Untargeted metabolomics analysis revealed changes in the composition of glycerolipids and phospholipids in Bacillus subtilis under 1-butanol stress.

1-Butanol has been utilized widely in industry and can be produced or transformed by microbes. However, current knowledge about the mechanisms of 1-butanol tolerance in bacteria remains quite limited. Here, we applied untargeted metabolomics to study Bacillus subtilis cells under 1-butanol stress and identified 55 and 37 ions with significantly increased and decreased levels, respectively. Using accurate mass determination, tandem mass spectra, and synthetic standards, 86 % of these ions were characterized. The levels of phosphatidylethanolamine, diglucosyldiacylglycerol, and phosphatidylserine were found to be upregulated upon 1-butanol treatment, whereas those of diacylglycerol and lysyl phosphatidylglycerol were downregulated. Most lipids contained 15:0/15:0, 16:0/15:0, and 17:0/15:0 acyl chains, and all were mapped to membrane lipid biosynthetic pathways. Subsequent two-stage quantitative real-time reverse transcriptase PCR analyses of genes in the two principal membrane lipid biosynthesis pathways revealed elevated levels of ywiE transcripts in the presence of 1-butanol and reduced expression levels of cdsA, pgsA, mprF, clsA, and yfnI transcripts. Thus, the gene transcript levels showed agreement with the metabolomics data. Lastly, the cell morphology was investigated by scanning electron microscopy, which indicated that cells became almost twofold longer after 1.4 % (v/v) 1-butanol stress for 12 h. Overall, the studies uncovered changes in the composition of glycerolipids and phospholipids in B. subtilis under 1-butanol stress, emphasizing the power of untargeted metabolomics in the discovery of new biological insights.

An alternative genome-integrated method for undomesticated Bacillus subtilis and related species.

Given their applicability in genetic engineering, undomesticated Bacillus strains are extensively used as non-natural hosts for chemical production due to their high tolerance of toxic substrates or products. However, they are difficult to genomically modify due to their low transformation efficiencies. In this study, the Bacillus-E. coli shuttle vector pHY300PLK, which is widely used in gram-positive bacteria, was adopted for genome integration in organic solvent-tolerant Bacillus isolates. The Bacillus-replicative vector was used to deliver homologous recombinant DNA and propagate itself inside the host cell, increasing the likelihood of genome integration of the recombinant DNA. Then, the unintegrated vectors were cured by cell cultivation in antibiotic-free medium with facilitation of nickel ions. The developed protocol was successfully demonstrated and validated by the disruption of amyE gene in B. subtilis 168. With an improved clonal selection protocol, the probability of clonal selection of the amyE::cat genome-integrated mutants was increased up to 42.0 ± 10.2%. Genome integration in undomesticated, organic solvent tolerant Bacillus strains was also successfully demonstrated with amyE as well as proB gene creating the gene-disrupted mutants with the corresponding phenotype and genotype. Not only was this technique effectively applied to several strains of undomesticated B. subtilis, but it was also successfully applied to B. cereus. This study validates the possibility of the application of Bacillus-replicative vector as well as the developed protocol in a variety of genome modification of undomesticated Bacillus species.

The significance of proline and glutamate on butanol chaotropic stress in Bacillus subtilis 168.

BACKGROUND: Butanol is an intensively used industrial solvent and an attractive alternative biofuel, but the bioproduction suffers from its high toxicity. Among the native butanol producers and heterologous butanol-producing hosts, Bacillus subtilis 168 exhibited relatively higher butanol tolerance. Nevertheless, organic solvent tolerance mechanisms in Bacilli and Gram-positive bacteria have relatively less information. Thus, this study aimed to elucidate butanol stress responses that may involve in unique tolerance of B. subtilis 168 to butanol and other alcohol biocommodities. RESULTS: Using comparative proteomics approach and molecular analysis of butanol-challenged B. subtilis 168, 108 butanol-responsive proteins were revealed, and classified into seven groups according to their biological functions. While parts of them may be similar to the proteins reportedly involved in solvent stress response in other Gram-positive bacteria, significant role of proline in the proline-glutamate-arginine metabolism was substantiated. Detection of intracellular proline and glutamate accumulation, as well as glutamate transient conversion during butanol exposure confirmed their necessity, especially proline, for cellular butanol tolerance. Disruption of the particular genes in proline biosynthesis pathways clarified the essential role of the anabolic ProB-ProA-ProI system over the osmoadaptive ProH-ProA-ProJ system for cellular protection in response to butanol exposure. Molecular modifications to increase gene dosage for proline biosynthesis as well as for glutamate acquisition enhanced butanol tolerance of B. subtilis 168 up to 1.8% (vol/vol) under the conditions tested. CONCLUSION: This work revealed the important role of proline as an effective compatible solute that is required to protect cells against butanol chaotropic effect and to maintain cellular functions in B. subtilis 168 during butanol exposure. Nevertheless, the accumulation of intracellular proline against butanol stress required a metabolic conversion of glutamate through the specific biosynthetic ProB-ProA-ProI route. Thus, exogenous addition of glutamate, but not proline, enhanced butanol tolerance. These findings serve as a practical knowledge to enhance B. subtilis 168 butanol tolerance, and demonstrate means to engineer the bacterial host to promote higher butanol/alcohol tolerance of B. subtilis 168 for the production of butanol and other alcohol biocommodities.