Putative Cellulose-binding Domain Research Articles

Characterization of a potential Listeria monocytogenes virulence factor associated with attachment to fresh produce.

A study to determine the attachment of L. monocytogenes serotype 4b strain F2365 on vegetables and fruits was conducted. In an initial study, we screened 32 genes encoding surface proteins and lipases of the strain to find highly expressed genes on lettuce leaves. The results showed that transcription levels of LMOf2365_0413, LMOf2365_0498, LMOf2365_0859, LMOf2365_2052, and LMOf2365_2812 were significantly upregulated on lettuce leaves. In silico analysis showed that LMOf2365_0859 contains a putative cellulose binding domain. Thus, we hypothesized that this gene may be involved in an attachment to vegetables, and named it lcp (gene encoding Listeria cellulose binding protein [LCP]). lcp mutant (Δlcp) and lcp complement (F2365::pMAD::cat::lcp) strains were generated by homologous recombination. The abilities of a wild-type (WT) strain, the Δlcp strain, and the complemented strain to attach to lettuce leaves were evaluated, which indicated that the attachment of the Δlcp strain to lettuce was significantly less than that of the WT and the complemented strains. Similar results were observed for baby spinach and cantaloupe. Fluorescence microscopy and field emission scanning microscopy analysis further supported these findings. The binding of L. monocytogenes to cellulose was determined using cellulose acetate-coated plates. The results showed that a binding ability of the Δlcp strain was significantly lower than that of the wild type. Combined, these results strongly suggest that LCP plays an important role in an attachment to vegetables and fruits.

Evaluation of the role of the endo-β-(1,4)-glucanase gene FaEG3 in strawberry fruit softening

In strawberry, Fragaria × ananassa Duch., fruit, two different endo-β-1,4-glucanase genes, FaEG1 and FaEG3, also named Cel1 and Cel2, are expressed during the softening process that occurs during fruit ripening. It has also been suggested that FaEG3, which contains a putative cellulose-binding domain, could play a key role in fruit development, since previous attempts to down-regulate this gene through transgenesis have been unsuccessful. In this investigation, we obtained transgenic strawberry plants containing an antisense sequence of the FaEG3 gene under the control of the 35S promoter. Ripened fruit from transgenic lines (Acel lines) showed large variation in FaEG3 silencing, but fruit firmness was similar to control fruit in all the lines. Two Acel lines showing almost 95% reductions in FaEG3 mRNA levels were selected for further study. In these lines, FaEG3 down-regulation was high, from 78 to 95%, at all fruit developmental stages, whereas FaEG1 was only slightly suppressed. In spite of the high FaEG3 silencing achieved, EGase activity was not modified in ripe fruit. At the cell wall level, walls from transgenic ripe fruit contained a significantly higher amount of the 4 M KOH fraction, which is enriched in hemicellulosic polymers. The analysis of this fraction by size exclusion chromatography showed that transgenic cell walls contained a smaller amount of higher molecular mass polymers than controls. Altogether, these results indicate that FaEG3 does not play a key role either in fruit development or fruit softening. However, its silencing affects the amount and, in a minor way, the size of hemicellulosic polymers.

Characterization of a cellulose binding domain from Clostridium cellulovorans endoglucanase-xylanase D and its use as a fusion partner for soluble protein expression in Escherichia coli

Different chimeric proteins combining the non-catalytic C-terminal putative cellulose binding domain of Clostridium cellulovorans endoglucanase-xylanase D (EngD) with its proline-threonine rich region PT-linker, PTCBD EngD, cellulose binding domain of C. cellulovorans cellulose binding protein A, CBD CbpA, cohesin domains Cip7, Coh6 and CipC1 from different clostridial species and recombinant antibody binding protein LG were constructed, expressed, purified and analyzed. The solubilities of chimeric proteins containing highly soluble domains Cip7, CipC1 and LG were not affected by fusion with PTCBD EngD. Insoluble domain Coh6 was solubilized when fused with PTCBD EngD. In contrast, fusion with CBD CbpA resulted in only a slight increase in solubility of Coh6 and even decreased solubility of CipC1 greatly. PTCBD EngD and Cip7-PTCBD EngD were shown to bind regenerated commercial amorphous cellulose Cuprophan. The purity of Cip7-PTCBD EngD eluted from Cuprophan was comparable to that purified by conventional ion exchange chromatography. The results demonstrated that PTCBD EngD can serve as a bi-functional fusion tag for solubilization of fusion partners and as a domain for the immobilization, enrichment and purification of molecules or cells on regenerated amorphous cellulose.

Expression and processing of a hormonally regulated beta-expansin from soybean.

