Allozyme variability in populations of trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) from the rivers of Iran

For the first time, an analysis was carried out of allozyme variability in trout (Salmo trutta) from three rivers of Iran. We studied 23 gene loci coding enzymes: glycerol-3-phosphate dehydrogenase (G3PDH), aspartate aminotransferase (AAT), malate dehydrogenase (MDH), lactate dehydrogenase (LDH), creatine kinase (CK), malic enzyme [NADP-dependent MDH] (MEP), superoxide dismutase (SOD), esterase (EST), and esterase D (EST-D). The obtained data demonstrate the similarity between the trout samples from different rivers of Iran according to genetic characteristics. Taking into account the differences by allozyme markers of allele frequencies and allele composition of some loci, we should expect that Iranian trout diverges significantly in genetics from the other trout populations of the Caspian Sea.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in Jeddah,Kingdom of Saudi Arabia

Background

The development of polymerase chain reaction (PCR)-based methods for the detection of known mutations has facilitated detecting specific red blood cell (RBC) enzyme deficiencies. We carried out a study on glucose-6-phosphate dehydrogenase (G6PD) deficient subjects in Jeddah to evaluate the molecular characteristics of this enzyme deficiency and the frequency of nucleotide1311 and IVS-XI-93 polymorphisms in the glucose-6-phosphate dehydrogenase gene.

Results

A total of 1584 unrelated Saudis (984 neonates and 600 adults) were screened for glucose-6-phosphate dehydrogenase deficiency. The prevalence of glucose-6-phosphate dehydrogenase deficiency was 6.9% (n = 110). G6PD Mediterranean mutation was observed in 98 (89.1%) cases, G6PD Aures in 11 (10.0%) cases, and G6PD Chatham in 1 (0.9%) case. None of the samples showed G6PD A￣ mutation. Samples from 29 deficient subjects (25 males and 4 females) were examined for polymorphism. The association of two polymorphisms of exon/intron 11 (c.1311T/IVS-XI-93C) was observed in 14 (42.4%) of 33 chromosomes studied. This association was found in 9 (31.0%) carriers of G6PD Mediterranean and in 4 (13.8%) carriers of G6PD Aures.

Conclusions

The majority of mutations were G6PD Mediterranean, followed by G6PD Aures and < 1% G6PD Chatham. We conclude that 1311T is a frequent polymorphism in subjects with G6PD Mediterranean and Aures variants in Jeddah.

     

Changes in the expression of key genes involved in the biosynthesis of menthol and menthofuran in <Emphasis Type="Italic">Mentha piperita</Emphasis> L. under drought stress

Peppermint (Mentha piperita) is known as an important medicinal plant throughout the world. In the present study, after exposing peppermint plants under drought stress, the qRT-PCR was use to analyze the expression of genes involved in menthol biosynthesis pathway and encoding: limonene synthase (lS), limon-3-hydroxylase (l3oh), trans-isopiperitenol dehydrogenase (ipd), isopiperitenone reductase (ipr), pulegone reductase (pr), menthol dehydrogenase (mdeh), and menthofuran synthase (mfs), which also evaluated the morphological and physiological traits. The results revealed that due to water stress, the gene expression levels of ipd, ipr, and mfs were increased, whereas the gene expression level of pr and mdeh was decreased under water stress conditions. The most of essential oil components (menthol, menthofuran, and plugene), which were analyzed by gas chromatography–mass spectrometry (GC–MS), was positively correlated with genes expression. Drought stress decreased morphological and induces increasing contents of pulegone and menthofuran and reduction in menthol percentages. Results from this study suggest that up-regulation of mfs might contribute to the altered of menthofuran as well as down-regulation of mdeh might cause the reduction of menthol. Furthermore, increasing ls gene expression levels might induce more essential oil yield, while reduction of mfs gene expression levels causes an improvement of essential oil quality.     

