Fig 1. Deletion of the gene encoding TRAM0076 reduces the growth rate of Methanococcus maripaludis . Homologous recombination replaced the MMP0076 gene of M . maripaludis S0001 with the pac cassette encoding puromycin resistance, and produced strain Δ 0076 . (A) Deletion of MMP0076 significantly reduces the growth rate at 37˚C from 0.15 ± 0.001 h -1 of the parental strain (WT-S0001) to 0.065 ± 0.003 h -1 of the mutant (MT- Δ 0076 ). The MMP0076 complementation ( MMP0076 -com) restored the growth of the mutant. Growth at 37˚C was followed by monitoring OD600nm, and the average and standard deviation from triplicate cultures are shown. (B) Western blotting assays of the TRAM0076 abundance throughout the entire growth phase. Total cellular protein (7 μ g) from the cultures at early (7h)-, middle (11h)- and late-exponential (15h), and stationary (17h) growth phases was used for Western blotting with the antibody against TRAM0076, and 9 ng of purified recombinant His 6 -tagged TRAM0076 protein was included as reference (recombinant). (C) Growth of the MMP0076 deletion mutant ( Δ 0076 ) was also impaired at 22˚C. The growth rates of M . maripaludis S0001 and the Δ 0076 mutant were 0.042 ± 0.002 h -1 and 0.023 ± 0.001 h -1 , respectively. (D) Cold-shock induced expression of TRAM0076. A mid-exponential phase culture of strain S0001 grown at 37˚C was incubated at 4˚C for up to 7 hours. TRAM0076 protein abundance was assayed by Western blotting as in Fig