Flow-Cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-Independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations

Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 μM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, α-Proteobacteria); Caulobacter (α-Proteobacteria); and Brachymonas and Xenophilus (β-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, α-Proteobacteria) and novel γ-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.     

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.     

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis^1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA³. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment⁴. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion^5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages⁹. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment¹⁰. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture^11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)^13,14. Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs^9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting¹⁶. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.     

Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting

Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13^NEGSSEA4^POSTra-1-60^POS on day 7–10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and “contaminating” partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.     

Identification of Nitrite-Reducing Bacteria Using Sequential mRNA Fluorescence In Situ Hybridization and Fluorescence-Assisted Cell Sorting

Sequential mRNA fluorescence in situ hybridization (mRNA FISH) and fluorescence-assisted cell sorting (SmRFF) was used for the identification of nitrite-reducing bacteria in mixed microbial communities. An oligonucleotide probe labeled with horseradish peroxidase (HRP) was used to target mRNA of nirS, the gene that encodes nitrite reductase, the enzyme responsible for the dissimilatory reduction of nitrite to nitric oxide. Clones for nirS expression were constructed and used to provide proof of concept for the SmRFF method. In addition, cells from pure cultures of Pseudomonas stutzeri and denitrifying activated sludge were hybridized with the HRP probe, and tyramide signal amplification was performed, conferring a strongly fluorescent signal to cells containing nirS mRNA. Flow cytometry-assisted cell sorting was used to detect and physically separate two subgroups from a mixed microbial community: non-fluorescent cells and an enrichment of fluorescent, nitrite-reducing cells. Denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of 16S ribosomal RNA (rRNA) genes were used to compare the fragments amplified from the two sorted subgroups. Sequences from bands isolated from DGGE profiles suggested that the dominant, active nitrite reducers were closely related to Acidovorax BSB421. Furthermore, following mRNA FISH detection of nitrite-reducing bacteria, 16S rRNA FISH was used to detect ammonia-oxidizing and nitrite-oxidizing bacteria on the same activated sludge sample. We believe that the molecular approach described can be useful as a tool to help address the longstanding challenge of linking function to identity in natural and engineered habitats.     

Combining Culture-Dependent and -Independent Methodologies for Estimation of Richness of Estuarine Bacterioplankton Consuming Riverine Dissolved Organic Matter

Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated β-proteobacteria to be RDOM consumers.     

Fluorescence Activated Cell Sorting of Rickettsia prowazekii-Infected Host Cells Based on Bacterial Burden and Early Detection of Fluorescent Rickettsial Transformants

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens.     

Exploring Bacterioplankton Growth and Protein Synthesis to Determine Conversion Factors Across a Gradient of Dissolved Organic Matter

The effect of bacterial specific growth rates of abundance (micro) and protein synthesis (b) on conversion factor (CF) variability was explored in order to provide an alternative approach to the controversial application of just one universal CF to field data. Nine regrowth cultures (RCs) were set up from very diverse aquatic ecosystems, controlling temperature and adding N and P to avoid mineral limitation and force organic carbon limitation. The values of micro varied one order of magnitude from 0.26 to 3.34 d(-1), whereas b values varied two orders of magnitude from 0.28 to 34.87 d(-1). We found no relationships between micro or b values and the dissolved organic carbon (DOC) concentration or the dissolved organic matter (DOM) quality indexes assayed. Abundance and protein synthesis increased exponentially and synchronously in four RCs, leading to balanced growth (micro = b). In contrast, abundance and protein synthesis increased logistically in the other five RCs and b values were significantly higher than g values, leading to unbalanced growth (micro not equal b). CFs ranged from 0.0062 to 0.0576 x 10(18) cells mol leucine(-1) with an average of 0.0305 x 10(18) cells mol leucine(-1). CFs obtained in RCs with balanced growth were generally higher than CFs obtained in RCs with unbalanced growth and were not alike, impeding the establishment of an upper limit for CFs. A positive and significant relationship (n = 8, p < 0.0 1, r2 = 0.71) was found between CFs and DOC concentration (CF (x10(18) cells mol leucine(-1)) = 0.0104 + 0.0094 DOC (mM)) when the value for the most productive system was excluded. This function permits the estimation of site-specific CFs based on DOC concentration instead of the controversial use of a single CF for different systems.     

