Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities.

A single gene (xylB) encoding both beta-D-xylosidase (EC 3.2.1.37) and alpha-L-arabinofuranosidase (EC 3.2.1.55) activities was identified and sequenced from the ruminal bacterium Butyrivibrio fibrisolvens. The xylB gene consists of a 1.551-bp open reading frame (ORF) encoding 517 amino acids. A subclone containing a 1.843-bp DNA fragment retained both enzymatic activities. Insertion of a 10-bp NotI linker into the EcoRV site within the central region of this ORF abolished both activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytoplasmic proteins from recombinant Escherichia coli confirmed the presence of a 60,000-molecular-weight protein in active subclones and the absence of this protein in subclones lacking activity. With p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinofuranoside as substrates, the specific activity of arabinosidase was found to be approximately 1.6-fold higher than that of xylosidase. The deduced amino acid sequence of the xylB gene product did not exhibit a high degree of identity with other xylan-degrading enzymes or glycosidases. The xylB gene was located between two incomplete ORFs within the 4,200-bp region which was sequenced. No sequences resembling terminators were found within this region, and these three genes are proposed to be part of a single operon. Based on comparison with other glycosidases, a conserved region was identified in the carboxyl end of the translated xylB gene which is similar to that of glucoamylase from Aspergillus niger.     

Identification of the Butyrivibrio fibrisolvens xylosidase gene (xylB) coding region and its expression in Escherichia coli

The gene encoding the principal Butyrivibrio fibrisolvens xylosidase (xylB) has been cloned and expressed in Escherichia coli under the control of the lac promoter. The coding region for this gene was localized within a 3.2-kilobase B. fibrisolvens DNA fragment in pUC18. A new protein band was observed in recombinant E. coli containing xylB. This protein (approximately 60,000 molecular weight) was presumed to be the xylosidase monomer. The optimal pH (5.5) and substrate range for the recombinant and native xylosidases appeared identical. Both enzymes hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 5 and both were inactive on xylan.     

Identification of the Butyrivibrio fibrisolvens xylosidase gene (xylB) coding region and its expression in Escherichia coli.

The gene encoding the principal Butyrivibrio fibrisolvens xylosidase (xylB) has been cloned and expressed in Escherichia coli under the control of the lac promoter. The coding region for this gene was localized within a 3.2-kilobase B. fibrisolvens DNA fragment in pUC18. A new protein band was observed in recombinant E. coli containing xylB. This protein (approximately 60,000 molecular weight) was presumed to be the xylosidase monomer. The optimal pH (5.5) and substrate range for the recombinant and native xylosidases appeared identical. Both enzymes hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 5 and both were inactive on xylan.     

Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a beta-glucosidase

The cloning, expression and nucleotide sequence of a 3.74 kb DNA segment on pLS215 containing a beta-glucosidase gene (bglA) from Butyrivibrio fibrisolvens H17c was investigated. The B. fibrisolvens bglA open reading frame (ORF) of 2490 bp encoded a beta-glucosidase of 830 amino acid residues with a calculated Mr of 91,800. In Escherichia coli C600(pLS215) cells the beta-glucosidase was localized in the cytoplasm and these cells produced an additional protein with an apparent Mr of approximately 94,000. The bglA gene was expressed from its own regulatory region in E. coli and a single mRNA initiation point was identified upstream of the bglA ORF and adjacent to a promoter consensus sequence. The primary structure of the beta-glucosidase showed greater than 40% similarity with a domain of 237 amino acids present in the beta-glucosidases of Kluyveromyces fragilis and Clostridium thermocellum. The B. fibrisolvens beta-glucosidase hydrolysed cellobiose to a limited extent, cellotriose to cellobiose and glucose, and cellotetraose and cellopentaose to predominantly glucose.     

Cloning of a gene encoding cinnamoyl ester hydrolase from the ruminai bacterium Butyrivibrio fibrisolvens E14 by a novel method

Abstract A gene ( cin I) encoding a cinnamoyl ester hydrolase (CEH) has been isolated from the ruminai bacterium, Butyrivibrio fibrisohens E14, using a model substrate, MUTMAC [4-methylumbelliferoyl ( p -trimethylammonium cinnamate chloride)]. CinI has significant amino-acid similarities with members of a large and diverse family of hydrolases with a serine/aspartic acid/ histidine catalytic triad. Our analyses identified two previously unclassified amino acid sequences, the amino-terminal domain of unknown function in XynZ from Clostridium thermocellum and XynC, an acetylxylan esterase from Caldicellulosiruptor saccharolyticus , as members of the same family of hydrolases. A previously described esterase with CEH activity, XylD from Pseudomonas fluorescens ssp. cellulosa , is not similar to CinI. CinI was expressed at high levels in the periplasmic fraction of E. coli TOPP2 and released ferulic acid from Fara [5- O -( trans -feruloyl)-arabinofuranose] prepared from wheat bran.     

