Cloning and sequencing of the gene encoding a 125-kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene

Abstract

Using the vector pGEM-4-blue, a 4,251-base-pair DNA fragment containing the gene for the surface (S)-layer protein of Bacillus sphaericus 2362 was cloned into Escherichia coli. Determination of the nucleotide sequence indicated an open reading frame (ORF) coding for a protein of 1,176 amino acids with a molecular size of 125 kilodaltons (kDa). A protein of this size which reacted with antibody to the 122-kDa S-layer protein of B. sphaericus was detected in cells of E. coli containing the recombinant plasmid. Analysis of the deduced amino acid sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The first amino acid of the N-terminal sequence of the 122-kDa S-layer protein followed the predicted cleavage site of the leader peptide in the 125-kDa protein. Bacillus sphaericus is a promising agent for the biological control of mosquitos which are vectors of important human and animal diseases (47). Toxicity for mosquito larvae has been associated with the formation, during sporulation, of a parasporal crystal and with proteins of 100 to 125 kilodaltons (kDa) which may be made during vegetative growth (3,4,7,9,12,32,33). In the case of B. sphaericus 2362, proteins with a molecular size of 42, 51, and 110 kDa have been shown to play a role in toxicity for mosquito larvae (2-4, 9). The genes for the 42-and 51-kDa proteins have recently been cloned and sequenced (2,3,21).A number of gram-positive and gram-negative bacteria possess a protein or glycoprotein surface (S) layer that forms a barrier between the cell and the environment (39, 40). These proteins may constitute between 5 and 10% of the total protein of cells in exponential growth (39,40). The high energy expenditure required for the synthesis of such a large amount of protein suggests that it has a vital function requiring its maintenance on the cell surface. Evidence has been presented indicating that the S layer acts as a protective barrier and plays a role in bacterial pathogenesis (22,40). In the larvicidal strains of B. sphaericus, the S layer consists of a linear array of glycoproteins (29), the monomer having a molecular size of 127 to 129 kDa (45). In this species, it may serve as a site for bacteriophage attachment (28). On the basis of susceptibility to different bacteriophages, the mosquito-pathogenic strains have been subdivided into groups (47) which are in agreement with the subdivisions established by serological studies of the S-layer protein (29,45).In the present study, we cloned and sequenced a gene which codes for a 125-kDa precursor of the 122-kDa B. presented indicating that the 122-kDa protein is the precursor of the 110-kDa larvicide and that both of these proteins are absent from the parasporal crystal of this species. The latter finding indicates that our previous conclusion that the 122-and 110-kDa proteins were constituents of the crystal is invalid since our "crystal" preparation (4) appears to have been contaminated with cell wall material containing S-layer proteins. The cryp...

Expression, secretion and antigenic variation of bacterial S‐layer proteins

“…It is unclear whether synthesis and/or secretion of S-protein is limiting for this species at specific growth conditions, or whether the secreted S-protein just fails to constitute an S-layer and is released into the culture fluid. (Bowditch et al, 1989). d. Based on protein analysis, it is postulated that additional gene(s) are present (Sára and Sleytr, 1994).…”

“…sphaericus 2362 contains a truncated S-protein gene located 11 kb downstream from the gene encoding the 125 kDa S-protein. This truncated gene, which lacks a translation start, can encode a polypeptide of 80 kDa (Bowditch et al, 1989). The N-terminal part of the hypothetical 80 kDa protein is almost identical to the middle part of the 125 kDa S-protein, while the C-terminal part of the hypothetical 80 kDa protein has a reduced similarity with the C-terminal part of the 125-kDa S-protein.…”

S-Layer Proteins

“…This secondary cell wall polymer is a teichuronic acid and is composed of disaccharide repeating units having the structure 33)-[4,6-O-(1-carboxyethylidene)] ϳ0.5 -␤-D-ManpNAc-(134)-␤-D-GlcpNAc-(13. In contrast to the SLH motifs of most S-layer proteins, which reveal a positive net charge, those of B. sphaericus strains (14,26) are net negatively charged, which explains why bivalent cations are required for binding of the S-layer subunits to the rigid cell envelope layer (51).…”

“…By sequence comparison, S-layer-homologous (SLH) motifs (74) have been identified at the N-terminal part of many Slayer proteins (14,26,33,39,40,51,65,71,72,85,97,144) and at the C-terminal end of cell-associated exoenzymes (71,78,79) and other exoproteins (71, 73) of gram-positive bacteria. According to their origin (S-layer proteins, cell-associated exoproteins, porins), SLH motifs have been divided into three main groups whose specific properties have recently been reviewed by Engelhardt and Peters (38).…”

Phylogenetic Analysis and Heterologous Expression of Surface Layer Protein SlpC ofBacillus sphaericusC3-41

“…12,13) Although S-layers have been identified in some B. sphaericus and their structures and re-assembly characteristics have been described, 3,6,14) the occurrence of S-layers is not well documented in insect pathogenic B. sphaericus strains, and there is no report on the relationship between S-layer of pathogenic B. sphaericus and its toxicity against mosquito larvae.…”