Bacterioplankton Growth on Fractions of Dissolved Organic Carbon of Different Molecular Weights from Humic and Clear Waters

The ability of fractions of dissolved organic carbon (DOC) of different molecular weights (MW) to support bacterial growth was studied in batch culture experiments. Natural pelagic bacteria were inoculated into particle-free (0.2-μm filtered) water, taken from 10 oligotrophic lakes of differing humic content, and either used without further treatments or ultrafiltered to remove DOC of >10,000 MW or >1,000 MW. Stationaryphase abundance of bacteria in the cultures was used as an estimate of bacterial carrying capacity. High-MW DOC (>10,000) comprised an increasing fraction of total DOC with increasing total DOC and humic content of the lakes. High-MW DOC was generally more available to bacteria (i.e., more bacteria were produced per unit of organic carbon initially present) than low-MW (<10,000) DOC. The availability to bacteria of this high-MW DOC decreased with increasing humic content. However, although less available in humic lakes than in clearwater lakes, the higher abundance of high-MW DOC made it quantitatively more important as a bacterial substrate; i.e., a larger fraction of the total bacterial yield of the cultures was due to high-MW DOC compounds in humic lakes than in clearwater lakes. On the average, 48% of bacterial growth occurred at the expense of DOC of <10,000 MW. DOC of <1,000 MW was responsible for an average of 22% of bacterial growth, with no significant correlation to humic content and DOC concentration of lakes. The DOC which supports bacterial growth, as well as the total DOC, is of different quality in humic and clearwater lakes.     

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.Download video file.^{(48M, mov)}     

Environmental Dissolved Organic Matter Governs Biofilm Formation and Subsequent Linuron Degradation Activity of a Linuron-Degrading Bacterial Consortium

It was examined whether biofilm growth on dissolved organic matter (DOM) of a three-species consortium whose members synergistically degrade the phenylurea herbicide linuron affected the consortium''s integrity and subsequent linuron-degrading functionality. Citrate as a model DOM and three environmental DOM (eDOM) formulations of different quality were used. Biofilms developed with all DOM formulations, and the three species were retained in the biofilm. However, biofilm biomass, species composition, architecture, and colocalization of member strains depended on DOM and its biodegradability. To assess the linuron-degrading functionality, biofilms were subsequently irrigated with linuron at 10 mg liter⁻¹ or 100 μg liter⁻¹. Instant linuron degradation, the time needed to attain maximal linuron degradation, and hence the total amount of linuron removed depended on both the DOM used for growth and the linuron concentration. At 10 mg liter⁻¹, the final linuron degradation efficiency was as high as previously observed without DOM except for biofilms fed with humic acids which did not degrade linuron. At 100 μg liter⁻¹ linuron, DOM-grown biofilms degraded linuron less efficiently than biofilms receiving 10 mg liter⁻¹ linuron. The amount of linuron removed was more correlated with biofilm species composition than with biomass or structure. Based on visual observations, colocalization of consortium members in biofilms after the DOM feed appears essential for instant linuron-degrading activity and might explain the differences in overall linuron degradation. The data show that DOM quality determines biofilm structure and composition of the pesticide-degrading consortium in periods with DOM as the main carbon source and can affect subsequent pesticide-degrading activity, especially at micropollutant concentrations.     

有机农药活性污泥中表面活性剂产生菌的筛选及鉴定

目的:筛选表面活性剂产生菌,用于降解有机农药,提高氧化塘处理效率.方法:以液体石蜡为惟一碳源进行选择性培养,通过测定发酵液的排油活性和表面张力,对有机农药厂的活性污泥进行产生物表面活性剂细菌筛选,并对筛得的细菌进行形态学、生理生化及 16SrDNA 试验和鉴定.结果:筛选出 3 株产生物表面活性剂的细菌,经鉴定均属于不动杆菌属.结论:证实了有机农药活性污泥中存在表面活性剂的产生菌,对有机农药降解存在助溶作用.     

