RecJ3/4-aRNase J form a Ubl-associated nuclease complex functioning in survival against DNA damage in Haloferax volcanii.

Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii, an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3'-5' exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5'-3' exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identified in the archaeon Haloferax volcanii, which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3'-5' exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5'-3' exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.

Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids.

Advances are needed in the site-directed mutagenesis of large plasmids for protein structure-function studies, as current methods are often inefficient, complicated and time-consuming. Here two new methods are reported that overcome these difficulties, namely the single primer extension reaction (SSPER) strategy that reaches 100% efficiency and the reduce recycle PCR (rrPCR) method that is advantageous in generating single and pairwise combinations of mutations. Both methods are distinguished from current technologies by the addition of a step that easily removes the oligonucleotide primer(s) after the first reaction, thus allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). High efficiency of the methods is demonstrated by generating single and paired combinations of the 11 site-directed mutations targeted on 5 different plasmid DNA templates ranging from 10 to 12 kb and 57-60% GC-content at a rate of 50-100%. Overall, the methods are demonstrated to be (i) highly accurate, allowing for screening of plasmids by DNA sequencing, (ii) streamlined to generate the mutations within a single day, (iii) cost-effective in requiring only two primers and two enzymes (DpnI and a proofreading DNA polymerase), (iv) straightforward in primer design, (v) applicable for both large and small plasmids, and (vi) easily implemented by entry level researchers.

High-level synthesis and secretion of laccase, a metalloenzyme biocatalyst, by the halophilic archaeon Haloferax volcanii.

Haloarchaea and their enzymes have extremophilic properties desirable for use as platform organisms and biocatalysts in the bioindustry. These GRAS (generally regarded as safe) designated microbes thrive in hypersaline environments and use a salt-in strategy to maintain osmotic homeostasis. This unusual strategy has resulted in the evolution of most of the intracellular and extracellular enzymes of haloarchaea to be active and stable not only in high salt (2-5M) but also in low salt (0.2M). This salt tolerance is correlated with a resilience to low water activity, thus, rendering the haloarchaeal enzymes active and stable in organic solvent and temperatures of 50-60°C used in the enzymatic biodelignification and saccharification of lignocellulosic materials. High-level secretion of haloarchaeal enzymes to the extracellular milieu is useful for many applications, including enzymes that deconstruct biomass to allow for lignin depolymerization and simultaneous fermentation of sugars released from hemicellulose and cellulose fractions of lignocellulosics. Here we detail strategies and methods useful for high-level secretion of a laccase, HvLccA, that mediates oxidation of various phenolics by engineering a recombinant strain of the haloarchaeon Haloferax volcanii.

Expression and tandem affinity purification of 20S proteasomes and other multisubunit complexes in Haloferax volcanii.

Tandem affinity purification is a useful strategy to isolate multisubunit complexes of high yield and purity but can be limited when working with halophilic proteins that are not properly expressed in Escherichia coli. Halophilic proteins are desirable for bioindustrial applications as they are often stable and active in organic solvents; however, these proteins can be difficult to express, fold, and purify by traditional technologies. Haloarchaea provide a useful alternative for expression of halophilic proteins. These microorganisms use a salt-in strategy to maintain homeostasis and express most of their proteins with halophilic properties and low pI. Here, we provide detailed protocols for the genetic modification, expression and tandem affinity purification of "salt-loving" multisubunit complexes from the haloarchaeon Haloferax volcanii. The strategy for isolation of affinity tagged 20S proteasomes that form cylindrical proteolytic nanomachines of α1, α2 and ß subunits is described.

Origin and adaptation of green-sensitive (RH2) pigments in vertebrates.

One of the critical times for the survival of animals is twilight where the most abundant visible lights are between 400 and 550 nanometres (nm). Green-sensitive RH2 pigments help nonmammalian vertebrate species to better discriminate wavelengths in this blue-green region. Here, evaluation of the wavelengths of maximal absorption (λmax s) of genetically engineered RH2 pigments representing 13 critical stages of vertebrate evolution revealed that the RH2 pigment of the most recent common ancestor of vertebrates had a λmax of 503 nm, while the 12 ancestral pigments exhibited an expanded range in λmax s between 474 and 524 nm, and present-day RH2 pigments have further expanded the range to ~ 450-530 nm. During vertebrate evolution, eight out of the 16 significant λmax shifts (or |Δλmax | ≥ 10 nm) of RH2 pigments identified were fully explained by the repeated mutations E122Q (twice), Q122E (thrice) and M207L (twice), and A292S (once). Our data indicated that the highly variable λmax s of teleost RH2 pigments arose from gene duplications followed by accelerated amino acid substitution.

Ciliary and rhabdomeric photoreceptor-cell circuits form a spectral depth gauge in marine zooplankton.

