The Study On The Mechanism Of Mother Cell Lysis During Sporulation Of Bacillus Thuringiensis

Bacillus thuringiensis is a Gram-positive, spore-forming bacterium belonging to the Bacillus cereus group that produces parasporal crystal proteins. These parasporal crystal proteins possess highly specialized insecticidal activity against a large number of insect species Because of the insecticidal properties of the crystal protein, B. thuringiensis has been used for many years as pesticide in biocontrol applications. However, one of the problems encountered after applying B. thuringiensis in the field is a lack of stability of the crystals on plant leaves due to inactivation by environmental factors, such as UV light. This is also the main factors which restrict the development of the B. thuringiensis products and also it need to be solved. The research of functional genomics provides a solid foundation for the development of microbiology, Bt HD73genome sequencing was completed in2013by our laboratory and it provides a train of thought for carrying out the following work of bioinformatics. A previous study showed that the genetic construction with aK disruption did not sporulate, still produced toxins, and did not result in lysis of the mother cell. The cell envelope was sufficient to protect against B. thuringiensis crystal degradation under UV and had no significant effect on insecticidal activity. However, disruption of the σK factor can decrease both the transcription of some cry genes during late sporulation and the biomass. Thus, developing an alternative method that will block mother cell lysis but have no effect on sporulation and Cry protein production is important. Peptidoglycan hydrolases, some of which are known as autolysins, play an essential role in mother cell lysis of Bacillus strains. Three autolysins were reported to be involved in the mother cell lysis of Bacillus subtilis including CwlB,CwlC and CwlH. Single inactivation of cwlB, cwlC, or cwlH has not been shown to affect mother cell lysis. However, mother cell lysis was found to be blocked in a mutant that had multiple inactivated genes. Based on this research, we analyze the mechanism of mother cell lysis and the function of hydrolase genes involved in mother cell lysis in Bacillus thuringiensis in this study.Reverse transcription-PCR （RT-PCR） results indicated that cwlB and cwlA formed one transcriptional unit5’ rapid amplification of cDNA ends （5’-RACE）-PCR and transcriptional fusions with the lacZ gene indicated that transcription of the operon was directed by a promoter and the transcription start site is a single5’-end nucleotide residue T located25nucleotides （bp） upstream from the cwlA translational start codon. The β-galactosidase assay showed that the promoter PcwlA is controlled by the σK factor. The activity of the PcwlA-lacZ fusion in the gerE mutant results showed that a-galactosidase activity in HD（AgerE） was significantly decreased compared to that in HD73. An electrophoresis mobility shift assay （EMSA） detected that GerE protein and PcwlA promoter is not combination in vitro; the results reveal that the promoter PcwlA is indirectly regulated by the GerE protein.To determine the function of cwlAB operon, we constructed cwlAB operon single mutant, double mutant and their complemented strains. Optical microscopy observations showed that HD（AcwlB） and HD（AcwlAAcwlB） completely released their spores at T24, compared with T20for HD73and HD（△cwlA）, which means that the mutation of cwlB delays spore release and cwlB plays a role in mother cell lysis. However, cwlA had nothing on mother cell lysis. Optical microscopy observations about genetically complemented strains of the cwlB deletion mutant results confirmed that the delay of spore release resulted from the disruption of the cwlB gene. The effects of cwlB mutation on sporulation and Cry protein production at T24were analyzed in B. thuringiensis. The results showed that the sporulation rate of HD（△cwlB） was slightly decreased compared to that of HD73. Cry protein production was determined in various strains by SDS-PAGE after crystal and spore release. The Cry protein production in HD（△cwlB） was similar to that in HD73To examine the cell wall binding ability of CwlB, the CwlB-His protein was purified. Western blot analysis showed that CwlB protein was bound to the B. thuringiensis cell walls. To determine the peptidoglycan cleavage site, the free amino groups of the digested sample were investigated by labeling free amino groups with1-fluoro-2,4-dinitrobenzene by RP-HPLC, these results indicate that CwlB is an N-acetylmuramoyl-L-alanine amidase. A fusion of the GFP gene with the cwlB gene at the5’terminus, GFP-cwlB was constructed, Cells were observed via laser confocal microscopy. The result suggests that the CwlB protein is localized on the cell envelope.Although the disruption of cwlB was demonstrated to delay spore and crystal release in B. thuringiensis, mother cells still lysed after T24. This suggests that other hydrolyses exist and participate in this cellular process. According to the complete genome sequence and RNAseq informations, the three candidate hydrolase genes which was controlled by sigmaK were found in Bacillus thuringiensis HD73, and also these genes were designated HD73₃410, HD73₃157,HD73₃966. To determine the function of the candidate genes, Single mutants and double mutants with cwlB were constructed. Optical microscopy observations showed that the mother cell lysis weren’t held back by the sigle mutants and double mutants in cwlB. Orthologs of the three candidate genes HD73₃157and HD73₃966were found in many B. cereus group strains, but shares low sequence similarity with the known function hydrolase genes of B. subtilis and B. cereus group. So these two genes were novel hydrolase and the functions were studied next step.The candidate genes were chosen by sigle-crossing recombination at the same time. The results showed that HD73₅344in Bacillus thuringiensis HD73was endopeptidase. β-galactosidase transcription activity results that the promoter of HD73₅344was transcripted during the late of sporulation. To determine the function of HD73₅344, we constructed HD73₅344single mutant and double mutant with cwlB. Optical microscopy observations showed that HD（△5344） delay the spores and crystal release, however, the double mutant HD（△5344△cwlB） could hold back the mother cell lysis, and it affect the formation of spore and crystal. The effects of HD73₅344mutation on sporulation and Cry protein production at T28were analyzed in B. thuringiensis. The results showed that the sporulation rate of HD（△5344） was decreased compared with that of HD73. The Cry protein production in HD（△5344） was similar with that in HD73,but spores and crystal were not detected in double mutant. So the mechanismin need to study.