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Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.

A thermophilic Anoxybacillus sp. strain JT-12, isolated from soil, produced acidic xylotriose, 4-O-methyl-α-D-glucuronosyl-xylotriose (MeGlcAX₃), as a main product from birchwood xylan and accumulated them in the culture under optimum conditions at pH 7.0 and 55 °C using 0.75% (w/v) birchwood xylan as a carbon source for 42-72 h. The acidic xylotriose was purified by ethanol precipitation and high-performance liquid chromatography using NH₂ Lichosher® 100 column. The results of electrospray ionization mass spectrometry, mass to charge ratio (m/z) 603.23, confirmed that the purified sample was acidic xylotriose that had benefits and applications in many fields.

In the global ocean nitrogen cycle, the anaerobic ammonium-oxidizing (anammox) process is recognized as important. In this study, we established an enrichment culture of marine anammox bacteria (MAB) in a column-type reactor. The reactor, which included a porous polyester non-woven fabric that had been placed at the sea floor in advance for enrichment, was continuously fed with NH(4)Cl and NaNO(2) for more than 1 year. Anammox activity in the MAB reactor was confirmed by (15)N tracer analysis using (15)NH(4)Cl and Na(14)NO(2). We identified two 16S rRNA genes in the amplified DNA fragments derived from MAB, which were highly homologous with those from Candidatus "Scalindua wagneri" and an uncultured planctomycete clone. Fluorescence in situ hybridization analysis using an anammox-specific probe also confirmed that MAB predominated in the reactor. To our knowledge, this is the first report on the establishment of an enrichment culture of anammox bacteria from the marine environment using a continuous culture system.

S-layer homology (SLH) module polypeptides were derived from Clostridium josui xylanase Xyn10A, Clostridium stercorarium xylanase Xyn10B, and Clostridium thermocellum scafoldin dockerin binding protein SdbA as rXyn10A-SLH, rXyn10B-SLH, and rSdbA-SLH, respectively. Their binding specificities were investigated using various cell wall preparations. rXyn10A-SLH and rXyn10B-SLH bound to native peptidoglycan-containing sacculi consisting of peptidoglycan and secondary cell wall polymers (SCWP) prepared from these bacteria but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWP, suggesting that SCWP are responsible for binding with SLH modules. In contrast, rSdbA-SLH interacted with HF-EPCS, suggesting that this polypeptide had an affinity for peptidoglycans but not for SCWP. The affinity of rSdbA-SLH for peptidoglycans was confirmed by a binding assay using a peptidoglycan fraction prepared from Escherichia coli cells. The SLH modules of SdbA must be useful for cell surface engineering in bacteria that do not contain SCWP.

Biomass is originally photosynthesized from inorgainic compounds such as CO2, minerals, water and solar energy. Recent studies have shown that anaerobic bacteria have the ability to convert recalcitrant biomass such as cellulosic or chitinoic materials to useful compounds. The biomass containing agricultural waste, unutilized wood and other garbage is expected to utilize as feed, food and fuel by microbial degradation and other metabolic functions. In this study we isolated several anaerobic, cellulolytic and chitinolytic bacteria from rumen fluid, compost and soil to study their related enzymes and genes. The anaerobic and cellulolytic bacteria,Clostridium thermocellum, Clostridium stercorarium, andClostridium josui, were isolated from compost and the chitinolyticClostridium paraputrificum from beach soil andRuminococcus albus was isolated from cow rumen. After isolation, novel cellulase and xylanase genes from these anaerobes were cloned and expressed inEscherichia coli. The properties of the cloned enzymes showed that some of them were the components of the enzyme (cellulase) complex,i.e., cellulosome which is known to form complexes by binding cohesin domains on the cellulase integrating protein (Cip: or core protein) and dockerin domains on the enzymes. Several dockerin and cohesin polypeptides were independently produced byE. coli and their binding properties were specified with BIAcore by measuring surface plasmon resonance. Three pairs of cohesin-dockerin with differing binding specificities were selected. Two of their genes encoding their respective cohesin polypeptides were combined to one gene and expressed inE. coli as a chimeric core protein, on which two dockerin-dehydrogenase chimeras, the dockerin-formaldehyde dehydrogenase and the dockerin-NADH dehydrogenase are planning to bind for catalyzing CO2 reduction to formic acid by feeding NADH. This reaction may represent a novel strategy for the reduction of the green house gases. Enzymes from the anaerobes were also expressed in tobacco and rice plants. The activity of a xylanase fromC. stercorarium was detected in leaves, stems, and rice grain under the control of CaMV35S promoter. The digestibility of transgenic rice leaves in goat rumen was slightly accelerated.C. paraputrificum was found to solubilize shrimp shells and chitin to generate hydrogen gas. Hydrogen productivity (1.7 mol H2/mol glucose) of the organism was improved up to 1.8 times by additional expression of the own hydrogenase gene inC. paraputrficum using a modified vector ofClostridium perfringens. The hydrogen producing microflora from soil, garbage and dried pelletted garbage, known as refuse derived fuel (RDF), were also found to be effective in converting biomass waste to hydrogen gas.

