Maintenance of the supercooled state in overwintering pyrochroid beetle larvae, Dendroides canadensis : role of hemolymph ice nucleators and antifreeze proteins

Freeze-avoiding fire-colored beetle larvae, Dendroides canadensis, were monitored seasonally to explore the role of endogenous hemolymph ice nucleators and antifreeze proteins on the maintenance of supercooling. In preparation for overwintering, D. canadensis depressed hemolymph ice nucleator activity and increased thermal hysteresis activity [mean value circa 0. 5 °C (summer) versus circa 5 °C (midwinter)] resulting in decreased larval and hemolymph supercooling points [−7 °C (summer) versus −20 °C (midwinter)]. Results of gel filtration chromatography, flotation ultracentifugation and quantitative investigation of ice nucleator activity using hemolymph from summer and winter collected larvae strongly suggest that highly active protein and lipoprotein ice nucleators are removed in preparation for overwintering. Additions of either purified antifreeze proteins or midwinter hemolymph with high antifreeze protein activity to a mixture of protein or lipoprotein ice nucleators isolated from D. canadensis hemolymph inhibited the activity of these nucleators. This suggests that in addition to seasonal removal, inhibition of hemolymph ice nucleators by antifreeze proteins contributes to seasonal increases in hemolymph supercooling capacity. Accepted: 8 August 1996     

Maintenance of the supercooled state in the gut fluid of overwintering pyrochroid beetle larvae, Dendroides canadensis : role of ice nucleators and antifreeze proteins

  The effect of gut fluid ice nucleators and antifreeze proteins on maintenance of supercooling was explored in fire-colored beetle larvae, Dendroides canadensis, via seasonal monitoring of supercooling points, antifreeze protein activity and ice nucleator activity of gut fluid and/or larvae. During cold hardening in the field, freeze-avoiding larvae evacuated their guts and depressed larval supercooling points. Analysis of gut fluid indicated supercooling points and ice nucleator activity decreased, whereas antifreeze protein activity increased as winter approached. Suspensions of bacteria isolated from guts of feeding larvae collected in spring/summer had higher supercooling points than those from midwinter-collected non-feeding larvae, suggesting bacterial ice nucleators are removed from midwinter gut fluid. The ice nucleation active bacterium Pseudomonas fluorescens was isolated from gut fluid of feeding larvae but was absent in winter. When mixed with purified D.␣canadensis hemolymph antifreeze proteins (structurally similar and/or identical to those in gut fluid), the cumulative ice nucleus spectra of P. fluorescens suspensions were shifted to lower temperatures indicating an inhibitory effect on the bacteria's ice-nucleating phenotype. By extending larval supercooling capacity, both gut clearing and masking of bacterial ice nucleators by antifreeze proteins may contribute to overwintering survival in supercooled insects. Accepted: 8 August 1996     

Cryopreservation of Doritaenopsis suspension culture by vitrification

 Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1–3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations. Received: 18 January 2000 / Revision received: 16 June 2000 / Accepted: 22 June 2000     

Harpin induces mitogen-activated protein kinase activity during defence responses in Arabidopsis thaliana suspension cultures

Elicitation of Arabidopsis thaliana (L.) Heynh. suspension cultures with the bacterial protein harpin (from Pseudomonas syringae pv. syringae) induced the activation of two kinases of 39 and 44 kDa, as demonstrated by in-gel kinase assays using myelin basic protein (MBP) as a substrate. Both these kinases appeared to be tyrosine-phosphorylated upon activation, as demonstrated by treatment with tyrosine phosphatase and immunoprecipitation using an anti-phosphotyrosine monoclonal antibody. An inhibitor of mammalian mitogen-activated protein kinase (MAPK) activation, PD98059, inhibited harpin-induced MBPK activation, but did not inhibit the activity of these kinases. PD98059 also inhibited harpin-induced programmed cell death and defence gene expression, suggesting the involvement of harpin-induced MAPKs in defence responses in Arabidopsis thaliana. Received: 23 February 1999 / Accepted: 22 July 1999     

Growth of Photorhabdus luminescens in batch and glucose fed-batch culture

Photorhabdus luminescens, a bacterial symbiont of entomopathogenic biocontrol nematodes, was grown in batch and glucose fed-batch culture. The cell density, bioluminescence, production of antibiotic substances, number of cells with inclusion bodies, glucose concentration and oxygen uptake rate were recorded. The addition of 12.4 g l⁻¹ glucose prolonged the growth, and the yield almost doubled, from 6.85 g l⁻¹ to 12.45 g l⁻¹ dry mass. The production of antibiotic substances increased by 140%. Bioluminescence was higher in the batch culture. A shift of P. luminescens to phase II variants was not detected. Received: 21 January 2000 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

Bioremediation of polychlorinated biphenyl-contaminated soil using carvone and surfactant-grown bacteria

Partial bioremediation of polychlorinated biphenyl (PCB)-contaminated soil was achieved by repeated applications of PCB-degrading bacteria and a surfactant applied 34 times over an 18-week period. Two bacterial species, Arthrobacter sp. strain B1B and Ralstonia eutrophus H850, were induced for PCB degradation by carvone and salicylic acid, respectively, and were complementary for the removal of different PCB congeners. A variety of application strategies was examined utilizing a surfactant, sorbitan trioleate, which served both as a carbon substrate for the inoculum and as a detergent for the mobilization of PCBs. In soil containing 100 μg Aroclor 1242 g⁻¹ soil, bioaugmentation resulted in 55–59% PCB removal after 34 applications. However, most PCB removal occurred within the first 9 weeks. In contrast, repeated addition of surfactant and carvone to non-inoculated soil resulted in 30–36% PCB removal by the indigenous soil bacteria. The results suggest that bioaugmentation with surfactant-grown, carvone-induced, PCB-degrading bacteria may provide an effective treatment for partial decontamination of PCB-contaminated soils. Received: 9 March 2000 / Received revision: 27 June 2000 / Accepted: 16 July 2000     

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     

Vertical distribution of bacteria in Arctic sea ice from the Barents and Laptev Seas

The vertical distribution of bacterial abundance and biomass was investigated in relation to algal biomass in ice cores taken from drifting ice floes in two Arctic shelf areas: the Barents Sea and the Laptev Sea. Bacteria were not homogeneously distributed throughout the cores but occurred in dense layers. Different types of distribution patterns were found: either a single maximum occurred inside or at the bottom of the ice floe or maxima were found in different parts of the floes. Bacterial concentrations ranged from 0.4 to 36.7 · 10⁵ cells ml⁻¹. The size spectra of sea-ice bacteria were determined by image analysis. Cell sizes showed considerable variation between the ice floes. In multi-year sea ice, the largest bacteria were observed in the area of an internal chlorophyll a maximum. No specific vertical distribution patterns were found in first-year ice floes. Bacterial biomass for the ice cores ranged from 19.2 to 79.2 mg C m⁻², and the ratio of bacterial:ice algal biomass ranged from 0.43 to 10.42. A comparison with data collected from fast ice revealed large differences in terms of cell size, abundance and biomass. Received: 7 September 1995 / Accepted: 10 September 1996     

Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures

The entomopathogenic nematode–bacterium complex Heterorhabditis megidis–Photorhabdus luminescens was cultured in 10-l internal loop bioreactors with marine impellers at aeration rates of 0.3 vvm and 0.7 vvm. Process parameters like impeller velocity and oxygen saturation were controlled at equal set points. The bacterial density was assessed at 24 h. Nematode dauer juveniles (DJ) were then inoculated and the development to adults after 8 days and final DJ yields after 16 days were recorded. The bacterial population density and the nematode inoculum development was variable and was not influenced by the aeration rate. A significant effect on the yield was recorded at the highest aeration rate. This result was confirmed by a direct comparison in two 5-l internal loop glass bioreactors at 0.3 vvm and 1.0 vvm, which were inoculated with nematode and bacterium pre-cultures from the same flask culture. Possible reasons for the positive correlation between aeration rate and DJ yield are discussed. Received: 27 September 1999 / Received revision: 21 January 2000 / Accepted: 23 January 2000     

