Rumen Cellulosomics: Divergent Fiber-Degrading

Strategies Revealed by Comparative Genome-Wide

Analysis of Six Ruminococcal Strains
Bareket Dassa
1
, Ilya Borovok
2
, Vered Ruimy-Israeli
1
, Raphael Lamed
2
, Harry J. Flint
3
, Sylvia H. Duncan
3
,
Bernard Henrissat
4
, Pedro Coutinho
4
, Mark Morrison
5,6
, Pascale Mosoni
7
, Carl J. Yeoman
8
,
Bryan A. White
9,10
, Edward A. Bayer
1
*
1Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel, 2Department of Molecular Microbiology and Biotechnology, Tel Aviv University,
Ramat Aviv, Israel, 3Microbial Ecology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, United Kingdom, 4Architecture et Fonction des
Macromolecules Biologiques, Aix-Marseille University and Centre National de la Recherche Scientifique (CNRS), Marseille, France, 5University of Queensland Diamantina
Institute, Woolloongabba, Queensland, Australia, 6Department of Animal Sciences, The Ohio State University, Columbus, Ohio, United States of America, 7The French
National Institute for Agricultural Research (INRA), UR454 Unite de Microbiologie, Saint-Gene` s-Champanelle, France, 8Department of Animal and Range Sciences,
Montana State University, Bozeman, Montana, United States of America, 9The Institute for Genomic Biology, University of Illinois, Urbana, Illinois, United States of
America, 10Department of Animal Sciences, University of Illinois, Urbana, Illinois, United States of America
Abstract
Background: A complex community of microorganisms is responsible for efficient plant cell wall digestion by many
herbivores, notably the ruminants. Understanding the different fibrolytic mechanisms utilized by these bacteria has been of
great interest in agricultural and technological fields, reinforced more recently by current efforts to convert cellulosic
biomass to biofuels.
Methodology/Principal Findings: Here, we have used a bioinformatics-based approach to explore the cellulosome-related
components of six genomes from two of the primary fiber-degrading bacteria in the rumen: Ruminococcus flavefaciens
(strains FD-1, 007c and 17) and Ruminococcus albus (strains 7, 8 and SY3). The genomes of two of these strains are reported
for the first time herein. The data reveal that the three R. flavefaciens strains encode for an elaborate reservoir of cohesin-
and dockerin-containing proteins, whereas the three R. albus strains are cohesin-deficient and encode mainly dockerins and
a unique family of cell-anchoring carbohydrate-binding modules (family 37).
Conclusions/Significance: Our comparative genome-wide analysis pinpoints rare and novel strain-specific protein
architectures and provides an exhaustive profile of their numerous lignocellulose-degrading enzymes. This work provides
blueprints of the divergent cellulolytic systems in these two prominent fibrolytic rumen bacterial species, each of which
reflects a distinct mechanistic model for efficient degradation of cellulosic biomass.
Citation: Dassa B, Borovok I, Ruimy-Israeli V, Lamed R, Flint HJ, et al. (2014) Rumen Cellulosomics: Divergent Fiber-Degrading Strategies Revealed by Comparative
Genome-Wide Analysis of Six Ruminococcal Strains. PLoS ONE 9(7): e99221. doi:10.1371/journal.pone.0099221
Editor: Mickae l Desvaux, INRA Clermont-Ferrand Research Center, France
Received February 9, 2014; Accepted May 12, 2014; Published July 3, 2014
Copyright: 2014 Dassa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The research described in this communication was supported by a grant (No. 24/11) issued to RL by The Sidney E. Frank Foundation through the Israel
Science Foundation (ISF) and by a grant (No. 1349/13) to EAB also from the ISF (http://www.isf.org.il/english/). This research was also supported by the
establishment of an Israeli Center of Research Excellence (I-CORE Center No. 152/11, EAB) managed by the ISF, grants from the United States-Israel Binational
Science Foundation (BSF), Jerusalem, Israel (http://www.bsf.org.il/BSFPublic/Default.aspx), by the Weizmann Institute of Science Alternative Energy Research
Initiative (AERI) and the Helmsley Foundation (http://helmsleytrust.org/), a project (FiberFuel) funded through the ERA-NET Scheme of the 7th EU Framework
Programme European Union Contract (within the framework of the Third ERA-IB Call). A grant to EAB and RL from the Israel Ministry of Science (http://most.gov.il/
english/Pages/default.aspx) is gratefully acknowledged. The North American Consortium for Genomics of Rumen Bacteria Consortium was supported by the
Initiative for Future Agriculture and Food Systems, Grant no. 2000-52100-9618 and Grant No 2001-52100-11330, from the USDA Cooperative State Research,
Education, and Extension Services National Research Initiative Competitive Grants Program (http://www.csrees.usda.gov/). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors wish to declare that BAW, a PLOS ONE editor, was involved in the work performed. This does not alter the authors adherence
to all the PLOS ONE policies on sharing data and materials.
* Email: [email protected]
Introduction
The bovine rumen hosts a wide range of strictly anaerobic and
some facultatively anaerobic microorganisms [15]. The rumen
microbiota is highly diverse, including both prokaryotic and
eukaryotic anaerobes, that maintains a mutualistic relationship
with its host [6]. On the one hand, the rumen flora is dynamic and
known to adapt to changes in the host diet and age [7,8]. On the
other, the rumen microbiota produces large quantities of short-
chain fatty acids that are absorbed across the rumen wall and used
as energy sources by the host [9]. Fermentation of plant material
by rumen fiber-degrading microorganisms in the rumen typically
provides 70% of the energy obtained from the diet [10]. Herbivore
health and productivity are greatly affected by the composition
PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e99221
and activity of the rumen microbiota and, in particular, by fiber-
degrading species. Relatively few rumen bacteria have been
identified as primary degraders of plant fiber, but cellulolytic
Ruminococcus and Fibrobacter species clearly play an important role
[11,12]. Knowledge of the fibrolytic mechanisms employed by
these specific rumen bacteria is of great importance for
manipulation of animal diet and for improvement of its
performance. Moreover, insights in this field may lead to
biotechnological applications related to biofuel production.
