Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface.

The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned in Escherichia coli, and the nucleotide sequence was determined. The reading frame of amyB consisted of 5,586 bp encoding an exceptionally large enzyme of 205,991 Da. Sequence analysis revealed a composite structure of the pullulanase consisting of catalytic and noncatalytic domains. The N-terminal half of the protein contained a leader peptide of 35 amino acid residues and the catalytic domain, which included the four consensus regions of amylases. Comparison of the consensus regions of several pullulanases suggested that enzymes like pullulanase type II from T. thermosulfurigenes EM1 which hydrolyze alpha-1,4- and alpha-1,6-glycosidic linkages have specific amino acid sequences in the consensus regions. These are different from those of pullulanases type I which only cleave alpha-1,6 linkages. The C-terminal half, which is not necessary for enzymatic function, consisted of at least two different segments. One segment of about 70 kDa contained two copies of a fibronectin type III-like domain and was followed by a linker region rich in glycine, serine, and threonine residues. At the C terminus, we found three repeats of about 50 amino acids which are also present at the N-termini of surface layer (S-layer) proteins of, e.g., Thermus thermophilus and Acetogenium kivui. Since the pullulanase of T. thermosulfurigenes EM1 is known to be cell bound, our results suggest that this segment serves as an S-layer anchor to keep the pullulanase attached to the cell surface. Thus, a general model for the attachment of extracellular enzymes to the cell surface is proposed which assigns the S-layer a new function and might be widespread among bacteria with S-layers. The triplicated S-layer-like segment is present in several enzymes of different bacteria. Upstream of amyB, another open reading frame, coding for a hypothetical protein of 35.6 kDa, was identified. No significant similarity to other sequences available in DNA and protein data bases was found.     

Domain structure of the Acetogenium kivui surface layer revealed by electron crystallography and sequence analysis.

The three-dimensional structure of the Acetogenium kivui surface layer (S-layer) has been determined to a resolution of 1.7 nm by electron crystallographic techniques. Two independent reconstructions were made from layers negatively stained with uranyl acetate and Na-phosphotungstate. The S-layer has p6 symmetry with a center-to-center spacing of approximately 19 nm. Within the layer, six monomers combine to form a ring-shaped core surrounded by a fenestrated rim and six spokes that point towards the axis of threefold symmetry and provide lateral connectivity to other hexamers in the layer. The structure of the A. kivui S-layer protein is very similar to that of the Bacillus brevis middle wall protein, with which it shares an N-terminal domain of homology. This domain is found in several other extracellular proteins, including the S-layer proteins from Bacillus sphaericus and Thermus thermophilus, Omp alpha from Thermotoga maritima, an alkaline cellulase from Bacillus strain KSM-635, and xylanases from Clostridium thermocellum and Thermoanaerobacter saccharolyticum, and may serve to anchor these proteins to the peptidoglycan. To our knowledge, this is the first example of a domain conserved in several S-layer proteins.     

Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface.

The nucleotide sequence was determined for a 9.4-kb region of Clostridium thermocellum DNA extending from the 3' end of the gene (now termed cipA), encoding the S1/SL component of the cellulosome. Three open reading frames (ORFs) belonging to two operons were detected. They encoded polypeptides of 1,664, 688, and 447 residues, termed ORF1p, ORF2p, and ORF3p, respectively. The COOH-terminal regions of the three polypeptides were highly similar and contained three reiterated segments of 60 to 70 residues each. Similar segments have been found at the NH2 terminus of the S-layer proteins of Bacillus brevis and Acetogenium kivui, suggesting that ORF1p, ORF2p, and ORF3p might also be located on the cell surface. Otherwise, the sequence of ORF1p and ORF2p gave little clue concerning their potential function. However, the NH2-terminal region of ORF3p was similar to the reiterated domains previously identified in CipA as receptors involved in binding the duplicated segment of 22 amino acids present in catalytic subunits of the cellulosome. Indeed, it was found previously that ORF3p binds 125I-labeled endoglucanase CelD containing the duplicated segment (T. Fujino, P. Béguin, and J.-P. Aubert, FEMS Microbiol. Lett. 94:165-170, 1992). These findings suggest that ORF3p might serve as an anchoring factor for the cellulosome on the cell surface by binding the duplicated segment that is present at the COOH end of CipA.     

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988).     

Evidence for tyrosine-linked glycosaminoglycan in a bacterial surface protein.

