Indole Acetic Acid: A Key Metabolite That Protects Marine Sulfitobacter mediterraneus Against Oxidative Stress

Abstract

For marine bacteria, the phycosphere is attractive as a major source of labile nutrients, but it also presents challenges due to the accumulation of stressors, such as reactive oxygen species (ROS) from algal metabolisms. Therefore, successful colonization of bacteria in the phycosphere requires an efficient mechanism to fight against oxidative stress, which is still a missing piece in studying bacteria-algae interactions. Here, we demonstrate that a common metabolite, indole acetic acid (IAA), enables the Roseobacter clade Sulfitobacter mediterraneus SC1-11, an IAA-producer, to resist hydrogen peroxide (H₂O₂) stress and that IAA biosynthesis can be activated by low concentrations of H₂O₂. Proteomics and metabolomics analyses revealed that bacteria consume high amino acid levels when exposed to H₂O₂ stress, while exogenous supplementation with IAA effectively protects bacteria from ROS damage and alleviates amino acid starvation by upregulating several proteins responsible for replication, recombination, and repair, as well as two proteins involved in amino acid transport and metabolism. Furthermore, the supplementation of some amino acids, such as arginine, also showed a significant protective effect on bacteria under H₂O₂ stress. This study highlights an unprecedented role of IAA in regulating amino acid metabolisms for resisting oxidative stress, which may be a specific strategy for adapting to the phycosphere.

Keywords: Sulfitobacter mediterraneus; amino acid metabolism; bacterial–algal interaction; indole acetic acid; marine microbiome; oxidative stress.

Figure 1

The responses of IAA biosynthesis to oxidative stress in S. mediterraneus SC1-11. ROS levels (A) and IAA production (B) of SC1-11 after exposure to varying concentrations of H₂O₂. Values are means ± SD, and lowercase letters indicate significant differences between treatments (by one-way analysis of variance (ANOVA)). (C) Overview of predicted IAA biosynthetic pathways in SC1-11. Enzymes indicated in red have been annotated in SC1-11, and other enzymes indicated in black or gray were reported or proposed in bacteria. Dashed lines indicate biochemical activities for which microbial enzymes have not been identified in bacteria. Abbreviations for metabolites and enzymes: Trp, tryptophan; IAOx, indole-3-acetaldoxime; IAN, indole-3-acetonitrile; IAM, indole-3-acetamide; IPyA, indole-3-pyruvate; IAAld, indole-3-acetaldehyde; TAM, tryptamine; AAT, amino acid aminotransferases; TMO, tryptophan 2-monoxygenase; IAH, indole-3-acetamide hydrolase; IPDC, indole-3-pyruvate dehydrogenase; ALD, indole-3-acetaldehyde dehydrogenase; TSO, tryptophan side chain oxidase. (D) RT-qPCR analysis of responses of IAA biosynthetic genes in SC1-11 under oxidative stress. Values are means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired t-test.

Figure 2

The protective role of IAA supplementation in S. mediterraneus SC1-11 against H₂O₂. (A) The growth of SC1-11 with the addition of IAA when exposed to 2 mM H₂O₂. (B) ROS and MDA levels of SC1-11 with incubation in varying concentrations of IAA and H₂O₂. Values are means ± SD, and lowercase letters indicate significant differences between treatments (ANOVA).

Figure 3

Proteomic responses of S. mediterraneus SC1-11 to 10 mM H₂O₂ before and after adding 0.57 μM IAA. (A) Differentially expressed proteins (DEPs) were categorized based on the Clusters of Orthologous Groups (COG) database. (B,C) Heatmaps of a subset of DEPs involved in amino acid transport and metabolism. (D) The DEPs enriched in amino acid transport and metabolism, lipid transport and metabolism, as well as replication, recombination and repair, are shown.

Figure 4

Changes in amino acid levels under oxidative stress without/with addition of varying concentrations of IAA. (A) Heatmap of normalized amino acid levels across different treatments. Abbreviations for amino acids: Ala, alanine; Asp, aspartic acid; Glu, glutamic acid; Phe, phenylalanine; Gly, glycine; His, histidine; Leu/Ile, leucine or isoleucine; Lys, lysine; Met, methionine; Asn, asparagine; Pro, proline; Gln, glutamine; Arg, arginine; Ser, serine; Thr, threonine; Val, valine; Trp, tryptophan; Tyr, tyrosine. (B) Heatmap of correlation between amino acids. Colors indicate the Pearson’s correlation value and significant correlations are marked with asterisks. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

The protective role of amino acid supplementation in S. mediterraneus SC1-11 against H₂O₂. (A) The survival of SC1-11 with the addition of a mixture of 20 amino acids (AAs) after incubation in 10 mM H₂O₂. (B) Heatmap of bacterial growth incubated with individual amino acids when exposed to 10 mM H₂O₂. Colors indicate growth strength of SC1-11 (white indicates no growth). Abbreviations for amino acids are as described above; Cys indicates cysteine. (C,E) Growth curves of SC1-11 with the addition of arginine (C) or both arginine and IAA (E). (D) The survival of SC1-11 with the addition of arginine and IAA after incubation in 10 mM H₂O₂.