Bacterial diversity from benthic mats of Antarctic lakes as a source of new bioactive metabolites

During the MICROMAT project, the bacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes was accessed for the discovery of novel antibiotics. In all, 723 Antarctic heterotrophic bacteria belonging to novel and/or endemic taxa in the α-, β- and γ-subclasses of the Proteobacteria, the Bacteroidetes branch, and of the high and low percentage G+C Gram-positives, were isolated, cultivated in different media and at different temperatures, and then screened for the production of antimicrobial activities. A total of 6348 extracts were prepared by solid phase extraction of the culture broths or by biomass solvent extraction. 122 bacteria showed antibacterial activity against the Gram-positives Staphylococcus aureus and to a lower extent Enterococcus faecium, and versus the Gram-negative Escherichia coli. Few of these strains showed also some antifungal activity against Cryptococcus neoformans, Aspergillus fumigatus and to a lower extent Candida albicans. LC–MS fractionation of extracts from a subset of strains (hits) that exhibited relatively potent antibacterial activities evidenced a chemical novelty that was further investigated. Two strains of Arthrobacter agilis produced potent antibacterial compounds with activity against Gram-positives and possibly related to novel cyclic thiazolyl peptides. To our knowledge, this is the first report of new antibiotics produced by bacteria from benthic microbial mats from Antarctic lakes. With no doubts these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as antibiotic compounds.     

Cyanobacteria from benthic mats of Antarctic lakes as a source of new bioactivities

Aims:  To exploit the cyanobacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes for the discovery of novel antibiotic and antitumour activities.
Methods and results:  In all, 51 Antarctic cyanobacteria isolated from benthic mats were cultivated in the laboratory by optimizing temperature, irradiance and mixing. Productivity was generally very low (≤60 mg l⁻¹ d⁻¹) with growth rates ( μ ) in the range of 0·02–0·44 d⁻¹. Growth rates were limited by photosensitivity, sensitivity to air bubbling, polysaccharide production or cell aggregation. Despite this, 126 extracts were prepared from 48 strains and screened for antimicrobial and cytotoxic activities. Seventeen cyanobacteria showed antimicrobial activity (against the Gram-positive Staphylococcus aureus , the filamentous fungus Aspergillus fumigatus or the yeast Cryptococcus neoformans ), and 25 were cytotoxic. The bioactivities were not in accordance with the phylogenetic grouping, but rather strain-specific. One active strain was cultivated in a 10-l photobioreactor.
Conclusions:  Isolation and mass cultivation of Antarctic cyanobacteria and LC-MS (liquid chromatography/mass spectrometry) fractionation of extracts from a subset of those strains (hits) that exhibited relatively potent antibacterial and/or antifungal activities, evidenced a chemical novelty worthy of further investigation.
Significance and impact of the study:  Development of isolation, cultivation and screening methods for Antarctic cyanobacteria has led to the discovery of strains endowed with interesting antimicrobial and antitumour activities.     

Diversity of 746 heterotrophic bacteria isolated from microbial mats from ten Antarctic lakes

Microbial mats, growing in Antarctic lakes constitute unique and very diverse habitats. In these mats microorganisms are confronted with extreme life conditions. We isolated 746 bacterial strains from mats collected from ten lakes in the Dry Valleys (lakes Hoare and Fryxell), the Vestfold Hills (lakes Ace, Druzhby, Grace, Highway, Pendant, Organic and Watts) and the Larsemann Hills (lake Reid), using heterotrophic growth conditions. These strains were investigated by fatty acid analysis, and by numerical analysis, 41 clusters, containing 2 to 77 strains, could be delineated, whereas 31 strains formed single branches. Several fatty acid groups consisted of strains from different lakes from the same region, or from different regions. The 16S rRNA genes from 40 strains, representing 35 different fatty acid groups were sequenced. The strains belonged to the alpha, beta and gamma subclasses of the Proteobacteria, the high and low percent G+C Gram-positives, and to the Cytophaga-Flavobacterium-Bacteroides branch. For strains representing 16 fatty acid clusters, validly named nearest phylogenetic neighbours showed pairwise sequence similarities of less than 97%. This indicates that the clusters they represent, belong to taxa that have not been sequenced yet or as yet unnamed new taxa, related to Alteromonas, Bacillus, Clavibacter, Cyclobacterium, Flavobacterium, Marinobacter, Mesorhizobium, Microbacterium, Pseudomonas, Saligentibacter, Sphingomonas and Sulfitobacter.     

