Transcriptional analysis of the effect of exogenous decanoic acid stress on Streptomyces roseosporus

Abstract

Background: Daptomycin is an important antibiotic against infections caused by drug-resistant pathogens. Its production critically depends on the addition of decanoic acid during fermentation. Unfortunately, decanoic acid (>2.5 mM) is toxic to daptomycin producer, Streptomyces roseosporus.

Results: To understand the mechanism underlying decanoic tolerance or toxicity, the responses of S. roseosporus was determined by a combination of phospholipid fatty acid analysis, reactive oxygen species (ROS) measurement and RNA sequencing. Assays using fluorescent dyes indicated a sharp increase in reactive oxygen species during decanoic acid stress; fatty acid analysis revealed a marked increase in the composition of branched-chain fatty acids by approximately 10%, with a corresponding decrease in straight-chain fatty acids; functional analysis indicated decanoic acid stress has components common to other stress response, including perturbation of respiratory functions (nuo and cyd operons), oxidative stress, and heat shock. Interestingly, our transcriptomic analysis revealed that genes coding for components of proteasome and related to treholase synthesis were up-regulated in the decanoic acid -treated cells.

Conclusion: These findings represent an important first step in understanding mechanism of decanoic acid toxicity and provide a basis for engineering microbial tolerance.

Figure 1

The chemical structure of daptomycin and other A21978C factors.

Figure 2

Cell growth profiles with the addition of 1 mM of decanoic acid at 48 h. Error bars represent the standard deviation of three biological replicates.

Figure 3

Phospho-lipid fatty acid profiles of S. roseosporus after two hours of decanoic acid-stress. Control and stressed cell cultures were grown in triplicate. Stressed samples were exposed to1 mM decanoic acid for two hours before being harvested. Cell cultures (50 ml each) were harvested, centrifuged, washed in PBS.

Figure 4

Measurement of intracellular reactive oxygen species using carboxy-H₂DCFDA. Stressed samples(1, 2) were exposed to 1 mM decanoic acid for 30 min. control cells treated with tertbutyl hydroperoxide (TBHP), known to produce intracellular H₂O₂ and serve as a positive control (PC); Cells without treatment serve as a negative control (NC). Measurements were carried out in triplicate.

Figure 5

Illustration about hypothesis about DA toxicity mechanism and the dynamic response of S. roseosporus. DA inserted into the cell membrane disrupted the membrane integrity and induced the generation of ROS by unknown mechanisms. Cells activated expression of a number of genes coding for antioxidative systems to degrade ROS (Superoxide dismutase and catalase), to repair or degrade the disfolded proteins (heat shock proteins, Clp and proteasome). In addition, the yield of stress protectants, such as trehalose may increase to help the cell to mitigate the toxicity of DA.