The gene encoding the catalytic domain of thermostable xylanase from Clostridium thermocellum F1 was expressed in rice plants under the control of a constitutive promoter. The gene encoding Xylanase A was modified to encode the catalytic domain of family 11 xylanase without the signal sequence (xynA1), and was introduced into rice plants and expressed under the control of a modified cauliflower mosaic virus 35S promoter. Zymogram analysis indicated that the recombinant xylanase was produced in rice plants. The xynA1 gene was stably expressed in rice straw and seed grains. No phenotypic effect of xylanase expression was noted. The enzyme was detected in the desiccated grain. High levels of enzyme activity were maintained in the cell-free extract during incubation at 60 degrees C for 24 h. The results indicated that high levels of xylanase can be produced in rice plants.

A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli. The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da. Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain. Nag3A was purified from recombinant E. coli and characterized. The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide [4-MU-(GlcNAc)], but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine. The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively. SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.

INTRODUCTIONConjugal plasmids of various incompatibilitygroups avoid restriction when transferred among var-ious bacterial species and genera. This is mainly dueto the ard ( a lleviation of r estriction of D NA) geneswhich are in the leader region of a plasmid and codefor antirestriction proteins [1–5]. The Ard proteins(ArdA, ArdB, ArdC, ArdU, etc.) are small (140–180amino acid residues), highly acidic (total charge –7 to–30), and nonhomologous to each other, sharing onlyan antirestriction motif [6–9]. The motif is commonlyin the C domain, 30–35 residues away from the C end[10]. Its core consists of nine amino acid residues,with a certain arrangement of negatively charged ones(commonly D/E, highly polar N/Q in one site of ArdBand ArdC) and hydrophobic residues located in thecenter and at the ends (Table 1).We have previously shown that restriction allevia-tion induced by conjugal plasmid R64 (incI1) in Escherichia coli K12 is due to arsR , which codes fora suppressor of the ars operon conferring resistance toarsenite and arsenate [13]. ArsR consists of 117 aminoacid residues, is nonhomologous to the Ard proteins,and contains the antirestriction motif 25–35 residuesaway from the N end [13]. Its antirestriction activity islower than in the Ard proteins: ArsR alleviates restric-tion of nonmodified phage λ DNA by a factor of 20,whereas the factor is about 10

A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74,511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49,883 Da. XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-II xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome.

Immediately (17 bp) upstream of the Clostridium paraputrificum chiB gene [J. Bacteriol. 179: 7306-7314 (1997)], we found another chitinase gene chiA encoding chitinase A (ChiA). The chiA gene consists of an open reading frame of 2496 nucleotides and encodes 832 amino acids with a deduced molecular mass of 92,585 Da. The mature ChiA is a modular enzyme composed of a family-18 catalytic domain responsible for chitinase activity, two cadherin-like domains, and a chitin-binding domain. The domain organization of ChiA is fundamentally identical to that of ChiB and the overall sequence identity between them is 35.4%. ChiA was purified from the periplasm fraction of Escherichia coli harboring the chiA gene. The molecular mass of purified ChiA (89,000 Da), determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis, was in good agreement with the value (89,119 Da) calculated from the deduced amino acid sequence, excluding the signal peptide. Immunological and N-terminal amino acid sequence analyses revealed that ChiA and ChiB are major chitinases of C. paraputrificum and their production is inducible by ball-milled chitin. Northern blot analysis indicated that the chiA and chiB genes constitute a polycistronic operon. Primer-extension analysis confirmed that the transcription of this operon starts upstream of chiA.

Saccharomyces cerevisiae rhk mutants were previously shown to have a phenotype that is resistant to HM-1 killer toxin secreted from Hansenula mrakii IFO 0895. The RHK1/ALG3 gene encodes a mannosyl-transferase that is involved in the synthesis of an oligosaccharide in protein N-glycosylation. Previously, this gene was cloned and shown to complement the rhk1 mutation. In this study, the RHK2 gene, which complements the rhk2 mutation, was cloned. The RHK2 gene was found to be identical to the essential gene STT3, which encodes a subunit of the oligosaccharyl-transferase complex. This complex transfers the core oligosaccharide to proteins. The rhk2 mutants showed supersensitivity to several drugs (Calcofluor White, caffeine and FK506), suggesting that these strains have cell-wall defects. Activity staining of invertase in an acrylamide gel indicated that it was underglycosylated. These results suggest that one or more mannoproteins are involved in the cytocidal process of HM-1.

The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall beta-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast. A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin. These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups. A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated. DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids. Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S. cerevisiae cells with complete killer resistance. A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of beta-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin.