Variation in the surface layer proteins of Clostridium difficile

Surface layers (S-layers) form regular crystalline structures on the outermost surface of many bacteria. Clostridium difficile possesses such an S-layer consisting of two protein subunits. Treatment of whole cells of C. difficile with 5 M guanidine hydrochloride revealed two major proteins of different molecular masses characteristic of the S-layer on SDS-PAGE. In this study 25 isolates were investigated. A high degree of variability in the molecular mass of the two S-layer proteins was evident. Molecular masses ranged from 48 to 56 kDa for the heavier protein and from 37 to 45 kDa for the lighter protein. A further protein component of 70 kDa was detectable in all isolates. No cross-reaction was seen between the two major proteins from isolates that produced different S-layer patterns, and most S-layer proteins from isolates with the same or similar banding patterns did not cross-react. The S-layer proteins, when detected by a combination of Coomassie blue staining and immunoblotting, are a useful marker for phenotyping.     

Fluorescence emission properties of S-Layer enhanced green fluorescent fusion protein as a function of temperature, pH conditions, and guanidine hydrochloride concentration

The fluorescent properties of the S-layer enhanced green fluorescent fusion protein (rSbpA31-1068/EGFP) were investigated as a function of temperature, pH conditions, and guanidine hydrochloride concentration. These results were compared to the fluorescent properties of the recombinant enhanced green fluorescent protein (EGFP) and an equimolar mixture of the S-layer protein rSbpA and EGFP. The intensity of the fluorescence emission of the EGFP at 510 nm, after excitation at 490 nm, is not affected by the presence of rSbpA, either as a fusion partner or as a free protein in solution. In each of the three protein systems, the emission intensity at 510 nm reaches its maximum value between pH 7 and 9 at 20 degrees C and at 0 M guanidine hydrochloride. No fluorescence could be measured at pH 4 and 6 M guanidine hydrochloride. These results show that the S-layer fusion protein (rSbpA31-1068/EGFP) is a suitable candidate for future applications in nanobiotechonology at a wide range of pH, temperature, and guanidine hydrochloride concentrations.     

Structural and chemical characterization of the S layer of a Pseudomonas-like bacterium.

Sections and freeze-fractured preparations showed an S layer on the surface of Pseudomonas-like strain EU2. Polyacrylamide gel electrophoresis of cell envelopes extracted with 1% sodium dodecyl sulfate (SDS) at room temperature showed three proteins (45K, 55K, and 110K). The 55K protein was identified as the S-layer protein. Incubation in 1.5 M guanidine hydrochloride removed the S layer from cell envelopes and dissociated the structure into subunits. The soluble 55K protein reassembled into planar sheets upon removal of the guanidine hydrochloride by dialysis. Electron microscopy and image processing indicated that these sheets had p4 symmetry in projection with a lattice constant of 13.2 +/- 0.1 nm (corresponding to 9.3 nm between adjacent fourfold axes). In some instances these reassemblies appeared to form small three-dimensional crystals which gave particularly clear views of the structure in projection because of the superimposition of information from a number of layers. A model is proposed with molecules having rounded lobes connected by a narrower linker region and joining at the lobes to form the fourfold axes of the array. The pattern superficially resembles those of other bacterial S layers, such as those of Aeromonas salmonicida, Aeromonas hydrophila, and Azotobacter vinelandii. Extraction of cell envelopes with 1% SDS at 50 degrees C released the 110K protein from the envelopes and removed an amorphous backing layer from the S layer. The 45K protein displayed heat-modifiable migration in SDS-polyacrylamide gel electrophoresis and was insoluble in SDS at 50 degrees C or in high concentrations of guanidine hydrochloride, suggesting that it was associated with the peptidoglycan.     

Characterization of a dynamic S layer on Bacillus thuringiensis.

