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.     

Decolorization of textile azo dye and Congo red by an isolated strain of the dissimilatory manganese-reducing bacterium Shewanella xiamenensis BC01

Shewanella xiamenensis BC01 (SXM) was isolated from sediment collected off Xiamen, China and was identified based on the phylogenetic tree of 16S rRNA sequences and the gyrB gene. This strain showed high activity in the decolorization of textile azo dyes, especially methyl orange, reactive red 198, and recalcitrant dye Congo red, decolorizing at rates of 96.2, 93.0, and 87.5 %, respectively. SXM had the best performance for the specific decolorization rate (SDR) of azo dyes compared to Proteus hauseri ZMd44 and Aeromonas hydrophila NIU01 strains and had an SDR similar to Shewanella oneidensis MR-1 in Congo red decolorization. Luria-Bertani medium was the optimal culture medium for SXM, as it reached a density of 4.69 g-DCW L^?1 at 16 h. A mediator (manganese) significantly enhanced the biodegradation and flocculation of Congo red. Further analysis with UV–VIS, Fourier Transform Infrared spectroscopy, and Gas chromatography–mass spectrometry demonstrated that Congo red was cleaved at the azo bond, producing 4,4′-diamino-1,1′-biphenyl and 1,2′-diamino naphthalene 4-sulfonic acid. Finally, SEM results revealed that nanowires exist between the bacteria, indicating that SXM degradation of the azo dyes was coupled with electron transfer through the nanowires. The purpose of this work is to explore the utilization of a novel, dissimilatory manganese-reducing bacterium in the treatment of wastewater containing azo dyes.     

Application of rRNA-targeted oligonucleotide probes in biotechnology

Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.     

Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.     

probeBase: an online resource for rRNA-targeted oligonucleotide probes

Ribosomal RNA-(rRNA)-targeted oligonucleotide probes are widely used for culture-independent identification of microorganisms in environmental and clinical samples. ProbeBase is a comprehensive database containing more than 700 published rRNA-targeted oligonucleotide probe sequences (status August 2002) with supporting bibliographic and biological annotation that can be accessed through the internet at http://www.probebase.net. Each oligonucleotide probe entry contains information on target organisms, target molecule (small- or large-subunit rRNA) and position, G+C content, predicted melting temperature, molecular weight, necessity of competitor probes, and the reference that originally described the oligonucleotide probe, including a link to the respective abstract at PubMed. In addition, probes successfully used for fluorescence in situ hybridization (FISH) are highlighted and the recommended hybridization conditions are listed. ProbeBase also offers difference alignments for 16S rRNA-targeted probes by using the probe match tool of the ARB software and the latest small-subunit rRNA ARB database (release June 2002). The option to directly submit probe sequences to the probe match tool of the Ribosomal Database Project II (RDP-II) further allows one to extract supplementary information on probe specificities. The two main features of probeBase, 'search probeBase' and 'find probe set', help researchers to find suitable, published oligonucleotide probes for microorganisms of interest or for rRNA gene sequences submitted by the user. Furthermore, the 'search target site' option provides guidance for the development of new FISH probes.     

Effects of nitrate and nitrite on dissimilatory iron reduction by Shewanella putrefaciens 200.

The inhibitory effects of nitrate (NO3-) and nitrite (NO2-) on dissimilatory iron (FE3+) reduction were examined in a series of electron acceptor competition experiments using Shewanella putrefaciens 200 as a model iron-reducing microorganism. S. putrefaciens 200 was found to express low-rate nitrate reductase, nitrite reductase, and ferrireductase activity after growth under highly aerobic conditions and greatly elevated rates of each reductase activity after growth under microaerobic conditions. The effects of NO3- and NO2- on the Fe3+ reduction activity of both aerobically and microaerobically grown cells appeared to follow a consistent pattern; in the presence of Fe3+ and either NO3- or NO2-, dissimilatory Fe3+ and nitrogen oxide reduction occurred simultaneously. Nitrogen oxide reduction was not affected by the presence of Fe3+, suggesting that S. putrefaciens 200 expressed a set of at least three physiologically distinct terminal reductases that served as electron donors to NO3-, NO2-, and Fe3+. However, Fe3+ reduction was partially inhibited by the presence of either NO3- or NO2-. An in situ ferrozine assay was used to distinguish the biological and chemical components of the observed inhibitory effects. Rate data indicated that neither NO3- nor NO2- acted as a chemical oxidant of bacterially produced Fe2+. In addition, the decrease in Fe3+ reduction activity observed in the presence of both NO3- and NO2- was identical to the decrease observed in the presence of NO2- alone. These results suggest that bacterially produced NO2- is responsible for inhibiting electron transport to Fe3+.     

