Fluorescence in situ hybridization of genes in environmental microbiology

Our knowledge concerning the metabolic potentials of as yet uncultured microorganisms has increased tremendously with the advance of sequencing technologies and the consequent discovery 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 main objective of the present study was to develop geneFISH - a protocol for linking gene presence with cell identity in environmental samples. This protocol combines rRNA-targeted Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) and in situ gene detection. The method of rRNA-targeted CARD-FISH was previously developed (Pernthaler et al., 2002a). The gene detection method was adapted from Pernthaler and Amann (2004). It uses multiple digoxigenin labeled polynucleotide probes to target genes, followed by the binding of HRP-conjugated antibodies and catalyzed reporter deposition (CARD), to amplify and visualize the gene signal. However, the specificity of polynucleotide probes has not been thoroughly investigated and a rational probe design concept is still missing, because the well established concept for oligonucleotide probe design cannot be transferred to polynucleotides. Therefore, we developed a concept and software (PolyPro) for rational design of polynucleotide probe mixes used to identify particular genes in defined taxa. PolyPro consists of three modules: a GenBank Taxonomy Extractor (GTE), a Polynucleotide Probe Designer (PPD) and a Hybridization Parameters Calculator (HPC). Applying this probe design concept to three metabolic marker genes revealed the following about the use of polynucleotide probes in FISH: (i) a single probe is not sufficient to detect all alleles of a gene; (ii) single probes can be used mostly at the genus level; (iii) probe mixes cannot be used to detect all alleles of a gene, because of differences in the melting temperature; (iv) probe mixes can be used for identifying a gene mostly at the genus and family level. The newly developed concept for polynucleotide probe design was further applied to the probe design for the geneFISH experiments. The geneFISH protocol was first developed and tested in Escherichia coli. In a second phase, it was applied on seawater samples from Benguela upwelling system on the Namibian shelf, in which the presence of putative amoA gene was directly visualized in crenarchaeotal cells. This involved a specially designed polynucleotide probe mix (amoA-Nam) that targets the crenarchaeotal putative amoA alleles present in these environmental samples. Additionally, geneFISH was applied on two more systems, an enrichment sample, targeting rdsrA genes, and an eukaryotic host - bacterial symbiont system, targeting hynL and aprA genes in the symbionts. Further development of this method will in the direction of improving the gene detection efficiency, from less than 50% to 100%. This will allow a quantitative use of the geneFISH protocol.