Relevance of charged groups for the integrity of the S-layer from Bacillus coagulans E38-66 and for molecular interactions.

In this paper, the importance of charged amino and carboxyl groups for the integrity of the cell surface layer (S-layer) lattice from Bacillus coagulans E38-66 and for the self-assembly of the isolated subunits was investigated. Amidination of the free amino groups which preserved their positive net charge had no influence on both. On the other hand, acetylation and succinylation, which converted the amino groups into either neutral or negatively charged groups, and amidation of carboxyl groups were accompanied by the disintegration or at least by the loss of the regular structure of the S-layer lattice. Treatment of S-layer monolayers with the zero-length cross-linker carbodiimide led to the introduction of peptide bonds between activated carboxyl groups and amino groups from adjacent subunits. This clearly indicated that in the native S-layer lattice the charged groups are located closely enough for direct electrostatic interactions. Under disrupting conditions in which the S-layer polypeptide chains were unfolded, 58% of the Asx and Glx residues could be amidated, indicating that they occur in the free carboxylic acid form. As derived from chemical modification of monolayer self-assembly products, about 90% of the lysine and 70% of the aspartic and glutamic acid residues are aligned on the surface of the S-layer protein domains. This corresponded to 45 amino groups and to 63 carboxyl groups per S-layer subunit. Labelling experiments with macromolecules with different sizes and charges and adsorption studies with ion-exchange particles revealed a surplus of free carboxyl groups on the inner and on the outer faces of the S-layer lattice. Since the carboxyl groups on the outer S-layer face were accessible only for protein molecules significantly smaller then the S-layer protomers or for positively charged, thin polymer chains extending from the surface of ion-exchange beads, the negatively charged sites must be located within indentations of the corrugated S-layer protein network. This was in contrast to the carboxyl groups on the inner S-layer face, which were found to be exposed on elevations of the S-layer protein domains (D. Pum, M. Sára, and U.B. Sleytr, J. Bacteriol. 171:5296-5303, 1989).     

Complete structure of the tyrosine-linked saccharide moiety from the surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70.

In this study, we have extended and completed a previous investigation (P. Messner, R. Christian, J. Kolbe, G. Schulz, and U. B. Sleytr, J. Bacteriol. 174:2236-2240, 1992) in which we demonstrated for the first time in prokaryotic organisms the presence of a novel O-glycosidic linkage via tyrosine. The surface layer glycoprotein of the eubacterium Clostridium thermohydrosulfuricum S102-70 is arranged in a hexagonal lattice, with center-to-center spacings of approximately 16.3 nm. Molecular weight determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both glycosylated and chemically deglycosylated surface layer glycoprotein showed values for the monomeric subunits of 94,000 and 87,500, respectively. Glycopeptide fractions obtained after exhaustive pronase digestion of purified, intact glycoprotein were isolated by reversed-phase liquid chromatography. One- and two-dimensional nuclear magnetic resonance studies, together with chemical analyses and plasma desorption time-of-flight mass spectrometry, were used to elucidate the structure of the hexasaccharide moiety linked by the novel O-glycosidic linkage to tyrosine. The combined evidence suggests the following structure: beta-D-Galf-(1-->3)-alpha-D-Galp- (1-->2)-alpha-L-Rhap-(1-->3)-alpha-D-Manp-(1--3)-alpha-L- Rhap-(1-->3)-beta- D-Glcp-(1-->4)-L-Tyr.     

Large-scale recrystallization of the S-layer of Bacillus coagulans E38-66 at the air/water interface and on lipid films.

S-layer protein isolated from Bacillus coagulans E38-66 could be recrystallized into large-scale coherent monolayers at an air/water interface and on phospholipid films spread on a Langmuir-Blodgett trough. Because of the asymmetry in the physiochemical surface properties of the S-layer protein, the subunits were associated with their more hydrophobic outer face with the air/water interface and oriented with their negatively charged inner face to the zwitterionic head groups of the dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine (DPPE) monolayer films. The dynamic crystal growth at both types of interfaces was first initiated at several distant nucleation points. The individual monocrystalline areas grew isotropically in all directions until the front edge of neighboring crystals was met. The recrystallized S-layer protein and the S-layer-DPPE layer could be chemically cross-linked from the subphase with glutaraldehyde.     

