A genetic transformation system for the heterotrophic diatom Nitzschia putrida (Bacillariophyceae).

Diatoms are important primary producers in aquatic ecosystems. Most of them are photoautotrophs and have evolved to thrive under diverse environmental conditions from the poles to the tropics. However, some diatom species such as Nitzschia putrida have lost photosynthesis and have therefore become free-living secondary heterotrophs. Thus, these diatoms provide unique opportunities to study the evolutionary processes required to thrive without photosynthesis and independent of a resource-providing host. They may also provide a chassis for reverse engineering photosynthesis in eukaryotic organisms. Here, we have developed a genetic transformation system for N. putrida using a biolistic approach. By leveraging genome and transcriptome data, we identified the nicotinamide adenine dinucleotide hydride (NADH)-ubiquinone reductase complex 1 promoter as a robust candidate for driving transgene expression. Through Golden Gate Cloning, we engineered plasmids, including the selectable marker nourseothricin and the reporter eGFP. An evaluation of transformation efficiency confirmed the successful integration and expression of the transgenes. Fluorescence microscopy demonstrated the expression of eGFP in the transformed cell lines, which retained a growth phenotype similar to that of the wild type cells. Thus, our work in combination with the available genome and transcriptome of N. putrida enables reverse genetics with a free-living secondary heterotroph.