Detection of the Nitrogen Fixing Bacteria UCYN-A using nifH geneFISH

Many marine bacteria are uncultivated and known only from gene sequences. As a result, little is known even about the cell morphology from which the sequences originated. Catalyzed reporter deposition-fluorescence in-situ hybridization (CARD-FISH) can be used to visualize marine bacteria with a known 16S rRNA gene sequence. However, the 16S rRNA gene is unknown for many uncultivated marine bacteria, including many nitrogen-fixing bacteria which have only been identified by the sequence of the nitrogenase gene, nifH. In this study, we employed geneFISH in order to link cell identity with the presence of the nifH gene in individual cells. We specifically tested if nitrogen-fixing organisms can be identified microscopically in environmental samples by designing geneFISH probes for the nifH gene. As proof of concept, we targeted the nitrogen-fixing unicellular cyanobacteria group A (UCYN-A) for which a whole genome has been sequenced, both the nifH and 16S rRNA sequences are known, and the cells have been visualized by CARD-FISH. GeneFISH allowed us to clearly identify the cyanobacteria UCYN-A using the nifH gene probe. Additionally, geneFISH provided novel information about UCYN-A2 as it showed there were multiple copies of the nifH gene in the cyanobacterial cells. It is known that there is a single nifH gene copy per genome, therefore, the geneFISH results suggest the presence of multiple UCYN-A2 cells or a single cell that is polyploid. GeneFISH promises to be a useful tool for studying a nitrogen fixing bacteria with unknown 16S rRNA genes.