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GeneFISH – an in situ technique for linking gene presence and cell identity in environmental microorganisms
Author(s) -
Moraru Cristina,
Lam Phyllis,
Fuchs Bernhard M.,
Kuypers Marcel M. M.,
Amann Rudolf
Publication year - 2010
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/j.1462-2920.2010.02281.x
Subject(s) - biology , gene , in situ , fluorescence in situ hybridization , computational biology , ribosomal rna , reporter gene , seawater , genetics , microbiology and biotechnology , gene expression , ecology , physics , meteorology , chromosome
Summary Our knowledge concerning the metabolic potentials of as yet to be cultured microorganisms has increased tremendously with the advance of sequencing technologies and the consequent discoveries of novel genes. On the other hand, it is often difficult to reliably assign a particular gene to a phylogenetic clade, because these sequences are usually found on genomic fragments that carry no direct marker of cell identity, such as rRNA genes. Therefore, the aim of the present study was to develop geneFISH – a protocol for linking gene presence with cell identity in environmental samples, the signals of which can be visualized at a single cell level. This protocol combines rRNA‐targeted catalysed reporter deposition – fluorescence in situ hybridization and in situ gene detection. To test the protocol, it was applied to seawater samples from the Benguela upwelling system. For gene detection, a polynucleotide probe mix was used, which was designed based on crenarchaeotal amo A clone libraries prepared from each seawater sample. Each probe in the mix was selected to bind to targets with up to 5% mismatches. To determine the hybridization parameters, the T m of probes, targets and hybrids was estimated based on theoretical calculations and in vitro measurements. It was shown that at least 30%, but potentially the majority of the Crenarchaeota present in these samples harboured the amo A gene and were therefore likely to be catalysing the oxidation of ammonia.