Major Cell Wall Protein Research Articles

Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain.

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus β-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology. KEY POINTS: • CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out. • CCW12 and CCW14 co-disruption improved the display efficiency of BGL. • The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.

Selection and characterization of two monoclonal antibodies specific for the Aspergillus flavus major antigenic cell wall protein Aflmp1

Aspergillus flavus is a major fungal pathogen of plants and an opportunistic pathogen of humans. In addition to the direct impact of infection, it produces immunosuppressive and carcinogenic aflatoxins. The early detection of A. flavus is therefore necessary to diagnose and monitor fungal infection, to prevent aflatoxin contamination of food and feed, and for effective antifungal therapy. Aspergillus-specific monoclonal antibodies (mAbs) are promising as diagnostic and therapeutic reagents for the tracking and treatment of Aspergillus infections, respectively. However, A. flavus has a complex cell wall composition and dynamic morphology, hindering the discovery of mAbs with well-characterized targets. Here we describe the generation and detailed characterization of mAb5.52 (IgG2aκ) and mAb17.15 (IgG1κ), which bind specifically to the highly immunogenic cell wall antigen A. flavus mannoprotein 1 (Aflmp1). Both mAbs were generated using hybridoma technology following the immunization of mice with a recombinant truncated version of Aflmp1 (ExD, including the homologous CR4 domain) produced in bacteria. We show that mAb5.52 and mAb17.15 bind specifically to A. flavus and A. parasiticus cell wall fragments (CWFs), with no cross-reaction to CWFs from other fungal pathogens. Immunofluorescence microscopy revealed that both mAbs bind to the surface of Aspergillus hyphae and that mAb17.15 also binds to spores. The epitope for both mAbs is localized within the CR4 region of the Aflmp1 protein. These Aspergillus-specific mAbs may be useful for the early detection of fungal infection in food/feed crops, for serodiagnosis in patients with invasive aspergillosis caused by A. flavus infection and for the development of antibody-expressing disease-resistant crops.

Overexpression of VviPGIP1 and NtCAD14 in Tobacco Screened Using Glycan Microarrays Reveals Cell Wall Reorganisation in the Absence of Fungal Infection.

The expression of Vitis vinifera polygalacturonase inhibiting protein 1 (VviPGIP1) in Nicotiana tabacum has been linked to modifications at the cell wall level. Previous investigations have shown an upregulation of the lignin biosynthesis pathway and reorganisation of arabinoxyloglucan composition. This suggests cell wall tightening occurs, which may be linked to defence priming responses. The present study used a screening approach to test four VviPGIP1 and four NtCAD14 overexpressing transgenic lines for cell wall alterations. Overexpressing the tobacco-derived cinnamyl alcohol dehydrogenase (NtCAD14) gene is known to increase lignin biosynthesis and deposition. These lines, particularly PGIP1 expressing plants, have been shown to lead to a decrease in susceptibility towards grey rot fungus Botrytis cinerea. In this study the aim was to investigate the cell wall modulations that occurred prior to infection, which should highlight potential priming phenomena and phenotypes. Leaf lignin composition and relative concentration of constituent monolignols were evaluated using pyrolysis gas chromatography. Significant concentrations of lignin were deposited in the stems but not the leaves of NtCAD14 overexpressing plants. Furthermore, no significant changes in monolignol composition were found between transgenic and wild type plants. The polysaccharide modifications were quantified using gas chromatography (GC–MS) of constituent monosaccharides. The major leaf polysaccharide and cell wall protein components were evaluated using comprehensive microarray polymer profiling (CoMPP). The most significant changes appeared at the polysaccharide and protein level. The pectin fraction of the transgenic lines had subtle variations in patterning for methylesterification epitopes for both VviPGIP1 and NtCAD14 transgenic lines versus wild type. Pectin esterification levels have been linked to pathogen defence in the past. The most marked changes occurred in glycoprotein abundance for both the VviPGIP1 and NtCAD14 lines. Epitopes for arabinogalactan proteins (AGPs) and extensins were notably altered in transgenic NtCAD14 tobacco.

Mechanistic studies on the biosorption of Pb(II) by Pseudomonas aeruginosa.