Structure, surface charge, and self-assembly of the S-layer lattice from Bacillus coagulans E38-66.

In freeze-etched preparations, whole cells from Bacillus coagulans E38-66 exhibited an oblique S-layer lattice (a = 9.4 nm; b = 7.4 nm; gamma = 80 degrees). The three-dimensional structure of the crystalline array was characterized by optical and computer image analysis. The lattice showed two distinctly shaped types of pores. In vitro self-assembly of isolated subunits yielded flat sheets and open-ended cylinders composed of two back-to-back monolayers. Unlike whole cells, in vitro self-assembly products were capable of binding polycationized ferritin (pI, approximately 11). This showed that only the inner S-layer face adhering to the peptidoglycan-containing layer in whole cells was net negatively charged. S-layer monomers and/or oligomers were capable of generating a closed monolayer with oblique symmetry on poly-L-lysine-coated supports. The monolayer had a typical crazy paving appearance, with numerous crystal boundaries. The handedness of the oblique lattice and ability to bind polycationized ferritin revealed that the subunits had bound with the outer, not net negatively charged face to the poly-L-lysine-coated supports. Carbodiimide-activated carboxyl groups on either cell wall fragments or self-assembly products could covalently bind high-molecular-weight nucleophiles such as ferritin. This confirmed the location of negatively charged carboxyl groups on the outermost surface of both S-layer faces. The difference in pH optimum for carbodiimide activation indicated a preponderance of alpha- and beta-carboxyl groups on the inner S-layer face and a preponderance of beta- and gamma-carboxyl groups on the outer S-layer face.     

The effect of S-layer protein adsorption and crystallization on the collective motion of a planar lipid bilayer studied by dynamic light scattering.

A dedicated dynamic light scattering (DLS) setup was employed to study the undulations of freely suspended planar lipid bilayers, the so-called black lipid membranes (BLM), over a previously inaccessible spread of frequencies (relaxation times ranging from 10(-2) to 10(-6) s) and wavevectors (250 cm(-1) < q < 38,000 cm(-1)). For a BLM consisting of 1,2-dielaidoyl-sn-3-glycero-phosphocholine (DEPC) doped with two different proportions of the cationic lipid analog dioctadecyl-dimethylammonium bromide (DODAB) we observed an increase of the lateral tension of the membrane with the DODAB concentration. The experimentally determined dispersion behavior of the transverse shear mode was in excellent agreement with the theoretical predictions of a first-order hydrodynamic theory. The symmetric adsorption of the crystalline bacterial cell surface layer (S-layer) proteins from Bacillus coagulans E38-66 to a weakly cationic BLM (1.5 mol % DODAB) causes a drastic reduction of the membrane tension well beyond the previous DODAB-induced tension increase. The likely reason for this behavior is an increase of molecular order along the lipid chains by the protein and/or partial protein penetration into the lipid headgroup region. S-layer protein adsorption to a highly cationic BLM (14 mol % DODAB) shows after 7 h incubation time an even stronger decrease of the membrane tension by a factor of five, but additionally a significant increase of the (previously negligible) surface viscosity, again in excellent agreement with the hydrodynamic theory. Further incubation (24 h) shows a drastic increase of the membrane bending energy by three orders of magnitude as a result of a large-scale, two-dimensional recrystallization of the S-layer proteins at both sides of the BLM. The results demonstrate the potential of the method for the assessment of the different stages of protein adsorption and recrystallization at a membrane surface by measurements of the collective membrane modes and their analysis in terms of a hydrodynamic theory.     

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.     