Two cellulolytic Firmicutes bacteria, Ruminococcus flavefaciens and
Ruminococcus albus, and the gram-negative Fibrobacter succinogenes are
important and culturable cellulose-degrading agents in the rumen
[2]. These three species are able to adhere and grow on cellulosic
polysaccharides as their primary carbon and energy sources and in
doing so breakdown plant cell wall material [13].
Efficient degradation of plant cell-wall polysaccharides by some
anaerobic bacteria is achieved by a multienzyme complex
specialized in cellulose degradation, known as the cellulosome,
which has been best studied in Clostridium thermocellum [1419]. The
cellulosome is a molecular platform that assembles a multiplicity of
carbohydrate-degrading enzymes, i.e., glycoside hydrolases (GHs),
polysaccharide lyases (PLs) and carbohydrate esterases (CEs).
These are degradative enzymes, such as endoglucanases, cellobio-
hydrolases, xylanases, etc., which attack heterogeneous, insoluble
cellulosic substrates in a synergistic manner [18,2022]. Unlike
other (notably aerobic) bacteria and fungi, these enzymes are not
freely diffusible, because they contain a dockerin module that
mediates their integration into the major cellulosome structural
subunits, termed scaffoldins. The dockerin strongly interacts with
multiple copies of cohesin modules located on the scaffoldins via a
high-affinity protein-protein interaction [2327]. In C. thermocellum,
the scaffoldin also contains a carbohydrate-binding module (CBM)
that binds the cellulosome complex to the plant cell wall substrate
[2831]. Thus, dockerin-containing enzymes are incorporated into
scaffoldin-borne cohesins, and a CBM-bearing scaffoldin targets
the assembly to the carbohydrate substrate. Moreover, the C.
thermocellum cellulosomes are attached to the bacterial cell surface
by virtue of an S-layer homology (SLH) domain [32].
One of the most elaborate cellulosomal architectures was
recently discovered in R. flavefaciens through extensive study of its
genome sequence and transcriptome [33,34]. R. flavefaciens codes
for more than a dozen cohesin-containing proteins that may
interact with an unprecedented number (,220) of dockerin-
containing proteins. These early studies on the cellulosome of this
bacterium established new features that deviate from those of the
canonical C. thermocellum cellulosome. In R. flavefaciens, the ScaC
protein bears both a cohesin and a dockerin module and serves as
an adaptor scaffoldin [35]. Additionally, the cellulosome is
attached to the bacterial cell surface in an unconventional manner,
whereby a singular type of scaffoldin, ScaE, is covalently fastened
to the cell-wall envelope via proteolytic cleavage and transfer by
sortase-mediated attachment [36]. Previous analysis of R.
flavefaciens dockerins [34] has served to classify the dockerins into
at least six major groups, according to their conserved sequence
profiles, and demonstrated the modular nature of the enzymes and
their association to the other non-catalytic proteins. The
characteristics of the cohesin-containing proteins and additional
elements have yet to be described in detail.
In contrast to the elaborate cellulosome evident in R. flavefaciens,
the system of R. albus remains puzzling. Despite the fact that R.
albus produces an array of dockerin-bearing proteins [37], no
genes encoding cohesin-containing proteins have been deter-
mined, and the presence of a defined cellulosome is thus in
question. In previous work, several of its dockerin-containing
endoglucanases were indeed characterized [38,39]. R. albus is also
known to adhere tightly to cellulose and appears to utilize several
types of cellulose-adhesion mechanisms for this purpose, such as
Pil proteins [4043] and an exopolysaccharide glycocalyx [4447].
Surprisingly, the major Cel48 exoglucanase that commonly
characterizes cellulosomes in other bacterial species was found to
bear a distinctive type of CBM rather than a dockerin at its C
terminus [48]. This family 37 CBM was found to bind to
numerous types of polysaccharides and was identified in several
enzymes with catalytic modules such as GHs, PLs and CEs
[49,50]. Subsequent studies indicated that R. albus utilizes
CBM37s to mediate bacterial cell surface attachment [51].
Moreover, CBM37 was shown to be exposed at the cell surface
of R. albus 20 by Rakotoarivonina [50], who proposed that the
adhesion and fibrolytic systems of R. albus are linked.
The recent availability of genomic data of R. flavefaciens and R.
albus strains has enabled us to unravel the blueprint of the
cellulolytic systems of ruminococci and to compare their
alternative fiber-degrading strategies. Comparative genome-wide
analysis has allowed the identification of structural elements of
each cellulosome, such as scaffoldins and CBMs, and to assess the
profile of dockerin-containing proteins and carbohydrate-degrad-
ing enzymes in each strain. This work provides a framework for
the cellulose-degrading systems of these two ruminococcal species,
thereby demonstrating both core elements and novel strain-
specific enzymes, which would either assemble into a multi-
enzyme cellulosome or comprise an array of cell-bound carbohy-
drate-active enzymes and associated proteins for R. flavefaciens and
R. albus, respectively.
Results
Six available Ruminococcus genomes
The ability of cellulolytic bacteria to degrade plant cell-wall
carbohydrates is encoded in their genomes. In this work, we
explored the genomes of three strains each of Ruminococcus
flavefaciens (FD-1, 17 and 007c) and Ruminococcus albus (7, 8 and
SY3). Using a comparative bioinformatics approach, we identified
their putative cellulolytic enzymes and, particularly for these two
ruminococcal species, their cellulosome-related components (Fig. 1
and Table 1). Two new genomes, R. flavefaciens 007c and R. albus
SY3, were sequenced and submitted to GenBank (see relevant
sections in Materials and Methods). Although each of the six
genomes was derived from bacteria obtained from a different cow
and isolated at different geographical locations and time periods, it
has been established that various species and strains coexist at the
same time in the rumen of a given host organism [52,53]. In an
attempt to profile the cellulose-degrading strategy of each
bacterium, each genome was examined in this work to identify
homologs of the primary building blocks of the cellulosome,
namely cohesin-containing proteins and dockerin-containing
proteins, together with CBMs. We further applied various
sequence analysis methods to identify and analyze the presence
of known carbohydrate-active enzymes (CAZymes, [54], i.e., GHs,
PLs and CEs) as detailed below. The following analyses were
based on draft genome sequences (except for R. albus 7), showing
an adequate level of genome coverage (see Materials and
Methods), yet may include sequence gaps which restrict some of
the information.
