Archaebacterial class I and class II aldolases from extreme halophiles

Both, class I (Schiff-base forming) and class II (metal requiring) fructose biphosphate aldolases were found to be distributed among halophilic archaebacteria. The aldolase activity fromHalobacterium halobium, H. salinarium, H. cutirubrum, H. mediterranei andH. volcanii exhibited properties of a bacterial class II aldolase as it was metal-dependent for activity and therefore inhibited by EDTA. In contrast, aldolase fromH. saccharovorum, Halobacterium R-113, H. vallismortis andHalobacterium CH-1 formed a Schiff-base intermediate with the substrate and therefore resembled to eukaryotic class I type. The type of aldolase did not vary by changes in the growth medium.     

TWO CLASS I ALDOLASES IN KLEBSORMIDIUM FLACCIDUM (CHAROPHYCEAE): AN EVOLUTIONARY LINK FROM CHLOROPHYTES TO HIGHER PLANTS1

Two fructose-bisphosphate aldolases(EC 4.1.2.13) from Klebsormidium flaccidum Silver, Mattox and Black-well were purified by affinity elution from phosphocellulose. The two enzymes were subsequently separated by HPLC on an anion-exchange column (QAE-silica). The aldolase eluting first represented 5% of the total activity; the other aldolase represented the remaining activity. The activity of the enzymes was not reduced by the presence of 1 mM EDTA or increased by 0.1 mM Zn²⁺, establishing their character as class I type (Me²⁺ independent) aldolases. The K_m(fructose-1,6-bisphosphate) values were 1.7 and 34.7 μM for the enzyme eluting first and second, respectively, from the QAE-silica column. The subunit molecular masses, as determined by SDS-PACE, were 40.5 and 37 kD; the specific activities of the purified enzymes were 7.9 and 24.7 · mg^?1 protein, respectively. The two aldolases of K. flaccidum are homologous to the cytosol and chloroplast specific isoenzymes of higher plants by several criteria and are therefore probably located in the same cellular compartments in K. flaccidum. The K_m and specific activity for the chloroplast aldolase of K. flaccidum are three times higher than for the chloroplast aldolase of higher plants, a remarkable difference. Immunotitration with specific antisera against the chloroplast aldolase of Chlamydomonas reinhardtii Dangeard and spinach showed that the chloroplast aldolase of K. flaccidum was immunochemically intermediate in structure to the respective aldolases of C. reinhardtii and higher plants. K. flaccidum is the second species of Charophyceae (besides Chara foetida Braun) with two class I aldolases as in higher plants whereas two species of Chlorophyceae have only one class I aldolase and, under some conditions, an additional class II (Me²⁺ dependent) aldolase. Thus, aldolases may turn out, in addition to the known enzymes of glycolate conversion and urea degradation, be a novel enzyme system to evaluate algal evolution along with cytological features.     

Triosephosphate isomerases and aldolases from light- and dark-grown Euglena gracilis

Euglena gracilis synthesizes two distinct types of triosephosphate isomerase which can be resolved by isoelectric focusing. The more acidic Type A isomerase (pI = 4.4) predominates when cells are grown photoautotrophically and is localized in the chloroplasts. The Type B isoenzyme exhibits a more basic isoelectric pH (pI = 4.8), predominates under heterotrophic growth conditions and is of cytoplasmic origin. The two isoenzymes exhibit similar molecular weights (56,000–60,000) and catalytic properties but can be distinguished by their pH activity profiles. The situation parallels that of fructose diphosphate aldolase where a chloroplastic Class I enzyme (pI = 4.6, M_r 120,000) found in autotrophically grown cells can be resolved from the cytoplasmic Class II (pI = 5.7, M_r 88,000) enzyme which predominates under heterotrophic conditions. Inhibition of chloroplastic 70S ribosomal synthesis by chloramphenicol blocks the formation of the Type A triosephosphate isomerase and the Class I aldolase.     

