Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria

Area of Science:

• Molecular Biology
• Microbiology
• Biotechnology

Background:

• Broad-host-range plasmid vectors are crucial for genetic manipulation in diverse bacteria.
• Controlling gene expression is essential for studying gene function and developing biotechnological applications.
• Existing vectors may have limitations in host range or inducibility.

Purpose of the Study:

• To construct and characterize a novel broad-host-range plasmid vector, pNM185, for regulated gene expression.
• To evaluate the efficacy of pNM185 in various gram-negative bacterial species.
• To assess the induction levels and regulation efficiency of cloned genes using pNM185.

Main Methods:

• Construction of the pNM185 plasmid vector based on pKT231, incorporating the pm twin promoters and xylS regulator.
• Cloning of the xylE gene (encoding catechol 2,3-dioxygenase) into pNM185.
• Testing regulated expression of xylE in a wide range of gram-negative bacterial strains.
• Quantification of enzyme levels and induction ratios.

Main Results:

• The pNM185 vector enabled regulated gene expression in most tested gram-negative bacteria.
• Induced enzyme levels reached up to 5% of total cellular protein.
• High induction ratios (up to 600-fold) were achieved with inexpensive coinducers (benzoate or m-toluate).
• Gene expression levels correlated with phylogenetic distance from Pseudomonas putida.

Conclusions:

• The pNM185 plasmid vector provides a versatile tool for regulated gene expression across diverse gram-negative bacteria.
• The vector system is efficient and responsive to low concentrations of inexpensive chemical inducers.
• pNM185 facilitates functional studies and biotechnological applications of genes in a broad bacterial range.