Nanoscale mono- and multi-layer cylinder structures formed by recombinant S-layer proteins of mosquitocidal Bacillus sphaericus C3-41.

The mature surface layer (S-layer) protein SlpC of mosquitocidal Bacillus sphaericus C3-41 comprises amino acids 31-1,176 and could recrystallize in vitro. The N-terminal SLH domain is responsible for binding function. Deletion of this part, S-layer proteins could not bind to the cell wall sacculi. To investigate the self-assembly ability of SlpC from B. sphaericus, nine truncations were constructed and their self-assembly properties were compared with the recombinant mature S-layer protein rSlpC31₋1,176. The results showed that rSbsC31₋1,176 and truncations rSlpC211₋1,176, rSlpC278₋1,176, rSlpC31₋1,100, and rSlpC31₋1,050 could assemble into multilayer cylinder structures, while N-terminal truncations rSlpC338₋1,176, rSlpC438₋1,176, and rSlpC498₋1,176 mainly showed monolayer cylinders in recombinant Escherichia coli BL21 (DE3) cells. Growth phase analysis of the self-assembly process revealed that rSlpC498₋1,176 mainly formed monolayer cylinders in the early stage (0.5 and 1 h induction of expression), but few double-layer or multilayer cylinders were also found with the cells growing, while rSlpC31₋1,176 could formed multilayer cylinders in all the growth stage in the E. coli cells. It is concluded that the deletion of the C-terminal 126 aa or the N-terminal 497 aa did not interfere with the self-assembly process, the fragment (amino acids 278 to 337) is essential for the multilayer cylinder formation in E. coli BL21 (DE3) cells in the early stage and the fragment (amino acids 338 to 497) is related to monolayer cylinder formation. The information is important for further studies on the assembly mechanism of S-layer proteins and forms a basis for further studies concerning surface display and nanobiotechnology.

Species-specific cell wall binding affinity of the S-layer proteins of mosquitocidal bacterium Bacillus sphaericus C3-41.

The binding affinities and specificities of six truncated S-layer homology domain (SLH) polypeptides of mosquitocidal Bacillus sphaericus strain C3-41 with the purified cell wall sacculi have been assayed. The results indicated that the SLH polypeptide comprised of amino acids 31 to 210 was responsible for anchoring the S-layer subunits to the rigid cell wall layer via a distinct type of secondary cell wall polymer and that a motif of the recombinant SLH polypeptide comprising amino acids 152 to 210 (rSLH(152-210)) was essential for the stable binding of the S-layer with the bacterial cell walls. The quantitative assays revealed that the K(D) (equilibrium dissociation constant) values of rSLH(152-210) and rSLH(31-210) with purified cell wall sacculi were 1.11 x 10(-6) M and 1.40 x 10(-6) M, respectively. The qualitative assays demonstrated that the SLH domain of strain C3-41 could bind only to the cell walls or the cells treated with 5 M guanidinium hydrochloride of both toxic and nontoxic B. sphaericus strains but not to those from other bacteria, indicating the species-specific binding of the SLH polypeptide of B. sphaericus with bacterial cell walls.

Improving the insecticidal activity against resistant Culex quinquefasciatus mosquitoes by expression of chitinase gene chiAC in Bacillus sphaericus.

Expression of a chitinase gene, chiAC, from Bacillus thuringiensis in B. sphaericus 2297 using the binary toxin promoter yielded a recombinant strain that was 4,297-fold more toxic than strain 2297 against resistant Culex quinquefasciatus. These results show that this chitinase can synergize the toxicity of the binary toxin against mosquitoes and thus may be useful in managing mosquito resistance to B. sphaericus.

A strain of Bacillus sphaericus causes slower development of resistance in Culex quinquefasciatus.

Two field-collected Culex quinquefasciatus colonies were subjected to selection pressure by three strains of Bacillus sphaericus, C3-41, 2362, and IAB59, under laboratory conditions. After 13 and 18 generations of exposure to high concentrations of C3-41 and IAB59, a field-collected low-level-resistant colony developed >144,000- and 46.3-fold resistance to strains C3-41 and IAB59, respectively. A field-collected susceptible colony was selected with 2362 and IAB59 for 46 and 12 generations and attained >162,000- and 5.7-fold resistance to the two agents, respectively. The pattern of resistance evolution in mosquitoes depended on continuous selection pressure, and the stronger the selection pressure, the more quickly resistance developed. The resistant colonies obtained after selection with B. sphaericus C3-41 and 2362 showed very high levels of cross-resistance to B. sphaericus 2362 and C3-41, respectively, but they displayed only low-level cross-resistance to IAB59. On the other hand, the IAB59-selected colonies had high cross-resistance to both strains C3-41 and 2362. Additionally, the slower evolution of resistance against strain IAB59 may be explained by the presence of another larvicidal factor. This is in agreement with the nontoxicity of the cloned and purified binary toxin (Bin1) of IAB59 for 2362-resistant larvae. We also verified that all the B. sphaericus-selected colonies showed no cross-resistance to Bacillus thuringiensis subsp. israelensis, suggesting that it would be a promising alternative in managing resistance to B. sphaericus in C. quinquefasciatus larvae.