Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).     

Exploring the in situ accessibility of small subunit ribosomal RNA of members of the domains Bacteria and Eukarya to oligonucleotide probes

The principle that the small subunit ribosomal RNA (ssu rRNA) is generally accessible to oligonucleotide probes designed to have high thermodynamic affinity was tested with Stenotrophomonas maltophilia, Rhodobacter sphaeroides, Bacillus subtilis, and Saccharomyces cerevisiae. Fluorescein-labeled probes, designed to have ΔG_overall° = −14 ± 1 and to avoid the potential of nucleobase-specific quenching, were used to target 20 randomly selected sites in each organism. A site was considered accessible if probe brightness was at least 10 times the background signal. With 30-h hybridizations, 71 out of 80 target sites passed the accessibility criterion. Three additional sites were demonstrated to be accessible with either longer hybridizations, which seemed to have a negative effect on some probes, or the addition of formamide to the hybridization buffer. The remaining 6 sites were demonstrated to be accessible by changing the fluorophore to Cy5, slightly modifying probe lengths, using dual-labeled fluorescein probes, or a combination of these approaches. Probe elongations were only needed in 4 probes, indicating a 95% success in correctly predicting ΔG_overall°, the key parameter for the design of high affinity probes. In addition, 94% of the fluorescein labeled probes yielded bright signals, demonstrating that nucleobase-specific quenching of fluorescein is an important factor affecting probe brightness that can be predicted during probe design. Overall, the results support the principle that with a rational design of probes, it is possible to make most target sites in the ssu rRNA accessible.     

Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization

In fluorescent in situ hybridization (FISH), the efficiency of hybridization between the DNA probe and the rRNA has been related to the accessibility of the rRNA when ribosome content and cell permeability are not limiting. Published rRNA accessibility maps show that probe brightness is sensitive to the organism being hybridized and the exact location of the target site and, hence, it is highly unpredictable based on accessibility only. In this study, a model of FISH based on the thermodynamics of nucleic acid hybridization was developed. The model provides a mechanistic approach to calculate the affinity of the probe to the target site, which is defined as the overall Gibbs free energy change (DeltaG degrees overall) for a reaction scheme involving the DNA-rRNA and intramolecular DNA and rRNA interactions that take place during FISH. Probe data sets for the published accessibility maps and experiments targeting localized regions in the 16S rRNA of Escherichia coli were used to demonstrate that DeltaG degrees overall is a strong predictor of hybridization efficiency and superior to conventional estimates based on the dissociation temperature of the DNA/rRNA duplex. The use of the proposed model also allowed the development of mechanistic approaches to increase probe brightness, even in seemingly inaccessible regions of the 16S rRNA. Finally, a threshold DeltaG degrees overall of -13.0 kcal/mol was proposed as a goal in the design of FISH probes to maximize hybridization efficiency without compromising specificity.     

Fingerprinting of prokaryotic 16S rRNA genes using oligodeoxyribonucleotide microarrays and virtual hybridization

An oligonucleotide microarray hybridization system to differentiate microbial species was designed and tested. Seven microbial species were studied, including one Bacillus and six Pseudomonas strains. DNA sequences near the 5′ end of 16S rRNA genes were aligned and two contiguous regions of high variability, flanked by highly conserved sequences, were found. The conserved sequences were used to design PCR primers which efficiently amplified these polymorphic regions from all seven species. The amplicon sequences were used to design 88 9mer hybridization probes which were arrayed onto glass slides. Single-stranded, fluorescence-tagged PCR products were hybridized to the microarrays at 15°C. The experimental results were compared with the ΔG° values for all matched and mismatched duplexes possible between the synthetic probes and the 16S target sequences of the seven test species, calculated using a ‘virtual hybridization’ software program. Although the observed hybridization patterns differed significantly from patterns predicted solely on the basis of perfect sequence matches, a unique hybridization fingerprint was obtained for each of the species, including closely related Pseudomonas species, and there was a reasonable correlation between the intensity of observed hybridization signals and the calculated ΔG° values. The results suggest that both perfect and mismatched pairings can contribute to microbial identification by hybridization fingerprinting.     