Multiple architectures of cohesin-bearing scaffoldins in R.
flavefaciens strains
We identified numerous cohesin-containing proteins in all three
R. flavefaciens strains. Specifically, 17, 11 and 10 scaffoldin subunits
Ruminococcal Cellulosomics
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Figure 1. Blueprints of the cellulosome-related proteins in the designated strains of (A) R. flavefaciens and (B) R. albus, studied in this
work. Schematic representation of scaffoldins, cohesin- and dockerin-containing proteins, which were identified in the genomes of each strain in this
work. Numbers indicated the copy number of each type of protein architecture, identified in the designated strain. Legend of pictograms is shown in
Panel B. See text for details.
doi:10.1371/journal.pone.0099221.g001
Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 3 July 2014 | Volume 9 | Issue 7 | e99221
were detected in strains FD-1, 17 and 007c, respectively (Table 1
and Fig. 1A). R. flavefaciens cellulosomes contain a unique spectrum
of type-III cohesin modules [36,55,56], which are different than
the type-I and type-II cohesins found in C. thermocellum and other
cellulosome-producing clostridia. Type-III cohesin-containing
proteins can be further catalogued into four functional groups
according to their architecture:
(i) As demonstrated in earlier publications for strains 17 and
FD-1, ScaA and ScaB serve as major scaffoldin subunits
with multiple non-identical repeats of cohesin modules
(Fig. 1A.1). ScaA harbors a unique type of C-terminal
dockerin and ScaB contains a C-terminal X-dockerin
(XDoc) modular dyad [56]. Notably, the composition of
the major cohesins in the ScaB scaffoldin is different
between the FD-1 strain (which contains two subtypes of
cohesins on the same scaffoldin) and the 17 strain (in which
all cohesins are of the same subtype) [57]. In addition, the
number of cohesin repeats in ScaB varies between the R.
flavefaciens strains, whereby strain 17 contains 7 cohesin
repeats and strain FD-1 contains 9 repeats. ScaB of strain
007c contains at least 4 cohesins, but since its ORF
(EWM54563) is located near the end of a contig in the
draft genome, its C-terminus sequence is incomplete by
definition (no stop codon was observed). Moreover, the
presence of an XDoc modular pair in this strain can thus
not be verified at this time. Yet it is clear that its sequenced
cohesins are of the ScaA variety that resemble those of
strain 17 as opposed to cohesins 14 of the FD-1 ScaB. We
therefore presume that the 007c ScaB bears a single
subtype of cohesin, the exact number of which is currently
unknown.
(ii) ScaE-like proteins (Fig. 1A.1) were identified in all three
genomes. As shown for strains 17 and FD-1 in previous
works, this type of scaffoldin has an important anchoring
function, due to its ability to anchor the ScaB and CttA
proteins [58] and to the presence of a C-terminal sortase
sequence, which is involved in the attachment of the
cellulosome to the bacterial cell surface [36]. In turn, CttA
attaches to cellulose through its two CBMs, and the
bacterial cell itself is thus attached to the substrate through
this mechanism [58].
(iii) The current work has revealed a third group of proteins
(511 copies, according to the strain), characterized by a
bi-modular theme, which includes both a single cohesin
module and a single dockerin in the same polypeptide
(Fig. 1A.2). As shown previously for ScaC in strain 17 [35],
this type of protein may serve as an adaptor protein to
regulate binding of either particular scaffoldins and/or
enzymes into cellulosome complexes, thereby altering the
repertoire of cellulosome content. Interestingly, this study
indicates that R. flavefaciens FD-1 exclusively contains a
second potential variation of this theme, in the form of two
proteins that bear a C-terminal dockerin with two cohesins
instead of one.
(iv) In addition, we identified several scaffoldins (13 copies per
strain) in the present research that bear a single cohesin
module, which is .90% similar between strains 17 and
007c and ,60% similar between strains FD-1 and 007c.
These cohesins lack a dockerin module but are fused to a
protein region whose function is as yet unknown (Fig. 1A.2).
In order to evaluate the sequence relatedness among the
cohesins from the different R. flavefaciens strains, we constructed a
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Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 4 July 2014 | Volume 9 | Issue 7 | e99221
phylogenetic tree (Fig. 2). The tree includes established cohesin
sequences, some of which were previously investigated experi-
mentally in strain FD-1 (i.e., ScaA, ScaB, ScaC and ScaE) as well
as a variety of putative cohesins (see Table S1). Many of the latter
cohesins are found only in strain FD-1 (e.g., ScaJ, ScaK, ScaL,
ScaM, ScaO and ScaP) as well as additional ORFs present in all
three strains. Whether or not these protein modules constitute
authentic cohesins remains an open question to be solved
experimentally in the future.
The cohesins of the scaffoldins expressed by the different genes
of the sca gene cluster, i.e., scaC, scaA, scaB and scaE (according to
their order on the genome) are in general conserved among the
strains according to previous findings ([57]). Thus, the ScaA
cohesins of the three strains all appeared on the same branch. As
anticipated, the first four ScaB cohesins of the FD-1 strain also co-
clustered with the ScaA cohesins. The other ScaB cohesins (i.e.,
the last five ScaB cohesins of the FD-1 strain and all of the
cohesins from strains 17 and 007c) co-clustered on a separate
branch. Similarly, the ScaE cohesins co-cluster on a separate
branch of the phylogenetic tree.
