Synthesis of the compatible solute ectoine in Virgibacillus pantothenticus is triggered by high salinity and low growth temperature.
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ABSTRACT:
SUBMITTER: Kuhlmann AU
PROVIDER: S-EPMC2493186 | biostudies-literature | 2008 Jul
REPOSITORIES: biostudies-literature
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Synthesis of the compatible solute ectoine in Virgibacillus pantothenticus is triggered by high salinity and low growth temperature.
Kuhlmann Anne U AU Bursy Jan J Gimpel Silvy S Hoffmann Tamara T Bremer Erhard E
Applied and environmental microbiology 20080516 14
The quantification of the intracellular concentration of ectoine in Virgibacillus pantothenticus revealed that the production of this compatible solute is triggered either by an increase in the external salinity or by a reduction in the growth temperature. This finding reflects increased transcription of the ectoine biosynthetic operon (ectABC) under both environmental conditions. ...[more]
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Project description:In order to explore the effect of introducing the ectoine synthesis module in Synechococcus elongatus PCC 7942 on the global regulation of the strain under high-salt environment. The ectoine synthesis module was introduced at the Synpcc7942_0808 locus to obtain the Syn7942/Δsps-ect strain that lacked sucrose synthesis and could synthesize ectoine. Comparative transcriptomic analysis was performed on WT and Syn7942/Δsps-ect under 0 mM and 300 mM NaCl conditions.
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Project description:By using natural-abundance (13)C-nuclear magnetic resonance spectroscopy and high-performance liquid chromatography (HPLC) analysis we have investigated the types of compatible solutes that are synthesized de novo in a variety of Bacillus species under high-osmolality growth conditions. Five different patterns of compatible solute production were found among the 13 Bacillus species we studied. Bacillus subtilis, B. licheniformis, and B. megaterium produced proline; B. cereus, B. circulans, B. thuringiensis, Paenibacillus polymyxa, and Aneurinibacillus aneurinilyticus synthesized glutamate; B. alcalophilus, B. psychrophilus, and B. pasteurii synthesized ectoine; and Salibacillus (formerly Bacillus) salexigens produced both ectoine and hydroxyectoine, whereas Virgibacillus (formerly Bacillus) pantothenticus synthesized both ectoine and proline. Hence, the ability to produce the tetrahydropyrimidine ectoine under hyperosmotic growth conditions is widespread within the genus Bacillus and closely related taxa. To study ectoine biosynthesis within the group of Bacillus species in greater detail, we focused on B. pasteurii. We cloned and sequenced its ectoine biosynthetic genes (ectABC). The ectABC genes encode the diaminobutyric acid acetyltransferase (EctA), the diaminobutyric acid aminotransferase (EctB), and the ectoine synthase (EctC). Together these proteins constitute the ectoine biosynthetic pathway, and their heterologous expression in B. subtilis led to the production of ectoine. Northern blot analysis demonstrated that the ectABC genes are genetically organized as an operon whose expression is strongly enhanced when the osmolality of the growth medium is raised. Primer extension analysis allowed us to pinpoint the osmoregulated promoter of the B. pasteurii ectABC gene cluster. HPLC analysis of osmotically challenged B. pasteurii cells revealed that ectoine production within this bacterium is finely tuned and closely correlated with the osmolality of the growth medium. These observations together with the osmotic control of ectABC transcription suggest that the de novo synthesis of ectoine is an important facet in the cellular adaptation of B. pasteurii to high-osmolarity surroundings.
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Project description:Salt stress is one of the essential abiotic stresses for the survival of cyanobacteria. However, the realization of large-scale cultivation of cyanobacteria is inseparable from the utilization of abundant seawater resources. Therefore, research on the regulatory mechanism, as well as the improvement of salt tolerance of cyanobacteria is fundamental. Ectoine, a compatible solute which was found in halophilic microorganisms, has potentiality to confer salt tolerance. Here in this article, the salt tolerance of Synechococcus elongatus PCC 7942 (Syn7942) was significantly improved via expressing the ectoine biosynthetic pathway, reaching an increased final OD750 by 20% under 300 mM NaCl and 80% under 400 mM NaCl than that of wild-type (WT), respectively. Encouragingly, the engineered strain could even survive under 500 mM NaCl which was lethal to WT. In addition, by introducing the ectoine synthetic pathway into the sucrose-deficient strain, the salt tolerance of the obtained strain Syn7942/Δsps-ect was restored to the level of WT under 300 mM NaCl stress, demonstrating that ectoine could substitute for sucrose to combat against salt stress in Syn7942. In order to study the difference in the regulation of mechanism on the salt adaptation process after replacing sucrose with ectoine, transcriptomic analysis was performed for Syn7942/Δsps-ect and WT. The differentially expressed gene analysis successfully identified 19 up-regulated genes and 39 down-regulated genes in Syn7942/Δsps-ect compared with WT under salt stress condition. The results also showed that the global regulation of Syn7942/Δsps-ect and WT had certain differences in the process of salt adaptation, in which Syn7942/Δsps-ect reduced the demand for the intensity of sulfur metabolism in this process. This study provides a valuable reference for further salt tolerance engineering in cyanobacteria.
