ABSTRACT
We developed an improved method for cultivation-independent sorting of bacterial cells. The technique is based on labeling the target cells by in situ hybridization with polynucleotide transcript probes. Due to the probes' length, part of the probe remains outside the cell and can subsequently be used to capture the cells. Target cells are immobilized during a second hybridization step in microplates that are coated with DNA that is complementary to the probe sequence. The method was applied successfully to artificial mixtures of cells with polynucleotide probes targeting either rRNA, a plasmid-borne beta-lactamase gene, or a chromosome-borne glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Cells could be separated based on phylogenetic parameters (using rRNA-targeted probes) as well as on other DNA-encoded traits.
Subject(s)
DNA, Bacterial/analysis , Gram-Negative Bacteria/cytology , Oligonucleotide Probes , Polynucleotides , Bacteriological Techniques , Cells, Immobilized , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Gram-Negative Bacteria/enzymology , Gram-Negative Bacteria/genetics , In Situ Hybridization , Plasmids/genetics , RNA, Ribosomal/genetics , beta-Lactamases/geneticsABSTRACT
Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 10(4)-10(5) copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (10(1)-10(3) copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method--a halo-like, ring-shaped concentration of fluorescence in the cell periphery--we coined the term RING-FISH (recognition of individual genes) to differentiate it from conventional FISH.
Subject(s)
Escherichia coli/genetics , Genes, Bacterial/genetics , In Situ Hybridization, Fluorescence/methods , Chromosomes, Bacterial/genetics , Chromosomes, Bacterial/ultrastructure , DNA Primers , DNA, Bacterial/genetics , DNA, Bacterial/ultrastructure , Escherichia coli/classification , Image Processing, Computer-Assisted , Microscopy, Fluorescence , Oligonucleotide Probes , Phylogeny , Polymerase Chain ReactionABSTRACT
An improved subtraction hybridization technique was developed and evaluated. The hybridization is performed in a microplate with the subtractor-DNA immobilized in the plate while the probe-DNA is in solution. After hybridization the probe-specific DNA can easily be removed from the microwell and submitted to further analysis. This new technique has been successfully applied to generate several strain-specific PCR-primers for Lactococcus lactis subsp. lactis, Pediococcus spec., Saccharomyces spec. and Listeria monocytogenes.
