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 diand 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.Salt stress conditions are known to cause major changes in the primary and secondary metabolism of bacterial cells (26,34). This is also the case for the Gram-positive soil bacteria of the genus Streptomyces, which are well known for their complex secondary metabolism and production of a wide range of industrially relevant metabolites, including various antibiotics (21). Upon salt stress, these species are known to increase the levels of various compatible solutes, including ectoine, alanine, glutamine, and proline (6,11,16,28). The genome sequence of Streptomyces indicates a largely unexplored capacity for production of secondary metabolites (2), some of which could also be involved in the salt stress response. Here we conducted an untargeted metabolomics analysis of salt-stressed Streptomyces coelicolor, the genome-sequenced model species of the genus (2), to investigate the complexity of the metabolite changes associated with the salt stress response in more detail and to increase our understanding of the mechanisms of osmoadaptation by metabolomic rearrangement. For this purpose, we used a high-resolution LTQ Orbitrap mass spectrometer coupled with hydrophobic-interaction liquid chromatography (HILIC) (23). The accurate mass determination and high resolving power of the Orbitrap (22, 40) have made the analysis of complex metabolite mixtures feasible, making the Orbitrap an attractive option for untargeted metabolomics screens (5). The use of HILIC ensures that the resolving power of the mass spectrometer is further extended by fractionation of the sample, resulting in a less complex mixture being injected in the mass spectrometer at each moment in time. In addition to wild-type S. coelicolor, we studied strains with disruptions in various ste...

A Comparative Metabolomics Analysis ofSaccharopolyspora spinosaWT, WH124, and LU104 Revealed Metabolic Mechanisms Correlated with Increases in Spinosad Yield

“…For example, the concentration of intracellular ectonie in Streptomyces coelicolor significantly increased when it was cultured in a highly osmotic environment. 37,38) Intracellular ectonie was also detected in our study. Notably, the concentration of intracellular ectonie increased during the fermentation process (Fig.…”

Genome Sequence of an Ammonia-Oxidizing Soil Archaeon, “Candidatus Nitrosoarchaeum koreensis” MY1

“…Also, instead of genes coding for the ectoine biosynthesis pathway to tolerate salt stress (7,14), MY1 keeps a number of genes to cope with oxidative stress and antibiotic resistance genes (17).…”

GlnR-Mediated Regulation of ectABCD Transcription Expands the Role of the GlnR Regulon to Osmotic Stress Management

“…In S. coelicolor, the bacterial tolerance to high osmolality is influenced by the biosynthesis of the intracellular ectoine and hydroxyectoine (26), and blocking of the ectoine biosynthesis, i.e., mutation in ectA, ectB, or ectC, leads to remarkably increased bacterial sensitivity to high salinity (27). Therefore, the intracellular production of ectoine and hydroxyectoine was measured both in the strain containing glnR and in the glnR null mutant strain (Fig.…”

“…High salinity and high temperature stimulate the biosynthesis of these compatible solutes in S. coelicolor, helping cells adapt to the extracellular stresses (26,27). However, unlike the regulation characterized in methylobacteria (18,21,22), no EctR1 homologue is found around the ect genes in S. coelicolor, and therefore the mechanism of regulation of ect expression remains uncharacterized.…”