The surfaces of three Bacillus thuringiensis strains possess an S layer composed of linear arrays of small particles arranged with p2 symmetry and with a = 8.5 nm, b = 7.2 nm, and gamma = 73 degrees. Platinum shadows of whole cells and S-layer fragments revealed the outer surface of the array to be smooth and the inner surface to be corrugated. Treatment with 2 M guanidine hydrochloride at pH 2.5 to 4 best removed the S layer for chemical characterization; it was a relatively hydrophilic 91.4-kilodalton protein with a pI of 5, no detectable carbohydrate, cysteine, methionine or tryptophan, and 21.2% nonpolar residues. No N-terminal homology with other S-layer proteins was evident. Antibody labeling experiments confirmed that the amount of S layer was proportional to the growth phase in broth cultures. Late-exponential- and stationary-growth-phase cells typically sloughed off fragments of S layer, and this may be the result of wall turnover. Indigenous autolytic activity in isolated walls rapidly digested the wall fabric, liberating soluble S-layer protein. At the same time, proteases frequently reduced the molecular weight of the 91.4-kilodalton protein, but these polypeptides could still be identified as S-layer components by immunoblotting. As cultures were serially subcultured, the frequency of appearance of the S layer diminished, and it was eventually lost. The dynamic nature of this S layer makes it atypical of most previously identified S layers and made it unusually difficult to characterize.     

Biochemical and molecular characterization of PepR, a dipeptidase, from Lactobacillus helveticus CNRZ32.

A dipeptidase with prolinase activity from Lactobacillus helveticus CNRZ32, which was designated PepR, was purified to gel electrophoretic homogeneity and characterized. The NH2-terminal amino acid sequence of the purified protein had 96% identity to the deduced NH2-terminal amino acid sequence of the pepR gene, which was previously designated pepPN, from L. helveticus CNRZ32. The purified enzyme hydrolyzed Pro-Met, Thr-Leu, and Ser-Phe as well as dipeptides containing neutral, nonpolar amino acid residues at the amino terminus. Purified PepR was determined to have a molecular mass of 125 kDa with subunits of 33 kDa. The isoelectric point of the enzyme was determined to be 4.5. The optimal reaction conditions, as determined with Pro-Leu as substrate, were pH 6.0 to 6.5 and 45 to 50 degrees C. The purified PepR had a Km of 4.9 to 5.2 mM and a Vmax of 260 to 270 mumol of protein per min/mg at pH 6.5 and 37 degrees C. The activity of purified PepR was inhibited by Zn2+ but not by other cations or cysteine, serine, aspartic, or metal-containing protease inhibitors or reducing agents. Results obtained by site-directed mutagenesis indicated that PepR is a serine-dependent protease. Gene replacement was employed to construct a PepR-deficient derivative of CNRZ32. This mutant did not differ from the wild-type strain in its ability to acidify milk. However, the PepR-deficient construct was determined to have reduced dipeptidase activity compared to the wild-type strain with all dipeptide substrates examined.     

Isolation and characterization of cytoplasmic fibrils from treponemes

Electron microscopy of Triton X-100-treated whole cells of an oral treponeme, Treponema sp. strain E-21, revealed that six cytoplasmic fibrils (CFs) helically wound as a bundle in the cytoplasm. The CFs were isolated and purified by disruption and solubilization of the cells followed by CsCl density gradient centrifugation. The purified CF preparation contained mostly fibrils of about 9 nm in width and very small amounts of thinner strands of about 3 nm in diameter. The CFs were apparently seen to be a tubular structure, but the isolated CFs had narrowed sites of about 4-5 nm in width lacking lumen-like images, possibly representing twisted sites. Thus, the CF did not seem to be a tubular structure. The purified CFs were composed of one major 82 kDa protein and a few minor proteins. The CFs were destructed by treatment with proteases, 8 M urea or 4 M guanidine hydrochloride. Very low tyrosine content (0.76 mol %) and lack of methionine were characteristic features for the 82 kDa protein. The CF preparations from the other five treponemes including Treponema phagedenis and T. denticola also had 82 kDa proteins as a major component, and the 82 kDa proteins of all of the treponemes had a common antigen when examined by using antiserum against the 82 kDa protein from Treponema sp. strain E-21. Furthermore, the 82 kDa protein was demonstrated to be a principal component of the CFs of all the treponemes by immunoelectron microscopy.     

