Detection of denitrification genes by in situ rolling circle amplification‐fluorescence in situ hybridization to link metabolic potential with identity inside bacterial cells

A target‐primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single‐copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene to target both DNA strands; the target DNA was cut by a restriction endonuclease close to the probe binding sites, which subsequently were made accessible by 5′‐3′ exonucleolysis. After hybridization, the padlock probe was circularized by ligation and served as template for in situ RCA, primed by the probe target site. Finally, the RCA product inside the cells was detected by standard fluorescence in situ hybridization (FISH). The optimized protocol showed high specificity and signal‐to‐noise ratio but low detection frequency (up to 15% for single‐copy genes and up to 43% for the multi‐copy 16S rRNA gene). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA‐FISH was demonstrated on activated sludge by the differential detection of two types of nirS‐defined denitrifiers; one of them was identified as Candidatus Accumulibacter phosphatis by combining in situ RCA‐FISH with 16S rRNA‐targeted FISH. While not suitable for quantification because of its low detection frequency, in situ RCA‐FISH will allow to link metabolic potential with 16S rRNA (gene)‐based identification of single microbial cells.     

Spatial structure in the “Plastisphere”: Molecular resources for imaging microscopic communities on plastic marine debris

Plastic marine debris (PMD) affects spatial scales of life from microbes to whales. However, understanding interactions between plastic and microbes in the “Plastisphere”—the thin layer of life on the surface of PMD—has been technology‐limited. Research into microbe–microbe and microbe–substrate interactions requires knowledge of community phylogenetic composition but also tools to visualize spatial distributions of intact microbial biofilm communities. We developed a CLASI‐FISH (combinatorial labelling and spectral imaging – fluorescence in situ hybridization) method using confocal microscopy to study Plastisphere communities. We created a probe set consisting of three existing phylogenetic probes (targeting all Bacteria, Alpha‐, and Gammaproteobacteria) and four newly designed probes (targeting Bacteroidetes, Vibrionaceae, Rhodobacteraceae and Alteromonadaceae) labelled with a total of seven fluorophores and validated this probe set using pure cultures. Our nested probe set strategy increases confidence in taxonomic identification because targets are confirmed with two or more probes, reducing false positives. We simultaneously identified and visualized these taxa and their spatial distribution within the microbial biofilms on polyethylene samples in colonization time series experiments in coastal environments from three different biogeographical regions. Comparing the relative abundance of 16S rRNA gene amplicon sequencing data with cell‐count abundance data retrieved from the microscope images of the same samples showed a good agreement in bacterial composition. Microbial communities were heterogeneous, with direct spatial relationships between bacteria, cyanobacteria and eukaryotes such as diatoms but also micro‐metazoa. Our research provides a valuable resource to investigate biofilm development, succession and associations between specific microscopic taxa at micrometre scales.     

Direct‐geneFISH: a simplified protocol for the simultaneous detection and quantification of genes and rRNA in microorganisms

Although fluorescence in situ hybridization (FISH) with specific ribosomal RNA (rRNA)‐targeted oligonucleotides is a standard method to detect and identify microorganisms, the specific detection of genes in bacteria and archaea, for example by using geneFISH, requires complicated and lengthy (> 30 h) procedures. Here we report a much improved protocol, direct‐geneFISH, which allows specific gene and rRNA detection within less than 6 h. For direct‐geneFISH, catalyzed amplification reporter deposition (CARD) steps are removed and fluorochrome‐labelled polynucleotide gene probes and rRNA‐targeted oligonucleotide probes are hybridized simultaneously. The protocol allows quantification of gene copy numbers per cell and the signal of the directly labelled probes enables a subcellular localization of the rRNA and target gene. The detection efficiencies of direct‐geneFISH were first evaluated on Escherichia coli carrying the target gene on a copy‐control vector. We could show that gene copy numbers correlated to the geneFISH signal within the cells. The new protocol was then applied for the detection of the sulfate thiolhydrolase (soxB) genes in cells of the gammaproteobacterial clade SUP05 in Lake Rogoznica, Croatia. Cell and gene detection efficiencies by direct‐geneFISH were statistically identical to those obtained with the original geneFISH, demonstrating the suitability of the simpler and faster protocol for environmental samples.     

