High-nutrient Media Research Articles

Microbial Community Structure in the Taklimakan Desert: The Importance of Nutrient Levels in Medium and Culture Methods.

Although the Taklimakan Desert lacks the necessary nutrients and conditions to support an extensive ecosystem, it is a treasure trove of extremophile resources with special structures and functions. We analyzed the bacterial communities using oligotrophic medium and velvet cloth replicate combined with an extended culture duration. We isolated numerous uncultured microorganisms and rare microorganisms belonging to genera not often isolated or recently described, such as Aliihoeflea, Halodurantibacterium, and Indioceanicola. A total of 669 strains were isolated from the soil of the Taklimakan Desert, which were classified into 5 phyla, 7 classes, 25 orders, 42 families, 83 genera, and 379 species. Among them, 148 strains were potential new species. Our data show that even when working with samples from extreme environments, simple approaches are still useful for cultivating stubborn microbes. Through comparing the isolation effects of different nutrient levels on microbial diversity and abundance, the results show that reducing the nutrient level of the medium was more conducive to improving the culturability of microorganisms in low-nutrient environments, while the high-nutrient medium was more suitable for the isolation of dominant fast-growing strains. This study helps to better reflect the diversity of microbial resources and lays a foundation for the further research and utilization of soil microbial resources in the Taklimakan Desert.

Comparing the Effects of Two Culture Methods to Determine the Total Heterotrophic Bacterial Colony Count in Hospital Purified Water

BackgroundAccurately detecting the quantity of microorganisms in hospital purified water is of significant importance for early identification of microbial contamination and reducing the occurrence of water-borne hospital infections. The choice of detection method is a prerequisite for ensuring accurate results. Traditional Plate Count Agar (PCA) belongs to a high-nutrient medium, and there may be limitations in terms of accuracy or sensitivity in detecting microorganisms in hospital purified water. On the other hand, Reasoner’s 2A agar (R2A) has characteristics, such as low-nutrient levels, low cultivation temperature, and extended incubation time, providing advantages in promoting the growth of aquatic microorganisms. This study, through comparing the differences in total colony counts between two detection methods, aims to select the method more suitable for the growth of aquatic microorganisms, offering new practical insights for accurately detecting the total count of heterotrophic bacteria in hospital purified water.MethodsThe most commonly used plate count agar (PCA) method, and the R2A agar culture were adopted to detect microorganisms and determine the total number of bacterial colonies in the water for oral diagnosis and treatment water and terminal rinse water for endoscopes in medical institutions. The two water samples were inoculated by pour plate and membrane filtration methods, respectively. Using statistical methods including Spearman and Pearson correlation, Wilcoxon signed-rank sum test, paired-Chi-square test, and linear regression, we analyze the differences and associations in the bacterial counts cultivated through two different methods.ResultsIn 142 specimens of the water, the median and interquartile range of the heterotrophic bacterial colony number under the R2A culture method and under the PCA culture method were 200 (Q1–Q3: 25–18,000) and 6 (Q1–Q3: 0–3700). The total number of heterotrophic bacteria colonies cultured in R2A medium for 7 days was more than that cultured in PCA medium for 2 days (P < 0.05). The linear regression results showed a relatively strong linear correlation between the number of colonies cultured by the R2A method and that cultured by the PCA method (R2 = 0.7264). The number of bacterial species detected on R2A agar medium is greater than that on PCA agar medium.ConclusionThe R2A culture method can better reflect the actual number of heterotrophic bacterial colonies in hospital purified water. After logarithmic transformation, the number of colonies cultured by the two methods showed a linear correlation.