Expansin proteins are essential components of acid-induced cell wall loosening in plants. Beta-expansins, which constitute a subfamily of related expansin proteins, include the group I grass pollen allergens. To provide a better description of beta-expansin expression, we have characterized a cytokinin-inducible beta-expansin from soybean (Glycine max cv Mandarin) called Cim1. Our results demonstrate that the hormones cytokinin and auxin act synergistically to induce the accumulation and proteolytic processing of Cim1. Carboxyl terminal truncation of a 35-kD form of Cim1 is predicted to remove the putative cellulose binding domain from the amino terminal cysteine-rich domain, resulting in a 20-kD form of the protein. Furthermore, the identical amino termini of the 35- and 20-kD forms of Cim1 correspond to a position 11 amino acids downstream of the predicted signal sequence cleavage site, suggesting proteolysis of a short amino terminal propeptide after removal of the signal peptide. This propeptide fragment contains a consensus site for N-glycosylation and our data suggest that it is glycosylated by a tunicamycin-sensitive mechanism in cultured soybean cells. The onset of Cim1 expression correlates with increased growth of soybean cultures. Ultimately, Cim1 is rapidly and specifically proteolyzed as soybean cultures reach stationary phase. These findings are consistent with the hypothesis that beta-expansin proteins are extensively modified by post-translational N-glycosylation and proteolysis.

Exploitation of a cellulose-binding domain from Neurospora crassa.

After eight decades as a purely research organism, Neurospora crassa is becoming a production system for heterologous peptides. The present work exploits the cbh-1 gene, which encodes a class C cellobiohydrolase (EC 3.2.1.91) and has, at its carboxy-terminus, a domain with homology to other fungal cellulose-binding domains. We describe the construction of two translational fusions of the putative cellulose-binding domain with a reporter gene, which is the catalytic domain of the gla-1 glucoamylase gene of the same species, their transformation back into the organism, and expression of the constructs as cellulose-binding glucoamylase activity. This adds to the developing biotechnology of the organism the potential for enzyme/protein immobilisation.

CelE, a multidomain cellulase from Clostridium cellulolyticum: a key enzyme in the cellulosome?

CelE, one of the three major proteins of the cellulosome of Clostridium cellulolyticum, was characterized. The amino acid sequence of the protein deduced from celE DNA sequence led us to the supposition that CelE is a three-domain protein. Recombinant CelE and a truncated form deleted of the putative cellulose binding domain (CBD) were obtained. Deletion of the CBD induces a total loss of activity. Exhibiting rather low levels of activity on soluble, amorphous, and crystalline celluloses, CelE is more active on p-nitrophenyl-cellobiose than the other cellulases from this organism characterized to date. The main product of its action on Avicel is cellobiose (more than 90% of the soluble sugars released), and its attack on carboxymethyl cellulose is accompanied by a relatively small decrease in viscosity. All of these features suggest that CelE is a cellobiohydrolase which has retained a certain capacity for random attack mode. We measured saccharification of Avicel and bacterial microcrystalline cellulose by associations of CelE with four other cellulases from C. cellulolyticum and found that CelE acts synergistically with all tested enzymes. The positive influence of CelE activity on the activities of other cellulosomal enzymes may explain its relative abundance in the cellulosome.

A novel E-type endo-beta-1,4-glucanase with a putative cellulose-binding domain is highly expressed in ripening strawberry fruits.

Two full-length cDNA clones (faEG1 and faEG3, respectively) have been isolated by screening a cDNA library representing transcripts from red strawberry fruits. Southern blot analysis of genomic DNA suggests that the strawberry endo-beta-1,4-glucanases (EGases) are encoded by a multigene family. The cognate genes are predominantly expressed during the ripening process proper, although, in the case of faEG3, some expression has also been observed in large green fruits and, at low amounts, in young vegetative green tissues. In agreement with other ripening-related genes in strawberry, also the expression of faEG1 and faEG3 is down-regulated by treatment with an auxin analogue (1-naphthaleneacetic acid, NAA). Differences in temporal expression of the two EGase genes in fruits are not accompanied by differences in spatial expression. The pattern of expression and the sequence characteristics of the two polypeptides suggest that the two strawberry EGases operate in a synergistic and coordinate manner. The protein encoded by faEG1 looks like one of the usual higher-plant EGases (average molecular mass of 54 kDa), while the protein encoded by faEG3 has a greater deduced molecular mass (about 68 kDa) due to the presence of an extra peptide of about 130 amino acids at the C-terminus. Such unusual peptide shows some features also found in microbial cellulases and contains a putative cellulose-binding domain. We propose that the faEG3-encoded EGase might especially hydrolyse the xyloglucans coating the cellulose microfibrils, thus rendering the cell wall more susceptible to the subsequent hydrolytic activity of the faEG1-encoded EGase.

Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.