Melting studies of dangling-ended DNA hairpins: effects of end length,loop sequence and biotinylation of loop bases

The effects of 3′ single-strand dangling-ends of different lengths, sequence identity of hairpin loop, and hairpin loop biotinylation at different loop residues on DNA hairpin thermodynamic stability were investigated. Hairpins contained 16 bp stem regions and five base loops formed from the sequence, 5′-TAGTCGACGTGGTCC-N₅-GGACCACGTCGACTAG-E_n-3′. The length of the 3′ dangling-ends (E_n) was n = 13 or 22 bases. The identities of loop bases at positions 2 and 4 were varied. Biotinylation was varied at loop base positions 2, 3 or 4. Melting buffers contained 25 or 115 mM Na⁺. Average t_m values for all molecules were 73.5 and 84.0°C in 25 and 115 mM Na⁺, respectively. Average two-state parameters evaluated from van’t Hoff analysis of the melting curve shapes in 25 mM Na⁺ were ΔH_vH = 84.8 ± 15.5 kcal/mol, ΔS_vH = 244.8 ± 45.0 cal/K·mol and ΔG_vH = 11.9 ± 2.1 kcal/mol. In 115 mM Na⁺, two-state parameters were not very different at ΔH_vH = 80.42 ± 12.74 kcal/mol, ΔS_vH = 225.24 ± 35.88 cal/K·mol and ΔG_vH = 13.3 ± 2.0 kcal/mol. Differential scanning calorimetry (DSC) was performed to test the validity of the two-state assumption and evaluated van’t Hoff parameters. Thermodynamic parameters from DSC measurements (within experimental error) agreed with van’t Hoff parameters, consistent with a two-state process. Overall, dangling-end DNA hairpin stabilities are not affected by dangling-end length, loop biotinylation or sequence and vary uniformly with [Na⁺]. Consider able freedom is afforded when designing DNA hairpins as probes in nucleic acid based detection assays, such as microarrays.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Quantification of Bacterial Groups within Human Fecal Flora by Oligonucleotide Probe Hybridization

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroides group-specific probe accounted for 37% ± 16% of the total rRNA, while the enteric group probe accounted for less than 1%. Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% ± 7% and 14% ± 6%, respectively, while Bifidobacterium and Lactobacillus groups made up less than 2%.     

Quantitative Molecular Analysis of the Microbial Community in Marine Arctic Sediments (Svalbard)

Fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI (4′,6′-diamidino-2-phenylindole) cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides δ-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts and up to 6.1% of prokaryotic rRNA. Furthermore, members of the order Planctomycetales accounted for up to 3.9% of total cell counts. In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments. Members of the γ-proteobacteria also constituted a significant fraction in this sediment (6.1% ± 2.5% of total cell counts, 14.4% ± 3.6% of prokaryotic rRNA). A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed. A significant number of cells was detected by this probe (2.1% ± 0.7% of total DAPI cell counts, 13.2% ± 4.6% of prokaryotic rRNA), showing no clear zonation along the vertical profile. Gram-positive bacteria and the β-proteobacteria were near the detection limit in all sediments.     

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).     

Community Composition of Marine Bacterioplankton Determined by 16S rRNA Gene Clone Libraries and Fluorescence In Situ Hybridization

We determined the compositions of bacterioplankton communities in surface waters of coastal California using clone libraries of 16S rRNA genes and fluorescence in situ hybridization (FISH) in order to compare the community structures inferred from these two culture-independent approaches. The compositions of two clone libraries were quite similar to those of clone libraries of marine bacterioplankton examined by previous studies. Clones from γ-proteobacteria comprised ca. 28% of the libraries, while approximately 55% of the clones came from α-proteobacteria, which dominated the clone libraries. The Cytophaga-Flavobacter group and three others each comprised 10% or fewer of the clone libraries. The community composition determined by FISH differed substantially from the composition implied by the clone libraries. The Cytophaga-Flavobacter group dominated 8 of the 11 communities assayed by FISH, including the two communities assayed using clone libraries. On average only 10% of DAPI (4′,6′-diamidino-2-phenylindole)-stained bacteria were detected by FISH with a probe for α-proteobacteria, but 30% of DAPI-stained bacteria appeared to be in the Cytophaga-Flavobacter group as determined by FISH. α-Proteobacteria were greatly overrepresented in clone libraries compared to their relative abundance determined by FISH, while the Cytophaga-Flavobacter group was underrepresented in clone libraries. Our data show that the Cytophaga-Flavobacter group can be a numerically dominant component of coastal marine bacterioplankton communities.     

Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [μ-dppzip(phen)₄Ru₂]⁴⁺ (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)₂dppz) and the distal subunit (Ru(phen)₂ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δx_eq, and the dynamic DNA structural deformations during ligand association x_on and dissociation x_off. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a K_d of 27 ± 3 nM for (Δ,Λ)-Piz to a K_d of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δx_eq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and x_on from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited x_off changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics.     