Cloning and sequencing of the celA gene encoding endoglucanase A of Butyrivibrio fibrisolvens strain A46

Genomic DNA from Butyrivibrio fibrisolvens strain A46 was digested with EcoRI and ligated into lambda gt11. Two recombinant phages isolated from the gene bank hydrolysed carboxymethylcellulose and were shown to contain the same 2.3 kb EcoRI restriction fragment, which was cloned into pUC12 to generate pBA46. Escherichia coli JM83 harbouring pBA46 expressed an endoglucanase (EGA) which hydrolysed a range of other substrates including barley beta-glucan, Avicel, filter paper and p-nitrophenyl beta-D-cellobioside. Nucleotide sequencing of the B. fibrisolvens strain A46 DNA cloned in pBA46 revealed a single open reading frame (ORF) of 1296 bp, encoding a protein of 48,863 Da. Confirmation that the ORF coded for EGA was obtained by comparing the N-terminal sequence of the purified endoglucanase with that deduced from the nucleotide sequence. EGA contains a typical prokaryotic signal peptide at its N-terminus and shows some homology with the Bacillus family of cellulases. The enzyme does not contain distinct functional domains, which are prevalent in cellulases from Pseudomonas fluorescens subsp. cellulosa and Cellulomonas fimi.     

Cloning, sequencing and expression of a gene encoding a 73 kDa xylanase enzyme from the rumen anaerobe Butyrivibrio fibrisolvens H17c

Summary The cloning, expression and nucleotide sequence of a 3 kb DNA segment on pLS206 containing a xylanase gene (xynB) from Butyrivibrio fibrisolvens H17c was investigated. The open reading frame (ORF) of 1905 by encoded a xylanase of 635 amino acid residues (M_r 73156). At least 850 by at the 3 end of the gene could be deleted without loss of xylanase activity. The deduced amino acid sequence was confirmed by purifying the enzyme and subjecting it to N-terminal amino acid sequence analysis. In Escherichia coli C600 (pLS206) cells the xylanase was localized in the cytoplasm. Its optimum pH for activity was between pH 5.4 and 6, and optimum temperature 55° C. The primary structure of the xylanase showed a significant level of identity with a cellobiohydrolase/endoglucanase of Caldocellum saccharolyticum, as well as with the xylanases of the alkaliphilic Bacillus sp. strain C-125, B. fibrisolvens strain 49, and Pseudomonas fluorescens subsp. cellulosa.Abbreviations ORF open reading frame - pNPCase p-nitrophen-yl--d-cellobiosidase - (xynB) gene coding for XynB - XynB xylanase     

Cloning, sequencing, and expression of a xylanase gene from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens.

A gene coding for xylanase activity, xynA, from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens 49 was cloned into Escherichia coli JM83 by using plasmid pUC19. The gene was located on a 2.3-kilobase (kb) DNA insert composed of two adjacent EcoRI fragments of 1.65 and 0.65 kb. Expression of xylanase activity required parts of both EcoRI segments. In E. coli, the cloned xylanase enzyme was not secreted and remained cell associated. The enzyme exhibited no arabinosidase, cellulase, alpha-glucosidase, or xylosidase activity. The isoelectric point of the cloned protein was approximately 9.8, and optimal xylanase activity was obtained at pH 5.4. The nucleotide sequence of the 1,535-base-pair EcoRV-EcoRI segment from the B. fibrisolvens chromosome that included the xynA gene was determined. An open reading frame was found that encoded a 411-amino-acid-residue polypeptide of 46,664 daltons. A putative ribosome-binding site, promoter, and leader sequence were identified. Comparison of the XynA protein sequence with that of the XynA protein from alkalophilic Bacillus sp. strain C-125 revealed considerable homology, with 37% identical residues or conservative changes. The presence of the cloned xylanase gene in other strains of Butyrivibrio was examined by Southern hybridization. The cloned xylanase gene hybridized strongly to chromosomal sequences in only two of five closely related strains.     

Characterization of the Butyrivibrio fibrisolvens glgB gene, which encodes a glycogen-branching enzyme with starch-clearing activity.

A Butyrivibrio fibrisolvens H17c glgB gene, was isolated by direct selection for colonies that produced clearing on starch azure plates. The gene was expressed in Escherichia coli from its own promoter. The glgB gene consisted of an open reading frame of 1,920 bp encoding a protein of 639 amino acids (calculated Mr, 73,875) with 46 to 50% sequence homology with other branching enzymes. A limited region of 12 amino acids showed sequence similarity to amylases and glucanotransferases. The B. fibrisolvens branching enzyme was not able to hydrolyze starch but stimulated phosphorylase alpha-mediated incorporation of glucose into alpha-1,4-glucan polymer 13.4-fold. The branching enzyme was purified to homogeneity by a simple two-step procedure; N-terminal sequence and amino acid composition determinations confirmed the deduced translational start and amino acid sequence of the open reading frame. The enzymatic properties of the purified enzyme were investigated. The enzyme transferred chains of 5 to 10 (optimum, 7) glucose units, using amylose and amylopetin as substrates, to produce a highly branched polymer.     

