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. 2024 Apr 9;121(15):e2401632121.
doi: 10.1073/pnas.2401632121. Epub 2024 Apr 3.

A mutualistic bacterium rescues a green alga from an antagonist

David Carrasco Flores  1 , Vivien Hotter  1 , Trang Vuong  1 , Yu Hou  1 , Yuko Bando  2 , Kirstin Scherlach  3 , Bertille Burgunter-Delamare  1 , Ron Hermenau  3 , Anna J Komor  3 , Prasad Aiyar  1 , Magdalena Rose  1   4 , Severin Sasso  1   4 , Hans-Dieter Arndt  2   5 , Christian Hertweck  3   5   6 , Maria Mittag  1   5
Affiliations

A mutualistic bacterium rescues a green alga from an antagonist

David Carrasco Flores et al. Proc Natl Acad Sci U S A. .

Abstract

Photosynthetic protists, known as microalgae, are key contributors to primary production on Earth. Since early in evolution, they coexist with bacteria in nature, and their mode of interaction shapes ecosystems. We have recently shown that the bacterium Pseudomonas protegens acts algicidal on the microalga Chlamydomonas reinhardtii. It secretes a cyclic lipopeptide and a polyyne that deflagellate, blind, and lyse the algae [P. Aiyar et al., Nat. Commun. 8, 1756 (2017) and V. Hotter et al., Proc. Natl. Acad. Sci. U.S.A. 118, e2107695118 (2021)]. Here, we report about the bacterium Mycetocola lacteus, which establishes a mutualistic relationship with C. reinhardtii and acts as a helper. While M. lacteus enhances algal growth, it receives methionine as needed organic sulfur and the vitamins B1, B3, and B5 from the algae. In tripartite cultures with the alga and the antagonistic bacterium P. protegens, M. lacteus aids the algae in surviving the bacterial attack. By combining synthetic natural product chemistry with high-resolution mass spectrometry and an algal Ca2+ reporter line, we found that M. lacteus rescues the alga from the antagonistic bacterium by cleaving the ester bond of the cyclic lipopeptide involved. The resulting linearized seco acid does not trigger a cytosolic Ca2+ homeostasis imbalance that leads to algal deflagellation. Thus, the algae remain motile, can swim away from the antagonistic bacteria and survive the attack. All three involved genera cooccur in nature. Remarkably, related species of Pseudomonas and Mycetocola also act antagonistically against C. reinhardtii or as helper bacteria in tripartite cultures.