Long chain polyunsaturated fatty acid production and partitioning to triacylglycerols in four microalgae

Gas chromatographic profiling of fatty acids was performed during the growth cycle of four marine microalgae in order to establish which, if any, of these could act as a reliable source of genes for the metabolic engineering of long chain polyunsaturated fatty acid (LC-PUFA) synthesis in alternative production systems. A high-throughput column based method for extraction of triacylglycerols (TAGs) was used to establish how much and at what stage in the growth phase LC-PUFAs partition to storage lipid in the different species. Differences in the time course of production and incorporation of docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) into TAGs were found in the marine microalgae Nannochloropsis oculata (Eustigmatophyceae), Phaeodactylum tricornutum and Thalassiosira pseudonana (Bacillariophyceae), and the Haptophyte Pavlova lutheri. Differences were not only observed between species but also during the different phases of growth within a species. A much higher percentage of the total cellular EPA was partitioned to TAGs in stationary phase cells of N. oculata compared to P. tricornutum. Although P. tricornutum produces DHA it does not partition it to TAGs. Both T. pseudonana and P. lutheri produce EPA and DHA and partition these to TAGs during the stationary phase of growth. These two species are therefore good candidates for further biochemical and molecular analysis, in order to understand and manipulate the processes that are responsible for the incorporation of LC-PUFAs into storage oils.     

Fructose diphosphate aldolase-class I (Schiff base) fromMycobacterium tuberculosis H37Rv

An aldolase was partially purified from fermenter grownMycobacterium tuberculosis H₃₇Rv cells. The aldolase has a molecular weight of 150,000, possesses a tetrameric structure and cleaves both fructose diphosphate and fructose-1-phosphate, the former being cleaved 17 times faster. The enzyme was inactivated by treatment with NaBH₄ in the presence of fructose diphosphate or dihydroxyacetone, phosphate suggesting Schiff base formation during its catalytic function. Thiol reagents, EDTA and metal ions had no apparent effect on the aldolase activity. These results show that aldolase is of Class I type. However, this enzyme, unlike the mammalian Class I aldolase, was unaffected by carboxypeptidase A. N-ethylmaleiniide and dithionitrobenzoic acid.     

Effect of Some Unicellular Algae on Escherichia coli Populations in Sea Water and Oysters

Acrylic acid was detected in sea water in which oysters had been maintained and was shown to be derived from unicellular algae containing dimethyl-β-propiothetin (DMPT). Anticoliform activity was noted in samples containing acrylic acid. Intensification of the effect was achieved in laboratory studies and Phaeodactylum tricornutum (DMPT present) caused a rapid decline of Escherichia coli populations in sea water and in oysters which had been fed this alga. Pavlova lutheri (DMPT absent) was ineffective against E. coli .     

Purification and characterization of a class I fructose 1,6-bisphosphate aldolase from Staphylococcus carnosus

Summary A fructose 1,6-bisphosphate aldolase (E.C.4.1.2.13) from Staphylococcus carnosus DSM 20501 was purified for the first time. The enzymatic activity was insensitive to high levels of EDTA indicating that the enzyme is a class I aldolase. This enzyme exhibits good stability at high temperatures and extreme stability over a wide pH range. The K _m for fructose 1,6-bisphosphate as substrate was 0.022 mm. The S. carnosus aldolase is a monomeric enzyme with a molecular mass of about 33 kDa. It exhibits a relatively broad pH optimum between pH 6.5 and 9.0. Furthermore, the aldolase accepts other aldehydes in place of its natural substrate, glyceraldehyde 3-phosphate, allowing the synthesis of various sugar phosphates. Offprint requests to: M. R. Kula     

Morphology of the petal inAconitum

The petals ofAconitum were classified into six types. Type I: the labium tubular at the base and no appendage inside. Type II: a lambda (A)-shaped enation present inside the limb. The upper part of the enation is situated at the lower edge of the spur mouth and both wings of the enation extend to margins of the labium. Type III resembles type II but both wings do not extend to the margins. Type IV: a small flap attached at the lower edge of the spur mouth. Type V: two auriculate appendages present on both lateral walls of the labium. Type VI: without inside appendage. Most species of sect.Lycoctonum have type I petal and those of sect.Aconitum have type V petal. Type I is distinctly cup-shaped or peltate with a well developed cross zone or adaxial wall and type II is a modification of type I. Type VI is distinctly flat or epeltate without the cross zone. Others are intermediate between cup-shaped and flat or peltate and epeltate types. Based on the observation of petal ontogeny onA. pterocaule var.glabrescens, A. vulparia andA. japonicum var.eizanense, the relation among these types was explained by the partial or total reduction of the adaxial meristematic regions.     

