DNA sequence and transcription of an endoglucanase gene fromPrevotella (Bacteroides) ruminicola AR20

Vercoe, Phil; Gregg, Keith

doi:10.1007/bf00587590

Cited by 41 publications

(20 citation statements)

References 49 publications

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“…Also, E. chrysanthemi (21) and P. solanacearlum (48) endoglucanases appear to be transcribed from their own promoters. To our knowledge, the only reported evidence for polycistronic transcription comes from primer extension experiments with P. ruminicola endoglucanase (56). So far, we have no data concerning genes being cotranscribed with eg/.…”

Section: Methodsmentioning

confidence: 93%

Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph

et al. 1993

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We screened members of a new genus of grass-associated diazotrophs (Azoarcus spp.) for the presence of cellulolytic enzymes. Out of five Azoarcus strains representing different species, only in the endorhizosphere isolate BH72, which is also capable of invading grass roots, was significant endoglucanase activity, in addition to 13-glucosidase and cellobiohydrolase activity, present. Reducing sugars were readily released from medium-viscosity carboxymethylcellulose (CMC), but neither CMC, cellulose filter strips, Avicel, cellobiose, nor D-glucose served as the sole carbon source for growth of Azoarcus spp. Clones from a plasmid library of strain BH72 expressed all three enzymes in Escherichia coli, apparently not from their own promoter. According to restriction endonuclease mapping and subclone analysis, ,-glucosidase and cellobiohydrolase activities were localized on a single 2.6-kb fragment not physically linked to a 1.45-kb fragment from which endoglucanase (egl) was expressed. Two isoenzymes of endoglucanase probably resulting from proteolytic cleavage had pl values of 6.4 and 6.1 and an apparent molecular mass of approximately 36 kDa. Cellobiohydrolase and ,I-glucosidase activity were conferred by one enzyme 41 kDa in size with a pl of 5.4, which we classified as an unspecific exoglycanase (exg) according to substrate utilization and specificity mapping; hydrolysis of various oligomeric substrates differentiated it from endoglucanase, which degraded substituted soluble cellulose derivatives but not microcrystalline cellulose. Both enzymes were not excreted but were associated with the surface of Azoarcus cells. Both activities were only slightly influenced by the presence of CMC or D-glucose in the growth medium but were enhanced by ethanol. egl was located on a large transcript 15 kb in size, which was detectable only in cells grown under microaerobic conditions on N2. Surface-bound exo-and endoglucanases with some unusual regulatory features, detected in this study in a strain which is unable to metabolize cellulose or sugars, might assist Azoarcus sp. strain BH72 in infection of grass roots.

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Section: Methodsmentioning

confidence: 93%

Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph

et al. 1993

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“…Despite its documented importance in rumen physiology, relatively little is known about the cellular machinery that P. ruminicola 23 employs to harvest energy from hemicellulosic substrates. Several studies have explored the carbohydrate active enzymes from the related taxon Prevotella bryantii B 1 4 (previously classified as P. ruminicola B 1 4) (25,27,28,50,52,66,73); however, less is known about the xylanolytic system that P. ruminicola 23 elaborates. These two species share 88.9% nucleotide identity in their 16S rRNA genes (1,473 nucleotides aligned); however, there is evidence that these two species exhibit differences in their xylanolytic systems (2).…”

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confidence: 99%

Biochemical Analysis of a β- d -Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

