Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30°C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170 kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity. Received: September 17, 1999 / Accepted: March 21, 2000     

Formate hydrogenlyase in the hyperthermophilic archaeon, <Emphasis Type="Italic">Thermococcus litoralis</Emphasis>

Background  

Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis.     

Cloning and characterization of EstC from Burkholderia gladioli, a novel-type esterase related to plant enzymes

By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated. High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv. syringae. The sequenced ORF encodes for a protein of 298 amino acids. The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities. The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed. Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized. Received: 8 January 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Site-directed chemical modification of archaeal Thermococcus litoralis Sh1B DNA polymerase: Acquired ability to read through template-strand uracils

We present site-directed chemical modification (SDCM), a tool for engineering U-resistant archaeal DNA polymerases of family B. The Thermococcus litoralis Sh1B DNA polymerase (GenBank: GQ891548) was chosen as the object of the study. Similar to D.Tok, Kod1, Pfu, Tgo and other archaeal members of this family, the T. litoralis Sh1B DNA polymerase is a domain structured, proofreading-proficient enzyme that has the polymerization and 3′→5′ DNA exonucleolytic activities and contains N-terminally located highly conserved template-strand U-binding pocket. The tight binding of template uracil in the enzyme pocket during polymerization blocks the replication of DNA containing uracils. This effect can be alleviated by mutations in key amino acids of the U-binding pocket. We altered T. litoralis Sh1B DNA polymerase's ability to read through the template-strand uracils by applying SDCM. Specific modification of individual cysteine residues in U-binding pocket — targets introduced into certain positions by site-directed mutagenesis — enables the enzyme to effectively replicate DNA containing uracils. We demonstrate that the acquired resistance of chemically modified T. litoralis Sh1B DNA polymerase to DNA uracil correlates with its decreased affinity for template-strand uracil.     

Identification of a Cytokinin, 6-(3 Methyl-2-butenylamino) purine, in Sea Water and the Effect of Cytokinins on Brown Algae

Using combined gas chromatography and mass spectrometry a growth-promoting, organic component of sea water has been quantitatively estimated and identified as the cytokinin 6-(3 methyl-2-butenylamino) purine. Additions of this cytokinin, kinetin or sea water from the Fucus-Ascophyllum zone increased the growth of Ectocarpus confervoides and Pylaiella litoralis in artificial sea water. Additions of kinetin to bacteria-free cultures of Ectocarpus fasciculatus also initiated the production of upright ectocarpoid filaments.     

Production of d-amino acids using immobilized d-hydantoinase from lentil, Lens esculenta, seeds

d-Hydantoinase from the lentil, Lens esculenta, seed is quite unstable, and has been immobilized on Diethyl amino ethyl (DEAE) cellulose by an adsorption and cross-linking method. The immboilized d-hydantoinase exhibited 80% enzyme activity and contained 86% protein. The immobilization of the enzyme preparation does not change its optimum pH, temperature or affinity constant, but increases its shelf-life, thermostability and stability in various organic solvents. This immobilized d-hydantoinase can be used effectively for the production of d-amino acids from the corresponding hydantoins and may therefore be of use in the chemical and pharmaceutical industries. Received: 28 April 1998 / Received last revision: 10 July 1998 / Accepted: 10 July 1998     

Characterization of Amylolytic Enzymes, Having Both α-1,4 and α-1,6 Hydrolytic Activity, from the Thermophilic Archaea Pyrococcus furiosus and Thermococcus litoralis

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90°C) and Pyrococcus furiosus (optimal growth temperature, 98°C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca²⁺, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140°C. The addition of Ca²⁺ also affected substrate binding, as evidenced by a decrease in K_m for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the α-1,6 linkages in pullulan, α-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller α-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.     

Identification and molecular characterisation of new Mexican isolates of Pochonia chlamydosporia for the management of Meloidogyne spp.