Continuous monitoring of plant sodium transport dynamics using clinical PET

BackgroundThe absorption, translocation, accumulation and excretion of substances are fundamental processes in all organisms including plants, and have been successfully studied using radiotracers labelled with 11C, 13N, 14C and 22Na since 1939. Sodium is one of the most damaging ions to the growth and productivity of crops. Due to the significance of understanding sodium transport in plants, a significant number of studies have been carried out to examine sodium influx, compartmentation, and efflux using 22Na- or 24Na-labeled salts. Notably, however, most of these studies employed destructive methods, which has limited our understanding of sodium flux and distribution characteristics in real time, in live plants. Positron emission tomography (PET) has been used successfully in medical research and diagnosis for decades. Due to its ability to visualise and assess physiological and metabolic function, PET imaging has also begun to be employed in plant research. Here, we report the use of a clinical PET scanner with a 22Na tracer to examine 22Na-influx dynamics in barley plants (Hordeum vulgare L. spp. Vulgare—cultivar Bass) under variable nutrient levels, alterations in the day/night light cycle, and the presence of sodium channel inhibitors.Results3D dynamic PET images of whole plants show readily visible 22Na translocation from roots to shoots in each examined plant, with rates influenced by both nutrient status and channel inhibition. PET images show that plants cultivated in low-nutrient media transport more 22Na than plants cultivated in high-nutrient media, and that 22Na uptake is suppressed in the presence of a cation-channel inhibitor. A distinct diurnal pattern of 22Na influx was discernible in curves displaying rates of change of relative radioactivity. Plants were found to absorb more 22Na during the light period, and anticipate the change in the light/dark cycle by adjusting the sodium influx rate downward in the dark period, an effect not previously described experimentally.ConclusionsWe demonstrate the utility of clinical PET/CT scanners for real-time monitoring of the temporal dynamics of sodium transport in plants. The effects of nutrient deprivation and of ion channel inhibition on sodium influx into barley plants are shown in two proof-of-concept experiments, along with the first-ever 3D-imaging of the light and dark sodium uptake cycles in plants. This method carries significant potential for plant biology research and, in particular, in the context of genetic and treatment effects on sodium acquisition and toxicity in plants.

Endophytic association between Alternaria oxytropis and Oxytropis kansuensis affected by nutritional conditions and temperature

Alternaria oxytropis has been reported as an endophyte of species in the genera Astragalus and Oxytropis (Fabaceae) in the USA and China. Infected plants contain the alkaloid swainsonine produced by the fungus, and this causes poisoning of grazing animals. Therefore, ecological investigations are important to clarify the extent of this endophytic association. Seeds of O. kansuensis were collected from a locality known for indigenous A. oxytropis in China and were cultured on artificial media under controlled conditions for about two months. Less than 50% of seeds germinated with colonies of A. oxytropis, however, germination and seedling growth were influenced by the type of culture media used as well as temperature. Seedling growth with colonies of the fungus was significantly better on low nutrient media at low temperature (20 °C) than that on a high nutrient media at high temperature (25 °C). Seedlings with colonies of the fungus on a high nutrient media were eventually killed by the fungus. It is suggested that the endophytic association between the fungus and plants is affected by nutritional conditions and temperature.

Atypical Cadherin FAT3 Is a Novel Mediator for Morphological Changes of Microglia.

Microglia are resident macrophages that are critical for brain development and homeostasis. Microglial morphology is dynamically changed during postnatal stages, leading to regulating synaptogenesis and synapse pruning. Moreover, it has been well known that the shape of microglia is also altered in response to the detritus of the apoptotic cells and pathogens such as bacteria and viruses. Although the morphologic changes are crucial for acquiring microglial functions, the exact mechanism which controls their morphology is not fully understood. Here, we report that the FAT atypical cadherin family protein, FAT3, regulates the morphology of microglial cell line, BV2. We found that the shape of BV2 becomes elongated in a high-nutrient medium. Using microarray analysis, we identified that FAT3 expression is induced by culturing with a high-nutrient medium. In addition, we found that purinergic analog, hypoxanthine, promotes FAT3 expression in BV2 and mouse primary microglia. FAT3 expression induced by hypoxanthine extends the time of sustaining the elongated forms in BV2. These data suggest that the hypoxanthine-FAT3 axis is a novel pathway associated with microglial morphology. Our data provide a possibility that FAT3 may control microglial transitions involved in their morphologic changes during the postnatal stages in vivo.

Phage resistance evolution in vitro is not reflective of in vivo outcome in a plant-bacteria-phage system.

