Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization

Smolina, Irina; Lee, Charles; Frank‐Kamenetskii, Maxim D.

doi:10.1128/aem.02038-06

Cited by 49 publications

(37 citation statements)

References 39 publications

(35 reference statements)

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“…We have recently proposed and tested on model bacteria a new, more versatile, isothermal approach for bacterial detection in a convenient FISH format that is based on exceedingly specific and sensitive recognition of short signature sites within bacterial genomes [6, 7]. Here we report our progress in using this approach for detection of several important bacterial pathogens: methicillin-sensitive and methicillin-resistant strains of S. aureus (MSSA and MRSA, respectively) and a common, often multidrug-resistant pathogen P. aeruginosa.…”

Section: Introductionmentioning

confidence: 99%

PNA-based microbial pathogen identification and resistance marker detection

Smolina¹,

Miller²,

Frank‐Kamenetskii³

2010

Artificial DNA: PNA & XNA

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Section: Introductionmentioning

confidence: 99%

PNA-based microbial pathogen identification and resistance marker detection

Smolina¹,

Miller²,

Frank‐Kamenetskii³

2010

Artificial DNA: PNA & XNA

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“…Five day old cells of the isolates grown in MV medium were harvested and suspended in phosphate-buffered saline (PBS) containing formalin up to 2% concentration and stored under refrigeration till further use. Four different permeabilization techniques were tried: (1) cells were incubated overnight with Triton X100 in order to reduce hydrophobic interactions (Thimm & Tebbe 2003); (2) cells were subjected to repeated freeze and thaw in liquid nitrogen to facilitate disruption of cells through ice crystal formation (Biegala et al 2003); (3) cells were incubated for 5 min with dithiothreitol (DTT), to reduce disulphide bonds of membrane proteins (Moter & Göbel 2000); cells permeabilized after these 3 treatments were then smeared on glass slides coated with double-sided adhesive tape (La Cono & Urzì 2003) and also on Polyprep slides (Sigma) (Smolina et al 2007) and dehydrated in an ethanol series; (4) cells fixed with formalin were treated with 1 M DTT for 5 min, followed by washing with distilled water and final suspension in 1 M sorbitol. These cells, which were suspended in sorbitol, were transferred in a cuvette and given an electric pulse of 2500 V for~5 ms in a Gene Pulser Xcell TM (BioRad, S. No.…”

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confidence: 99%

Association of the stramenopilan protists, the aplanochytrids, with zooplankton of the equatorial Indian Ocean

Damare¹,

Raghukumar²

2010

Mar. Ecol. Prog. Ser.