Role of Aldolase in Photosynthesis. II Demonstration of Aldolase Types in Photosynthetic Organisms

Spinach leaves and photoautotrophically grown Euglena and Chlorella possess fructose 1,6-diphosphate aldolases inhibited by p-chloromercuribenzoate but insensitive to K⁺ or ethylenediamine tetraacetate (Type I). Dark grown Euglena and Chlorella have aldolases inhibited by p-chloromercuribenzoate and ethylenediamine tetraacetate but stimulated by K⁺ (Type II). The red alga, Chondrus, and the golden-brown alga, Ochromonas, appear to possess both types. Bean, pea, and spinach seeds and the leaves and cotyledons of etiolated bean seedlings contain a p-chloromercuribenzoate insensitive, apparently non-sulfhydryl variant of Type I. Sensitivity of leaf aldolase to p-chloromercuribenzoate occurs in etiolated bean seedlings only after an extended period of illumination. Type II aldolase activity in cell-free extracts of 4 blue-green algae has been demonstrated.     

Modification of the PthA4 effector binding elements in Type I CsLOB1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit alleviating XccΔpthA4:dCsLOB1.3 infection

Citrus canker caused by Xanthomonas citri subspecies citri (Xcc) is a severe disease for most commercial citrus cultivars and responsible for significant economic losses worldwide. Generating canker‐resistant citrus varieties will provide an efficient and sustainable solution to control citrus canker. Here, we report our progress in generating canker‐resistant grapefruit by modifying the PthA4 effector binding elements (EBEs) in the CsLOB1 Promoter (EBE_PthA4‐CsLOBP) of the CsLOB1 (Citrus sinensis Lateral Organ Boundaries) gene. CsLOB1 is a susceptibility gene for citrus canker and is induced by the pathogenicity factor PthA4, which binds to the EBE_PthA4‐CsLOBP to induce CsLOB1 gene expression. There are two alleles, Type I and Type II, of CsLOB1 in Duncan grapefruit. Here, a binary vector was designed to disrupt the PthA4 EBEs in Type I CsLOB1 Promoter (TI CsLOBP) via epicotyl transformation of Duncan grapefruit. Four transgenic Duncan plants with targeted modification of EBE_PthA4‐T1 CsLOBP were successfully created. As for Type I CsLOB1 promoter, the mutation rate was 15.63% (#D13), 14.29% (#D17), 54.54% (#D18) and 81.25% (#D22). In the presence of wild‐type Xcc, transgenic Duncan grapefruit developed canker symptoms similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not the mutated Type I CsLOBP or Type II CsLOBP, was developed to infect Duncan transformants. Consequently, #D18 had weakened canker symptoms and #D22 had no visible canker symptoms in the presence of XccΔpthA4:dCsLOB1.3. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by PthA4 is sufficient to induce citrus canker disease, and mutation in the promoters of both alleles of CsLOB1 is probably required to generate citrus canker‐resistant plants. This work lays the groundwork to generate canker‐resistant citrus varieties via Cas9/sgRNA in the future.     

Role of isozyme group-specific sequence 4 in the isozyme-specific properties of human aldolase C

To assess which regions of the aldolase C molecule are required for exhibiting isozyme-specific kinetic properties, we have constructed nine chimeric enzymes of human aldolases A and C. Kinetic studies of these chimeric enzymes revealed that aldolase C absolutely required its own isozyme group-specific sequences (IGS), particularly IGS-4, for exhibiting the characteristics of aldolase C which differ significantly from those of isozymes A and B (Kusakabe T, Motoki K, Hori K. Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli. J Biochem (Tokyo) 1994;115:1172–7). Whereas human aldolases A and B required their own isozyme group-specific sequences-1 and -4 (IGS-1 and -4) as the main determinants of isozyme-specific kinetic properties (Motoki K, Kitajima Y, Hori K. Isozyme-specific modules on human aldolase A molecule. J Biol Chem 1993;268:1677–83; Kusakabe T, Motoki K, Sugimoto Y, Takasaki Y, Hori K. Human aldolase B: liver-specific properties of the isoenzyme depend on type B isozyme group-specific sequence. Prot. Eng. 1994;7:1387–93), the present studies indicate that the IGS-1 is principally substitutable between aldolases A and C. The kinetic data also suggests that the connector-2 (amino acid residues 243–306) may modulate the interaction of IGS units with the α/β barrel of the aldolase molecule.     