The evolution of resistance to parasites is fundamentally important to disease ecology, yet we remain unable to predict when and how resistance will evolve. This is largely due to the context-dependent nature of host-parasite interactions, as the benefit of resistance will depend on the abiotic and biotic environment. Through experimental evolution of the plant pathogenic bacterium Pseudomonas syringae and two lytic bacteriophages across two different environments (high-nutrient media and the tomato leaf apoplast), we demonstrate that de novo evolution of resistance is negligible in planta despite high levels of resistance evolution in vitro. We find no evidence supporting the evolution of phage-selected resistance in planta despite multiple passaging experiments, multiple assays for resistance, and high multiplicities of infection. Additionally, we find that phage-resistant mutants (evolved in vitro) did not realize a fitness benefit over phage-sensitive cells when grown in planta in the presence of phage, despite reduced growth of sensitive cells, evidence of phage replication in planta, and a large fitness benefit in the presence of phage observed invitro. Thus, this context-dependent benefit of phage resistance led to different evolutionary outcomes across environments. These results underscore the importance of studying the evolution of parasite resistance in ecologically relevant environments.

A survey on cultivable heterotrophic bacteria inhabiting a thermally unstratified water column in an Atlantic Rainforest lake.

Due to the importance of heterotrophic bacteria in biogeochemical cycles and their influence on water quality, many studies have assessed the composition of the bacterial community. Most of these were made in temperate freshwaters. Eighteen heterotrophic bacteria communities distributed over time and space in the water column of Carioca Lake, not exposed to anthropogenic activities, were analyzed to characterize their composition. A polyphasic approach was used, including 16S rDNA restriction analysis, 16S rRNA gene sequence analysis, BIOLOG Ecoplates and statistical methods. The physiological profiles among the 18 microbial communities were diverse. Clustering analysis and the metabolic fingerprint of the Biolog EcoplateTM system data separated the communities based on temporal scale. A set of 673 isolates were recovered on high nutrient medium. The 673 isolates obtained yielded 360 Amplified Ribosomal DNA Restriction Analysis (ARDRA) Operational Taxonomic Units (OTUs). Most (313) of the ARDRA patterns, OTUs, were from isolates obtained in a single sampling point, in temporal and spatial scales, indicating changes in the bacterial community. A subset of representative isolates for each ARDRA OTU was identified by 16S rRNA gene fragment sequencing and categorized into five phyla, Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Deinococcus-Thermus, represented by 38 genera. The results of this work contribute to a better understanding about the phylogeny of tropical freshwater heterotrophic bacteria.

The Yersinia enterocolitica Ysa type III secretion system is expressed during infections both in vitro and in vivo

Yersinia enterocolitica biovar 1B maintains two type III secretion systems (T3SS) that are involved in pathogenesis, the plasmid encoded Ysc T3SS and the chromosomally encoded Ysa T3SS. In vitro, the Ysa T3SS has been shown to be expressed only at 26°C in a high-nutrient medium containing an exceptionally high concentration of salt – an artificial condition that provides no clear insight on the nature of signal that Y. enterocolitica responds to in a host. However, previous research has indicated that the Ysa system plays a role in the colonization of gastrointestinal tissues of mice. In this study, a series of Ysa promoter fusions to green fluorescent protein gene (gfp) were created to analyze the expression of this T3SS during infection. Using reporter strains, infections were carried out in vitro using HeLa cells and in vivo using the mouse model of yersiniosis. Expression of green fluorescent protein (GFP) was measured from the promoters of yspP (encoding a secreted effector protein) and orf6 (encoding a structural component of the T3SS apparatus) in vitro and in vivo. During the infection of HeLa cells GFP intensity was measured by fluorescence microscopy, while during murine infections GFP expression in tissues was measured by flow cytometry. These approaches, combined with quantification of yspP mRNA transcripts by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), demonstrate that the Ysa system is expressed in vitro in a contact-dependent manner, and is expressed in vivo during infection of mice.

Hydrophobic Liquid-Infused Porous Polymer Surfaces for Antibacterial Applications

Biofilms represent a fundamental problem in environmental biology, water technology, food hygiene as well as in medical and technical systems. Recently introduced slippery liquid-infused porous surface (SLIPS) showed great promise for preventing biofilm formation owing to the low surface energy of such surface in combination with its self-cleaning properties. In this study we demonstrated a novel hydrophobic liquid-infused porous poly(butyl methacrylate-co-ethylene dimethacrylate) surface (slippery BMA-EDMA) with bacteria-resistance in BM2 mineral medium and long-term stability in aqueous environments. We showed that the slippery BMA-EDMA surface prevents biofilm formation of different strains of opportunistic pathogen Pseudomonas aeruginosa for at least up to 7 days in low nutrient medium. Only ∼1.8% of the slippery surface was covered by the environmental P. aeruginosa PA49 strain under investigation. In uncoated glass controls the coverage of surfaces reached ∼55% under the same conditions. However, in high nutrient medium, more relevant to physiological conditions, the biofilm formation on the slippery surface turned out to be highly dependent on the bacterial strain. Although the slippery surface could prevent biofilm formation of most of the P. aeruginosa strains tested (∼1% surface coverage), the multiresistant P. aeruginosa strain isolated from wastewater was able to cover up to 12% of the surface during 7 days of incubation. RAPD-PCR analysis of the used P. aeruginosa strains demonstrated their high genome variability, which might be responsible for their difference in biofilm formation on the slippery BMA-EDMA surface. The results show that although the slippery BMA-EDMA surface has a great potential against biofilm formation, the generality of its bacteria resistant properties is still to be improved.

Proposal for a method to estimate nutrient shock effects in bacteria.

BackgroundPlating methods are still the golden standard in microbiology; however, some studies have shown that these techniques can underestimate the microbial concentrations and diversity. A nutrient shock is one of the mechanisms proposed to explain this phenomenon. In this study, a tentative method to assess nutrient shock effects was tested.FindingsTo estimate the extent of nutrient shock effects, two strains isolated from tap water (Sphingomonas capsulata and Methylobacterium sp.) and two culture collection strains (E. coli CECT 434 and Pseudomonas fluorescens ATCC 13525) were exposed both to low and high nutrient conditions for different times and then placed in low nutrient medium (R2A) and rich nutrient medium (TSA).The average improvement (A.I.) of recovery between R2A and TSA for the different times was calculated to more simply assess the difference obtained in culturability between each medium. As expected, A.I. was higher when cells were plated after the exposition to water than when they were recovered from high-nutrient medium showing the existence of a nutrient shock for the diverse bacteria used. S. capsulata was the species most affected by this phenomenon.ConclusionsThis work provides a method to consistently determine the extent of nutrient shock effects on different microorganisms and hence quantify the ability of each species to deal with sudden increases in substrate concentration.

Preservation of encapsulated shoot tips and nodes of the tropical hardwoods Corymbia torelliana × C. citriodora and Khaya senegalensis

Alginate encapsulation is a simple and cost-effective technique to preserve plant germplasm but there are only a few reports available on preservation of encapsulated explants of two highly valuable groups of tropical trees, the eucalypts (Myrtaceae) and mahoganies (Meliaceae). This study investigated alginate encapsulation for preservation of the eucalypt hybrid, Corymbia torelliana × C. citriodora, and the African mahogany, Khaya senegalensis. We assessed shoot regrowth of encapsulated shoot tips and nodes after storage for 0, 3, 6 and 12 months on media varying in sucrose and nutrient content, under storage conditions of 14°C and zero-irradiance. Encapsulated explants of both trees were preserved most effectively on high-nutrient (half-strength Murashige and Skoog) medium containing 1% sucrose, which provided very high frequencies of shoot regrowth (92–100% for Corymbia and 71–98% for Khaya) and excellent shoot development after 12 months’ storage. This technique provides an extremely efficient means for storage and exchange of eucalypts and mahoganies, ideally suited for incorporation into plant breeding and germplasm conservation programs.

