Improved In Situ Tracking of Rhizosphere Bacteria Using Dual Staining with Fluorescence-Labeled Antibodies and rRNA-Targeted Oligonucleotides

Abstract Fluorescence-labeled antibodies and oligonucleotides were used simultaneously for the in situ identification of bacteria in mixed cultures, as well as in the rhizosphere of inoculated plants. Counterstaining was performed with 4′-6-diamidino-2-phenylindole (DAPI), and scanning confocal laser microscopy or epifluoresence microscopy with a charge-coupled device (CCD) camera were used for detection of individual cells. This strategy gave insight into the relative abundance of an inoculated strain, enabled the exact localization of single cells, and allowed the estimation of the metabolic activity of the bacteria in a complex specimen. Using a strain-specific monoclonal antibody for Azospirillum brasilense Wa3, we could identify this particular strain in root samples of inoculated wheat plantlets. Strain Wa3, as well as other bacteria colonizing the rhizosphere, were stained simultaneously with rRNA-targeted, fluorescence-labeled oligonucleotide probes. In a co-inoculation experiment with A. brasilense strains Sp7 and Wa3, it was demonstrated by in situ identification and quantitative chemoluminescence ELISA that strain Sp7 outcompeted strain Wa3. The combined application of fluorescently labeled antibodies and oligonucleotides should be generally applicable for monitoring specific bacterial strains, within the background of the same species, in relation to the total microbiota. Received: 18 March 1996; Accepted: 22 March 1996     

Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA

A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences was labeled with Cy3 fluor, and the probe specific to the tom genes was labeled with Alexa647 fluor. Probe specificity was first determined, and hybridization temperature was optimized using three rhizosphere bacteria pure cultures as controls, along with the P. putida TOM20 strain. The probes were highly specific to the respective targets, with minimal non-specific binding. The recombinant strain was inoculated into wheat seedling rhizosphere. Colonization of P. putida TOM20 was confirmed by extraction of root biofilm and growth of colonies on selective agar medium. Confocal microscopy of hybridized root biofilm detected P. putida TOM20 cells emitting both Cy3 and Alexa647 fluorescence signals.     

A straightforward DOPE (double labeling of oligonucleotide probes)-FISH (fluorescence in situ hybridization) method for simultaneous multicolor detection of six microbial populations

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is an essential tool for the cultivation-independent identification of microbes within environmental and clinical samples. However, one of the major constraints of conventional FISH is the very limited number of different target organisms that can be detected simultaneously with standard epifluorescence or confocal laser scanning microscopy. Recently, this limitation has been overcome via an elegant approach termed combinatorial labeling and spectral imaging FISH (CLASI-FISH) (23). This technique, however, suffers compared to conventional FISH from an inherent loss in sensitivity and potential probe binding biases caused by the competition of two differentially labeled oligonucleotide probes for the same target site. Here we demonstrate that the application of multicolored, double-labeled oligonucleotide probes enables the simultaneous detection of up to six microbial target populations in a straightforward and robust manner with higher sensitivity and less bias. Thus, this newly developed technique should be an attractive option for all researchers interested in applying conventional FISH methods for the study of microbial communities.     

Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster

The genera Azospirillum, Skermanella and Rhodocista form a phylogenetic subgroup within the alfa subclass of Proteobacteria. Based on comparative 16S rRNA sequence analysis a nested set of new oligonucleotide probes was designed. It comprises probes for the whole genus cluster Azospirillum-Skermanella-Rhodocista, for the Azospirilli subcluster I including A. lipoferum, A. doebereinerae, A. largimobile, A. brasilense and A. halopraeferens, for the Azospirilli subcluster II including A. amazonense, A. irakense and the genus Skermanella, for the genus Rhodocista as well as for all Azospirilli species or species cluster. The new probes allow a fast and reliable in situ identification of bacteria belonging to the Azospirillum-Skermanella-Rhodocista-cluster at different phylogenetic levels. The specificity of the new probes was tested with 56 strains of the Azospirillum-Rhodocista-Skermanella-cluster and selected reference cells from other genera by hybridising with the complete probe set. In addition, applications of the fluorescently labelled probes for in situ identification of isolates and for the in situ localisation of A. brasilense on maize roots were demonstrated using confocal laser scanning microscopy.     

Combination of fluorescent in situ hybridization and microautoradiography-a new tool for structure-function analyses in microbial ecology

A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli and Herpetosiphon aurantiacus under aerobic incubation conditions with added [3H]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([14C]acetate, [14C]butyrate, [14C]bicarbonate, and 33Pi) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities.     

