Heterologous Expression and Characterization of an Alcohol Dehydrogenase from the Archeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Analysis of the Thermoplasma acidophilum DSM 1728 genome identified two putative alcohol dehydrogenase (ADH) open reading frames showing 50.4% identity against each other. The corresponding genes Ta0841 and Ta1316 encode proteins of 336 and 328 amino acids with molecular masses of 36.48 and 36.01 kDa, respectively. The genes were expressed in Escherichia coli and the recombinant enzymes were functionally assessed for activity. Throughout the study only Ta1316 ADH resulted active in the oxidative reaction in the pH range 2–8 (optimal pH 5.0) and temperatures from 25 to 90°C (optimal 75°C). This ADH catalyzes the oxidation of several alcohols such as ethanol, methanol, 2-propanol, butanol, and pentanol during the reduction of the cofactor NAD⁺. The highest activity was found in the presence of ethanol producing optically pure acetaldehyde. The specific enzyme activity of the purified Ta1316 ADH with ethanol as a substrate in the optimal conditions was 628.7 U/mg.     

<Emphasis Type="Italic">Pichia pastoris</Emphasis> is a valuable host for the expression of genes encoding membrane proteins from the hyperthermophilic Archeon <Emphasis Type="Italic">Pyrococcus abyssi</Emphasis>

We present here the experimental strategies, first results and identified bottlenecks of a structural genomics initiative on membrane proteins of the hyperthermophilic archaea Pyrococcus abyssi. Five ORFs coding for putative membrane proteins have been cloned and expressed in the methylotrophic Pichia pastoris expression system, using two different constructs, with or without the signal sequence α-mating factor of Saccharomyces cerevisiae. A c-myc epitope and 6 His codons were added at the 3′-end of the targeted genes to allow immunodetection of the recombinant proteins and to facilitate their further purification. We have selected at least one producer clone for each protein of interest and for almost every construction. All the membrane proteins were produced in Erlenmeyer flasks culture and in fed-batch cultivation for large-scale preparation. The proteins were detected in the membrane fractions of P. pastoris. Production efficiencies were relatively low in both production conditions but the quantities of biomass obtained during fed-batch cultivation have allowed us to collect sufficient amount of material for further purification. The proteins were extracted, solubilized and partially purified. Large-scale purification will be necessary for further structural work.     

Identification of <Emphasis Type="Italic">S</Emphasis>-haplotype-specific F-box gene in Japanese plum (<Emphasis Type="Italic">Prunus salicina</Emphasis> Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility controlled by a single polymorphic locus containing at least two linked genes, i.e., the S-RNase gene and the pollen-expressed SFB/SLF (S-haplotype-specific F-box/S-locus F-box) gene. However, the SFB gene in Japanese plum (Prunus salicina Lindl.) has not yet been identified. We determined eight novel sequences homologous to the SFB genes of other Prunus species and named these sequences PsSFB. The gene structure of the SFB genes and the characteristic domains in deduced amino acid sequences were conserved. Three sequences from 410 to 2,800 bp of the intergenic region between the PsSFB sequences and the S-RNase alleles were obtained. The eight identified PsSFB sequences showed S-haplotype-specific polymorphism, with 74–83% amino acid identity. These alleles were exclusively expressed in the pollen. These results suggest that the PsSFB alleles are the pollen S-determinants of GSI in Japanese plum. Nucleotide sequence data reported are available in the NCBI database under the accession numbers DQ849084–DQ849090 and DQ849118.     

Characterization of Novel Orange Fluorescent Protein Cloned from Cnidarian Tube Anemone <Emphasis Type="Italic">Cerianthus</Emphasis> sp.