Cottoquinazoline A and Cotteslosins A and B, Metabolites from an Australian Marine-Derived Strain of Aspergillus versicolor

An Australian marine-derived isolate of Aspergillus versicolor (MST-MF495) yielded the known fungal metabolites sterigmatocystin, violaceol I, violaceol II, diorcinol, (-)-cyclopenol, and viridicatol, along with a new alkaloid, cottoquinazoline A (1), and two new cyclopentapeptides, cotteslosins A (2) and B (3). Structures for 1-3 and the known compounds were determined by spectroscopic analysis. The absolute configurations of 1-3 were addressed by chemical degradation and application of the C(3) Marfey's method. The use of "cellophane raft" high-nutrient media as a device for up-regulating secondary metabolite diversity in marine-derived fungi is discussed. The antibacterial properties displayed by A. versicolor (MST-MF495) were attributed to the phenols violaceol I, violaceol II, and diorcinol, while cotteslosins 2 and 3 were identified as weak cytotoxic agents.

Ultraviolet radiation sensitivity in cave bacteria: evidence of adaptation to the subsurface?

We hypothesize that a reduced capacity to withstand or repair cellular damage from ultraviolet radiation may be present in cave-adapted microorganisms that never experience such conditions. However, a small number of previous studies have shown that some subsurface bacteria do not show greater sensitivity to ultraviolet radiation (UVR) than surface bacteria. To estimate UVR sensitivity in cave bacteria, bacterial isolates were collected from Carlsbad Cavern, New Mexico, U.S.A., and percent survival following exposure to various UVC and UVA radiation doses was determined. Cave bacteria from Left Hand Tunnel in Carlsbad Cavern and surface bacteria from soil and rocks above Carlsbad Cavern were grown on low and high nutrient media then exposed to 0, 10,000 and 20,000 μWs/ cm2 of UVR in a laboratory biological safety cabinet. Incubations were conducted at 15°C or 37ºC, in accordance with the isolates’ natural temperature environments. In addition, DNA repair capacity was estimated by exposing the organisms to various doses of UVC radiation and measuring survivability. Gram status and pigmentation also were determined. Results showed that most of the cave isolates were more sensitive to UVR than the surface isolates, but survivability data suggest that cave microbes retain some of their capacity to repair UV-induced DNA damage. Selection appears to have favored bacteria that can survive in this low nutrient environment, while not maintaining (or paying the cost of maintaining) unneeded traits such as UVR resistance. Cave bacteria appear to have maintained DNA repair capacity, most likely because of the need to repair damage to their DNA from other environmental stressors found in caves.

Cultivable Bacterial Community from South China Sea Sponge as Revealed by DGGE Fingerprinting and 16S rDNA Phylogenetic Analysis

The cultivable bacterial communities associated with four South China Sea sponges-Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis in mixed cultures-were investigated by microbial community DNA-based DGGE fingerprinting and 16S rDNA phylogenetic analysis. Diverse bacteria such as alpha-, gamma-, delta-Proteobacteria, Bacteroidetes, and Firmicutes were cultured, some of which were previously uncultivable bacteria, potential novel strains with less than 95% similarity to their closest relatives and sponge symbionts growing only in the medium with the addition of sponge extract. According to 16S rDNA BLAST analysis, most of the bacteria were cultured from sponge for the first time, although similar phyla of bacteria have been previously recognized. The selective pressure of sponge extract on the cultured bacterial species was suggested, although the effect of sponge extract on bacterial community in high nutrient medium is not significant. Although alpha- and gamma-Proteobacteria appeared to form the majority of the dominant cultivable bacterial communities of the four sponges, the composition of the cultivable bacterial community in the mixed culture was different, depending on the medium and sponge species. Greater bacterial diversity was observed in media C and CS for Stelletta tenuis, in media F and FS for Halichondria rugosa and Craniella australiensis. S. tenuis was found to have the highest cultivable bacterial diversity including alpha-, gamma-, delta-Proteobacteria, Bacteroidetes, and Firmicutes, followed by sponge Dysidea avara without delta-Proteobacteria, sponge Halichondria rugosa with only alpha-, gamma-Proteobacteria and Bacteroidetes, and sponge C. australiensis with only alpha-, gamma-Proteobacteria and Firmicutes. Based on this study, by the strategy of mixed cultivation integrated with microbial community DNA-based DGGE fingerprinting and phylogenetic analysis, the cultivable bacterial community of sponge could be revealed effectively.

