In situ classification and image cytometry of pelagic bacteria from a high mountain lake (gossenkollesee, austria)

We describe a procedure to measure the cell sizes of pelagic bacteria after determinative hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes. Our approach is based on established image analysis techniques modified for objects simultaneously stained with two fluorescent dyes. It allows the estimation of biomass and cell size distribution and the morphological characterization of different bacterial taxa in plankton samples. The protocol was tested in a study of the bacterioplankton community of a high mountain lake during and after the ice break period. Cells that hybridized with a probe for the domain Bacteria accounted for 70% of the bacterial abundance (range, 49 to 83%) as determined by 4(prm1),6(prm1)-diamidino-2-phenylindole staining (K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980), but for >85% of the total biomass (range, 78 to 99%). The size distribution for members of the beta subclass of the Proteobacteria shifted toward larger cells and clearly distinguished this group from the total bacterial assemblage. In the surface water layer beneath the winter cover, bacteria belonging to the beta 1 subgroup constituted about one-half of the beta subclass abundance. The mean cell volume of the beta 1 subgroup bacteria was significantly less than that of the beta subclass proteobacteria, and the beta 1 subgroup accounted for less than 30% of the total beta subclass biovolume. Two weeks later, the biovolume of the beta Proteobacteria had decreased to the level of the beta 1 subgroup, and both the biovolume size distributions and cell morphologies of the beta Proteobacteria and the beta 1 subgroup were very similar. We could thus quantify the disappearance of large, morphologically distinct beta subclass proteobacteria which were not members of the beta 1 subgroup during the ice break period. Our results demonstrate that changes in biovolumes and cell size distributions of different bacterial taxa, and eventually of individual populations, reveal hitherto unknown processes within aquatic bacterial assemblages and may open new perspectives for the study of microbial food webs.     

High similarity between bacterioneuston and airborne bacterial community compositions in a high mountain lake area

The bacterioneuston (bacteria inhabiting the air–water interface) is poorly characterized and possibly forms a unique community in the aquatic environment. In high mountain lakes, the surface film is subjected to extreme conditions of life, suggesting the development of a specific and adapted bacterioneuston community. We have studied the surface film of a remote high mountain lake in the Pyrenees by cloning the PCR-amplified 16S rRNA gene and comparing with bacteria present in underlying waters (UW), and airborne bacteria from the dust deposited on the top of the snow pack. We did not detect unusual taxa in the neuston but rather very common and widespread bacterial groups. Betaproteobacteria and Actinobacteria accounted for >75% of the community composition. Other minor groups were Gammaproteobacteria (between 8% and 12%), Alphaproteobacteria (between 1% and 5%), and Firmicutes (1%). However, we observed segregated populations in neuston and UW for the different clades within each of the main phylogenetic groups. The soil bacterium Acinetobacter sp. was only detected in the snow–dust sample. Overall, higher similarities were found between bacterioneuston and airborne bacteria than between the former and bacterioplankton. The surface film in high mountain lakes appears as a direct interceptor of airborne bacteria useful for monitoring long-range bacterial dispersion.     

Spectral distribution of light under a subarctic winter lake cover

This study examines the spectral distribution of light penetrating the snow and ice cover of a lake in subarctic Labrador. Using a large sample under a range of cover conditions, the role of distinct layers, (snow, white or snow ice, black or clear ice) commonly found in subartic and temperate lake winter covers, was investigated. Measurement and analysis were restricted to wavelengths between 450 nm and 720 nm. Snow and white ice light transmittance was greatest at 450 and 470 to 500 nm respectively. Absorption was greatest in the red portion of the measured spectrum; white ice showing greater red absorption than snow. Black ice light transmittance showed only a slight wavelength dependence. The differential spectral absorption of light by snow and white ice layers produced a marked spectral distribution of the light that reaches the lake water surface. The importance of a spectral, rather than polychromatic, approach to the study of light, in winter biological limnology studies, is stressed.     

In situ analysis of the bacterial communities associated to farmed eel by whole-cell hybridization

Bacterial communities in water samples and eel slime were investigated by fluorescence in situ hybridization of whole bacterial cells in an eel intensive culture system over 1 year. A newly developed probe, matching 27 Vibrio spp., and a specific probe for Vibrio vulnificus were used. Phylogenetic probes complementary to selected regions of the 16S and 23S ribosomal RNA revealed that Proteobacteria of the alpha and beta subclass were predominant in water and eel slime. Members of the gamma subclass (e.g. vibrios and aeromonads) were more abundant in eel slime, although no V. vulnificus was detected.     

