Mmp10 is required for post-translational methylation of arginine at the active site of methyl-coenzyme M reductase

Catalyzing the key step for anaerobic methane production and oxidation, methyl-coenzyme M reductase or Mcr plays a key role in the global methane cycle. The McrA subunit possesses up to five post-translational modifications (PTM) at its active site. Bioinformatic analyses had previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To examine its role, MMP1554, the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool, resulting in the specific loss of the 5-(S)-methylarginine PTM of residue 275 in the McrA subunit and a 40~60 % reduction in the maximal rates of methane formation by whole cells. Methylation was restored by complementations with the wild-type gene. However, the rates of methane formation of the complemented strains were not always restored to the wild type level. This study demonstrates the importance of Mmp10 and the methyl-Arg PTM on Mcr activity.

S1). Then 0.5~1 kb of upstream and downstream sequences of the target gene were PCR 147 amplified with the same overhangs at the 3'-end of the upstream and 5'-end of the downstream 148 sequence, respectively. The PCR products were digested with SfiI and ligated to create the initial 149 construct, which was either used directly for transformation of M. maripaludis or PCR amplified 150 to increase the amount of DNA. The p5L-R procedure (Fig. S3) (Table S2 and Fig. S4). In contrast, the peptide containing the methyl-Arg PTM was not 179 observed in the enzyme from the deletion strain S0030, even though the unmodified RR form of 180 the partial digestion product increased to 8 % of the fully digested peptide (Table S2 and Fig. 181 S5). Tryptic digestions tend to be incomplete at dibasic sites such as RR, RK and KK (29, 30). 182 Thus, removal of the Arg modification was expected to increase the rate of partial cleavage as 183 observed. In the Mcr from the complementation strain S0031, the unmodified peptide was not 184 observed, and the fraction of partially digested peptide was reduced again to 4 %, as seen in the 185 wild-type (Table S2 and Fig. S6). 186 While these results suggested that mmp10 was required for the PTM of Arg 275 , the partial 187 digestion by trypsin made quantification of the extent of modification difficult. For that reason, a 188 pepsin degradation was developed that yielded the peptide PGRr 275 ARGPNEPGGIRF and 189 enabled unambiguous quantification of the methyl-Arg 275 . Based on peak area integration, Me-more than 96-98% of the Arg 275 was methylated in the complementation and wild type strains 192 ( Table 2 and Fig. S8 to S10). These results confirmed that mmp10 was required for the Arg 275 193 methylation.

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Growth phenotype of ∆mmp10 mutants 196 The growth of the ∆mmp10 deletion strain S0030 was severely inhibited compared to the 197 wild type. Not only did the lag phase increase, but the growth rate was reduced from 0.27 + 0.03 198 h -1 (average + standard deviation, n = 3) in the wild-type to 0.17 ± 0.03 h -1 in S0030 (Fig. 3). 199 Complementation of the deletion failed to restore wild-type growth. Strain S0031 had a 200 prolonged lag and its growth rate was further reduced to 0.14 + 0.01 h -1 . While strain S0034 201 grew somewhat better than the deletion mutant, the lag was longer and the growth rate, 0.19 ± 202 0.03 h -1 , was still slower than wild type. To examine if the levels of Mcr expression were 203 different among the strains, whole cell extracts were separated by SDS PAGE (Fig. 4). Although  0.13 by One-way ANOVA), and they were 13.6 ± 1.8 % (average and standard deviation of three 208 measurements, S0001, the wild type), 14.2 ± 1.2 % (S0030, the Δ mutant), 14.5 ± 1.0 % (S0034, 209 the PhmvA-mmp10 complement) and 12.6 ± 1.5 % (S0031, the Pnat -mmp10 complement). The 210 similar SDS profiles among the strains also suggested similar expression levels for most other 211 proteins (Fig. 4). Thus, the ∆mmp10 deletion and complementation did not appear to affect the 212 levels of Mcr. However, one band at the position of ~25 kDa showed substantial increase in intensity in 214 strain S0031 (p < 0.0001 by One-way ANOVA). The relative abundances were 2.0 ± 0.1 % 215 (average and standard deviation of three measurements, S0001), 1.6 ± 0.5 % (S0030), 2.8 ± 0.2 216 % (S0034) and 7.8 ± 0.7 % (S0031), respectively. MALDI sequencing analysis of the band from 217 S0031 revealed that it was the puromycin N-acetyltransferase, which was encoded on the 218 complementation plasmid and provided puromycin resistance.