Influence of culture conditions on Escherichia coli O157:H7 biofilm formation by atomic force microscopy

Biofilms are complex microbial communities that are resistant against attacks by bacteriophages and removal by drugs and chemicals. In this study, biofilms of Escherichia coli O157:H7, a bacterial pathogen, were investigated using atomic force microscopy (AFM) in terms of the dynamic transition of morphology and surface properties of bacterial cells over the development of biofilms. The physical and topographical properties of biofilms are different, depending on nutrient availability. Compared to biofilms formed in a high nutrient medium, biofilms form faster and a higher number of bacterial cells were recovered on glass surface in a low nutrient medium. We demonstrate that AFM can obtain high-resolution images and the elastic information about biofilms. As E. coli biofilm becomes mature, the magnitude of the force between a tip and the surface of the biofilm gets stronger, suggesting that extracellular polymeric substances (EPSs), sticky components of biofilms, accumulate over the surface of cells upon the initial attachment of bacterial cells to surfaces.

Genetic Variation of the Bloom-Forming CyanobacteriumMicrocystis aeruginosawithin and among Lakes: Implications for Harmful Algal Blooms

To measure genetic variation within and among populations of the bloom-forming cyanobacterium Microcystis aeruginosa, we surveyed a suite of lakes in the southern peninsula of Michigan that vary in productivity (total phosphorus concentrations of approximately 10 to 100 microg liter(-1)). Survival of M. aeruginosa isolates from lakes was relatively low (i.e., mean of 7% and maximum of 30%) and positively related to lake total phosphorus concentration (P = 0.014, r2 = 0.407, n = 14). In another study (D. F. Raikow, O. Sarnelle, A. E. Wilson, and S. K. Hamilton, Limnol. Oceanogr. 49:482-487, 2004), survival rates of M. aeruginosa isolates collected from an oligotrophic lake (total phosphorus of approximately 10 mug liter(-1) and dissolved inorganic nitrogen:total phosphorus ratio of 12.75) differed among five different medium types (G test, P of <0.001), with higher survival (P = 0.003) in low-nutrient media (28 to 37% survival) than in high-nutrient media. Even with the relatively low isolate survivorship that could select against detecting the full range of genetic variation, populations of M. aeruginosa were genetically diverse within and among lakes (by analysis of molecular variance, Phi(sc) = 0.412 [Phi(sc) is an F-statistic derivative which evaluates the correlation of haplotypic diversity within populations relative to the haplotypic diversity among all sampled populations], P = 0.001), with most clones being distantly related to clones collected from lakes directly attached to Lake Michigan (a Laurentian Great Lake) and culture collection strains collected from Canada, Scotland, and South Africa. Ninety-one percent of the 53 genetically unique M. aeruginosa clones contained the microcystin toxin gene (mcyA). Genotypes with the toxin gene were found in all lakes, while four lakes harbored both genotypes possessing and genotypes lacking the toxin gene.

Identification and Characterization of Potentially Algal‐lytic Marine Bacteria Strongly Associated with the Toxic Dinoflagellate Alexandrium catenella

The toxic dinoflagellate Alexandrium catenella isolated from fjords in Southern Chile produces several analogues of saxitoxin and has been associated with outbreaks of paralytic shellfish poisoning. Three bacterial strains, which remained in close association with this dinoflagellate in culture, were isolated by inoculating the dinoflagellate onto marine agar. The phenotypically different cultivable bacterial colonies were purified. Their genetic identification was done by polymerase chain reaction amplification of the 16S rRNA genes. Partial sequence analysis suggested that the most probable affiliations were to two bacterial phyla: Proteobacteria and the Cytophaga group. The molecular identification was complemented by morphological data and biochemical profiling. The three bacterial species, when grown separately from phytoplankton cells in high-nutrient media, released algal-lytic compounds together with aminopeptidase, lipase, glucosaminidase, and alkaline phosphatase. When the same bacteria, free of organic nutrients, were added back to the algal culture they displayed no detrimental effects on the dinoflagellate cells and recovered their symbiotic characteristics. This observation is consistent with phylogenetic analysis that reveals that these bacteria correspond to species distinct from other bacterial strains previously classified as algicidal bacteria. Thus, bacterial-derived lytic activities are expressed only in the presence of high-nutrient culture media and it is likely that in situ environmental conditions may modulate their expression.

