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Importance of Inducible Enzymes in the Utilization of Inulin and Starch

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Importance of Inducible Enzymes in the Utilization of Inulin and Starch Substrate-driven gene expression in Roseburia inulinivorans: Importance of inducible enzymes in the utilization of inulin and starch Karen P. Scotta,1, Jenny C. Martina, Christophe Chassarda, Marlene Clergeta, Joanna Potrykusa, Gill Campbella, Claus-Dieter Mayerb, Pauline Younga, Garry Rucklidgea, Alan G. Ramsaya, and Harry J. Flinta aRowett Institute of Nutrition and Health, University of Aberdeen, Bucksburn, Aberdeen AB21 9SB, United Kingdom; and bBiomathematics and Statistics Scotland, Rowett Institute of Nutrition and Health, University of Aberdeen, Bucksburn, Aberdeen AB21 9SB, United Kingdom Edited by Todd R. Klaenhammer, North Carolina State University, Raleigh, NC, and approved June 24, 2010 (received for review February 8, 2010) Roseburia inulinivorans is a recently identified motile representative by inulin and FOS (4). This may be caused by bacterial cross- of the Firmicutes that contributes to butyrate formation from a va- feeding (5, 8), the direct stimulation of butyrate-producing bac- riety of dietary polysaccharide substrates in the human large intes- teria by the prebiotics (3, 4) or the butyrogenic effect of reduced tine. Microarray analysis was used here to investigate substrate- pH (9). Rossi et al. (4) established that only 8 of 55 bifido- driven gene-expression changes in R. inulinivorans A2-194. A cluster bacterial strains tested were able to grow on inulin in pure cul- of fructo-oligosaccharide/inulin utilization genes induced during ture, although virtually all of the 55 strains used FOS for growth. growth on inulin included one encoding a β-fructofuranosidase They concluded that the elevated butyrate production observed protein that was prominent in the proteome of inulin-grown cells. in mixed fecal cultures indicated that other bacterial groups were This cluster also included a 6-phosphofructokinase and an ABC able to degrade inulin and that the bifidobacteria were able to transport system, whereas a distinct inulin-induced 1-phosphofruc- use the mono- and oligosaccharide (FOS) products released by tokinase was linked to a fructose-specific phosphoenolpyruvate- the primary inulin degraders (4). Kleessen et al. (10) used fluo- dependent sugar phosphotransferase system (PTS II transport en- rescent in situ hybridization (FISH) to investigate changes in zyme). Real-time PCR analysis showed that the β-fructofuranosidase specific bacterial groups in human flora-associated rats fed diets and adjacent ABC transport protein showed greatest induction containing various mixtures of short- and long-chain fructans. A during growth on inulin, whereas the 1-phosphofructokinase en- mix of oligofructose with long-chain inulin or inulin alone en- zyme and linked sugar phosphotransferase transport system were hanced the numbers of the Clostridium coccoides/Eubacterium most strongly up-regulated during growth on fructose, indicating rectale group. This bacterial group includes Roseburia inulini- that these two clusters play distinct roles in the use of inulin. The vorans and other butyrate-producing bacteria (11) and was un- R. inulinivorans β-fructofuranosidase was overexpressed in Escher- ichia coli affected by FOS supplementation. In contrast, numbers of and shown to hydrolyze fructans ranging from inulin down bifidobacteria were only stimulated by FOS (10). In a human to sucrose, with greatest activity on fructo-oligosaccharides. Genes study, numbers of the butyrate-producing species Faecalibacte- induced on starch included the major extracellular α-amylase and rium prausnitzii were significantly increased after inulin–oligo- two distinct α-glucanotransferases together with a gene encoding fructose consumption (12). The chain length of the fructan a flagellin protein. The latter response may be concerned with im- molecule seems to be critical in defining the stimulatory effect. proving bacterial access to insoluble starch particles. Longer chain molecules have a greater and longer-lasting effect on bacterial fermentation in in vitro models (13). anaerobic gut bacteria | differential gene expression | fructo- The simple linear structure of FOS and inulin means that only oligosaccharides | butyrate | prebiotic a few enzymes are required for their degradation. The β- fructosidase superfamily includes all enzymes involved in the lant cell wall polysaccharides, storage polysaccharides such as hydrolysis of nonreducing β-D,fructosidic bonds to release fruc- Presistant starch and inulin, and many oligosaccharides of tose (14). Despite the diversity in nomenclature of this group plant origin remain undigested in the upper gastrointestinal tract of enzymes (β-fructosidases, β-fructofuranosidases, sucrose-6- and become important substrates for the growth of colonic phosphate hydrolases, endo-inulinases, exo-inulinases, invertases, bacteria. Prebiotics are defined as dietary substrates that reach fi and saccharases), they all contain highly conserved residues and the colon where they selectively stimulate the growth of bene - motifs and are