A Polyhydroxybutyrate-Producing <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Isolated from Antarctic Environments with High Stress Resistance

Pseudomonas sp. 14-3, a strain that accumulates large quantities of polyhydroxybutyrate (PHB) when grown on octanoate, was isolated from Antarctic environments. This isolate was characterized on the basis of phenotypic features and partial sequencing of its 16S ribosomal RNA gene. Pseudomonas sp. 14-3 showed increased tolerance to both thermal and oxidative stress compared with three other Pseudomonas species. Stress tolerance of Pseudomonas sp. 14-3 was analyzed in polyhydroxyalkanoate accumulating and non-accumulating conditions, and increased levels of stress resistance were observed when PHB was produced. Pseudomonas sp. 14-3 was isolated from Antarctic regions, a habitat normally exposed to extreme conditions. An association between high PHB accumulation and high stress resistance in bacteria adapted to extreme environments is suggested.     

Vacuolating,lethal, collagenase and clostripain activities of six reference ATCC <Emphasis Type="Italic">Clostridium histolyticum</Emphasis> strains

We have previously demonstrated that C. histolyticum reference strain ATCC 19401 produces not only lethal factor but also hitherto unrecognized vacuolating toxin. The aim of this study was to compare vacuolating and lethal activities of six reference C. histolyticum strains (ATCC 6282, 8034, 17859, 17860, 19401 and 25770) and to determine whether production of vacuolating toxin is strain-dependent and how the amounts of both toxins produced by the same strain are related to each other and also to protease, collagenase and clostripain activities. All strains produced vacuolating and lethal toxins as well as collagenase, clostripain and proteases, but with different yield. Strain ATCC 19401 demonstrated considerable vacuolating and lethal activities and low activity of collagenase, clostripain and proteases. In other strains such relationship was not evident. Positive correlations were observed in collagenase and clostripain activities of all studied C. histolyticum strains (r = 0.71). Positive correlations were detected also in vacuolating activities of studied strains and clostripain (r = 0.62) and collagenase (r = 0.87) production, and this effect was statistically significant (P < 0.05). Negative non-significant correlations were detected: (a) in lethal activities of studied strains and clostripain, or collagenase activities, (b) in vacuolating activities and protease production.     

Production of enzymes and antimicrobial compounds by halophilic Antarctic <Emphasis Type="Italic">Nocardioides</Emphasis> sp. grown on different carbon sources

This study demonstrated the potential of microbial isolates from Antarctic soils to produce hydrolytic enzymes by using specific substrates. The results revealed potential of the strains to produce a broad spectrum of hydrolytic enzymes. Strain A-1 isolated from soil samples in Casey Station, Wilkes Land, was identified as Nocardioides sp. on the basis of morphological, biochemical, physiological observations and also chemotaxonomy analysis. Enzymatic and antimicrobial activities of the cell-free supernatants were explored after growth of strain A-1 in mineral salts medium supplemented with different carbon sources. It was found that the carbon sources favored the production of a broad spectrum of enzymes as well as compounds with antimicrobial activity against Gram-positive and Gram-negative bacteria, especially Staphylococcus aureus and Xanthomonas oryzae. Preliminary analysis showed that the compounds with antimicrobial activity produced by the strain A-1 are mainly glycolipids and/or lipopeptides depending on the used carbon source. The results revealed a great potential of the Antarctic Nocardioides sp. strain A-1 for biotechnological, biopharmaceutical and biocontrol applications as a source of industrially important enzymes and antimicrobial/antifungal compounds.     