Biogeographical distribution and ecological ranges of benthic cyanobacteria in East Antarctic lakes

For the first time, the cyanobacterial diversity from microbial mats in lakes of Eastern Antarctica was investigated using microscopic and molecular approaches. The present study assessed the biogeographical distribution of cyanobacteria in Antarctica. Five samples were taken from four lakes spanning a range of different ecological environments in Larsemann Hills, Vestfold Hills and Rauer Islands to evaluate the influence of lake characteristics on the cyanobacterial diversity. Seventeen morphospecies and 28 16S rRNA gene-based operational taxonomic units belonging to the Oscillatoriales, Nostocales and Chroococcales were identified. The internal transcribed spacer was evaluated to complement the 16S rRNA gene data and showed similar but more clear-cut tendencies. The molecular approach suggested that potential Antarctic endemic species, including a previously undiscovered diversity, are more abundant than has been estimated by morphological methods. Moreover, operational taxonomic units, also found outside Antarctica, were more widespread over the continent than potential endemics. The cyanobacterial diversity of the most saline lakes was found to differ from the others, and correlations between the sampling depth and the cyanobacterial communities can also be drawn. Comparison with database sequences illustrated the ubiquity of several cyanobacterial operational taxonomic units and their remarkable range of tolerance to harsh environmental conditions.     

Light climate and phytoplankton photosynthesis in maritime Antarctic lakes

The responses of phytoplankton populations to seasonal changes in radiation flux in two Antarctic lakes with extensive winter ice-cover are described. A phytoplankton capable of photosynthesis was found throughout the year in both systems. During winter, low incident radiation combined with thick layers of snow and ice prevented in situ photosynthesis becoming detectable. The beginning of spring was marked by a reduction in snow cover which resulted in a considerable increase in surface penetrating radiation. Planktonic algae rapidly adapted to utilise these increased levels efficiently, though they still showed characteristics of strong shade adaptation.Loss of ice cover at the start of the short open water period further increased the radiation levels and a summer population developed which was much less shade adapted. Saturation and photoinhibition effects were widespread during this period as the algae proved unable to utilise high radiation levels efficiently. They were however effective at the radiation fluxes prevalent in the lower part of the rapidly circulating water columns.     

Extraction of chlorophyll and carotenoid pigments from Antarctic benthic mats for analysis by HPLC

The efficiency of 9:1 acetone-water, DMSO and boiling 9:1 ethanol-water in extracting chlorophyll and carotenoid pigments from benthic cyanobacterial mats from Antarctica for HPLC (high performance liquid chromatography) analysis was examined. Considerable breakdown of chlorophylla was observed after 5 min extraction in boiling ethanol and 2 h extraction in DMSO. Over 50% of the chlorophylla was degraded to chlorophyllidea and there was substantial loss of carotenoids after a 15 h exposure of ground cells to cold 9:1 acetone-water.Mild sonication of ground mat material in 9:1 acetone-water followed by a 4 h extraction at 4 ° C was found to minimise chlorophylla breakdown and dramatically improved the extraction efficiency of chlorophylla, myxoxanthophyll and -carotene.     

Microbial mats of twenty-six lakes from Potter Peninsula,King George Island,Antarctica

The taxonomic composition and ecology of microbial mats were investigated in 26 lentic environments of Potter Peninsula (King George Island, Antarctica) during the summer of 1995/96. These have different limnological characteristics according to the location of the basins. Among the 139 algal taxa registered, 10 are new records for Antarctica. Bacillariophyceae was the dominant class in terms of species richness, followed by the Cyanobacteria and Chlorophyceae. TWINSPAN classification was used to describe six epilithic algal assemblages; Canonical Correspondence Analysis ordination showed that soluble reactive phosphorus, phytoplanktonic chlorophyll a concentration and conductivity were the main sources for the variation in the data.     

Viral dynamics and patterns of lysogeny in saline Antarctic lakes

Antarctic lakes are extreme ecosystems with microbially dominated food webs, in which viruses may be important in controlling community dynamics. A year long investigation of two Antarctic saline lakes (Ace and Pendant Lakes) revealed high concentrations of virus like particles (VLP) (0.20–1.26 × 10⁸ ml⁻¹), high VLP: bacteria ratios (maximum 70.6) and a seasonal pattern of lysogeny differing from that seen at lower latitudes. Highest rates of lysogeny (up to 32% in Pendant Lake and 71% in Ace Lake) occurred in winter and spring, with low or no lysogeny in summer. Rates of virus production (range 0.176–0.823 × 10⁶ viruses ml⁻¹ h⁻¹) were comparable to lower latitude freshwater lakes. In Ace Lake VLP did not correlate with bacterial cell concentration or bacterial production but correlated positively with primary production, while in Pendant Lake VLP abundance correlated positively with both bacterial cell numbers and bacterial production but not with primary production. In terms of virus and bacterial dynamics the two saline Antarctic lakes studied appear distinct from other aquatic ecosystems investigated so far, in having very high viral to bacterial ratios (VBR) and a very high occurrence of lysogeny in winter.     