Variation in relative substrate specificity of bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase by single-site mutations: roles of substrate distortion and recognition

To probe differential control of substrate specificities for 4-nitrophenyl-alpha-l-arabinofuranoside (4NPA) and 4-nitrophenyl-beta-d-xylopyranoside (4NPX), residues of the glycone binding pocket of GH43 beta-d-xylosidase/alpha-l-arabinofuranosidase from Selenomonas ruminantium were individually mutated to alanine. Although their individual substrate specificities (kcat/Km)(4NPX) and (kcat/Km)(4NPA) are lowered 330 to 280,000 fold, D14A, D127A, W73A, E186A, and H248A mutations maintain similar relative substrate specificities as wild-type enzyme. Relative substrate specificities (kcat/Km)(4NPX)/(kcat/Km)(4NPA) are lowered by R290A, F31A, and F508A mutations to 0.134, 0.407, and 4.51, respectively, from the wild type value of 12.3 with losses in (kcat/Km)(4NPX) and (kcat/Km)(4NPA) of 18 to 163000 fold. R290 and F31 reside above and below the C4 OH group of 4NPX and the C5 OH group of 4NPA, where they can serve as anchors for the two glycone moieties when their ring systems are distorted to transition-state geometries by raising the position of C1. Thus, whereas R290 and F31 provide catalytic power for hydrolysis of both substrates, the native residues are more important for 4NPX than 4NPA as the xylopyranose ring must undergo greater distortion than the arabinofuranose ring. F508 borders C4 and C5 of the two glycone moieties and can serve as a hydrophobic platform having more favorable interactions with xylose than arabinofuranose.     

Cell-cycle-dependent expression of the STK-1 gene encoding a novel murine putative protein kinase

We have cloned a novel putative serine/threonine kinase-encoding gene, designated STK-1, from murine embryonic stem (ES) cell and testis cDNA libraries. The kinase most closely related to STK-1 is Xenopus laevis XLP46 protein kinase which shows 71% amino-acid identity to STK-1 between their kinase domains. Nevertheless, STK-1 is conserved throughout phylogeny with hybridizing sequences being detected in DNA from mammals, amphibians, insects and yeast. STK-1 mRNA is detected in testis, intestine and spleen, tissues that contain a large number of proliferating cells, but not in other tissues. All cell lines tested expressed STK-1 mRNA with levels being dependent upon proliferation rates. In NIH 3T3 cells, STK-1 is expressed in a cell-cycle-dependent fashion. These findings suggest a role for STK-1 in cell growth     

Sequencing, expression, and genetic characterization of the Helicobacter pylori ftsH gene encoding a protein homologous to members of a novel putative ATPase family.

In this study, we isolated and sequenced a Helicobacter pylori gene, designated ftsH, coding for a 632-amino-acid protein which displayed striking similarity throughout its full length to FtsH proteins identified in Escherichia coli, Lactococcus lactis, and Bacillus subtilis. H. pylori FtsH also possessed approximately 200-amino-acid region containing a putative ATPase module which is conserved among members of the AAA protein family (AAA, ATPase associated with diverse cellular activities). The H. pylori ftsH product was overexpressed in E. coli and reacted immunologically with an anti-E. coli FtsH serum (T. Tomoyasu, K. Yamanaka, K. Murata, T. Suzaki, P. Bouloc, A. Kato, H. Niki, S. Hiraga, and T. Ogura, J. Bacteriol. 175:1352-1357, 1993). FtsH was also shown to be present in the membrane fraction of H. pylori, suggesting that it is membrane bound. Disruption of the ftsH gene led to the loss of viability of H. pylori, demonstrating that this gene is essential for cell growth. Overproduction of both H. pylori FtsH and E. coli FtsH together tremendously reduced the growth rate of the E. coli host cells, whereas the growth of the E. coli cells carrying the wild-type E. coli ftsH operon on the chromosome was not significantly affected by overproduction of H. pylori FtsH itself. This result suggests that the abnormal growth of cells results from interaction between H. pylori FtsH and E. coli FtsH.     

Identification of the pgmG gene, encoding a bifunctional protein with phosphoglucomutase and phosphomannomutase activities, in the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461

The pgmG gene of Sphingomonas paucimobilis ATCC 31461, the industrial gellan gum-producing strain, was cloned and sequenced. It encodes a 50,059-Da polypeptide that has phosphoglucomutase (PGM) and phosphomannomutase (PMM) activities and is 37 to 59% identical to other bifunctional proteins with PGM and PMM activities from gram-negative species, including Pseudomonas aeruginosa AlgC. Purified PgmG protein showed a marked preference for glucose-1-phosphate (G1P); the catalytic efficiency was about 50-fold higher for G1P than it was for mannose-1-phosphate (M1P). The estimated apparent K(m) values for G1P and M1P were high, 0.33 and 1.27 mM, respectively. The pgmG gene allowed the recovery of alginate biosynthetic ability in a P. aeruginosa mutant with a defective algC gene. This result indicates that PgmG protein can convert mannose-6-phosphate into M1P in the initial steps of alginate biosynthesis and, together with other results, suggests that PgmG may convert glucose-6-phosphate into G1P in the gellan pathway.     

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.