Trimethylated homoserine functions as the major compatible solute in the globally significant oceanic cyanobacterium Trichodesmium

Abstract

The oceanic N₂-fixing cyanobacterium Trichodesmium spp. form extensive surface blooms and contribute significantly to marine carbon and nitrogen cycles in the oligotrophic subtropical and tropical oceans. Trichodesmium grows in salinities from 27 to 43 parts per thousand (ppt), yet its salt acclimation strategy remains enigmatic because the genome of Trichodesmium erythraeum strain IMS101 lacks all genes for the biosynthesis of any known compatible solute. Using NMR and liquid chromatography coupled to mass spectroscopy, we identified the main compatible solute in T. erythraeum strain IMS101 as the quaternary ammonium compound N,N,N-trimethyl homoserine (or homoserine betaine) and elucidated its biosynthetic pathway. The identification of this compatible solute explains how Trichodesmium spp. can thrive in the marine system at varying salinities and provides further insight into the diversity of microbial salt acclimation.

Keywords: compatible solute; cyanobacteria; salt stress.

Fig. 1.

Salt-dependent growth and accumulation of compatible solute in Trichodesmium spp. (A) Growth curve based on cell concentration of four Trichodesmium IMS101 cultures, each grown in artificial seawater medium supplemented with 30, 35, 43, or 48 ppt NaCl over the course of 9 d. (B) Intracellular homoserine betaine concentration in Trichodesmium IMS101, which was grown in artificial seawater medium supplemented with 30, 35, or 43 ppt NaCl as determined by HPLC-MS.

Fig. 2.

Homoserine betaine detection by HPLC-MS in vivo, in vitro, and in situ. (A and B) HPLC-MS analysis of the cell extract of Trichodesmium IMS101 grown in YCBII medium in the presence of 35 ppt NaCl. (C–J) HPLC-MS analysis of cell extracts from Trichodesmium spp. collected from the Gulf of Eilat/Aqaba and the Red Sea (C and D), of the standard compound carnitine (E and F), of synthesized homoserine betaine (G and H), and the reaction products of the enzyme assay using heterologously expressed Tery_2447 (I and J).

Fig. 3.

Structure elucidation of homoserine betaine by NMR. ¹H-NMR spectra (500 MHz, 300 K) recorded in D₂O (calibrated externally against acetone 5% (vol/vol) in D₂O; 2.25 ppm): cell extract of Trichodesmium IMS101 grown in YCBII medium (A) and synthesized (L)-homoserine betaine (B). Approximately 5 mg homoserine betaine were dissolved per ml D₂O to obtain spectrum B. The final formula of homoserine betaine is shown in the upper right corner.

Fig. S1.

¹³C-NMR spectra (125.8 MHz, 300 K) recorded in D₂O (calibrated externally against acetone 5% in D₂O; 30.5 ppm): cell extract of Trichodesmium IMS101 grown in YCBII medium (A) and synthesized (L)-homoserine betaine (B). Both spectra show five distinct signals at defined chemical shifts (δ 29.5, 52, 58, 76.2, and 171.8), which were assignable to different C-atoms present in the different functional groups of homoserine betaine. The distribution of carbon chemical shifts associated with different functional groups, which were differentially positioned within the molecule, is indicated by the Greek letters. These analyses give information about the structure of our molecule homoserine betaine, which is shown in the middle of the figure.

Fig. S2.

The 2D NMR showing ¹H,¹³C-HSQC spectrum (500 MHz, D₂O) for a cell extract of Trichodesmium IMS101 grown in YCBII medium. This analysis correlates chemical shifts of directly bound nuclei. The resulting spectrum is 2D, showing the proton (¹H) spectrum at one axis and the ¹³C spectrum on the other axis. The spectrum contains a peak for each unique proton attached to the heteronucleus being considered. The final formula of homoserine betaine is shown in the lower right corner.

Fig. 4.

Verification of the compatible nature of homoserine betaine. Growth rates of E. coli wild-type MC4100 and mutant FF4169 in minimal medium containing 400 mM NaCl. Growth rates were obtained in control medium and in medium supplemented with compatible solutes, 1 mM concentration of either glycine betaine or homoserine betaine.

Fig. S3.

HPLC-MS–based detection of homoserine betaine in extracts of the E. coli wild-type or mutant FF4169 cells. HPLC-MS analysis of the cell extract of E. coli (wild type and mutant) grown in M63 minimal medium in the presence of 400 mM NaCl. (A–D) HPLC-MS analysis of cell extracts from E. coli wild type in the presence of 1 mM glycine betaine (A and B) or in the presence of homoserine betaine (C and D). (E–H) HPLC-MS analysis of cell extracts from E. coli mutant in presence of 1 mM glycine betaine (E and F) or in the presence of homoserine betaine (G and H).

Fig. S4.

Salt-dependent expression of the gene tery_2447 in T. erythraeum IMS101. (A) Total RNA for cDNA synthesis was isolated from cells grown at different salt concentrations. The relative expression (rnpB level was used as internal loading control) of tery_2447 was estimated by qPCR. The expression at the lowest tolerable salinity (30 ppt or 513 mM NaCl) was set to 1. Data are the means ± SD of duplicate cultures. Statistically significant differences to 30 ppt are marked by asterisks. ***P < 0.001. (B) Data from the transcriptome analysis of Trichodesmium IMS101 (26) cultivated for 9 d at different salinities (30 and 43 ppt). The number of reads mapping to the methyltransferase-coding gene tery_2447 (WP_011612023) are shown that were associated with the respective transcriptional start site (average of duplicate cultures).

Fig. S5.

Purity of recombinant Tery_2447. SDS/PAGE of heterologous expressed, purified and desalted protein Tery_2447. M, unstained protein molecular weight marker. The lane was loaded with 10 µg of the protein and the SDS/PAGE gel was stained with Coomassie Blue.

Fig. S6.

Biosynthetic pathway of homoserine betaine. The methyltransferase Tery_2447 of Trichodesmium is capable to transfer subsequently all three methyl groups to the precursor l-homoserine (HOMOSER) in an in vitro enzyme assay.