The cell surfaces of several Lactobacillus species are covered by a regular layer composed of a single species of protein, the S-protein. The 43-kDa S-protein of the neotype strain Lactobacillus acidophilus ATCC 4356, which originated from the pharynx of a human, was purified. Antibodies generated against purified S-protein were used to screen a lambda library containing chromosomal L. acidophilus ATCC 4356 DNA. Several phages showing expression of this S-protein in Escherichia coli were isolated. A 4.0-kb DNA fragment of one of those phages hybridized to a probe derived from an internal tryptic fragment of the S-protein. The slpA gene, coding for the surface layer protein, was located entirely on the 4.0-kb fragment as shown by deletion analysis. The nucleotide sequence of the slpA gene was determined and appeared to encode a protein of 444 amino acids. The first 24 amino acids resembled a putative secretion signal, giving rise to a mature S-protein of 420 amino acids (44.2 kDa). The predicted isoelectric point of 9.4 is remarkably high for an S-protein but is in agreement with the data obtained during purification. The expression of the entire S-protein or of large, C-terminally truncated S-proteins is unstable in E. coli.

SummaryThe function of the S-layer, a regularly arranged structure on the outside of numerous bacteria, appears to be different for bacteria living in different environments. Almost no similarity exists between the primary sequences of S-proteins, although their amino acid composition is comparable. S-protein production is directed by single or multiple promoters in front of the S-protein gene, yielding stable mRNAs. Most bacteria secrete S-proteins via the general secretory pathway (GSP). Translocation of S-protein across the outer membrane of Gram-negative bacteria sometimes occurs by S-protein-specific branches of the GSP. O-polysaccharide side-chains of the lipopolysaccharide component of the cell wall of Gramnegative bacteria appear to function as receptors for attachment of the S-layer. Silent S-protein genes have been found in Campylo-bacter fetus and Lactobacillus acidophilus. These silent genes are placed in the expression site in a fraction of the bacterial population via inversion of a chromosomal segment.

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The bacterial S-layer forms a regular structure, composed of a monolayer of one (glyco)protein, on the surfaces of many prokaryotic species. S-layers are reported to fulfil different functions, such as attachment structures for extracellular enzymes and major virulence determinants for pathogenic species. Lactobacillus acidophilus ATCC 4356, which originates from the human pharynx, possesses such an S-layer. No function has yet been assigned to the S-layer of this species. Besides the structural gene (slpA) for the S-layer protein (S-protein) which constitutes this S-layer, we have identified a silent gene (slpB), which is almost identical to slpA in two regions. From the deduced amino acid sequence, it appears that the mature S B -protein (44,884 Da) is 53% similar to the S A -protein (43,636 Da) in the N-terminal and middle parts of the proteins. The C-terminal parts of the two proteins are identical except for one amino acid residue. The physical properties of the deduced S-proteins are virtually the same. Northern (RNA) blot analysis shows that only the slpA gene is expressed in wild-type cells, in line with the results from sequencing and primer extension analyses, which reveal that only the slpA gene harbors a promoter, which is located immediately upstream of the region where the two genes are identical. The occurrence of in vivo chromosomal recombination between the two S-protein-encoding genes will be described elsewhere.Up to 300 different species of eubacteria and archaebacteria have been reported to be covered with a regular structure, the S-layer. This S-layer is built up entirely of one species of (glyco)protein known as the S-layer protein (S-protein) (for reviews, see references 2 and 23). Despite the common property of forming a regular layer on the outside of the bacteria, not much overall similarity between the primary structures of S-proteins can be identified. No general function seems to be associated with the presence of an S-layer, as S-layers of different species are reported to have different functions, e.g., as a cell shape determinant in Thermoproteus tenax (41), a phage receptor-masking layer in Aquaspirillum serpens (15), a major determinant for virulence in Aeromonas salmonicida (13), and an attachment structure for amylase in Bacillus stearothermophilus (11).Several but not all species of the genus Lactobacillus possess an S-layer (21). The S-proteins of Lactobacillus helveticus (19) and Lactobacillus buchneri and Lactobacillus plantarum (24) have been characterized, and the genes of two Lactobacillus species, Lactobacillus brevis (38) and Lactobacillus acidophilus ATCC 4356 (5), have been cloned and sequenced. Lactobacilli are gram-positive bacteria which play a major role in human and animal food production. For many years it has been believed that some Lactobacillus strains in the gastrointestinal and female urogenital tracts have a beneficial effect on human and animal health and thus may be used for therapeutic purposes. Several explanations for this effect are possible, e.g....

The capsid proteins VP2 and VP3 of infectious bursal disease virus, a birnavirus, are derived from the processing of a large polyprotein: NH2-pVP2-VP4-VP3-COOH. Although the primary cleavage sites at the pVP2-VP4 and VP4-VP3 junctions have been identified, the proteolytic cascade involved in the processing of this polyprotein is not yet fully understood, particularly the maturation of pVP2. By using different approaches, we showed that the processing of pVP2 (residues 1 to 512) generated VP2 and four small peptides (residues 442 to 487, 488 to 494, 495 to 501, and 502 to 512). We also showed that in addition to VP2, at least three of these peptides (residues 442 to 487, 488 to 494, and 502 to 512) were associated with the viral particles. The importance of the small peptides in the virus cycle was assessed by reverse genetics. Our results showed that the mutants lacking the two smaller peptides were viable, although the virus growth was affected. In contrast, The birnaviruses are a family of small icosahedral viruses infecting insects, fish, and birds (15). Only five proteins, generally referred to as VP1, VP2, VP3, VP4, and VP5, are encoded by the viral genome. The Tϭ13 icosahedral birnavirus capsids are made by the VP2 and VP3 proteins. They contain the two double-stranded RNA genomic segments (A and B) and the VP1 protein, a putative RNA-dependent RNA polymerase. Translation of genomic segment A yields a polyprotein, pVP2-VP4-VP3, and a small protein, VP5, of unknown function. The B segment encodes VP1. The polyprotein processing gives rise to VP4, the viral protease, and VP2 and VP3. VP2 carries all the neutralizing epitopes, suggesting that it is at least partly exposed at the outer surface of the capsid. VP3, which interacts with VP1 (16, 24), is thought to be located on the inner surface of the capsid (6). VP3 contains charged residues at its carboxy-terminal domain, a domain suggested to be involved in the genomic RNA interaction. As found for other virus families, the capsid assembly seems to be regulated by polyprotein processing.The infectious bursal disease virus (IBDV), an avian birnavirus, is of major importance to the poultry industry. It causes an immunosuppressive disease in young chickens. After infection, IBDV multiplies rapidly in the B lymphocytes of the bursa of Fabricius, leading to increased susceptibility to other diseases. Very virulent strains have resulted in high rates of mortality in many countries.The first step governing the IBDV capsid assembly is the autoproteolytic cleavage of the polyprotein (1,012 amino acids). This process generates pVP2, VP4, and VP3. The pVP2-to-VP2 conversion involves several proteolytic cleavages at the carboxy end of pVP2 (1,14,20). Based on mutagenesis studies, the putative cleavage site was proposed as defined by the (Thr/ Ala)-X-Ala2Ala motif; three potential sites are present in the C-terminal domain of pVP2 (14).In the present study, we further analyzed the maturation process of VP2. By using mass spectrometry and N-terminal sequence analysis, we s...