Substrate uptake by uncultured bacteria from the genus Achromatium determined by microautoradiography.

Microautoradiography was used to investigate substrate uptake by natural communities of uncultured bacteria from the genus Achromatium. Studies of the uptake of (14)C-labelled substrates demonstrated that Achromatium cells from freshwater sediments were able to assimilate (14)C from bicarbonate, acetate, and protein hydrolysate; however, (14)C-labelled glucose was not assimilated. The pattern of substrate uptake by Achromatium spp. was therefore similar to those of a number of other freshwater and marine sulfur-oxidizing bacteria. Different patterns of radiolabelled bicarbonate uptake were noted for Achromatium communities from different geographical locations and indicated that one community (Rydal Water) possessed autotrophic potential, while the other (Hell Kettles) did not. Furthermore, the patterns of organic substrate uptake within a single population suggested that physiological diversity existed in natural communities of Achromatium. These observations are consistent with and may relate to the phylogenetic diversity observed in Achromatium communities. Incubation of Achromatium-bearing sediment cores from Rydal Water with (35)S-labelled sulfate in the presence and absence of sodium molybdate demonstrated that this bacterial population was capable of oxidizing sulfide to intracellular elemental sulfur. This finding supported the role of Achromatium in the oxidative component of a tightly coupled sulfur cycle in Rydal Water sediment. The oxidation of sulfide to sulfur and ultimately to sulfate by Achromatium cells from Rydal Water sediment is consistent with an ability to conserve energy from sulfide oxidation.     

Use of microautoradiography combined with fluorescence in situ hybridization to determine dimethylsulfoniopropionate incorporation by marine bacterioplankton taxa

The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU). In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from [(35)S]DMSP ranged between 27 and 51% of 4',6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating [(3)H]leucine. In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from [(35)S]DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom. At the same time, the proportion of cells incorporating [(3)H]leucine increased from 21 to 40%. The combination of AU and fluorescence in situ hybridization (FISH) revealed that the Roseobacter clade (alpha-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating [(35)S]DMSP at both sampling sites. Significant uptake of sulfur from DMSP was also found among members of the gamma-proteobacteria and Cytophaga-Flavobacterium groups. Roseobacter and gamma-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating (35)S from DMSP (around 50%). Altogether, the application of AU with [(35)S]DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization. These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton.     

Substrate uptake in extremely halophilic microbial communities revealed by microautoradiography and fluorescence in situ hybridization

The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR approach) was applied to brine samples of a solar saltern crystallizer pond from Mallorca (Spain) where the simultaneous occurrence of Salinibacter spp. and the conspicuous square Archaea had been detected. Radioactively labeled bicarbonate, acetate, glycerol, and an amino acid mixture were tested as substrates for the microbial populations inhabiting such brines. The results indicated that hitherto uncultured 'square Archaea' do actively incorporate amino acids and acetate. However, Salinibacter spp. only showed amino acid incorporation in pure culture, but no evidence of such activity in their natural environment could be demonstrated. No glycerol incorporation was observed for any component of the microbial community.Communicated by W.D. Grant     

Quantification of cell-specific substrate uptake by probe-defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization

A technique based on quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) was developed and evaluated in order to determine the quantitative uptake of specific substrates in probe-defined filamentous bacteria directly in a complex system. The technique, QMAR-FISH, has a resolution of a single cell and is based on an improved fixation protocol and the use of an internal standard of bacteria with known specific radioactivity. The method was used to study the in situ ecophysiology of the filamentous bacteria 'Candidatus Meganema perideroedes' and Thiothrix sp. directly in an activated sludge system. The cellular uptake rate of tritium-labelled substrates revealed an average cell-specific uptake rate of 4.1 yen 10-15 mol of acetate cell-1 h-1 and 3.1 yen 10-15 mol of acetate cell-1 h-1 for the two filamentous species respectively. The two filamentous species had very similar activity in all cells along each filament. Surprisingly, the filaments within both probe-defined populations had threefold variation in activity between the different filaments, demonstrating a large variation in activity level within a single population in a complex system. The substrate affinity (Ks) for uptake of acetate of the cells within the two filamentous bacteria was determined by incubation with variable concentrations of labelled acetate. The Ks values of the 'Candidatus Meganema perideroedes' and the Thiothrix filamentous bacteria were determined to be 1.8 micro M and 2.4 micro M acetate respectively.     

