title: Identification and functional analysis of the gene cluster for fructan utilization in Prevotella intermedia.
abstract: Fructanase enzymes hydrolyze the β-2,6 and β-2,1 linkages of levan and inulin fructans, respectively. We analyzed the influence of fructan on the growth of Prevotella intermedia. The growth of P. intermedia was enhanced by addition of inulin, implying that P. intermedia could also use inulin. Based on this finding, we identified and analyzed the genes encoding a putative fructanase (FruA), sugar transporter (FruB), and fructokinase (FruK) in the genome of strain ATCC25611. Transcript analysis by RT-PCR showed that the fruABK genes were co-transcribed as a single mRNA and semi-quantitative analysis confirmed that the fruA gene was induced in response to fructose and inulin. Recombinant FruA and FruK were purified and characterized biochemically. FruA strongly hydrolyzed inulin, with slight degradation of levan via an exo-type mechanism, revealing that FruA is an exo-β-d-fructanase. FruK converted fructose to fructose-6-phosphate in the presence of ATP, confirming that FruK is an ATP-dependent fructokinase. These results suggest that P. intermedia can utilize fructan as a carbon source for growth, and that the fructanase, sugar transporter, and fructokinase proteins we identified are involved in this fructan utilization.
authors.affiliation: Department of Pediatric Dentistry, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Fuse
authors.forename: Haruka
authors.initials: H
authors.lastname: Fukamachi
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Inoue
authors.forename: Takeshi
authors.initials: T
authors.lastname: Igarashi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002874:Chromosome Mapping; D003470:Culture Media; D005630:Fructans / Q000378:metabolism*; D005631:Fructokinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005632:Fructose; D015870:Gene Expression; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007444:Inulin / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D018713:Prevotella intermedia / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D005630:Fructans; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D005632:Fructose; D007444:Inulin; C072599:levan; D005631:Fructokinases; C046173:fructokinase; D006026:Glycoside Hydrolases; C021512:levanase