Many of the analogous scaffoldin sequences of strains 17 and
007c are remarkably similar and generally differ from their
counterparts in strain FD-1. These include the cohesins of ScaG
and ScaI as well as the cohesin sequence homologues of ScaC,
ScaA, ScaB and ScaE. In contrast, the protein sequences of the
ScaF cohesin are identical in all three strains. In addition, strains
17 and 007c contain an additional ScaF-like cohesin that differs
somewhat from the ScaF cohesin. Strain FD-1 lacks the second
ScaF-like cohesin.
Intriguingly, despite the near identity among most of the
homologous cohesins of strains 17 and 007c, the ScaC cohesin in
all three R. flavefaciens strains are conspicuously different in their
sequences, thus reinforcing the notion that they may be used as a
marker of the parent strain.
Exceptional features of R. flavefaciens dockerins
We identified an unusually large and diverse pool of dockerin-
containing proteins in all R. flavefaciens strains, compared with
other cellulosome-containing species of Clostridiales, which ranges
between 180 and 223 proteins (Table 1; 223, 180 and 183
dockerin-containing proteins in strains FD-1, 17 and 007c,
respectively). These proteins bear a signal peptide, suggesting that
they are secreted from the bacterium, and are often composed of
cellulose-degrading catalytic modules as well as putative proteases,
serpins, leucine-rich repeats and other unknown conserved protein
modules as described earlier for strain FD-1 [34].We extensively
explored the sequence conservation of each dockerin-containing
protein, and identified its catalytic modules according to the CAZy
database (see Materials and Methods). We profiled all modules of
known GHs, PLs and CEs and classified them into family types,
for both dockerin-containing proteins (Table 2) and other non-
cellulosomal proteins (Table 3). Another group of dockerin-
containing proteins contain non-catalytic modules, such as CBMs
and domains of unknown function [34]. Of note are the catalytic
modules that are unique to R. flavefaciens and absent in R. albus,
such as GH families 18, 24, 42 and 97; CE families 13 and 15; and
CBM families 32 and 63.
Table 4 describes a group of dockerin-containing enzymes that
contains more than one type of catalytic module on the same
polypeptide chain. R. flavefaciens codes for a relatively large number
of such multifunctional enzymes. One of the dominant modules
is GH43, which has been recently shown to be abundant in the
rumen in metagenomic studies [59,60] and is one of the more
abundant GH enzyme families in the genomes of common
hemicellulolyic rumen bacteria [61,62]. The GH43 family exhibits
broad substrate specificity and promiscuous characteristics
[61,63]. It is clear that strains 17 and 007c share numerous
protein architectures, many of which are different from those of
strain FD-1. This observation may indeed reflect the relatedness
between strains 17 and 007c and their distinction from strain FD-
1.
Compared with other rumen bacteria we noted a group of
exclusive enzymes, which are unique to the R. flavefaciens strains
and are absent or underrepresented in the genomes of R. albus
strains and other fibrolytic rumen species, e.g., Fibrobacter
succinogenes subsp. succinogenes S85. These include b-galactosidas-
es (GH42), a-glucosidases (GH97), xylanases (GH11) and proteins
with an unusual number of PLs from family 11 (Table 2).
The conserved sequence pattern of R. flavefaciens FD-1 dockerins
was examined previously [33,34], and the data supported the
classification of all dockerins in that genome into six major groups.
Subtypes of dockerins with unique features were described, that
included atypical lengths of the second calcium-binding repeat,
different sequence insertions and different linkers within the
dockerin module. When comparing dockerins from the three R.
flavefaciens strains we observed a similar trend of diversity and
heterogeneity in the sequences of dockerins (Fig. S1). Interestingly,
there are only three identical dockerins between strain FD-1
dockerins and those of strain 17 or 007c. Strain FD-1 dockerins
are on average 46% similar to homologues in 007c and 67%
similar to those of strain 17. BLAST searches with dockerin
members from FD-1 groups as queries revealed homologous
dockerins (e-value ,10
210
) in strains 17 and 007c, except for
group 4 b dockerins which were exclusive to strain FD-1.
Overall, we identified genes coding for an elaborate and
sophisticated cellulosome in all three R. flavefaciens strains. Notably,
we observed particular variations in the composition and in the
number of key cellulosomal elements between the different strains.
Of the major novel architectures is a multi-dockerin protein
(EWM52407 in R. flavefaciens 007c and WP_019680459 in R.
flavefaciens 17), which contains seven tandem non-identical dock-
erin repeats and appears in strains 007c and 17 but not FD-1. This
novel protein architecture has yet to be observed in any other
cellulosome-producing bacterium. In addition, another rare
protein arrangement of two non-tandem repeats of a dockerin in
the same polypeptide was observed in these strains (EWM52383 in
R. flavefaciens 007c and orf03158 in R. flavefaciens 17), and joins a
recent observation of this type of protein in Acetivibrio cellulolyticus
[64].
R. albus is cohesin-deficient yet encodes for dockerins
and cell-anchoring modules
In order to further understand the cellulosomics of R. albus, we
sequenced the genome of R. albus SY3 and compared it to the two
publicly available genomes of R. albus, strains 7 and 8 (Fig. 1B and
Table 1). Genome-wide analysis of the three R. albus strains
revealed 90, 62 and 58 dockerin-containing proteins in strains 7, 8
and SY3, respectively. Unlike R. flavefaciens, these dockerins are
generally conserved and could not be divided into significant
subgroups. The predominant predicted recognition residues in all
three R. albus strains were V(I), T, A and A in positions 10, 11, 17
and 18 of the repeated segment.
Surprisingly, only one cohesin-containing protein was deter-
mined in the genomes of R. albus strains 7 and SY3, and none in
strain 8 (GI number 317056975 and EXM40378, respectively).
The single cohesin module is supplemented by a C-terminal
dockerin module and a linker between the two, thus resembling an
adaptor cohesin-dockerin protein, similar to that of ScaC in R.
Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 5 July 2014 | Volume 9 | Issue 7 | e99221
Figure 2. Phylogenetic relationship among cohesin modules of R. flavefaciens and R. albus. The names of the different cohesins are color
coded according to the given strains. The various cohesins from the different strains were named based on the sequence similarity to those of the R.
flavefaciens FD-1 strain (Table S1). The single cohesins identified in the two R. albus strains (arrows) cluster with those of the ScaF cohesins of R.
flavefaciens and were hence labeled ScaF. Branches with bootstrap values below confidence level 0.7 were collapsed.
doi:10.1371/journal.pone.0099221.g002
Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 6 July 2014 | Volume 9 | Issue 7 | e99221
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Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 7 July 2014 | Volume 9 | Issue 7 | e99221
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Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 8 July 2014 | Volume 9 | Issue 7 | e99221
flavefaciens. The two homologous R. albus cohesin-containing
proteins are 92% similar. Comparison of the cohesin module
with R. flavefaciens cohesins showed 69% similarity (with R.
flavefaciens 17) and 79% (with R. flavefaciens FD-1). This single R.
albus cohesin is orthologous to the R. flavefaciens ScaF protein
(Fig. 2). The apparent presence of a lone cohesin in R. albus
represents a puzzling deviation from the classical cellulosome
architecture, where dockerins are anchored onto multiple cohesin-
containing scaffoldins. These observations suggest an alternative
mechanism for immobilization of dockerin-containing enzymes
onto carbohydrates or their anchoring to the cell surface.
R. albus contains CBMs belonging to several family types
(Table 2), two of which (family 2 and 37) are absent in R.
flavefaciens. The cellulose-binding CBM2 (common in numerous
non-cellulosomal cellulolytic bacteria) appears in only one or two
copies in proteins that also contain a GH5 module. More
intriguingly, all three R. albus genomes contain multiple copies of
a family 37 sugar-binding module (CBM37), which is unique to
this species (77, 51 and 102 copies in R. albus 7, 8 and SY3,
respectively). The CBM37 module is absent in R. flavefaciens, and
has not been detected in any other sequenced genome. This
special CBM is integrated into various carbohydrate-active
Table 4. Cellulosomal and non-cellulosomal multifunctional proteins in R. flavefaciens.
Domain architecture (cellulosome-related domains) R. flavefaciens accession numbers
Strain FD-1 17 007c
Shared by all strains:
CBM13-Doc-GH43-GH43 ZP_06142338 WP_019678907 orf03036
CBM22-GH10-CBM22-Doc-GH43-CBM6 orf03865 WP_019680029 EWM52826
CE12-CBM13-Doc-CBM35-CE12 orf02983, orf03219 WP_019678069 EWM54325
CE8-PL1-Doc orf02371 WP_00998568 EWM52494
GH11-CBM22-GH10-Doc-CBM22-CE4 orf01222 WP_019679223 EWM54891
GH25-GH25 ZP_06141601 WP_019678757 EWM53404
GH43-CBM22-Doc-CE1 orf00341 WP_009983072 EWM54432
GH43-CBM6-CBM22-Doc-CE1 orf00764 WP_019678371 EWM53765
Shared by two strains:
CBM22-Doc-CE1-CE1 WP_019678253 EWM55310
CE3-Doc-CE15 WP_019679655, CAB55348 EWM52579, EWM54090
GH11-CBM22-Doc-GH16 AAB26620 (reported in [82]) EWM53768
GH11-CE1 orf01851 orf04775
GH11-CE4 orf02455 orf00919
GH11-GH10 P29126 (reported in [83]) orf01418
GH11-GH11-Doc WP_019679180 EWM54934
GH9-GH16 orf02516 orf00858
Strain specific:
CBM35-CE3- Doc-CBM35-GH26 orf03447
CBM35-CE3-GH5-Doc orf00227
CE3-CBM22-Doc-CE15 orf02390
Doc-GH16-GH16-GH16 orf00265
GH11-CBM13-CE1- Doc orf00775
GH11-CBM22-Doc-GH11-CE1 orf03180
GH11-CBM22-Doc-GH11-CE3 orf01315
GH11-CBM22-GH10- Doc-GH11 orf00468
GH11-CBM22-GH10- Doc-GH11-CE4 orf03896
GH11-CE3-Doc orf01321
GH53-CE3-Doc orf01739
GH5-GH5-Doc orf01388
PL1-PL9-X215-Doc orf00696
PL11- Doc-CBM35-CE12 orf03451
GH11-GH16 orf01699
GH11-CBM22-CE3- Doc CAB93667 (reported in [84])
doi:10.1371/journal.pone.0099221.t004
Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 9 July 2014 | Volume 9 | Issue 7 | e99221
proteins, in association with catalytic modules such as GHs, CEs,
as well as non-catalytic proteins, but very rarely with dockerins
only observed once per strain. In several cases in all three
organisms, the CBM37 module appears in a tandem repeat (13,
11, and 18 in strains 7, 8 and SY3, respectively).
We examined the co-appearance of two modules, CBM37 and
GHs, in the same protein (Table 3). CBM37 was associated with
11 different GH families, including cellulases (GH5, GH9, GH48)
and hemicellulases (GH5, GH10, GH11, GH26, GH43). Inter-
estingly, some of the GH families appear both in R. flavefaciens and
in R. albus, the latter of which are also associated with CBM37
(with one exception, GH98).
The distribution of GH modules within the dockerin-containing
enzymes (Table 2) shows that R. albus codes for modules from
unique GH families, which are exclusive to that species, such as
family 4 (acetyl xylan esterase), family 23, family 27, family 28
(polygalacturonase), family 32, family 39 (a-L-iduronidase and b-
xylosidase), family 51 (endoglucanase/endoxylanase), family 67
(glucuronidase), family 98 (endo-b-galactosidase) and family 113
(b-mannanase). The R. albus genome also codes for PL10 and CE9
modules, which are absent in R. flavefaciens.
R. albus codes for 48 multifunctional proteins (Table 5), some of
which have a common protein architecture in two of the strains,
while others are strain-specific. Five of these proteins contain
GH11-CBM22 modules, with a different C-terminal variation on
the protein. Strain 7 and SY3 share more multifunctional protein
architectures with each other than with strain 8. The number of
multifunctional proteins in R. albus is significantly less than those of
R. flavefaciens.
Discussion
The microbial community of the rumen shares a rich source of
novel plant cell wall degrading enzymes, which include cellulases,
xylanases and other hemicellulases, as well as pectinases [65].