<Emphasis Type="Italic">Escherichia coli ydiO</Emphasis> and <Emphasis Type="Italic">ydiQRST</Emphasis> genes encode components of acyl-CoA dehydrogenase complex of anaerobic fatty acid β-oxidation pathway

Escherichia coli open reading frames ydiO and ydiQRST were identified as genes encoding components of the acyl-CoA dehydrogenase complex of anaerobic fatty acid β-oxidation. Individual or concomitant inactivation of fadE gene, encoding known aerobic acyl-CoA dehydrogenase, and ydiO and/or ydiQRST genes did not affect cellular growth on glucose as a sole carbon source. Aerobic growth on sodium oleate was observed only for the cells with intact fadE gene. With an alternative electron acceptor, the cells possessing intact fadE gene demonstrated anaerobic growth on sodium oleate irrespective of the presence or absence of ydiO and ydiQRST genes. For the fadE-deficient mutants, anaerobic growth on sodium oleate was observed only for cells with intact ydiO and ydiQRST genes, while the fadE/ydiO and fadE/ydiQRST mutants failed to grow under the similar conditions.     

Effective improvement of the activity of membrane-bound alcohol dehydrogenase by overexpression of <Emphasis Type="Italic">adhS</Emphasis> in <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis>

Objectives

To investigate the roles of adhS, which encodes the AdhS subunit of membrane-bound alcohol dehydrogenase (mADH) in Gluconobacter oxydans DSM2003, and to rationally improve mADH activity.

Results

adhS was identified and overexpressed in G. oxydans DSM2003. Its overexpression promoted the AdhA subunit which serves as the primary dehydrogenase transfer from the periplasmic space to the periplasmic surface of the membrane thereby increasing the amount of active mADH and thus enhancing mADH activity up to 1.96-fold. The increased mADH activity significantly altered product selectivity (glyceric acid/dihydroxyacetone) during glycerol oxidation and increased the glyceric acid production by 7.6-fold. By comparison, overexpression of adhS and adhABS was equally effective in increasing the mADH activity and glyceric acid production.

Conclusions

adhS overexpression effectively improved mADH activity, indicating that for mADH, adhS might be a limiting component. The findings provide a guide for the efficient application of Gluconobacter spp. in hydroxy acid production.

     

Asymmetric reduction of ketopantolactone using a strictly (<Emphasis Type="Italic">R</Emphasis>)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy

Objectives

To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL].

Results

The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%.

Conclusions

Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

     

A panel of the most suitable reference genes for RT-qPCR expression studies of coffee: screening their stability under different conditions

The reliability of analyses using real-time quantitative polymerase chain reaction (RT-qPCR) depends on the selection of appropriate reference genes to correct for sample-to-sample and run-to-run variations. The aim of the present study was to select the most suitable reference genes for gene expression analyses in tissue samples from coffee, Coffea arabica L. (Arabica) grown under well-watered (WW) and water-deficit (WD) conditions and C. canephora Pierre ex A. Froehner (Robusta) grown under WW conditions. Expression profiles and stabilities were evaluated for 12 reference genes in different tissues from C. arabica and for 8 genes in tissues from C. canephora. The web-based RefFinder tool, which combines the geNorm, NormFinder, Bestkeeper, and Delta-Ct algorithms, was employed to assess the stability of the tested genes. The most stable reference genes identified for all tissues grouped (WW/WD) of C. arabica were clathrin adaptor protein medium subunit (AP47), ubiquitin (UBQ), 60S ribosomal protein L39 (RPL39), and elongation factor 1α (EF1α), while class III alcohol dehydrogenase (ADH2), β-actin (ACT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and ubiquitin (UBQ) genes were the most stable for all tissues grouped (WW) of C. canephora tissues. Validation by the expression level analysis of CaACO-like demonstrated that the use of the best and the worst set of reference genes produced different expression results. The results reinforce the general assumption that there is no universal reference gene and that it is essential to select the most appropriate gene for each individual experiment to apply adequate normalization procedures of RT-qPCR data.     