Arctic Sea ice biota: design and evaluation of a mesocosm experiment

A mesocosm experiment (enclosure volume 220 l) was designed such that sea ice inhabited by Arctic Sea ice organisms was formed and maintained under natural conditions at 66°N in Rovaniemi, Finland. The experiment was run from natural freezing in December 1994 to melting in April 1995. The ice was inhabited by diatoms, chlorophyceae, heterotrophic flagellates, ciliates, nematodes and turbellarians. Biomass in the ice, expressed as Chlorophyll a concentration, was 20–110 μg l⁻¹; total cell densities varied from 5 × 10⁶ to 35 × 10⁶ cells l⁻¹. Amongst phototrophic organisms, a succession from a flagellate-dominated community (Chlamydomonas sp.) to a multi-species diatom-dominated community was observed. Typical Arctic species such as Nitzschia frigida and Melosira arctica were present in the ice. Bacterial concentration varied between 2 × 10⁸ and 7 × 10⁸ cells l⁻¹. At least two trophic levels were present in the ice. Received: 3 April 1997 / Accepted: 9 September 1997     

Incompatible pathogen infection results in enhanced reactive oxygen and cell death responses in transgenic tobacco expressing a hyperactive mutant calmodulin

Transgenic tobacco (Nicotiana tabacum L. cv. Wisconsin 38) lines expressing a mutant calmodulin (VU-3) that hyperactivates NAD kinase exhibit an enhanced elicitor-stimulated oxidative-burst reaction (S.A. Harding et al., 1997, EMBO J. 16: 1137–1144). VU-3 transgenic tobacco was used in the present study to investigate the relationship between calmodulin signalling, the production of active oxygen species and cell death in response to infection with an incompatible pathogen. Following P. syringae pv. syringae 61 infection, suspension cells derived from VU-3 transgenic plants exhibited a stronger oxidative burst (3- to 4-fold higher primary and secondary burst reactions), greater media alkalinization (3-fold) and more rapid cell death (4-fold greater mortality at 20 h post infection) than did infected control tobacco cells. Infection of leaf tissues with P. syringae pv. syringae 61 also resulted in an enhanced cell death response compared to control tobacco tissues. This cell death response of VU-3 leaf tissues, but not control leaf tissues, was further enhanced by the presence of 50 μM salicylic acid, suggesting that this transgenic line is more sensitive to the effects of this agent. Overall, the data support the model that calmodulin signalling pathways are involved in the plant oxidative burst and contribute to the regulation of cell death in infected plant tissues undergoing the hypersensitive response. Received: 6 January 1998 / Accepted: 7 March 1998     

Factors influencing the biosorption of gadolinium by micro-organisms and its mobilisation from sand

The present work was devoted to the study of the biosorption capacities of various microbial species (Bacillus subtilis, Pseudomonas aeruginosa, Ralstonia metallidurans CH34 previously Alcaligenes eutrophus CH34, Mycobacterium smegmatis, Saccharomyces cerevisiae) for ions of the lanthanide gadolinium (Gd³⁺). The uptake by sand of this element was also measured. Saturation curves and Scatchard models were established for all biosorbants used in this work. The results enabled us to determine the binding affinities and the maximum capacities for biosorption of Gd³⁺, which ranged from 350 μmol g⁻¹ for B. subtilis to 5.1 μmol g⁻¹ for S. cerevisiae. This study demonstrated the usefulness of optimisation of experimental conditions in biosorption investigations. Experimental results showed that biosorption could be influenced by the growth stage and by the composition of the growth medium of microbial cells. Finally, particular attention was given to the transfer of gadolinium ions from a loaded sand to a bacterial suspension. Received: 8 November 1999 / Received revision: 3 February 2000 / Accepted: 4 February 2000     

The effect of feeding on specific soil algae on the cold-hardiness of two Antarctic micro-arthropods (Alaskozetes antarcticus and Cryptopygus antarcticus)

