Genome-wide high-throughput screening of interactive bacterial metabolite in the algal population using Escherichia coli K-12 Keio collection

Algae-bacteria interaction is one of the main factors underlying the formation of harmful algal blooms (HABs). The aim of this study was to develop a genome-wide high-throughput screening method to identify HAB-influenced specific interactive bacterial metabolites using a comprehensive collection of gene-disrupted E. coli K-12 mutants (Keio collection). The screening revealed that a total of 80 gene knockout mutants in E. coli K-12 resulted in an approximately 1.5-fold increase in algal growth relative to that in wild-type E. coli. Five bacterial genes (lpxL, lpxM, kdsC, kdsD, gmhB) involved in the lipopolysaccharide (LPS) (or lipooligosaccharide, LOS) biosynthesis were identified from the screen. Relatively lower levels of LPS were detected in these bacteria compared to that in the wild-type. Moreover, the concentration-dependent decrease in microalgal growth after synthetic LPS supplementation indicated that LPS inhibits algal growth. LPS supplementation increased the 2,7-dichlorodihydrofluorescein diacetate fluorescence, as well as the levels of lipid peroxidation-mediated malondialdehyde formation, in a concentration-dependent manner, indicating that oxidative stress can result from LPS supplementation. Furthermore, supplementation with LPS also remarkably reduced the growth of diverse bloom-forming dinoflagellates and green algae. Our findings indicate that the Keio collection-based high-throughput in vitro screening is an effective approach for the identification of interactive bacterial metabolites and related genes.