The biosorption of Pb(II) ions from aqueous solution has been studied using both the intact and thermolyzed cells of Pseudomonas aeruginosa. Further, the role of the major cell wall components, namely DNA, protein, polysaccharide, and lipid, in Pb(II) binding has been assessed using an enzymatic treatment method. The Pb(II) bioremediation capability of P. aeruginosa cells has been investigated by varying the parameters of pH, time of interaction, amount of biomass, and concentration of Pb(II). The complete bioremoval of Pb(II) using intact cells has been achieved for an initial Pb(II) concentration of 12.4 mg L-1 at pH 6.2 and temperature 29 ± 1 °C. The biosorption isotherm follows Langmuirian behavior with a Gibbs free energy of -30.7 kJ mol-1, indicative of chemisorption. The biosorption kinetics is consistent with a pseudo-second-order model. The possible Pb(II) binding mechanisms of P. aeruginosa cells are discussed based on characterization using zeta potential measurements, Fourier transform infra-red spectroscopy, and energy dispersive X-ray spectroscopy. The results confirm that among the major cell wall components studied, polysaccharide shows the highest contribution towards Pb(II) binding, followed by DNA, lipid, and protein. Similar studies using thermolyzed cells show higher Pb(II) uptake compared to the intact cells both before and after enzymatic treatment.

Crystal structure of kiwellin, a major cell-wall protein from kiwifruit.

Kiwellin is a cysteine-rich, cell wall-associated protein with no known structural homologues. It is one of the most abundant proteins in kiwifruit (Actinidia spp.), and has been shown to be recognised by IgE of some patients allergic to kiwifruit. Cleavage of kiwellin into an N-terminal 4 kDa peptide called kissper and a core domain called KiTH is mediated by actinidin in vitro, and isolation of the kissper peptide from green-fleshed kiwifruit extracts suggested it may result from in vivo processing of kiwellin. In solution, kissper is highly flexible and displays pore-forming activity in synthetic lipid-bilayers. We present here the 2.05 Å resolution crystal structure of full-length kiwellin, purified from its native source, Actinidia chinensis (gold-fleshed kiwifruit). The structure confirms the modularity of the protein and the intrinsic flexibility of kissper and reveals that KiTH harbours a double-psi β-barrel fold hooked to an N-terminal β hairpin. Comparisons with structurally-related proteins suggest that a deep gorge located at the protein surface forms a binding site for endogenous ligands.

α-1,6-Mannosylation of N-Linked Oligosaccharide Present on Cell Wall Proteins Is Required for Their Incorporation into the Cell Wall in the Filamentous Fungus Neurospora crassa

The enzyme α-1,6-mannosyltransferase (OCH-1) is required for the synthesis of galactomannans attached to the N-linked oligosaccharides of Neurospora crassa cell wall proteins. The Neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. A carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. Analysis of the integral cell wall protein from wild-type and och-1 mutant cells showed that the mutant cell wall had reduced protein content. The och-1 mutant was found to secrete 18-fold more protein than wild-type cells. Proteomic analysis of the proteins released by the mutant into the growth medium identified seven of the major cell wall proteins. Western blot analysis of ACW-1 and GEL-1 (two glycosylphosphatidylinositol [GPI]-anchored proteins that are covalently integrated into the wild-type cell wall) showed that high levels of these proteins were being released into the medium by the och-1 mutant. High levels of ACW-1 and GEL-1 were also released from the och-1 mutant cell wall by subjecting the wall to boiling in a 1% SDS solution, indicating that these proteins are not being covalently integrated into the mutant cell wall. From these results, we conclude that N-linked mannosylation of cell wall proteins by OCH-1 is required for their efficient covalent incorporation into the cell wall.

A screen for deficiencies in GPI‐anchorage of wall glycoproteins in yeast

Many of the genes and enzymes critical for assembly and biogenesis of yeast cell walls remain unidentified or poorly characterized. Therefore, we designed a high throughput genomic screen for defects in anchoring of GPI-cell wall proteins (GPI-CWPs), based on quantification of a secreted GFP-Sag1p fusion protein. Saccharomyces cerevisiae diploid deletion strains were transformed with a plasmid expressing the fusion protein under a GPD promoter, then GFP fluorescence was determined in culture supernatants after mid-exponential growth. Variability in the amount of fluorescent marker secreted into the medium was reduced by growth at 18 degrees C in buffered defined medium in the presence of sorbitol. Secondary screens included immunoblotting for GFP, fluorescence emission spectra, cell surface fluorescence, and cell integrity. Of 167 mutants deleted for genes affecting cell wall biogenesis or structure, eight showed consistent hyper-secretion of GFP relative to parental strain BY4743: tdh3 (glyceraldehyde-3-phosphate dehydrogenase), gda1 (guanosine diphosphatase), gpi13 and mcd4 (both ethanolamine phosphate-GPI-transferases), kre5 and kre1 (involved in synthesis of beta1,6 glucan), dcw1(implicated in GPI-CWP cross-linking to cell wall glucan), and cwp1 (a major cell wall protein). In addition, deletion of a number of genes caused decreased secretion of GFP. These results elucidate specific roles for specific genes in cell wall biogenesis, including differentiating among paralogous genes.

