Transcriptional analysis of the effect of exogenous decanoic acid stress on Streptomyces roseosporus
© Liao et al.; licensee BioMed Central Ltd. 2013
Received: 26 December 2012
Accepted: 19 February 2013
Published: 21 February 2013
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.
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.
These findings represent an important first step in understanding mechanism of decanoic acid toxicity and provide a basis for engineering microbial tolerance.
KeywordsDaptomycin Decanoic acid Toxic Tolerance Streptomyces roseosporus
The mechanism of toxicity of free fatty acids (FFA) varies with the length, branching and saturation status of the carbon backbone . The degree of toxicity of a fatty acid also varies across bacteria, with some bacteria being more affected by the length of the carbon backbone while others are more affected by saturation. Their antibacterial mode of action is poorly understood, but most toxicity studies have proposed the cell membrane as the most affected target of fatty acids. In yeast, it has been proposed that DA inserts itself into the lipid bilayer of membrane and physically disturbs the membrane, resulting in increased fluidity of the membrane, leading to conformational changes in membrane proteins, the release of intracellular components . It has been observed that increase of membrane fluidity induced by free fatty acid is accompanied by an increase of ROS production . It can also be hypothesized that the same mechanism may be true for DA.
To elucidate the cytotoxicity mechanism of DA, we combine phospholipid fatty acid analysis, ROS measurement and RNA sequencing technologies to characterize the physiological response to DA and found that resistance to DA likely involves a functional shift of cell membrane composition, increase the gene expression involved in oxidative stress response and oxidative phospholytion. Our findings represent an important advance to understand the mechanism of DA and also provide a list of potential gene targets for further engineering DA tolerance in S. roseosporus.
Results and discussion
The effect of decanoic acid on the growth of S. roseosporus
The concentration of DA that caused stress but not significant cell death was found to be 1 mM and was used in the other growth assay and gene expression analysis.
Effect of decanoic acid on S. roseosporus phospholipid fatty acid composition
Effect of decanoic on the generation of ROS in S. roseosporus
Effect of decanoic acid on S. roseosporus transcriptome
To elucidate molecular mechanisms underlying tolerance, global gene expression changes during SR growth with DA were analyzed using Illumina RNA deep sequencing (RNA-seq) technology. Tanscriptome libraries were constructed using SR cells grown in the absence (control) or the presence of DA (1 mM).
RNA-seq data revealed a small subset of genes with differential transcription; 134 genes were up-regulated and 12 genes were down-regulated. The presence of DA at 1 mM resulted in transcriptional reprogramming of genes in three major discernible categories, including: energy production and conversion, posttranslational modification and protein turnover, and carbohydrate metabolism (Additional file 1).
Changes in energy metabolism
Selected genes involved in energy production and conversion with significant change in expression in DA-stress relative to control
F0F1 ATP synthase subunit alpha
H(+)-transporting ATP synthase
cytochrome b subunit
succinate dehydrogenase iron-sulfur subunit
In addition, the increase in the transcripts in genes encoding the member of the ATP synthase complex (SSGG_04986 and SSGG_04898) was observed. ATP synthase is responsible for generation of ATP through oxidative phosphorylation . It uses energy stored in the pH and potential gradients, created by pumping of protons across the membrance by enzymes of the respiratory chian, to synthesize ATP. Similar results were observed in yeast, where exposure of cells to octanoic acid (C8) led to the activation of plasma membrane H + −ATPase . These results suggested that an increased requirement for energy was required to deal with the DA stress.
Induction of oxidative stress response
Selected genes involved in oxidative stress response with significant change in expression in DA-stress relative to control
phage shock protein A
bifunctional thioredoxin reductase/thioredoxin
In addition, expression levels of genes coding for components of proteasome (pafA, pup, pfafA2, mpa) were enhanced. Bacterial proteasomes could only be found in actinomycetes . Mpa assembles into a hexameric ATPase and Pup, a prokaryotic ubiquitin-like protein, is ligated by PafA to substrate proteins. Subsequently, proteins tagged with Pup were targeted for degradation by the proteasome . Recently, it was shown that proteasome is important for defense against reactive nitrogen intermediates (RNI) in Mycobacterium tuberculosis. Increased expression of proteasome and clpB may degrade the misfolded proteins impaired by ROS.
In streptomyces, the expression of genes coding for proteins involved in antioxidative defense systems was under the control of several key regulators, such as OxyR . However, the homologue of OxyR, master regulator of oxidative response, was absent in S. roseosporus. FurS (SSGG_00190) is a zinc-containing redox regulator of S. reticuli which binds to an operator upstream of the furS-cepB. Under oxidative stress conditions, an internal S-S bridge formed FurS abrogated its capability to block the transcription of furS-cpeB. SSGG_00191 shared 80% amino acid identity to cpeB of S. reticuli and was induced during DA stress. Further studies are required to determine the roles of FurS in oxidative response.