Ciliary and rhabdomeric photoreceptor cells represent two main lines of photoreceptor-cell evolution in animals. The two cell types coexist in some animals, however how these cells functionally integrate is unknown. We used connectomics to map synaptic paths between ciliary and rhabdomeric photoreceptors in the planktonic larva of the annelid Platynereis and found that ciliary photoreceptors are presynaptic to the rhabdomeric circuit. The behaviors mediated by the ciliary and rhabdomeric cells also interact hierarchically. The ciliary photoreceptors are UV-sensitive and mediate downward swimming in non-directional UV light, a behavior absent in ciliary-opsin knockout larvae. UV avoidance overrides positive phototaxis mediated by the rhabdomeric eyes such that vertical swimming direction is determined by the ratio of blue/UV light. Since this ratio increases with depth, Platynereis larvae may use it as a depth gauge during vertical migration. Our results revealed a functional integration of ciliary and rhabdomeric photoreceptor cells in a zooplankton larva.

Engineering a xylanase from Streptomyce rochei L10904 by mutation to improve its catalytic characteristics.

Protein engineering was performed by N-terminal region replacement and site-directed mutagenesis in the cord of a xylanase (Srxyn) from Streptomyce rochei L10904 to improve its catalytic characteristics. Three mutants SrxynF, SrxynM and SrxynFM displayed 2.1-fold, 3.2-fold and 5.3-fold higher specific activities than that of Srxyn, respectively. Moreover, all of the mutants showed greater substrate affinity and kcat/Km than the native Srxyn. In addition, the enzymes showed improved hydrolysis characteristics, of which the most noteworthy is the enhanced ability of producing xylobiose (X2) and xylotriose (X3) from polymeric substrates. The engineered xylanases have greater potential for applications in oligosaccharide preparation industry.

Purification and characterization of a naringinase from a newly isolated strain of Bacillus amyloliquefaciens 11568 suitable for the transformation of flavonoids.

An intracellular naringinase from Bacillus amyloliquefaciens 11568 isolated from soil was purified, identified, and characterized. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified enzyme gave a single protein band corresponding to a molecular mass of 32kDa. The optimum pH and temperature for naringinase and its α-l-rhamnosidase and ß-d-glucosidase activities were pH 7.5 and 45°C, respectively. The enzymes were stable below 45°C between pH 3.5 and 8.5. The Km and the Vmax of the isolated naringinase were 0.95mmol/L and 3847.3mmol/(L·min), respectively. The isolated naringinase was capable of hydrolyzing naringin, neohesperidin, and other glycosides. Additionally, a concentration of 4U/mL of the enzyme in citrus juice was sufficient to remove the naringin and alleviate the bitterness of the juice. These results provide an in-depth insight into the structure of the naringinase and the hydrolysis of naringin and other flavonoids.

Adaptive evolutionary paths from UV reception to sensing violet light by epistatic interactions.

Ultraviolet (UV) reception is useful for such basic behaviors as mate choice, foraging, predator avoidance, communication, and navigation, whereas violet reception improves visual resolution and subtle contrast detection. UV and violet reception are mediated by the short wavelength-sensitive (SWS1) pigments that absorb light maximally (λmax) at ~360 nm and ~395 to 440 nm, respectively. Because of strong nonadditive (epistatic) interactions among amino acid changes in the pigments, the adaptive evolutionary mechanisms of these phenotypes are not well understood. Evolution of the violet pigment of the African clawed frog (Xenopus laevis, λmax = 423 nm) from the UV pigment in the amphibian ancestor (λmax = 359 nm) can be fully explained by eight mutations in transmembrane (TM) I-III segments. We show that epistatic interactions involving the remaining TM IV-VII segments provided evolutionary potential for the frog pigment to gradually achieve its violet-light reception by tuning its color sensitivity in small steps. Mutants in these segments also impair pigments that would cause drastic spectral shifts and thus eliminate them from viable evolutionary pathways. The overall effects of epistatic interactions involving TM IV-VII segments have disappeared at the last evolutionary step and thus are not detectable by studying present-day pigments. Therefore, characterizing the genotype-phenotype relationship during each evolutionary step is the key to uncover the true nature of epistasis.

Spectral Tuning of Phototaxis by a Go-Opsin in the Rhabdomeric Eyes of Platynereis.

Phototaxis is characteristic of the pelagic larval stage of most bottom-dwelling marine invertebrates. Larval phototaxis is mediated by simple eyes that can express various types of light-sensitive G-protein-coupled receptors known as opsins. Since opsins diversified early during metazoan evolution in the marine environment, understanding underwater light detection could elucidate this diversification. Opsins have been classified into three major families, the r-opsins, the c-opsins, and the Go/RGR opsins, a family uniting Go-opsins, retinochromes, RGR opsins, and neuropsins. The Go-opsins form an ancient and poorly characterized group retained only in marine invertebrate genomes. Here, we characterize a Go-opsin from the marine annelid Platynereis dumerilii. We found Go-opsin1 coexpressed with two r-opsins in depolarizing rhabdomeric photoreceptor cells in the pigmented eyes of Platynereis larvae. We purified recombinant Go-opsin1 and found that it absorbs in the blue-cyan range of the light spectrum. To characterize the function of Go-opsin1, we generated a Go-opsin1 knockout Platynereis line by zinc-finger-nuclease-mediated genome engineering. Go-opsin1 knockout larvae were phototactic but showed reduced efficiency of phototaxis to wavelengths matching the in vitro Go-opsin1 spectrum. Our results highlight spectral tuning of phototaxis as a potential mechanism contributing to opsin diversity.