Although cellulolytic enzyme systems have been investigated over
the years, the mechanisms by which bacteria achieve efficient
plant cell wall breakdown are still obscure. In this work we have
described a multi-dimensional perspective on the cellulolytic
potential of the two dominant fibrolytic ruminococci, R. flavefaciens
and R. albus by comparing the cellulase system of three different
strains from each species. Divergent mechanisms of fiber
degradation were revealed by integrating the data, which involved
(i) the outlining of their scaffoldins and dockerin-containing
proteins, (ii) the profiling of cellulose-degrading enzymes in each
species and strain, and (iii) the identification of protein architec-
tures of complex multifunctional enzymes of each strain.
All R. flavefaciens strains code for particularly elaborate
cellulosome systems, having multiple cohesin-containing proteins
that may assemble into defined cellulosomal structures, which
exhibit various combinations of dockerin-containing cellulases on
their surface. Distinct differences in the number of enzymes
(Table 2) or their modular architectures (Table 4) were observed
among the different R. flavefaciens strains. Based on these
observations it is likely strains 17 and 007c are more closely
related to one another than either is to FD-1. This is also reflected
by the phylogenetic relatedness of the cohesin sequences of the
former two strains versus those of the latter. It is also clear that
strain FD-1 bears the most elaborate cellulosome system.
Sequence variability in the structural sca gene cluster (scaC-scaA-
scaB-cttA-scaE) was also supported by a previous work [53],
suggesting that other R. flavefaciens strains may reflect such strain-
related plasticity. Indeed, recent work, which explored the
diversity of R. flavefaciens strains in the rumen using the
polymorphic nature of ScaC [52], revealed spatial and temporal
differences among strains that may relate to functional differences
among R. flavefaciens strains.
Analysis of the cellulolytic gene complement of R. albus raises
questions regarding its approach to degrade cellulose fibers. Each
genome contains several dozens of dockerins. Surprisingly,
however, only a single cohesin-containing protein was detected
in strains 7 and SY3, and a cohesin counterpart was not detected
in strain 8. These findings do not coincide with the classical
cellulosome paradigm, whereby multiple cohesin-bearing scaffol-
dins are essential for enzyme assembly, and it is thus difficult to
assign a functional role for the dozens of dockerins that are
Table 5. Cellulosomal and non-cellulosomal multifunctional proteins in R. albus.
Domain architecture (cellulosome-related domains) R. albus accession numbers
Strain 7 8 SY3
Shared by two strains:
CBM35-GH26-CE3-CBM37 YP_004103508 ZP_08158982
CE12-CBM13-Doc-CBM35-CE12 YP_004103674 EXM39991
GH11-CBM22-CBM37-CE1 YP_004105842 EXM39976
GH11-CBM22-CBM37-CE4 YP_004104068
GH11-CBM22-CE4 YP_004103272 EXM39050
GH11-CBM22-GH10-CBM37 YP_004090078 EXM37450
GH43-CBM22-CBM22-Doc-CE1 YP_004104621 EXM37569
PL1-PL1-CBM37 YP_004105710 EXM39993
Strain specific:
CE12-CBM13-Doc-CBM35-CE12 ZP_08160451
PL10-CE8-Doc ZP_08159991
PL11-CE12-CBM13-CBM13-CBM37 ZP_08159623
PL10-CE8-CBM37 EXM38121
Protein domain architecture is described, including only cellulosome-related domains.
doi:10.1371/journal.pone.0099221.t005
Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 10 July 2014 | Volume 9 | Issue 7 | e99221
conserved in the R. albus genomes. Indeed, a broad range of non-
cellulolytic microbes that lack appropriate GH and other
CAZymes have been found to possess numerous genes encoding
dockerin-containing proteins, and in many cases genes for cohesins
are either lacking or appear in only a single copy [66]. This clearly
implies that the latter microbes (mainly bacteria and archaea) do
not produce bona fide cellulosome-like structures, which raises the
question as to what is the exact role of the dockerin in these
proteins. It was previously suggested that such dockerins may bind
an as-yet undetermined protein component or they may be
involved in other reactions [66]. Nevertheless, in R. albus many of
the dockerins are borne by CAZymes, and the rich rumen
ecosystem may provide appropriate scaffoldins in an interspecies
manner (e.g., those of R. flavefaciens) that may accept them
symbiotically. Thus, an alternative mechanism might involve a
collaborative usage of cohesins and dockerins of both R. flavefaciens
and R. albus for putative hybrid cellulosomes where R. flavefaciens
cohesins would incorporate both its own dockerin-bearing
components and those of R. albus. Interestingly, some dockerin-
containing proteins in R. albus are encoded by plasmid genes (e.g.
in strain 7, two plasmid, pRUMAL01 and pRUMAL02 encode
nine such proteins). It is thus possible that the ruminal microbial
communities adjust to environmental changes by sharing and
acquisition of advantageous components, such as dockerin-
containing proteins, via interspecies exchange of plasmids [67].
Despite the lack of a genuine cellulosome, R. albus is known to
degrade cellulosic substrates to levels similar to those of R.
flavefaciens [68]. In this context, our analyses highlight a key role for
a dominant and unique protein module in R. albus, CBM37, that
appears to provide an alternative strategy for this bacterium.
CBM37s appear in high copy number in all three R. albus strains,
and their numbers vary greatly among them. Indeed, this
particular module has been shown definitively to attach enzymes
directly to bacterial cell wall carbohydrates [51]. Interestingly,
CBM37s are distributed in many R. albus enzymes whose orthologs
in R. flavefaciens are instead equipped with dockerins. Notably, the
critically important family 48 cellulase bears a CBM37 in all three
R. albus strains, as does the family 74 xyloglucanase and the family
11 xylanases. This observation raises the intriguing possibility that
CBM37 is the major mechanism for cell-surface anchoring of the
cellulolytic and associated enzymes instead of the classical type of
scaffoldin that positions them in close proximity to the bacterial
cell. Of note is the disproportionate number of dockerins and
CBM37s in strain SY3 versus the other two strains, mainly due to
a higher copy number of GHs with CBM37 modules (Table 1).