The S-layer protein of Acetogenium kivui was subjected to proteolysis with different proteases and several high molecular mass glycosaminoglycan peptides containing glucose, galactosamine and an unidentified sugar-related component were separated by molecular sieve chromatography and reversed-phase HPLC and subjected to N-terminal sequence analysis. By methylation analysis glucose was found to be uniformly 1,6-linked, whereas galactosamine was exclusively 1,4-linked. Hydrazinolysis and subsequent amino-acid analysis as well as two-dimensional NMR spectroscopy were used to demonstrate that in these peptides carbohydrate was covalently linked to tyrosine. As all of the four Tyr-glycosylation sites were found to be preceded by valine, a new recognition sequence for glycosylation is suggested.     

Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies.

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate [HPI])-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.     

Characterization of the genes for the hexagonally arranged surface layer proteins in protein-producing Bacillus brevis 47: complete nucleotide sequence of the middle wall protein gene.

Bacillus brevis 47 contains two surface (S)-layer proteins, termed the outer wall protein (OWP) and the middle wall protein (MWP), which form a hexagonal array in the cell wall. The MWP and OWP genes are contained in the 9-kilobase-pair (kbp) BclI fragment and constitute an operon under coordinate control of their expression. The nucleotide sequence of a 3.8-kbp EcoRI-SacI fragment containing the entire MWP gene has been determined in this study. Together with the DNA sequence of the promoter region for the MWP-OWP gene operon (H. Yamagata, T. Adachi, A. Tsuboi, M. Takao, T. Sasaki, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 169:1239-1245, 1987) and that of the OWP gene (A. Tsuboi, R. Uchihi, R. Tabata, Y. Takahashi, H. Hashiba, T. Sasaki, H. Yamagata, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 168:365-373, 1986), the complete nucleotide sequence of the MWP-OWP gene operon has been determined. The MWP gene encodes a secretory precursor of the MWP, consisting of a total of 1,053 amino acid residues with a signal peptide of 23 amino acid residues at its amino-terminal end. Bacillus subtilis harboring the MWP gene synthesized an immunoreactive polypeptide with almost the same molecular weight as the authentic MWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid compositions deduced from the MWP and OWP genes were similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic amino acids compared with basic amino acids and in the very low content of sulfur-containing amino acids. The acidic nature of the MWP and OWP was confirmed by isoelectric focusing on polyacrylamide gels. In addition, circular dichroism spectra indicated that the S-layer proteins in B. brevis 47 were composed of approximately 30% beta-sheet and 5% alpha-helical structures, with the remainder of the polypeptide backbone being aperiodic in nature.     

Characterization of an S-layer glycoprotein produced in the course of S-layer variation of<Emphasis Type="Italic"> Bacillus stearothermophilu</Emphasis>s ATCC 12980 and sequencing and cloning of the<Emphasis Type="Italic"> sbsD</Emphasis> gene encoding the protein moiety

The cell surface of Bacillus stearothermophilus ATCC 12980 is completely covered by an oblique lattice which consists of the S-layer protein SbsC. On SDS-polyacrylamide gels, the mature S-layer protein migrates as a single band with an apparent molecular mass of 122 kDa. During cultivation of B. stearothermophilus ATCC 12980 at 67 degrees C instead of 55 degrees C, a variant developed that had a secondary cell wall polymer identical to that of the wild-type strain, but it carried an S-layer glycoprotein that could be separated on SDS-polyacrylamide gels into four bands with apparent molecular masses of 92, 118, 150 and 175 kDa. After deglycosylation, only a single protein band with a molecular mass of 92 kDa remained. The complete nucleotide sequence encoding the protein moiety of this S-layer glycoprotein, termed SbsD, was established by PCR and inverse PCR. The sbsD gene of 2,709 bp is predicted to encode a protein of 96.2 kDa with a 30-amino-acid signal peptide. Within the 807 bp encoding the signal peptide and the N-terminal sequence (amino acids 31-269), different nucleotides for sbsD and sbsC were observed in 46 positions, but 70% of these mutations were silent, thus leading to a level of identity of 95% for the N-terminal parts. The level of identity of the remaining parts of SbsD and SbsC was below 10%, indicating that the lysine-, tyrosine- and arginine-rich N-terminal region in combination with a distinct type of secondary cell wall polymer remained conserved upon S-layer variation. The sbsD sequence encoding the mature S-layer protein cloned into the pET28a vector led to stable expression in Escherichia coli HMS174(DE3). This is the first example demonstrating that S-layer variation leads to the synthesis of an S-layer glycoprotein.     

Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.     

