Abstract
BackgroundLysinibacillus sphaericus has been widely used in integrated mosquito control program and it is one of the minority bacterial species unable to metabolize carbohydrates. In consideration of the high genetic conservation at genomic level and difficulty of genetic horizontal transfer, it is hypothesized that effective restriction-modification (R-M) systems existed in mosquitocidal L. sphaericus.ResultsIn this study, six type II R-M systems including LspC3–41I were predicted in L. sphaericus C3–41 genome. It was found that the cell free extracts (CFE) from this strain shown similar restriction and methylation activity on exogenous Bacillus/Escherichia coli shuttle vector pBU4 as the HaeIII, which is an isoschizomer of BspRI. The Bsph_0498 (encoding the predicted LspC3–41IR) knockout mutant Δ0498 and the complement strain RC0498 were constructed. It was found that the unmethylated pBU4 can be digested by the CFE of C3–41 and RC0498, but not by that of Δ0498. Furthermore, the exogenous plasmid pBU4 can be transformed at very high efficacy into Δ0498, low efficacy into RC0498, but no transformation into C3–41, indicating that LspC3–41I might be a major determinant for the genetic restriction barrier of strain C3–41. Besides, lspC3–41IR and lspC3–41IM genes are detected in other two strains besides C3–41 of the tested 16 L. sphaericus strains, which all belonging to serotype H5 and MLST sequence type (ST) 1. Furthermore, the three strains are not horizontal transferred, and this restriction could be overcome by in vitro methylation either by the host CFE or by commercial methytransferase M. HaeIII. The results provide an insight to further study the genetic restriction, modification and evolution of mosquitocidal L. sphaericus, also a theoretical basis and a method for the genetic manipulations of L. sphaericus.ConclusionsLspC3–41I is identified as the major determinant for the restriction barrier of L. sphaericus C3–41. Only three strains of the tested 16 L. sphaericus strains, which all belonging to serotype H5 and ST1 by MLST scheme, contain LspC3–41I system. Two different methods can be used to overcome the restriction barrier of the three isolates to get transformants efficiently: 1) to methylate plasmid DNA prior to the electroporation; and 2) to delete the major restriction endonuclease encoding gene lspC3–41IR.
Highlights
Lysinibacillus sphaericus has been widely used in integrated mosquito control program and it is one of the minority bacterial species unable to metabolize carbohydrates
The results provide an insight to further study the genetic restriction, modification and evolution of mosquitocidal L. sphaericus, a theoretical basis and a method for the genetic manipulations of L. sphaericus
LspC3–41I is identified as the major determinant for the restriction barrier of L. sphaericus C3–41
Summary
Lysinibacillus sphaericus has been widely used in integrated mosquito control program and it is one of the minority bacterial species unable to metabolize carbohydrates. In consideration of the high genetic conservation at genomic level and difficulty of genetic horizontal transfer, it is hypothesized that effective restriction-modification (R-M) systems existed in mosquitocidal L. sphaericus. Lysinibacillus sphaericus is a spore-forming bacterium widely distributed in soil and other environment. Nine serotypes (H1, H2, H3, H5, H6, H9, H25, H26, and H48) can produce different types of insecticidal proteins against mosquito larvae: the binary toxin (Bin) and two-component toxin (Cry and Cry49) formed during sporulation, and mosquitocidal toxins (Mtx toxins) produced during the vegetative growth [4,5,6,7]. E.g. 16S rRNA and MLST analysis, revealed that the mosquitocodal L. sphaericus are highly conserved. Comparative genomics revealed that toxic strains form a well-defined lineage that could be considered as a separate species from non-toxic strain [10]
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