The rumen microbial population is dynamic and complex in
terms of its biodiversity, exhibiting both competitive and symbiotic
types of relationship [69]. The conditions in the rumen may thus
allow the variety of R. flavefaciens strains to share substrates as well
as promote cross-strain symbiosis, whereby the strains can share
cellulosomal components and/or benefit together from their
degraded products. Thus, closely related strains of R. flavefaciens
have homologous dockerin and cohesin components, which raises
the hypothesis that such structural components and enzymes may
be interchangeable when secreted. This may expand the number
of combinations for building a cellulosome and increase its
diversity. In spite of the benefits that may be derived from the
exchange of components, there is evidence for competition in the
utilization of either cellulose or cellobiose in co-cultures of R. albus
and R. flavefaciens [70]. The nature of the catalytic enzyme may be
another tool employed by the bacterium for a competitive
advantage and efficient cellulose degradation. Both R. flavefaciens
and R. albus code for various carbohydrate-degrading enzymes, yet
each species also codes for exclusive families of GHs, PLs and CEs
(Table 2). This trend is also reflected in the arrangement of the
multifunctional proteins, which are very abundant in R. flavefaciens
compared to other known Firmicutes, and compared to R. albus.
An additional species dominant in the fibrolytic consortium of
the rumen is Fibrobacter succinogenes. Its genome does not code for
known cellulosomal components, yet it codes for over a hundred
predicted carbohydrate-active enzymes [71], exhibiting catalytic
activities of cellulases, xylanases, PLs and CEs. A comparison of
the enzymatic profile between this genome and all six ruminal
genomes shows that F. succinogenes exclusively codes for GH
families which neither appear in R. flavefaciens nor R. albus, such as
family 45 (endoglucanases), family 54 (a-L-arabinofuranosidases
and b-xylosidases), family 57 (a-amylases and others) and family
116 (b-glucosidases and b-xylosidases). Interestingly, endocellu-
lases from GH family 45 are rare in bacteria, and are more
common in eukarya. F. succinogenes also contains PL family14 and
CE family 6, which are absent in the ruminococci. Of note is the
unique profile of CBMs in the F. succinogenes genome. The presence
of family 6 CBMs is expanded in its genome to 25 copies, while
CBMs important for crystalline cellulose degradation (families 2
and 3) are absent. Most of its CBMs (5 types out of 7) belong to
families which are absent in R. flavefaciens and R. albus genomes.
One possible mechanism for F. succinogenes fiber degradation has
been suggested by Brumm et al [71], who proposed a molecular
motor which removes glucan chains from cellulose crystals and
transports them, using energy derived from cellulolysis.
The present work surveys the different strategies by which two
ruminococcal species can degrade cellulose fibers, by analyzing the
encoded cellulosomal and enzymatic proteins from their genomes.
The extreme diversity of enzymes and structural scaffoldins was
demonstrated within R. flavefaciens and R. albus strains, and also
between these species. It is yet to be understood how the elaborate
arsenal of CAZymes and the different cohesin-containing compo-
nents are being regulated in the rumen. This work highlights the
need for more extensive experimental studies to assess the spatial
and temporal organization of the multiple cohesins, dockerins and
enzyme activities of these species in the rumen.
Materials and Methods
Genome sources
Six genomes were explored in this work, three strains each of
Ruminococcus flavefaciens (FD-1, 17 and 007c) and Ruminococcus albus
(7, 8 and SY3) (Table 6). R. flavefaciens FD-1 was isolated by M.
Bryant from a pill containing ruminal organisms in 1953 in
Maryland, US [1] and R. flavefaciens 17 was isolated from the
rumen of a Friesian cow that received a diet of grass cubes, hay,
and concentrates at the Rowett Institute in Aberdeen, UK [72]. R.
flavefaciens 007c is another Rowett strain isolated from rumen
contents of a cannulated cow that was fed hay and starchy
concentrates, and shares with strain 17 the ability to degrade
dewaxed cotton cellulose [73,74]. R. albus SY3 was also isolated at
the Rowett, in 1976 [74]. R. albus 7 (a type strain, ATCC 27210,
DSM 20455) was isolated in 1951 by M. Bryant from a Holstein
cow fed alfalfa hay-grain [1]; R. albus 8 is an isolate from the
rumen of an alfalfa hay-fed cow [75]. The genomes of R. albus 8
and F. succinogenes S85 were sequenced by the North American
Consortium for Rumen Bacteria at The Institute for Genome
Research (now the J. C. Venter Institute). Standard methods used
at TIGR during this period for library construction, DNA
sequencing (Sanger-based technologies) and data assembly were
employed [62].
Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 11 July 2014 | Volume 9 | Issue 7 | e99221
Genome sequencing of R. albus SY3
R. albus SY3 was sequenced at the W.M. Keck Center for
Comparative and Functional Genomics (University of Illinois at
Urbana-Champaign). Total sequence data was generated from
both a paired-ended 500-nt insert library sequenced on a single
lane of HiSeq (Illumina) and a paired ended 3-kb insert library
sequenced on a full plate of 454 sequencing (Roche Diagnostics).
These approaches yielded 47 million 100-nt reads (4.7 billion
bases) and 1.4 million reads with an average read length of 402 nt
(577 million bases; 71% true paired end, actual paired distance
was 2386+597 nt), respectively. The 454 sequence data was
assembled using Newbler v2.5.3 and the Illumina was assembled
using Velvet v1.1. The assemblies were combined using Mini-
mus2. The sequence assembled to 4 scaffolds
(N50 =1,120,630 bp) and 97 contigs (N50 =114,193). 99.95% of
bases were .Q40 and all others (1808 bp) were Q39. The total
sequence produced was 3,832,777 nt and the genome was
estimated to be 4.1 Mb, giving us 93.5% coverage. The modal
sequence coverage depth was 1316. The sequence was annotated
using subsystems in RAST.