Book reviews

Book reviewed in this article:
E lectrochemical D etection T echniques I n T he A pplied B iosciences Volume 2 (1988). By G-A. Junter
P lant R esistance T o V iruses (1987). Edited by D. Evered & S. Harnett
M icrobial R econtamination I n D airy P rocessing –P roceedings O f T he I nternational C ongress.
D evelopments I n F ood M icrobiology –4 (1988). Edited by R.K. Robinson.
C ellulose H ydrolysis (1987). Edited by L. T. Fan, M.M. Gharpuray & Y.-H. Lee
I mmobilization O f C ells (1988). By C.R. Phillips & Y.C. Poon
C rystalline B acterial C ell S urface L ayers (1988). Edited by U.B. Sleytr, P. Messner, D. Pum & M. Sara     

Antigenic differences among Campylobacter fetus S-layer proteins.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of S-layer proteins extracted from Campylobacter fetus strains by using acid glycine buffer showed that the predominant S-layer proteins of different strains had subunit molecular weights in the range of 90,000 to 140,000. Electron microscopy revealed oblique S-layer lattices with a spacing of approximately 5.6 nm (gamma = 75 degrees) on wild-type strains VC1, VC119, VC202, and VC203. Three variants of C. fetus VC119 producing a predominant S-layer subunit protein of different molecular weight (Mr) from that of the parent were also examined. Each variant produced an oblique lattice morphologically indistinguishable from that of the parent. Amino-terminal sequence analysis showed that the S-layer proteins of the VC119 parent and variants were identical up to residue 18 and that this sequence differed from but was related to the first 16 N-terminal residues shared by the S-layer proteins of the three other wild-type C. fetus isolates. Western immunoblot analysis with an antiserum prepared to the VC119 protein and an antiserum prepared to C. fetus 84-40 LP (Z. Pei, R. T. Ellison, R. V. Lewis, and M. J. Blaser, J. Biol. Chem. 263:6416-6420, 1988) showed that strains of C. fetus were capable of producing S-layer proteins with at least four different antigenic specificities. Immunoelectron microscopy with antiserum to the VC119 S-layer protein showed that C. fetus cultures contained cells with immunoreactive oblique S-layer lattices as well as cells with oblique S-layer lattices which did not bind antibody. This suggests that C. fetus S-layer proteins undergo antigenic variation. Thermal denaturation experiments indicated that the antigenicity conferred by the surface-exposed C. fetus S-layer epitopes was unusually resistant to heat, and the thermal stability appeared to be due to the highly organized lattice structure of the S. layer. Protease digestion of purified VC119 S-layer protein revealed a trypsin-, chymotrypsin-, and endoproteinase Glu-C-resistant domain with an apparent Mr of 110,000, which carried the majority of the epitopes of the S-layer protein, and a small enzyme-sensitive domain. The trypsin- and chymotrypsin-resistant polypeptides shared an overlapping sequence which differed from the N-terminal sequence of the intact S-layer protein.     

A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus.

There is experimental evidence to suggest that the 100-kDa S-layer protein from Thermus thermophilus HB8 binds to the peptidoglycan cell wall. This property could be related to the presence of a region (SLH) of homology with other S-layer proteins and extracellular enzymes (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). By using specific monoclonal antibodies, we show that similar regions are present in different members of the Deinococcus-Thermus phylogenetic group. To analyze the role that the SLH domain plays in vivo and in vitro in T. thermophilus, we have obtained a mutant form (slpA.X) of the S-layer gene (slpA) in which the SLH domain was deleted. The slpA.X gene was inserted into the chromosome of the thermophile by gene replacement, resulting in a mutant which expressed a major membrane protein with the size expected from the construction (90 kDa). This protein was identified as the product of slpA.X by its differential reaction with monoclonal antibodies. Mutants expressing the SlpA.X protein grow as groups of cells, surrounded by a common external envelope of trigonal symmetry that contains the SlpA.X protein as a main component, thus showing the inability of the SLH-defective protein to attach to the underlying material in vivo. In addition, averaged images of SlpA.X-rich fractions showed a regular arrangement, identical to that built up by the wild-type (SlpA) protein in the absence of peptidoglycan. Finally, we demonstrate by Western blotting (immunoblotting) the direct role of the SLH domain in the binding of the S-layer of T. thermophilus HB8 to the peptidoglycan layer.     

