Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii

Xiaolei Ze, Ben David, Jenny Laverde Gomez, Bareket Dassa, Paul O Sheridan, Sylvia H Duncan, Petra Gisela Helen Louis, Bernard Henrissat, Natahlie Juge, Nicole M Koropatkin, Edward A. Bayer, Harry J Flint

Research output: Contribution to journalArticle

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Abstract

Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energy
from dietary starches that escape digestion by host enzymes via its exceptional activity against particulate “resistant” starches.
Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family
(GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructose
as an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptides
were detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted to
carry dockerin modules, with 1, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediate
the formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerins
from R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymes
Amy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wallanchoring
protein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylases
Amy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, as
its dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides the
first example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.”
Original languageEnglish
Article numbere01058-15
JournalmBio
Volume6
Issue number5
DOIs
Publication statusPublished - 29 Sep 2015

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Ruminococcus
Amylases
Starch
Bacteria
Proteins
Enzymes
Recombinant Fusion Proteins
Glycoside Hydrolases
cohesins
Firmicutes
Proteomics
Digestion

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Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii. / Ze, Xiaolei; David, Ben; Laverde Gomez, Jenny; Dassa, Bareket; Sheridan, Paul O; Duncan, Sylvia H; Louis, Petra Gisela Helen; Henrissat, Bernard; Juge, Natahlie ; Koropatkin, Nicole M; Bayer, Edward A.; Flint, Harry J.

In: mBio, Vol. 6, No. 5, e01058-15, 29.09.2015.

Research output: Contribution to journalArticle

Ze, Xiaolei ; David, Ben ; Laverde Gomez, Jenny ; Dassa, Bareket ; Sheridan, Paul O ; Duncan, Sylvia H ; Louis, Petra Gisela Helen ; Henrissat, Bernard ; Juge, Natahlie ; Koropatkin, Nicole M ; Bayer, Edward A. ; Flint, Harry J. / Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii. In: mBio. 2015 ; Vol. 6, No. 5.
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abstract = "Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energyfrom dietary starches that escape digestion by host enzymes via its exceptional activity against particulate “resistant” starches.Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family(GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructoseas an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptideswere detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted tocarry dockerin modules, with 1, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediatethe formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerinsfrom R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymesAmy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wallanchoringprotein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylasesAmy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, asits dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides thefirst example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.”",
author = "Xiaolei Ze and Ben David and {Laverde Gomez}, Jenny and Bareket Dassa and Sheridan, {Paul O} and Duncan, {Sylvia H} and Louis, {Petra Gisela Helen} and Bernard Henrissat and Natahlie Juge and Koropatkin, {Nicole M} and Bayer, {Edward A.} and Flint, {Harry J}",
note = "ACKNOWLEDGMENTS We acknowledge support from BBSRC grant no. BB/L009951/1, from the Scottish government Food, Land and People program, and from the Society for Applied Microbiology. E.A.B. is supported by a grant (no. 1349/13) from the Israel Science Foundation (ISF), Jerusalem, Israel, and by a grant from the United States-Israel Binational Science Foundation (BSF). E.A.B. is the incumbent of the Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry. Thanks are due to Fergus Nicol for proteomic analysis and to Auriane Bernard for enzyme assays on stationary-phase cultures. We also thank Julian Parkhill and Keith Turner (Wellcome Trust Sanger Institute, Cambridge, United Kingdom) for making the R. bromii L2-63 genome sequence available for analysis.",
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AU - Ze, Xiaolei

AU - David, Ben

AU - Laverde Gomez, Jenny

AU - Dassa, Bareket

AU - Sheridan, Paul O

AU - Duncan, Sylvia H

AU - Louis, Petra Gisela Helen

AU - Henrissat, Bernard

AU - Juge, Natahlie

AU - Koropatkin, Nicole M

AU - Bayer, Edward A.

AU - Flint, Harry J

N1 - ACKNOWLEDGMENTS We acknowledge support from BBSRC grant no. BB/L009951/1, from the Scottish government Food, Land and People program, and from the Society for Applied Microbiology. E.A.B. is supported by a grant (no. 1349/13) from the Israel Science Foundation (ISF), Jerusalem, Israel, and by a grant from the United States-Israel Binational Science Foundation (BSF). E.A.B. is the incumbent of the Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry. Thanks are due to Fergus Nicol for proteomic analysis and to Auriane Bernard for enzyme assays on stationary-phase cultures. We also thank Julian Parkhill and Keith Turner (Wellcome Trust Sanger Institute, Cambridge, United Kingdom) for making the R. bromii L2-63 genome sequence available for analysis.

PY - 2015/9/29

Y1 - 2015/9/29

N2 - Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energyfrom dietary starches that escape digestion by host enzymes via its exceptional activity against particulate “resistant” starches.Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family(GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructoseas an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptideswere detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted tocarry dockerin modules, with 1, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediatethe formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerinsfrom R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymesAmy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wallanchoringprotein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylasesAmy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, asits dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides thefirst example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.”

AB - Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energyfrom dietary starches that escape digestion by host enzymes via its exceptional activity against particulate “resistant” starches.Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family(GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructoseas an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptideswere detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted tocarry dockerin modules, with 1, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediatethe formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerinsfrom R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymesAmy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wallanchoringprotein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylasesAmy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, asits dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides thefirst example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.”

U2 - 10.1128/mBio.01058-15

DO - 10.1128/mBio.01058-15

M3 - Article

VL - 6

JO - mBio

JF - mBio

SN - 2161-2129

IS - 5

M1 - e01058-15

ER -