Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea

Industrial overproducing strains present unique hosts for expression of heterologous gene clusters encoding secondary metabolite biosynthesis. For this purpose, efficient gene expression tools and methods are needed. A robust and versatile reporter system based on the rppA gene from Saccharopolyspora erythraea is presented as the method of choice when studying gene expression in actinomycete hosts. The method is easily scalable to accommodate high-throughput procedure, and collected samples can be easily stored and re-tested when needed. The product of RppA is an inert 1,3,6,8-tetrahydroxynaphthalene which spontaneously oxidises to a dark-red quinone flaviolin providing a qualitative visual assessment of gene expression on an agar plate as well as a quantitative spectrophotometric measurement in liquid broth without the need for invasive procedures or external substrate addition. The applicability of the reporter system has been demonstrated by expressing the rppA gene under the control of the heterologous promoters actII-ORF4/P_actI, ermE and its upregulated variant ermE*. The model streptomycete Streptomyces coelicolor, and three industrially important species, Streptomyces tsukubaensis (FK506), Streptomyces cinnamonensis (monensin) and Streptomyces rimosus (oxytetracycline) were used as hosts. The reporter system has shown its utility independently of cultivation conditions or composition of growth medium, from simple laboratory to complex industrial media. The simplicity and robustness of the system, demonstrated even in industrial settings, shows great potential for wider use in different microbial hosts and applications, and may thus represent a new generic and versatile tool useful to a wider scientific community.     

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.     

Genotypic and phenotypic diversity of rhizobia isolated from Lathyrus japonicus indigenous to Japan

Sixty-one rhizobial strains from Lathyrus japonicus nodules growing on the seashore in Japan were characterized and compared to two strains from Canada. The PCR-based method was used to identify test strains with novel taxonomic markers that were designed to discriminate between all known Lathyrus rhizobia. Three genomic groups (I, II, and III) were finally identified using RAPD, RFLP, and phylogenetic analyses. Strains in genomic group I (related to Rhizobium leguminosarum) were divided into two subgroups (Ia and Ib) and subgroup Ia was related to biovar viciae. Strains in subgroup Ib, which were all isolated from Japanese sea pea, belonged to a distinct group from other rhizobial groups in the recA phylogeny and PCR-based grouping, and were more tolerant to salt than the isolate from an inland legume. Test strains in genomic groups II and III belonged to a single clade with the reference strains of R. pisi, R. etli, and R. phaseoli in the 16S rRNA phylogeny. The PCR-based method and phylogenetic analysis of recA revealed that genomic group II was related to R. pisi. The analyses also showed that genomic group III harbored a mixed chromosomal sequence of different genomic groups, suggesting a recent horizontal gene transfer between diverse rhizobia. Although two Canadian strains belonged to subgroup Ia, molecular and physiological analyses showed the divergence between Canadian and Japanese strains. Phylogenetic analysis of nod genes divided the rhizobial strains into several groups that reflected the host range of rhizobia. Symbiosis between dispersing legumes and rhizobia at seashore is discussed.     

Unusual Thermal Disassembly of the SPFH Domain Oligomer from Pyrococcus horikoshii

Stomatin, prohibitin, flotillin, and HflK/C (SPFH) domain proteins are membrane proteins that are widely conserved from bacteria to mammals. The molecular functions of these proteins have not been established. In mammals, the domain is often found in raft-associated proteins such as flotillin and podocin. We determined the structure of the SPFH domain of PH0470 derived from Pyrococcus horikoshii using NMR. The structure closely resembles that of the SPFH domain of the paralog PH1511, except for two C-terminal helices. The results show that the SPFH domain forms stable dimers, trimers, tetramers, and multimers, although it lacks the coiled-coil region for oligomerization, which is a highly conserved region in this protein family. The oligomers exhibited unusual thermodynamic behavior, as determined by circular dichroism, NMR, gel filtration, chemical cross-linking, and analytical ultracentrifugation. The oligomers were converted into monomers when they were heated once and then cooled. This transition was one-way and irreversible. We propose a mechanism of domain swapping for forming dimers as well as successive oligomers. The results of this study provide what to our knowledge are new insights into the common molecular function of the SPFH domain, which may act as a membrane skeleton through oligomerization by domain swapping.     

