Identification of Binding Partners of CsaA - An Archaeal Chaperonic Protein of Picrophilus torridus.

BACKGROUND: CsaA is among the few chaperones which are present in both bacteria and archaea, but absent in eukaryotes. There are no reports on interactome analysis of CsaA from archaea, till date. Identification of binding partners of CsaA might be helpful in understanding CsaA-associated processes in Picrophilus torridus an extreme thermoacidophilic euryarchaeon. OBJECTIVES: The present study was conducted to identify the binding partners of CsaA of P. torridus (PtCsaA). METHODS: The binding partners of PtCsaA were isolated and identified using a pull down assay and liquid chromatography-mass spectrometry (LC-MS). RESULTS: The results revealed twelve potential binding partners of CsaA. These were thermosome subunits (Q6KZS2 and Q6L132), nascent polypeptide-associated complex protein (Q6L1N3), elongation factor 1-alpha (Q6L202), uncharacterized protein (Q6L0Y6), citrate synthase (Q6L0M8), asparaginyl- tRNA synthetase (Q6L0M5), succinyl-CoA synthetase beta chain (Q6L0B4), pyruvate ferredoxin oxidoreductase alpha and beta chain proteins (Q6KZA7 and Q6KZA6, respectively), malate dehydrogenase (Q6L0C3) and reversed fumarylacetoacetase (Q6KZ97). Functional categorization revealed that of these, six proteins were involved in energy metabolic pathways, three were archaeal chaperones, two were involved in translation and one might be a transcription regulator. STRING-based analysis of the protein-protein interactions of the experimental interactome revealed strong interactions among them. CONCLUSION: PtCsaA might be a multifaceted protein which besides translation might also play important role in metabolic processes of P. torridus. However, further experiments investigating the binding partners of CsaA in other archaea are required for a better understanding of CsaA-associated processes in archaea.

Structural basis for unique color tuning mechanism in heliorhodopsin.

Microbial rhodopsins comprise an opsin protein with seven transmembrane helices and a retinal as the chromophore. An all-trans retinal is covalently bonded to a lysine residue through the retinal Schiff base (RSB) and stabilized by a negatively charged counterion. The distance between the RSB and counterion is closely related to the light energy absorption. However, in heliorhodopsin-48C12 (HeR-48C12), while E107 acts as the counterion, E107D mutation exhibits an identical absorption spectrum to the wild-type, suggesting that the distance does not affect its absorption spectra. Here we present the 2.6 Å resolution crystal structure of the Thermoplasmatales archaeon HeR E108D mutant, which also has an identical absorption spectrum to the wild-type. The structure revealed that D108 does not form a hydrogen bond with the RSB, and its counterion interaction becomes weaker. Alternatively, the serine cluster, S78, S112, and S238 form a distinct interaction network around the RSB. The absorption spectra of the E to D and S to A double mutants suggested that S112 influences the spectral shift by compensating for the weaker counterion interaction. Our structural and spectral studies have revealed the unique spectral shift mechanism of HeR and clarified the physicochemical properties of HeRs.

Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

Diversity of "Ca. Micrarchaeota" in Two Distinct Types of Acidic Environments and Their Associations with Thermoplasmatales.

"Candidatus Micrarchaeota" are widely distributed in acidic environments; however, their cultivability and our understanding of their interactions with potential hosts are very limited. Their habitats were so far attributed with acidic sites, soils, peats, freshwater systems, and hypersaline mats. Using cultivation and culture-independent approaches (16S rRNA gene clonal libraries, high-throughput amplicon sequencing of V3-V4 region of 16S rRNA genes), we surveyed the occurrence of these archaea in geothermal areas on Kamchatka Peninsula and Kunashir Island and assessed their taxonomic diversity in relation with another type of low-pH environment, acid mine drainage stream (Wales, UK). We detected "Ca. Micrarchaeota" in thermophilic heterotrophic enrichment cultures of Kunashir and Kamchatka that appeared as two different phylotypes, namely "Ca. Mancarchaeum acidiphilum"-, and ARMAN-2-related, alongside their potential hosts, Cuniculiplasma spp. and other Thermoplasmatales archaea without defined taxonomic position. These clusters of "Ca. Micrarchaeota" together with three other groups were also present in mesophilic acid mine drainage community. Present work expands our knowledge on the diversity of "Ca. Micrarchaeota" in thermophilic and mesophilic acidic environments, suggests cultivability patterns of acidophilic archaea and establishes potential links between low-abundance species of thermophilic "Ca. Micrarchaeota" and certain Thermoplasmatales, such as Cuniculiplasma spp. in situ.

Ultrafast Dynamics of Heliorhodopsins.

