Different Proteins Mediate Step-wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis

Archaeal species encode a variety of distinct lineage-specific chromosomal proteins. We have previously shown that in Thermococcus kodakarensis, histone, Alba, and TrmBL2 play distinct roles in chromosome organization. Although our understanding of individual archaeal chromosomal proteins has been advancing, how archaeal chromosomes are folded into higher-order structures and how they are regulated are largely unknown. Here, we investigated the primary and higher-order structures of archaeal chromosomes from different archaeal lineages. Atomic force microscopy of chromosome spreads out of Thermoplasma acidophilum and Pyrobaculum calidifontis cells revealed 10-nm fibers and 30–40-nm globular structures, suggesting the occurrence of higher-order chromosomal folding. Our results also indicated that chromosome compaction occurs toward the stationary phase. Micrococcal nuclease digestion indicated that fundamental structural units of the chromosome exist in T. acidophilum and T. kodakarensis but not in P. calidifontis or Sulfolobus solfataricus. In vitro reconstitution showed that, in T. acidophilum, the bacterial HU protein homolog HTa formed a 6-nm fiber by wrapping DNA, and that Alba was responsible for the formation of the 10-nm fiber by binding along the DNA without wrapping. Remarkably, Alba could form different higher-order complexes with histone or HTa on DNA in vitro. Mass spectrometry detected HTa in the T. acidophilum chromosome but not in other species. A putative transcriptional regulator of the AsnC/Lrp family (Pcal_1183) was detected on the P. calidifontis chromosome, but not on that of other species studied. Putative membrane-associated proteins were detected in the chromosomes of the three archaeal species studied, including T. acidophilum, P. calidifontis, and T. kodakarensis. Collectively, our data show that Archaea use different combinations of proteins to achieve chromosomal architecture and functional regulation.

Exploring long-range cooperativity in the 20S proteasome core particle from Thermoplasma acidophilum using methyl-TROSY–based NMR

The 20S core particle (CP) proteasome is a molecular assembly catalyzing the degradation of misfolded proteins or proteins no longer required for function. It is composed of four stacked heptameric rings that form a barrel-like structure, sequestering proteolytic sites inside its lumen. Proteasome function is regulated by gates derived from the termini of α-rings and through binding of regulatory particles (RPs) to one or both ends of the barrel. The CP is dynamic, with an extensive allosteric pathway extending from one end of the molecule to catalytic sites in its center. Here, using methyl-transverse relaxation optimized spectroscopy (TROSY)-based NMR optimized for studies of high–molecular-weight complexes, we evaluate whether the pathway extends over the entire 150-Å length of the molecule. By exploiting a number of different labeling schemes, the two halves of the molecule can be distinguished, so that the effects of 11S RP binding, or the introduction of gate or allosteric pathway mutations at one end of the barrel can be evaluated at the distal end. Our results establish that while 11S binding and the introduction of key mutations affect each half of the CP allosterically, they do not further couple opposite ends of the molecule. This may have implications for the function of so-called “hybrid” proteasomes where each end of the CP is bound with a different regulator, allowing the CP to be responsive to both RPs simultaneously. The methodology presented introduces a general NMR strategy for dissecting pathways of communication in homo-oligomeric molecular machines.

The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog

Histones are a principal constituent of chromatin in eukaryotes and fundamental to our understanding of eukaryotic gene regulation. In archaea, histones are widespread but not universal: several lineages have lost histone genes. What prompted or facilitated these losses and how archaea without histones organize their chromatin remains largely unknown. Here, we elucidate primary chromatin architecture in an archaeon without histones, Thermoplasma acidophilum, which harbors a HU family protein (HTa) that protects part of the genome from micrococcal nuclease digestion. Charting HTa-based chromatin architecture in vitro, in vivo and in an HTa-expressing E. coli strain, we present evidence that HTa is an archaeal histone analog. HTa preferentially binds to GC-rich sequences, exhibits invariant positioning throughout the growth cycle, and shows archaeal histone-like oligomerization behavior. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

The Main (Glyco) Phospholipid (MPL) of Thermoplasma acidophilum

The main phospholipid (MPL) of Thermoplasma acidophilum DSM 1728 was isolated, purified and physico-chemically characterized by differential scanning calorimetry (DSC)/differential thermal analysis (DTA) for its thermotropic behavior, alone and in mixtures with other lipids, cholesterol, hydrophobic peptides and pore-forming ionophores. Model membranes from MPL were investigated; black lipid membrane, Langmuir-Blodgett monolayer, and liposomes. Laboratory results were compared to computer simulation. MPL forms stable and resistant liposomes with highly proton-impermeable membrane and mixes at certain degree with common bilayer-forming lipids. Monomeric bacteriorhodopsin and ATP synthase from Micrococcus luteus were co-reconstituted and light-driven ATP synthesis measured. This review reports about almost four decades of research on Thermoplasma membrane and its MPL as well as transfer of this research to Thermoplasma species recently isolated from Indonesian volcanoes.

Characterisation of the First Archaeal Mannonate Dehydratase from Thermoplasma acidophilum and Its Potential Role in the Catabolism of D-Mannose

Mannonate dehydratases catalyse the dehydration reaction from mannonate to 2-keto-3-deoxygluconate as part of the hexuronic acid metabolism in bacteria. Bacterial mannonate dehydratases present in this gene cluster usually belong to the xylose isomerase-like superfamily, which have been the focus of structural, biochemical and physiological studies. Mannonate dehydratases from archaea have not been studied in detail. Here, we identified and characterised the first archaeal mannonate dehydratase (TaManD) from the thermoacidophilic archaeon Thermoplasma acidophilum. The recombinant TaManD enzyme was optimally active at 65 °C and showed high specificity towards D-mannonate and its lactone, D-mannono-1,4-lactone. The gene encoding for TaManD is located adjacent to a previously studied mannose-specific aldohexose dehydrogenase (AldT) in the genome of T. acidophilum. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that the mannose-specific AldT produces the substrates for TaManD, demonstrating the possibility for an oxidative metabolism of mannose in T. acidophilum. Among previously studied mannonate dehydratases, TaManD showed closest homology to enzymes belonging to the xylose isomerase-like superfamily. Genetic analysis revealed that closely related mannonate dehydratases among archaea are not located in a hexuronate gene cluster like in bacteria, but next to putative aldohexose dehydrogenases, implying a different physiological role of mannonate dehydratases in those archaeal species.

Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production

Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in Nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene pathway (ADH) serve to leverage consecutive kinases to convert exogenous alcohols to pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway, isopentenyl phosphate kinase from Thermoplasma acidophilum, towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide non-natural pyrophosphates and provide a critical first step in characterizing the rate limiting steps in the artificial ADH pathway.<br>