STUDIES OF CARBON AND NITROGEN METABOLISM BY THREE MARINE PHYTOPLANKTON SPECIES IN NITRATE-LIMITED CONTINUOUS CULTURE1,2

Characteristics of carbon production, excretion and dark respiration, and nitrate uptake kinetics were studied using continuous culture techniques for Thalassiosira allenii Takano, Monorhrysis lutheri Droop and Dunaliella tertiolccta Butcher. Fur T. allenii. the ratio of dark C loss to daytime net C production varied between 0.1 and 0.2 over a growth rate range from ca. 0.005 to 0.06 h^-1. For M. lutheri and D. tertiolecta. this same ratio varied belween 0.2 and 0.3 between growth rates of ca. 0.005 and 0.025 h^-1, but declined at higher growth rates when the dark nitrate uptake capacity of the cells was exceeded by the pumping rate. Carbon excretion rates averaged less than 1.5% of daytime net C production rates. Productivity indices showed little correlation with growth rate, due to the significant poisitive correlation between chl a:C ratios and growth rate. Chlorophyll a:C ratios for T. allenii were less than 0.01 al growth rates less than 0.03 h^-1, and appoached zero at zero growth rate. Dark nitrate maximum uptake rates for M. lutheri, D. tertiolecta and T. allenii averaged 23, 64 and 120%, respectively, of light nitrate maximum uptake rates. Excretion of nitrite was observed during most nitrate uptake experiments. This excretion reduced net uptake of nitrate spikes in the dark for M. lutheri and D. tertiolecta by 79 and 23%, respectively.     

Localization of the enzyme 3-deoxy-d-manno-2-octulosonic acid aldolase

Summary Several strains of Gram-negative microorganisms were screened for maximum 3-deoxy-d-manno-2-octulosonic acid (KDO) aldolase (EC 4.1.2.23) activity. Although this enzyme has been noted to be inducible on special medium, no induction was found. By centrifugation studies the KDO aldolase was found to be localized in the cell wall or membrane fraction. The enzyme activity was very susceptible to small amounts of detergent in solution. Offprint requests to: M.-R. Kula     

Mutations of the structural gene of 2-keto-3-desoxy-6-P-gluconate aldolase in E. coli K 12

Summary In order to determine the nature of KDPG-aldolase negative mutations (described in a recent paper) we have studied revertants to wild type. The structure of restored KDPG-aldolase in two revertants is very different with regard to wild type aldolase activity (modified thermosensibility, K_m and V_M). These restored aldolases like the wild type aldolase are under the control of the regulator gene (kdg R). The observation that one of these revertants maps in the eda locus demonstrates that this locus is the structural gene of KDPG-aldolase in E. coli K 12.     

Molecular and morphological analyses revealed a cryptic species of dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) in Japan

Although it has been reported that populations of the Japanese dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) belong to two distinct mitochondrial (mt)DNA (Type I and Type II), the taxonomic status of the species remains unresolved. To address this question, nuclear DNA and morphological analyses were performed on M. anguillicaudatus population in the Nakaikemi Wetland, where Type I and Type II lineages are sympatric. Results suggest the existence of a cryptic species (Type I) within the Japanese dojo loach.     

D-Glucosaminate Aldolase Activity of D-Glucosaminate Dehydratase from Pseudomonas fluorescens and Its Requirement for Mn2+ Ion

When D-glucosaminate dehydratase (GADH) was incubated with D-glucosaminate (GlcNA) in veronal buffer (VB; 0.01 M, pH 8.0), GlcNA was converted stoichiometrically to glyceraldehyde, pyruvate, and ammonia (aldolase reaction A). This reaction occurred in addition to the dehydratase reaction (conversion of GlcNA to 2-keto-3-deoxy-o-gluconate and ammonia: α,β-elimination reaction, B). The ratio of the activities (A:B) was about 1:4. However, in potassium phosphate buffer (KPB; 0.04 M, pH 8.0), the aldolase reaction was inhibited to 3–4% of that in VB, and also inhibited by various derivatives of glycerol, in particular, glycerol-3-phosphate (glycerol-3-P) and glyceraldehyde-3-phosphate (glyceraldehyde-3-P) in VB. The native enzyme was inhibited by incubation with 0.1 M EDTA, and the activity was restored by incubation of the EDTA-treated enzyme with (Mn²⁺ + pyridoxal 5′-phosphate (PLP)). When the EDTA-treated enzyme was incubated with (Mn²⁺ + PLP + glycerol-3-P), the activity of reaction B increased to 131% but that of reaction A decreased to 21%. These results suggested that Mn²⁺, PLP, and the phosphate group of glycerol-3-P are involved in formation of the active enzyme. In the case of the aldolase reaction, Mn²⁺ ion, which might be essential for the reaction, is chelated by the phosphate group of glycerol-3-P with resultant inhibition of the aldolase reaction.     

