RESUMO
Methanococcus maripaludis type strain JJ (DSM 2067) is an important organism because it serves as a model for primary energy metabolism and hydrogenotrophic methanogenesis and is amenable to genetic manipulation. The complete genome (1.7 Mb) harbors 1,815 predicted protein-encoding genes, including 9 encoding selenoproteins.
RESUMO
BACKGROUND: The archaeon Methanococcus maripaludis strain JJ employs several selenocysteine (Sec)-containing proteins in its primary energy metabolism, methanogenesis. Upon selenium deprivation, or when the pathway for selenoprotein synthesis is disrupted, they are replaced by cysteine (Cys)-containing isoforms, thus allowing for selenium-independent growth. METHODS: Expression of a fusion of the promoter region of frcA (encoding a subunit of the selenium-independent hydrogenase Frc) and bla [encoding ß-lactamase (Bla)] in M. maripaludis JJ was assessed in response to the selenium supply, growth substrate, and growth phase. Random transposon mutants of the reporter strain were screened for deregulated bla expression, which identified HrsM, a LysR-type transcriptional regulator (LTTR). Its involvement in selenium-dependent gene regulation was further assessed by analyzing in vivo transcription, synthesis of selenoproteins and of HrsM, and by analyzing in vitro binding of HrsM to DNA. RESULTS: HrsM, which is not required for selenoprotein synthesis, acts as a positive effector of selenoprotein gene expression and as a negative effector of Cys-encoding isogene expression, but its own expression is independent of the selenium availability. Specific binding in vitro of HrsM to a promoter region under in vivo HrsM control verified its role in selenium-dependent gene regulation. CONCLUSIONS: HrsM exerts a key role in regulating expression of selenoprotein genes and their Cys-encoding isogenes in M. maripaludis in a selenium-dependent fashion. However, this activity is not achieved via autoregulation but probably by a mechanism, which modulates the DNA-binding of HrsM. GENERAL SIGNIFICANCE: Although LTTRs are abundant in Bacteria, HrsM represents only the second characterized member of this group in Archaea.
RESUMO
BACKGROUND: The major biological form of selenium is that of the co-translationally inserted amino acid selenocysteine (Sec). In Archaea, the majority of proteins containing Sec, selenoproteins, are involved in methanogenesis. However, the function of this residue is often not known because selenium-independent homologs of the selenoproteins can be employed, sometimes even in one organism. SCOPE OF REVIEW: This review summarizes current knowledge about the selenoproteins of Archaea, the metabolic pathways where they are involved, and discusses the (potential) function of individual Sec residues. Also, what is known about the "archaeal" way of selenoprotein synthesis, and the regulatory mechanism leading to the replacement of the selenoproteins with selenium-independent homologs, will be presented. Where appropriate, similarities with (and differences to) the respective steps employed in the other two domains, Bacteria and Eukarya, will be emphasized. MAJOR CONCLUSIONS: Genetic and biochemical studies guided by analysis of genome sequences of Sec-encoding archaea has revealed that the pathway of Sec synthesis in Archaea and Eukarya are principally identical and that Sec insertion in Eukarya probably evolved from an archaeal mechanism employed prior to the separation of the archaeal and eukaryal lines of decent. GENERAL SIGNIFICANCE: In light of the emerging close phylogenetic relationship of Eukarya and Archaea, archaeal models may be highly valuable tools for unraveling "eukaryotic" principles in molecular and cell biology.
RESUMO
Six Hyp maturation proteins (HypABCDEF) are conserved in micro-organisms that synthesize [NiFe]-hydrogenases (Hyd). Of these, the HypC chaperones interact directly with the apo-form of the catalytically active large subunit of Hyd enzymes and are believed to transfer the Fe(CN)2CO moiety of the bimetallic cofactor from the Hyp machinery to this large subunit. In E. coli, HypC is specifically required for maturation of Hyd-3 while its paralogue, HybG, is specifically required for Hyd-2 maturation; either HypC or HybG can mature Hyd-1. In this study, we demonstrate that the products of the hypABFCDE operon from the deeply branching hydrogen-dependent and obligate organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 were capable of maturing and assembling active Hyd-1, Hyd-2 and Hyd-3 in an E. coli hyp mutant. Maturation of Hyd-1 was less efficient, presumably because HypB of E. coli was necessary to restore optimal enzyme activity. In a reciprocal maturation study, the highly O2-sensitive H2-uptake HupLS [NiFe]-hydrogenase from D. mccartyi CBDB1 was also synthesized in an active form in E. coli. Together, these findings indicated that HypC from D. mccartyi CBDB1 exhibits promiscuity in its large subunit interaction in E. coli. Based on these findings, we generated amino acid variants of E. coli HybG capable of partial recovery of Hyd-3-dependent H2 production in a hypC hybG double null mutant. Together, these findings identify amino acid regions in HypC accessory proteins that specify interaction with the large subunits of hydrogenase and demonstrate functional compatibility of Hyp accessory protein machineries.