Heliorhodopsins (HeR) constitute a new rhodopsin family and show only <15% sequence identities with type-1 and type-2 rhodopsins. The large difference in amino acid sequence between HeRs and other rhodopsins raises a question whether their biological function is triggered by efficient and rapid photoisomerization of the retinal chromophore as in the case of other rhodopsins. We performed femtosecond time-resolved absorption measurements of two HeRs, HeR 48C12 and HeR from Thermoplasmatales archaeon SG8-52-1. Both HeRs exhibit excited-state absorption around 480 nm and stimulated emission in the >650 nm region, and these transient signals decay concomitantly with appearance of photoproduct absorption on a subpicosecond time scale. The observed spectral change indicates that ultrafast retinal photoisomerization proceeds in the femtosecond time region. The transient spectra and dynamics of HeRs are surprisingly similar to those of type-1 rhodopsins, despite remarkable differences in amino acid arrangement in the hydrophobic region of the retinal binding site.

Resonance Raman Investigation of the Chromophore Structure of Heliorhodopsins.

Heliorhodopsins (HeRs) are a new category of retinal-bound proteins recently discovered through functional metagenomics analysis that exhibit obvious differences from type-1 microbial rhodopsins. We conducted the first detailed structural characterization of the retinal chromophore in HeRs using resonance Raman spectroscopy. The observed spectra clearly show that the Schiff base of the chromophore is protonated and forms a strong hydrogen bond to a species other than a water molecule, highly likely a counterion residue. The vibrational mode of the Schiff base of HeRs exhibits similarities with that of photosensory microbial rhodopsins, that is consistent with the previous proposal that HeRs function as photosensors. We also revealed unusual spectral features of the in-plane chain vibrations of the chromophore, suggesting an unprecedented geometry of the Schiff base caused by a difference in the retinal pocket structure of HeRs. These data demonstrate structural characteristics of the photoreceptive site in this novel type of rhodopsin family.

The hidden treasure in your data: phasing with unexpected weak anomalous scatterers from routine data sets.

Single-wavelength anomalous dispersion (SAD) utilizing anomalous signal from native S atoms, or other atoms with Z ≤ 20, generally requires highly redundant data collected using relatively long-wavelength X-rays. Here, the results from two proteins are presented where the anomalous signal from serendipitously acquired surface-bound Ca atoms with an anomalous data multiplicity of around 10 was utilized to drive de novo structure determination. In both cases, the Ca atoms were acquired from the crystallization solution, and the data-collection strategy was not optimized to exploit the anomalous signal from these scatterers. The X-ray data were collected at 0.98 Šwavelength in one case and at 1.74 Šin the other (the wavelength was optimized for sulfur, but the anomalous signal from calcium was exploited for structure solution). Similarly, using a test case, it is shown that data collected at ∼1.0 Šwavelength, where the f'' value for sulfur is 0.28 e, are sufficient for structure determination using intrinsic S atoms from a strongly diffracting crystal. Interestingly, it was also observed that SHELXD was capable of generating a substructure solution from high-exposure data with a completeness of 70% for low-resolution reflections extending to 3.5 Šresolution with relatively low anomalous multiplicity. Considering the fact that many crystallization conditions contain anomalous scatterers such as Cl, Ca, Mn etc., checking for the presence of fortuitous anomalous signal in data from well diffracting crystals could prove useful in either determining the structure de novo or in accurately assigning surface-bound atoms.

Environmental, biogeographic, and biochemical patterns of archaea of the family Ferroplasmaceae.

About 10 years ago, a new family of cell wall-deficient, iron-oxidizing archaea, Ferroplasmaceae, within the large archaeal phylum Euryarchaeota, was described. In this minireview, I summarize the research progress achieved since then and report on the current status of taxonomy, biogeography, physiological diversity, biochemistry, and other research areas involving this exciting group of acidophilic archaea.

Affinity covalent immobilization of glucoamylase onto ρ-benzoquinone-activated alginate beads: II. Enzyme immobilization and characterization.

A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K (m) values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V (m) value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.

Affinity covalent immobilization of glucoamylase onto ρ-benzoquinone activated alginate beads: I. Beads preparation and characterization.

ρ-Benzoquinone-activated alginate beads were presented as a new carrier for affinity covalent immobilization of glucoamylase enzyme. Evidences of alginate modification were extracted from FT-IR and thermal gravimetric analysis and supported by morphological changes recognized through SEM examination. Factors affecting the modification process such as ρ-benzoquinone (PBQ) concentration, reaction time, reaction temperature, reaction pH and finally alginate concentration, have been studied. Its influence on the amount of coupled PBQ was consequently correlated to the changes of the catalytic activity and the retained activity of immobilized enzyme, the main parameters judging the success of the immobilization process. The immobilized glucoamylase was found kept almost 80% of its native activity giving proof of non-significant substrate, starch, diffusion limitation. The proposed affinity covalent immobilizing technique would rank among the potential strategies for efficient immobilization of glucoamylase enzyme.