Esterase from the oil-degrading Acinetobacter lwoffii RAG-1: Sequence analysis and over-expression in Escherichia coli

Abstract The antigenic properties of the surface layer (S-layer) proteins of various Campylobacter rectus strains including 24 clinical isolates and the type strain ATCC 33238 were examined. S-layer proteins were extracted from whole cells by acid treatment according to the method of McCoy et al. (Infect. Immun. 11, 517–525, 1975). The acid extracts from 23 of the isolates and ATCC 33238 contained two major proteins with molecular masses of 130 kDa and 150 kDa, both of which were identified as subunits of the S-layer after comparison with the protein profiles of acid-treated (S-layer-deficient) cells. An S-layer protein from one isolate (CI-808) demonstrated a different molecular mass (160 kDa). Both the 150-kDa proteins of ATCC 33238 and isolate CI-306 and the 160-kDa protein of CI-808 were purified by ion-exchange chromatography in the presence of urea. In Ouchterlony immunodiffusion experiments with these purified proteins and rabbit antiserum raised to each purified protein, both common and strain-specific antigenic determinants were identified in the C. rectus S-layer proteins.     

Isolation and characterization of a xylose-glucose isomerase from a new strain Streptomyces thermovulgaris 127, var. 7-86.

A thermostable D-xylose-glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by gamma-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE-Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12 columns. The N-terminal amino acid sequence and amino acid analysis shows 73-92% homology with xylose-glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12 column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60-85 degrees C at pH 7.0, using D-glucose, and up to 50-60 degrees C at pH 7.6, using D-xylose as substrates. The activation energy (Ea = 46 kJ x mol(-1)) and the critical temperature (Tc = 60 degrees C) were determined by fluorescence spectroscopy. Tc is in close coincidence with the melting temperature of denaturation (Tm = 59 degrees C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 k x mol(-1). The specific activity (km values) for D-xylose-glucose isomerase at 70 degrees C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, respectively.     

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.     

Denaturation of thymidylate synthetase from amethopterin-resistant Lactobacillus casei.

The effects of various concentrations of urea and guanidine hydrochloride on enzyme activity and on subunit association were determined. Incubation of thymidylate synthetase with buffered solutions of 3M to 3.5M guanidine hydrochloride or 5 M to 6 M urea resulted in the loss of about 90% of the enzyme activity. Under these denaturing conditions a red shift of the fluorescence emission maximum from 340 nm to 351 nm was observed together with a significant decrease in the relative fluorescence intensity of the protein. Studies at both 4 degrees C and 25 degrees C indicated that the enzyme was in the dimer form in 2 M guanidine hydrochloride but was dissociated into monomers in concentrations of this denaturant of 3 M and above. Although only monomeric species were evident at 4 degrees C in 6 M urea, at 25 25 degrees C this denaturant caused protein aggregation which increased with decreasing phosphate buffer concentration. Enzyme (5 mg/ml) in 0.5 M potassium phosphate buffer, pH 6.8, containing 4 M guanidine hydrochloride gave a minimum S20, w value of 1.22S at 25 degrees C. Sedimentation behavior of the native enzyme in the range of 5 to 20 mg/ml was only slightly concentration-dependent (4.28 S to 4.86 S) but extensive aggregation occurred above 20 mg/ml.     

Intrinsic viscosity of ovomucoid in random coil conformation.

Ovomucoid is denatured by concentrated solutions of guanidine hydrochloride. The intrinsic viscosities of the glycoprotein in 6 M guanidine hydrochloride in the absence and presence of beta-mercaptoethanol were found to be 8.1 and 16.0 ml/g, respectively. Ovomucoid with disulphide bonds reduced exists in linear random coil conformation. However, the intrinsic viscosity of the randomly coiled protein was less than that predicted from the empirical equations describing the molecular weight dependence of intrinsic viscosities of random coil proteins in 6 M guanidine hydrochloride. On excluding the carbohydrate content of the protein, which is theoretically justified, the calculated intrinsic viscosity interestingly became closer to the measured one. The temperature dependence of the intrinsic viscosity of ovomucoid in linear random coil conformation was studied in the temperature range, 25-55 degrees. The features of the intrinsic viscosity-temperature profile are not comparable with those exhibited by other linear random coil proteins in 6 M guanidine hydrochloride.     