Digital mapping of bacterial artificial chromosomes by fluorescence in situ hybridization

The bacterial artificial chromosome (BAC) has become the most popular tool for cloning large DNA fragments. The inserts of most BAC clones average 100-200 kilobases (kb) and molecular characterization of such large DNA fragments is a major challenge. Here we report a simple and expedient technique for physical mapping of BAC inserts. Individual BAC molecules were immobilized on glass slides coated with Poly-L-lysine. The intact circular BAC molecules were visualized by fluorescence in situ hybridization using BAC DNA as a probe. The 7.4 kb BAC vector was extended to approximately 2.44 kb per micrometer. Digitally measured linear distances can be transformed into kilobases of DNA using the extension of BAC vector as a standard calibration. We mapped DNA fragments as small as 2 kb directly on circular BAC molecules. A rice BAC clone containing both tandem and dispersed repeats was analyzed using this technique. The distribution and organization of the different repeats within the BAC insert were efficiently determined. The results showed that this technique will be especially valuable for characterizing BAC clones that contain complex repetitive DNA sequences.     

Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization(1)

We report here on novel groups of Archaea in the bacterioplankton of a small boreal forest lake studied by the culture-independent analysis of the 16S rRNA genes amplified directly from lake water in combination with fluorescent in situ hybridization (FISH). Polymerase chain reaction products were cloned and 28 of the 160 Archaea clones with around 900-bp-long 16S rRNA gene inserts, were sequenced. Phylogenetic analysis, including 642 Archaea sequences, confirmed that none of the freshwater clones were closely affiliated with known cultured Archaea. Twelve Archaea sequences from lake Valkea Kotinen (VAL) belonged to Group I of uncultivated Crenarchaeota and affiliated with environmental sequences from freshwater sediments, rice roots and soil as well as with sequences from an anaerobic digestor. Eight of the Crenarchaeota VAL clones formed a tight cluster. Sixteen sequences belonged to Euryarchaeota. Four of these formed a cluster together with environmental sequences from freshwater sediments and peat bogs within the order Methanomicrobiales. Five were affiliated with sequences from marine sediments situated close to marine Group II and three formed a novel cluster VAL III distantly related to the order Thermoplasmales. The remaining four clones formed a distinct clade within a phylogenetic radiation characterized by members of the orders Methanosarcinales and Methanomicrobiales on the same branch as rice cluster I, detected recently on rice roots and in anoxic bulk soil of flooded rice microcosms. FISH with specifically designed rRNA-targeted oligonucleotide probes revealed the presence of Methanomicrobiales in the studied lake. These observations indicate a new ecological niche for many novel 'non-extreme' environmental Archaea in the pelagic water of a boreal forest lake.     

Vertical and seasonal variation of phytoplankton photosynthesis in a brown-water lake with winter ice cover

SUMMARY. Unlike previously studied lakes with prolonged winter ice and snow cover, Lake Paajarvi, southern Finland, has a high humus content and consequently differs in both the quantity and quality of light penetration into its waters. Moreover, the range of temperature fluctuation and the degree of development of thermal stratification are greater in Paajarvi, and this increased environmental heterogeneity apparently stimulates diversity in the phytoplankton community, especially in the seasonal succession of species. Differences in the photosynthetic capacity of algae from different depths in the water column were not great. This is attributed to the extremely shallow euphotic zone, algae circulating freely through the steep light gradient and sedimenting rapidly once they pass through the thermocline into the hypolimnion. It is suggested that 'adaptation' of phytoplankton to the great seasonal changes in irradiance is achieved largely by successive growths of different species in the community, and that the adaptations and vertical migrations by individual algal species, which have been reported from polar and high alpine lakes, may be of secondary importance in Pääjärvi. The species successions in Pääjärvi produce changes in the pigment content of algae similar to those reported from polar and high alpine lakes, confirming that change in pigmentation is an important mechanism in light adaptation, whether at community or individual level. Algal pigment content was particularly high at the end of the long period of winter ice cover, indicating a degree of adaptation to the prolonged low-light conditions, which produced the extremely high photosynthetic capacities measured at this time. However, phytoplankton production at any irradiance was primarily determined by biomass.     