Enrichment of previously uncultured bacteria from natural complex communities by adhesion to solid surfaces

The adhesion to inert solid surfaces was explored as a novel approach for the enrichment of previously uncultured bacteria from natural microbial communities. Enrichments on solid steel, glass and synthetic polymeric surfaces were established using samples from five freshwater lakes, a marine microbial mat and an alpine soil, and were subsequently analysed by molecular fingerprinting and sequencing of their 16S rRNA gene fragments. The majority of the enriched phylotypes grouped with the Alphaproteobacteria, Betaproteobacteria or Bacteroidetes and in several cases were related to typical biofilm‐forming species and genera. Most enrichments were most closely related to previously uncultured phylotypes and none had previously been cultivated from the original environments even when applying improved high throughput liquid cultivation techniques. Of the 13 phylotypes enriched from freshwater samples, seven were previously unknown, three matched so‐far uncultured environmental clones, and three were identical to previously cultivated bacteria. Of the 17 phylotypes recovered from soil, 12 were previously unknown with five of these phylotypes representing novel genera, whereas five phylotypes were identical to previously cultured soil bacteria. The feasibility of the biofilm‐enrichment approach was exemplified by the successful isolation of a not‐yet cultured Betaproteobacterium that constituted a discernible component of the alpine soil microbial community in situ and exhibited only 93% similarity to its closest cultured relative. Based on these results, cultivation on solid surfaces represents a promising approach to recover isolates that have so far escaped cultivation as suspended cultures in liquid media.     

Quantifying substrate uptake by individual cells of marine bacterioplankton by catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography

Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking up (3)H-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucine-assimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions.     

Combining catalyzed reporter deposition-fluorescence in situ hybridization and microautoradiography to detect substrate utilization by bacteria and Archaea in the deep ocean

The recently developed CARD-FISH protocol was refined for the detection of marine Archaea by replacing the lysozyme permeabilization treatment with proteinase K. This modification resulted in about twofold-higher detection rates for Archaea in deep waters. Using this method in combination with microautoradiography, we found that Archaea are more abundant than Bacteria (42% versus 32% of 4',6'-diamidino-2-phenylindole counts) in the deep waters of the North Atlantic and that a larger fraction of Archaea than of Bacteria takes up l-aspartic acid (19% versus 10%).     

Calcite-accumulating large sulfur bacteria of the genus Achromatium in Sippewissett Salt Marsh

Large sulfur bacteria of the genus Achromatium are exceptional among Bacteria and Archaea as they can accumulate high amounts of internal calcite. Although known for more than 100 years, they remain uncultured, and only freshwater populations have been studied so far. Here we investigate a marine population of calcite-accumulating bacteria that is primarily found at the sediment surface of tide pools in a salt marsh, where high sulfide concentrations meet oversaturated oxygen concentrations during the day. Dynamic sulfur cycling by phototrophic sulfide-oxidizing and heterotrophic sulfate-reducing bacteria co-occurring in these sediments creates a highly sulfidic environment that we propose induces behavioral differences in the Achromatium population compared with reported migration patterns in a low-sulfide environment. Fluctuating intracellular calcium/sulfur ratios at different depths and times of day indicate a biochemical reaction of the salt marsh Achromatium to diurnal changes in sedimentary redox conditions. We correlate this calcite dynamic with new evidence regarding its formation/mobilization and suggest general implications as well as a possible biological function of calcite accumulation in large bacteria in the sediment environment that is governed by gradients. Finally, we propose a new taxonomic classification of the salt marsh Achromatium based on their adaptation to a significantly different habitat than their freshwater relatives, as indicated by their differential behavior as well as phylogenetic distance on 16S ribosomal RNA gene level. In future studies, whole-genome characterization and additional ecophysiological factors could further support the distinctive position of salt marsh Achromatium.     

Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria

We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.     

Abundance and phylogenetic affiliation of iron reducers in activated sludge as assessed by fluorescence in situ hybridization and microautoradiography

Microautoradiography (MAR) was used to enumerate acetate-consuming bacteria under Fe(III)-reducing conditions in activated sludge. This population is believed to consist of dissimilatory iron-reducing bacteria, because the applied incubation conditions and the use of specific inhibitors excluded consumption of radiolabeled acetate by other physiological groups such as sulfate reducers. By use of this approach, dissimilatory iron reducers were found in a concentration of 1.1 x 10(8) cells per ml, corresponding to approximately 3% of the total cell count as determined by DAPI (4',6'-diamino-2-phenylindoledihydrochloride-dilactate) staining. The MAR enumeration method was compared to the traditional most-probable-number (MPN) method (FeOOH-MPN) and a modified MPN method, which contains Ferrozine directly within the MPN dilutions to determine the production of small amounts of ferrous iron (Ferrozine-MPN). The Ferrozine-MPN method yielded values 6 to 10 times higher than those obtained by the FeOOH-MPN method. Nevertheless, the MAR approach yielded counts that were 100 to 1,000 times higher than those obtained by the Ferrozine-MPN method. Specific in situ Fe(III) reduction rates per cell (enumerated by the MAR method) were calculated and found to be comparable to the respective rates for pure cultures of dissimilatory iron-reducing bacteria, suggesting that the new MAR method is most reliable. A combination of MAR and fluorescence in situ hybridization was used for phylogenetic characterization of the putative iron-reducing bacteria. All activated-sludge cells able to consume acetate under iron-reducing conditions were targeted by the bacterial oligonucleotide probe EUB338. Around 20% were identified as gamma Proteobacteria, and 10% were assigned to the delta subclass of Proteobacteria.     

Rapid confirmation of previously detected prenatal mosaicism by fluorescence in situ hybridization in interphase uncultured amniocytes

Fluorescence in situ hybridization (FISH) of chromosome-specific probes to interphase uncultured amniocytes was performed in cases in which follow-up amniocenteses were done for confirmation of previously detected mosaicism. FISH results were informative in all seven cases included in the study, and confirmed by subsequent cytogenetic analysis. FISH analysis provides rapid results for referral physicians and in most cases reassurance for patients within 24 hours of the follow-up aminocentesis. Although FISH studies are not considered accurate in determining a primary diagnosis of mosaicism in uncultured cells, the analysis is accurate and clinically useful when the diagnosis is known and mosaicism involving a specific chromosome needs to be confirmed in follow-up testing.     

荧光原位杂交技术对土壤石油降解菌的检测

从辽河油田样品中筛选出一株高效石油降解菌,经鉴定为地衣芽孢杆菌。针对其16S rRNA设计寡核苷酸探针。荧光原位杂交(FISH)技术利用寡核苷酸探针检测特定细胞内的互补核苷酸序列。通过对纯菌和泥浆中地衣芽孢杆菌的FISH进行优化,得到泥浆中地衣芽孢杆菌的荧光原位杂交实验条件：样品固定时间17 h,杂交温度46 ℃,杂交时间3 h,杂交液中去离子甲酰胺浓度35%,冲洗缓冲液中与去离子甲酰胺对应的NaCl的浓度88 mmol·L^-1。运用上述FISH技术监测生物泥浆反应器中地衣芽孢杆菌量的变化,并与泥浆中含油率的变化进行比较,二者的变化情况符合微生物降解石油的趋势,为监测含油污泥中微生物的变化提供了一种可行的技术。     

Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization

The 17s-5.8s-25s ribosomal RNA gene (rDNA) loci in Oryza spp. were identified by the fluorescence in-situ hybridization (FISH) method. The rDNA loci were located on one-to-three chromosomes (two-to-six sites) within the eight diploid Oryza spp. One of the rDNA loci gave the weakest hybridization signal. This locus is reported for the first time in the genus Oryza. The chromosomes containing the rDNA loci were determined to be numbers 9, 10 and 11 in descending order of the copy number of rDNA. The application of image analysis methods, after slide preparation treatments (post-treatments), and the use of a thermal cycler, greatly improved the reproducibility of the results. The evolutionary significance of the variability of rDNA loci among the Oryza spp. is discussed.     

Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH).

Herein we report the results of the first major prospective study directly comparing aneuploidy detection by fluorescence in situ hybridization of interphase nuclei with the results obtained by cytogenetic analysis. We constructed probes derived from specific subregions of human chromosomes 21, 18, 13, X, and Y that give a single copy-like signal when used in conjunction with suppression hybridization. A total of 526 independent amniotic fluid samples were analyzed in a blind fashion. All five probes were analyzed on 117 samples, while subsets of these five probes were used on the remaining samples (because of insufficient sample size), for a total of over 900 autosomal hybridization reactions and over 400 sex chromosome hybridization reactions. In this blind series, 21 of 21 abnormal samples were correctly identified. The remaining samples were correctly classified as disomic for these five chromosomes. The combination of chromosome-specific probe sets composed primarily of cosmid contigs and optimized hybridization/detection allowed accurate chromosome enumeration in uncultured human amniotic fluid cells, consistent with the results obtained by traditional cytogenetic analysis.     

Detection of ingested bacteria in benthic ciliates using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.     

Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue

Widefield deconvolution epifluorescence microscopy (WDEM) combined with fluorescence in situ hybridization (FISH) was performed to identify and characterize single bacterial cells within sections of the mediterranean sponge Chondrosia reniformis. Sponges were embedded in paraffin wax or plastic prior to the preparation of thin sections, in situ hybridization and microscopy. Serial digital images generated by widefield epifluorescence microscopy were visualized using an exhaustive photon reassignment deconvolution algorithm and three-dimensional rendering software. Computer processing of series of images taken at different focal planes with the deconvolution technique provided deblurred three-dimensional images with high optical resolution on a submicron scale. Results from the deconvolution enhanced widefield microscopy were compared with conventional epifluorescent microscopical images. By the application of the deconvolution algorithm on digital image data obtained with widefield epifluorescence microscopy after FISH, the occurrence and spatial arrangement of Desulfovibrionaceae closely associated with micropores of Chondrosia reniformis could be visualized.     

Protocol for rapid fluorescence in situ hybridization of bacteria in cryosections of microarthropods

A protocol was developed to detect bacteria inhabiting microarthropods by means of small-subunit rRNA-targeted fluorescence in situ hybridization and microscopy. The protocol is based on cryosections of whole specimens. In contrast to more commonly applied paraffin-embedding techniques, the protocol is quicker and reduces the number of manipulations which might damage the microscopic material. The method allowed the study of the bacterial colonization of Folsomia candida (Collembola) and the detection of bacteria in both the gut and tissue.     

厌氧生境体系中产氢产乙酸细菌的FISH定量解析

产氢产乙酸细菌是一类在有机物厌氧降解过程中起重要作用的细菌。以基于16S rRNA序列设计的特异性寡核苷酸探针为基础,优化FISH实验条件,确定该技术检测产氢产乙酸细菌的实验条件为样品固定19h、乙醇脱水5min,杂交缓冲液中甲酰胺浓度55%。运用建立的FISH技术检测了几种厌氧消化体系中产氢产乙酸细菌的数量,并与用传统MPN方法的结果进行了比较。结果表明,产氢产乙酸细菌分布广泛,废水处理UASB反应器和动物消化道,特别是反刍动物瘤胃中的产氢产乙酸细菌数量较高,其丰度分别为1.70×109 cells/mL样品,6.50×108 cells/mL样品。湖底沉积物中产氢产乙酸细菌数量较少,仅占整个微生物群落的0.4%,含量为1.20×108 cells/mL样品。