Two Groups Of S-Layer Proteins Co-Exist In S-Layer And Parasporal Inclusions

S-layer proteins (SLPs) are typically thought to be constituents of the bacterial cell surface layer (S-layer). Previously we reported that SLPs can form parasporal inclusions in some Bacillus thuringiensis strains, and these inclusions consist of S-layer protein. But it is still unknown about whether these proteins are encoded by typical S-layer protein genes in phylogenetic level and whether S-layer protein gene directly lead to the formation of parasporal inclusions or S-layer. And it is need to know if S-layer and parasporal inclusions are encoded by the same gene. In this study, we screened four B. thuringiensis strains whose parasporal inclusions consist of S-layer protein (PICS strain) and cloned8slp genes from these strains. It is interesting that each strain contained two adjacent slp genes in each genome. Phylogenetic tree analysis indicated these SLPs could be divided into two groups, designated SLP1s and SLP2s. To confirm which SLPs were present in the S-layer or as a parasporal inclusion, two strains, CTC and BMB1152, were chosen for further study. Western blots using whole-cell associated proteins from strain CTC and BMB1152showed that SLP1s and SLP2s were both constituents of the S-layer. Immunofluorescence reaction with S-layer of strain CTC and BMB1152showed two phynotypes that in strain CTC, SLP1and SLP2expressed in similar grade to form a homologous S-layer in both exponential and stationary phases, but in strain BMB1152, SLP1expression was switched by SLP2gradually so a homologous SLP1-S-layer in exponential phase was replaced by SLP2-S-layer in stationary phase. Immunofluorescence reaction with spore-inclusion mixtures of strain CTC and BMB1152showed SLP1s and SLP2s were also both constituents of parasporal inclusions. When heterogeneously expressed in a crystal negative strain BMB171, four SLPs from strain CTC and BMB1152could also form parasporal inclusions. These mean both groups of SLPs are constituents of the S-layer and parasporal inclusions during different phases (vegetative or sporulation). This temporal and spatial expression is not an occasional phenomenon but ubiquitous in B. thuringiensis strains. Bacillus thuringiensis subsp finitimus has two types of parasporal inclusions one of which are inside exosporium and coded by cry26and cry28genes that located in plasmids. Another type of inclusions is outside exosporium and constituents are unknown. In order to know the constitute of parasporal inlusions and S-layer of YBT-020, Western blots with S-layer and parasporal inclusions associated proteins were made and results showed that in YBT-020, SLP1expressed early and steady in exponential phase but SLP2expressed considerably late at about10hour so SLP1is the dominant proteins in S-layer formation of YBT-020. In sporulation phase, two S-layer proteins of YBT-020were unsteady and degraded into70kDa proteins different to BMB1152that SLP2protein expressed highly and only SLP1also degraded into70kDa. So in YBT-020free inclusions that coded by S-layer proteins were not steady like in BMB1152and so were not easy to be found like inclusions that codede by cry genes. SDS-PAGE of S-layer proteins of YBT-020and its variant strain BMB1152(crued all plasmids) and BMB1151(crued pBMB26) showed that only in YBT-020,100kDa S-layer protein was expressed lowly and inhibited compared with other two variants, which means that pBMB26may play roles in negatively regulation of S-layer protein expression in YBT-020. Our work showed that in YBT-020, there were two types of parasporal inclusions, one of which were free and consist of S-layer proteins. We also found that the expression of S-layer proteins in YBT-020was negatively regulated by the plasmid pBMB26.