Abstract
Bacillus thuringiensis formulation losing its activity under field conditions due to UV radiation and photoprotection of B. thuringiensis based on melanin has attracted the attention of researchers for many years. Here, a single amino acid substitution (G272E) in homogentisate 1,2-dioxygenase was found to be responsible for pigment overproduction in B. thuringiensis BMB181, a derivative of BMB171. Disrupting the gene encoding homogentisate dioxygenase in BMB171 induced the accumulation of the homogentisic acid and provoked an increased pigment formation. To gain insights into homogentisate 1,2-dioxygenase in B. thuringiensis, we constructed a total of 14 mutations with a single amino acid substitution, and six of the mutant proteins were found to affect the melanin production when substituted by alanine. This study provides a new way to construct pigment-overproducing strains by impairing the homogentisate dioxygenase with a single mutation in B. thuringiensis, and the findings will facilitate a better understanding of this enzyme.
Highlights
Bacillus thuringiensis, a gram-positive spore-forming soil bacterium, has been widely used in biological pest control due to the formation of parasporal crystal proteins that are toxic to the larvae of various insect pests (Sudakin, 2003)
We found that the pigment produced by the strain BMB181 has nothing to do with DOPA
The peak with the same chromatography retention time as homogentisic acid (HGA) was identified in the culture supernatants of the strain BMB181 (Figure 2C), suggesting that homogentisate could be secreted by the pigmented strain BMB181 and the pigment produced by the strain probably resulted from the accumulation and polymerization of homogentisate
Summary
A gram-positive spore-forming soil bacterium, has been widely used in biological pest control due to the formation of parasporal crystal proteins that are toxic to the larvae of various insect pests (Sudakin, 2003). Melanins are polymers of phenolic and/or indolic compounds and classified into three main categories: eumelanins, pheomelanins, and allomelanins (Plonka and Grabacka, 2006) These black pigments are widely distributed in nature and can be found in species of all biological kingdoms, including humans, fungi, and bacteria (Plonka and Grabacka, 2006). Melanins provide free-living species a survival advantage in the environment by protecting against different exogenous stresses, such as UV-irradiation, reactive oxygen species (ROS), metals, and defensins (Nosanchuk and Casadevall, 2003, 2006; Heinekamp et al, 2012) Both eumelanins and pheomelanins are produced from the oxidation of tyrosine or phenylalanine to o-dihydroxyphenylalanine (DOPA) and dopaquinone via tyrosinases or laccases. Allomelanins include a heterogeneous group of polymers formed through the oxidation and polymerization of the intermediates such as dihydroxynaphthalene, homogentisic acid (HGA), γ-glutaminyl4-hydroxybenzene, catechols, and 4-hydroxyphenylacetic acid (Plonka and Grabacka, 2006)
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