Bromodeoxyuridine (BrdU) template and thymodine pool effects on high frequencies of sister-chromatid exchange (SCE) in Bloom syndrome cells and a mutant cell line (AsHa) originated from ataxia telangiectasia

The effect of bromodeoxyuridine (BrdU)-substituted DNA template and thymidine (dT) pool on excess sister-chromatid exchanges (SCEs) was studied in Bloom syndrome (BS) cells and an ataxia telangiectasia (AT)-derived mutant cell line (AsHa). When BS endomitotic cells were labeled with low and high (or high and low) BrdU concentrations during S₁ and S₂, only the BrdU concentration during S₁ phase affected the observed SCE. In BS cells about a 10-fold increase in SCEs occurs during or following replication on a BrdU-substituted template (high-high and high-low BrdU labeling) relative to the normal DNA template. SCEs decreased to about half in AsHa cells labeled with various BrdU doses (40, 60, 80 and 100 μg/ml) during only S₁, compared with those labeled during S₁ and S₂. Co-cultivation of AsHa and BS cells resulted in a significant reduction in SCE level from 70 to 13–17 in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential (SCD) staining from 40 to 10 μg/ml with normal SCE level and resulted in decreased level of SCEs at high BrdU concentrations (80–100 μg/ml) 12–14 SCE) in AsHa cells, compared with the originally increased SCE level (36.65 SCE at 100 μg/ml) without co-culture. However, co-cultivation between AsHa and normal cells lowered the BrdU dose necessary for SCD staining from 40 to 30 μg/ml; the dT pool possibly balanced at this level, which is clearly higher than that at co-cultivation between AsHa and BS cells. The reason for the very high BrdU doses needed to achieve SCD would seem to be that AsHa cells have high levels of thymidylate (TMP) synthetase, which maintain a large endogenous thymidine pool. This has been confirmed by direct measurement. These findings strongly support that excess and decreased dT pools are closely related to the condition necessary for high SCE induction.     

Acid Ethanol Fixation and Polyester Wax Embedding Combines Preservation of Antigenic Determinants with Good Morphology and Enables Simultaneous Bromodeoxyuridine (BRDU) Labeling

Lymphoid tissue, and/or isolated peripheral mononuclear blood cells were fixed in acid ethanol and embedded in polyester wax (melting point 37 C). The excellent cytomorphol-ogy obtained allowed distinguishing different types of individual lymphoid and nonlymphoid cells. Furthermore, this procedure was satisfactory in the immunophenotyping of histiocytes, endothelial, mesenchymal, epithelial cells, different (sub-) types of lymphocytes and also of lym-phokine activated killer (LAK) cells. The staining patterns obtained with the different poly- and monoclonal antibodies on polyester wax sections were not only analogous to those obtained on frozen sections, but cells which had incorporated bromodeoxyuridine could be double labeled with specific antiserum.     

Elemental Composition (C, N, P) and Cell Volume of Exponentially Growing and Nutrient-Limited Bacterioplankton

Marine bacterioplankton were isolated and grown in batch cultures until their growth became limited by organic carbon (C), nitrogen (N), or phosphorus (P). Samples were taken from the cultures at both the exponential and stationary phases. The elemental composition of individual bacterial cells was analyzed by X-ray microanalysis with an electron microscope. The cell size was also measured. The elemental content was highest in exponentially growing cells (149 ± 8 fg of C cell⁻¹, 35 ± 2 fg of N cell⁻¹, and 12 ± 1 fg of P cell⁻¹; average of all isolates ± standard error). The lowest C content was found in C-limited cells (39 ± 3 fg of C cell⁻¹), the lowest N content in C- and P-limited cells (12 ± 1 and 12 ± 2 fg of N cell⁻¹, respectively), and the lowest P content in P-limited cells (2.3 ± 0.6 fg of P cell⁻¹). The atomic C:N ratios varied among treatments between 3.8 ± 0.1 and 9.5 ± 1.0 (average ± standard error), the C:P ratios between 35 ± 2 and 178 ± 28, and the N:P ratios between 6.7 ± 0.3 and 18 ± 3. The carbon-volume ratios showed large variation among isolates due to different types of nutrient limitation (from 51± 4 to 241 ± 38 fg of C μm⁻¹; average of individual isolates and treatments ± standard error). The results show that different growth conditions and differences in the bacterial community may explain some of the variability of previously reported elemental and carbon-volume ratios.     