A novel orange fluorescent protein (OFP) was cloned from the tentacles of Cnidarian tube anemone Cerianthus sp. It consists of 222 amino acid residues with a calculated molecular mass of 25.1 kDa. A BLAST protein sequence homology search revealed that native OFP has 81% sequence identity to Cerianthus membranaceus green fluorescent protein (cmFP512), 38% identity to Entacmaea quadricolor red fluorescent protein (eqFP611), 37% identity to Discosoma red fluorescent protein (DsRed), 36% identity to Fungia concinna Kusabira-orange fluorescent protein (KO), and a mere 21% identity to green fluorescent protein (GFP). It is most likely that OFP also adopts the 11-strand β-barrel structure of fluorescent proteins. Spectroscopic analysis indicated that it has a wide absorption spectrum peak at 548 nm with two shoulders at 487 and 513 nm. A bright orange fluorescence maximum at 573 nm was observed when OFP was excited at 515 nm or above. When OFP was excited well below 515 nm, a considerable amount of green emission maximum at 513 nm was also observed. It has a fluorescence quantum yield (Φ) of 0.64 at 25°C. The molar absorption coefficients (ɛ) of folded OFP at 278 and 548 nm are 47,000 and 60,000 M-1⁻¹ • cm-1⁻¹, respectively. Its fluorescent brightness (ɛ Φ) at 25°C is 38,400 M⁻¹-1 • cm⁻¹-1. Like other orange-red fluorescent proteins, OFP is also tetrameric. It was readily expressed as soluble protein in Escherichia coli at 37°C, and no aggregate was observed in transfected HeLa cells under our experimental conditions. Fluorescent intensity of OFP is detectable over a pH range of 3 to 12.     

Analysis of two promoters that control the expression of the <Emphasis Type="Italic">GTP cyclohydrolase I</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5′-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between −98 and +31, and between −73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between −98 and −56 and between −73 and −41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilis GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.     

New proteins orthologous to cerato-platanin in various <Emphasis Type="Italic">Ceratocystis</Emphasis> species and the purification and characterization of cerato-populin from <Emphasis Type="Italic">Ceratocystis populicola</Emphasis>

Natural variants of cerato-platanin (CP), a pathogen associated molecular pattern (PAMP) protein produced by Ceratocystis platani (the causal agent of the plane canker stain), have been found to be produced by other four species of the genus Ceratocystis, including five clones of Ceratocystis fimbriata isolated from different hosts. All these fungal strains were known to be pathogenic to plants with considerable importance in agriculture, forestry, and as ornamental plants. The putative premature proteins were deduced on the basis of the nucleotide sequence of genes orthologous to the cp gene of C. platani; the deduced premature proteins of Ceratocystis populicola and Ceratocystis variospora reduced the total identity of all the others from 87.3% to 60.3%. Cerato-populin (Pop1), the CP-orthologous protein produced by C. populicola, was purified and characterized. Pop1 was a well-structured α/β protein with a different percentage of the α-helix than CP, and it self-assembled in vitro in ordered aggregates. Moreover, Pop1 behaved as PAMP, since it stimulated poplar leaf tissues to activate defence responses able to reduce consistently the C. populicola growth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular cloning and functional characterization of fumarases C in <Emphasis Type="Italic">Neisseria</Emphasis> species

Fumarase is one of the key enzymes in the TCA cycle and has been implicated in virulence and survival of some microorganisms under suboptimal environmental conditions. In this study, the fumC genes that encode fumarase C (FUMCs) from Neisseria meningitidis, N. gonorrhoeae and N. subflava were identified by homology-based analysis, cloned by polymerase chain reactions and fully sequenced. The inferred primary sequence of neisserial FUMCs showed a high degree of conservation with 97.8–98.7% amino acid identity. However, phylogenetic analysis revealed that these neisserial FUMCs are divergent from class II fumarases found in other microorganisms, rat and human. The putative fumC genes were subcloned into the expression vector, pGEX-6P-1 and efficiently expressed in Esherichia coli BL21. The purified recombinant fusion proteins obtained by affinity chromatography demonstrated high catalytic activities (120–180 U/mg), thus authenticating the identities and functionalities of the cloned genes. Whether FUMC has any physiological relevance to the pathogenesisity of Neisseriae must await future gene disruption or mutagenesis studies.     

A Novel Phytase appA from <Emphasis Type="Italic">Citrobacter amalonaticus</Emphasis> CGMCC 1696: Gene Cloning and Overexpression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436–amino-acid protein, which had a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg·mL⁻¹, and the enzyme activity level reached 15,000 U·mL⁻¹, which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and characterized. The specific activity of r-appA for sodium phytate was 3548 U·mg⁻¹. The optimum pH and temperature for enzyme activity were 4.5 and 55°C, respectively. r-appA was highly resistant to pepsin or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed industry.     