Suitability of Selected Marine Algae for Growing the Marine Heterotrich Ciliate Fabrea salina1

ABSTRACT. Forty-five axenically grown algal (sensu lato) species representing six divisions—that is. 13 Chlorophyceae, 14 Chrysophycophyta, five Dinophycophyta, seven Cryptophycophyta, two Rhodophycophyta, and four Cyanochloronta—were aseplicaily presented separately as potential food sources to the marine helerotrich ciliate Fabrea salina under standardized algal number, medium, lighting, and temperature. The algae can be placed into three groups based on their effect on the intrinsic growth rate of the ciliate. Nutritious: Rhodomonas lens, cryptomonad LIS₁, Dunaliella parva, Prasinodadus marinus, Chroomonas salina, D. tertiolecta, Chaeloceros galvestonensis, D. primolecta, Phaeodactylum tricornutum, D. salina, Isochrysis galbana, Cylindrothecaclosterium, cryptomonad strains M₂, WH₂ & FSA, Chroomonas sp., P. lubricus, and Peridinium trochoideum. Maintamers: Cyanobacterium strain Tigriopus blue green, P. triquetum. Monochrysis lutheri, Exuviella gracilis, Platymonas tetrathele. Cyclotella caspa, Crypthecodinium cohnii, Prasinocladus C₅ strain, D. viridis, Nannochloris occulata, Tetraselmis gracilis, Anacystis marinum, Rhodosorus marinum, and Thalassiosira pseudonana. Nonnutritious: Stichococcus immobilis, Hymenomonas sp. strain 150, Syracosphaera sp. strain 181, Tetraselmis verrucosa, Thalassiosira fluviatilis, Microcoleus chthonoplastes, Synechococcus sp., Pavlova gyrans, Prymnesium parvum, Coccolithus huxleyi, Olisthodiscus luteus, Amphidinium carterii, and Porphyridium aerugineum. There was no apparent relationship between a given taxon and the nutritional value of the group, with the possible exception of the Cryptophycophyta.     

Relationship of species composition of tropical seagrass meadows to multiple physical environmental factors

The purpose of this study was to examine the relationship between species composition of tropical seagrasses and various physical environmental factors: depth, sediment thickness and silt–clay content in the sediments. We investigated species composition and abundance of seagrasses as well as the physical environmental factors for six transects around Ishigaki Island, southwest Japan. Eight species occurred in the quadrat census. The occurrence frequencies ranged from 66.8% (Thalassia hemprichii) to 4.5% (Enhalus acoroides). Both canonical correspondence analysis (CCA) and cluster analysis elucidated that depth was mainly responsible for the distributions of species and assemblage type. Monte Carlo permutation for partial CCA revealed that 37.5% of the variance was explained by depth, 10.3% by sediment thickness and 4.6% by silt–clay content in the sediment. Twenty-six sites were categorized into four assemblage types by a cluster analysis using the leaf area index (LAI; the ratio of total leaf area to bottom area) as a measure of species abundance. Type I was dominated by T. hemprichii and Cymodocea rotundata, Type II by C. serrulata, Type III by E. acoroides, and Type IV by Halodule pinifolia and Halophila ovalis. Type I occurred mostly in the intertidal zone (91.3±30.5 cm below MSL, mean sea level), Type II in the subtidal zone (179.1±75.0 cm below MSL) and Type IV in both shallow sites (between 47.8 and 75.6 cm below MSL) and in those with low silt–clay contents (between 2.0 and 3.8%).     

Identification of a fructose-1,6-bisphosphate aldolase gene and association of the single nucleotide polymorphisms with growth traits in the clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis>

This study investigated whether there were single nucleotide polymorphisms (SNPs) in fructose-1,6-bisphosphate aldolase (FBA) gene associated with growth traits of the clam Meretrix meretrix. A FBA gene was identified in M. meretrix and its deduced amino acid residues shared high identity with type I aldolase. The FBA (MmeFBA) mRNA expression profile was examined by real-time PCR in different tissues and the significantly high expression level in foot and adduct muscle suggests that MmeFBA is a muscle type aldolase which functions in glycolytic pathway. In the MmeFBA gene, we identified four intron SNPs and three exon SNPs including a nonsynonymous SNP (mmfbae-2). These SNPs were genotyped in 205 clams from two clam populations with significantly different growth performance. Results showed that allele frequencies of three SNPs (mmfbai-1, mmfbai-3 and mmfbae-2) and the genotype frequency of mmfbai-1 were all significantly different between the two populations. The haplotype analysis further supported the three SNPs distributed differently between the two populations. This study successively characterized three growth-related SNPs in a gene involved in energy metabolism of M. meretrix. These findings could contribute the development of phenotype-selective breeding program in M. meretrix.