Viruses in the plankton of freshwater and saline Antarctic lakes

1. Virus‐like particle (VLP) abundances in nine freshwater to saline lakes in the Vestfold Hills, Eastern Antarctica (68° S) were determined in December 1999. In the ultra‐oligotrophic to oligotrophic freshwater lakes, VLP abundances ranged from 1.01 to 3.28 × 10⁶ mL^–1 in the top 6 m of the water column. In the saline lakes the range was between 6.76 and 36.5 × 10⁶ mL^–1. The lowest value was found in meromictic Ace Lake and the highest value in hypersaline Lake Williams. Virus to bacteria ratios (VBR) were lowest in the freshwater lakes and highest in the saline lakes, with a maximum of 23.4 in the former and 50.3 in the latter. 2. A range of morphologies among VLP was observed, including phages with short (Podoviridae) and long tails, icosahedric viruses of up to 300 nm and star‐like particles of about 80 nm diameter. 3. In these microbially dominated ecosystems there was no correlation between VLP and either bacterial numbers or chlorophyll a. There was a significant correlation between VLP abundances and dissolved organic carbon concentration (r=0.845, P < 0.01). 4. The data suggested that viruses probably attack a spectrum of bacteria and protozoan species. Virus‐like particle numbers in the freshwater lakes were lower than values reported for lower latitude systems. Those in the saline lakes were comparable with abundances reported from other Antarctic lakes, and were higher than most values published for lower latitude lakes and many marine systems. Across the salinity spectrum from freshwater through brackish to hypersaline, VLP concentrations increased roughly in relation to increasing trophy. 5. Given that Antarctic lakes have a plankton almost entirely made up of bacteria and protists, and that VLP abundances are high, it is likely that viruses play a pivotal role in carbon cycling in these extreme ecosystems.     

小麦种子内携真菌多样性研究

为揭示小麦种子所携带真菌的种类和多样性,丰富小麦相关微生物的可利用资源,我们对来自俄罗斯、澳大利亚、哈萨克斯坦、加拿大以及中国的57份小麦种子样品进行内携真菌分离并对其中的21份种子利用高通量测序进行多样性分析.结果显示,分离获得564株真菌,经ITS序列分析结合形态观察鉴定为31属69种,其中菌株数量相对多度最高的5...     

Virus and microbial loop dynamics over an annual cycle in three contrasting Antarctic lakes

1. Viral and microbial loop dynamics were investigated over an annual cycle in three contrasting saline Antarctic lakes – Highway Lake (salinity 4‰), Pendant Lake (salinity 19‰) and Ace Lake, a meromictic system (with a mixolimnion salinity of 18‰) in order to assess the importance of viruses in extreme, microbially dominated systems. 2. Virus like particles (VLP) showed no clear seasonal pattern, with high concentrations occurring in both winter and summer (range 0.89 × 10⁷ ± 0.038 to 12.017 × 10⁷ ± 1.28 mL^?1). VLP abundances reflected lake productivity based on chlorophyll a concentrations. Bacterial abundances and biomass did not correlate with VLP numbers except in Pendant Lake, the most productive of the three lakes studied. 3. Pendant Lake supported the highest bacterial biomass (range Highway: 18.44 ± 1.35 to 59.43 ± 2.80 ng C mL^?1; Ace: 14.42 ± 2.69 to 68.39 ± 2.95 ng C mL^?1; Pendant: 31.36 ± 3.94 to 115.95 ± 4.49 ng C mL^?1) so that virus to bacteria ratios (VBR) (range 30.48 ± 7.96 to 96.67 ± 8.21) were higher in Ace Lake (range 30.58 ± 3.98 to 80.037 ± 1.60) and Highway Lake (range 18.63 ± 3.12 to 126.74 ± 6.50). 4. Negative correlations occurred between VLP and cryptophytes (dominant phototrophic nanoflagellates), suggesting that they were not hosts to lytic viruses. Among the other protists only the heterotrophic nanoflagellates of Highway Lake (dominated by the marine choanoflagellate Diaphanoeca grandis) showed a positive correlation with VLP. 5. The VLP was negatively correlated with photosynthetically active radiation (PAR) and temperature, both of which increased with ice thinning and breakout, increasing viral decay. In winter VLP probably persisted in cold, dark water. 6. High VLP concentrations and high VBR (values at the upper end of those reported for marine and lacustrine systems) indicated that viruses, most of which were probably bacteriophage, are a major element within the microbial communities in extreme, saline lakes.     