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Thraustochytrids and aplanochytrids, belonging to the Labyrinthulomycetes of the Kingdom Stramenopila, have been frequently reported to occur as parasites or symbionts in a number of coastal invertebrates. Given the fact that these protists are widespread in coastal and oceanic waters, it is possible that they are also associated with pelagic zooplankton. This study examines their occurrence in zooplankton from equatorial waters of the Indian Ocean. A total of 171 of 2100 individual specimens of zooplankton yielded Labyrinthulomycetes in culture, the colony morphology and/or life cycles of which corresponded to aplanochytrids. Small-subunit ribosomal DNA (SSU rDNA) sequence analysis of 8 of the isolates placed them in a distinct clade among aplanochytrids, but closest to Aplanochytrium yorkensis or A. kerguelensis. The 8 isolates were further segregated into 2 clusters, corresponding to isolates obtained from 2 different seasons. Biotinylated probes for 2 isolates of A. kerguelensis from the 2 different seasons were developed based on internal transcribed spacer (ITS) sequences. In situ hybridization (ISH) of zooplankton using the probes with streptavidin-alkaline phosphatase showed that aplanochytrid cells fed to copepods were grazed and could subsequently be detected in the animals. ISH on natural samples of zooplankton yielded a positive but diffuse reaction in copepods, while cells resembling aplanochytrids were detected within chaetognaths. ISH using streptavidin-peroxidase conjugate lent further support for their presence within chaetognaths. This study suggests the predominant occurrence of A. kerguelensis in association with mesozooplankton of the equatorial Indian Ocean, particularly with chaetognaths. Further studies are suggested to determine whether genetically distinct populations of A. kerguelensis are associated with chaetognaths in oceanic waters and if such an association is parasitic, mutualistic or commensalistic.KEY WORDS: Stramenopila · Labyrinthulomycetes · Aplanochytrids · Zooplankton · Internal transcribed spacer · In situ hybridization · Biotinylated probes Resale or republication not permitted without written consent of the publisherMar Ecol Prog Ser 399: [53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68] 2010 require these fatty acids for their growth and reproduction but possess a limited or no ability to synthesize them (Kanazawa et al. 1979a,b, Lubzens et al. 1985, Nichols & Nichols 2008, Labyrinthulomycetes present in the water column might play an important role in the marine food web by serving as sources of these fatty acids for the zooplankton (Fell & Newell 1998, Kimura et al. 1999, Raghukumar 2002.Thraustochytrids and aplanochytrids are known to be associated with invertebrates in a variety of ways. observed thraustochytrids in the faecal pellets of salps. They were found to be endolithic in mollusk shell fragments, especially mussel shells and clam shells (Porter & Lingle 1992), and were isolated from oyster tissue (Perkins 1973) and surface mucus...

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“…Recently, there have been an increasing number of reports describing different approaches for successfully detecting cells containing specific functional genes in mixed microbial communities [17][18][19][20]. These studies are important examples of the remarkable improvement in sensitivity recently introduced to FISH approaches and show that it is now possible to detect single copy genes.…”

Section: Introductionmentioning

confidence: 97%

Identification of Nitrite-Reducing Bacteria Using Sequential mRNA Fluorescence In Situ Hybridization and Fluorescence-Assisted Cell Sorting

Mota

Reyes

2012

Microb Ecol

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Sequential mRNA fluorescence in situ hybridization (mRNA FISH) and fluorescence-assisted cell sorting (SmRFF) was used for the identification of nitrite-reducing bacteria in mixed microbial communities. An oligonucleotide probe labeled with horseradish peroxidase (HRP) was used to target mRNA of nirS, the gene that encodes nitrite reductase, the enzyme responsible for the dissimilatory reduction of nitrite to nitric oxide. Clones for nirS expression were constructed and used to provide proof of concept for the SmRFF method. In addition, cells from pure cultures of Pseudomonas stutzeri and denitrifying activated sludge were hybridized with the HRP probe, and tyramide signal amplification was performed, conferring a strongly fluorescent signal to cells containing nirS mRNA. Flow cytometry-assisted cell sorting was used to detect and physically separate two subgroups from a mixed microbial community: non-fluorescent cells and an enrichment of fluorescent, nitrite-reducing cells. Denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of 16S ribosomal RNA (rRNA) genes were used to compare the fragments amplified from the two sorted subgroups. Sequences from bands isolated from DGGE profiles suggested that the dominant, active nitrite reducers were closely related to Acidovorax BSB421. Furthermore, following mRNA FISH detection of nitrite-reducing bacteria, 16S rRNA FISH was used to detect ammonia-oxidizing and nitrite-oxidizing bacteria on the same activated sludge sample. We believe that the molecular approach described can be useful as a tool to help address the longstanding challenge of linking function to identity in natural and engineered habitats.

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Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization

Cited by 49 publications

References 39 publications

PNA-based microbial pathogen identification and resistance marker detection

PNA-based microbial pathogen identification and resistance marker detection

Association of the stramenopilan protists, the aplanochytrids, with zooplankton of the equatorial Indian Ocean

Identification of Nitrite-Reducing Bacteria Using Sequential mRNA Fluorescence In Situ Hybridization and Fluorescence-Assisted Cell Sorting

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