The effect of simulated microgravity on bacteria from the mir space station

The effects of simulated microgravity on two bacterial isolates, Sphingobacterium thalpophilium and Ralstonia pickettii (formerly Burkholderia pickettii), originally recovered from water systems aboard the Mir space station were examined. These bacteria were inoculated into water, high and low concentrations of nutrient broth and subjected to simulated microgravity conditions. S. thalpophilium (which was motile and had flagella) showed no significant differences between simulated microgravity and the normal gravity control regardless of the method of enumeration and medium. In contrast, for R. pickettii (that was non-motile and lacked flagella), there were significantly higher numbers in high nutrient broth under simulated microgravity compared to normal gravity. Conversely, when R. pikkettii was inoculated into water (i.e., starvation conditions) significantly lower numbers were found under simulated microgravity compared to normal gravity. Responses to microgravity depended on the strain used (e.g., the motile strain exhibited no response to microgravity, while the non-motile strain did), the method of enumeration, and the nutrient concentration of the medium. Under oligotrophic conditions, non-motile cells may remain in geostationary orbit and deplete nutrients in their vicinity, while in high nutrient medium, resources surrounding the cell may be sufficient so that high growth is observed until nutrients becoming limiting.

Double-stranded RNA transmission through basidiospores of Heterobasidion annosum

A search for double-stranded RNA (dsRNA) was conducted among 106 isolates of the pathogenic basidiomycete Heterobasidion annosum. Of these isolates, 47 were tissue isolates from fruit bodies and 59 were isolated from decayed wood. Nucleic acids were extracted from freeze-dried mycelia and dsRNA was separated by cellulose CF-11 chromatography and confirmed by digestions with specific nucleases. dsRNA was present in 19 and 14% of the tissue and wood isolates, respectively. From five of the fruit bodies containing dsRNA basidiospores were investigated and 10-84% of the germinated basidiospores contained dsRNA. On high nutrient media, the germination frequency of basidiospores was reduced by presence of dsRNA in the fruit body (P < 0.05); germination frequencies were 34 and 78% for spores from fruit bodies with and without dsRNA, respectively. The same trend was present also on low nutrient media, although not statistically significant; germination was 3 and 10% for spores from infected and dsRNA free fruit bodies, respectively. Transmission of dsRNA in H. annosum from mycelia into basidiospores together with the lowered germination frequency are likely to play a significant role in the life cycle of the fungus. The relative importance of different transmission routes for dsRNA in H. annosum is discussed.

Cultivation and growth characteristics of a diverse group of oligotrophic marine Gammaproteobacteria.

Forty-four novel strains of Gammaproteobacteria were cultivated from coastal and pelagic regions of the Pacific Ocean using high-throughput culturing methods that rely on dilution to extinction in very low nutrient media. Phylogenetic analysis showed that the isolates fell into five rRNA clades, all of which contained rRNA gene sequences reported previously from seawater environmental gene clone libraries (SAR92, OM60, OM182, BD1-7, and KI89A). Bootstrap analyses of phylogenetic reliability did not support collapsing these five clades into a single clade, and they were therefore named the oligotrophic marine Gammaproteobacteria (OMG) group. Twelve cultures chosen to represent the five clades were successively purified in liquid culture, and their growth characteristics were determined at different temperatures and dissolved organic carbon concentrations. The isolates in the OMG group were physiologically diverse heterotrophs, and their physiological properties generally followed their phylogenetic relationships. None of the isolates in the OMG group formed colonies on low- or high-nutrient agar upon their first isolation from seawater, while 7 of 12 isolates that were propagated for laboratory testing eventually produced colonies on 1/10 R2A agar. The isolates grew relatively slowly in natural seawater media (1.23 to 2.63 day(-1)), and none of them grew in high-nutrient media (>351 mg of C liter(-1)). The isolates were psychro- to mesophilic and obligately oligotrophic; many of them were of ultramicrobial size (<0.1 micro m(3)). This cultivation study revealed that sporadically detected Gammaproteobacteria gene clones from seawater are part of a phylogenetically diverse constellation of organisms mainly composed of oligotrophic and ultramicrobial lineages that are culturable under specific cultivation conditions.