The genus Stauroneis (Bacillariophyceae) in the Antarctic region

Aim To provide a detailed biogeography of the diatom genus Stauroneis in the Antarctic and sub‐Antarctic regions and to establish the biogeographical relationships between the different constituent locations to test the application of a precise and refined taxonomy in generating accurate polar biogeographies. Location The Antarctic and sub‐Antarctic region comprising the islands South Georgia, Crozet, Kerguelen, Marion, Heard and the Antarctic Peninsula. Methods Diatom samples from different habitats in a large part of the sub‐Antarctic and Antarctic region were investigated for their Stauroneis content. Presence/absence data were investigated using Sørensen's similarity index. An additional 500 samples from Arctic locations were used to provide a bipolar comparison. Using reliable literature data, gaps in the distribution of the Stauroneis taxa were filled. Results The Stauroneis flora of the Antarctic and sub‐Antarctic regions is quite distinct from its Arctic equivalent, with only five species (out of 60) common to both areas. Within the (sub‐)Antarctic group, the islands of the Indian Ocean have the most diverse Stauroneis composition, which is clearly separated from the rest of the region. The South Georgia Stauroneis composition has some affinities with the Antarctic Peninsula but the latter has far fewer species. These results are in clear contrast to older data showing no biogeographical difference between the Arctic and Antarctic regions. Main conclusions Using only a single genus, a clear biogeography of the (sub‐)Antarctic region can be produced that separates the Indian Ocean islands from other sampling locations. When based on a precise taxonomy, biogeographical relationships between locations in the region become much more reliable. Contrary to previous work, there is almost no similarity in the diatom floras of the Arctic and Antarctic regions.     

Chemical Characterization of Exopolysaccharides from Antarctic Marine Bacteria

Exopolysaccharides (EPS) may have an important role in the Antarctic marine environment, possibly acting as ligands for trace metal nutrients such as iron or providing cryoprotection for growth at low temperature and high salinity. Ten bacterial strains, isolated from Southern Ocean particulate material or from sea ice, were characterized. Whole cell fatty acid profiles and 16S rRNA gene sequences showed that the isolates included representatives of the genera Pseudoalteromonas, Shewanella, Polaribacter, and Flavobacterium as well as one strain, which constituted a new bacterial genus in the family Flavobacteriaceae. The isolates are, therefore, members of the “Gammaproteobacteria” and Cytophaga-Flexibacter-Bacteroides, the taxonomic groups that have been shown to dominate polar sea ice and seawater microbial communities. Exopolysaccharides produced by Antarctic isolates were characterized. Chemical composition and molecular weight data revealed that these EPS were very diverse, even among six closely related Pseudoalteromonas isolates. Most of the EPS contained charged uronic acid residues; several also contained sulfate groups. Some strain produced unusually large polymers (molecular weight up to 5.7 MDa) including one strain in which EPS synthesis is stimulated by low temperature. This study represents a first step in the understanding of the role of bacterial EPS in the Antarctic marine environment.     

Characterization of rhizospheric bacteria isolated from Deschampsia antarctica Desv.

Deschampsia antarctica Desv. is the only gramineae capable of colonizing the Antarctic due to the region’s extreme climate and soil environment. In the present research, bacteria colonizing the rhizospheric soil of D. antarctica were isolated and characterized. The soil studies showed that D. antarctica possesses a wide spectrum of psychrotolerant bacteria with extensive and varied antibiotic resistance, as well as heavy metal tolerance. The bacterial strains isolated from the rhizosphere of D. antarctica also produced a diverse pattern of enzymes. Based on the strain identification with partial characterization of the 16S rRNA gene, the majority of the isolates correspond to different Pseudomonas species, and species of the genus Flavobacterium sp. and Arthrobacter sp. The isolated strains collected from this research constitute a unique collection for future, more detailed taxonomic analysis and physiological characterization, contributing to the search for potential biotechnological uses. These findings and others have great potential for developing new biotechnological products from Antarctic microorganisms.     

COMPARATIVE STUDY ON THE PHOTOSYNTHETIC PROPERTIES OF PRASIOLA (CHLOROPHYCEAE) AND NOSTOC (CYANOPHYCEAE) FROM ANTARCTIC AND NON‐ANTARCTIC SITES1