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t ₅₀ of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).     

Expression and purification of two major outer membrane proteins from <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Vibrio alginolyticus, a Gram-negative bacterium, is one of Vibrio pathogens common to human and marine animals. Outer membrane proteins of bacteria play an important role during infection and induction of host immune response. In present research, two outer membrane protein genes (OmpK and OmpW) of V. alginolyticus were cloned and expressed. The open reading frames of OmpK and OmpW contain 846 bp and 645 bp, respectively, the mature proteins consist of 261 and 193 amino acid residues. At the signal peptides positions −3 to −1, the amino acids were V-M-A in OmpK and V-F-A in OmpW, which consistented with the observed sequence V-X-A of the signal peptides of transmembrane OMP. The alignment analysis indicated that both proteins were highly conserved, which could serve as surface antigens for vaccine candidates. SDS-PAGE indicated two genes over-expressed in E. coli BL21 (DE3). By affinity chromatography on Ni²⁺-nitriloaceate resin, the recombinant proteins were purified from inclusion bodies. Western blot analysis revealed that both proteins had immunoreactivity, which provided a base for further study on the evaluation of diagnostication and vaccine candidates.     

印度块菌-云南松菌根际土壤细菌的种群组成和群落结构

以印度块菌-云南松菌根际土壤细菌为研究对象,研究其种群组成和结构特征。(1)稀释平板法分离得到印度块菌-云南松菌根际土壤细菌的纯培养菌株,对菌株的16S rRNA序列测序分析,对测序的菌株数量和得到的OTUs数量绘制物种累积曲线,当物种累积曲线趋于平缓时,对OTUs进行系统发育分析,揭示可培养细菌的种群组成和结构特征。(2)对印度块菌-云南松菌根际土壤细菌16S rRNA基因的V3—V4区进行高通量测序,分析全部细菌类群的种群组成和结构特征。(1)分离得到菌根际可培养细菌793株,分属于3个属的61个OTUs,其中假单胞菌属(Pseudomonas)序列占总序列的86%,不动杆菌属(Acinetobacter)序列占总序列的9.8%,链霉菌属(Streptomyces)序列占总序列的6.5%。假单胞菌是印度块菌-云南松菌根际土壤可培养细菌的绝对优势类群。(2)高通量测序得到菌根际细菌序列8937条,分属于20个门、198属、2073个OTUs。隶属于变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和酸杆菌门(Acidobacteria)的OTUs占总OTUs的65.9%,变形菌门、放线菌门和酸杆菌门细菌是印度块菌-云南松菌根际土壤细菌的优势细菌。隶属于黄杆菌属(Flavobacterium)、根瘤菌属(Rhizobium)和假黄色单胞菌属(Pseudoxanthomona)的OTUs占总OTUs的33%,黄杆菌属、根瘤菌属和假黄色单胞菌属细菌是印度块菌-云南松菌根际土壤细菌的优势属。印度块菌-云南松菌根际土壤可培养细菌多样性较低,假单胞菌属细菌占据绝对优势地位。印度块菌-云南松菌根际土壤细菌类群具有较高的多样性,物种种类丰富,优势菌群集中。     

Effects of constant light on circadian rhythmicity in mice lacking functional <Emphasis Type="Italic">cry</Emphasis> genes: dissimilar from <Emphasis Type="Italic">per</Emphasis> mutants

Mutations in each of the genes mPer1, mPer2, mCry1 and mCry2 separately cause deviations from the wild type circadian system. Differences between these mutant strains have inspired the hypothesis that the duality of circadian genes (two mPer and two mCry genes involved) is related to the existence of two components in the circadian oscillator (Daan et al., J Biol Rhythms 16:105–116, 2001). We tested the predictions from this theory that the circadian period (τ) lengthens under constant illumination (LL) in mCry1 and mPer1 mutant mice, while it shortens in mCry2 and mPer2 mutants. mCry1 ^−/− and mCry2 ^−/− knockout mice both consistently increased τ with increasing light intensity, as did wild type mice. With increasing illumination, rhythmicity is reduced in mCry1, mCry2 and mPer1, but not in mPer2 deficient mice. Results for mPer mutant mice are in agreement with data reported on these strains earlier by Steinlechner et al. (J Biol Rhythms 17:202–209, 2002), and also with the predictions from the model. The increase in cycle length of the circadian system by light in the mCry2 deficient mice violates the predictions. The model is thereby rejected: the mCry genes do not play a differential role, although the opposite responses of mPer mutants to light remain consistent with a functional Evening–Morning differentiation.     