Community analysis of the bacterial assemblages in the winter cover and pelagic layers of a high mountain lake by in situ hybridization

The bacterial community structure in the winter cover and pelagic zone of a high mountain lake was analyzed by in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes. Cells fixed on membrane filters were hybridized with a probe specific for the domain Bacteria as well as with probes for the alpha, beta, and gamma subclasses of the class Proteobacteria and the Cytophaga-Flavobacterium group. The fraction of bacteria detectable after hybridization with the bacterial probe EUB ranged from 40 to 81% of 4(prm1),6-diamidino-2-phenylindole (DAPI) counts. The bacterial assemblage varied considerably between and within different habitats (snow, slush, and lake water) but was in most cases dominated by members of the beta subclass (6.5 to 116% of bacteria detectable with probe EUB). The sum of bacteria hybridizing with group-specific probes was usually lower than the fraction detectable with probe EUB. Image analysis was used to characterize morphology and the size-specific biomass distribution of bacterial assemblages, which clearly separated the three habitats. Although the measured secondary production parameters and the fraction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride-reducing bacteria varied by more than an order of magnitude in the different slush and pelagic layers, detectability with the fluorescent probe EUB was constantly high. Physiological strategies of bacteria under nutrient limitation and at low temperatures are discussed in the context of the ribosome content of single cells. This study confirms the suitability of fluorescently labeled rRNA-targeted probes for the characterization of bacterial population structures even in oligotrophic habitats.     

CLARITY组织透明化技术在蜜蜂肠道组织上的运用

【目的】以西方蜜蜂Apis mellifera工蜂肠道为例探究组织透明化技术--丙烯酰胺交联替换脂质透明硬化成像/免疫染色/原位杂交兼容组织水凝胶(clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridization-compatible tissue-hYdrogel, CLARITY)在昆虫组织上的应用,确定CLARITY与荧光原位杂交(FISH)相结合在昆虫肠道组织透明化中的适用性。【方法】依照CLARITY技术操作程序,用水凝胶固定西方蜜蜂肠道,并以被动方式透明化,再用靶向东方蜜蜂微孢子虫Nosema ceranae 16S rRNA带异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记和靶向真核细胞18S rRNA带Texas RED标记的寡核苷酸荧光探针进行肠道组织的荧光原位杂交,然后用DAPI(蓝色)进行细胞核复染,通过激光共聚焦显微镜观察透明化的染色组织。【结果】首次成功将西方蜜蜂肠道组织透明化。在激光共聚焦显微镜下,观察到马氏管的原始分布形态,以及东方蜜蜂微孢子虫在中肠末端分布更密集的空间分布特征,并实现了对肠道组织的3D重构。【结论】CLARITY能应用于蜜蜂肠道组织透明化,透明化组织能进行原位杂交和激光共聚焦观察。CLARITY和FISH相结合免除抗体制备和石蜡切片的麻烦,直观展示肠道内部的真实状态,为昆虫生理病理研究提供了一种可靠特异的标记方法。     

Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification.

The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with horseradish peroxidase to improve the sensitivity of in situ hybridization of whole fixed bacterial cells. Flow cytometry and quantitative microscopy of cells hybridized by this technique showed 10- to 20-fold signal amplifications relative to fluorescein-monolabeled probes. The application of the new technique to the detection of natural bacterial communities resulted in very bright signals; however, the number of detected cells was significantly lower than that detected with fluorescently monolabeled, rRNA-targeted oligonucleotide probes.     

Determination of the structure of the repeated unit of the Azospirillum brasilense SR75 O-specific polysaccharide and homology of the lps loci in the plasmids of Azospirillum brasilense strains SR75 and Sp245

The structural identity of the repeated unit in O-specific polysaccharides (OPSs) present in the outer membrane of strain SR75 of the bacterium Azospirillum brasilense, isolated from wheat rhizosphere in Saratov oblast, and the OPSs of previously studied A. brasilense strain Sp245, isolated from surface-sterilized wheat roots in Brazil, has been demonstrated. Plasmid profiles, DNA restriction, and hybridization assays suggested that A. brasilense strains SR75 and Sp245 have different genomic structures. It was shown that homologous lps loci of both strains was localized in their plasmid DNA. This fact allows us to state that, despite their different origin, the development of the strains studied was convergent. Presumably, the habitation of these bacteria in similar ecological niches influenced this process in many respects.     