Genome sequence of the dissimilatory metal ion-reducing bacterium Shewanella oneidensis

Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities, conferred in part by multicomponent, branched electron transport systems. Here we report the sequencing of the S. oneidensis genome, which consists of a 4,969,803-base pair circular chromosome with 4,758 predicted protein-encoding open reading frames (CDS) and a 161,613-base pair plasmid with 173 CDSs. We identified the first Shewanella lambda-like phage, providing a potential tool for further genome engineering. Genome analysis revealed 39 c-type cytochromes, including 32 previously unidentified in S. oneidensis, and a novel periplasmic [Fe] hydrogenase, which are integral members of the electron transport system. This genome sequence represents a critical step in the elucidation of the pathways for reduction (and bioremediation) of pollutants such as uranium (U) and chromium (Cr), and offers a starting point for defining this organism's complex electron transport systems and metal ion-reducing capabilities.     

Design of 16S rRNA-targeted oligonucleotide probes for detecting cultured and uncultured archaeal lineages in high-temperature environments

In order to facilitate the evaluation of archaeal community diversity and distribution in high-temperature environments, 14 16S rRNA oligonucleotide probes were designed. Adequate hybridization and wash conditions of the probes encompassing most known hyperthermophilic Archaea, members of the orders Thermococcales, Desulfurococcales and Sulfolobales, of the families Methanocaldococcaceae, Pyrodictiaceae and Thermoproteaceae, of the genera Archaeoglobus, Methanopyrus and Ignicoccus, and of the as yet uncultured lineages Korarchaeota, Crenarchaeota marine group I, deep-sea hydrothermal vent euryarchaeotic group 2 (DHVE 2), and deep-sea hydrothermal vent euryarchaeotic group 8 (DHVE 8) were determined by dot-blot hybridization from target and non-target reference organisms and environmental clones. The oligonucleotide probes were also used to evaluate the archaeal community composition in nine deep-sea hydrothermal vent samples. All probes, except those targeting members of Sulfolobales, Thermoproteaceae, Pyrodictiaceae and Korarchaeota, gave positive hybridization signals when hybridized against 16S rDNA amplification products obtained from hydrothermal DNA extracts. The results confirmed the widespread occurrence of Thermococcales, Desulfurococcales, Methanocaldococcaceae and Archaeoglobus in deep-sea hydrothermal vents, and extended the known ecological habitats of uncultured lineages. Despite their wide coverage, the probes were unable to resolve the archaeal communities associated with hydrothermally influenced sediments, suggesting that these samples may contain novel lineages. This suite of oligonucleotide probes may represent an efficient tool for rapid qualitative and quantitative characterization of archaeal communities. Their application would help to provide new insights in the future into the composition, distribution and abundance of Archaea in high-temperature environments.     

Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes.

A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.     

Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes.

A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.     

Morphological and metabolic responses to starvation by the dissimilatory metal-reducing bacterium Shewanella alga BrY.

The response of the dissimilatory metal-reducing bacterium Shewanella alga BrY to carbon and nitrogen starvation was examined. Starvation resulted in a gradual decrease in the mean cell volume from 0.48 to 0.2 micron 3 and a dramatic decrease in Fe(III) reductase activity. Growth of starved cultures was initiated with O2, ferric oxyhydroxide, Co(III)-EDTA, or Fe(III)-bearing subsurface materials as the sole electron acceptor. Microbially reduced subsurface materials reduced CrO(4)2-. Starvation of dissimilatory metal-reducing bacteria may provide a means of delivering this metabolism to contaminated subsurface environments for in situ bioremediation.     

Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.     

Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.

Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.     

Identification of lactococci and enterococci by colony hybridization with 23S rRNA-targeted oligonucleotide probes.

Specific sequences of 23S rRNA of Lactococcus lactis, Enterococcus faecalis, Enteroccus faecium, and Enterococcus malodoratus/Enterococcus avium were identified, and complementary oligonucleotide probes were synthesized. The specificity of the probes was evaluated by dot blot and colony hybridizations. The probes can be used for the specific detection and identification of colonies of the corresponding species in mixed cultures.     

Order-specific 16S rRNA-targeted oligonucleotide probes for (hyper)thermophilic archaea and bacteria

New oligonucleotide probes were designed and evaluated for application in fluorescence in situ hybridization (FISH) studies on (hyper)thermophilic microbial communities—Arglo32, Tcoc164, and Aqui1197 target the 16S rRNA of Archaeoglobales, Thermococcales, and Aquificales, respectively. Both sequence information and experimental evaluation showed high coverage and specificity of all three probes. The signal intensity of Aqui1197 was improved by addition of a newly designed, unlabeled helper oligonucleotide, hAqui1045. It was shown that in addition to its function as a probe for Aquificales, Aqui1197 is suitable as a supplementary probe to extend the coverage of the domain-specific bacterial probe EUB338. In sediments from two hydrothermal seeps on Vulcano Island, Italy, the microbial community structure was analyzed by FISH with both established and the new oligonucleotide probes, showing the applicability of Arglo32, Tcoc164, and Aqui1197/hAqui1045 to natural samples. At both sites, all major groups of (hyper)thermophiles, except for methanogens, were detected: Crenarchaeota (19%, 16%), Thermococcales (14%, 22%), Archaeoglobales (14%, 12%), Aquificales (5%, 8%), Thermotoga/Thermosipho spp. (12%, 9%), Thermus sp. (12%, none), and thermophilic Bacillus sp. (12%, 8%).     