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The relative transcription rates of the Pnat and PhmvA promoters were also examined 220 using an mCherry reporter system in strains S0032 and S0033, respectively. The mCherry 221 fluorescence for S0032 was around 30-fold lower than S0033, i.e., arbitrary fluorescence values 222 OD -1 mL -1 were 19.3 ± 1.5 (average and standard deviation of three measurements) versus 618 ± 223 40, respectively. Therefore, the differences in growth and methanogenesis observed between 224 strains S0031 and S0034 could at least in part be attributed to the differences in expressional 225 levels of mmp10 between the two strains.

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Collectively, these results suggested that the poor growth of the complementation strains 227 was due to pleiotropic effects unrelated to the mmp10 deletion. To test this hypothesis, the 228 complementation plasmids were transformed into the wild type strain S0001, resulting in strains 229 S0035 and S0036. Both strains grew poorly in comparison to the wild type and ∆mmp10 deletion 230 strains (Fig. 5A). Moreover, the inhibition was even more profound than the corresponding 231 complementation strains S0031 and S0034 in the ∆mmp10 background (Fig. 5B). Thus, the poor 60% of the wild type rate. To confirm that these results were not due to fluctuations in the 244 medium composition during growth, the rates of methanogenesis were also measured in resting 245 cells after washing and resuspension in fresh medium (Fig. 6B), where a 60% reduction in the 246 maximal rates of methanogenesis was observed in the ∆mmp10 strain S0030. These results 247 confirmed that the rate of methanogenesis was severely impaired in the ∆mmp10 mutant.

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In the complementation strains, the rate of methanogenesis was restored to the wild-type 249 level in strain S0034, which possessed mmp10 under the control of the strong PhmvA promoter, 250 but the rate of methanogenesis for S0031, which possessed the Pnat promoter, was similar to that 251 of the deletion mutant ( Fig. 6A and 6B). In a separate experiment, the rates of methanogenesis 252 for strains S0035 and S0036, which possessed the complementation plasmids in the wild-type 253 background, were comparable to the wild-type level (data not shown). Pmcr is also used to drive expression of puromycin N-transacetylase in the pac cassette of the 300 complementation plasmids, and strain S0031 contained three-fold higher levels of this protein than S0034, which contained PhmvA. Thus, the poor growth and methanogenesis of strain S0031 302 could have been a consequence of the high expression of puromycin N-acetyltransferase. 303 Although the rate of methanogenesis in the PhmvA complementation strain S0034 was 304 similar to that of wild type, the growth was somewhat slower. Because Mmp10 is probably  Importantly, hydrogenase is a key enzyme in methanogenesis, and this gene is likely essential for 312 growth (33, 38, 39). Therefore, spurious methylation of MMP0140 might well inhibit growth, 313 and further investigation will be needed to address this possibility. Plasmids and PCR primers are listed in Table 3 and Table S3. Cloning was performed in 339 E. coli Top10, and selection was conducted with 100 µg mL -1 ampicillin. Genomic DNA  primers 1554-F/R to confirm complete removal of the markers (Fig S11A). This conclusion was 352 further confirmed by the sensitivity of the mutant to puromycin and the inability of primers 353 1554w-F/R, which target an internal region of mmp10, to yield a product. As a positive control,

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PCR amplification under the same conditions detected 0.1% of wild-type DNA when mixed with 355 the mutant DNA (Fig S11B). 356 Expression plasmid vectors pM10 and p4MK10 were made to complement the ∆mmp10 357 in strain S0030, producing strains S0031 and S0034, respectively. The same two plasmids were 358 also transformed into the wild-type strain S0001, resulting in strains S0035 and S0036,      instead of the conserved acidic E residue (Fig. S1). The legend for each structural feature is