The terrestrial cyanobacterium Nostoc commune Vaucher ex Bornet et Flahault occurs worldwide, including in Japan and on the Antarctic continent. The terrestrial green alga Prasiola crispa (Lightf.) Kütz. is also distributed in Antarctica. These two species need to acclimate to the severe Antarctic climate including low ambient temperature and desiccation under strong light conditions. To clarify this acclimation process, the physiological characteristics of the photosynthetic systems of these two Antarctic terrestrial organisms were assessed. The relative rate of photosynthetic electron flow in N. commune collected in Japan and in Antarctica reached maxima at 900 and 1,100 μmol photons · m^?2 · s^?1, respectively. The difference seemed to reflect the presence of high amounts of UV‐absorbing substances within the Antarctic cyanobacterium. On the other hand, the optimal temperatures for photosynthesis at the two locations were 30°C–35°C and 20°C–25°C, respectively. This finding suggested a decreased photosynthetic thermotolerance in the Antarctic strain. P. crispa exhibited desiccation tolerance and dehydration‐induced quenching of PSII fluorescence. Re‐reduction of the photooxidized PSI reaction center, P700, was also inhibited at fully dry states. Photosynthetic electron flow in P. crispa reached a maximum at 20°C–25°C and at a light intensity of 700 μmol photons ? m^?2 ? s^?1. Interestingly, the osmolarity of P. crispa cells suggested that photosynthesis is performed using water absorbed in a liquid form rather than water absorbed from the air. Overall, these data suggest that these two species have acclimated to optimally photosynthesize under conditions of the highest light intensity and the highest temperature for their habitat in Antarctica.     

Gas Chromatographic Analysis of Volatile Esters in Wines

We isolated a new aerobic gram negative collagenolytic bacterium, strain CRZV₁. This strain is yellow pigmented, non motile; it poorly degrades the sugars and its proteolytic activity corresponds to one enzyme, a collagenase which is produced in media with collagen, or collagen like substrates. The strain CRZV_l, presents many similarities with Flavobacteria. However, the GC content of the DNA, which is 65%, excludes our collagenolytic strain from the genus Flavobacterium, where all the species have GC content between 30 and 42%. All the strains, already described as Flavobacteria, the GC contents of which are situated between 55 and 70%, are actually unclassified. They could be included in the genus Empedobacter. This genus is not in the approved list of bacterial names, so we only consider this new collagenolytic bacteria as a gram negative, aerobic, yellow pigmented bacteria.     

Effects of products from macrophages, blood mononuclear cells and of retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied in cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of ³H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of α₁ chains; the amount of α₂ chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. α₂ chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen and, therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Analysis of the Phospholipase C Gene of Clostridium perfringens KZ1340 Isolated from Antarctic Soil

Clostridium perfringens KZ1340 isolated from Antarctic soil was first classified as Clostridium plagarum and later as a lecithinase-negative variant of C. perfringens. Although the strain produced no detectable lecithinase (phospholipase C, PLC) activity in the culture supernatant, it was shown by Southern blot hybridization to possess a PLC-encoding gene (plc). To determine the cause of the PLC deficiency, we cloned and sequenced the plc gene from KZ1340. The deduced amino acid sequence consists of 398 amino acid residues, coinciding with those of the plc genes previously determined. Tyrosine was substituted for histidine at amino acid position 148, which is thought to bind a zinc ion essential for PLC activity. Northern blot analysis revealed that KZ1340 expressed the plc gene at an extremely low level. Furthermore, the plc gene cloned from C. perfringens strain 13 into a plasmid was expressed weakly in KZ1340, compared to that in strain 13. This indicates that the former strain represses plc gene expression in trans. When a phylogenetic tree of plc genes was constructed, the KZ1340 plc gene formed a monophyletic branch along with those of various other C. perfringens strains, supporting the classification of the strain as a variant of C. perfringens.     

Flavobacterium frigidimaris sp. nov., isolated from Antarctic seawater

We described the polyphasic characterization of the psychrotolerant isolated from Antarctic seawater. The strain was closely related to Flavobacterium hydatis, F. pectinovorum, and F. saccharophilum on the basis of the 16S rDNA sequence analysis. However, DNA–DNA hybridization experiments showed that the DNA-similarities between strain KUC-1^T and the reference strains of Flavobacterium were less than 30%. Therefore, we can definite a new species of Flavobacterium phylogenetically, and strain KUC-1^T can be considered to be a new species of Flavobacterium. i.e. F. frigidimaris (KUC-1^T: JCM 12218^T and DSM 15937^T; mol% G+C of DNA of the type strain is 34.5 mol%). Useful phenotypical features for discrimination of F. frigidimaris from other Flavobacterium species, such as a resistance to NaCl, optimum growth temperature, and cellular fatty acid composition, were also determined.     

