Disruption and complementation of the selenocysteine biosynthesis pathway reveals a hierarchy of selenoprotein gene expression in the archaeon Methanococcus maripaludis

Abstract

:selenocysteine synthase with the corresponding genes from M. maripaludis S2 restored the mutant's ability to synthesize selenoproteins. However, only partial restoration of the wild-type selenoproteome was observed as only selenocysteine-containing formate dehydrogenase was synthesized. Quantification of transcripts showed that disrupting the pathway of selenocysteine synthesis leads to downregulation of selenoprotein gene expression, concomitant with upregulation of a selenium-independent backup system, which is not re-adjusted upon complementation. This transcriptional arrest was independent of selenophosphate but depended on the 'history' of the mutants and was inheritable, which suggests that a stable genetic switch may cause the resulting hierarchy of selenoproteins synthesized.

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…In contrast, both Ffd variants in Mc. maripaludis are selenoproteins (266); hence, the organism requires the presence of selenium to grow on formate (267,268). Genetic dissection has demonstrated that each homolog confers a competitive growth advantage, with single mutants impaired and double mutants unviable for formatotrophic growth (242).…”

Selenocysteine-independent suppression of UGA codons in the archaeon Methanococcus maripaludis

“…The mutant strains had the same genotypes as reported previously for the selC, pstK, and spcS mutations in the wild type [22] and were, thus, verified. With H 2 +CO 2 as the energy source the …”

“…M. maripaludis strain JJ [20], for which no genome sequence is available, encodes, like strain S2, two sec-containing Fdhs, Fdh1 and Fdh2, but no selenium-independent isoform. While in M. maripaludis JJ fdhA2 is barely expressed under laboratory conditions and cannot complement an inactivated fdhA1 gene [21], fdhA1 is expressed at a high level regardless if the organism grows on H 2 +CO 2 or formate [22].…”

“…Since Fdh is a prerequisite for formate-dependent growth in M. maripaludis [21,42] [22]. Because the other selenoproteins involved in methanogenesis and their cys-containing isoforms are strongly regulated at the transcriptional level in response to the availability of sec, the constitutive expression of fdhA1was interpreted as a means to ensure preferential incorporation of selenium into Fdh, for which no cys-containing "back-up" system is present [22].…”

“…Next, the genomic region encoding FdhA2 (a selenoprotein) and FdhB2 in a putative transcriptional unit was removed from strain J901 using pCRupt∆fdhA2B2 (Table 1) via markerless deletion [41]. Although fdhA2 is expressed in M. maripaludis JJ only at very low levels and does not complement disruptions of the respective isogene fdhA1 [21,22], it was removed to avoid potential ambiguities of results during subsequent analysis of FdhA1. The resulting strain, JdF2 (Table 1) was verified by PCR (Supplementary Figure S1B) and used to replace the 3'-region of the fdhA1 gene with the hexahistidine encoding allele of plasmid pCRuptfdhA1-his (Table 1) via markerless exchange [18].…”