Comparative analysis of bacterioplankton assemblages from maritime Antarctic freshwater lakes with contrasting trophic status

The bacterioplankton assemblages of eight maritime Antarctic lakes with a wide range of trophic status and geographic span (six lakes from Hope Bay, Antarctic Peninsula and two from Potter Peninsula, King George Island) were described using denaturing gradient gel electrophoresis and band sequencing during two consecutive austral summers (2003–2004). Analyses of the gels identified a total of 230 bands spread across 57 different positions. Among those bands, 14 were shared between lakes from Hope Bay and Potter Peninsula, 17 were observed only in particular lakes, and 17 were registered both years in the same lake. We successfully reamplified and sequenced 43 bands located in 36 different positions belonging to Bacteroidetes, Actinobacteria, Betaproteobacteria and Cyanobacteria. The closest matches for 63% of the sequenced bands were from Antarctic or from other cold environment clones and sequences already in the databases, suggesting the widespread dominance of microbial communities adapted to cold habitats. The results of the multivariate analyses (Cluster Analysis and CCA) indicated that the nutrient status of the lake influences the bacterioplankton assemblages. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chemolithotrophic sulfur bacteria in sediments,mats, and stromatolites of western Australian saline lakes

Samples of stromatolites, microbial mats, and sediments from four saline lakes (approximate seasonal salinity ranges 20–220%o) in Western Australia were used to establish enrichments for elective cultures of aerobic and anaerobic denitrifying chemolithoautotrophs that could grow with thiosulfate as sole energy source. Organisms of these types were obtained from all sources tested. Twenty‐four pure cultures were isolated, all of which were gram‐negative, rod‐shaped bacteria exhibiting a considerable diversity of metabolic capability. Isolation of these obligate and facultative sulfur‐oxidizing chemolithotrophs from the stromatolite and mat habitats indicates the possibility that these rod‐shaped bacteria contribute to the oxidative phase of the sulfur cycle in these habitats, in addition to oxidation by phototrophs or Beggiatoa. Only four of the pure cultures could grow without salt, but all 24 showed significant halophily, some tolerating 3 M NaCl. Three novel isolates of NaCl‐dependent, thiosulfate‐oxidizing, aerobic and denitrifying obligate chemolithotrophs are described. In addition, a facultatively heterotrophic halophilic strain growing either methylotrophically on methylamine or chemolithotrophically on thiosulfate aerobically or with anaerobic denitrification was found.     

The screening and selection of inoculant arbuscular-mycorrhizal and ectomycorrhizal fungi

The initiation of a programme of screening and selection of arbuscular-mycorrhizal fungi (AM fungi) and ectomycorrhizal fungi (ECM fungi) for use as inoculant fungi in agriculture, horticulture of forestry will depend on whether inoculation is more appropriate as a management option than manipulation of the indigenous mycorrhizal populations. The greatest immediate potential for the successful use of mycorrhizal inoculants will be in soils and growth media where phosphorus (P) limits plant growth and where the indigenous mycorrhizal fungi are either ineffective at increasing P uptake by the plant or their numbers have been drastically reduced by human influence or natural disturbance. In recent investigations, however, additional benefits to the plant following colonization of roots by effective AM or ECM fungi have been demonstrated. These additional benefits of an effective mycorrhizal association have necessitated a re-evaluation of the optimum screening procedures for isolates of AM and ECM fungi. Both current methodologies and suggestions for alternative approaches to the screening of AM and ECM fungi are discussed in this paper. The problems inherent in choosing suitable experimental conditions to compare different isolates at the screening stage are also addressed. The review also stresses the importance of continued monitoring of introduced mycorrhizal fungi to learn more about their longer-term ecological role, particularly within reforestation or revegetation studies.     

