Expression of a cloned cellulase/xylanase gene from Prevotella ruminicola in Bacteroides vulgatus, Bacteroides uniformis and Prevotella ruminicola

A S Daniel, J Martin, I Vanat, T R Whitehead, Harry James Flint, Jennifer Cynthia Martin

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

A new shuttle vector, pRH3 (8.7 kb), was constructed for use in Prevotella/Bacteroides host strains. This vector combines the pRRI2 replicon from P. ruminicola, pBluescript sequences and a tetQ marker gene for selection in Prevotella/Bacteroides hosts. Following insertion of a fragment carrying an endoglucanase/xylanase gene from P. ruminicola 23 into the multiple cloning site, the resulting construct, pRH3X, was introduced into B. vulgatus 1447, B. uniformis 1100 and P. ruminicola 2202. This resulted in increases of between 4 and 50-fold in CM-cellulase and xylanase activities in cells grown with glucose. In contrast activities were barely detectable for the same construct in E. coli DH5 alpha. Most of the total xylanase activity produced was found within the cell in P. ruminicola 2202 and B. vulgatus 1447 transformed with pRH3X, and in P. ruminicola 23. An osmotic shock experiment indicated that a significant proportion of the xylanase activity in B. vulgatus 1447 cells carrying pRH3X was periplasmic.

Original languageEnglish
Pages (from-to)417-24
Number of pages8
JournalJournal of Applied Bacteriology
Volume79
Issue number4
Publication statusPublished - Oct 1995

Fingerprint

Prevotella ruminicola
Bacteroides
Cellulase
Prevotella
Genes
Genetic Vectors
Replicon
Osmotic Pressure
Organism Cloning
Escherichia coli
Glucose

Keywords

  • Bacteroides
  • Carboxymethylcellulose Sodium
  • Cellulase
  • Cloning, Molecular
  • Gene Expression Regulation, Bacterial
  • Genes, Bacterial
  • Genetic Vectors
  • Prevotella
  • Xylan Endo-1,3-beta-Xylosidase
  • Xylosidases

Cite this

Expression of a cloned cellulase/xylanase gene from Prevotella ruminicola in Bacteroides vulgatus, Bacteroides uniformis and Prevotella ruminicola. / Daniel, A S; Martin, J; Vanat, I; Whitehead, T R; Flint, Harry James; Martin, Jennifer Cynthia.

In: Journal of Applied Bacteriology, Vol. 79, No. 4, 10.1995, p. 417-24.

Research output: Contribution to journalArticle

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abstract = "A new shuttle vector, pRH3 (8.7 kb), was constructed for use in Prevotella/Bacteroides host strains. This vector combines the pRRI2 replicon from P. ruminicola, pBluescript sequences and a tetQ marker gene for selection in Prevotella/Bacteroides hosts. Following insertion of a fragment carrying an endoglucanase/xylanase gene from P. ruminicola 23 into the multiple cloning site, the resulting construct, pRH3X, was introduced into B. vulgatus 1447, B. uniformis 1100 and P. ruminicola 2202. This resulted in increases of between 4 and 50-fold in CM-cellulase and xylanase activities in cells grown with glucose. In contrast activities were barely detectable for the same construct in E. coli DH5 alpha. Most of the total xylanase activity produced was found within the cell in P. ruminicola 2202 and B. vulgatus 1447 transformed with pRH3X, and in P. ruminicola 23. An osmotic shock experiment indicated that a significant proportion of the xylanase activity in B. vulgatus 1447 cells carrying pRH3X was periplasmic.",
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AU - Daniel, A S

AU - Martin, J

AU - Vanat, I

AU - Whitehead, T R

AU - Flint, Harry James

AU - Martin, Jennifer Cynthia

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N2 - A new shuttle vector, pRH3 (8.7 kb), was constructed for use in Prevotella/Bacteroides host strains. This vector combines the pRRI2 replicon from P. ruminicola, pBluescript sequences and a tetQ marker gene for selection in Prevotella/Bacteroides hosts. Following insertion of a fragment carrying an endoglucanase/xylanase gene from P. ruminicola 23 into the multiple cloning site, the resulting construct, pRH3X, was introduced into B. vulgatus 1447, B. uniformis 1100 and P. ruminicola 2202. This resulted in increases of between 4 and 50-fold in CM-cellulase and xylanase activities in cells grown with glucose. In contrast activities were barely detectable for the same construct in E. coli DH5 alpha. Most of the total xylanase activity produced was found within the cell in P. ruminicola 2202 and B. vulgatus 1447 transformed with pRH3X, and in P. ruminicola 23. An osmotic shock experiment indicated that a significant proportion of the xylanase activity in B. vulgatus 1447 cells carrying pRH3X was periplasmic.

AB - A new shuttle vector, pRH3 (8.7 kb), was constructed for use in Prevotella/Bacteroides host strains. This vector combines the pRRI2 replicon from P. ruminicola, pBluescript sequences and a tetQ marker gene for selection in Prevotella/Bacteroides hosts. Following insertion of a fragment carrying an endoglucanase/xylanase gene from P. ruminicola 23 into the multiple cloning site, the resulting construct, pRH3X, was introduced into B. vulgatus 1447, B. uniformis 1100 and P. ruminicola 2202. This resulted in increases of between 4 and 50-fold in CM-cellulase and xylanase activities in cells grown with glucose. In contrast activities were barely detectable for the same construct in E. coli DH5 alpha. Most of the total xylanase activity produced was found within the cell in P. ruminicola 2202 and B. vulgatus 1447 transformed with pRH3X, and in P. ruminicola 23. An osmotic shock experiment indicated that a significant proportion of the xylanase activity in B. vulgatus 1447 cells carrying pRH3X was periplasmic.

KW - Bacteroides

KW - Carboxymethylcellulose Sodium

KW - Cellulase

KW - Cloning, Molecular

KW - Gene Expression Regulation, Bacterial

KW - Genes, Bacterial

KW - Genetic Vectors

KW - Prevotella

KW - Xylan Endo-1,3-beta-Xylosidase

KW - Xylosidases

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EP - 424

JO - Journal of Applied Bacteriology

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