High-level expression of a xylanase gene from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris.

A xylanase gene from Paecilomyces thermophila was functionally expressed in Pichia pastoris. The recombinant xylanase (xynA) was predominantly extracellular; in a 5 l fermentor culture, the total extracellular protein was 8.1 g l(-1) with an activity of 52,940 U ml(-1). The enzyme was purified to homogeneity with a recovery of 48 %. The recombinant xynA was optimally active at 75 °C, as measured over 10 min, and at pH 7. The enzyme was stable up to 80 °C for 30 min. It hydrolyzed birchwood xylan, beechwood xylan and xylooligosaccharides to produce xylobiose and xylotriose as the main products.

Engineering a thermostable ß-1,3-1,4-glucanase from Paecilomyces thermophila to improve catalytic efficiency at acidic pH.

To fulfill the need for acid-tolerant and thermostable ß-1,3-1,4-glucanases, an error-prone PCR and DNA-shuffling approach was employed to enhance the activity of thermostable ß-1,3-1,4-glucanases from Paecilomyces thermophila (PtLic16A) at acidic pH. Mutant PtLic16AM2 was selected and characterized, and showed optimal activity at pH 5.0, corresponding to an acidic shift of 2.0 pH units relative to the wild-type enzyme. Other properties of PtLic16A such as temperature optimum and substrate specificity that are beneficial for industrial applications did not change. Based on the substituted residues of PtLic16AM2, three site-directed mutations, D56G, D221G and C263S, were designed to study these residues' roles. The amino acid residues at positions 56 and 263 were found to be important in determining optimal pH activity. Activity of the D221G variant showed no significant difference from the wild-type. Thus, it appears that the change in optimal pH for PtLic16AM2 was mainly caused by the combination of substitutions D56G and C263S. This study provides a ß-1,3-1,4-glucanase (PtLic16AM2) with high potential for industrial applications.

Characterization of a protease-resistant α-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides.

The α-galactosidase gene, RmGal36, from Rhizomucor miehei was cloned and expressed in Escherichia coli. The gene has an open reading frame of 2256bp encoding 751 amino acid residues. RmGal36 was optimally active at pH 4.5 and 60°C, but is stable between pH 4.5 and 10.0 and at a temperature of up to 55°C for 30min retaining more than 80% of its relative activity. It displayed remarkable resistance to proteases and its activity was not inhibited by galactose concentrations of 100mM. The relative specificity of RmGal36 towards various substrates is in the order of p-nitrophenyl α-galactopyranoside>melibiose>stachyose>raffinose, with a K(m) of 0.36, 16.9, 27.6, and 47.9mM, respectively. The enzyme completely hydrolyzed raffinose and stachyose present in soybeans and kidney beans at 50°C within 60min. These features make RmGal36 useful in the food and feed industries and in processing of beet-sugar.

High-level expression of extracellular secretion of a ß-xylosidase gene from Paecilomyces thermophila in Escherichia coli.

A novel ß-xylosidase gene (designated as PtXyl43) from thermophilic fungus Paecilomycesthermophila was cloned and extracellularly expressed in Escherichia coli. PtXyl43 belonging to glycoside hydrolase (GH) family 43 has an open reading frame of 1017 bp, encoding 338 amino acids without a predicted signal peptide. No introns were found by comparison of the PtXyl43 genomic DNA and cDNA sequences. The recombinant ß-xylosidase (PtXyl43) was secreted into the culture medium in E. coli with a yield of 98.0 U mL(-1) in shake-flask cultures. PtXyl43 was purified 1.2-fold to homogeneity with a recovery yield of 61.5% from the cell-free culture supernatant. It appeared as a single protein band on SDS-PAGE with a molecular mass of approx 52.3 kDa. The enzyme exhibited an optimal activity at 55 °C and pH 7.0, respectively. This is the first report on the cloning and expression of a GH family 43 ß-xylosidase gene from thermophilic fungi.

[Improvement of catalytic capability of Paecilomyces thermophila J18 thermostable beta-1,3-1,4-glucanase under acidic condition by directed evolution].

Directed evolution was used to improve the performance of beta-1,3-1,4-glucanase (designated as PtLicl6A) from Paecilomyces thermophila J18 under acidic condition. A mutant library was constructed by error-prone PCR and DNA shuffling, and positive clones were screened by Congo red staining. More than 1 500 mutants were selected. One mutant (PtLic16AM1) exhibited an optimal activity at pH 5.5, while the optimal pH of the wild-type enzyme was 7.0. The mutant PtLic16AM1 kept the high specific activity and thermotolerence of the wild-type enzyme. Sequence analysis revealed that the mutant enzyme has four sense substitutions which caused four amino acid substitutions - namely T58S, Y110N, G195E and D221G.. Homology modeling showed that among the four amino acid substitutions, Y110N was near the active site of the enzyme, while the other three was distant. T58S and G195E may play key roles in the change of optimal pH. This study provided a new perspective of obtaining applicable 3-1,3-1,4-glucanase for industrial use.