Psychro-tolerant nematophagous fungi from the maritime Antarctic

Summary The present investigation examines the comparative growth rates, at various temperatures between 4 and 30°C, of two nematophagous fungiMonacrosporium ellipsosporum (Preuss), (Grove), Cooke and Dickinson andM. cionapagum (Drechsler), (Subramanian), Cooke and Dickinson, both isolated from the Antarctic and from Britain. No psychrophilic species were found although the results clearly show that both the Antarctic isolates were psychro-tolerant, displaying lower minimum, optimum and maximum temperatures for growth than the British isolates. A modified form ofM. ellipsosporum isolated from the Antarctic grew only between 4 and 15°C, indicating it to be much better adapted to such cold habitats than the other isolates examined.     

Planktonic microbial assemblages and the potential effects of metazooplankton predation on the food web of lakes from the maritime Antarctica and sub-Antarctic islands

Antarctica is the continent with the harshest climate on the Earth. Antarctic lakes, however, usually presents liquid water, at least during part of the year or below the ice cover, especially those from the sub-Antarctic islands and the maritime Antarctic region where climatic conditions are less extreme. Planktonic communities in these lakes are mostly dominated by microorganisms, including bacteria and phototrophic and heterotrophic protists, and by metazooplankton, usually represented by rotifers and calanoid copepods, the latter mainly from the genus Boeckella. Here I report and discuss on studies performed during the last decade that show that there is a potential for top–down control of the structure of the planktonic microbial food web in sub-Antarctic and maritime Antarctic lakes. In some of the studied lakes, the effect of copepod grazing on protozoa, either ciliates or flagellates, depending on size of both the predator and the prey, could promote cascade effects that would be transmitted to the bacterioplankton assemblage.     

Development of a convenient and supersensitive high-throughput screening system for genetically encoded fluorescent probes of small molecules using a confocal microscope

Monitoring the dynamic patterns of intracellular signaling molecules, such as inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺, that control many diverse cellular processes, provides us significant information to understand the regulatory mechanism of cellular functions. For searching more sensitive and higher dynamic range probes for signaling molecules, convenient and supersensitive high throughput screening systems are required. Here we show the optimal “in Escherichia coli (E. coli) colony” screening method based on the twin-arginine translocase (Tat) pathway and introduce a novel application of a confocal microscope as a supersensitive detection system to measure changes in the fluorescence intensity of fluorescent probes in E. coli grown on an agar plate. To verify the performance of the novel detection system, we compared the changes detected in the fluorescent intensity of genetically encoded Ca²⁺ indicator after Ca²⁺ exposure to two kinds of conventional fluorescence detection systems (luminescent image analyzer and fluorescence stereomicroscope). The rate of fluorescence change between Ca²⁺ binding and unbinding detected by novel supersensitive detection system was almost double than those measured by conventional detection systems. We also confirmed that the Tat pathway-based screening method is applicable to the development of genetically encoded probes for IP₃. Our convenient and supersensitive screening system improves the speed of developing florescent probes for small molecules.     

Diversity & tractability revisited in collaborative small molecule phenotypic screening library design

Identification of purposeful chemical matter on a broad range of drug targets is of high importance to the pharmaceutical industry. However, disease-relevant but more complex hit-finding plans require flexibility regarding the subset of the compounds that we screen. Herein we describe a strategy to design high-quality small molecule screening subsets of two different sizes to cope with a rapidly changing early discovery portfolio. The approach taken balances chemical tractability, chemical diversity and biological target coverage. Furthermore, using surveys, we actively involved chemists within our company in the selection process of the diversity decks to ensure current medicinal chemistry principles were incorporated. The chemist surveys revealed that not all published PAINS substructure alerts are considered productive by the medicinal chemistry community and in agreement with previously published results from other institutions, QED scores tracked quite well with chemists’ notions of chemical attractiveness.     

喜树内生真菌的分离及其抗肿瘤活性代谢产物的筛选方法

从喜树(Camptotheca acuminata Decne.)的根、枝条、叶和果实中分离纯化了48株内生真菌,通过对各个菌株的少量发酵培养,HPLC分析结合色谱峰紫外扫描检测方法,以紫外扫描图谱的相似性为依据,对喜树内生真菌产生喜树碱结构类似物进行初步筛选,并进一步以抑瘤实验确证其抗肿瘤活性.结果证明,以该方法筛选到的10个内生菌株中有7个菌株发酵液对HL-60细胞增殖具有显著的抑制活性.相对于常规的生物活性筛选,高效液相色谱结合色谱峰紫外光谱的方法,在药用植物内生真菌活性次生代谢产物筛选研究中具有快速、高效的特点.