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

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. A sequence characteristic of promoters expressed during vegetative growth was found within a 177-base-pair region upstream from the ORF coding for the 125-kDa protein. This putative promoter may account for the expression of this gene during the vegetative growth of B. sphaericus and E. coli. The gene for the 125-kDa protein was followed by an inverted repeat characteristic of terminators. Downstream from this gene (11.2 kilobases) was an ORF coding for a putative 80-kDa protein having a high sequence similarity to the 125-kDa protein. Evidence was presented indicating that this gene is cryptic.     

Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis

Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliC(H7) gene of Escherichia coli. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of L. brevis ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies.     

The regular surface layer of Acetogenium kivui: some structural, developmental and evolutionary aspects

Abstract The structure of the regular surface layer of Acetogenium kivui has been investigated by electron microscopy in conjunction with image processing techniques. From the averaged unit cell structure a tentative model of the subunit organization is derived. The occurrence of mono- and polycrystalline S-layer sheets is interpreted in terms of transitions related to the growth cycle of cell. The question is raised as to whether the striking structural similarity of S-layers, even from evolutionary rather distant species, is due to divergent or convergent evolution.     

The S-layer protein from Campylobacter rectus: sequence determination and function of the recombinant protein

The gene encoding the crystalline surface layer (S-layer) protein from Campylobacter rectus , designated slp , was sequenced and the recombinant gene product was expressed in Escherichia coli . The gene consisted of 4086 nucleotides encoding a protein with 1361 amino acids. The N-terminal amino acid sequence revealed that Slp did not contain a signal sequence, but that the initial methionine residue was processed. The deduced amino acid sequence displayed some common characteristic features of S-layer proteins previously reported. A homology search showed a high similarity to the Campylobacter fetus S-layer proteins, especially in their N-terminus. The C-terminal third of Slp exhibited homology with the RTX toxins from Gram-negative bacteria via the region including the glycine-rich repeats. The Slp protein had the same N-terminal sequence as a 104-kDa cytotoxin isolated from the culture supernatants of C. rectus . However, neither native nor recombinant Slp showed cytotoxicity against HL-60 cells or human peripheral white blood cells. These data support the idea that the N-terminus acts as an anchor to the cell surface components and that the C-terminus is involved in the assembly and/or transport of the protein.     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

Nucleotide sequence of cloned cDNA coding for pumpkin 11-S globulin beta subunit

cDNA coding for preproglobulin beta, a precursor protein of 11-S globulin beta subunit, was cloned and the nucleotide sequence has been determined. The sequence covers the whole coding region (1440 base pairs) with 5' and 3' noncoding region (30 and 214 base pairs, respectively). The deduced amino acid sequence of preproglobulin beta consists of a 21-amino-acid N-terminal signal peptide, preceding the acidic gamma polypeptide region (275 amino acids) and the subsequent basic delta region (184 amino acids). The site for post-translational cleavage of the precursor polypeptide to make the gamma and delta chains is estimated to be located between the asparagine-glycine residues. The N-terminal amino acid of the gamma chain of mature 11-S globulin beta subunit was reported to be blocked by 5-oxoproline (pyroglutamic acid) [Ohmiya et al. (1980) Plant Cell Physiol. 21, 157-167]. It was shown that the blocked N-terminal amino acid is coded as a glutamine residue. The derived amino acid sequence was also compared with those of precursor proteins of other 11-S globulins such as soybean glycinin, cotton beta globulin, pea legumin and rape 11-S globulin by dot matrix analysis.     

Characterization and regulation of a second gene encoding thioredoxin from the fission yeast

A genomic DNA encoding a second thioredoxin (TRX2) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The cloned sequence contains 1823 bp and encodes a protein of 121 amino acids. It has extra N-terminal 17 amino acid residues compared to previously identified thioredoxin (TRX1), which are positively charged and hydrophobic amino acids. The additional N-terminal region contains a plausible prepeptidase cleavage site, indicating that the TRX2 protein exists in mitochondria. The cloned TRX2 gene produced functional TRX estimated with insulin reduction assay. The upstream region of the TRX2 gene was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357R. The 782 bp sequence in the region further upstream of the TRX2 gene was found to be inhibitory in its expression. Synthesis of beta-galactosidase from the fusion plasmid pYFX135-HRL was enhanced by the addition of aluminum chloride and ferrous chloride, indicating that the TRX2 protein is involved in stress response.     

Molecular characterization of the S-layer gene,sbpA, of Bacillus sphaericus CCM 2177 and production of a functional S-layer fusion protein with the ability to recrystallize in a defined orientation while presenting the fused allergen

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5' end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA(31-1068)). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA(31-1068). Labeling of the square S-layer lattice formed by recrystallization of rSbpA(31-1068)/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.