Fluorescence in situ hybridization for intracellular localization of nifH mRNA

Few reports on in situ mRNA detection in bacteria have been published, even though a major aim in environmental microbiology is to link function/activity to the identity of the organisms. This study reports a reliable approach for the in situ detection of nifH mRNA using fluorescence hybridization based on a previously described protocol for pmoA. nifH codes for a dinitrogenase reductase, a key enzyme in dinitrogen fixation. nifH mRNA was hybridized with a digoxigenin-labelled polynucleotide probe. The hybrid was detected with an anti-DIG-antibody labelled with horseradish peroxidase. Subsequently, the signal was amplified by catalyzed reporter deposition (CARD) with fluorochrome-labelled tyramides. Furthermore, the imaged organisms were identified using standard fluorescence in situ hybridization of rRNA. Thus, the approach enabled us specifically to link in situ the information from the dinitrogen fixation activity of an organism to its identity. Unexpectedly, the signals derived from nifH mRNA hybridization showed a distinct uneven pattern within the cells. This indicated that the method used could even give insights about the localization of the detected mRNA within the cell, which is a potential use of mRNA fluorescence in situ hybridization (FISH) that has not been reported up to now for bacterial cells.     

Molecular identification of toxic Alexandrium tamiyavanichii (Dinophyceae) using two DNA probes

To improve labeling-intensity of whole-cell fluorescence in situ hybridization (FISH) in the molecular identification of toxic Alexandrium tamiyavanichii, two DNA probes (TAMID2 plus TAMIS1 designed from the LSU and SSU rDNA regions, respectively) were used to test the labeling intensity of targeted cultured A. tamiyavanichii cells. The cross-reactivity of the DNA probe to natural seawater samples and six Alexandrium species: A. affine, A. catenella, A. fraterculus, A. insuetum, A. pseudogonyaulax and A. tamarense, was also tested. The labeling intensity of the DNA probe TAMID2S1, a combination of two separate probes that target different regions of the rRNA, was 1.7–2.7 times higher than that of the single DNA probe TAMID2. With cultured A. tamiyavanichii cells in the dead growth phase at 30 days, the TAMID2S1 intensity was 1.9 times higher than that of TAMID2. During a 30-day culture, the labeling intensity of A. tamiyavanichii cells hybridized with TAMID2S1 decreased to 49.4% of the original intensity. No cross-reactivity to various microorganisms in natural seawater samples was found. The two DNA probes together, designated as TAMID2S1, readily detected A. tamiyavanichii added to natural seawater samples, even aged cultured cells.     

Development of an Argopecten-Specific 18S rRNA Targeted Genetic Probe

Comparison of 18S ribosomal RNA gene sequences between diverse bivalve species, including eight scallop species, allowed the design of an 18S rRNA targeted oligonucleotide probe (BS-1364) that was specific for scallops belonging to the genus Argopecten (bay and calico scallops). The high sequence similarity of the 18S rRNA gene between Argopecten irradians and Argopecten gibbus (98.8%) prevented the design of an A. irradians species-specific probe. Hybridization studies using amplified 18S rDNA from a diverse collection of bivalve species demonstrated that the specificity of the digoxygenin-labeled probe was consistent with the predicted specificity indicated by sequence comparison. Hybridization studies using laboratory-spawned bay scallop veligers indicated that a single veliger could be detected by probe hybridization in a blot format, and that probe hybridization signal was proportional (r ²= .99) to the abundance of veligers. Methods for rRNA extraction and blotting were developed that allowed bay scallop veligers to be specifically and quantitatively identified in natural plankton samples. Preliminary studies conducted in Tampa Bay, Florida, suggest that introduced scallops can successfully spawn and produce veligers under in situ conditions. The Argopecten-specific probe and methods developed in this study provide the means to study the production and fate of bay scallop larvae in nature and provide evidence that scallops introduced into Tampa Bay have the potential for successful reproduction and enhancement of scallop stocks. Received January 25, 1999; accepted May 7, 1999.     

Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65°C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r² = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.     

Abundance of Zetaproteobacteria within crustal fluids in back‐arc hydrothermal fields of the Southern Mariana Trough

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back‐arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture‐independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on‐ and off‐ridge of the back‐arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide‐oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron‐oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.     

Non‐target sites with single nucleotide insertions or deletions are frequently found in 16S rRNA sequences and can lead to false positives in fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) has impacted profoundly on our knowledge of the in situ ecophysiology and biodiversity of bacteria in natural communities. However, it has many technical challenges including the possibility of false positives from the binding of probes to non‐target rRNA sequences. We show here that probe target sites containing single‐base insertions or deletions can lead to false FISH positives, the result of hybridization with a bulge around the missing base. Experimental and in silico data suggest this situation occurs at a surprisingly high frequency. The existence of such sites is not currently considered during most FISH probe design processes. We describe software to identify potential non‐target sites resulting from single‐base insertions or deletions in rRNA sequences. This software also provides an estimate of the FISH probe hybridization efficiency to these sites.     

A species-specific probe and a PCR assay for the marine bacterium,Pseudomonas stutzeri strain Zobell

The cloning, sequencing, and analysis of a Pseudomonas stutzeri Zobell 23S rRNA gene is described. Three variable regions were identified, and oligonucleotides homologous to portions of these regions were synthesized. The oligonucleotides were used as probes to screen DNA from various cultured bacteria to identify a species-specific probe. All probes were found to hybridize strongly with P. stutzeri Zobell DNA under stringent conditions and did not hybridize with other Pseudomonas species. One probe showed slight cross-reactivity with DNA from four other bacteria under the hybridization conditions used. Finally, PCR conditions were optimized for detection of P. stutzeri Zobell in mixed culture with a detection limit of 400 cells. The assay detected P. stutzeri Zobell rDNA in coastal seawater samples sampled over a 20-month period. In the future, these probes could be used to quantify the 23S rRNA and rDNA from P. stutzeri Zobell in mixed culture and in environmental samples.     

Development of a 16S rRNA-targeted fluorescence in situ hybridization probe for quantification of the ammonia-oxidizer Nitrosotalea devanaterra and its relatives

The Thaumarchaeota SAGMCG-1 group and, in particular, members of the genus Nitrosotalea have high occurrence in acidic soils, the rhizosphere, groundwater and oligotrophic lakes, and play a potential role in nitrogen cycling. In this study, the specific oligonucleotide fluorescence in situ hybridization probe SAG357 was designed for this Thaumarchaeota group based on the available 16S rRNA gene sequences in databases, and included the ammonia-oxidizing species Nitrosotalea devanaterra. Cell permeabilization for catalyzed reporter deposition fluorescence in situ detection and the hybridization conditions were optimized on enrichment cultures of the target species N. devanaterra, as well as the non-target ammonia-oxidizing archaeon Nitrosopumilus maritimus. Probe specificity was improved with a competitor oligonucleotide, and fluorescence intensity and cell visualization were enhanced by the design and application of two adjacent helpers. Probe performance was tested in soil samples along a pH gradient, and counting results matched the expected in situ distributions. Probe SAG357 and the CARD-FISH protocol developed in the present study will help to improve the current understanding of the ecology and physiology of N. devanaterra and its relatives in natural environments.     