The effect of consuming terrestrial algae on the cold tolerance of two Antarctic micro-arthropods was examined. From the results of preferential feeding experiments, seven species of Antarctic terrestrial micro-algae were chosen and fed to two common, freeze-avoiding Antarctic micro-arthropods: the springtail Cryptopygus antarcticus (Collembola: Isotomidae), and the mite Alaskozetes antarcticus (Acari: Oribatida). Mites were very selective in their choice of food whereas the springtails were less discriminating. The ice nucleating activity of each species of alga was measured using an ice nucleator spectrometer and a differential scanning calorimeter. Pure cultures of individual species of algae had characteristic supercooling points ranging from ca. −5 to −18 °C. The effect of eating a particular alga on the supercooling point of individual micro-arthropods cultured at two different temperatures (0 and 10 °C) was examined. Neither species showed a preference for algae with low ice-nucleating activity and there was no clear correlation between the supercooling point of food material and that of the whole animal. However, feeding on certain algae such as Prasiola crispa, which contained the most active ice nucleators, decreased the cold tolerance of both species of arthropods. Accepted: 6 May 2000     

Heterologous protein production in methylotrophic yeasts

The facultative methylotrophic yeasts Candida boidinii, Pichia methanolica, Pichia pastoris and Hansenula polymorpha have been developed as systems for heterologous gene expression. They are based on strong and regulatable promoters for expression control derived from methanol metabolism pathway genes. An increasing number of biotechnological applications attest to their status as preferred options among the various gene expression hosts. The well-established P. pastoris and H. polymorpha systems have been utilized in especially competitive and consistent industrial-scale production processes. Pharmaceuticals and technical enzymes produced in these methylotrophs have either already entered the market or are expected to do so in the near future. The article describes the present status of the methylotrophic yeasts as expression systems, focusing on applied examples of the recent past. Received: 9 May 2000 / Received revision: 20 June 2000 / Accepted: 23 June 2000     

Evidence for active microbial nitrogen transformations in sea ice (Gulf of Bothnia, Baltic Sea) in midwinter

Nutrient concentrations, chlorophyll-a, bacterial biomass and relative activity of denitrifying organisms were investigated from ice-core, brine and underlying water samples in February 1998 in the Gulf of Bothnia, Baltic Sea. Examined sea ice was typical for the Baltic Sea; ice bulk salinity varied from 0.1 to 1.6 psu, and in underlying water salinity was from 4.2 to 4.7 psu. In 2- to 3-months-old sea ice (thickness 0.4–0.6 m), sea-ice communities were at the winter stage; chl-a concentrations were generally below 1 mg m⁻³ and heterotrophic organisms composed 7–20% of organism assemblage. In 1-month-old ice (thickness 0.2–0.25 m), an ice spring bloom was already developing and chl-a concentrations were up to 5.6 mg m⁻³. In relation to low salinity, high concentrations of NH⁺ ₄, NO⁻ ₂, PO³⁺ ₄ and SiOH₄ were found in the ice column. The results suggest that the upper part of ice accumulates atmospheric nutrient load during the ice season, and nutrients in the upper 10–20 cm of ice are mainly of atmospheric origin. The most important biological processes controlling the sea-ice nutrient status are nutrient regeneration, nutrient uptake and nitrogen transformations. Nutrient regeneration is specially active in the middle parts of the 50- to 60-cm-thick ice and subsequent accumulation of nutrients probably enhances the ice spring bloom. Nitrite accumulation and denitrifying activity were located in the same ice layers with nutrient regeneration, which together with the observed significant correlation between the concentrations of nitrogenous nutrients points to active nitrogen transformations occurring in the interior layers of sea ice in the Baltic Sea. Accepted: 12 June 2000     

Biosynthesis of Cephalosporins

Abstract

Certain aerobic, Gram-negative bacteria, including the epiphytic plant pathogen, Pseudomonas syringae, possess a membrane protein that enables them to nucleate crystallization in supercooled water. Currently, these ice-nucleating (IN) bacteria are being used in snow making and have potential applications in the production and texturing of frozen foods, and as a replacement of silver iodide in cloud seeding. A negative aspect of these IN bacteria is frost damage to plant surfaces. Thus, of the various types of biological ice nucleators, bacteria have been the subject of most research and also appear relevant to the anticipated practical uses. The intent of this review is to explain the identification and ecology of the ice-nucleating bacteria, as well as to discuss aspects of molecular biology related to ice nucleation and consider existing and potential applications of this unique phenomenon.     