InvB is a type III secretion-associated chaperone for the Salmonella enterica effector protein SopE

SopE is a bacteriophage-encoded effector protein of Salmonella enterica serovar Typhimurium that is translocated into the cytosol of eukaryotic cells by a type III secretion system (TTSS) (W.-D. Hardt, H. Urlaub, and J. E. Galán, Proc. Natl. Acad. Sci. USA 95:2574-2579, 1998; M. W. Wood, R. Rosqvist, P. B. Mullan, M. H. Edwards, and E. E. Galyov, Mol. Microbiol. 22:327-338, 1996). In this study, we provide evidence that an unlinked gene carried within the Salmonella pathogenicity island 1 (SPI-1), invB (K. Eichelberg, C. Ginocchio, and J. E. Galán, J. Bacteriol. 176:4501-4510, 1994), is required for the secretion of SopE through the SPI-1 TTSS. Furthermore, far-Western blotting analysis shows that SopE directly interacts with InvB through a domain located at its amino terminus. We conclude that InvB is the TTSS-associated chaperone for SopE.     

Heterogeneity of S-layer proteins of Lactobacillus acidophilus strains.

An S-layer (surface regular array) was found in the cell wall from six out of ten strains of Lactobacillus acidophilus examined by electron microscopic observations. All of the six strains which were shown to carry the S-layers belonged to the deoxyribonucleic acid (DNA) homology group A, but not to B, which had been classified by Johnson et al (Int. J. Syst. Bacteriol. 30: 53-68, 1980). On the other hand, the other four strains which possessed no S-layers were in the homology group B. The apparent molecular weights of the S-layer proteins ranged from 41 to 49 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the S-layer proteins from the six strains, three were susceptible to chemical cleavage with N-chlorosuccinimide, giving different peptide maps. All of the six S-layer proteins were fragmented by limited proteolysis with Staphylococcus aureus V8 protease, and gave markedly different peptide patterns by the subsequent peptide mapping analysis, except that the peptide maps of the S-layer proteins from the two strains which were in the same subgroup were identical.     

Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli.

CytA, a 27-kDa cytolytic crystal protein of Bacillus thuringiensis subsp. israelensis, is produced only at very low levels by recombinant Escherichia coli cells unless a 20-kDa B. thuringiensis subsp. israelensis protein is also present (K. M. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987; L. F. Adams, J. E. Visick, and H. R. Whiteley, J. Bacteriol. 171:521-530, 1989). However, the data reported here demonstrate that the 20-kDa protein is not required for high-level CytA production in E. coli strains carrying mutations in rpoH, groEL, or dnaK, all of which affect the proteolytic ability of the cells. The 20-kDa protein also increases the amount of CryIVD (another B. thuringiensis subsp. israelensis crystal protein) and LacZX90 (a mutant of beta-galactosidase) made by E. coli. The latter phenomenon is attributable to an increase in the half-life of LacZX90, suggesting that the 20-kDa protein may stabilize this protein. The effect of the 20-kDa protein was also examined in vitro and in a T7 RNA polymerase expression system, and the possible significance of these results for the timing of proteolysis and of 20-kDa protein activity is discussed. Finally, the ability of a single antibody to coimmunoprecipitate CytA and the 20-kDa protein from E. coli extracts provides evidence for a protein-protein interaction that may be related to the mechanism of action of the 20-kDa protein.     

Generation of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody

Crystalline bacterial cell surface layer (S-layer) proteins are composed of a single protein or glycoprotein species. Isolated S-layer subunits frequently recrystallize into monomolecular protein lattices on various types of solid supports. For generating a functional protein lattice, a chimeric protein was constructed, which comprised the secondary cell wall polymer-binding region and the self-assembly domain of the S-layer protein SbpA from Bacillus sphaericus CCM 2177, and a single variable region of a heavy chain camel antibody (cAb-Lys3) recognizing lysozyme as antigen. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-Lys3. The functionality of the fused cAb-Lys3 in the S-layer fusion protein was proved by surface plasmon resonance measurements. Dot blot assays revealed that the accessibility of the fused functional sequence for the antigen was independent of the use of soluble or assembled S-layer fusion protein. Recrystallization of the S-layer fusion protein into the square lattice structure was observed on peptidoglycan-containing sacculi of B. sphaericus CCM 2177, on polystyrene or on gold chips precoated with thiolated secondary cell wall polymer, which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Thereby, the fused cAb-Lys3 remained located on the outer S-layer surface and accessible for lysozyme binding. Together with solid supports precoated with secondary cell wall polymers, S-layer fusion proteins comprising rSbpA(31)(-)(1068) and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology.     