两种免疫磁珠分离法分选小鼠骨髓粒-单核祖细胞的效率比较

摘要 目的：提取小鼠骨髓细胞（bone marrow cell, BMC）,用两种不同的免疫磁珠分离（magnetic activated cell sorting, MACS）试剂盒从小鼠BMC中分选提纯粒-单核祖细胞（granulocyte-monocyte progenitor, GMP）,比较这两种免疫磁珠的分选效率。方法：从小鼠股骨和胫骨中提取BMC,通过两种不同的MACS试剂盒,即Lineage阳性细胞清除试剂盒和CD117阳性细胞分选试剂盒,分别得到Lineage-细胞群和CD117⁺细胞群,用代表GMP细胞表面标志物的荧光抗体标记,孵育后通过流式细胞荧光分选技术得到GMP细胞,并且对比得到GMP细胞的效率。结果：每2只野生型C57BL/6J小鼠可共收集骨髓细胞（7.02±1.24）×10⁷个,细胞活力为（91.86±5.24）%。经过Lineage阳性细胞清除试剂盒得到的细胞数量为（5.71±2.86）×10⁶个;经过CD117阳性细胞分选试剂盒得到的细胞数量为（2.70±0.56）×10⁶个。Lineage磁珠分选纯化得到的GMP细胞数占总细胞数的比例为（10.90±1.37）%,CD117磁珠分选纯化得到的GMP细胞数占总细胞数的比例为（4.83±2.08）%。结论：Lineage阳性细胞清除试剂盒能更有效分选小鼠骨髓细胞中的粒-单核祖细胞。     

Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility

Fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted oligonucleotide probes is widely applied for direct identification of microbes in the environment or in clinical specimens. Here we show that a replacement of singly labeled oligonucleotide probes with 5′-, 3′-doubly labeled probes at least doubles FISH signal intensity without causing specificity problems. Furthermore, Cy3-doubly labeled probes strongly increase in situ accessibility of rRNA target sites and thus provide more flexibility for probe design.Since its introduction almost 20 years ago (2, 7), the identification of microorganisms by fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted probes has found widespread application in environmental and medical microbiology (1, 16). Despite being a methodologically robust technique, standard FISH suffers from several limitations (26) that may prevent successful detection of target microorganisms. One of the most frequently reported FISH problems is a low signal intensity of the detected microbes.Dim signals can be caused by a low cellular concentration of the target molecules (16S rRNA or 23S rRNA), a feature typically found in microorganisms thriving in oligotrophic environments (19). In order to increase the sensitivity of FISH and make it suitable for the detection of microbes with a low ribosome content, several strategies have been developed (6, 13, 18, 19, 21, 25), of which catalyzed reporter deposition (CARD)-FISH has found the most widespread application. The CARD-FISH technique (18, 21) uses horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification and achieves a 26- to 41-fold-higher sensitivity than standard FISH (11). However, CARD-FISH is rather expensive, requires enzymatic pretreatment to allow the large horseradish peroxidase-labeled probes to penetrate the target cells (17), and causes a dramatically altered melting behavior of the probes (11).Another frequently encountered FISH problem is the low in situ accessibility of many regions of the 16S and 23S rRNA for singly labeled probes (9, 10). Probes targeting such regions, which comprise about one-third of the Escherichia coli 16S rRNA (10), confer signals which are very dim or even below the detection limit. In order to avoid the selection of poorly accessible target sites for FISH probe design, a consensus 16S rRNA accessibility map for prokaryotes has been established based on detailed accessibility maps of two bacterial model organisms and one archaeal model organism (3). Considering this consensus map during probe design is recommended, but it excludes many probes with useful specificities from FISH applications. Furthermore, accessibility of many 16S and 23S rRNA target sites varies between different microorganisms and thus cannot yet be reliably predicted in silico. Accessibility of target sites to probes can be improved by the following: (i) use of unlabeled helper probes (8), (ii) elongation of the hybridization time up to 96 h (30), (iii) elongation of the probes, resulting in an altered ΔG°_overall (30), or (iv) use of peptide nucleic acid probes (reference 26 and references therein). However, all these strategies have specific limitations. For example, the design of helper probes is often impossible for probes with broader specificities, the extension of the hybridization time might lead to unspecific probe or dye binding in complex samples, probe elongation is often not possible without narrowing its specificity, and previously published oligonucleotide probes cannot simply be converted into the expensive peptide nucleic acid probes without a dramatically changed specificity (26).In principle, using oligonucleotide probes labeled with multiple fluorescent dyes should provide a simple means to increase the FISH signal intensity. The first experiments with multilabeled oligonucleotides were performed briefly after the introduction of the technique in microbiology but resulted in a pronounced increase in unspecific staining of nontarget organisms and/or an unexpected decrease in the signal intensity of the target organism which was attributed to quenching effects (28). Inconsistent with these data, Spear et al. (23) reported a successful increase in the signal-to-noise ratio of FISH-detected fungal cells by application of a multilabeled 18S rRNA-targeted oligonucleotide probe. In this work, we systematically evaluated the effect of 5′- and 3′-doubly labeled oligonucleotide probes (which were not included in the previously published study of multilabeled probes [28]) on the FISH signal intensity of Gram-negative and Gram-positive cells and studied the influence of double labeling on the in situ accessibility of rRNA target sites.The double-labeling-of-oligonucleotide-probes (DOPE)-FISH approach was initially tested with four bacterial pure cultures. These included the gamma- and betaproteobacterial Gram-negative species Escherichia coli (DSM 498) and Burkholderia cepacia (DSM 7288), respectively. In addition, the two Gram-positive bacteria Bacillus subtilis (DSM 10) and Listeria monocytogenes (strain LO28) were used. E. coli, B. cepacia, and B. subtilis were grown according to the DSMZ instructions until they reached their stationary growth phase and were fixed with paraformaldehyde (E. coli