Evaluation of the Clearview<Superscript>®</Superscript> malaria pLDH malaria rapid diagnostic test in a non-endemic setting

Background

Malaria Rapid Diagnostic Tests (RDTs) are widely used to diagnose malaria. The present study evaluated a new RDT, the Clearview^® Malaria pLDH test targeting the pan-Plasmodium antigen lactate dehydrogenase (pLDH).

Methods

The Clearview^® Malaria pLDH test was evaluated on fresh samples obtained in returned international travellers using microscopy corrected by PCR as the reference method. Included samples were Plasmodium falciparum (139), Plasmodium vivax (22), Plasmodium ovale (20), Plasmodium malariae (7), and 102 negative.

Results

Overall sensitivity for the detection of Plasmodium spp was 93.2%. For P. falciparum, the sensitivity was 98.6%; for P. vivax, P. ovale and P. malariae, overall sensitivities were 90.9%, 60.0% and 85.7% respectively. For P. falciparum and for P. vivax, the sensitivities increased to 100% at parasite densities above 100/μl. The specificity was 100%. The test was easily to perform and the result was stable for at least 1 hour.

Conclusion

The Clearview^® Malaria pLDH was efficient for the diagnosis of malaria. The test was very sensitive for P. falciparum and P. vivax detection. The sensitivities for P. ovale and P. malariae were better than other RDTs

     

Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production

Background

The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis.

Results

The pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain.

Conclusions

Pyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.

     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

Comparison of L-lactate dehydrogenases of different origins produced in the cells of yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The expression of L-lactate dehydrogenase genes ldh1 (Bos taurus), ldhA (Homo sapiens), ldhA (Rhizopus oryzae), ldh1 (Lactobacillus plantarum), and ldh1 (Lactobacillus pentosus) in the cells of yeast Schizosaccharomyces pombe VKPM U-3106 has been investigated. The catalytic characteristics of the enzymes encoded by these genes have been compared, and the intensity of lactic acid synthesis by the recombinant strains obtained has been evaluated. The enzymatic activity of L-lactate dehydrogenases from L. plantarum and L. pentosus was the highest (approximately 2 to 2.5 times higher than that of the mammalian enzymes), and these enzymes therefore appear to have the highest potential for the development of lactic-acid producing strains of yeast S. pombe.     

No need to breed for enhanced colonization by arbuscular mycorrhizal fungi to improve low-P adaptation of West African sorghums

Background and aims

Herbaspirillum seropedicae (Hs) Z67 a diazotrophic endophyte was genetically engineered for secretion of 2-keto-D-gluconic acid by heterologous expression of genes for pqq synthesis and gluconate dehydrogenase to study its beneficial effect on plants.

Methods

Two plasmids, pJNK5, containing a 5.1 Kb pqq gene cluster of Acinetobacter calcoaceticus and pJNK6, carrying in addition the Pseudomonas putida KT2440 gluconate dehydrogenase (gad) operon were constructed in pUCPM18Gm^r under Plac promoter. H. seropedicae Z67 transformants were monitored for P and K solubilization, cadmium (Cd) tolerance and rice growth promotion.

Results

Hs (pJNK5) secreted 23.5 mM gluconic acid and Hs (pJNK6) secreted 3.79 mM gluconic acid and 15.8 mM 2-ketogluconic acid respectively. Under aerobic conditions, Hs (pJNK5) and Hs (pJNK6) solubilized 239.7 μM and 457.7 μM P on HEPES rock phosphate and, 76.7 μM and 222.7 μM K on HRPF (feldspar), respectively, in minimal medium containing 50 mM glucose. Under N free minimal medium, similar effects of P and K solubilization were obtained. Hs (pJNK5) and Hs (pJNK6) inoculation increased the biomass, N, P, K content of rice plants (Gujarat – 17). These plants also accumulated 0.73 ng/g PQQ, and had improved growth and tolerance to CdCl₂.