Horizontal arsC gene transfer among microorganisms isolated from arsenic polluted soil

The study of recent evolution of the arsC genes amplified from microorganisms inhabiting a Colombian oil-polluted soil with high concentrations of arsenic was performed through the isolation of 26 bacterial morphotypes resistant to 10 mM of arsenate. A 353 bp fragment of the gene coding for arsenate-reductase enzyme (i.e. arsC), and a 500 bp 16S rDNA partial sequence were sequenced for 16 morphotypes of the 26 previously isolated. arsC sequences clustered on the same clade with previously reported arsC chromosomal genes of Escherichia coli and Shigella sp.; while 16S rDNA sequences grouped within the genus Pseudomonas and Bacillus. The GC content and the Codon Adaptation Index (CAI) were calculated and statistically compared, both supported the previous results. The Isolation-Migration model (IM model) was applied to calculate the genetic flux between each clade defined by the phylogenetic analysis. In general, the existence of recent horizontal gene transfer (HGT) events was confirmed, and the presence of the arsC gene in Bacillus sphaericus is reported for the first time.     

Expression by Chlamydomonas reinhardtii of a chloroplast ATP synthase with polyhistidine-tagged beta subunits

The green alga Chlamydomonas reinhardtii is a model organism for the study of photosynthesis. The chloroplast ATP synthase is responsible for the synthesis of ATP during photosynthesis. Using genetic engineering and biolistic transformation, a string of eight histidine residues has been inserted into the amino-terminal end of the β subunit of this enzyme in C. reinhardtii. The incorporation of these amino acids did not impact the function of the ATP synthase either in vivo or in vitro and the resulting strain of C. reinhardtii showed normal growth. The addition of these amino acids can be seen through altered gel mobility of the β subunit and the binding of a polyhistidine-specific dye to the subunit. The purified his-tagged CF1 has normal Mg²⁺-ATPase activity, which can be stimulated by alcohol and detergents and the enzyme remains active while bound to a nickel-coated surface. Potential uses for this tagged enzyme as a biochemical tool are discussed.     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

Molecular evolution and functional specialization of chalcone synthase superfamily from Phalaenopsis Orchid

Plant genomes appear to exploit the process of gene duplication as a primary means of acquiring biochemical and developmental flexibility. The best example is the gene encoding chalcone synthase (CHS, EC2.3.1.74), the first committed step in flavonoid biosynthesis. In this study, we examined the molecular evolution of three CHS family members of Phalaenopsis including a novel chs gene (phchs5), which is slowly evolved. The inferred phylogeny of the chs genes of Phalaenopsis with other two orchid plants, Bromoheadia finlaysoniana and Dendrobium hybrid, suggested that gene duplication and divergence have occurred before divergence of these three genera. Relatively quantitative RT-PCR analysis identified expression patterns of these three chs genes in different floral tissues at different developmental stages. Phchs5 was the most abundantly expressed chs gene in floral organs and it was specifically transcribed in petal and lip at the stages when anthocyanin accumulated (stage1–4). Phchs3 and phchs4 were expressed at much lower levels than phchs5. Phchs3 was expressed in pigmented tissue (including lip, petal and sepal) at middle stages (stages 2–4) and in colorless reproductive tissue at late stage (stage 5). Phchs4 was only expressed in petal at earlier stages (stage 1–3) and in lip at middle stage (stage 4). These results present new data on differentiation of gene expression among duplicate copies of chs genes in Phalaenopsis.     