New Mexican isolates of the nematophagous fungus Pochonia chlamydosporia were obtained from nematode infested fields in the vegetable growing area of Tepeaca Valley, Puebla State, Mexico. Based on macro and microscopic morphology, seven ‘putative’ P. chlamydosporia isolates were selected and the DNA extracted for polymerase chain reaction (PCR). Three new isolates of P. chlamydosporia were identified: Pcp2, Pcp21 and Pcp31. The amplification reaction of the internal transcribed spacer (ITS) region revealed a 650 bp amplicon which was used in a maximum likelihood phylogenetic inference analysis. Three groups were recovered in the tree topology, supported by a > 90% bootstrap value. Nucleotide identity values were > 83.6% between the test sequences and the reference sequence. In addition, using specific primers for two existing varieties of P. chlamydosporia, restriction fragment length polymorphism on the ITS products in conjunction with the phylogenetic inferences and the molecular test for detection of P. chlamydosporia vcp1 gene, it was found that all three isolates belong to a new variety which we have named P. chlamydosporia var. mexicana. We compared the chlamydospore production rate, rhizosphere colonisation and egg parasitism percentages of the three native isolates in Meloidogyne spp. with a reference isolate (Pc10). Native isolates produced > 1×10⁶ chlamydospores/50 g of substrate (of which more than 80% were viable), colonised > 80% of the rhizosphere, and parasitised > 60% of Meloidogyne incognita and Meloidogyne arenaria eggs. Meloidogyne hapla egg parasitism was < 60%. Isolates Pcp2 and Pcp21 were identified as potential biological control agents of Meloidogyne spp. to be tested further in greenhouse and field tests.     

Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi

Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn²⁺ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a ¹⁴C-ring-labelled synthetic lignin (¹⁴C-DHP). The fungi mineralised around 25% of the lignin to ¹⁴CO₂ within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn²⁺ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature. Received: 20 April 2000 / Received revision: 12 July 2000 / Accepted: 16 July 2000     

Molecular cloning and characterization of a chitosanase from the chitosanolytic bacterium Burkholderia gladioli strain CHB101

A chitosanase was purified from the culture fluid of the chitino- and chitosanolytic bacterium Burkholderia gladioli strain CHB101. The purified enzyme (chitosanase A) had a molecular mass of 28 kDa, and catalyzed the endo-type cleavage of chitosans having a low degree of acetylation (0–30%). The enzyme hydrolyzed glucosamine oligomers larger than a pentamer, but did not exhibit any activity toward N-acetyl-glucosamine oligomers and colloidal chitin. The gene coding for chitosanase A (csnA) was isolated and its nucleotide sequence determined. B. gladioli csnA has an ORF encoding a polypeptide of 355 amino acid residues. Analysis of the N-terminal amino acid sequence of the purified chitosanase A and comparison with that deduced from the csnA ORF suggests post-translational processing of a putative signal peptide and a possible substrate-binding domain. The deduced amino acid sequence corresponding to the mature protein showed 80% similarity to the sequences reported from Bacillus circulans strain MH-K1 and Bacillus ehimensis strain EAG1, which belong to family 46 glycosyl hydrolases. Received: 30 July 1999 / Revised revision: 17 February 2000 / Accepted: 25 February 2000     

Microbial formation, biotechnological production and applications of 1,2-propanediol

This short review covers metabolic pathways, genetics and metabolic engineering of 1,2-propanediol formation in microbes. 1,2-Propanediol production by bacteria and yeasts has been known for many years and two general pathways are recognized. One involves the metabolism of deoxyhexoses, where lactaldehyde is formed during the glycolytic reactions and is then reduced to 1,2-propanediol. The second pathway derives from the formation of methylglyoxal from dihydroxyacetonephosphate and its subsequent reduction to 1,2-propanediol. The enzymes involved in the reduction of methylglyoxal can generate isomers of lactaldehyde or acetol, which can be further reduced by specific reductases, giving chiral 1,2-propanediol as the product. The stereospecificity of the enzymes catalyzing the two reduction steps is important in deriving a complete pathway. Through genetic engineering, appropriate combinations of enzymes have been brought together in Escherichia coli and yeast to generate 1,2-propanediol from glucose. The optimization of these strains may yield microbial processes for the production of this widely used chemical. Received: 25 May 2000 / Received revision: 24 July 2000 / Accepted: 25 July 2000     

Two new species of Isochrysis with remarks on the genus Ruttnera

Two marine strains of Chrysophyceae have been studied with the aid of light and electron microscopy. The benthic stages show definite similarities with two marine Gloeochrysis spp. (G. maritima and G. litoralis) described by Anand (1937), but the swarmers do not agree with the definition of this genus. The reason why they also cannot be assigned to the genus Ruttnera is discussed and their inclusion within the Isochrysidales is justified by the observation of haptophycean scales on the motile cells. The names Isochrysis maritima sp. nov. and Isochrysis litoralis sp. nov. are proposed in view of the fact that these strains might prove to be the species observed by Anand.     