Conclusions

Incorporation of pqq and gad gene clusters in H. seropedicae Z67 imparted additional plant growth promoting traits of P and K solubilization and ability to alleviate Cd toxicity to the host plant.

     

Isozyme Analysis in a Genetic Collection of Amaranths (<Emphasis Type="Italic">Amaranthus</Emphasis> L.)

In various populations of the cultivated and weedy amaranth species, the electrophoretic patterns of alcohol dehydrogenase (ADH), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and malic enzyme (Me) were studied. In total, 52 populations and two varieties (Cherginskii and Valentina) have been examined. Allozyme variation of this material was low. Irrespective of species affiliation, 26 populations and two varieties were monomorphic for five enzymes; a slight polymorphism of three, two, and one enzymes was revealed in three, nine, and fourteen populations, respectively. A single amaranth locus, Adh, with two alleles, Adh F and Adh S, controls amaranth ADH. Two alleles, common Gdh S and rare Gdh F, control GDH; no heterozygotes at this locus were found. The MDH pattern has two, the fast- and slow-migrating, zones of activity (I and II, respectively). Under the given electrophoresis conditions, the fast zone is diffuse, whereas slow zone is controlled by two nonallelic genes, monomorphic Mdh 1 and polymorphic Mdh 2 that includes three alleles: Mdh 2-F, Mdh 2-N, and Mdh 2-S. Low polymorphism of IDH and Me was also found, though their genetic control remains unknown.     

Identification of D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum.     

The mitochondrial DNA of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>: complete sequence,gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564?bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4?kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii.     

Use of the yeast-like cells of <Emphasis Type="Italic">Tremella fuciformis</Emphasis> as a cell factory to produce a <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> hydrophobin

Objectives

To obtain hydrophobin, a Class I hydrophobin gene, Po.hyd from Pleurotus ostreatus, was transformed into the yeast-like cells of Tremella fuciformis using Agrobacterium tumefaciens.

Results

The hydrophobin Po.HYD from P. ostreatus was heterogeneously expressed by the yeast-like cells of T. fuciformis. Plasmids harboring the Po.hyd gene driven by endogenous glyceraldehyde-3-phosphate dehydrogenase promoter were transformed by A. tumefaciens. The integration and expression of the rPo.HYD in the T. fuciformis cells were confirmed by PCR, Southern blot, fluorescence microscopy and quantitative real-time PCR. SDS-PAGE demonstrated that the rPo.HYD was extracted with the expected MW of 14 kDa. The yield of purified rPo.HYD was 0.58 mg/g dry wt. The protein, with its ability to stabilize oil droplets, exhibited a better emulsifying activity than the typical food emulsifiers Tween 20 and sodium caseinate.

Conclusion

Tremella fuciformis can be used as a cell factory to produce hydrophobin on a large scale for the food industry.

     

Phylogenetic study of clavicipitaceous fungi using acetaldehyde dehydrogenase gene sequences

Aciculosporium and Heteroepichloë (Clavicipitaceae) are characteristic bambusicolous fungi in east Asia. In this study, we examined their intergeneric relationships based on the ALDH1-1 gene, which encodes a member of the aldehyde dehydrogenase family. In the clavicipitaceous fungi examined in this study, the nucleotide sequence of the third exon of ALDH1-1 (Exon-3) is 889 bp in length and has no insertion/deletion. A phylogenetic tree based on Exon-3 indicated that the clavicipitaceous fungi could be divided into two large groups: Cordyceps, Nomuraea, and Ustilaginoidea species formed a paraphyletic group, and the other grass biotrophic species formed a monophyletic group. This monophyletic group was further divided into three groups with high bootstrap support: i.e., species with Neotyphodium anamorphs (e.g., Epichloë), species with Ephelis anamorphs (e.g., Heteroepichloë), and Aciculosporium-Claviceps species. We discuss the relationships among Aciculosporium, Heteroepichloë, and other clavicipitaceous fungi.