Substrate-driven gene expression in Roseburia inulinivorans: importance of inducible enzymes in the utilization of inulin and starch.
Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene-expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization genes induced during growth on inulin included one encoding a β-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis showed that the β-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked sugar phosphotransferase transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the use of inulin. The R. inulinivorans β-fructofuranosidase was overexpressed in Escherichia coli and shown to hydrolyze fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on starch included the major extracellular α-amylase and two distinct α-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.
Scott
Karen P
KP
Rowett Institute of Nutrition and Health, University of Aberdeen, Bucksburn, Aberdeen AB21 9SB, United Kingdom. kps@rowett.ac.uk
Martin
Jenny C
JC
Chassard
Christophe
C
Clerget
Marlene
M
Potrykus
Joanna
J
Campbell
Gill
G
Mayer
Claus-Dieter
CD
Young
Pauline
P
Rucklidge
Garry
G
Ramsay
Alan G
AG
Flint
Harry J
HJ
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D001483:Base Sequence; D003470:Culture Media / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D016937:Gram-Positive Endospore-Forming Rods / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D007420:Intestine, Large / Q000382:microbiology*; D007444:Inulin / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D010732:Phosphofructokinase-1 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013213:Starch / Q000378:metabolism* / Q000494:pharmacology; D043324:beta-Fructofuranosidase / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D003470:Culture Media; D017931:DNA Primers; D013213:Starch; D007444:Inulin; D010732:Phosphofructokinase-1; D043324:beta-Fructofuranosidase