Estimation of the phylogenetic diversity of prokaryotic microorganisms in Sphagnum bogs with the use of fluorescence in situ hybridization (FISH)

The microbial population of Sphagnum bogs of northern Russia was analyzed with respect to the presence and cell numbers of representatives of particular phylogenetic groups of prokaryotes by means of in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes with broad detection spectra. The total number of cells that hybridized with universal Archaea- or Bacteria-specific probes varied, in peat samples of different bogs, from 45 to 83% of the number of cells revealed by DAPI staining. Down the bog profiles, the total number of prokaryotes and the fraction of archaea among them increased. Application of a set of oligonucleotide probes showed that the number of microorganisms belonging to such phylogenetic lineages of the domain Bacteria as the phyla Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Acidobacteria, and Planctomycetes constituted, in total, 14.0-26.5% of the number of eubacteria detected in the samples. Among the bacteria identified in the peat samples, the most abundant were representatives of the classes Alphaproteobacteria and Betaproteobacteria and the phyla Acidobacteria, Bacteroidetes, and Actinobacteria.     

Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization

The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4′,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of the Cytophaga-Flavobacterium cluster were most abundant in all layers, followed by the sulfate-reducing bacteria.     

Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules

16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35 degrees C) and thermophilic (55 degrees C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells. Methanosaeta-, Methanobacterium-, Methanospirillum-, and Methanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus Methanosaeta. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655-2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related to Syntrophobacter species. The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges.     

In situ visualization of high genetic diversity in a natural microbial community.

Simultaneous in situ visualization of seven distinct bacterial genotypes, all affiliated with the phylogenetically narrow group of beta-1 Proteobacteria, was achieved in activated sludge. This finding indicates that the high diversity found in the same sample by direct rRNA sequence retrieval was indeed present in this complex community. By the combination of comparative rRNA sequence analysis, in situ hybridization with fluorescently labeled, rRNA-targeted oligonucleotides and confocal laser scanning microscopy several microbial populations can be analyzed for abundance, relative spatial distribution and phylogeny directly at their site of action without prior cultivation.     

Enhanced micropropagation response and biocontrol effect of Azospirillum brasilense Sp245 on Prunus cerasifera L. clone Mr.S 2/5 plants

Inoculation with Azospirillum brasilense Sp245 exerts beneficial effects on micropropagated plants of Prunus cerasifera L. clone Mr.S 2/5, as seen in the results of a comparative analysis of inoculated and non-inoculated explants, during both the rooting and acclimatation phases. The presence of Azospirillum brasilense Sp245 increased root system, root hair biomass production and apical activity. Although the presence of the bacteria had a positive effect on rooting, the addition of indolebutyric acid (IBA) to Murashige and Skoog (MS) medium was seen as indispensable in order to promote the rooting of explants. Aside from the promotion of plant growth, A. brasilense Sp245 protects plants against pathogen attacks, such as Rhizoctonia spp., with a plant survival rate of nearly 100% vs. 0% as seen in the negative control. The biocontrol effect of A. brasilense Sp245 on the fungal rhizospheric community has been confirmed by denaturing gradient gel electrophoresis (DGGE) profiles of the rhizospheric microbial community. This study indicates that A. brasilense Sp245 could be employed as a tool in plant biotechnology.     

In situ detection of rhodococci associated with activated sludge foams

Genus-specific 16S rRNA targeted oligonucleotide probes, Rco1 and Rco2, were designed and used to detect rhodococci in activated sludge foam samples by confocal laser scanning microscopy. Pure cultures were used to find the optimal hybridisation conditions which were determined by comparing the mean fluorescent intensities of target and non-target cells from images captured using a confocal laser scanning microscope (CLSM). The combination of fluorescent in situ hybridisation with rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy provides an effective way of detecting rhodococci in environmental samples.     

Root Colonization by Pseudomonas sp. DSMZ 13134 and Impact on the Indigenous Rhizosphere Bacterial Community of Barley

Over the last few decades, the ability of rhizosphere bacteria to promote plant growth has been considered to be of scientific, ecological, and economic interest. The properties and mechanisms of interaction of these root-colonizing bacteria have been extensively investigated, and plant protection agents that are based on these bacterial strains have been developed for agricultural applications. In the present study, the root colonization of barley by Pseudomonas sp. DSMZ 13134, that is contained in the commercially available plant protection agent Proradix®, was examined using the fluorescence in situ hybridization method with oligonucleotide probes and specific gfp-tagging of the inoculant strain in combination with confocal laser scanning microscopy. In the first phase of root colonization, the inoculant strain competed successfully with seed and soil-borne bacteria (including Pseudomonads) for the colonization of the rhizoplane. Pseudomonas sp. DSMZ 13134 could be detected in all parts of the roots, although it did not belong to the dominant members of the root-associated bacterial community. Gfp-tagged cells were localized particularly in the root hair zone, and high cell densities were apparent on the root hair surface. To investigate the impact of the application of Proradix® on the structure of the dominant root-associated bacterial community of barley, T-RFLP analyses were performed. Only a transient community effect was found until 3 weeks post-application.     