Isolation and characterization of porins from <Emphasis Type="Italic">Desulfovibrio piger</Emphasis> and <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>: structure and gene sequencing

The outer membrane proteins of Desulfovibrio piger and Bilophila wadsworthia (Omp-DP and Omp-BW, respectively) and the genes encoding them (omp-DP and omp-BW) were isolated and characterized. Native Omp-DP and Omp-BW form a trimeric structure of approximately 120 kDa. These proteins disaggregated into monomers with a molecular weight of approximately 53 kDa after heating at 95°C for 10 min. The pore-forming abilities of these oligomeric proteins demonstrated that they form small nonspecific channels with an exclusion limit of 260–300 Da. The omp-DP and omp-BW genes were cloned and sequenced. Sequence analyses revealed an open reading frame of 1,512 bp for omp-DP and 1,440 bp for omp-BW. The mature Omp-DP protein consisted of 480 amino acids and had a calculated MW of 53,290 Da. The mature Omp-BW protein consisted of 456 amino acids and had a calculated MW of 50.050 Da. Alignment of Omp-DP with Omp-BW revealed 54% homology, whereas alignment with other known porins showed a low level of homology. Analysis of the secondary structures indicated that both proteins span the outer membrane 18 times with amphipathic β-strands. This research presents porins which were isolated and characterized for the first time from bacteria belonging to the Desulfovibrionaceae family. O. Avidan and E. Kaltageser have contributed equally to this work.     

Cloning and characterization of three epoxide hydrolases from a marine bacterium, <Emphasis Type="Italic">Erythrobacter litoralis</Emphasis> HTCC2594

Previously, we reported that ten strains belonging to Erythrobacter showed epoxide hydrolase (EHase) activities toward various epoxide substrates. Three genes encoding putative EHases were identified by analyzing open reading frames of Erythrobacter litoralis HTCC2594. Despite low similarities to reported EHases, the phylogenetic analysis of the three genes showed that eeh1 was similar to microsomal EHase, while eeh2 and eeh3 could be grouped with soluble EHases. The three EHase genes were cloned, and the recombinant proteins (rEEH1, rEEH2, and rEEH3) were purified. The functionality of purified proteins was proved by hydrolytic activities toward styrene oxide. EEH1 preferentially hydrolyzed (R)-styrene oxide, whereas EEH3 preferred to hydrolyze (S)-styrene oxide, representing enantioselective hydrolysis of styrene oxide. On the other hand, EEH2 could hydrolyze (R)- and (S)-styrene oxide at an equal rate. The optimal pH and temperature for the EHases occurred largely at neutral pHs and 40–55 °C. The substrate selectivity of rEEH1, rEEH2, and rEEH3 toward various epoxide substrates were also investigated. This is the first representation that a strict marine microorganism possessed three EHases with different enantioselectivity toward styrene oxide.     

Cloning and heterologous expression of nitrate reductase genes from <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.     

Cyclodextrin production and genetic characterization of cyclodextrin glucanotranferase of <Emphasis Type="Italic">Paenibacillus graminis</Emphasis>

Paenibacillus graminis strains were described recently as cyclodextrin (CD) producers. Cyclodextrins are produced by cyclodextrin glucanotransferase (CGTase) which has not been characterized in P. graminis. Similar amounts of α- and β-CDs were produced by P. graminis (MC22.13) and P. macerans (LMD24.10^T). Primers were designed to sequence the gene encoding CGTase from P. graminis. A phylogenetic tree was constructed and P. graminis CGTase protein showed to be closer (79.4% protein identity) to P. macerans |P31835|. Hybridization studies suggested that the gene encoding CGTase is located in different positions in the genomes of P. macerans and P. graminis.