Thermogymnomonas acidicola gen. nov., sp. nov., a novel thermoacidophilic, cell wall-less archaeon in the order Thermoplasmatales, isolated from a solfataric soil in Hakone, Japan.

A novel thermoacidophilic, cell wall-less archaeon, strain IC-189T, was isolated from a solfataric field in Ohwaku-dani, Hakone, Japan. The cells were irregular cocci, sometimes lobed, club-shaped or catenated, and were highly variable in size, ranging from 0.8 to 8.0 microm in diameter. The strain grew at temperatures in the range 38-68 degrees C (optimally at 60 degrees C) and at pH 1.8-4.0 (optimally at around pH 3.0). Strain IC-189T was obligately aerobic and heterotrophic, requiring yeast extract for growth. Yeast extract, glucose and mannose served as carbon and energy sources. The polar lipids consisted mainly of cyclic or acyclic glycerol-bisdiphytanyl-glycerol tetraethers, and the predominant quinone was a menaquinone with seven isoprenoid units (MK-7). The G+C content of total DNA was 56.1 mol%. 16S rRNA gene sequence analysis revealed that strain IC-189T was a member of the order Thermoplasmatales, but diverged from the hitherto known species of the genera Thermoplasma, Picrophilus and Ferroplasma (86.2-91.0% sequence similarity). These phenotypic and phylogenetic properties clearly support a separate taxonomic status for this strain. Therefore, strain IC-189T represents a novel genus (order Thermoplasmatales) and species, for which the name Thermogymnomonas acidicola gen. nov., sp. nov. is proposed, with type strain IC-189T (=JCM 13583T=DSM 18835T).

Community proteomics of a natural microbial biofilm.

Using genomic and mass spectrometry­based proteomic methods, we evaluated gene expression, identified key activities, and examined partitioning of metabolic functions in a natural acid mine drainage (AMD) microbial biofilm community. We detected 2033 proteins from the five most abundant species in the biofilm, including 48% of the predicted proteins from the dominant biofilm organism, Leptospirillum group II. Proteins involved in protein refolding and response to oxidative stress appeared to be highly expressed, which suggests that damage to biomolecules is a key challenge for survival. We validated and estimated the relative abundance and cellular localization of 357 unique and 215 conserved novel proteins and determined that one abundant novel protein is a cytochrome central to iron oxidation and AMD formation.

A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O6-alkylguanine-DNA alkyltransferase and endonuclease V activities.

A recently discovered DNA repair protein of 303 aa from the archaeal organism Ferroplasma acidarmanus was studied. This protein (AGTendoV) consists of a fusion of the C-terminal active site domain of O(6)-alkylguanine-DNA alkyltransferase (AGT) with an endonuclease V domain. The AGTendoV recombinant protein expressed in Escherichia coli and purified to homogeneity repaired O(6)-methylguanine lesions in DNA via alkyl transfer action despite the complete absence of the N-terminal domain and some differences in key active site residues present in known AGTs. The AGTendoV recombinant protein also cleaved DNA substrates that contained the deaminated bases uracil, hypoxanthine, or xanthine in a similar manner to E. coli endonuclease V. Expression of AGTendoV in E. coli GWR109, a strain that lacks endogenous AGT activity, protected against both the killing and mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine and was more effective in preventing mutations than human alkyltransferase, suggesting that the endonuclease V activity may also repair a promutagenic lesion produced by this alkylating agent. Expression of AGTendoV in a DNA repair-deficient E. coli nfi(-)alkA(-) strain protected from spontaneous mutations arising in saturated cultures and restored the mutation frequency to that found in the nfi(+) alkA(+) strain. These results demonstrate the physiological occurrence of two completely different but functional DNA repair activities in a single polypeptide chain.

Beta-D-glucopyranosyl caldarchaetidylglycerol is the main lipid of the acidophilic, mesophilic, ferrous iron-oxidising archaeon Ferroplasma acidiphilum.

Chloroform-methanol-extractable lipids account for about 5% by weight of dry cells of the acidophilic, autotrophic, mesophilic, ferrous compound-oxidising, cell wall-less archaeon Ferroplasma acidiphilum strain Y(T), about 90% of these being contributed by phospholipids and glycophospholipids. The most abundant constituent (about 55% of total lipids) was purified by DEAE cellulose and silica gel column chromatography. By means of matrix-assisted laser desorption ionisation mass spectrometry, infrared spectroscopy, (1)H-nuclear magnetic resonance spectroscopy, and chemical degradation experiments it was established to be beta-D-glucopyranosyl caldarchaetidylglycerol, the isopranyl chains of which have a cyclopentane ring each.