干酪乳杆菌LC2W表面黏附相关蛋白的提取与鉴定

目的提取和鉴定干酪乳杆菌LC2W表面黏附相关蛋白,初步探索LC2W对胃癌细胞MKN-45细胞的黏附机制。方法LiCl处理、Sephadex G-75柱层析分离提取LC2W的表面蛋白,用黏附试验、电镜观察和SDS-PAGE电泳进行黏附相关蛋白的鉴定。结果LC2W经LiCl处理后,扫描电镜结果发现菌体表面粗糙但仍完整,黏附试验表明其对MKN-45细胞的黏附能力显著降低。提取到的表面蛋白的分子量分别为41.6、63.5、66.2 kDa。粗提物经柱层析后发现分子量为41.6 kDa的组分可以明显增强经LiCl处理过的菌体的黏附,而与未经处理的菌体黏附情况类似。结论表面蛋白参与了LC2W对MKN-45细胞的黏附,其主要活性成分的分子量为41.6 kDa。     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli.

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

Structural studies of apolipoprotein B: physical properties of the protein in guanidine hydrochloride

Apolipoprotein B was isolated from human plasma low-density-lipoprotein without precipitation by diethyl ether/ethanol extraction of the protein in 6 M guanidine hydrochloride. The physical properties of this protein, which contained a residuum of approximately 7% phospholipid, were examined in 6 M guanidine solution under reducing conditions. The circular dichroism spectrum was indistinguishable from that of a random coil protein. Sedimentation equilibrium analyses of apolipoprotein B by the meniscus depletion method of Yphantis (1984, Biochemistry 3, 297-317) were complicated by heterogeneity and nonideality despite the low concentrations employed. 63 analyses of the weight average (Mw) and z average (Mz) molecular weight were made on the apolipoprotein B from 12 subjects. The Mw observed was a function of initial concentration, rotor speed, and a heterogeneity index (Mz/Mw). Multiple linear regression of apolipoprotein B molecular mass against these parameters suggested that an Mw of 540,000 +/- 110,000 would be observed under apparently ideal and homogeneous conditions. The sedimentation coefficient and intrinsic viscosity of the reduced protein at 25 degrees C in 6 M guanidine were 2.13 S and 116 ml/g, respectively; these values predict molecular weights of 640,000 and 250,000, respectively, if apolipoprotein B was fully denatured into a random coil. Lack of agreement between these estimates and with the sedimentation equilibrium analysis can best be explained by compactness of structure and incomplete denaturation to a random coil state. Furthermore, an irreversible temperature dependence of apolipoprotein B reduced viscosity indicated that residual structure remained in solutions of 6 M guanidine hydrochloride/20 mM dithiothreitol. Taken together, the physical data demonstrate that apolipoprotein is a single polypeptide of approximately 540 kDa, whose structure resists denaturation under conditions where most proteins exist as random coils.     

Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis

A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 m guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nm), cathepsin L (K(i) = 0.41 nm), cathepsin B (K(i) = 0.48 micrometer), and bromelain (K(i) = 0.16 micrometer) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).     

Genes and derived amino acid sequences of S-layer proteins from mesophilic,thermophilic, and extremely thermophilic methanococci

Cells of methanococci are covered by a single layer of protein subunits (S-layer) in hexagonal arrangement, which are directly exposed to the environment and which cannot be stabilized by cellular components. We have isolated S-layer proteins from cells of Methanococcus vannielii ( T(opt.)=37 degrees C), Methanococcus thermolithotrophicus ( T(opt.)=65 degrees C), and Methanococcus jannaschii ( T(opt.)=85 degrees C). The primary structure of the S-layer proteins was determined by sequencing the corresponding genes. According to the predicted amino acid sequence, the molecular masses of the S-layer proteins of the different methanococci are in a small range between 59,064 and 60,547 Da. Compared with its mesophilic counterparts, it is worth noting that in the S-layer protein of the extreme thermophile Mc. jannaschii the acidic amino acid Asp is predominant, the basic amino acid Lys occurs in higher amounts, and Cys and His are only present in this organism. Despite the differences in the growth optima and the predominance of some amino acids, the comparative total primary structure revealed a relatively high degree of identity (38%-45%) between the methanococci investigated. This observation indicates that the amino acid sequence of the S-layer proteins is significantly conserved from the mesophilic to the extremely thermophilic methanococci.     

