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Background    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 .    Results  In 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  H aeIII, 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.  Hae III. 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.Conclusions  LspC3–41I 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 .

The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41