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Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli.

ABSTRACT: The plasmid-borne raf operon encodes functions required for inducible uptake and utilization of raffinose by Escherichia coli. Raf functions include active transport (Raf permease), alpha-galactosidase, and sucrose hydrolase, which are negatively controlled by the Raf repressor. We have defined the order and extent of the three structural genes, rafA, rafB, and rafD; these are contained in a 5,284-base-pair nucleotide sequence. By comparisons of derived primary structures with known subunit molecular weights and an N-terminal peptide sequence, rafA was assigned to alpha-galactosidase (708 amino acids), rafB was assigned to Raf permease (425 amino acids), and rafD was assigned to sucrose hydrolase (476 amino acids). Transcription was shown to initiate 13 nucleotides upstream of rafA; a putative promoter, a ribosome-binding site, and a transcription termination signal were identified. Striking similarities between Raf permease and lacY-encoded lactose permease, revealed by high sequence conservation (76%), overlapping substrate specificities, and similar transport kinetics, suggest a common origin of these transport systems. alpha-Galactosidase and sucrose hydrolase are not related to host enzymes but have their counterparts in other species. We propose a modular origin of the raf operon and discuss selective forces that favored the given gene organization also found in the E. coli lac operon.

SUBMITTER: Aslanidis C

PROVIDER: S-EPMC210573 | biostudies-other | 1989 Dec

REPOSITORIES: biostudies-other

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Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli.

Aslanidis C C Schmid K K Schmitt R R

Journal of bacteriology 19891201 12

The plasmid-borne raf operon encodes functions required for inducible uptake and utilization of raffinose by Escherichia coli. Raf functions include active transport (Raf permease), alpha-galactosidase, and sucrose hydrolase, which are negatively controlled by the Raf repressor. We have defined the order and extent of the three structural genes, rafA, rafB, and rafD; these are contained in a 5,284-base-pair nucleotide sequence. By comparisons of derived primary structures with known subunit mole ...[more]

PMID: 2556373

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Project description:Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E. coli. The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na(+)/melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K. oxytoca and recombinant E. coli. Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E. coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons. Based on similarities in protein sequence, the xynTB genes in K. oxytoca appear to have originated from a gram-positive ancestor related to L. lactis. Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant E. coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant E. coli than K. oxytoca M5A1 (the source of xynT and xynB). XynB exhibited weak arabinosidase activity, 3% that of xylosidase.

Dataset Information

Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli.

Publications

Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli.

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