RESUMEN
DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and also occur in select archaea. In the model archaeon Haloferax volcanii, previous work implicated GlpR, a DeoR-type transcriptional regulator, in the transcriptional repression of glpR and the gene encoding the fructose-specific phosphofructokinase (pfkB) during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here, we compared transcriptomes of wild-type and ΔglpR mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with in vivo and in vitro DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes involved in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under the indirect influence of GlpR. In vitro experiments demonstrated that GlpR purifies to function as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that H. volcanii GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators.IMPORTANCE Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents the broad use of archaea as microbial factories for industrial products. Here, we characterize how sugar uptake and use are regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in this archaeal species functions using molecular mechanisms conserved with distantly related bacterial species.
Asunto(s)
Proteínas Arqueales/genética , Fructosa/metabolismo , Regulación de la Expresión Génica Arqueal , Haloferax volcanii/genética , Proteínas Represoras/genética , Proteínas Arqueales/metabolismo , Regulación Enzimológica de la Expresión Génica , Glucosa/metabolismo , Glicerol/metabolismo , Haloferax volcanii/enzimología , Redes y Vías Metabólicas , Mutación , Regulón , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Glycerol is a primary energy source for heterotrophic haloarchaea and a major component of "salty" biodiesel waste. Glycerol is catabolized solely by glycerol kinase (encoded by glpK) to glycerol-3-phosphate (G3P) in Haloferax volcanii. Here we characterized the next critical step of this metabolic pathway: the conversion of G3P to dihydroxyacetone phosphate by G3P dehydrogenase (G3PDH). H. volcanii harbors two putative G3PDH operons: (i) glpA1B1C1, located on the chromosome within the neighborhood of glpK, and (ii) glpA2B2C2, on megaplasmid pHV4. Analysis of knockout strains revealed that glpA1(and not glpA2) is required for growth on glycerol. However, both glpA1 and glpA2 could complement a glpA1 knockout strain (when expressed from a strong promoter in trans) and were required for the total G3PDH activity of cell lysates. The glpA1B1C1, glpK, glpF(encoding a putative glycerol facilitator), and ptsH2(encoding a homolog of the bacterial phosphotransferase system protein Hpr) genes were transcriptionally linked and appeared to be under the control of a strong, G3P-inducible promoter upstream of glpA1. Overall, this study provides fundamental insights into glycerol metabolism in H. volcanii and enhances our understanding of central metabolic pathways of haloarchaea.
Asunto(s)
Proteínas Arqueales/genética , Cromosomas de Archaea/genética , Glicerol/metabolismo , Glicerolfosfato Deshidrogenasa/genética , Haloferax volcanii/genética , Proteínas Arqueales/metabolismo , Cromatografía Líquida de Alta Presión , ADN de Archaea/genética , Regulación de la Expresión Génica Arqueal , Técnicas de Inactivación de Genes , Genes Arqueales , Glicerolfosfato Deshidrogenasa/metabolismo , Haloferax volcanii/efectos de los fármacos , Haloferax volcanii/enzimología , Plásmidos , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
This review identifies the various pathways responsible for modulating hepatic protein synthesis following acute and chronic alcohol intoxication and describes the mechanism(s) responsible for these changes. Alcohol intoxication induces a defect in global protein synthetic rates that is localized to impaired translation of mRNA at the level of peptide-chain initiation. Translation initiation is regulated at two steps: formation of the 43S preinitiation complex [controlled by eukaryotic initiation factors 2 (eIF2) and 2B (eIF2B)] and the binding of mRNA to the 40S ribosome (controlled by the eIF4F complex). To date, alcohol-induced alterations in eIF2 and eIF2B content and activity are best investigated. Ethanol decreases eIF2B activity when ingested either acutely or chronically. The reduced eIF2B activity most likely is a consequence of twofold increased phosphorylation of the alpha-subunit of eIF2 on Ser(51) following acute intoxication. The increase in eIF2alpha phosphorylation after chronic alcohol consumption is the same as that induced by acute ethanol intoxication, and protein synthesis is not further reduced by long-term alcohol ingestion despite additional reduced expression of initiation factors and elongation factors. eIF2alpha phosphorylation alone appears sufficient to maximally inhibit hepatic protein synthesis. Indeed, pretreatment with Salubrinal, an inhibitor of eIF2alpha(P) phosphatase, before ethanol treatment does not further inhibit protein synthesis or increase eIF2alpha phosphorylation, suggesting that acute ethanol intoxication causes maximal eIF2alpha phosphorylation elevation and hepatic protein synthesis inhibition. Ethanol-induced inhibition of hepatic protein synthesis is not rapidly reversed by cessation of ethanol consumption. In conclusion, sustained eIF2alpha phosphorylation is a hallmark of excessive alcohol intake leading to inhibition of protein synthesis. Enhanced phosphorylation of eIF2alpha represents a unique response of liver to alcohol intoxication, because the ethanol-induced elevation of eIF2alpha(P) is not observed in skeletal muscle or heart.