Detection of nitrifying bacteria in activated sludge by fluorescent in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for eubacteria (EUB338), ammonium-oxidizing bacteria (Nsm156) and nitrite-oxidizing bacteria (Nb1000) were used for the rapid detection of nitrifying bacteria in the activated sludge of a pilot nitrifying reactor by whole-cell, fluorescent in situ hybridization (FISH). Emission scanning and synchronous scanning fluorescence spectrometry were used to measure the hybridization. The binding of the probes at a temperature significantly lower than the melting temperature of the hybrids was conventionally considered as non-specific. Total binding of the probes at a temperature significantly higher than the melting temperature of the hybrids was conventionally considered as the sum of non-specific and specific binding (hybridization). Non-specific binding of the oligonucleotide probes with a biomass of activated sludge was 37% of the total binding of the EUB338 probe, 54% of the total binding of the Nsm156 probe, and 69% of the total binding of the Nb1000 probe. The ratio of the specific binding of the Nsm156 and Nb1000 probes was 2.3:1. The ratio of the numbers of ammonium-oxidizing bacteria to nitrite-oxidizing bacteria, determined by microbiological methods, was 2.4:1. Measuring fluorescent in situ hybridization by fluorescence spectrometry appears to be a practical tool for monitoring the microbial communities that contain nitrifying bacteria. However, a method that accounts for the non-specific binding of the probes more easily and reliably should be developed for practical application.     

The microbial community composition of a nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analyzed by the full-cycle rRNA approach

The composition of the microbial community present in the nitrifying-denitrifying activated sludge of an industrial wastewater treatment plant connected to a rendering facility was investigated by the full-cycle rRNA approach. After DNA extraction using three different methods, 94 almost full-length 16S rRNA gene clones were retrieved and analyzed phylogenetically. 59% of the clones were affiliated with the Proteobacteria and clustered with the beta- (29 clones), alpha- (24), and delta-class (2 clones), respectively. 15 clones grouped within the green nonsulfur (GNS) bacteria and 11 clones belonged to the Planctomycetes. The Verrucomicrobia, Acidobacteria, Nitrospira, Bacteroidetes, Firmicutes and Actinobacteria were each represented by one to five clones. Interestingly, the highest 'species richness' [measured as number of operational taxonomic units (OTUs)] was found within the alpha-class of Proteobacteria, followed by the Planctomycetes, the beta-class of Proteobacteria, and the GNS-bacteria. The microbial community composition of the activated sludge was determined quantitatively by using 36 group-, subgroup-, and OTU-specific rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH), confocal laser scanning microscopy and digital image analysis. 89% of all bacteria detectable by FISH with a bacterial probe set could be assigned to specific divisions. Consistent with the 16S rRNA gene library data, members of the beta-class of Proteobacteria dominated the microbial community and represented almost half of the biovolume of all bacteria detectable by FISH. Within the beta-class, 98% of the cells could be identified by the application of genus- or OTU-specific probes demonstrating a high in situ abundance of bacteria related to Zoogloea and Azoarcus sensu lato. Taken together, this study provides the first encompassing, high-resolution insight into the in situ composition of the microbial community present in a full-scale, industrial wastewater treatment plant.     

Mapping nonisotopically labeled DNA probes to human chromosome bands by confocal microscopy.

A method for mapping nonisotopically labeled probes to human metaphase chromosomes that can be used with laser scanning confocal microscopy has been developed. Only a limited number of wavelengths are available from the argon ion lasers used in most commercial instruments and therefore a method that allowed the visualization of bands on human chromosomes stained with propidium iodide and, simultaneously, the detection of hybridization signals using FITC-labeled antibodies was developed. The confocal microscope was used to map single-copy probes to chromosome bands and the positions of the probes on the R-banded chromosomes corresponded to map positions previously determined on Hoechst 33258-stained chromosomes (G-banded). A comparison of confocal imaging of single-copy hybridization signals with conventional fluorescence microscopy and high-sensitivity video cameras revealed little difference in sensitivity but greater resolution of chromosome bands with the confocal microscope. The polymerase chain reaction was used to prepare nonisotopically labeled probes for in situ hybridization and to amplify Alu and KpnI family repeats from cloned DNA to be used to suppress hybridization of these repeat sequences so that a cosmid probe could be mapped to a chromosome band.     

Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector

To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.