巴西橡胶树 HbERFB4-1 基因的克隆和表达分析

根据EST序列信息,利用RACE技术分离了巴西橡胶树(Hevea brasiliensis)的1个AP2/ERF类基因,命名为HbERFB4-1。该基因cDNA全长732 bp,含有完整的开放阅读框架,编码147个氨基酸,不含内含子。推导的HbERFB4-1氨基酸序列与杨树(Populus trichocarpa)、蓖麻(Ricinus communis)、拟南芥(Arabidopsis thaliana)和大豆(Glycine max)中相应蛋白氨基酸序列的一致性分别为78%、71%、62%和55%,具有典型的AP2类蛋白结构特征。聚类分析表明,HbERFB4-1属于ERF家族B4亚族。半定量RT-PCR分析结果表明,该基因在胶乳中的表达量相对较低,但乙烯利刺激后其在胶乳中的表达量迅速增加,推测该基因可能参与了胶乳中乙烯信号的转导。     

Protein splicing of the three Pyrococcus abyssi ribonucleotide reductase inteins

An intein is a polypeptide that interrupts the functional domains of a protein, called the exteins. The intein can facilitate its own excision from the exteins, concomitant with the ligation of the exteins, in a process called protein splicing. The alpha subunit of the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi is interrupted by three inteins in separate insertion sites. Each intein can facilitate protein splicing when over-expressed in Escherichia coli, with affinity domains serving as the exteins. The influence of the N-terminal flanking residue on the efficiency of splicing is specific to each intein. Each intein has a different downstream nucleophilic residue, and cannot tolerate substitution to a residue of lesser or equal nucleophilicity. The influence of the conserved penultimate His also differs between the inteins.     

Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans

Fungal methionine synthase catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to create methionine. The enzyme, called Met6p in fungi, is required for the growth of the pathogen Candida albicans, and is consequently a reasonable target for antifungal drug design. In order to understand the mechanism of this class of enzyme, we created a three-dimensional model of the C. albicans enzyme based on the known structure of the homologous enzyme from Arabidopsis thaliana. A fusion protein was created and shown to have enzyme activity similar to the wild-type Met6p. Fusion proteins containing mutations at eight key sites were expressed and assayed in this background. The D614 carboxylate appears to ion pair with the amino group of homocysteine and is essential for activity. Similarly, D504 appears to bind to the polar edge of the folate and is also required for activity. Other groups tested have lesser roles in substrate binding and catalysis.     

Purification and properties of tobacco ferredoxin-dependent glutamate synthase,and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C₁₆) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C₃₅) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C₁₆ as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C₃₅ was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp base pairs - Fd-GOGAT ferredoxin-dependent glutamate synthase - GS glutamine synthetase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.     

Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens

ent-Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal ent-kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified ent-kaurene synthase (PpCPS/KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101-kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the ent-kaurene skeleton from GGDP. PpCPS/KS has two aspartate-rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to ent-kaurene and ent-16alpha-hydroxykaurene).     

A molecular analysis of some Eastern Atlantic grouper from the Epinephelus and Mycteroperca genus

Mitochondrial cytochrome b (397 bp) and 16S rDNA (516 bp) sequences analysis was used to investigate the phylogenetic relationships among some Eastern Atlantic Epinephelinae species. Six species of Epinephelus (E. aeneus, E. caninus, E. costae, E. haifensis, E. marginatus and E. tauvina) and two species of Mycteroperca (M. rubra and M. fusca) were analysed. Neighbour-joining and maximum-parsimony analysis support the paraphyletic grouping of the Epinephelus and Mycteroperca analysed. The maximum pairwise nucleotide divergence value in cyt b among all taxa was 0.196 between E. aeneus and E. marginatus and the minimum value was 0.006 between E. costae and M. rubra. Meanwhile, in 16S sequence analysis, the maximum value is 0.093 between E. aeneus and E. tauvina and the minimum value is 0.011 between E. marginatus and M. rubra. Molecular clock estimates for the species suggest a divergence time of 20-24 mya, which coincides with the Miocene period. A molecular analysis was also conducted, using other Epinephelinae sequences from GenBank in order to improve our understanding of the phyletic status of the Epinephelus and Mycteroperca species analysed.