Transcriptome revealed the molecular mechanism of Glycyrrhiza inflata root to maintain growth and development, absorb and distribute ions under salt stress

Background Soil salinization extensively hampers the growth, yield, and quality of crops worldwide. The most effective strategies to counter this problem are a) development of crop cultivars with high salt tolerance and b) the plantation of salt-tolerant crops. Glycyrrhiza inflata, a traditional Chinese medicinal and primitive plant with salt tolerance and economic value, is among the most promising crops for improving saline-alkali wasteland. However, the underlying molecular mechanisms for the adaptive response of G. inflata to salinity stress remain largely unknown. Result G. inflata retained a high concentration of Na+ in roots and maintained the absorption of K+, Ca2+, and Mg2+ under 150 mM NaCl induced salt stress. Transcriptomic analysis of G. inflata roots at different time points of salt stress (0 min, 30 min, and 24 h) was performed, which resulted in 70.77 Gb of clean data. Compared with the control, we detected 2645 and 574 differentially expressed genes (DEGs) at 30 min and 24 h post-salt-stress induction, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that G. inflata response to salt stress post 30 min and 24 h was remarkably distinct. Genes that were differentially expressed at 30 min post-salt stress induction were enriched in signal transduction, secondary metabolite synthesis, and ion transport. However, genes that were differentially expressed at 24 h post-salt-stress induction were enriched in phenylpropane biosynthesis and metabolism, fatty acid metabolism, glycerol metabolism, hormone signal transduction, wax, cutin, and cork biosynthesis. Besides, a total of 334 transcription factors (TFs) were altered in response to 30 min and 24 h of salt stress. Most of these TFs belonged to the MYB, WRKY, AP2-EREBP, C2H2, bHLH, bZIP, and NAC families. Conclusion For the first time, this study elucidated the salt tolerance in G. inflata at the molecular level, including the activation of signaling pathways and genes that regulate the absorption and distribution of ions and root growth in G. inflata under salt stress conditions. These findings enhanced our understanding of the G. inflata salt tolerance and provided a theoretical basis for cultivating salt-tolerant crop varieties.

Klebsiella Pneumoniae Metabolic Regulation and its Application in 1,3-Propanediol Production

Klebsiella pneumoniae is a well-known model organism for glycerol metabolism to produce 1,3-propanediol (1,3-PD), a valuable chemical intermediate for materials, such as polyesters. However, the relatively low conversion rate and productivity, as well as the accumulation of by-products such as lactic acid, ethanol and acetic acid, inhibit the production of 1,3-PD. Hereby, the 1,3-PD metabolism in K. pneumoniae was regulated through pathway engineering by using CRISPR-Cas9 technology for the first time to knock out the ldhA gene of lactate dehydrogenase,the adhE gene of alcohol dehydrogenase and the ack gene of acetate kinase respectively as needed and constructed recombinant bacteria ldhA(−), ldhA(−)-ack(−), ldhA(−)-adhE(−) and ldhA(−)-adhE(−)-ack(−), all of which showed a decrease in by-product production, leading to a higher NADH availability, and 1,3-BD production was significantly increased. In the shake flask fermentation, the 1,3-PD yield and conversion rate of the recombinant strain ldhA(−), ldhA(−)-ack(−), ldhA(−)-adhE(−), ldhA(−)-adhE(−)-ack(−) were higher than those of the parent strain. In the fed-batch fermentation, the 1,3-PD yield and conversion rate of the recombinant strain ldhA(−) were higher than those of the parent strain. The biomass of the recombinant strain ldhA(−)-adhE(−)-ack(−) was reduced due to the accumulation of acetic acid, but its 1,3-PD conversion rate was still higher than that of the parent strain. The higher productivity and fewer by-products concluded that the four Klebsiella pneumoniae recombinant strains could be promising industrial strain for economical production of 1,3-PD.