Thermodynamic characterization of the multivalent binding of chartreusin to DNA

Characterization of the thermodynamics of DNA– drug interactions is a very useful part in rational drug design. Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and UV melting experiments have been used to analyze the multivalent (intercalation plus minor groove) binding of the antitumor antibiotic chartreusin to DNA. Using DNA UV melting studies in the presence of the ligand and the binding enthalpy determined by ITC, we determined that the binding constant for the interaction was 3.6 × 10⁵ M^–1 at 20°C, in a solution containing 18 mM Na⁺. The DNA–drug interaction was enthalpy driven, with a ΔH_b of –7.07 kcal/mol at 20°C. Binding enthalpies were determined by ITC in the 20–35°C range and used to calculate a binding-induced change in heat capacity (ΔCp) of –391 cal/mol K. We have obtained a detailed thermodynamic profile for the interaction of this multivalent drug, which makes possible a dissection of ΔG_obs into the component free energy terms. The hydrophobic transfer of the chartreusin chromophore from the solution to the DNA intercalating site is the main contributor to the free energy of binding.     

Sequence effects of aminofluorene-modified DNA duplexes: thermodynamic and circular dichroism properties

Circular dichroism (CD) and UV-melting experiments were conducted with 16 oligodeoxynucleotides modified by the carcinogen 2-aminofluorene, whose sequence around the lesion was varied systematically [d(CTTCTNG[AF]NCCTC), N = G, A, C, T], to gain insight into the factors that determine the equilibrium between base-displaced stacked (S) and external B-type (B) duplex conformers. Differing stabilities among the duplexes can be attributed to different populations of S and B conformers. The AF modification always resulted in sequence-dependent thermal (T_m) and thermodynamic (−ΔG°) destabilization. The population of B-type conformers derived from eight selected duplexes (i.e. -AG*N- and -CG*N-) was inversely proportional to the −ΔG° and T_m values, which highlights the importance of carcinogen/base stacking in duplex stabilization even in the face of disrupted Watson–Crick base pairing in S-conformation. CD studies showed that the extent of the adduct-induced negative ellipticities in the 290–350 nm range is correlated linearly with −ΔG° and T_m, but inversely with the population of B-type conformations. Taken together, these results revealed a unique interplay between the extent of carcinogenic interaction with neighboring base pairs and the thermodynamic properties of the AF-modified duplexes. The sequence-dependent S/B heterogeneities have important implications in understanding how arylamine–DNA adducts are recognized in nucleotide excision repair.     

New insights into DNA triplexes: residual twist and radial difference as measures of base triplet non-isomorphism and their implication to sequence-dependent non-uniform DNA triplex

DNA triplexes are formed by both isomorphic (structurally alike) and non-isomorphic (structurally dissimilar) base triplets. It is espoused here that (i) the base triplet non-isomorphism may be articulated in structural terms by a residual twist (Δt°), the angle formed by line joining the C1′…C1′ atoms of the adjacent Hoogsteen or reverse Hoogsteen (RH) base pairs and the difference in base triplet radius (Δr Å), and (ii) their influence on DNA triplex is largely mechanistic, leading to the prediction of a high (t + Δt)° and low (t − Δt)° twist at the successive steps of Hoogsteen or RH duplex of a parallel or antiparallel triplex. Efficacy of this concept is corroborated by molecular dynamics (MD) simulation of an antiparallel DNA triplex comprising alternating non-isomorphic G*GC and T*AT triplets. Conformational changes necessitated by base triplet non-isomorphism are found to induce an alternating (i) high anti and anti glycosyl and (ii) BII and an unusual BIII conformation resulting in a zigzag backbone for the RH strand. Thus, base triplet non-isomorphism causes DNA triplexes into exhibiting sequence-dependent non-uniform conformation. Such structural variations may be relevant in deciphering the specificity of interaction with DNA triplex binding proteins. Seemingly then, residual twist (Δt°) and radial difference (Δr Å) suffice as indices to define and monitor the effect of base triplet non-isomorphism in nucleic acid triplexes.     

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).      

Pure-Culture Growth of Fermentative Bacteria, Facilitated by H2 Removal: Bioenergetics and H2 Production

We used an H₂-purging culture vessel to replace an H₂-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (ΔG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H₂ production changed such that ΔG remained nearly constant for each organism (−11.1 ± 1.4 kJ mol butyrate⁻¹ for S. lipocalidus and −58.2 ± 1.0 kJ mol alanine⁻¹ for A. colombiense). The cellular maintenance energy, determined from the ΔG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for S. lipocalidus at 55°C and 6.2 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for A. colombiense at 37°C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.     

Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10⁶ cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10⁴ type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10⁶ type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 10⁶ cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.     

The First Identification of Carbohydrate Binding Modules Specific to Chitosan

Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T_m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)₆. The T_m elevation (ΔT_m) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)₆ and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔT_m = 13.6 °C), when (GlcN)₆ was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)_n; ΔG_r° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)_n with the lower affinities (ΔG_r° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)₆ for individual DD1 and DD2 sites (ΔG_r° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)_n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.