Changes in carbohydrate transport and metabolism
Selected genes related to carbohydrate metabolism and transport with significant change in expression in DA-stress relative to control
putative maltose ABC transporter permease
Genes encoding proteins involved in the TCA cycle were nearly unchanged. Interestingly, expression of genes involved in pyruvate production was up-regulated. Genes coding for fructose-bisphosphate aldolase (SSGG_03685), glyceraldehyde-3-phosphate dehydrogenase (SSGG_06343), phosphopyruvate hydratase (SSGG_02477) and pyruvate kinase (SSGG_01114) were upregulated after exposure to DA stress. Pyruvate is a key intermediate involved in a number of metabolic pathways. It was recently reported that pyruvate is involved into octanoic acid (C8) stress of E. coli. Addition of pyruvate into media helps the cell partially recover from stress, but the exact mechanism was unclear. Similarly, enhanced production of pyruvate may help S. roseosporus to recover from DA stress.
Preliminary model for the mechanism of DA toxicity and cell response
Multiple mechanisms are involved in the mitigation of the toxicity of DA. The relative contribution of particular mechanism to the toxicity of fatty acid remains elusive. Taken together, our study provided insights into the toxicity or tolerance mechanism underlying DA exposure and several candidates that may be targeted for further engineering to mitigate the toxicity of DA.
For toxicity assays, Streptomyces roseosporus (NRRL11579) was grown in TSB medium. To determine the effect of DA on S. roseosporus growth, wide range of concentrations was tested first (data not shown) and then narrowed to a range that caused stress but not significant cell death. Growth assays to test the effect of different concentrations of DA on S. roseosporus were performed in 250 ml shake flasks with 25 ml of TSB medium with a 2% inoculation culturing at 200 rpm at 28°C. Unless specified otherwise, all subsequent DA assays were conducted at 1 mM DA.
Phospholipid fatty acid analysis
Cells were harvested in control culture and DA-stressed S. rosoeporus cultures (2 h after exposure to 1 mM DA) of growth by centrifugation at 3000 × g and 4 for 15 min, and the pellet was washed three times with distilled water. The fatty acids in the cells (40–50 mg in wet weight) were saponified and methylated. The methyl ester mixtures were separated using an Agilent 5890 dual tower gas chromatograph. Fatty acids were identified by the MIDI microbial identification system (Sherlock 4.5 microbial identification system) . Minor fatty acids (<0.6% of the total) are not reported.
Reactive oxygen species assay
Control and DA-stressed S. rosoeporus cultures were grown in TSB medium as described with various concentrations of DA. Positive controls for oxidative stressed cells were prepared by adding 10 ul of 7.78 M tert-butyl hydroperoxide (TBHP) (Invitrogen, USA) to one set of control cells before incubation. Ten microliters of 25 mM carboxy-H2DCFDA was added to all cells. Florescence at 535 nm was measured after 30 min.
S. roseosprous was cultured in TSB to exponential phase (48 h). DA was added to a final concentration of 1 mM, and biomass was collected after treatment of 30 min. the cultures were centrifuged at 3000 × g and 4 for 15 min, and cell pellets were immediately frozen in liquid nitrogen and stored at −80 for subsequent RNA isolation. Total RNA was extracted using Trizol (Invitrogen), following manufacturer’s protocols. RNA preparations were treated with RNase-free DNase (Promega) and the integrity of the RNA was determined using Bioanalyzer 2100 (Agilent Technologies). mRNA was enriched by removing the rRNAs using MICROBExpress kit. The mRNA remaining in the supernatant was recovered by ethanol precipitation and quantified by Bioanalyzer 2100. A cDNA library was constructed and sequenced by Illumina Hiseq™ 2000 .
Data processing and analysis
Raw sequencing reads were mapped against the S. roseosprous genome. Reads that mapped to more than one region of the genome (5 to 8% of the total) failed to be unambiguously mapped were excluded for subsequent analyses. Analyses of differential expression including FDR calculations were performed using DESeq [26, 27]. Only P values of <0.01, FDR ≤ 0.01 were considered to be significant.
This work was supported by grants from National Natural Science Foundation of China (Grant No. 31100069), the Natural Science Foundation Project of CQ CSTC (cstc2012jjA10149) and the Fundamental Research Funds for the Central Universities (XDJK2013A010), New Century Excellent Talents in Universities (NCET-11-0703).
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