Glucose Metabolism and Acetate Switch in Archaea: the Enzymes in Haloferax volcanii

Abstract

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. Following our previous studies on key enzymes of this pathway, we now focus on the characterization of enzymes involved in 3-phosphoglycerate conversion to pyruvate, in anaplerosis and in acetyl-CoA formation from pyruvate. These enzymes include phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenolpyruvate carboxylase and pyruvate: ferredoxin oxidoreductase. The essential function of these enzymes were shown by transcript analyses and growth experiments with respective deletion mutants. Further, it is shown that H. volcanii - during aerobic growth on glucose - excreted significant amounts of acetate, which was consumed in the stationary phase (acetate switch). The enzyme catalyzing the conversion of acetyl-CoA to acetate as part of the acetate overflow mechanism, an ADP-forming acetyl-CoA synthetase (ACD), was characterized. The functional involvement of ACD in acetate formation and of AMP-forming acetyl-CoA synthetases (ACSs) in activation of excreted acetate was proven by using respective deletion mutants. Together, the data provide a comprehensive analysis of enzymes of the spED pathway, of anaplerosis, and report first genetic evidence of the functional involvement of enzymes of the acetate switch in archaea. Importance: In this work we provide a comprehensive analysis of glucose degradation via the semiphosphorylative Entner-Doudoroff pathway in the haloarchaeal model organism Haloferax volcanii. The study includes transcriptional analyses, growth experiments with deletion mutants and characterization of all enzymes involved in the conversion of 3-phosphoglycerate to acetyl-CoA and in anaplerosis. Phylogenetic analyses of several enzymes indicate various lateral gene transfer events from bacteria to haloarchaea. Further, we analyzed the key players involved in the acetate switch, i.e in the formation (overflow) and subsequent consumption of acetate during aerobic growth on glucose. Together, the data provide novel aspects on glucose degradation, anaplerosis and acetate switch in H. volcanii and thus expand our understanding of the unusual sugar metabolism in archaea.

Isolation of a virus causing a chronic infection in the archaeal model organism Haloferax volcanii reveals antiviral activities of a provirus

“…On the other hand, several genes that showed significant up-regulation, with more than a 30-fold increase in expression, are related to energy production and conversion (i.e., HVO_B0291, HVO_2411, HVO_1083, and HVO_2251) and two universal stress proteins (i.e., HVO_2337 and HVO_0401) ( Dataset S2 ). Also, an acetate-CoA ligase (HVO_1000), which is a key enzyme that catalyzes the recycling of acetate to acetyl-CoA, to compensate starvation during stationary phase in H. volcanii ( 85 ), is threefold up-regulated. The data suggest that, upon infection with HFPV-1, the infected cells are forced to fulfill at least the energy requirements of the machinery dedicated to virus production.…”

TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii

“…Genes in this cluster are enriched for functions involved in amino acid transport (FDR adjusted p < 2.78 × 10 −2 , hypergeometric test), carbohydrate transport ( p < 2.16 × 10 −16 ), and lipid transport and metabolism ( p < 1.57 × 10 −05 ). Notably, this cluster includes three paralogous genes encoding acetyl coA synthase that were previously shown to be involved in acetate secretion ( acs1 [HVO_0894], acs2 [HVO_0896], and acs4 [HVO_1374] Kuprat et al., 2021). Cluster 2 contains four genes: HVO_0034, encoding an acyl‐CoA thioesterase; two genes encoding DMT family putative carbohydrate transporters (HVO_1791 and HVO_B0276); and HVO_2375, encoding a component of a putative phosphate ABC transporter.…”

“…Because algJ expression is not glucose dependent, it is unlikely the sole gene underlying ∆tbsP hypermotility. Comparison with proteomics data enabled the identification of several candidates, namely acs2 and acs4 , involved in long‐chain fatty acid synthesis (Kuprat et al., 2021). However, none exhibit expression patterns concordant with the observed phenotypes (Figure S5).…”

Open Issues for Protein Function Assignment in Haloferax volcanii and Other Halophilic Archaea

“…Additionally, a conditional lethal porAB mutant was unable to grow on glucose or pyruvate, thus excluding that alternative enzymes for the conversion of pyruvate to acetyl-CoA exist in Hfx. volcanii [ 22 ]. Nonetheless, despite experimental results to the contrary, pyruvate has been assigned as a substrate for some of the homologs of the pyruvate dehydrogenase complex in KEGG (as of April 2021).…”

“…Its genome has been sequenced and carefully annotated [ 1 , 10 , 11 ]. A plethora of biological aspects have been successfully tackled in this species, with examples including DNA replication [ 4 ]; cell division and cell shape [ 12 , 13 , 14 , 15 , 16 ]; metabolism [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ]; protein secretion [ 26 , 27 , 28 , 29 ]; motility and biofilms [ 30 , 31 , 32 , 33 , 34 , 35 ]; mating [ 36 ]; signaling [ 37 ]; virus defense [ 38 ]; proteolysis [ 39 , 40 , 41 , 42 , 43 , 44 ]; posttranslational modification (N-glycosylation; SAMPylation) [ 45 , 46 , 47 , 48 , 49 , 50 ]; gene regulation [ 21 , 25 , 51 , 52 , 53 , 54 , 55 ]; microproteins [ 56 , 57 , 58 ] and small noncoding RNAs (sRNAs) […”