Molecular studies on isopenicillin N synthases

The isopenicillin N synthases isolated thus far are related to oxidases from other microorganisms and plants. These enzymes maintain a non-heme monoferrous-dependent catalytic centre comprising a HisXAsp(53–57)XHis motif and a crucial substrate-binding pocket with an ArgXSer motif for their functionality. The elucidation of these motifs was dependent on information collated from studies on structural chemistry, structural biology, site-directed engineered mutations and biochemical experiments. It is envisaged that these enzymes can potentially be improved through molecular breeding and protein engineering. Received: 15 December 1999 / Received revision: 26 January 2000 / Accepted: 27 January 2000     

Continuous production of enantiopure 1,2-epoxyhexane by yeast epoxide hydrolase in a two-phase membrane bioreactor

A two-phase membrane bioreactor was developed to continuously produce enantiopure epoxides using the epoxide hydrolase activity of Rhodotorula glutinis. An aqueous/organic cascade, hydrophilic, hollow-fiber membrane bioreactor was used: (1) to carry out large-scale resolution of epoxides, (2) to continuously extract residual enantiopure epoxides from the aqueous phase, and (3) to separate inhibitory formed diol from the yeast cells contained in the aqueous phase. Dodecane was employed to dissolve-feed epoxide as well as to extract residual epoxide. 1,2-Epoxyhexane was used as a model substrate. By use of this membrane bioreactor, enantiopure (S)-1,2-epoxyhexane (>98% enantiomeric excess) was obtained with a volumetric productivity of 3.8 g l⁻¹ h⁻¹. The continuous-production system was operated for 12 days and resulted in 38 g enantiopure (S)-1,2-epoxyhexane. Received: 14 February 2000 / Received revision: 15 June 2000 / Accepted: 18 June 2000     

Immobilisation of Thiobacillus ferrooxidans cells on nickel alloy fibre for ferrous sulfate oxidation

The immobilisation of the iron-oxidising bacteria Thiobacillus ferrooxidans on nickel alloy fibre as support is described. This matrix showed promise for application in iron oxidation under strongly acidic conditions. The influence on the colonisation process of T. ferrooxidans exerted by the initial pH of the medium and by temperature has also been studied. Results showed that immobilisation of T. ferrooxidans cells was affected by changes of temperature between 30 °C and 40 °C and in pH from 1.4 to 2.0. Received: 25 January 2000 / Received version: 20 April 2000 / Accepted: 1 May 2000     

Influence of the starting microbial nucleus type on the anaerobic granulation dynamics

 The influence of four different granulation precursors, syntroph-enriched methanogenic consortia, Methanosaeta-enriched, Methanosarcina-enriched nuclei and acidogenic flocs, on the time course of complex granule development and the lag time for start-up was investigated in four upflow anaerobic sludge-bed and filter reactors. Although the operational conditions allowed the maintenance of the same specific growth rate of biomass in the four reactors, granulation proceeded rapidly with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei. However, granulation was significantly retarded when acidogenic flocs were used as precursors. The granule mean Sauter diameter increased rapidly in the reactor inoculated with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei and reached, at the end of the experiment, 3.1, 2.7 and 2.4 mm compared to 1.1 mm in that inoculated with acidogenic flocs. This corresponded to a rate of granule size increase of 31, 21, 18 μm/day in syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei, respectively, compared to 7 μm/day in acidogenic flocs. Biomass specific activities (i.e. acidogenic, syntrophic and methanogenic activities) increased stepwise in all reactors with time, especially in those inoculated with syntroph/methanogenic consortia and Methanosaeta nuclei. From these results it appears that syntrophs and Methanosaeta spp. play an important role in the anaerobic granulation process. Received: 25 January 1996 / Received revision: 3 September 1996 / Accepted: 13 September 1996