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A new IS4 family insertion sequence, IS4Bsu1, responsible for genetic instability of poly-gamma-glutamic acid production in Bacillus subtilis.

ABSTRACT: Certain Bacillus subtilis strains, such as B. subtilis (natto) starter strains for the manufacture of natto (fermented soybeans), produce capsular poly-gamma-glutamate (gammaPGA). In B. subtilis (natto), gammaPGA synthesis is controlled by the ComP-ComA two-component regulatory system and thereby induced at the beginning of the stationary growth phase. We have found a new insertion sequence (IS), designated IS4Bsu1, in the comP gene of a spontaneous gammaPGA-negative mutant of B. subtilis (natto) NAF4. IS4Bsu1 (1,406 bp), the first IS discovered in B. subtilis, encodes a putative transposase (Tpase) with a predicted M(r) of 34,895 (374 residues) which displays similarity to the Tpases of IS4 family members. Southern blot analyses have identified 6 to 11 copies of IS4Bsu1, among which 6 copies were at the same loci, in the chromosomes of B. subtilis (natto) strains, including NAF4, three commercial starters, and another three gammaPGA-producing B. subtilis (natto) strains. All of the eight spontaneous gammaPGA(-) mutants, which were derived from five independent NAF4 cultures, had a new additional IS4Bsu1 copy in comP at six different positions within 600 bp of the 5'-terminal region. The target sites of IS4Bsu1 were determined to be AT-rich 9-bp sequences by sequencing the flanking regions of IS4Bsu1 in mutant comP genes. These results indicate that IS4Bsu1 transposes by the replicative mechanism, in contrast to other IS4 members that use the conservative mechanism, and that most, if not all, of spontaneous gammaPGA(-) mutants appear to have resulted from the insertion of IS4Bsu1 exclusively into comP. The presence of insertion hot spots in comP, which is essential for gammaPGA synthesis, as well as high transposition activity, would account for the high frequency of spontaneous gammaPGA(-) mutation by IS4Bsu1 in B. subtilis (natto).

SUBMITTER: Nagai T

PROVIDER: S-EPMC111298 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

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A new IS4 family insertion sequence, IS4Bsu1, responsible for genetic instability of poly-gamma-glutamic acid production in Bacillus subtilis.

Nagai T T Tran L S LS Inatsu Y Y Itoh Y Y

Journal of bacteriology 20000501 9

Certain Bacillus subtilis strains, such as B. subtilis (natto) starter strains for the manufacture of natto (fermented soybeans), produce capsular poly-gamma-glutamate (gammaPGA). In B. subtilis (natto), gammaPGA synthesis is controlled by the ComP-ComA two-component regulatory system and thereby induced at the beginning of the stationary growth phase. We have found a new insertion sequence (IS), designated IS4Bsu1, in the comP gene of a spontaneous gammaPGA-negative mutant of B. subtilis (natto ...[more]

PMID: 10762236

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Project description:BackgroundGenome-scale metabolic models (GEMs) allow predicting metabolic phenotypes from limited data on uptake and secretion fluxes by defining the space of all the feasible solutions and excluding physio-chemically and biologically unfeasible behaviors. The integration of additional biological information in genome-scale models, e.g., transcriptomic or proteomic profiles, has the potential to improve phenotype prediction accuracy. This is particularly important for metabolic engineering applications where more accurate model predictions can translate to more reliable model-based strain design.ResultsHere we present a GEM with Enzymatic Constraints using Kinetic and Omics data (GECKO) model of Bacillus subtilis, which uses publicly available proteomic data and enzyme kinetic parameters for central carbon (CC) metabolic reactions to constrain the flux solution space. This model allows more accurate prediction of the flux distribution and growth rate of wild-type and single-gene/operon deletion strains compared to a standard genome-scale metabolic model. The flux prediction error decreased by 43% and 36% for wild-type and mutants respectively. The model additionally increased the number of correctly predicted essential genes in CC pathways by 2.5-fold and significantly decreased flux variability in more than 80% of the reactions with variable flux. Finally, the model was used to find new gene deletion targets to optimize the flux toward the biosynthesis of poly-γ-glutamic acid (γ-PGA) polymer in engineered B. subtilis. We implemented the single-reaction deletion targets identified by the model experimentally and showed that the new strains have a twofold higher γ-PGA concentration and production rate compared to the ancestral strain.ConclusionsThis work confirms that integration of enzyme constraints is a powerful tool to improve existing genome-scale models, and demonstrates the successful use of enzyme-constrained models in B. subtilis metabolic engineering. We expect that the new model can be used to guide future metabolic engineering efforts in the important industrial production host B. subtilis.

Project description:BackgroundPoly-γ-glutamic acid (γ-PGA) is a valuable polymer with glutamate as its sole precursor. Enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-PGA production, especially for those glutamate-independent γ-PGA producing strains. Corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate γ-PGA production.ResultsIn this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent γ-PGA producing Bacillus amyloliquefaciens NK-1 strain. After introducing the energy-saving NADPH-dependent glutamate dehydrogenase (NADPH-GDH) pathway, the NK-1 (pHT315-gdh) strain showed slightly increase (by 9.1%) in γ-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of ɑ-oxoglutarate dehydrogenase complex (ODHC) was introduced into the NK-1 strain, because it was previously shown that the ODHC in C. glutamicum was completely inhibited when glutamate was actively produced. The obtained NK-PO1 (pHT01-xylR) strain showed 66.2% higher γ-PGA production than the NK-1 strain. However, the further combination of these two strategies (introducing both NADPH-GDH pathway and the metabolic toggle switch) did not lead to further increase of γ-PGA production but rather the resultant γ-PGA production was even lower than that in the NK-1 strain.ConclusionsWe proposed new metabolic engineering strategies to improve the γ-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in γ-PGA production. The NK-PO1 (pHT01-xylR) strain with the introduction of a metabolic toggle switch for controlling the expression of ODHC showed 66.2% higher γ-PGA production than the NK-1 strain. This work proposed a new strategy for improving the target product in microbial cell factories.

Dataset Information

A new IS4 family insertion sequence, IS4Bsu1, responsible for genetic instability of poly-gamma-glutamic acid production in Bacillus subtilis.

Publications

A new IS4 family insertion sequence, IS4Bsu1, responsible for genetic instability of poly-gamma-glutamic acid production in Bacillus subtilis.

Similar Datasets

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