Melanin Synthesis Mechanism Of Bacillus Thuringiensis BMB171

Melanin is widely existed in nature and can be found in microorganisms,animals,plants and humans.Studies have shown that the melanin synthesized by organisms has many important physiological functions,such as anti-oxidation,anti-ultraviolet radiation,and combining with heavy metal ions,and so on,which can improve the living capacity of organisms and the environmental adaptability.Bacillus thuringiensis?Bt?is a kind of gram-positive bacteria that forms crystalline proteins with sporulation and is the most widely used biological insecticide in the world.As the main active ingredient of Bt preparations,Bt with insecticidal activity can be easily affected by the environment,such as ultraviolet radiation.The researchers have used various methods to improve the anti-ultraviolet ability of Bt formulations to increase the potency of Bt insecticide,one of which is to use melanin as the uv protective agent of Bt.Previous studies have shown that most Bt strains could obtain the melanogenic phenotype through high temperature induction.The mutant BMB181 with high melanin production of BMB171 was obtained by high temperature induction and subculture in the early stage of our laboratory.The series results of experiments proved that BMB181could produce melanin in LB medium,ICPM culture medium and fermentation culture medium without inducer and keep the good performance characteristics and expression of BMB171.And biological test results also showed that melanin produced by BMB181could protect the crystal protein from uv damage.However,the exact mechanism of melanin production of Bt is unclear.The analysis of melanin production mechanism of Bt can not only increase our understanding of Bt,but also provide a theoretical basis for the construction of Bt engineering bacteria with anti-ultraviolet ability,which is of great significance for the application of Bt preparation in the agricultural production process.Melanin in nature are mainly classified as eumelanin and pheomelanin and allomelanin,among which DHN melanin,HPQ melanin and pyomelanin are belonging to allomelanin.At present,the biosynthesis pathways of eumelanin,DHN melanin,HPQ melanin and pyomelanin have been reported.The genes involved in these melanin synthetic pathways are known.Previous studies have found that the gene coding for tyrosinase was exist in the BMB171 genome sequence,but the mutant strain BMB181,derivative of BMB171,still produced melanin after knocking out the gene coding for tyrosinase.Based on this,we guess that there is other melanin production pathway in Bt that is different from the melanin synthesis pathway catalyzed by tyrosinase.In this study,the mechanism for the production of melanin by Bt strain BMB181was studied.The main work and results are as follows:1.Bacillus thuringiensis strains can produce melanin by inactivation of homogentisate dioxygenaseAnalyzing the BMB171 genome sequence,we found that the tyrosine metabolic pathway via homogentisate was located on the genome.This tyrosine metabolic pathway mediated by homogentisate requires six enzymes in humans.Among them,homogentisate dioxygenase is a key enzyme that catalyzes the ring opening reaction of homogentisate.When the homogentisate dioxygenase is inactivated,homogentisate will accumulate acid and oxidation and polymerization to form pyomelanin,causing the metabolic disease alkaptonuria?AKU?.We thus speculated that Bt strains could produce melanin from homogentisate.Temperature sensitive plasmid was used to construct the hmgA gene insertion mutant BMB171?hmgA.Mutant strain BMB171?hmgA showed the same melanin production phenotype as the strain BMB181.HPLC results showed that homogentisate could be produced and accumulation during the life cycle of the mutant strain BMB171?hmgA and BMB181.DNA sequencing results indicated that there was a nucleotide substitution?815C?T?in the hmgA gene of BMB181,resulting in a missense mutation?272G?E?in the homogentisate dioxygenase of BMB181.The complementation tests confirmed that the expression of homogentisate dioxygenase of BMB171 in BMB181 could restore the non-pigmented phenotype of Bt strain.We found the HmgA in CT137 had missense mutation?G241D?by sequencing the hmgA gene of the melanogenic strain CT137.HmgA in the melanogenic strain CT153 had a stop mutation due to the insertion of one nucleotide.The complement strains of CT137 and CT153 did not produce melanin.All of these indicated that single point mutation of homogentisate dioxygenase could lead to the production of melanin in Bt.2.Melanin is produced when a single point mutation occurs in 6 amino acid residues in HmgA seperatelyThe secondary structure of HmgA showed that amino acid residue Gly272 was located in the?19-?20 loop.As G272E mutation of HmgA could lead to the generation of melanin in BMB181,we constructed 14 alanine scanning mutants of BMB171 HmgA?G89A,N116A,G119A,D120A,G128A,F136A,K219A,G241A,P245A,H261A,N300A,G323A,H334A and P336A?.All these residues were located in?