Petraliellidae Harmer, 1957 (Bryozoa: Cheilostomata) from Queensland,Australia

Several species of the family Petraliellidae were first described from the coast of Queensland, including the type species of Sinupetraliella, S. litoralis. These species are redescribed from type, or topotype specimens, and include Petraliella concinna, which has not been certainly found since its introduction in 1891; lectotype material is designated for P. buski and P. magna. Six other species are described from Queensland, P. crassocirca, P. dentilabris, P. dorsiporosa, Mucropetraliella bennetti, M. serrata and M. tuberosa. The species Mucropetraliella tuberosa is a new addition to the Queensland fauna and is described and illustrated here for the first time since its introduction in 1884. The characters of the family Petraliellidae are briefly discussed and the genera to which the Queensland species are assigned is reviewed. A taxonomic key to the ten Queensland petraliellid species described is also provided.     

Enzymes and proteins from extremophiles as hyperstable probes in nanotechnology: the use of D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus litoralis</Emphasis> for sugars monitoring

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose (Mal) uptake system. In the hyperthermophilic archaeon Thermococcus litoralis, this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds sugars, such as maltose, trehalose and glucose, with high affinity. In this work, we have immobilized TMBP on a porous silicon wafer. The immobilization of TMBP to the chip was monitored by reflectivity and Fourier Transformed Infrared spectroscopy. Furthermore, we have tested the optical response of the protein-Chip complex to glucose binding. The obtained data suggest the use of this protein for the design of advanced optical non-consuming analyte biosensors for glucose detection. The authors wish to dedicate this work to Prof. Ignacy Gryczynski, University of North Texas, TX, USA, for his outstanding contribution to the development of new sensing methodologies.     

Genetic transformation of the C3–C4 intermediate plant, Flaveria pubescens (Asteraceae)

The dicot genus Flaveria (Asteraceae), besides species with C₃ or C₄ photosynthesis, contains taxa with a broad range of different states of transition between the two major photosynthetic types. We have developed a reproducible and efficient Agrobacterium-mediated method for the stable genetic transformation of the C₃–C₄ intermediate species F. pubescens. Fusion constructs of the reporter gene β-glucuronidase (uidA, GUS) to several plant promoters, mainly derived from genes encoding subunits of the glycine cleavage system (gdcs), have been used to confirm the reproducibility and efficiency of the method. The stable integration of the foreign DNA has been examined by Southern analysis, kanamycin resistance, GUS enzyme activities and histochemical staining. Transformed shoots can be routinely obtained within 8–10 weeks after co-cultivation with A. tumefaciens. Received: 16 April 1996 / Revision received: 12 July 1996 / Accepted: 28 July 1996     

Enzymology of Phanerochaete chrysosporium with respect to the degradation of recalcitrant compounds and xenobiotics

The archetypal white-rot fungus Phanerochaete chrysosporium has been shown to degrade a variety of persistent environmental pollutants. Many of the enzymes responsible for pollutant degradation, which are normally involved in the degradation of wood, are extracellular. Thus, P. chrysosporium is able to degrade toxic or insoluble chemicals more efficiently than other microorganisms. P. chrysosporium has a range of oxidative and reductive mechanisms and uses highly reactive, nonspecific redox mediators which increase the number of chemicals that can be effectively degraded. This review gives an overview of the enzymes that are believed to be important for bioremediation and briefly discusses the degradation of some individual chemicals. Received: 25 April 2000 / Received revision: 05 June 2000 / Accepted: 04 July 2000     

Foraging behavior of the American oystercatcher Haematopus palliatus pitanay (Murphy 1925) on the intertidal ascidian Pyura praeputialis (Heller 1878) in the Bay of Antofagasta, Chile

Oystercatcher foraging behavior has been described for diverse intertidal prey such as limpets, mussels, and oysters. This paper describes foraging behavior of the American oystercatcher, Haematopous palliatus pitanay, on attached and wave-dislodged ascidians, Pyura praeputialis (prey with a restricted geographic range of 70 km) in the Bay of Antofagasta, Chile. Stabbed holes on the top of the ascidian's tunic, probing excursions, handling time, and five prey-handling sequential stages (striking, hammering, prying, cavity food searching, and swallowing) are described and measured. The need to determine ascidian profit-ability for oystercatcher species in Australia and Chile is highlighted. Received: January 19, 2000 / Accepted: July 23, 2000