Cold-adapted yeasts from Antarctica and the Italian Alps—description of three novel species: Mrakia robertii sp. nov., Mrakia blollopis sp. nov. and Mrakiella niccombsii sp. nov.

Worldwide glaciers are annually retreating due to global overheating and this phenomenon determines the potential lost of microbial diversity represented by psychrophilic microbial population sharing these peculiar habitats. In this context, yeast strains, all unable to grow above 20°C, consisting of 42 strains from Antarctic soil and 14 strains isolated from Alpine Glacier, were isolated and grouped together based on similar morphological and physiological characteristics. Sequences of the D1/D2 and ITS regions of the ribosomal DNA confirmed the previous analyses and demonstrated that the strains belong to unknown species. Three new species are proposed: Mrakia robertii sp. nov. (type strain CBS 8912), Mrakia blollopis sp. nov. (type strain CBS 8921) and a related anamorphic species Mrakiella niccombsii sp. nov. (type strain CBS 8917). Phylogenetic analysis of the ITS region revealed that the new proposed species were closely related to each other within the Mrakia clade in the order Cystofilobasidiales, class Tremellomycetes. The Mrakia clade now contains 8 sub-clades. Teliospores were observed in all strains except CBS 8918 and for the Mrakiella niccombsii strains.     

Gut evacuation rates of Antarctic copepods during austral spring

The results of gut evacuation experiments performed on Antarctic copepods during the austral spring are presented and discussed. Four species of large copepods commonly occurring in the Indian sector of the Antarctic Ocean were studied: Calanus propinquus, Calanoides acutus, Rhincalanus gigas and Pleuromamma robusta. For each species two experiments were carried out, one in daytime and one in night-time, except for Calanoides acutus, which was only studied at night. P. robusta showed pigment gut retention in all experiments. The results showed that all species studied had a longer gut passage time than that previously recorded and that gut evacuation rate appears to decrease during daytime. Accepted: 1 October 1998     

A halotolerantPlanococcus from Antarctic Dry Valley soil

A halotolerantPlanococcus (strain A4a) was isolated from saline Antarctic Dry Valley soil.Planococcus strain A4a grew over wide ranges of temperature (0°C–40°C) and NaCl concentrations (0–2.0M). When the NaCl concentration of the growth medium was increased, the total intracellular free amino acid concentration increased; however, the intracellular potassium concentration did not increase. This result suggested that intracellular free amino acids functioned as compatible solutes for growth ofPlanococcus strain A4a at elevated NaCl concentrations. The halotolerant and psychrotolerant nature ofPlanococcus strain A4a would appear to provide it with the capacity for growth in the saline Antarctic Dry Valley soil environment from which it was isolated.     

Adaptation of fungi,including yeasts,to cold environments

A wide range of cold environments exist, with an equally broad variety of fungi and yeasts that have adapted to such environments. These adaptations, which affect membranes, enzymes and other cellular components, such as radical scavenging molecules, display a great potential for exploitation in biotechnology. Alterations have been detected in membrane lipids, with an increase in fatty acid unsaturated bonds that enhance their fluidity. We report new data on the different phospholipid composition in membrane lipids in the same fungal species from both Antarctic and temperate regions. The decrease in temperature causes intracellular oxidative stress by inducing the generation of reactive oxygen species. We report the results of the first analysis of the non-enzymatic antioxidant response and phenolic compound production by an Antarctic strain of Geomyces pannorum. A survey on yeasts from the cryosphere is reported with a focus on their adaptation to a cold environment. Some studies have shown that the number of macrofungi in glacier forefronts rises as deglaciation increases. The survival success of many plants in such areas may be attributed to their mycorrhizal associations. We highlighted the macrofungal biodiversity of some Italian alpine habitats, in which we Inocybe microfastigiata, Laccaria montana and Lactarius salicis-herbaceae were recorded for the first time in Lombardy (Italy).     

