Biotic countermeasures that rescue Nannochloropsis gaditana from a Bacillus safensis infection.

The natural assemblage of a symbiotic bacterial microbiome (bacteriome) with microalgae in marine ecosystems is now being investigated as a means to increase algal productivity for industry. When algae are grown in open pond settings, biological contamination causes an estimated 30% loss of the algal crop. Therefore, new crop protection strategies that do not disrupt the native algal bacteriome are needed to produce reliable, high-yield algal biomass. Bacteriophages offer an unexplored solution to treat bacterial pathogenicity in algal cultures because they can eliminate a single species without affecting the bacteriome. To address this, we identified a highly virulent pathogen of the microalga Nannochloropsis gaditana, the bacterium Bacillus safensis, and demonstrated rescue of the microalgae from the pathogen using phage. 16S rRNA amplicon sequencing showed that phage treatment did not alter the composition of the bacteriome. It is widely suspected that the algal bacteriome could play a protective role against bacterial pathogens. To test this, we compared the susceptibility of a bacteriome-attenuated N. gaditana culture challenged with B. safensis to a N. gaditana culture carrying a growth-promoting bacteriome. We showed that the loss of the bacteriome increased the susceptibility of N. gaditana to the pathogen. Transplanting the microalgal bacteriome to the bacteriome-attenuated culture reconstituted the protective effect of the bacteriome. Finally, the success of phage treatment was dependent on the presence of beneficial bacteriome. This study introduces two synergistic countermeasures against bacterial pathogenicity in algal cultures and a tractable model for studying interactions between microalgae, phages, pathogens, and the algae microbiome.

High-Density Guide RNA Tiling and Machine Learning for Designing CRISPR Interference in Synechococcus sp. PCC 7002.

While CRISPRi was previously established in Synechococcus sp. PCC 7002 (hereafter 7002), the design principles for guide RNA (gRNA) effectiveness remain largely unknown. Here, 76 strains of 7002 were constructed with gRNAs targeting three reporter systems to evaluate features that impact gRNA efficiency. Correlation analysis of the data revealed that important features of gRNA design include the position relative to the start codon, GC content, protospacer adjacent motif (PAM) site, minimum free energy, and targeted DNA strand. Unexpectedly, some gRNAs targeting upstream of the promoter region showed small but significant increases in reporter expression, and gRNAs targeting the terminator region showed greater repression than gRNAs targeting the 3' end of the coding sequence. Machine learning algorithms enabled prediction of gRNA effectiveness, with Random Forest having the best performance across all training sets. This study demonstrates that high-density gRNA data and machine learning can improve gRNA design for tuning gene expression in 7002.