-?loop or in one end of?sheet.The effect of these mutants on melanin production were studied through the complementation analysis,and it was found that only 6 mutants?G128A,F136A,G241A,H261A,H334A and P336A?had an effect on the formation of melanin.It indicated that these six mutants could cause HmgA to be inactivated,allowing Bt to produce melanin.Although these 14 sites were found to be highly conserved in HmgA by multiple sequence alignment,the sites G128,F136,G241,H334 and P336 were more conserved in terms of the relationships between structure and function.Analysis of more than 40 Bt strains whose whole genome sequences have been published in the NCBI database,we found that the genes?hmgA,hppD and fahA?that encoding the related enzymes which took part in the tyrosine metabolic pathway via homogentisate were prevalent in all these Bt strains.At the same time,we found that hmgA genes were existed in 70 Bt strains of different H serotypes.It suggested that Bt had the potential to produce melanin with homogentisate as the precursor by inactivating the homogentisate dioxygenase.The characteristics of homogentisate dioxygenase including hydrophobicity,conserved structural domain,phylogenetic analysis and polymorphism of amino acids,and the three-dimensional structure of HmgA were analyzed by bioinformatics methods.We found that HmgA was a hydrophilic protein,belonging to the cupin-like super family protein.The three-dimensional structure showed that HmgA was associated as a hexamer arranged as a dimer of trimers.The active site iron ion was coordinated near the interface between subunits in the HmgA trimer by a Glu and two His side chains?His298,Glu305 and His334?.Amino acid polymorphism analysis showed that there were 19 polymorphic sites?S23A,S54A,L56V,S71A,S80N,S83T,I87V,I93M,S105T,D110N,T138M,V169L,L199I,E213D,H227Y,A343G,Q384E,Q384D,Y387H,T388N?.However,these different amino acid residues in these sites had no effect on melanin production,all these related strains have not been reported to produce melanin.This indicates that the function of homogentisate dioxygenase in Bt is conservative,so that the vast majority of Bt strains isolated from nature do not produce melanin.3.Other melanin production pathway that different from pyomelanin formation may exist in Bt.It has been reported that Bt can produce melanin by tyrosine induction at high temperature.However,the mechanism of the melanin production is not very clear,it is speculated that the melanin is produced by tyrosine catalyzed by tyrosinase at high temperature.However,tyrosinase genes were rare in Bt strain,indicating that other proteins with similar activity to tyrosinase might exist in Bt.The coding region of the genome sequence of BMB171 was analyzed and a Laccase-like protein with multiple Cu oxidase domain was found.The three-dimensional structure and active sites of the protein were analyzed,and the structure domain contained Zn²⁺binding active sites,and Cys181,Cys182,Cys239 and Cys242 ligated with Zn²⁺.The results of genomic analysis showed that this gene was widely found in Bt.The gene of laccase-like protein was expressed and purified and laccase activity was studied.The relationship between this protein and melanin needs further study.In addition,by screening the interfering mutant of YBT-1518,we found that the three mutant strains produced melanin.The three mutants were interfered with genes YBT1518₀7140?enyl-?acyl carrier protein?reductase?Fab??,YBT1518₂1755?XRE?Xenobiotic Response Element?family regulation factor?and YBT1518₂3810?site-specific integrase?.The relationship between these proteins and melanin has not been reported.This study focused on the high melanin yielding Bt strain BMB181 and elucidated the formation mechanism of melanin in Bt strains.It will not only enrich the understanding of diversity of the melanin formation mechanism of Bt,but also provide a theoretical basis for construction of genetic engineering bacteria with high melanin production by genetic methods.This provides a theoretical basis for the point mutation in HmgA to construct a Bt strain resistant to UV,and also provides a meaningful model for the study of human genes responsible for alkaptonuria.