Fluoride content of Antarctic marine animals caught off Elephant Island

Summary A number of marine animals were collected off Elephant Island (South Shetland Islands) and analyzed for fluoride (mg/kg wet weight). In fish muscle from six Antarctic species the content was low (approx. 2) not exceeding the content of fishes from other waters. In bone-tissue a concentration of 616–1207 was found. In some benthic living animals the amount of fluoride (determined in the whole body) was different depending on the species investigated. Low amounts of approx. 10 were encountered in a pycnogonid, an octopodid and a polychaet. Very high values of 200–600 were determined in isopods, amphipods, ophiuroids and asteroids, these concentrations exceeded sometimes the value found in Antarctic krill.Results of the Antarctic Expedition 1981 of the Federal Republic of Germany with FRV Walther Herwig     

Polyhydroxyalkanoates are essential for maintenance of redox state in the Antarctic bacterium Pseudomonas sp. 14-3 during low temperature adaptation

Polyhydroxyalkanoates (PHAs) are highly reduced bacterial storage compounds that increase fitness in changing environments. We have previously shown that phaRBAC genes from the Antarctic bacterium Pseudomonas sp. 14-3 are located in a genomic island containing other genes probably related with its adaptability to cold environments. In this paper, Pseudomonas sp. 14-3 and its PHA synthase-minus mutant (phaC) were used to asses the effect of PHA accumulation on the adaptability to cold conditions. The phaC mutant was unable to grow at 10°C and was more susceptible to freezing than its parent strain. PHA was necessary for the development of the oxidative stress response induced by cold treatment. Addition of reduced compounds cystine and gluthathione suppressed the cold sensitive phenotype of the phaC mutant. Cold shock produced very rapid degradation of PHA in the wild type strain. The NADH/NAD ratio and NADPH content, estimated by diamide sensitivity, decreased strongly in the mutant after cold shock while only minor changes were observed in the wild type. Accordingly, the level of lipid peroxidation in the mutant strain was 25-fold higher after temperature downshift. We propose that PHA metabolism modulates the availability of reducing equivalents, contributing to alleviate the oxidative stress produced by low temperature.     

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic <Emphasis Type="Italic">Cobetia marina</Emphasis> strain L-2

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5°C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5–15°C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5°C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5°C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5°C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5°C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.     

Benthic hydroids (Cnidaria: Hydrozoa) from the Spanish Antarctic expedition Bentart 95

A total of 61 species of hydroids, belonging to 13 families and 23 genera, were found during the Spanish Antarctic expedition Bentart 95 with the RV Hespérides. Ten of the species were identified only to generic level. The dominance of the subclass Leptothecatae, with 57 species, was remarkable. The remaining four species belong to the subclass Anthoathecatae. By far the most diverse family was the Sertulariidae, with 25 species (41%), followed by Haleciidae with nine species (15%) and Kirchenpaueriidae with six (10%). The family Plumulariidae, represented by one species of Nemertesia, is recorded for the first time from Antarctic waters. Eudendrium scotti, Perarella clavata and Symplectoscyphus hero are each recorded for the second time. Symplectoscyphus with 11 species was the dominant genus. Almost 60% of the species diversity is concentrated in just a little more than the 20% of genera. Nearly 70% of the species are endemic to Antarctic waters and 90% of them are restricted to Antarctic or Antarctic/sub-Antarctic waters.     

Comparative investigations of growth and solvent formation in ‘Clostridium saccharoperbutylacetonicum’ DSM 2152 and Clostridium acetobutylicum DSM 792

The butanol and acetone-producing strain DSM 2152, invalidly described as ‘Clostridium saccharoperbutylacetonicum’ is compared with the type strain C. acetobutylicum, DSM 792, with respect to solvent and acid formation at varying pH values and growth rates. Batch cultures, product-limited chemostat and pH-auxostat cultures were used for characterization. Under all conditions strain DSM 2152 produced much lower amounts of butyric and acetic acids than the type strain. The pH optimum for solvent formation was higher, ie 5.5 instead of 4.5. Solvent formation occurred at higher dilution rates, but below 0.1 h⁻¹ a lower solvent concentration was obtained, indicating that acid production was too low to provide a sufficient amount for acetone formation. The results are discussed in the light of recent publications on the taxonomy of butanol-acetone producing clostridia using 16S rRNA sequence analysis and other nucleic acid data. The presently suggested ‘phylogenetic’ classification of the collective species, C. acetobutylicum, is also reflected in the fermentation characteristics. Received 21 December 1998/ Accepted in revised form 22 January 1999