Dodd

Kocherginskaya²,

Spies

et al. 2009

J Bacteriol

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Prevotella ruminicola 23 is an obligate anaerobic bacterium in the phylum Bacteroidetes that contributes to hemicellulose utilization within the bovine rumen. To gain insight into the cellular machinery that this organism elaborates to degrade the hemicellulosic polymer xylan, we identified and cloned a gene predicted to encode a bifunctional xylanase-ferulic acid esterase (xyn10D-fae1A) and expressed the recombinant protein in Escherichia coli. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-␤-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 ␤-Dglucosidase gene, with a novel PA14-like insertion sequence, was identified two genes downstream of xyn10D-fae1A. Biochemical analyses of the purified recombinant protein revealed that the putative ␤-D-glucosidase has activity for pNP-␤-D-xylopyranoside, pNP-␣-L-arabinofuranoside, and xylo-oligosaccharides; thus, the gene was designated xyl3A. When incubated in combination with Xyn10D-Fae1A, Xyl3A improved the release of xylose monomers from a hemicellulosic xylan substrate, suggesting that these two enzymes function synergistically to depolymerize xylan. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. When a mutation was introduced into the putative catalytic site for the xylanase domain (E280S), the ferulic acid esterase activity increased threefold, which suggests that the two catalytic domains for Xyn10D-Fae1A are functionally coupled. Directed mutagenesis of conserved residues for Xyl3A resulted in attenuation of activity, which supports the assignment of Xyl3A as a GH family 3 ␤-D-xylosidase.␤-1,4-linked xylopyranose is the principal component of plant cell wall hemicellulose, which represents the second largest reservoir of fixed carbon in the biosphere (11,30,34,38,42,72). The catabolic breakdown of hemicellulose thus represents a critical step in the recycling of carbon in nature and has been targeted as a subject of intense research with respect to renewable energy resources. Two enzymes of principal importance for recycling hemicellulosic material are endo-1,4-␤-xylanases (EC 3.2.1.8), which cleave the xylan backbone, and ␤-D-xylosidases (EC 3.2.1.37), which cleave xylose monomers from the nonreducing end of xylo-oligosaccharides (17).Prevotella ruminicola 23 is an important member of the anaerobic rumen microbiota (60) that contributes to the utilization of noncellulosic polysaccharides, such as starch and xylan (15,25,35,55). Despite its documented importance in rumen physiology, relatively little is known about the cellular machinery that P. ruminicola 23 employs to harvest energy from hemicellulosic substrates. Several studies have explored the carbohydrate active enzymes from the related taxon Prevotella bryantii B 1 4 (previously classified as P. ruminicola B 1 4) (25,27,28,50,52,66,73); however, less is known about the xylanolytic system that P. rumi...

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“…dAR29 DNA was ligated to the phagemid pTZl9U (T4 DNA ligase, Promega), using methods described by Vercoe & Gregg (1992), and cloned in E. coli strain K803 by electroporation (Dower et a/., 1988). The sequence of cloned dAR29 DNA was determined by the dideoxynucleotide chain-termination method, using both single-and doublestranded sequencing techniques (Vercoe & Gregg, 1992).…”

Section: Dna Manipulationmentioning

confidence: 99%

Cloning and DNA sequence analysis of the region containing attP of the temperate phage AR29 of Prevotella ruminicola AR29

Gregg

Kennedy²,

Klieve³

1994

Microbiology

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Phage 4AR29 was shown to exist as a prophage integrated into the chromosome of Prevotella ruminicola AR29. By DNA hybridization studies, the point of integrative recombination on the phage genome (am) was located on a 4 5 kb EcoRV fragment. After preliminary mapping with restriction endonucleases, a 2.8 kb EcoRVIHindlll fragment was isolated, cloned in Escherichia coli and sequenced. DNA hybridization localized the attP site to the vicinity of an internal Dral site. Sequence analysis showed the presence of several direct and inverted repeats around the attP site, with consensus core sequences similar to the integrase binding sites of phage 1. Two open reading frames are present adjacent to attP (ORF1 and ORF2). The predicted polypeptide product of ORFl has a region of structural similarity to known integrases. Although the predicted product of ORF2 shows a t best weak homology with known excisionases, no other ORFs occur in the sequence upstream from ORF1, leaving ORF2 as the most likely candidate for this role. However, if ORF2 does represent an xis gene, then this putative integration module would possess a notable difference from that of other temperate phages in the inversion of the positions of int and xis relative to a m . The proposed 4AR29 integration module is being used to develop phage-based integrative vector systems for the genetic manipulation of rumen bacteria.

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DNA sequence and transcription of an endoglucanase gene fromPrevotella (Bacteroides) ruminicola AR20

Cited by 41 publications

References 49 publications

Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph

Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph

Biochemical Analysis of a β- d -Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

Cloning and DNA sequence analysis of the region containing attP of the temperate phage AR29 of Prevotella ruminicola AR29

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