Identification Of The Germination Related Genes In Bacillus Thuringiensis And Confirmation Of Incidental Variation During Transposon Mutagenesis

Bacillus thuringiensis(Bt)is a gram-positive soil bacterium,which produces insecticidal crystal proteins during sporulation process.The insecticidal crystal proteins have been shown high toxicity against more than 500 kinds of insects,a variety of parasitic nematodes of plants and animals and plasmodium.Spores exhibit significant synergistic effects on the crystal proteins during the insecticidal process of Bt.Spores were found to accelerate the death of insects by germinating and growing in the gut of target insects.Recent studies have also found that Bt spores play an important synergistic role in killing nematodes.The germination characteristics of Bt spores potentially have an influence on the effect of insecticidal avtivity.However,few systematic researches were carried out to explore the mechanism of spore germination in B.thuringiensis.The knowledge of germination mechanism of B.thuringiensis and infection related genes not only could increase our understanding the killing mechanism of Bt,but provide a guiding role for us to apply Bt in industrial and agricultural production.In this study,we found that spores of B.thuringiensis YBT-1518 completed the infectious life cycle in Caenorhabditis elegans,and then analyzes the relationship between the germination of Bacillus receptor diversity and bacteria adaptability.By transposon mutagenesis,we identified several genes required for the germination triggered by L-alanine、inosine and tissue extract of C.elegans.In addition,we found that additional mutations occur frequently during transposon mutagenesis by genome sequencing of 19 transposon mutants and transposon insertion library.Furthermore,we found that high temperature and antibiotics leads to occurrence of additional mutations.The main results of this work were displayed as following:1.By continuous observation under the phase contrast microscope,we found that spores of B.thuringiensis YBT-1518 can complete the infectious life cycle in C.elegans.The spores of YBT-1518 began to germinate in the gut after they were eaten by C.elegans for 24 hours,and then germinated spores rapidly enter the vegetative stage with nutrients from the nematode’s gut(48h);B.thuringiensis grew and reproduced rapidly by digesting the intestinal tissue of nematodes(60h),it made the nematodes form a phenotype known as“bag of bacteria”(Undigested cuticles of the nematodes represents the bag that contains the bacteria).B.thuringiensis YBT-1518 began to enter the sporulation stage with the nutrients depletion(72h),and the newly formed spores and Crystal proteins were able to be seen clearly at the 84 hours;Finally the spores and Crystal proteins were released after the nematodes were completely decomposed.2.Based on the analysis of the germination receptors of 110 Bacillus strains,we found that 52 strains of B.cereus group(B.anthracis,B.thuringiensis and B.cereus)contain abundant germination receptors,moreover,the germination receptors Ger I,Ger S and Ger Y were found to be mainly distributed in these strains and no homologous receptors were found in the strains belonging to the Bacillus subtilis group,so we deduced that strains of B.cereus group may be more adapted to germinate in the C.elegans constrast to the strains of B.subtilis group.Then we found that germination ability of B.thuringiensis YBT-1518 and BMB171 is four times that of B.subtilis 168when the spores were incubated in tissue extract of C.elegans,and we also found that the composition of tissue extract of C.elegans were more suitable for the germination of B.thuringiensis spores(tissue extract could act on more spore germination receptors of B.thuringiensis).Through gene deletion,we found that germination receptors Ger I,Ger S and Ger Y play an important role in the germination of B.thuringiensis YBT-1518and the pathogenesis against the C.elegans.The deletion of these three receptors resulted in a decrease in the germination ability of YBT-1518 triggered by L-alanine,inosine and nematode extracts,as well as the insecticidal activity against C.elegans.3.Through germination screening of the transposon insertion libraries(about five thousands mutants),We obtained 26 mutants with germination defect,and then we selected 13 new genes disrupted by transposon insertion for functional verification,and these genes encode three channel proteins(met P、glp T and K~+channel protein)、seven regulator proteins(HTH type transcription regulator、RNA helicase、Adaptor protein,and so on)、two glycolytic protein(fb A and glp D)and one electron transfer terminal receptors(cyt D);we found that the absence of 12 genes reduced the germination ability of YBT-1518 triggered by L-alanine、inosine and tissue extract of Caenorhabditis elegans,and these genes include met P、glp T、HTH type transcription regulator、fb A、glp D and cyt D.4.Spores of mutant MT1518-1 showed severe germination defect triggered by several germinants.Genome sequencing identified 186 unlinked mutations on the chromosome of the mutants MT1518-1,and found that these additional mutation rather than the gene gnt P disrupted by transposon insertion led to the germination defect of mutant MT1518-1.To understand the presence of additional genetic mutations,we identified 2343 SNPs,three insertion mutations and a deletion mutation in 19 randomly selected transposon mutants,and we also identified a large number of genetic mutations in four mini-Tn10 transposon libraries.In addition,we found that these additional mutations potentially lead to changes in amino acids or the phenotypes of transposon mutants.Finally,we selected the rifampin resistance mutations as an index to refelect the effect of high temperature and antibiotic on the genetic mutations,and found that the high temperature(42℃)and antibiotics used in constructing the transposon libraries have a significant effect on rifampin resistance mutations.In this work,we explored the germination characterization of Bacillus thuringenesis from multiple perspective,found several genes required for the germination triggered by L-alanine、inosine and tissue extract of Caenorhabditis elegans,and also found that spores of YBT-1518 can complete the infectious life cycle in Caenorhabditis elegans.These findings provide many new insights about the germination mechanism of Bt and its related species,enhance the understanding the role of spores in Bt toxicity.The finding that incidental mutations occur frequently during the process of transposon mutagenesis provides a good explanation for the phenomenon that phenotype changes unrelated to the genes disrupted by transposon,and this result also reminds researchers to pay more attention to the potential effect of additional mutations during the use of transposon mutagenesis.