title: GeneFISH--an in situ technique for linking gene presence and cell identity in environmental microorganisms.
abstract: 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 amoA 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 amoA gene and were therefore likely to be catalysing the oxidation of ammonia.
authors.affiliation: Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany.
authors.forename: Cristina
authors.initials: C
authors.lastname: Moraru
authors.forename: Phyllis
authors.initials: P
authors.lastname: Lam
authors.forename: Bernhard M
authors.initials: BM
authors.lastname: Fuchs
authors.forename: Marcel M M
authors.initials: MM
authors.lastname: Kuypers
authors.forename: Rudolf
authors.initials: R
authors.lastname: Amann
mesh_terms: D000353:Africa, Southern; D059001:Aquatic Organisms / Q000235:genetics / Q000302:isolation & purification / Q000382:microbiology; D001267:Atlantic Ocean; D001483:Base Sequence; D019615:Crenarchaeota / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D019766:Genes, Archaeal; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D015345:Oligonucleotide Probes; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011119:Polynucleotides; D012336:RNA, Ribosomal, 16S; D012623:Seawater / Q000382:microbiology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D015345:Oligonucleotide Probes; D011119:Polynucleotides; D012336:RNA, Ribosomal, 16S; D010088:Oxidoreductases; C045582:ammonia monooxygenase