Cell wall glycans and soluble factors determine the interactions between the hyphae ofCandida albicansandPseudomonas aeruginosa

The fungus, Candida albicans, and the bacterium, Pseudomonas aeruginosa, are opportunistic human pathogens that have been coisolated from diverse body sites. Pseudomonas aeruginosa suppresses C. albicans proliferation in vitro and potentially in vivo but it is the C. albicans hyphae that are killed while yeast cells are not. We show that hyphal killing involves both contact-mediated and soluble factors. Bacterial culture filtrates contained heat-labile soluble factors that killed C. albicans hyphae. In cocultures, localized points of hyphal lysis were observed, suggesting that adhesion and subsequent bacteria-mediated cell wall lysis is involved in the killing of C. albicans hyphae. The glycosylation status of the C. albicans cell wall affected the rate of contact-dependent killing because mutants with severely truncated O-linked, but not N-linked, glycans were hypersensitive to Pseudomonas-mediated killing. Deletion of HWP1, ALS3 or HYR1, which encode major hypha-associated cell wall proteins, had no effect on fungal susceptibility.

Anti-IgG antibodies in rheumatic diseases cross-react withStreptococcus mutansSR antigen

We have previously shown that SR protein, a S. mutans major cell wall protein, as well as the recombinant protein SR (rSR) share common epitopes with human IgG. Since this antigenic mimicry could play a role in the induction of anti IgG, we have examined, in k-ELISA, the presence of antibodies reacting with S. mutans SR proteins and S. mutans whole cells in sera from 36 patients with rheumatic diseases. The majority of the 36 sera showed a high reactivity with rSR when compared with control sera. Eight highly positive sera were further purified on rSR and human IgG sorbents and tested against both rSR and IgG in ELISA and Western blotting. The affinity-purified antibodies reacted strongly with rSR, IgG and IgG Fab fragments but failed to react with IgG Fc fragment. In Western blotting the addition of unlabelled IgG abolished the reactivity of affinity-purified biotinylated antibodies with all antigens, confirming the existence of a common epitope shared by rSR and human IgG heavy chain. We show the existence in rheumatic diseases of high titres of anti-human IgG antibodies cross-reactive with S. mutans SR proteins. Those antibodies are principally IgG and react with the Fd part of the Fab fragment. We can hypothesize from the above data that this antigenic mimicry existing between S. mutans SR-related antigens and human IgG could play a role in the synthesis of at least a part of the anti-IgG antibodies present in rheumatic diseases sera.

Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae.

The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.

Identification of major glucan-associated cell wall proteins of Candida albicans and their role in fluconazole resistance.

Identification of major glucan-associated proteins (GAPs) of the cell wall of a number of Candida albicans isolates susceptible or resistant to fluconazole (FLC) was addressed by direct sequencing of the protein bands resolved by unidimensional gel electrophoresis. Changes in the GAP compositions of the different strains grown in the presence of the drug were also investigated. In the FLC-susceptible strains, the major (more abundant) GAPs were enolase (46 kDa), two isoforms of phosphoglyceromutase (32 and 29 kDa), and two beta-(1-3)-exoglucanases (44 and 34 kDa), one of which (the 34-kDa component) was glycosylated. When these strains were grown in the presence of FLC there were substantial decreases in the intensities of the two enzymes of the glycolytic pathway (enolase and the phosphoglyceromutases), which were apparently replaced by enhancement of the exoglucanase constituents, particularly the 44-kDa one. This GAP pattern closely mimicked that observed in the FLC-resistant strains whether they were grown in the presence or in the absence of the drug. Both the enolase and the exoglucanase constituents were detected in the culture supernatants of FLC-treated cells, together with substantial amounts of highly glycosylated, probably mannoprotein secretory material, suggesting that FLC may cause marked alterations of GAP incorporation into the cell wall. Altogether, we were able to identify all major GAP constituents and monitor their distributions in the cell wall of C. albicans during treatment with FLC. The near equivalence of the GAP profile for the FLC-susceptible strain grown in the presence of FLC to that for the FLC-resistant strain suggests that the effects of the drug on GAPs may be stably incorporated into the cell wall of the fungus upon acquisition of resistance.

Efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis.

Expression/secretion vectors for the production of Fab' and single-chain (sc) Fab' by Bacillus brevis have been constructed. For the production of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab', the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab' was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab' remained at a level of a few milligrams per liter in the culture medium. The Fab' produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd' fragment, constituting the Fab', had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.

Identification and analysis of a static culture-specific cell wall protein, Tir1p/Srp1p in Saccharomyces cerevisiae.

A 100-kDa protein was found to be a major cell wall protein in Saccharomyces cerevisiae cells cultured without shaking, but was not present in cells cultured with shaking. The amino acid sequence of this protein was identical to the sequence of Tir1p/Srp1p. TIR1/SRP1 has previously been identified as a gene induced by glucose, cold shock or anaerobiosis and was believed to be a cell membrane protein but not a cell wall protein. However, we found that beta-1,3-glucanase solubilized Tir1p/Srp1p from the cell wall and the purified Tir1p/Srp1p reacted with antiserum to beta-1,6-glucan and contained glucose. These results suggest that Tir1p/Srp1p is a major structural cell wall protein in the static-cultured yeast cells and is bound to the cell wall through beta-1,6-glucan. TIR1/SRP1 mRNA was transcribed only in the static culture and its transcription was regulated by the ROX1 repressor.

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

Organization of the outer layers of the cell envelope of Corynebacterium glutamicum: A combined freeze-etch electron microscopy and biochemical study

The cell surface of Corynebacterium glutamicum grown on solid medium was totally covered with a highly ordered, hexagonal surface layer. Also, freeze-fracture revealed two fracture surfaces which were totally covered with ordered arrays displaying an hexagonal arrangement and the same unit cell dimension as the surface layer. The ordered arrays on the concave fracture surface, closest to the cell surface, were due to the presence of particles while those on the convex fracture surface were their imprints. The same cells grown on liquid medium displayed a cell surface and fracture surfaces only partially covered with ordered arrays. In this case, the ordered regions had the same relative position on the cell surface and on the fracture surfaces. All ordered arrays were totally absent in a mutant for cspB, the gene encoding PS2, one of the two major cell wall proteins. Treatment of the cells with proteinase K caused the gradual alteration of PS2 into a slightly lower molecular mass form. This was accompanied by a concomitant disappearance of the ordered fracture surfaces followed by the detachment of the ordered surface layer from the cell as large ordered patches displaying the same lattice symmetry and dimension as those of the surface layer. The ordered patches were isolated. They contained the totality of PS2 initially associated with the cell. We conclude that the highly ordered surface layer of the intact cell was composed of PS2 interacting strongly with some cell wall material leading to its organization. This organized cell wall material produced the ordered fracture surfaces. We show that in the absence of intact PS2 protein on the cell wall, the same cell wall material was not organized and formed a structureless smooth layer.

Altered expression of surface protein WI-1 in genetically related strains of Blastomyces dermatitidis that differ in virulence regulates recognition of yeasts by human macrophages.

The molecular basis for pathogenicity and virulence of the dimorphic fungus Blastomyces dermatitidis remains unknown. WI-1 is a major cell wall protein of B. dermatitidis yeasts and is a recognition target of both humoral and cell-mediated immunity. As an initial study to determine if WI-1 might be linked to virulence of B. dermatitidis, we quantified WI-1 expression on three genetically related strains that differ in their virulence for mice: wild-type virulent ATCC strain 26199, mutant ATCC strain 60915 (which is 10,000-fold reduced in virulence), and mutant ATCC strain 60916 (which is avirulent). Two principal alterations in WI-1 expression were observed in the mutants. First, the mutants express more WI-1 on their surface, as quantified by flow cytometry with monoclonal antibody to WI-1 and by radioimmunoassay, but the WI-1 on their cell wall is less extractable than that on the wild-type strain. Second, the mutants shed less WI-1 during culture and demonstrate impaired processing of shed WI-1. Surface alterations in WI-1 were accompanied by significant differences in the binding of the virulent and mutant strains to human monocyte-derived macrophages. Attachment of yeasts to macrophages paralleled and was proportional to the expression of WI-1. Compared with wild-type yeasts, both mutants bound to macrophages more rapidly and in two- to threefold-greater magnitude. Furthermore, about 75% of yeast binding to macrophages was inhibited by a Fab anti-WI-1 monoclonal antibody. These results suggest that altered WI-1 expression on attenuated and avirulent mutant B. dermatitidis yeasts greatly facilitates macrophage recognition and binding of yeasts and, in turn, may contribute to more rapid ingestion and killing in the host.

