The bacterium Pseudomonas protegens antagonizes the microalga Chlamydomonas reinhardtii using a blend of toxins

Abstract

The unicellular alga Chlamydomonas reinhardtii and the bacterium Pseudomonas protegens serve as a model to study the interactions between photosynthetic and heterotrophic microorganisms. P. protegens secretes the cyclic lipopeptide orfamide A that interferes with cytosolic Ca 2+ homeostasis in C. reinhardtii resulting in deflagellation of the algal cells. Here, we studied the roles of additional secondary metabolites secreted by P. protegens using individual compounds and co-cultivation of algae with bacterial mutants. Rhizoxin S2, pyrrolnitrin, pyoluteorin, 2,4-diacetylphloroglucinol (DAPG) and orfamide A all induce changes in cell morphology and inhibit the growth of C. reinhardtii. Rhizoxin S2 exerts the strongest growth inhibition, and its action depends on the spatial structure of the environment (agar versus liquid culture). Algal motility is unaffected by rhizoxin S2 and is most potently inhibited by orfamide A (IC 50 = 4.1 μM). Pyrrolnitrin and pyoluteorin both interfere with algal cytosolic Ca 2+ homeostasis and motility whereas high concentrations of DAPG immobilize C. reinhardtii without deflagellation or disturbance of Ca 2+ homeostasis. Co-cultivation with a regulatory mutant of bacterial secondary metabolism (ΔgacA) promotes algal growth under spatially structured conditions. Our results reveal how a single soil bacterium uses an arsenal of secreted antialgal compounds with complementary and partially overlapping activities.

Algicidal Bacteria: A Review of Current Knowledge and Applications to Control Harmful Algal Blooms

“…This is exemplified by the genomic analysis of algicidal bacterium Brevibacillus laterosporus Bl-zj, which included a number of genes putatively involved in algicidal activity, including virulence factors, proteases and antibiotic resistance genes ( Zhang et al, 2021a ). While a full description of algicidal compounds that have been identified is beyond the scope of this review, it is worthy to note the growing realization that a number of algicidal chemicals produced by bacteria act additively or synergistically ( Harvey et al, 2016 ; Wang and Seyedsayamdost, 2017 ; Zhuang et al, 2018 ; Ternon et al, 2019 ; Jeong and Son, 2021 ; Rose et al, 2021 ). These groups of chemicals may act together to overwhelm one physiological target, and/or affect multiple systems simultaneously.…”

Total Synthesis and Structure Correction of the Cyclic Lipodepsipeptide Orfamide A

“…To compare the biological response of synthetic CLPs 2 and 3 to isolated orfamide A in C. reinhardtii, we used the transgenic AEQ34 cell line that expresses apo-aequorin for measuring cytosolic Ca 2 + levels (for details see Supporting Information). [9,10] CLP 3 showed identical characteristics to the natural product, with a rapidly occurring initial Ca 2 + release signal and a secondary Ca 2 + -release that persisted over several minutes. These data additionally confirmed that compound 3 has the correct structure of orfamide A. CLP 2, which differs from CLP 3 only in the configuration of two residues, showed a distinct Ca 2 + signature, where the initial signal was strongly reduced and the secondary release was absent (Figure 5).…”

“…To compare the biological response of synthetic CLPs 2 and 3 to isolated orfamide A in C. reinhardtii , we used the transgenic AEQ34 cell line that expresses apo‐aequorin for measuring cytosolic Ca 2+ levels (for details see Supporting Information) [9,10] . CLP 3 showed identical characteristics to the natural product, with a rapidly occurring initial Ca 2+ release signal and a secondary Ca 2+ ‐release that persisted over several minutes.…”

“…[9] Intriguingly, orfamide A triggers an increase in cytosolic Ca 2 + in the green alga Chlamydomonas reinhardtii, causing its deflagellation, and thus leads to its immobilization (IC 50 = 4.1 μM). [9,10] Orfamide A immobilizes several flagellate algae from the class of Chlorophyceae but not select algae from other classes. [9] A total synthesis of orfamide A has not been reported to date, except for a patent claim.…”

“…Orfamide A from Pseudomonas protegens Pf‐5 or CHA0 [4,5] has been characterized as an insecticidal, [6,7] an antifungal, [8] and as an algicidal agent [9] . Intriguingly, orfamide A triggers an increase in cytosolic Ca 2+ in the green alga Chlamydomonas reinhardtii , causing its deflagellation, and thus leads to its immobilization ( IC 50 =4.1 μM) [9,10] . Orfamide A immobilizes several flagellate algae from the class of Chlorophyceae but not select algae from other classes [9] .…”