Analysis of bacterial community structure in bulk soil by in situ hybridization

In situ hybridization with rRNA-targeted, fluorescent (Cy3-labeled) oligonucleotide probes was used to analyze bacterial community structure in ethanol- or paraformaldehyde-fixed bulk soil after homogenization of soil samples in 0.1% pyrophosphate by mild ultrasonic treatment. In ethanol-fixed samples 37 ± 7%, and in paraformaldehyde 41 ± 8% of the 4′, 6-diamidino-2-phenylindole(DAPI)-stained cells were detected with the bacterial probe Eub338. The yield could not be increased by enzymatic and/or chemical pretreatments known to enhance the permeability of bacterial cells for probes. However, during storage in ethanol for 7 months, the detectability of bacteria increased in both ethanol- and paraformaldehyde-fixed samples to up to 47 ± 8% due to an increase in the detection yield of members of the α-subdivision of Proteobacteria from 2 ± 1% to 10 ± 3%. Approximately half of the bacteria detected by probe Eub338 could be affiliated to major phylogenetic groups such as the α-, β-, γ-, and δ-subdivisions of Proteobacteria, gram-positive bacteria with a high G+C DNA content, bacteria of the Cytophaga-Flavobacterium cluster of the CFB phylum, and the planctomycetes. The analysis revealed that bacteria of the α- and δ-subdivision of Proteobacteria and the planctomycetes were predominant. Here, members of the α-subdivision of Proteobacteria accounted for approximately 10 ± 3% of DAPI-stained cells, which corresponded to 44 ± 16 × 10⁸ cells (g soil, dry wt.)^–1, while members of the δ-subdivision of Proteobacteria made up 4 ± 2% of DAPI-stained cells [17 ± 9 × 10⁸ cells (g soil, dry wt.)^–1]. A large population of bacteria in bulk soil was represented by the planctomycetes, which accounted for 7 ± 3% of DAPI-stained cells [32 ± 12 × 10⁸ cells (g soil, dry wt.)^–1]. The detection of planctomycetes in soil confirms previous reports on the occurrence of planctomycetes in soil and indicates a yet unknown ecological significance of this group, which to date has never been isolated from terrestrial environments. Received: 29 March 1997 / Accepted: 28 May 1997     

Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells. By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S rRNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria within the tissue with molecular analysis of 16S rRNA gene or other genes of interest. This method is widely applicable for the identification of noncultivable bacteria and their gene pool from formalin-fixed paraffin-embedded tissue samples.     

Grazing by a dominant rotifer Conochilus unicornis Rousselet in a mountain lake: in situ measurements with synthetic microspheres

Grazing rates of zooplankton were analysed in the summer of 1999 in Yellow Belly Lake, an oligotrophic system in the Sawtooth Mountains of Idaho (U.S.A.). The colonial rotifer Conochilus unicornis was a dominant species in the epilimnion, with densities reaching 20 colonies l^–1 (ca. 400 ind. l^–1). Clearance rates were measured with an in situ Haney Grazing chamber and synthetic microspheres 5, 9 and 23m in diameter. At epilimnetic temperatures of around 14 °C, mean clearance rates for 5m particles ranged from 30 to 65 l ind.^–1 h ^–1. Clearance rates were 2–9 times higher on the 5m spheres than on the 9 m spheres, and C. unicornis almost never fed on the 23 m spheres. Grazing rates did not change over the diel cycle. Clearance rates declined more than 10-fold as temperatures declined from 14 °C in the epilimnion to 7 °C in the metalimnion. In the epilimnion, grazing by C. unicornis was more important than grazing by crustaceans in the community, at least on particles 9m. The results show the importance of grazing by rotifers in lakes, and the significance of spatial variations that influence grazing rates.     