members of glycosyl hydrolase (GH) families 32 or cial gut bacteria (1), with the most widely used prebiotics being the fructans inulin and fructo-oligosaccharides (FOS). Inulin is a linear polymer [degree of polymerization (DP) = 3–60] of β This paper results from the Arthur M. Sackler Colloquium of the National Academy of -2,1-linked fructose monomers with a terminal glucose residue, Sciences, "Microbes and Health" held November 2–3, 2009, at the Arnold and Mabel whereas FOS have the same backbone but a maximal chain Beckman Center of the National Academies of Sciences and Engineering in Irvine, CA. length of 8 monomeric units. The increasing use of prebiotics to The complete program and audio files of most presentations are available on the NAS enhance gut health is driving research into their mode of action. Web site at http://www.nasonline.org/SACKLER_Microbes_and_Health. Many studies have shown that both inulin and FOS selectively Author contributions: K.P.S., C.-D.M., and H.J.F. designed research; K.P.S., J.C.M., C.C., M.C., fi J.P., G.C., P.Y., G.R., and A.G.R. performed research; J.P., G.C., P.Y., G.R., and A.G.R. contri- stimulate the growth of potentially bene cial gut bacteria such as buted new reagents/analytic tools; K.P.S., J.C.M., C.C., M.C., J.P., and C.-D.M. analyzed data; bifidobacteria and lactobacilli (reviewed in ref. 2). However, only and K.P.S., J.P., and H.J.F. wrote the paper. a few studies have considered the potential for stimulation of The authors declare no conflict of interest. fi – other bacterial groups that may also be bene cial (3 6). This article is a PNAS Direct Submission. The bifidogenic dose of inulin has been defined as 5–8 g/d (7), fi Data deposition: The DNA sequences reported in this paper have been deposited in the yet the effect of such supplementation on other bene cial groups GenBank database (accession nos. GU591772M–GU591787). The microarray data reported of gut bacteria and the differences relating to different compo- in this paper have been deposited in the NCBI GEO repository (accession no. GSE22245). sitions of inulin have received little attention. For example, the 1To whom correspondence should be addressed. E-mail: [email protected]. fi stimulation of the Bi dobacterium genus by inulin and FOS This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. prebiotics does not explain why levels of butyrate are enhanced 1073/pnas.1000091107/-/DCSupplemental. 4672–4679 | PNAS | March 15, 2011 | vol. 108 | suppl. 1 www.pnas.org/cgi/doi/10.1073/pnas.1000091107 Downloaded by guest on September 29, 2021 68. This superfamily also includes fungal enzymes (15, 16). Exo- a highly regulated β-fructofuranosidase confirmed its role in acting β-fructofuranosidases cleave the terminal β,2–1 bonds using both mixed-chain length FOS and long-chain inulin. linking the fructose monomers, but most enzymes also have some invertase activity against the β,2–6 bonds between the terminal Results glucose–fructose units (17). Thus, activities of specific enzymes Microarray Detection of Genes Differentially Expressed During against sucrose, FOS, and inulin vary (18). β-fructofuranosidases Growth on Inulin Compared with Starch. A shotgun genomic have been identified in the genome sequences of Bifidobacterium microarray of random R. inulinivorans A2-194 DNA fragments longum (19), Bi. breve (20), Bi. lactis (21, 22), and Lactobacillus (27) was used to assess changes in gene expression during growth plantarum WCFS1 (23). The enzyme in Bi. breve had greatest on inulin compared with starch. Bacterial mRNA was purified activity against β,2–1 glucose–fructose links and no activity against from exponentially growing cells [optical density (OD)650 = 0.4] β,2–1 fructose–fructose links (20). Few published enzymes have and hybridized as described previously (27). Spots that were up- considerable activity against long-chain inulin molecules. regulated on either substrate were ranked based on the log2 R. inulinivorans, a butyrate producing low G+C Firmicute normalized data (Materials and Methods). Only eight clones were bacterium (3, 24), is a Clostridium cluster XIVa bacterium that induced more than 5-fold on inulin compared with starch (and 12 can comprise up to 2% of the gut microbiota (25) and is able to induced >3-fold), whereas 22 clones were up-regulated more grow on some sugars (24) and the complex substrates inulin and than 5-fold on starch compared with inulin. These 30 clones were starch in pure culture (3, 26). Inulin also enhanced the survival of sequenced, and the gene functions based on the closest database R. inulinivorans A2-194 against a background of total fecal matches are shown in Table 1 with full descriptions in Table S1. bacteria after introduction into a fermentor system designed to An additional microarray analysis was done to compare genes simulate the colon (3). In the present study, we identified specific expressed during growth on fructose,short-chain FOS (scFOS; P95), genes involved in inulin and starch utilization by R. inulinivorans and inulin to distinguish those required for degradation of long- and A2-194 using microarray analysis and were able to show the in- short-chain fructan substrates.
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