Shotgun proteomic profiling of dormant, 'non-culturable' Mycobacterium tuberculosis

Dormant cells of Mycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by 'non-culturability' – a state of the inability of the cells to grow on solid media. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, 'non-culturable' M. tuberculosis cells in a model of self-acidification of mycobacteria in the post-stationary phase, simulating the in vivo persistence conditions. This approach revealed the accumulation of a significant number of proteins after 4 months of storage in dormancy; among them, 468 proteins were significantly different from those in the actively growing cells and bore a positive fold change. Differential analysis revealed the proteins of the pH-dependent regulatory system phoP and allowed the reconstruction of the reactions of central carbon/glycerol metabolism, as well as revealing the salvaged pathways of mycothiol and UMP biosynthesis, establishing the cohort of survival enzymes of dormancy. The annotated pathways mirror the adaptation of the mycobacterial metabolic machinery to life within lipid-rich macrophages, especially the involvement of the methyl citrate and glyoxylate pathways. Thus, the current model of M. tuberculosis reflects the biochemical adaptation of these bacteria to persistence in vivo. Comparative analysis with published proteins with antigenic properties makes it possible to distinguish immunoreactive proteins among the proteins bearing a positive FC, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored in the dormant state were annotated.

Genetic and biochemical characterization of the Na + /H + antiporters of Pseudomonas aeruginosa

Pseudomonas aeruginosa has four Na + /H + antiporters that interconvert and balance Na + and H + gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we have constructed four strains, each of which has only one antiporter: NhaB, NhaP, NhaP2, and Mrp. We also constructed a quadruple deletion mutant that has no Na + /H + antiporters. Although the antiporters of P. aeruginosa have previously been studied, the strains constructed here present the opportunity to characterize their kinetic properties in their native membranes and their roles in the physiology of P. aeruginosa . The strains expressing only NhaB or Mrp, the two electrogenic antiporters, are able to grow essentially as the wild type across a range of [Na + ] and pH. Strains with only NhaP or NhaP2, which are electroneutral, grow more poorly at increasing [Na + ], especially at high pH, with NhaP the most sensitive. The strain with no Na + /H + antiporters is extremely sensitive to [Na + ] and shows essentially no Na + (Li + )/H + antiporter activity but retains most K + /H + antiporter activity of the wild type at pH 7.5 and approximately half at pH 8.5. We also used the four strains that each express one of the four antiporters to characterize the kinetic properties of each transporter. RNA-seq analysis of the quadruple deletion strain showed widespread changes, including pyocyanin synthesis, biofilm formation, and nitrate and glycerol metabolism. Thus, the strains constructed for this study will open a new door to understanding the physiological role of these proteins and their activities in P. aeruginosa . Importance Pseudomonas aeruginosa has four Na + /H + antiporters that connect and interconvert its Na + and H + gradients. We have constructed four deletion mutants, each of which has only one of the four Na + /H + antiporters. These strains made it possible to study the properties and physiological roles of each antiporter independently in its native membrane. Mrp and NhaB are each able to sustain growth over a wide range of pH and [Na + ], whereas the two electroneutral antiporters, NhaP and NhaP2, are most effective at low pH. We also constructed a quadruple mutant, lacking all four antiporters in which the H + and Na + gradients are disconnected. This will make it possible to study the role of the two gradients independently.

Survival mechanism of a novel marine multistress-tolerant Meyerozyma guilliermondii GXDK6 under high NaCl stress as revealed by integrative omics analysis

A novel strain named Meyerozyma guilliermondii GXDK6 was provided in this work, which was confirmed to survive independently under high salt stress (12% NaCl) or co-stress condition of strong acid (pH 3.0) and high salts (10% NaCl) without sterilization. Its survival mechanism under high salt stress was revealed by integrated omics for the first time. Whole-genome analysis showed that 14 genes (e.g., GPD1 and FPS1) of GXDK6 relevant to salt tolerance were annotated and known to belong to various salt-resistant mechanisms (e.g., regulation of cell signal transduction and glycerol metabolism controls). Transcriptome sequencing results indicated that 1220 genes (accounting for 10.15%) of GXDK6 were differentially transcribed (p < 0.05) when GXDK6 growth was under 10% stress for 16 h, including important novel salt-tolerant-related genes (e.g., RTM1 and YHB1). Proteomics analysis demonstrated that 1005 proteins (accounting for 27.26%) of GXDK6 were differentially expressed (p < 0.05) when GXDK6 was stressed by 10% NaCl. Some of the differentially expressed proteins were defined as the novel salt-tolerant related proteins (e.g., sugar transporter STL1 and NADPH-dependent methylglyoxal reductase). Metabolomic analysis results showed that 63 types of metabolites (e.g., D-mannose, glycerol and inositol phosphate) of GXDK6 were up- or downregulated when stressed by 10% NaCl. Among them, D-mannose is one of the important metabolites that could enhance the salt-tolerance survival of GXDK6.

Elucidating the Antimycobacterial Mechanism of Action of Decoquinate Derivative RMB041 Using Metabolomics

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), still remains one of the leading causes of death from a single infectious agent worldwide. The high prevalence of this disease is mostly ascribed to the rapid development of drug resistance to the current anti-TB drugs, exacerbated by lack of patient adherence due to drug toxicity. The aforementioned highlights the urgent need for new anti-TB compounds with different antimycobacterial mechanisms of action to those currently being used. An N-alkyl quinolone; decoquinate derivative RMB041, has recently shown promising antimicrobial activity against Mtb, while also exhibiting low cytotoxicity and excellent pharmacokinetic characteristics. Its exact mechanism of action, however, is still unknown. Considering this, we used GCxGC-TOFMS and well described metabolomic approaches to analyze and compare the metabolic alterations of Mtb treated with decoquinate derivative RMB041 by comparison to non-treated Mtb controls. The most significantly altered pathways in Mtb treated with this drug include fatty acid metabolism, amino acid metabolism, glycerol metabolism, and the urea cycle. These changes support previous findings suggesting this drug acts primarily on the cell wall and secondarily on the DNA metabolism of Mtb. Additionally, we identified metabolic changes suggesting inhibition of protein synthesis and a state of dormancy.

Elucidating the Antimycobacterial Mechanism of Action of Ciprofloxacin Using Metabolomics

In the interest of developing more effective and safer anti-tuberculosis drugs, we used a GCxGC-TOF-MS metabolomics research approach to investigate and compare the metabolic profiles of Mtb in the presence and absence of ciprofloxacin. The metabolites that best describe the differences between the compared groups were identified as markers characterizing the changes induced by ciprofloxacin. Malic acid was ranked as the most significantly altered metabolite marker induced by ciprofloxacin, indicative of an inhibition of the tricarboxylic acid (TCA) and glyoxylate cycle of Mtb. The altered fatty acid, myo-inositol, and triacylglycerol metabolism seen in this group supports previous observations of ciprofloxacin action on the Mtb cell wall. Furthermore, the altered pentose phosphate intermediates, glycerol metabolism markers, glucose accumulation, as well as the reduction in the glucogenic amino acids specifically, indicate a flux toward DNA (as well as cell wall) repair, also supporting previous findings of DNA damage caused by ciprofloxacin. This study further provides insights useful for designing network whole-system strategies for the identification of possible modes of action of various drugs and possibly adaptations by Mtb resulting in resistance.

Glycerol promoted the anaerobic production of rhamnolipids by different Pseudomonas aeruginosa strains

Background: Rhamnolipids is the most widely studied and applied biosurfactants. The anaerobic biosynthesis of rhamnolipids has important research and practical significance, such as meeting the in situ production of biosurfactant in anoxic environments and the foamless fermentation of biosurfactants. A few studies have reported the anaerobic biosynthesis of rhamnolipids from rare Pseudomonas aeruginosa strains. What did promote the anaerobic biosynthesis of rhamnolipids, the specificity of the rare strains or the effect of specific substrates? Here, anaerobic production of rhamnolipids by different P. aeruginosa strains was investigated using diverse substrates. The anaerobic biosynthesis mechanism of rhamnolipids were also discussed from the substrate point of view.Results: All P. aeruginosa strains anaerobically grew well using the tested substrates. But all P. aeruginosa strains anaerobically produced rhamnolipids only using substrates containing glycerol and nitrate. Fourier transform infrared (FTIR) spectra analysis confirmed the anaerobic production of rhamnolipids from all P. aeruginosa strains. All the anaerobically produced rhamnolipids decreased air-water surface tension from 72.6 mN/m to below 29.0 mN/m and emulsified crude oil with EI24 above 65%. Using crude glycerol as low-cost substrate, all P. aeruginosa strains can anaerobically grow and produce rhamnolipids to reduce the culture surface tension below 35 mN/m. The glycerol metabolic intermediate, 1, 2-propylene glycol, can also achieve the anaerobic production of rhamnolipids by all P. aeruginosa strains.Conclusions: Not the specificity of the rare P. aeruginosa strains but the effect of specific substrates promote the anaerobic biosynthesis of rhamnolipids by P. aeruginosa. Glycerol and nitrate are the excellent substrates for anaerobic production of rhamnolipids from all P. aeruginosa strains. Results indicated that glycerol metabolism involveed the anaerobic biosynthesis of rhamnolipids in P. aeruginosa. Results also showed the feasibility of using crude glycerol as low cost substrate to anaerobically biosynthesize rhamnolipids by P. aeruginosa.