and B. cepacia) or ethanol (B. subtilis) as described elsewhere (5). L. monocytogenes strain LO28 was grown on brain heart infusion for 5 h and fixed with ethanol as outlined previously (27). The oligonucleotide probes EUB338 (targeting the 16S rRNA of most but not all bacteria), NonEUB338 (a nonsense probe), GAM42a (targeting the 23S rRNA of many members of the Gammaproteobacteria), and five E. coli-targeted probes with a low 16S rRNA accessibility (10) were obtained as singly and doubly labeled derivatives from Thermo Hybaid (Interactiva Division, Ulm, Germany). More information about the applied probes can be found at probeBase (14) and in the publication by Fuchs et al. (10). FISH was performed by following the standard protocol (5) under the conditions recommended for each probe (14). If not stated otherwise, all hybridizations were carried out with identical hybridization (4 h) and washing times (10 min), respectively. Probe-conferred signal intensities were quantified using a confocal laser scanning microscope (CLSM) (LSM 510 Meta; Zeiss, Oberkochen, Germany) and the software program daime (4) by analyzing at least 1,000 single cells per experiment. For these measurements, individual cells were detected by image segmentation via edge detection.Regardless of the dye used (Cy3, Cy5, or FLUOS) and the respective target organism analyzed, hybridization with the doubly labeled probe EUB338 resulted in an increase in the FISH signal compared to the use of the singly labeled probe EUB338. For three of the four reference organisms, this increase was about 2-fold, while an even stronger signal amplification was observed for B. cepacia (Fig. ​(Fig.1A).1A). Consequently, the distance between the two dye molecules in 18-nucleotide probes labeled at both ends is sufficient to avoid quenching. This is in contrast to oligonucleotides which are multiply labeled at one end or within the probe (28). Hybridization of the four reference organisms with the singly and doubly labeled derivatives of probe GAM42a confirmed these results and demonstrated that double labeling does not increase the background fluorescence of nontarget microorganisms (Fig. ​(Fig.1B).1B). Consistent with these findings, hybridization of all reference organisms with a doubly labeled nonsense probe (with FLUOS, Cy3, and Cy5) resulted in signals below the detection limit of the CLSM if standard FISH settings were applied (data not shown). We also evaluated the influence of the hybridization time on the signal intensity achievable by DOPE-FISH by varying the hybridization time between 1 and 6 h. In these experiments, no significant difference in DOPE-FISH signal intensities of the Gram-positive and Gram-negative reference organisms were observed, indicating that the additional label of the DOPE-FISH probes does not dramatically influence the hybridization kinetics (data not shown).Open in a separate windowFIG. 1.(A) Effect of double labeling of the EUB338 probe on the FISH signal intensity of four reference organisms. For each organism, the signal intensity conferred by a doubly labeled EUB338 probe was normalized to the signal intensity obtained with the same probe as a singly labeled derivative. Hatched, light-gray, and dark-gray bars depict results with the Cy3-, Cy5- and FLUOS-labeled probe EUB338, respectively. (B) Effect of double labeling of the probe Gam42a (in the presence of the unlabeled competitor probe Bet42a, specific for most members of the Betaproteobacteria [14]) on the FISH signal intensities of four reference organisms. For each organism, the signal intensity conferred by the doubly labeled probe Gam42a was normalized to the signal intensity obtained for E. coli with the same probe as a singly labeled derivative. Hatched, light-gray, and dark-gray bars depict results with the Cy3-, Cy5-, and FLUOS-labeled probe, respectively. The weak unspecific signals observed with some DOPE-FISH probes for B. subtilis are also detectable at comparable intensities with singly labeled probes (data not shown). (C) Cy3-doubly labeled but not FLUOS-doubly labeled probes improve in situ accessibility of E. coli 16S rRNA target sites. E. coli was hybridized with five probes representing brightness classes V and VI (3). FISH signals were recorded for Cy3-singly and -doubly labeled probes and normalized to the FISH signal obtained for E. coli with the singly labeled probe EUB338. Light-gray and dark-gray bars depict results with Cy3-singly and doubly labeled probes, respectively. FLUOS-singly and -doubly labeled probes showed no signal. For all panels, all experiments were performed in triplicate. Error bars indicate the standard deviation. ND, not detectable.Although DOPE-FISH worked well with all tested reference organisms, it should be noted that the Gram-positive species L. monocytogenes showed a signal amplification only if it was treated with lysozyme (according to Wagner et al. [27]) prior to the application of the doubly labeled probe. Without this enzymatic pretreatment, application of the singly labeled probe EUB338 resulted in a stronger signal than was seen with the doubly labeled probe EUB338, indicating that for some Gram-positive microorganisms with dense cell walls, double labeling impairs probe penetration. The observation that lysozyme treatment of L. monocytogenes also enhanced