Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii

Proteasomes are composed of 20S core particles (CPs) of α- and β-type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeon Haloferax volcanii as a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α1 and α2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including α1 Thr147, α2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α1, thus, revealing a new type of proteasomal modification. Probing the biological role of α1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α1 variants including Thr147Ala, Thr158Ala and Ser58Ala. An H. volcanii Rio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α1. The α1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation

Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii

Background: Thiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms. Results: Based on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant. Conclusions: Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis

Engineering Thermotolerant Biocatalysts for Biomass Conversion to Products

Lignocellulosic biomass is a promising feedstock for producing renewable chemicals and transportation fuels as petroleum substitutes. Fermentation of the cellulose in biomass in an SSF process requires that the properties of the microbial biocatalyst match the fungal cellulase activity optima for cost-effective production of products. Fermentation of the pentose sugars derived from hemicellulose in biomass is an additional asset of an ideal biocatalyst. The microbial biocatalyst used by the industry, yeast, lacks the ability to ferment pentose sugars. The optimum temperature for growth and fermentation of yeast is about 35°C. The optimum temperature for commercially available cellulase enzymes for depolymerization of cellulose in biomass to glucose for fermentation is 50-55 °C. Because of the mismatch in the temperature optima for the enzyme and yeast, SSF of cellulose to ethanol (cellulosic ethanol) with yeast is conducted at a temperature that is close to the optimum for yeast. We have shown that by increasing the temperature of SSF to 50-55 °C using thermotolerant B. coagulans, the amount of cellulase required for SSF of cellulose to products can be reduced by 3-4 –fold compared to yeast-based SSF at 35°C with a significant cost savings due to lower enzyme loading. Thermotolerant Bacillus coagulans strains ferment hemicellulose-derived pentose sugars completely to L(+)-lactic acid, the primary product of fermentation. We have developed genetic tools to engineer B. coagulans for fermentation of all the sugars in biomass to ethanol. Using these tools, we have altered the fermentation properties of B. coagulans to produce ethanol as the primary product. The thermotolerant property of B. coagulans has been shown to also lower the cellulase requirement and associated cost in SSF of cellulose to lactic acid compared to lactic acid bacteria. Lactic acid is a potential petroleum substitute for bio-based renewable plastics production. This study has led to the development of B. coagulans as a thermotolerant microbial biocatalyst for production of ethanol as a transportation fuel and lactic acid as a starting material for bio-based plastics in a cost-effective manner from renewable biomass

A widespread riboswitch candidate that controls bacterial genes involved in molybdenum cofactor and tungsten cofactor metabolism

We have identified a highly conserved RNA motif located upstream of genes encoding molybdate transporters, molybdenum cofactor (Moco) biosynthesis enzymes, and proteins that utilize Moco as a coenzyme. Bioinformatics searches have identified 176 representatives in γ-Proteobacteria, δ-Proteobacteria, Clostridia, Actinobacteria, Deinococcus-Thermus species and DNAs from environmental samples. Using genetic assays, we demonstrate that a Moco RNA in Escherichia coli associated with the Moco biosynthetic operon controls gene expression in response to Moco production. In addition, we provide evidence indicating that this conserved RNA discriminates against closely related analogues of Moco. These results, together with extensive phylogenetic conservation and typical gene control structures near some examples, indicate that representatives of this structured RNA represent a novel class of riboswitches that sense Moco. Furthermore, we identify variants of this RNA that are likely to be triggered by the related tungsten cofactor (Tuco), which carries tungsten in place of molybdenum as the metal constituent

Evidence That Two ATP-Dependent (Lon) Proteases in Borrelia burgdorferi Serve Different Functions

The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH2-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host

Uncovering Ubiquitin and Ubiquitin-like Signaling Networks

Microscopic imaging and technolog

Characterization of the cdhD and cdhE genes encoding subunits of the corrinoid/iron-sulfur enzyme of the CO dehydrogenase complex from Methanosarcina thermophila.

The CO dehydrogenase enzyme complex from Methanosarcina thermophila contains a corrinoid/iron-sulfur enzyme composed of two subunits (delta and gamma). The cdhD and cdhE genes, which encode the delta and gamma subunits, respectively, were cloned and sequenced. The cdhD gene is upstream of and separated by 3 bp from cdhE. Both genes are preceded by apparent ribosome-binding sites. Northern (RNA) blot and primer extension analyses indicated that cdhD and cdhE are cotranscribed from a promoter located several kilobases upstream of cdhD. The putative CdhD and CdhE sequences are 37% identical to the sequences deduced from the genes encoding the beta and alpha subunits of the corrinoid/iron-sulfur enzyme from Clostridium thermoaceticum. The CdhE sequence had a four-cysteine motif with the potential to bind a 4Fe-4S cluster previously identified in the corrinoid/iron-sulfur enzyme by electron paramagnetic resonance spectroscopy. A T7 RNA polymerase/promoter system was used to produce CdhD and CdhE independently in Escherichia coli. The purified CdhD protein was reconstituted with hydroxocobalamin in the base-on configuration. The purified CdhE protein exhibited an Fe-S center and base-off cobalamin binding in which the benzimidazole base nitrogen atom was no longer a lower axial ligand to the cobalt atom

Subunit Topology of Two 20S Proteasomes from Haloferax volcanii

Haloferax volcanii, a halophilic archaeon, synthesizes three different proteins (α1, α2, and β) which are classified in the 20S proteasome superfamily. The α1 and β proteins alone form active 20S proteasomes; the role of α2, however, is not clear. To address this, α2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant H. volcanii. The α2 protein copurified with α1 and β in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described α1-β proteasome. Supplementing buffers with 10 mM CaCl(2) stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis. This facilitated the discovery that wild-type H. volcanii synthesizes more than one type of 20S proteasome. Two 20S proteasomes, the α1-β and α1-α2-β proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the α1-β proteasome was a symmetrical cylinder with α1 rings on each end. In contrast, the α1-α2-β proteasome appeared to be asymmetrical with homo-oligomeric α1 and α2 rings positioned on separate ends. Inter-α-subunit contacts were only detected when the ratio of α1 to α2 was perturbed in the cell using recombinant technology. These results support a model that the ratio of α proteins may modulate the composition and subunit topology of 20S proteasomes in the cell