Isolation and characterization of two glycoproteins from hyaline cartilage

Two acidic glycoproteins of molecular mass 92 kDa and 56 kDa were purified from 4 M guanidine hydrochloride extracts of chick sternal cartilage, by density gradient centrifugation, ion-exchange chromatography, gel chromatography and SDS/PAGE. The glycoproteins differed in their amino acid and carbohydrate compositions. They were identified by the immunoblotting technique in extracts of chick articular cartilage from various sites and in extracts of cartilage from other species. The proteins are synthesized by the chondrocytes and show a partial cross-reactivity between their antisera.     

苏云金芽胞杆菌CTC菌株的S-层蛋白可以形成伴胞晶体

苏云金芽胞杆菌(Bacillus thuringiensis)CTC菌株产生卵圆形伴胞晶体,晶体蛋白分子量为100kD;透射电子显微镜观察结果表明该菌株有S—层结构,而且在母细胞内可以形成伴胞晶体和S—层的初体结构;其蛋白基因导入苏云金芽胞杆菌无晶体突变株BMB171后,扫描电子显微镜观察结果表明转化子能形成晶体,而其形状与CTC菌株的相同;转化子晶体蛋白的分子量大小也与CTC菌株的相同,为100kD。以上实验结果结合以前晶体蛋白N—末端测序和基因核苦酸序列,表明苏云金芽胞杆菌CTC菌株的S—层蛋白可以形成伴胞晶体。     

Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92

AIMS: To investigate the functional role of surface layer proteins (S-layer) in probiotic strain Lactobacillus acidophilus M92, especially its influence on adhesiveness to mouse ileal epithelial cells. METHODS AND RESULTS: Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, ca at 45 kDa in L. acidophilus M92. Southern blot with pBK1 plasmid, containing slpA gene, gave a positive signal, suggesting that L. acidophilus M92 has a slpA gene coding for the S-layer proteins. S-layer proteins of this strain are present during all phases of growth. The S-layer proteins appeared when cells treated with 5 mol l(-1) LiCl were allowed to grow again. Removal of the S-layer proteins reduced adhesion of L. acidophilus M92 to mouse ileal epithelial cells. Furthermore, the viability of cells without S-layer were reduced in simulated gastric juice at low pH range (2, 2.5, 3) and simulated pancreatic juice with bile salts (1.5 and 3 g l(-1)). S-layer proteins of L. acidophilus M92 were resistant to pepsin and pancreatin, in contrast, the treatment with proteinase K led to a significant proteolysis of the S-layer proteins. CONCLUSIONS: These results demonstrated functional role of S-layer; it is responsible for adhesiveness of Lactobacillus acidophilus M92 to mouse ileal epithelial cells and has a protective role for this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: S-layer proteins have an important role in the establishment of probiotic strain Lactobacillus acidophilus M92 in the gastrointestinal tract.     

Recombinant agrobacterium AgaE-like protein with fructosyl amino acid oxidase activity

Agrobacterium tumefaciens AgaE-like protein had a similar sequence to that of a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1. To characterize the AgaE-like protein, we produced the enzyme in Escherichia coli, and purified it to homogeneity. The molecular mass of recombinant AgaE-like protein was 42 kDa on SDS-PAGE and 85 kDa on gel filtration. The protein acted on N-fructosyl valine and N-fructosyl glycine as substrates, but not on glycated protein or N(epsilon)-fructosyl lysine. Apparent Km for N-fructosyl valine and N-fructosyl glycine were 1.64 and 0.31 mM, respectively. The AgaE-like protein had maximum activity at pH 7.8 and 35 degrees C in 0.1 M potassium phosphate, but more than 80% of its activity was lost at 40 degrees C or more. In contrast to eukaryotic fructosyl amino acid oxidases, the AgaE-like protein contained noncovalently bound FAD as a cofactor and was inactive against N(epsilon)-fructosyl N(alpha)-Z(benzyloxycarbonyl)-lysine. These characteristics were similar to a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1, suggesting that these prokaryotic enzymes comprise a new family of fructosyl amino acid oxidases.