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: Proteins containing selenocysteine (sec) are found in Bacteria, Eukarya, and Archaea. While selenium-dependence of methanogenesis from H(2)+CO(2) in the archaeon Methanococcus maripaludis JJ is compensated by induction of a set of cysteine-containing homologs, growth on formate is abrogated in the absence of sec due to the dependence of formate dehydrogenase (Fdh) on selenium. Despite this dependence, formate-dependent growth occurs after prolonged incubation of M. maripaludis mutants lacking sec. METHODS: To study this phenomenon, a M. maripaludis strain with only one Fdh isoform and an FdhA selenoprotein C-terminally tagged for affinity enrichment was constructed. Factors required for sec synthesis were deleted in this strain and translation of UGA in fdhA was analyzed physiologically, enzymatically, immunologically, and via mass spectrometry. RESULTS: M. maripaludis JJ mutants lacking sec synthesis grew at least five times more slowly than the wild type on formate due to a 20-35-fold reduction of Fdh activity. The enzyme in the mutant strains lacked sec but was still produced as a full-length protein. Peptide mass spectrometry revealed that both cysteine (cys) and tryptophan (trp) were inserted at the UGA encoding sec without apparent mutations in tRNA(cys) or tRNA(trp), respectively. CONCLUSIONS: We demonstrate that M. maripaludis has the inherent capacity to translate UGA with cys and trp; other mechanisms to replace sec with cys in the absence of selenium could thereby be ruled out. GENERAL SIGNIFICANCE: This study exemplifies how an organism uses the inherent flexibility in its canonical protein synthesis machinery to recover some activity of an essential selenium-dependent enzyme in the absence of sec.
Assuntos
Códon , Formiato Desidrogenases/fisiologia , Mathanococcus/genética , Selenocisteína/fisiologia , Sequência de Aminoácidos , Dados de Sequência Molecular , Biossíntese de ProteínasRESUMO
Methane is a key intermediate in the carbon cycle and biologically produced by methanogenic archaea. Most methanogens are able to conserve energy by reducing CO2 to methane using molecular hydrogen as electron donor (hydrogenotrophic methanogenesis), but several hydrogenotrophic methanogens can also use formate as electron donor for methanogenesis. Formate dehydrogenase (Fdh) oxidizes formate to CO2 and is involved in funneling reducing equivalents into the methanogenic pathway, but details on other factors relevant for formate-dependent physiology of methanogens are not available. To learn more about the factors involved in formate-dependent growth of Methanococcus maripaludis strain JJ, we used a recently developed system for random in vitro mutagenesis, which is based on a modified insect transposable element to create 2,865 chromosomal transposon mutants and screened them for impaired growth on formate. Of 12 M. maripaludis transposon-induced mutants exhibiting this phenotype, the transposon insertion sites in the chromosome were mapped. Among the genes, apparently affecting formate-dependent growth were those encoding archaeal transcription factor S, a regulator of ion transport, and carbon monoxide dehydrogenase/acetyl-CoA synthase. Interestingly, in seven of the mutants, transposons were localized in a 10.2 kb region where Fdh1, one of two Fdh isoforms in the organism, is encoded. Two transcription start sites within the 10.2 kb region could be mapped, and quantification of transcripts revealed that transposon insertion in this region diminished fdhA1 expression due to polar effects.