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Project description:Mutations in the CFTR gene lead to cystic fibrosis, a genetic disease associated with chronic infection and inflammation and ultimately respiratory failure. The most common CF-causing mutation is F508del and CFTR modulators (correctors and potentiators) are being developed to rescue its trafficking and activity defects. However, there are currently no modulators that stabilize the rescued membrane F508del-CFTR which is endocytosed and quickly degraded resulting in a shorter half-life than wild-type (WT). We previously reported that the extracellular signal-regulated kinase (ERK) MAPK pathway is involved in CFTR degradation upon cigarette smoke exposure. Interestingly, we found that ERK phosphorylation was increased in CF human bronchial epithelial (HBE) cells (CF-HBE41o- and primary CF-HBE) compared to non-CF controls, and this was likely due to signaling by the epidermal growth factor receptor (EGFR). EGFR can be activated by several ligands, and we provide evidence that amphiregulin (AREG) is important for activating this signaling axis in CF. The natural osmolyte ectoine stabilizes membrane macromolecules. We show that ectoine decreases ERK phosphorylation, increases the half-life of rescued CFTR, and increases CFTR-mediated chloride transport in combination with the CFTR corrector VX-661. Additionally, ectoine reduces production of AREG and interleukin-8 by CF primary bronchial epithelial cells. In conclusion, EGFR-ERK signaling negatively regulates CFTR and is hyperactive in CF, and targeting this axis with ectoine may prove beneficial for CF patients.
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Project description:Improved salt tolerance of Synechococcus elongatus PCC 7942 by heterologous synthesis of compatible solute ectoine
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Project description:To maintain the turgor pressure of the cell under high osmolarity, bacteria accumulate small organic compounds called compatible solutes, either through uptake or biosynthesis. Vibrio parahaemolyticus, a marine halophile and an important human and shellfish pathogen, has to adapt to abiotic stresses such as changing salinity. Vibrio parahaemolyticus contains multiple compatible solute biosynthesis and transporter systems, including the ectABC-asp_ect operon required for de novo ectoine biosynthesis. Ectoine biosynthesis genes are present in many halotolerant bacteria; however, little is known about the mechanism of regulation. We investigated the role of the quorum sensing master regulators OpaR and AphA in ect gene regulation. In an opaR deletion mutant, transcriptional reporter assays demonstrated that ect expression was induced. In an electrophoretic mobility shift assay, we showed that purified OpaR bound to the ect regulatory region indicating direct regulation by OpaR. In an aphA deletion mutant, expression of the ect genes was repressed, and purified AphA bound upstream of the ect genes. These data indicate that AphA is a direct positive regulator. CosR, a Mar-type regulator known to repress ect expression in V. cholerae, was found to repress ect expression in V. parahaemolyticus In addition, we identified a feed-forward loop in which OpaR is a direct activator of cosR, while AphA is an indirect activator of cosR Regulation of the ectoine biosynthesis pathway via this feed-forward loop allows for precise control of ectoine biosynthesis genes throughout the growth cycle to maximize fitness.IMPORTANCE Accumulation of compatible solutes within the cell allows bacteria to maintain intracellular turgor pressure and prevent water efflux. De novo ectoine production is widespread among bacteria, and the ect operon encoding the biosynthetic enzymes is induced by increased salinity. Here, we demonstrate that the quorum sensing regulators AphA and OpaR integrate with the osmotic stress response pathway to control transcription of ectoine biosynthesis genes in V. parahaemolyticus We uncovered a feed-forward loop wherein quorum sensing regulators also control transcription of cosR, which encodes a negative regulator of the ect operon. Moreover, our data suggest that this mechanism may be widespread in Vibrio species.
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