Biochemical characterization of archaeal homocitrate synthase from Sulfolobus acidocaldarius.

The hyperthermophilic archaeon, Sulfolobus, synthesizes lysine via the α-aminoadipate pathway; however, the gene encoding homocitrate synthase, the enzyme responsible for the first and committed step of the pathway, has not yet been identified. In the present study, we identified saci_1304 as the gene encoding a novel type of homocitrate synthase fused with a Regulation of Amino acid Metabolism (RAM) domain at the C terminus in Sulfolobus acidocaldarius. Enzymatic characterization revealed that Sulfolobus homocitrate synthase was inhibited by lysine; however, the mutant enzyme lacking the RAM domain was insensitive to inhibition by lysine. The present results indicated that the RAM domain is responsible for enzyme inhibition.

Characterization of two ß-decarboxylating dehydrogenases from Sulfolobus acidocaldarius.

Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two ß-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg2+, thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.

The one-component system ArnR: a membrane-bound activator of the crenarchaeal archaellum.

Linking the motility apparatus to signal transduction systems enables microbes to precisely control their swimming behaviour according to environmental conditions. Bacteria have therefore evolved a complex chemotaxis machinery, which has presumably spread through lateral gene transfer into the euryarchaeal subkingdom. By contrast Crenarchaeota encode no chemotaxis-like proteins but are nevertheless able to connect external stimuli to archaellar derived motility. This raises fundamental questions about the underlying regulatory mechanisms. Recently, we reported that the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius becomes motile upon nutrient starvation by promoting transcription of flaB encoding the filament forming subunits. Here we describe two transcriptional activators as paralogous one-component-systems Saci_1180 and Saci_1171 (ArnR and ArnR1). Deletions of arnR and arnR1 resulted in diminished flaB expression and accordingly the deletion mutants revealed impaired swimming motility. We further identified two inverted repeat sequences located upstream of the flaB core promoter of S. acidocaldarius. These cis-regulatory elements were shown to be critical for ArnR and ArnR1 mediated flaB gene expression in vivo. Finally, bioinformatic analysis revealed ArnR to be conserved not only in Sulfolobales but also in the crenarchaeal order of Desulfurococcales and thus might represent a more general control mechanism of archaeal motility.

Insights into FlaI functions in archaeal motor assembly and motility from structures, conformations, and genetics.

Superfamily ATPases in type IV pili, type 2 secretion, and archaella (formerly archaeal flagella) employ similar sequences for distinct biological processes. Here, we structurally and functionally characterize prototypical superfamily ATPase FlaI in Sulfolobus acidocaldarius, showing FlaI activities in archaeal swimming-organelle assembly and movement. X-ray scattering data of FlaI in solution and crystal structures with and without nucleotide reveal a hexameric crown assembly with key cross-subunit interactions. Rigid building blocks form between N-terminal domains (points) and neighboring subunit C-terminal domains (crown ring). Upon nucleotide binding, these six cross-subunit blocks move with respect to each other and distinctly from secretion and pilus ATPases. Crown interactions and conformations regulate assembly, motility, and force direction via a basic-clamp switching mechanism driving conformational changes between stable, backbone-interconnected moving blocks. Collective structural and mutational results identify in vivo functional components for assembly and motility, phosphate-triggered rearrangements by ATP hydrolysis, and molecular predictors for distinct ATPase superfamily functions.

Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus.

LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.

Diversity, assembly and regulation of archaeal type IV pili-like and non-type-IV pili-like surface structures.