Ammonia-oxidizing archaea and ammonia-oxidizing bacteria in six full-scale wastewater treatment bioreactors

In this study, dideoxy sequencing and 454 high-throughput sequencing were used to analyze diversities of the ammonia monooxygenase (amoA) genes and the 16S rRNA genes of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in six municipal wastewater treatment plants. The results showed that AOB amoA genes were quite diverse in different wastewater treatment plants while the 16S rRNA genes were relatively conserved. Based on the observed complexity of amoA and 16S rRNA genes, most of the AOB can be assigned to the Nitrosomonas genus, with Nitrosomonas ureae, Nitrosomonas oligotropha, Nitrosomonas marina, and Nitrosomonas aestuarii being the four most dominant species. From the sequences of the AOA amoA genes, most AOA observed in this study belong to the CGI.1b group, i.e., the soil lineage. The AOB amoA and 16S rRNA genes were quantified by quantitative PCR and 454 high-throughput pyrosequencing, respectively. Although the results from the two approaches show some disconcordance, they both indicated that the abundance of AOB in activated sludge was very low.     

Use of Bifidobacterium dentium as an Indicator of the Origin of Fecal Water Pollution

A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described. The procedure is based on the detection of certain Bifidobacterium species in the samples. Two 16S rRNA gene-targeted probes are described. One of these probes (BDE) has as its target a region of the 16S rRNA gene of Bifidobacterium dentium, a Bifidobacterium species of exclusively human origin. The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several Bifidobacterium species related with animal origins. The specificity of both probes was evaluated by using 24 Bifidobacterium species, and their threshold detection limit was established by DNA-DNA hybridization. DNA-DNA hybridization with the BDE probe showed it to be specific for B. dentium, whereas that with the BAN probe showed it to be specific for B. animalis, B. asteroides, B. coryneforme, B. cuniculi, B. globosum, B. magnum, B. minimum, and B. subtile. A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces). DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. Thus, this probe is not suitable for the identification of any animal fecal pollution. Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin.     

Detection of Anaerobic Ammonium-Oxidizing Bacteria in Ago Bay Sediments

We identified 16S rRNA gene sequences in sediment samples from Ago Bay in Japan, forming a new branch of the anammox group or closely related to anaerobic ammonium oxidizing (anammox) bacterial sequences. Anammox activity in the sediment samples was detected by ¹⁵N tracer assays. These results, along with the results of fluorescence in situ hybridization (FISH) analysis, suggest the presence of anammox bacteria in the marine sediments.     

100-year-old enigma solved: identification,genomic characterization and biogeography of the yet uncultured Planctomyces bekefii

The first representative of the phylum Planctomycetes, Planctomyces bekefii, was described nearly one century ago. This morphologically conspicuous freshwater bacterium is a rare example of as-yet-uncultivated prokaryotes with validly published names and unknown identity. We report the results of molecular identification of this elusive bacterium, which was detected in a eutrophic boreal lake in Northern Russia. By using high-performance cell sorting, P. bekefii-like cell rosettes were selectively enriched from lake water. The retrieved 16S rRNA gene sequence was nearly identical to those in dozens of metagenomes assembled from freshwater lakes during cyanobacterial blooms and was phylogenetically placed within a large group of environmental sequences originating from various freshwater habitats worldwide. In contrast, 16S rRNA gene sequence similarity to all currently described members of the order Planctomycetales was only 83%–92%. The metagenome assembled for P. bekefii reached 43% genome coverage and showed the potential for degradation of peptides, pectins, and sulfated polysaccharides. Tracing the seasonal dynamics of P. bekefii by Illumina paired-end sequencing of 16S rRNA gene fragments and by fluorescence in situ hybridization revealed that these bacteria only transiently surpass the detection limit, with a characteristic population peak of up to 10⁴ cells ml⁻¹ following cyanobacterial blooms.     

Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture

A rapidly growingTriticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome.In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TakB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. Thein situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. Anin situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.     

Graphical representation of ribosomal RNA probe accessibility data using ARB software package

Background  

Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments.