doubling time of 11.1 h and 11.3 h, respectively (Welte and Deppenmeier, 2011a). Hence, in a Ms. mazei mutant missing FpoO, a severe growth deficiency should be measurable if the loss of FpoO had consequences for energy conservation in methylotrophic methanogenesis.

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TMA or acetate, and the optical density at 600 nm (OD600) was measured. As a control, the

wild type strain was used. Growing on complex medium with TMA, the wild type had a doubling time of 6.5 ± 0.5 h. The doubling time of the Ms. mazei ∆fpoO mutant was 6.7 ± 0.7 h (Figure 19). Hence, the difference between mutant and wild type was in the range of standard deviation. Another aspect of growth experiments is the final OD600. If a mutant

reaches a lower final OD600 than the wild type but consumes the same amount of substrate,

this points to less efficient energy conservation. However, in this experiment the final OD600

reached by Ms. mazei wt and ∆fpoO mutant was similar (Figure 19). Thus, the same amount of biomass was produced from the same amount of substrate and there was no evidence for an impaired energy conservation system in the mutant.

Figure 19: Growth curves for Ms. mazei wild type (■) and Ms. mazei ∆fpoO mutant (□). TMA was used

as a substrate. There were no significant differences in doubling time and final OD600 between the

mutant and wild type.

To exclude the possibility that a slight defect in the mutant was overlooked due to rich nutrient supply in the complex medium, growth experiments were performed in minimal medium. Again, TMA was used as a substrate. Doubling time in the wild type and mutant was slightly increased (8.6 h for the mutant, 7.9 h for the wild type), probably due to limited supply of biosynthetic precursors. Yet, again no significant difference in doubling time between Ms. mazei ∆fpoO mutant and wild type could be observed. Thus, using TMA as a substrate, growth of Ms. mazei was not impaired by the lack of FpoO.

In contrast to methylotrophic methanogenesis where reducing equivalents are transferred to F420 and Fd, in aceticlastic methanogenesis only Fdred is generated. It has been proposed

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Deppenmeier, 2011a, b, 2014). Therefore, acetate was used as substrate to investigate a possible role of FpoO in Fdred oxidation without interference of F420H2. As already mentioned

above, the F420H2-oxidizing subunit FpoF can dissociate from the complex and is active in the

cytoplasm as F420:Fd oxidoreductase. One hypothesis was that FpoO might be responsible

for the interaction between FpoF and the membrane-associated subunits FpoBCDI. FpoO could serve as redox sensor to recognize the level of Fdred. Thus, if the Fd pool becomes fully

reduced in aceticlastic methanogenesis, FpoO could mediate dissociation of FpoF from the Fpo complex. Subsequently, other subunits of the Fpo complex would be accessible for Fdred

and the complex could thus contribute to Fdred oxidation. Moreover, cytoplasmic FpoF could

transfer electrons to F420 and F420H2 would act as a temporary electron sink. These two

mechanisms would guarantee a steady supply of Fd for methanogenesis and allow a higher acetate turnover rate and thus faster growth. Therefore, if FpoO indeed acts as a trigger for the dissociation of FpoF, a phenotype with decreased doubling time could be expected for the Ms. mazei ∆fpoO mutant.

Figure 20: Growth curves for Ms. mazei wild type (■) and Ms. mazei ∆fpoO mutant (□). Acetate was

used as a substrate. There was no significant difference in doubling time for mutant and wild type.

Using acetate as substrate, doubling time increased to 35.4 h for the wild type and 36.5 h for the mutant. The overall increase in doubling time is due to the fact that acetate is a poor substrate for methanogenesis. Yet, again there was no difference in doubling time between the Ms. mazei ∆fpoO mutant and the wild type (Figure 20).

The lack of a phenotype in the growth experiments led to the conclusion that subunit FpoO does not have an important role in energy conservation in methylotrophic or aceticlastic methanogenesis. However, to gain deeper insight into electron transport in the

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Ms. mazei ∆fpoO mutant, enzymatic assays were performed with membrane fractions of the mutant and the wild type. The functionality of the Fpo complex itself was measured in a test with F420H2 as electron donor and the artificial electron acceptors methylviologen

(MV)/metronidazol (MTZ). MV/MTZ can accept electrons directly from the Fpo complex and thus no other membrane-bound enzymes are involved in the respective assay. The enzymatic activity of membrane fractions was 40 mU mg-1 _{for the mutant, which is in the}

range of literature values for the wild type ((Welte and Deppenmeier, 2011c), Table 12). Thus, the Fpo complex was functional without subunit FpoO.

Table 12: Activity of membrane fractions of Ms. mazei wt and the ∆fpoO mutant.

Electron acceptor Ms. mazei wt Ms. mazei ∆fpoO

MV/MTZ1 _{50 mU/mg}2 _{40 mU/mg}

Heterodisulfide 65 mU/mg 60 mU/mg

F420H2 was used as electron donor. Electrons were transferred either to MV/MTZ1 or to the

heterodisulfide. 1_{: Methylviologen / metronidazol;} 2_{: (Welte and Deppenmeier, 2011c)}

Another test was performed using F420H2 as electron donor and the heterodisulfide as

electron acceptor. In this test electrons are transferred from the Fpo complex to the membrane-bound heterodisulfide reductase (Hdr). The electron transfer is mediated by MP, therefore in this experiment the possible role of FpoO in electron transfer to MP was tested. Yet, similar enzymatic activities were measured in the wild type and in the mutant strain (65 mU mg-1 _{in the wild type, 60 mU mg}-1 _{for the mutant, Table 12). Hence, it could be shown}

that subunit FpoO most likely is not involved in electron transfer to MP.

To sum up, a Ms. mazei ∆fpoO mutant was employed to investigate the role of subunit FpoO from the Fpo complex. Due to the presence of one [2Fe2S] cluster it was feasible that FpoO is involved in electron transfer to MP. In growth experiments, the mutant did not show a clear phenotype in comparison to the wild type. Additionally, electron transfer to MP was tested with enzymatic activity assays in membrane fractions of mutant and wild type. Again, no phenotype was observed. It was concluded that electron transfer processes are not impaired by the lack of FpoO. It was furthermore conceivable that FpoO might serve as a redox sensor that facilitates dissociation of subunit FpoF from the Fpo complex, making other subunits accessible for Fdred. Growth experiments were performed with acetate as a

substrate, as in aceticlastic methanogenesis, reducing equivalents are transferred solely to Fd. However, no phenotype was observed in growth experiments performed with acetate as a substrate.

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