Keywords: Chlamydomonas reinhardtii; Mycetocola; Pseudomonas; cyclic lipopeptide; microbial interactions.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
While M. lacteus needs methionine and some B vitamins from C. reinhardtii, it enhances algal growth. (A) Axenic M. lacteus cannot grow in TAP medium with 0.2% (w/v) glucose, but cocultivation with C. reinhardtii enables bacterial growth. The two organisms were cocultivated at a ratio of 1:250 (algae:bacteria) and grown for 7 d in TAP medium supplemented with 0.2% (w/v) glucose. Bacterial cell densities were calculated by serially diluting and plating on LB medium daily. Colonies were counted after 2 d of growth, and cell densities were determined considering the dilution factor. Mla: M. lacteus. Asterisks indicate significant differences between mono- and cocultures (A and B) and between negative controls and supplemented cultures (C–E) as calculated by Student’s t test (*P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001). The error bars indicate SDs with n = 3 biological replicates per time point and culture. (B) M. lacteus significantly enhances the growth of C. reinhardtii in TAP medium with 0.2% (w/v) glucose. Algal cells were counted daily using a Thoma cell counting chamber. Cre: C. reinhardtii; see legend of (A) for further details. (C) M. lacteus needs cysteine or methionine for its growth. TAP medium with 0.2% (w/v) glucose and a mixture of the nine B-vitamins was used as medium. To investigate the bacterium’s need for amino acids, each M. lacteus culture was supplemented with either no amino acids (−aa), a mixture of the 20 amino acids (+aa), or one amino acid at a time. For details, see SI Appendix, Methods. The experiments represent the average of three independent biological replicates with the error bars denoting the SD. (D) M. lacteus needs certain B vitamins for its growth. TAP medium supplemented with 0.2% (w/v) glucose and methionine was used as medium. To test the bacterial B vitamin requirements, M. lacteus cultures were grown with either no B vitamins (−B-Vit), the mixture of the nine B vitamins (+B-Vit) or eight out of the nine B vitamins (one different vitamin always absent). See legend (C) for further details. (E) C. reinhardtii does not provide glucose for M. lacteus. TP medium without acetate and glucose was used as growth medium for a monoculture of M. lacteus and a coculture together with C. reinhardtii. D0, inoculation; D5, 5 d of growth. (F) C. reinhardtii spent medium provides methionine and thus can complement an E. coli auxotroph mutant to produce B12. Comparison of the growth of E. coli K12 DH5α wild type (WT) in M9 MM with an E. coli auxotroph mutant for the production of vitamin B12 in M9 MM supplemented with either nothing, vitamin B12, methionine (Met), or C. reinhardtii spent medium (supernatant, Sup); see SI Appendix, Methods for details.
Fig. 2.
Fig. 2.
The helper bacteria M. lacteus and M. tolaasinivorans, respectively, aid C. reinhardtii in recovering from the antagonistic bacteria P. protegens and P. tolaasii. (A): Algal and bacterial monocultures as well as bi- and tripartite cultures. The organisms were cocultivated in TAP medium containing 0.1 mM phosphate supplemented with 0.2% (w/v) glucose at a ratio of 1:250:250 (Cre:Ppr:Mla). Equally treated cultures of C. reinhardtii (Cre), P. protegens (Ppr), and M. lacteus (Mla) served as references for the growth of the axenic cultures. Pictures from days 10 until 19 that equal day 9 can be seen in SI Appendix, Fig. S6 along with further replicates. (B): Algal and bacterial cell densities in a bipartite culture of C. reinhardtii and P. protegens over 28 d. For cultivation, see legend of (A). Cells from 10 mL cell suspension were harvested on days 0 to 4 postinoculation and then every fourth day. Genomic DNA was extracted and used for qPCR to determine cell densities [see (A) and SI Appendix, Methods for details as well as SI Appendix, Fig. S7]. (C): Algal and bacterial cell densities in a tripartite culture of C. reinhardtii, P. protegens, and M. lacteus over the course of 28 d. See (B) for further details. (D) Algal and bacterial monocultures as well as bi- and tripartite cultures. The organisms were cocultivated as described in (A), but P. tolaasii (Pto) and M. tolaasinivorans (Mto) were used as bacteria. Two further independent biological replicates are shown in SI Appendix, Fig. S8.
Fig. 3.
Fig. 3.
M. lacteus produces enzymes cleaving orfamide A into a linear form at its C-terminal ester bond and in a double-cleaved form lacking the three C-terminal amino acids. (A) Orfamide A (35) and putative degradation products of orfamide A that were chemically synthesized. (B): LC-HRMS showing the degradation of intact orfamide A (m/z 1295.84 [M + H]+; compound 1) to either orfamide A with one cleavage (m/z 1313.85 [M + H]+; compound 2 or 3) and orfamide A with two cleavages missing the C-terminal residues Leu-Ser-Val (m/z 1014.66 [M + H]+, compound 4a) after 24 h incubation with the spent media (Mla supernatant) of the overnight culture of M. lacteus in LB or its cell lysate (see SI Appendix, Methods for details). (C): The Upper part shows LC-HRMS of chemically synthesized compounds, including intact orfamide A (m/z 1295.84 [M + H]+) and variants of the single cleaved degradation products (m/z 1313.85 [M + H]+) at the ester or amide bond as well as the double-cleaved degradation product missing the C-terminal residues Leu-Ser-Val (m/z 1014.66 [M + H]+). Below are cleaved orfamide A produced in bacterial coculture with M. lacteus (Mla) and P. protegens (Ppr) compared to a Ppr monoculture.
Fig. 4.
Fig. 4.
M. lacteus biologically inactivates orfamide A by cleavage. (A): The algal Ca2+ signature was investigated using a C. reinhardtii aequorin reporter line (see SI Appendix, Methods for details). Orfamide A (compound 1 from Fig. 3A) and its chemically synthesized degradation products (ester-cleaved compound 3 and double-cleaved compound 4a from Fig. 3A) were dissolved in DMSO and further diluted in TAP medium to a final concentration of 5 μM. As control, DMSO proportional to 5 μM of compound was used. Each line represents the mean of three independent biological replicates, and each biological replicate includes three technical replicates. (B) Deflagellation rates of wild-type algal cells treated with 5 μM intact orfamide A and its degradation products (see panel A for details). DMSO proportional to 5 μM of compounds was used as control. More than 100 cells were counted for each sample. Each column represents the mean of at least three independent biological replicates. Error bars represent SDs; Student’s t test was performed; n.s., not significant and ***P < 0.001.
Fig. 5.
Fig. 5.
The helper bacterium M. lacteus uses metabolites secreted by C. reinhardtii for its growth, enhances algal growth, and rescues the alga from the attack by P. protegens. The antagonistic bacterium P. protegens deflagellates, bleaches, and lyses the alga (orange arrow). Pictures are illustrated based on a 24 h bacterial attack of the algae (21). M. lacteus has a dual function: i) it promotes the growth of C. reinhardtii (yellow arrow), and it ii) rescues the alga from P. protegens (dashed orange arrow) by cleaving orfamide A (yellow arrow), and thus inactivating this CLiP (dashed orange arrow). Chlamydomonas releases specific B vitamins and methionine, which can be utilized as an organic sulfur source to support the growth of M. lacteus (green arrow).
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