Archaea have evolved fascinating surface structures allowing rapid adaptation to changing environments. The archaeal surface appendages display such diverse biological roles as motility, adhesion, biofilm formation, exchange of genetic material and species-specific interactions and, in turn, increase fitness of the cells. Intriguingly, despite sharing the same functions with their bacterial counterparts, the assembly mechanism of many archaeal surface structures is rather related to assembly of bacterial type IV pili. This review summarizes our state-of-the-art knowledge about unique structural and biochemical properties of archaeal surface appendages with a particular focus on archaeal type IV pili-like structures. The latter comprise not only widely distributed archaella (formerly known as archaeal flagella), but also different highly specialized archaeal pili, which are often restricted to certain species. Recent findings regarding assembly mechanisms, structural aspects and physiological roles of these type IV pili-like structures will be discussed in detail. Recently, first regulatory proteins involved in transition from both planktonic to sessile lifestyle and in assembly of archaella were identified. To conclude, we provide novel insights into regulatory mechanisms underlying the assembly of archaeal surface structures.

Regulation of archaella expression by the FHA and von Willebrand domain-containing proteins ArnA and ArnB in Sulfolobus acidocaldarius.

The ability of microorganisms to sense and respond to sudden changes in their environment is often based on regulatory systems comprising reversible protein phosphorylation. The archaellum (former: archaeal flagellum) is used for motility in Archaea and therefore functionally analogous to the bacterial flagellum. In contrast with archaellum-mediated movement in certain members of the Euryarchaeota, this process, including its regulation, remains poorly studied in crenarchaeal organisms like Sulfolobus species. Recently, it was shown in Sulfolobus acidocaldarius that tryptone limiting conditions led to the induction of archaella expression and assembly. Here we have identified two proteins, the FHA domain-containing protein ArnA and the vWA domain-containing protein ArnB that are involved in regulating archaella expression in S. acidocaldarius. Both proteins are phosphorylated by protein kinases in vitro and interact strongly in vivo. Phenotypic analyses revealed that these two proteins are repressors of archaella expression. These results represent the first step in understanding the networks that underlie regulation of cellular motility in Crenarchaeota and emphasize the importance of protein phosphorylation in the regulation of cellular processes in the Archaea.

Versatile Genetic Tool Box for the Crenarchaeote Sulfolobus acidocaldarius.

For reverse genetic approaches inactivation or selective modification of genes are required to elucidate their putative function. Sulfolobus acidocaldarius is a thermoacidophilic Crenarchaeon which grows optimally at 76°C and pH 3. As many antibiotics do not withstand these conditions the development of a genetic system in this organism is dependent on auxotrophies. Therefore we constructed a pyrE deletion mutant of S. acidocaldarius wild type strain DSM639 missing 322 bp called MW001. Using this strain as the starting point, we describe here different methods using single as well as double crossover events to obtain markerless deletion mutants, tag genes genomically and ectopically integrate foreign DNA into MW001. These methods enable us to construct single, double, and triple deletions strains that can still be complemented with the pRN1 based expression vector. Taken together we have developed a versatile and robust genetic tool box for the crenarchaeote S. acidocaldarius that will promote the study of unknown gene functions in this organism and makes it a suitable host for synthetic biology approaches.

Molecular analysis of the crenarchaeal flagellum.

The ability to move towards favourable conditions provides fundamental advantages to organisms. Interestingly, flagella as motility structures evolved independently in the bacterial and the archaeal kingdom. Whereas bacterial flagella have been intensively studied, our knowledge regarding the archaeal counterpart is mostly restricted to Euryarchaeota rather than crenarchaeal flagella. We therefore investigated the flagellar assembly system of the crenarchaeal model organism Sulfolobus acidocaldarius in vivo. Promoter studies and qRT-PCR analyses of the flagella gene cluster provided evidence that the expression of the fla genes was induced by tryptone starvation. Moreover, we confirmed presence of a secondary fla promoter within the flaB gene that regulates the transcription of downstream genes flaX-J. Markerless in-frame deletions for all fla genes encoded in the fla gene cluster were constructed. Western blot analysis of all fla deletion strains suggested hierarchical protein interactions during the archaeal flagella assembly. Moreover, functional analysis by thermomicroscopy revealed non-motile cells for each of the mutant strains. Electron micrographs demonstrated that lack of motility coincided with the loss of flagellar assembly. Thus we demonstrated that all seven fla genes are essential for crenarchaeal flagellum assembly and function.