Effects of ultraviolet radiation and nutrients on the structure-function of phytoplankton in a high mountain lake

The combined effect of high solar ultraviolet radiation (UVR) and nutrient supply in a phytoplankton community of a high mountain lake is analyzed in a in situ experiment for 6 days with 2 × 2 factorial design. Interactive UVR × nutrient effects on structural and functional variables (algal biomass, chlorophyll a (chl a), primary production (PP), maximal electron transport rate (ETR(max)), and alkaline phosphatase activity (APA)), as well as stoichiometric ones (sestonic N per cell and N:P ratio) were found. Under non-nutrient enriched conditions, no deleterious effects of UVR on structural variables, PP, photosynthetic efficiency and ETR(max) were observed, whereas only particulate and total APA were affected by UVR. However, percentage excreted organic carbon (%EOC), dissolved APA and sestonic C and P per cell increased under UVR, leading to a decrease in algal C:P and N:P ratios. After nutrient enrichment, chl a, total algal biomass and PP were negatively affected by UVR whereas %EOC, ETR(max) and internal C, P and N content increased. We suggest that the mechanism of algal acclimation to UVR in this high UVR flux ecosystem seems to be related to the increase of internal algal P-content mediated by physiological mechanisms to save P and by a stimulatory UVR effect on dissolved extracellular APA. The mechanism involved in the unmasking effect of UVR after nutrient-enrichment may be the result of a greater sensitivity to UVR-induced cell damage, making the negative UVR effects more evident.     

Fluorescence in situ hybridization and spectral imaging of coral-associated bacterial communities

Microbial communities play important roles in the functioning of coral reef communities. However, extensive autofluorescence of coral tissues and endosymbionts limits the application of standard fluorescence in situ hybridization (FISH) techniques for the identification of the coral-associated bacterial communities. This study overcomes these limitations by combining FISH and spectral imaging.     

Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues

Accurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, secondary cell walls non-specifically bind probes used for in situ hybridization thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techniques are used. Here we describe an in situ hybridization technique which uses fast freezing and freeze substitution to cryoimmobilize the mRNA followed by embedding in a methacrylate resin for high-resolution analysis of gene expression. By using a transgenic poplar line harbouring rolC:uidA, rolC:iaaM, the gene expression pattern could be compared with histochemical GUS staining. This in situ hybridization technique results in superior preservation of cellular contents, retention of mRNA in all cell types in the poplar stem, a significant reduction of non-specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of genes involved in xylogenesis and wood formation.     

Completely distinguishing individual A-genome chromosomes and their karyotyping analysis by multiple bacterial artificial chromosome - fluorescence in situ hybridization

Twenty bacterial artificial chromosome (BAC) clones that could produce bright signals and no or very low fluorescence in situ hybridization (FISH) background were identified from Gossypium arboreum cv. JLZM, and G. hirsutum accession (acc.) TM-1 and 0-613-2R. Combining with 45S and 5S rDNA, a 22-probe cocktail that could identify all 13 G. arboreum chromosomes simultaneously was developed. According to their homology with tetraploid cotton, the G. arboreum chromosomes were designated as A1-A13, and a standard karyotype analysis of G. arboreum was presented. These results demonstrated an application for multiple BAC-FISH in cotton cytogenetic studies and a technique to overcome the problem of simultaneous chromosome recognition in mitotic cotton cells.     

Characterizing bacterial assemblages in sediments and aerosols at a dry lake bed in Australia using high-throughput sequencing

Dust storms are responsible for the transport of a large quantity of bacteria from arid regions. A severe drought in the first decade of the new millennium in Australia increased the incidence of dust transport further. The major aims of this study were to characterize the bacterial communities in aerosols and their associated source sediments using high-throughput sequencing (HTS) and to investigate the possibility of using HTS to link dust to its source, which has not been previously performed in this way. Four field campaigns were conducted at the recently evaporated saline playa Lake Gnarpurt in the Australian state of Victoria between 2008 and 2010 (3 in the austral summer, 1 in winter) to collect aerosol and sediment samples. Aerosol samples were collected on filters up to 150 m above the lake bed using a tethered helium-filled balloon. DNA was extracted from all samples using commercial kits, and the bacterial communities were examined using 454 HTS on the 16S rRNA gene. Over 200,000 sequences from 29 samples were analysed. In both sediment and aerosol samples, Salinimicrobium was the most abundant taxon; however, there was great variation and diversity across all samples. Analysis of similarities of the bacterial communities indicated that there was a significant overlap between the sediment samples and the aerosols collected above that location, showing that the bacteria in the air was derived from a subset of dust from a nearby source. The challenge remains to use bacterial profiling to link an aerosol sample to a distant source.     

Telomere analysis by fluorescence in situ hybridization and flow cytometry.

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.