Specific location of penicillin-binding proteins within the cell envelope of Escherichia coli.

This communication deals with the location of penicillin-binding proteins in the cell envelope of Escherichia coli. For this purpose, bacterial cells have been broken by various procedures and their envelopes have been fractioned. To do so, inner (cytoplasmic) and outer membranes were separated by isopycnic centrifugation in sucrose gradients. Some separation methods (Osborn et al., J. Biol. Chem. 247:3962-3972, 1972; J. Smit, Y. Kamio, and H. Nikaido, J. Bacteriol. 124:942-958, 1975) revealed that penicillin-binding proteins are not exclusively located in the inner membrane. They are also found in the outer membrane (A. Rodríguez-Tébar, J. A. Barbas, and D. Vásquez, J. Bacteriol. 161:243-248, 1985). Under the milder conditions for cell rupture used in this work, an intermembrane fraction, sedimenting between the inner and outer membrane, can be recovered from the gradients. This fraction has a high content of both penicillin-binding proteins and phospholipase B activity and may correspond to the intermembrane adhesion sites (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-769, 1982). We postulate that this intermembrane fraction is a labile structure that contains a high amount of all penicillin-binding proteins which are usually found in both the inner and outer membranes when the adhesion sites are destroyed by the cell breakage and fractionation procedures.     

OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope.

Several proteins of Clostridium thermocellum possess a C-terminal triplicated sequence related to bacterial cell surface proteins. This sequence was named the SLH domain (for S-layer homology), and it was proposed that it might serve to anchor proteins to the cell surface (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). This hypothesis was investigated by using the SLH-containing protein ORF1p from C. thermocellum as a model. Subcellular fractionation, immunoblotting, and electron microscopy of immunocytochemically labeled cells indicated that ORF1p was located on the surface of C. thermocellum. To detect C. thermocellum components interacting with the SLH domains of ORF1p, a probe was constructed by grafting these domains on the C terminus of the MalE protein of Escherichia coli. The SLH domains conferred on the chimeric protein (MalE-ORF1p-C) the ability to bind noncovalently to the peptidoglycan of C. thermocellum. In addition, 125I-labeled MalE-ORF1p-C was shown to bind to SLH-bearing proteins transferred onto nitrocellulose, and to a 26- to 28-kDa component of the cell envelope. These results agree with the hypothesis that SLH domains contribute to the binding of exocellular proteins to the cell surface of bacteria. The gene carrying ORF1 and its product, ORF1p, are renamed olpB and OlpB (for outer layer protein B), respectively.     

Phylogenetic analysis and heterologous expression of surface layer protein SlpC of Bacillus sphaericus C3-41

The surface layer protein encoding genes from five mosquito-pathogenic Bacillus sphaericus isolates were amplified and sequenced. Negative staining of the S-layer protein extracted from the cell wall of wild-type B. sphaericus C3-41 was prepared. It showed a flat-sheet crystal lattice structure. Two genes encoding the entire and N-terminally truncated S-layer protein (slpC and DeltaslpC respectively), were ligated into plasmid pET28a and expressed in Escherichia coli. SDS-PAGE revealed that about 130 KD and 110 KD proteins could be expressed in the cytoplasm of recombinant E. coli BL21(pET28a/slpC) and E. coli BL21(pET28a/DeltaslpC) respectively. Furthermore, an intracellular sheet-like or fingerprint-shape structure was investigated in two recombinant strains, which expressed SlpC and DeltaSlpC protein respectively, by ultrathin microscopy study, but bioassay results suggested that the S-layer protein of wild B. sphaericus C3-41 and recombinant E. coli BL21 (pET28a/slpC) have no direct toxicity against mosquito larvae. These results should provide information for further understanding of the function of S-layer protein of pathogenic B. sphaericus.     

Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens.

FliI is a Salmonella typhimurium protein that is needed for flagellar assembly and may be involved in a specialized protein export pathway that proceeds without signal peptide cleavage. FliI shows extensive sequence similarity to the catalytic beta subunit of the F0F1 ATPase (A. P. Volger, M. Homma, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 173:3564-3572, 1991). It is even more similar to the Spa47 protein of Shigella flexneri (M. M. Venkatesan, J. M. Buysse, and E. V. Oaks, J. Bacteriol. 174:1990-2001, 1992) and the HrpB6 protein of Xanthomonas campestris (S. Fenselau, I. Balbo, and U. Bonas, Mol. Plant-Microbe Interact. 5:390-396, 1992), which are believed to play a role in the export of virulence proteins. Site-directed mutagenesis of residues in FliI that correspond to catalytically important residues in the F1 beta subunit resulted in loss of flagellation, supporting the hypothesis that FliI is an ATPase. FliI was overproduced and purified almost to homogeneity. It demonstrated ATP binding but not hydrolysis. An antibody raised against FliI permitted detection of the protein in wild-type cells and an estimate of about 1,500 subunits per cell. An antibody directed against the F1 beta subunit of Escherichia coli cross-reacted with FliI, confirming that the proteins are structurally related. The relationship between three proteins involved in flagellar assembly (FliI, FlhA, and FliP) and homologs in a variety of virulence systems is discussed.     

Wheat-germ agglutinin mimics metabolic effects of insulin without increasing receptor autophosphorylation

Expression of insulin metabolic effects can be obtained by anti-receptor antibodies without activation of the tyrosine kinase activity [O'Brien R. M., Soos M. A. and Siddle K. (1987) EMBO J. 6, 4003-4010; Forsayeth J. R., Caro J. F., Sinha M. K., Maddux B. A. and Goldfine I. D. (1987) Proc. natn. Acad. Sci. U.S.A. 84, 34,448-34,514; Ponzio G., Contreres J. O., Debant A., Baron V., Gautier N., Dolais-Kitabgi J. and Rossi B. (1988) EMBO J. 7, 4111-4117; Hawley D. M., Maddux B. A., Patel R. G., Wong K. Y., Mamula P. W., Firestone G. L., Brunetti A., Verspohl E. and Goldfine I. D. (1989) J. biol. Chem. 264, 2438-2444; Soos M. A., O'Brien R. M., Brindle N. P. J., Stigter J. M., Okamoto A. K., Whittaker J. and Siddle K. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 5217-5221.]. Recently, we have proposed that receptor cross-linking is sufficient in itself to stimulate glycogen synthesis, even if aggregation was performed on receptors mutated on Tyr 1162 and Tyr 1163 and thus devoid of tyrosine kinase activity [Debant A., Ponzio G., Clauser E., Contreres J. O. and Rossi B. (1989) Biochemistry 28, 14-17]. The aim of this study was to gain information on the involvement of receptor clustering in the expression of the different insulin biological effects. To this end, we studied the mimetic effects of wheat-germ agglutinin, which is likely to induce receptor aggregation without interacting with the receptor protein moiety. Wheat-germ agglutinin failed to promote DNA synthesis, whereas the lectin behaved as a potent mimicker of insulin on tyrosine aminotransferase activity and amino-acid transport. However, this stimulatory effect did not parallel the activation of receptor autophosphorylation. Our data reinforce the idea that the expression of the metabolic effects of insulin are not strictly dependent on a general tyrosine kinase activation.     

Affinity cross-flow filtration: purification of IgG with a novel protein a affinity matrix prepared from two-dimensional protein crystals

In this article, we describe the use of 1- to 2-mum sized affinity microparticles for the isolation and purification of IgG from artificial IgG-human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross-flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111-69, in which the peptidoglycan-containing layer was completely covered with a hexagonally ordered S-layer lattice. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S-layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S-layer protein release under cross-flow conditions between pH 2 to 12. The IgG-binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes. (c) 1994 John Wiley & Sons, Inc.