Metabolomic characterization of the salt stress response in Streptomyces coelicolor

Abstract

The humicolous actinomycete Streptomyces coelicolor routinely adapts to a wide variety of habitats and rapidly changing environments. Upon salt stress, the organism is also known to increase the levels of various compatible solutes. Here we report the results of the first high-resolution metabolomics time series analysis of various strains of S. coelicolor exposed to salt stress: the wild type, mutants with progressive knockouts of the ectoine biosynthesis pathway, and two stress regulator mutants (with disruptions of the sigB and osaB genes). Samples were taken from cultures at 0, 4, 8, and 24 h after salt stress treatment and analyzed by liquid chromatography-mass spectrometry with an LTQ Orbitrap XL mass spectrometer. The results suggest that a large fraction of amino acids is upregulated in response to the salt stress, as are proline/glycine-containing di- and tripeptides. Additionally we found that 5'-methylthioadenosine, a known inhibitor of polyamine biosynthesis, is downregulated upon salt stress. Strikingly, no major differences between the wild-type cultures and the two stress regulator mutants were found, indicating a considerable robustness of the metabolomic response to salt stress, compared to the more volatile changes in transcript abundance reported earlier.

FIG. 1.

Biosynthesis pathway for ectoine. l-Aspartate-β-semialdehyde (ASA) is converted into l-2,4-diaminobutyrate (DABA) by the enzyme DABA aminotransferase, encoded by the ectB gene. DABA is converted into N-γ-acetyl-l-2,4-diaminobutyrate (ADABA) by DABA acetyltransferase, encoded by the ectA gene, and l-ectoine (Ect) is formed from ADABA by ectoine synthetase, encoded by the ectC gene (8, 33, 35). Ectoine can then be hydroxylated to form 5-hydroxyectoine (EctOH) by the enzyme ectoine hydroxylase, encoded by the ectD gene (7, 17). CoA, coenzyme A.

FIG. 2.

Ectoine biosynthesis disruption mutants display a salt-sensitive phenotype, which is complemented by the extracellular addition of ectoines. S. coelicolor spore stocks of the M145, ectA::Tn, ectC::Tn, ectD::Tn, ΔsigB, and osaB::Tn strains were diluted so that a spot of 2.5 μl contained approximately 10⁵, 10⁴, 10³, 10², 10¹, or 10⁰ spores. Spots were plated on SMMS without any addition (A, left) or with the addition of 1 M NaCl (A, right), 1 M NaCl and 20 μM ectoine (B, left), 1 M NaCl and 20 μM hydroxyectoine (B, right), or 1 M NaCl, 20 μM ectoine, and 20 μM hydroxyectoine (C). Plates were incubated at 30°C for 3 days in the absence of salt and for 6 days in the presence of salt.

FIG. 3.

Levels of ectoine and related compounds detected under continuous salt stress reflect the ectoine mutants. The presence of DABA, ADABA, ectoine, and hydroxyectoine was determined in wild-type cells and in mutants lacking ectA, ectC, or ectD grown under continuous salt stress. DABA was detected only in the etcA::Tn strain, which is expected to be deficient in the conversion of DABA into ADABA and therefore accumulates the precursor. ADABA, another intermediate compound, accumulates in the ectC::Tn strain and to a lesser degree in the ectD::Tn strain, as expected. The etcD::Tn strain, which is deficient in the conversion of ectoine to hydroxyectoine, accumulated large amounts of ectoine, while hydroxyectoine accumulated in the M145 strain, as expected.

FIG. 4.

Overview of the putatively identified metabolites detected in the global metabolome screen. A total of 229 metabolites could be assigned putative identities based on database matching. By far the largest class of detected metabolites were lipids (107 metabolites) and in particular a large number of short- and medium-chain fatty acids. The second-largest class contained amino acids (AA) and their derivatives (66 metabolites), including a large number of di- and tripeptides, many of which contained proline and glycine residues.

FIG. 5.

Clustering analysis uncovers the salt stress response. Clustering analysis has been used to group metabolites showing similar temporal behaviors into clusters. For each of the 4 clusters, the mean behaviors are shown, with error bars depicting the standard deviations. Cluster 1 contains compounds that accumulate after the salt shock, including the well-known osmoprotectant proline. Cluster 4 contains compounds that decrease after the salt shock. Interestingly, these clusters are the most homogenous of all clusters, as indicated by the quality score, showing that the salt-responsive metabolites react as a single group.

FIG. 6.

Comparison of time trends for putatively identified salt stress-responsive metabolites. The level of the major osmoprotectant proline, as well as those of several other putatively identified amino acids, shows an increase in response to salt shock. The detected proline/glycine-containing di- and tripeptides also accumulate in response to salt shock (ArgGlyPro is shown as a representative example), as do a few nucleotide-related compounds, including adenosine. 5′-Methylthioadenosine is one of the few compounds reproducibly showing the opposite pattern, an accumulation under low-salt conditions only. Levels of histidine and its catabolite urocanate decrease in all of the cultures, showing no response to the salt shock. For an overview of all 20 reproducible metabolites, see Fig. SE in the supplemental material. Results are shown for samples taken at 0, 4, 8, and 24 h after salt shock.