Characterization of a major cell wall antigen and potential adhesin in three strains of Candida albicans.

Selected strains of Candida albicans were examined to reveal the surface antigenicity and biochemical nature of major cell wall proteins that also were shown to serve as cellular adhesins on human buccal epithelial cells. Confirmation of the adhesive properties of these cells was made by scanning electron microscopy and immunofluorescence microscopy. Particular attention was directed at the clinical isolate KM-302. By means of indirect immunofluorescence staining, the KM-302 blastoconidia absorbed rabbit anti-C. albicans ATCC-32354 serum, revealing specific localization of surface antigens on germ tubes and pseudohyphae. Extracellular polymeric material and the cell wall extract of C. albicans KM-302 blastoconidia were found to contain a major surface antigen of 49 kDa that exhibited 42% adhesion inhibition in vitro. Of considerable significance is that immunogold localization by electron microscopy showed the antigen to be almost exclusively cell wall bound. This major antigen, identified in affinity and gel filtration chromatography fractions, was composed of 4% carbohydrate and 95.7% protein and had an isoelectric point of 6.1. The major antigen also showed a high level of similarity with that of C. albicans strain SC-5314 inasmuch as the major antigen of that strain had carbohydrate and protein compositions of 4 and 95.5%, respectively. Both of these strains also possessed the same percent of adhesion inhibition on human buccal epithelial cells.

Production of human protein disulfide isomerase by Bacillus brevis

Human protein disulfide isomerase (PDI; EC 5.3.4.1) was expressed and secreted into the culture medium using Bacillus brevis as host and pNU200 which codes the promoter and signal sequence of major cell wall protein of B. brevis as vector. The accumulation of recombinant human PDI (rhPDI) reached about 5 mg l −1 in the late exponential phase of the bacterial growth. The purified rhPDI was found to be exactly processed at the carboxyl terminus of the signal sequence. It was as active as natural PDI derived from human placenta as determined by its ability to reactivate scrambled ribonuclease that was a fully oxidized mixture containing randomly formed disulfide bonds. The activity was significantly accelerated in the presence of dithiothreitol or a mixture of reduced and oxidized glutathione. These indicate that the characteristics of rhPDI are similar to those reported for mammalian PDI and that it can be used for refolding inactive proteins having incorrect disulfide bonds.

Induction Patterns of an Extensin Gene in Tobacco upon Nematode Infection.

When sedentary endoparasitic nematodes infect plants, they induce complex feeding sites within the root tissues of their host. To characterize cell wall changes induced within these structures at a molecular level, we studied the expression of an extensin gene (coding for a major structural cell wall protein) in nematode-infected tobacco roots. Extensin gene expression was observed to be induced very early upon infection. This induction was weak, transient, and probably due to wounding during penetration and migration of the tobacco cyst nematode Globodera tabacum ssp solanacea-rum. In contrast, high extensin gene expression was observed during the whole second larval stage (an ~2-week-long phase of establishment of the feeding site) of the root knot nematode Meloidogyne javanica. During later stages of this interaction, expression gradually decreased. Extensin gene expression was found in at least three different tissues of the gall. We propose that distinct mechanisms lead to induced expression in these different cell types. The significance of these results for the understanding of plant-nematode interactions as well as the function of structural cell wall proteins, such as extensin, is discussed.

Very efficient extracellular production of cholera toxin B subunit using Bacillus brevis.

We have constructed a very efficient synthesis and secretion system for cholera toxin B subunit (CTB) of Vibrio cholerae 569B using Bacillus-brevis. The constructed expression-secretion vector has the multiple promoters and the signal peptide coding region of the mwp gene, a structural gene for one of the major cell wall proteins of B. brevis strain 47, directly followed by the gene encoding the mature CTB. A large amount of mature CTB (1.4 g per liter of culture) was secreted into the medium. It had the same amino terminal amino acid sequence as that of authentic CTB and was fully active in GM1 ganglioside binding assay.