16S rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum Cytophaga-Flavobacterium-Bacteroides

Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically important members of many microbial communities. A suite of five 16S rRNA-targeted oligonucleotide probes for members of this group is described which was designed to dominantly target bacteria of the CFB-phylum that are found in particular habitats. For this we initially performed a literature survey-for the sources and sites of isolation of hitherto described members of the CFB-phylum. Probe CFB286 is mostly complementary to the 16S rRNA of species originally isolated from freshwater habitats, however, detects some marine and soil isolates and is the only probe which includes some food isolates. Probe CFB563 detects marine as well as animal-associated isolates. Probe CFB719, which also detects some environmental isolates, and probe CFB972 are mostly targeting animal-associated isolates. All probes are complementary to a variety of human-associated species within the CFB-phylum which, in the data set investigated (October 1998), made up 46% of all 16S rRNA sequences from the CFB-phylum. We conclude that it is difficult to find habitat-specific probes for members of the CFB-phylum and that the design of probes for monophyletic groups should remain the standard approach. Applicability of the probes for fluorescence in situ hybridization and specificity for single cell detection were evaluated for the four mentioned probes whereas the fifth, probe CFB1082, which almost exclusively targets human-associated species, was not further characterized. The new probes are of limited determinative value and should be used together with the already established probes for the CFB-phylum. It is the hybridization pattern observed for a given cell or culture with the enlarged probe set that is suggestive for its affiliation with a defined group within the CFB-phylum.     

In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes

Individual cyanobacterial cells are normally identified in environmental samples only on the basis of their pigmentation and morphology. However, these criteria are often insufficient for the differentiation of species. Here, a whole-cell hybridization technique is presented that uses horseradish peroxidase (HRP)-labeled, rRNA-targeted oligonucleotides for in situ identification of cyanobacteria. This indirect method, in which the probe-conferred enzyme has to be visualized in an additional step, was necessary since fluorescently monolabeled oligonucleotides were insufficient to overstain the autofluorescence of the target cells. Initially, a nonfluorescent detection assay was developed and successfully applied to cyanobacterial mats. Later, it was demonstrated that tyramide signal amplification (TSA) resulted in fluorescent signals far above the level of autofluorescence. Furthermore, TSA-based detection of HRP was more sensitive than that based on nonfluorescent substrates. Critical points of the assay, such as cell fixation and permeabilization, specificity, and sensitivity, were systematically investigated by using four oligonucleotides newly designed to target groups of cyanobacteria.     

The influence of chelating agents upon the dissimilatory reduction of Fe(III) by Shewanella putrefaciens

The ability of S. putrefaciens to reduce Fe(III) complexed by a variety of ligands has been investigated. All of the ligands tested caused the cation to be more susceptible to reduction by harvested whole cells than when uncomplexed, although some complexes were more readily reduced than others. Monitoring rates of reduction by a ferrozine assay for Fe(II) formation proved inadequate using Fe(III) ligands giving Fe(II) complexes of low kinetic lability (e.g. EDTA). A more suitable assay for Fe(III) reduction in the presence of such ligands proved to be the observation of associated cytochrome oxidation and re-reduction. Where possible, an assay for Fe(III) reduction based upon the disappearance of Fe(III) complex was also employed. Reduction of all Fe(III) complexes tested was totally inhibited by the presence of O₂, partially inhibited by HQNO and slower in the absence of a physiological electron donor. Upon cell fractionation, Fe(III) reductase activity was detected exclusively in the membranes. Using different physiological electron donors in assays on membranes, relative reduction rates of Fe(III) complexes complemented the data from whole cells. The differences in susceptibility to reduction of the various complexes are discussed, as is evidence for the respiratory nature of the reduction.     

Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes

Isolates of lactobacilli from infant faeces phenotypically characterized as Lactobacillus paracasei subsp. paracasei (six strains), Lact. rhamnosus (six strains), Lact. gasseri (three strains), Lact. acidophilus (one strain) and Lact. fermentum/reuteri (three strains) according to recent classification systems were subjected to SDS-PAGE of whole cell proteins and rRNA-targeted oligonucleotide probe hybridization, in order to confirm the phenotypic characterization and elucidate the exact taxonomic position of the three strains that had properties between fermentum and reuteri. Results suggested a good agreement between the phenotypic characterization, SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization for strains of all species except for the Lact. fermentum/reuteri strains. Results obtained by rRNA probes suggested a possible phylogenetic relatedness of the strains to Lact. reuteri. Isolates from infant faeces with interesting probiotic properties could be used as components of fermented milk products.