RESUMO
Extrachromosomal DNA maintenance (EDM) is an important process in molecular breeding and for various applications in the construction of genetically engineered microbes. Here we describe a novel Bacillus subtilis gene involved in EDM function called edmS (formerly pgsE). Functional gene regions were identified using molecular genetics techniques. We found that EdmS is a membrane-associated protein that is crucial for EDM. We also determined that EdmS can change a plasmid vector with an unstable replicon and worse-than-random segregation into one with better-than-random segregation, suggesting that the protein functions in the declustering and/or partitioning of episomes. EdmS has two distinct domains: an N-terminal membrane-anchoring domain and a C-terminal assembly accelerator-like structure, and mutational analysis of edmS revealed that both domains are essential for EDM. Further studies using cells of Bacillus megaterium and itsedmS (formerly capE) gene implied that EdmS has potential as a molecular probe for exploring novel EDM systems.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Modelos Moleculares , Plasmídeos/genética , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Mutação da Fase de Leitura/genética , Dados de Sequência MolecularRESUMO
The Bacillus subtilis structural gene pgsE was investigated as a tool for extrachromosomal DNA maintenance (EDM). It ameliorated the stability of high-copy-number vectors, regardless of whether they were derived from rolling-cycle or theta-mode replicons, without any selective pressure. This unique EDM phenomenon may occur via a trans-acting mechanism.
Assuntos
Bacillus subtilis/genética , DNA Bacteriano/genética , Glutamato Sintase/genética , Bacillus subtilis/metabolismo , Sequência de Bases , Cromossomos Bacterianos , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Glutamato Sintase/metabolismo , Ácido Poliglutâmico/biossíntese , Ácido Poliglutâmico/genética , Replicon/genética , Análise de Sequência de DNARESUMO
Poly-γ-glutamate (PGA) is a versatile nylon-like material, and enhanced production of PGA is required for various bio-industrial applications. In this study, we first examined the effects of available sugars on the production of Bacillus subtilis PGA, and demonstrated the good applicability of pentoses (e.g., D-xylose). Then, we characterized the pgsE gene of B. subtilis, which encodes a 6.5-kDa protein of 55 amino acids (PgsE), as a genetic tool for increasing the yield of PGA without changing its structural features (e.g., polymer stereochemistry and molecular size distribution). In the presence of Zn(2+), the induction of PgsE tripled the PGA productivity of B. subtilis subsp. chungkookjang. This finding will contribute to the establishment of an improved PGA-production system.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Ácido Poliglutâmico/metabolismo , Zinco/metabolismoRESUMO
It is generally thought that natural strains of Bacillus subtilis produce poly-gamma-glutamate (PGA) as a large exopolymer (over 1,000 kDa) with high water solubility. However, extracellular PGA (ePGA) of B. subtilis is actually diverse in molecular size and configuration. In this study, we identified membranous PGA (mPGA) from both natural and domestic strains of B. subtilis. In contrast to ePGA, mPGA was relatively small and consistently l-glutamate-rich. Genetic analysis revealed that the pgs operon of B. subtilis is responsible for mPGA production as well as ePGA production. Biochemical analyses using the membranous fractions from B. subtilis ssp. chungkookjang indicated that the presence of zinc ions (Zn(2+)) affected both the membrane association of mPGA and in vitro synthesis (elongation) of PGA. Our observations highlighted three important factors that will affect the structural diversity of B. subtilis PGA, namely the occurrence of mPGA, the effects of Zn(2+), and the configuration of glutamate substrate.