Tuning Gene Expression by Phosphate in the Methanogenic Archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a rapidly growing, hydrogenotrophic, and genetically tractable methanogen with unique capabilities to convert formate and CO2 to CH4. The existence of genome-scale metabolic models and an established, robust system for both large-scale and continuous cultivation make it amenable for industrial applications. However, the lack of molecular tools for differential gene expression has hindered its application as a microbial cell factory to produce biocatalysts and biochemicals. In this study, a library of differentially regulated promoters was designed and characterized based on the pst promoter, which responds to the inorganic phosphate concentration in the growth medium. Gene expression increases by 4- to 6-fold when the medium phosphate drops to growth-limiting concentrations. Hence, this regulated system decouples growth from heterologous gene expression without the need for adding an inducer. The minimal pst promoter is identified and contains a conserved AT-rich region, a factor B recognition element, and a TATA box for phosphate-dependent regulation. Rational changes to the factor B recognition element and start codon had no significant impact on expression; however, changes to the transcription start site and the 5' untranslated region resulted in the differential protein production with regulation remaining intact. Compared to a previous expression system based upon the histone promoter, this regulated expression system resulted in significant improvements in the expression of a key methanogenic enzyme complex, methyl-coenzyme M reductase, and the potentially toxic arginine methyltransferase MmpX.

The Nbp35/ApbC homolog acts as a nonessential [4Fe-4S] transfer protein in methanogenic archaea.

The nucleotide binding protein 35 (Nbp35)/cytosolic Fe-S cluster deficient 1 (Cfd1)/alternative pyrimidine biosynthetic protein C (ApbC) protein homologs have been identified in all three domains of life. In eukaryotes, the Nbp35/Cfd1 heterocomplex is an essential Fe-S cluster assembly scaffold required for the maturation of Fe-S proteins in the cytosol and nucleus, whereas the bacterial ApbC is an Fe-S cluster transfer protein only involved in the maturation of a specific target protein. Here, we show that the Nbp35/ApbC homolog MMP0704 purified from its native archaeal host Methanococcus maripaludis contains a [4Fe-4S] cluster that can be transferred to a [4Fe-4S] apoprotein. Deletion of mmp0704 from M. maripaludis does not cause growth deficiency under our tested conditions. Our data indicate that Nbp35/ApbC is a nonessential [4Fe-4S] cluster transfer protein in methanogenic archaea.

Methanogenesis.

Methanogenesis is an anaerobic respiration that generates methane as the final product of metabolism. In aerobic respiration, organic matter such as glucose is oxidized to CO2, and O2 is reduced to H2O. In contrast, during hydrogenotrophic methanogenesis, H2 is oxidized to H+, and CO2 is reduced to CH4. Although similar in principle to other types of respiration, methanogenesis has some distinctive features: the energy yield is very low, ≤1 ATP per methane generated, and only methanogens - organisms capable of this specialized metabolism - carry out biological methane production. Methanogens, like the process they catalyze, are similarly distinctive. Methanogens are comprised exclusively of archaea. They are obligate methane producers, that is, they do not grow using fermentation or alternative electron acceptors for respiration. Finally, methanogens are strict anaerobes and do not grow in the presence of O2. Historically, methanogenesis has been viewed as a highly specialized metabolism restricted to a narrow group of prokaryotes. However, recent developments have revealed enormous diversity within the methanogens and suggest that this metabolism is one of the most ancient on earth.