Characterisation of Lysinibacillus sphaericus strains

• Main Author: Purwanto, Hari
• Published: Cardiff University 2016
• Subjects: 572; QL Zoology
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.699376

Some strains of Lysinibacillus sphaericus bacteria produce proteins toxic to insects, in particular mosquitoes. The bacteria have been used on a large scale to control these disease vectors. Unfortunately, there have been some reports of insect resistance to L. sphaericus. Interestingly, there are also reports of new isolates that may provide an opportunity to exploit these bacteria in other ways. To achieve better control of insect vectors, studies of various aspects of the L. sphaericus and its toxins are needed. This dissertation reports properties of a newly isolated L. sphaericus isolate namely, Fang Large. Based on its morphological features and 16S RNA DNA sequence analysis, the Fang Large isolate was confirmed as a Lysinibacillus sphaericus strain. This isolate exerts its pathogenicity to Wax moth larvae through septicaemia. Next, regulation of toxin gene expression was performed through analysis of 5’ untranslated regions of L. sphaericus toxin genes of strain IAB59 using a 5’ RLM-RACE method to reveal the promoters of the toxin genes. These investigations did not always produce the expected results but 5’UTR analysis of the mtx2 sequence confirmed the previously predicted promoter, while for two other genes –binB and cry48– results indicated that the actual promoters are not those previously predicted and that unreported promoter sequences appear to act in regulating these genes. To study the genomic arrangement of L. sphaericus strain NHA15b and its toxin protein genes, next generation sequencing technology was employed. This showed that the chromosomal structure of strain NHA15b is similar to the previously sequenced L. sphaericus genomes, but lacked a 35 kb fragment that in strain C3-41 is known to harbour binA/B and mtx4 genes. There are at least five (possibly six) plasmids in L. sphaericus strain NHA15b. Two of them, pLsph100 and pLsph200 are similar to other plasmids reported from other L. sphaericus strains. Plasmids pLsph300, pLsph400 and pLsph500, in contrast, have never been reported in any L. sphaericus strain. The sixth plasmid, pLsph600 was predicted based on the presence of a T4SS system and DNA replication protein genes. The genes may be responsible for the conjugation ability of the particular plasmid. It is predicted that the cry48/49 genes of the strain NHA15b may be located in this putative plasmid. The prediction is supported by indications from physical separation efforts using Pulsed Field Gel Electrophoresis, that the cry genes were located in a plasmid with mobility equivalent to 750 kb linear DNA. This prediction still needs further verification.