scholarly journals Use of Microautoradiography Combined with Fluorescence In Situ Hybridization To Determine Dimethylsulfoniopropionate Incorporation by Marine Bacterioplankton Taxa

2004 ◽  
Vol 70 (8) ◽  
pp. 4648-4657 ◽  
Author(s):  
Maria Vila ◽  
Rafel Simó ◽  
Ronald P. Kiene ◽  
Jarone Pinhassi ◽  
José M. González ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Gulf Of Mexico ◽  
Fluorescence In Situ Hybridization ◽  
Marine Bacteria ◽  
Heterotrophic Bacteria ◽  
Phytoplankton Bloom ◽  
Mediterranean Coast ◽  
Marine Bacterioplankton ◽  
Significant Uptake

ABSTRACT The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU). In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from [35S]DMSP ranged between 27 and 51% of 4′,6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating [3H]leucine. In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from [35S]DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom. At the same time, the proportion of cells incorporating [3H]leucine increased from 21 to 40%. The combination of AU and fluorescence in situ hybridization (FISH) revealed that the Roseobacter clade (α-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating [35S]DMSP at both sampling sites. Significant uptake of sulfur from DMSP was also found among members of the γ-proteobacteria and Cytophaga-Flavobacterium groups. Roseobacter and γ-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating 35S from DMSP (around 50%). Altogether, the application of AU with [35S]DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization. These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton.

Molecular analyses of the diversity in marine bacterioplankton assemblages along the coastline of the northeastern Gulf of Mexico

2010 ◽  
Vol 56 (10) ◽  
pp. 853-863 ◽  
Author(s):  
Ola A. Olapade
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gulf Of Mexico ◽  
Fluorescence In Situ Hybridization ◽  
Community Diversity ◽  
Rrna Gene ◽  
Marine Bacterioplankton ◽  
Northeastern Gulf Of Mexico

Bacterial community diversity in marine bacterioplankton assemblages were examined in 3 coastal locations along the northeastern Gulf of Mexico (GOM) using 16S rRNA gene libraries and fluorescence in situ hybridization approaches. The majority of the sequences (30%–60%) were similar to the 16S rRNA gene sequences of unknown bacteria; however, the operational taxonomic units from members of the Cyanobacteria, Proteobacteria, and Bacteroidetes were also present at the 3 GOM sites. Overall, sequence diversity was more similar between the Gulf sites of Carrabelle and Ochlockonee than between either of the Gulf sites and Apalachicola Bay. Fluorescence in situ hybridization analyses revealed the quantitative predominance of members of the Alphaproteobacteria subclass and the Cytophaga – Flavobacterium cluster within the bacterioplankton assemblages. In general, the study further reveals the presence of many bacterial taxa that have been previously found to be dominant in coastal marine environments. Differences observed in the representation of the various bacterial phylogenetic groups among the GOM coastal sites could be partly attributed to dynamic variations in several site-specific conditions, including intermittent tidal events, nutrient availability, and anthropogenic influences.

scholarly journals Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria

2002 ◽  
Vol 68 (6) ◽  
pp. 3094-3101 ◽  
Author(s):  
Annelie Pernthaler ◽  
Jakob Pernthaler ◽  
Rudolf Amann
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Heterotrophic Bacteria ◽  
Cell Loss ◽  
Bacterial Cells ◽  
Detection Rates ◽  
Cell Counts ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

ABSTRACT Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml−1 at 37°C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.

Imaging Marine Bacteria with Unique 16S rRNA V6 Sequences by Fluorescence in situ Hybridization and Spectral Analysis

2010 ◽  
Vol 27 (3) ◽  
pp. 251-260 ◽  
Author(s):  
Yuko Hasegawa ◽  
Jessica L. Mark Welch ◽  
Alex M. Valm ◽  
Christopher W. Rieken ◽  
Mitchell L. Sogin ◽  
...  
Keyword(s):  
Spectral Analysis ◽  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Marine Bacteria

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

2000 ◽  
Vol 66 (12) ◽  
pp. 5116-5122 ◽  
Author(s):  
Matthew T. Cottrell ◽  
David L. Kirchman
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Community Composition ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clone Libraries ◽  
Marine Bacterioplankton ◽  
Bacterioplankton Communities

ABSTRACT 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. TheCytophaga-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 theCytophaga-Flavobacter group can be a numerically dominant component of coastal marine bacterioplankton communities.

scholarly journals Effects of Compost on Colonization of Roots of Plants Grown in Metalliferous Mine Tailings, as Examined by Fluorescence In Situ Hybridization

2008 ◽  
Vol 75 (3) ◽  
pp. 842-847 ◽  
Author(s):  
Sadie L. Iverson ◽  
Raina M. Maier
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Mine Tailings ◽  
Root Colonization ◽  
Heterotrophic Bacteria ◽  
Plant Biomass ◽  
Bacterial Colonization ◽  
Community Research ◽  
Compost Amendment

ABSTRACT The relationship between compost amendment, plant biomass produced, and bacterial root colonization as measured by fluorescence in situ hybridization was examined following plant growth in mine tailings. Mine tailings can remain devoid of vegetation for decades after deposition due to a combination of factors that include heavy metal toxicity, low pH, poor substrate structure and water-holding capacity, and a severely impacted heterotrophic microbial community. Research has shown that plant establishment, a desired remedial objective to reduce eolian and water erosion of such tailings, is enhanced by organic matter amendment and is correlated with significant increases in rhizosphere populations of neutrophilic heterotrophic bacteria. Results show that for the acidic metalliferous tailings tested in this study, compost amendment was associated with significantly increased bacterial colonization of roots and increased production of plant biomass. In contrast, for a Vinton control soil, increased compost had no effect on root colonization and resulted only in increased plant biomass at high levels of compost amendment. These data suggest that the positive association between compost amendment and root colonization is important in the stressed mine tailings environment where root colonization may enhance both microbial and plant survival and growth.

scholarly journals Ecophysiological Interaction between Nitrifying Bacteria and Heterotrophic Bacteria in Autotrophic Nitrifying Biofilms as Determined by Microautoradiography-Fluorescence In Situ Hybridization

2004 ◽  
Vol 70 (3) ◽  
pp. 1641-1650 ◽  
Author(s):  
Tomonori Kindaichi ◽  
Tsukasa Ito ◽  
Satoshi Okabe
Keyword(s):  
Acetic Acid ◽  
In Situ Hybridization ◽  
Bacterial Community ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Heterotrophic Bacteria ◽  
Nitrifying Bacteria ◽  
Community Members ◽  
Biofilm Community

ABSTRACT Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH4 + as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (α-Proteobacteria), 23%; γ-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%; Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. α- and γ-Proteobacteria dominated the utilization of [14C]acetic acid and 14C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-d-[1-14C]glucosamine (NAG). The GNSB accounted for 9% of the 14C-amino acid-consuming bacteria and 27% of the [14C]NAG-consuming bacteria but did not utilize [14C]acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.

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