the probe-conferred signal of the singly labeled EUB338 probe confirmed that the cell wall of this organism after ethanol fixation is also not freely permeable to singly labeled probes (27; also data not shown). Enzymatic pretreatment of fixed microbial cells is also routinely applied for successful application of CARD-FISH, but since this method uses peroxidase-labeled probes which are much larger than DOPE-FISH probes, such treatments are also used for Gram-negative bacteria (11). Although many microorganisms are detectable by CARD-FISH with enhanced signal intensities, appropriate pretreatment protocols are not yet available for all microbes. For example, the sheathed filamentous methane oxidizer Crenothrix polyspora can easily be detected by standard FISH (24), but only very few cells within the filaments show a signal after CARD-FISH even if harsh permeabilization pretreatments are applied (see Fig. S1A in the supplemental material), a phenomenon known for sheathed microorganisms (12). In contrast, detection of all Crenothrix cells with more than 2-fold-increased signal intensity is readily possible by DOPE-FISH (see Fig. S1B).Prior research has demonstrated that probe labeling with horseradish peroxidase for CARD-FISH dramatically alters the melting behavior of oligonucleotide probes (11). Therefore, we recorded melting curves for Cy3-, Cy5-, and FLUOS-singly and -doubly labeled probes EUB338 and Gam42a by applying increasingly stringent conditions in the hybridization and wash steps (5). Interestingly, these experiments showed that the FLUOS singly labeled probes formed less-stable duplexes with their target sequences than the respective Cy3- and Cy5-labeled probes (Fig. ​(Fig.2).2). This effect, which is consistent with recent data on the stabilizing effect of various fluorophores on model probe-target duplexes (15), indicates that FLUOS-labeled FISH probes are generally applied under more-stringent conditions than Cy3- or Cy5-labeled probes. Cy3- and Cy5-doubly labeled probes displayed with their target organisms probe dissociation profiles similar to those of the respective FLUOS singly labeled probes, demonstrating that Cy3 or Cy5 double labeling does not further stabilize but rather moderately weakens the probe-target hybrid. Consistent with these findings, doubly FLUOS-labeled probes showed the lowest T_m (Fig. ​(Fig.2).2). Importantly, double labeling of probe GAM42a did not adversely affect mismatch discrimination, as shown by its dissociation profiles if in situ hybridizations were performed at various stringencies with B. cepacia containing a single mismatch in the probe target site of its 23S rRNA. (Fig. ​(Fig.2B).2B). These results indicate that the specificities of DOPE-FISH probes can be regarded as identical to those of standard singly labeled FISH probes.Open in a separate windowFIG. 2.Comparison of probe dissociation profiles of singly and doubly labeled probes. For each profile, the microscopic settings were adjusted for the lowest formamide concentration and subsequently kept constant. Dashed and solid lines represent sigmoid fittings for singly and doubly labeled probes, respectively. (A) Dissociation profiles of the singly and doubly labeled probe Gam42a with E. coli as the target organism. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe GAM42a, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe GAM42a, respectively. (B) Dissociation profiles of the singly and doubly labeled probe Gam42a with B. cepacia as a nontarget organism having a single mismatch to probe GAM42a. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe GAM42a, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe GAM42a, respectively. The melting curves for FLUOS-singly labeled and Cy5-doubly labeled probes are almost identical and thus overlap. In the presence of the unlabeled probe Bet42a as a competitor, no probe-conferred signal was recordable for both singly and doubly labeled GAM42a probes. (C) Dissociation profiles of the singly and doubly labeled probe EUB338 with E. coli as the target organism. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe EUB338, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe EUB338, respectively. For all panels, error bars are not shown since they were always smaller than the symbols.In order to analyze whether the in situ accessibility of rRNA target sites to doubly labeled probes differs from that to those labeled with only one dye, we tested five probes targeting E. coli 16S rRNA. These probes were described as yielding only very dim signals with standard FISH as a consequence of limited target site accessibility and were thus assigned to the lowest brightness classes, V or VI (3, 10). Consistently, standard FISH with these five singly labeled probes (Cy3 and FLUOS) gave no or very weak signals (Fig. ​(Fig.1C).1C). Unexpectedly, however, Cy3-doubly labeled derivatives of these probes produced much brighter signals (Fig. ​(Fig.1C),1C), and for some of the probes (Eco468 and Eco1310), the DOPE-FISH signal intensity was higher than that measured for the Cy3-singly labeled probe EUB338 (Fig. ​(Fig.1C).1C). One could speculate that a Cy3 label at the 3′ end and not the double labeling might be responsible for the improved accessibility of rRNA target sites for Cy3 DOPE-FISH probes. However, since selected probes (Eco262, Eco468, and Eco1310) labeled with a single Cy3 molecule at the 3′ end did not result in increased fluorescence, this hypothesis can be rejected (data not shown). Interestingly, FLUOS-labeled DOPE-FISH probes did not show increased fluorescence, strongly indicating that the improved accessibility of Cy3 DOPE-FISH probes depends on the chemical structure of the fluorophore. This is consistent with the observation that Cy5 double labeling of the five E. coli probes also resulted in improved probe accessibilities (data not shown). While Cy3 and Cy5 double labeling decreases the probe-target duplex stability (Fig. ​(Fig.2),2), it apparently helps to resolve secondary or tertiary structures responsible for poor in situ accessibility of rRNA target sites. It is tempting to speculate that binding of Cy3 or Cy5 to double-stranded rRNA regions, analogous to the previously reported intercalation of certain cyanine class dyes in DNA (29) or other modes of nucleic acid binding by cyanine dyes (15), contributes to this phenomenon.The improved accessibility of rRNA target sites for Cy3 DOPE-FISH probes offers more flexibility for probe design because it enables the use of probes with excellent specificity but low standard FISH signal intensity for the successful in situ detection of microbes. This advantage of DOPE-FISH is nicely demonstrated by the probe Ntspa175 (5′-GAC CAG GAG CCG TAT GCG-3′), which targets the 16S rRNA (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU229885","term_id":"281398158","term_text":"GU229885"}}GU229885) of an uncultured nitrite oxidizer of the genus Nitrospira thriving in activated sludge. At 25% formamide in the standard FISH hybridization buffer (5), this probe is highly specific as demonstrated by Clone-FISH (22) using another activated sludge-derived 16S rRNA Nitrospira-like sequence with a single mismatch to probe Ntspa175 as a nontarget control (data not shown). Standard FISH of activated sludge with the Cy3-labeled probe Ntsp175, which targets the 175-to-193 region in the 16S rRNA, resulted in the detection of Nitrospira microcolonies with very variable FISH signal intensities. A considerable number of stained microcolonies had extremely dim FISH signals, indicating that these cells had a ribosome content too low to be reliably detectable by a standard FISH probe of a low brightness class. DOPE-FISH of the same sample with the Cy3-doubly labeled probe Ntspa175 led to a pronounced increase in signal intensity of the target cells (see Fig. S2 in the supplemental material) without causing increased background fluorescence if standard confocal-microscope settings were applied. In accordance with this observation, the relative biovolume-abundance of the detectable Ntspa175-stained population compared to the biovolume of those cells labeled by the Nitrospira genus-specific probe Ntspa662 (14) in the activated sludge increased by a factor of 1.81 ± 0.1 if a doubly labeled Ntspa175 probe was used (measurements made by the software package daime using confocal-microscope images as described previously [4]).In summary, DOPE-FISH with commercially available doubly labeled oligonucleotide probes is a straightforward modification of the standard FISH procedure which increases the signal intensity of standard FISH probes by at least a factor of 2 without causing specificity problems. Importantly, the influence of DOPE-FISH on the dissociation profile of probes is not larger than that caused by a dye switch from Cy3 to FLUOS if singly labeled probes are used for FISH. Thus, previously optimized hybridization and washing conditions for published probes can be applied for DOPE-FISH. Since DOPE-FISH unlocks previously inaccessible target sites on the rRNA, this new FISH approach offers more options for the design of specific probes, a task which becomes increasingly difficult with the rapid growth of rRNA databases (20).      

Importance of Viral Lysis and Dissolved DNA for Bacterioplankton Activity in a P-Limited Estuary,Northern Baltic Sea

Through lysis of bacterioplankton cells, viruses mediate an important, but poorly understood, pathway of carbon and nutrients from the particulate to the dissolved form. Via this activity, nutrient-rich cell lysates may become available to noninfected cells and support significant growth. However, the nutritional value of lysates for noninfected bacteria presumably depends on the prevailing nutrient limitation. In the present study, we examined dynamics of dissolved DNA (D-DNA) and viruses along a transect in the phosphorus (P)-limited Ore Estuary, northern Baltic Sea. We found that viruses were an important mortality factor for bacterioplankton and that their activity mediated a significant recycling of carbon and especially of P. Uptake of dissolved DNA accounted for up to 70% of the bacterioplankton P demand, and about a quarter of the D-DNA pool was supplied through viral lysis of bacterial cells. Generally, the importance of viral lysates and uptake of D-DNA was highest at the estuarine and offshore stations and was positively correlated with P limitation measured as alkaline phosphatase activity. Our results highlight the importance of viral activity for the internal recycling of principal nutrients and pinpoints D-DNA as a particularly relevant compound in microbial P dynamics.