Genome sequencing of R. flavefaciens 007c
Genome sequencing of strain 007c was performed at the
Wellcome Trust Sanger Institute, Cambridge UK, courtesy of
Keith and Julian Parkhill, based on 454 pyrosequencing, with
paired-end reads. Ruminococcus flavefaciens 007 was isolated from
rumen contents of a cannulated cow that was fed hay and starchy
concentrate, at the Rowett Institute in Scotland, as reported by
Stewart CS et al (1981) [76]. This was the only one of 54 single
colony isolates selected by their ability to form clear zones in
cellulose agar roll tubes (all reported to be ruminococci) that was
able to cause significant weight loss from dewaxed cotton fiber.
Thus it is one of the most active Ruminococcus strains to have been
isolated with respect to this highly recalcitrant form of cellulose.
This paper reported 78.1% weight loss from cotton fiber within 7
days for R. flavefaciens 007, compared with 81.4% for Fibrobacter
succinogenes BL2 (which was the most active Fibrobacter strain
isolated). Fibrobacter strains do not form clear zones in cellulose
agar, but were isolated from enrichment cultures. Subsequently,
subcultivation on medium containing cellobiose but no cellulose
was found to result in a loss of cotton-degrading activity by 007,
but this activity could be regained by serial subculture on cotton.
The derivative strains retaining, or lacking, cotton-degrading
activity were referred to as 007c and 007s, respectively [73]. The
proteomes of these two strains have been compared recently and
exhibit some potentially key differences [77]. This Whole Genome
Shotgun project has been deposited at GenBank under the
accession ATAX00000000. The version described in this paper is
version ATAX01000000.
Sequence identification of cohesins and dockerins
A genome-wide survey was conducted to predict cohesion- and
dockerin-containing proteins. Proteins were subjected to BLAST
[78] searches, using sequences of known cohesin and dockerin
modules as queries. Retrieved hits below E-value of 10
24
were
individually inspected by examining their characteristic sequence
features and protein architecture. Obvious dockerin modules were
expected to contain two Ca
+2
-binding repeats, putative helices and
linker regions. Low-scoring hits of dockerins and cohesins were
examined by comparing them against known dockerin or cohesin
sequences, respectively. Multiple sequence alignments were
obtained using CLUSTALW [79], with manual corrections when
needed. The cohesin dendrogram was generated using PhyML
algorithms (with LG substitution model, and default parameters of
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Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 12 July 2014 | Volume 9 | Issue 7 | e99221
the Approximate Likelihood-Ratio test) [80] and visualized using
TreeView [81].
Annotation of CAZymes
Both cellulosomal and non-cellulosomal proteins were annotat-
ed by the CAZy pipeline (http://www.cazy.org) [54], in order to
predict their catalytic modules. This includes identification of the
catalytic modules and their classification into family types,
according to sequence conservation, for glycoside hydrolases,
carbohydrate esterases, polysaccharide lyases, carbohydrate-bind-
ing modules and glycosyl transferases. Additional conserved
domains of the proteins were analyzed using the CD-search
website (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)
and the Pfam database (http://pfam.sanger.ac.uk/).
Supporting Information
Figure S1 Alignments of homologous R. flavefaciens dockerins.
(PDF)
Table S1 Protein architectures of identified scaffoldins.
(PDF)
Acknowledgments
The authors appreciate the contributions of Dr. Inna Rozman Grinberg
(Tel Aviv University), Dr. Sadanari Jindou (Meijo University) and Dr.
Itzhak Mizrahi (Volcani Center, Agricultural Research Organization). The
efforts of the North American Consortium for Genomics of Rumen
Bacteria in producing the first genome datasets for rumen bacteria and its
preliminary annotation is gratefully acknowledged. The Consortium
included I. K. O. Cann, R. I. Mackie, and B. A. White (University of
Illinois), C. W. Forsberg (University of Guelph), E. Mongodin and S.
Daugherty (TIGR/University of Maryland), J. B. Russell (deceased) and D.
B. Wilson (Cornell University), W. C. Nelson (TIGR/UCLA), Karen E.
Nelson (TIGR/JCVI) and Mark Morrison (The Ohio State University).
E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of
Bio-organic Chemistry. The authors appreciate the support of the
European Union, Area NMP.2013.1.1-2: Self-assembly of naturally
occurring nanosystems Project number: 604530 (http://www.2020-
horizon.com/Self-assembly-of-naturally-ocurring-nanosystems-i913.html).
Author Contributions
Conceived and designed the experiments: BD IB VR-I RL HJF BH PC
CJY BAW EAB. Performed the experiments: BD IB VR-I RL HJF BH PC
CJY BAW EAB. Analyzed the data: BD IB VR-I RL HJF BH PC MM PM
CJY BAW EAB. Contributed reagents/materials/analysis tools: BD IB
VR-I RL HJF SD BH PC MM PM CJY BAW EAB. Wrote the paper: BD
IB VR-I RL HJF BH PC MM PM CJY BAW EAB.
References
1. Bryant MP, Small N, Bouma C, Robinson IM (1958) Characteristics of ruminal
anaerobic cellulolytic cocci and Cilliobacterium cellulosolvens n. sp. J Bacteriol 76:
529537.
2. Hungate RE (1966) The rumen and its microbes. New York and London:
Academic Press.
3. Jayne-Williams DJ (1979) The bacterial flora of the rumen of healthy and
bloating calves. J Appl Bacteriol 47: 271284.
4. Fonty G, Jouany JP, Thivend P, Gouet P, Senaud J (1983) A descriptive study of
rumen digestion in meroxenic lambs according to the nature and complexity of
the microflora. Reprod Nutr Dev 23: 857873.
5. Varel VH, Richardson AJ, Stewart CS (1989) Degradation of barley straw,
ryegrass, and alfalfa cell walls by Clostridium longisporum and Ruminococcus albus.
Appl Environ Microbiol 55: 30803084.
6. Dougal K, Harris PA, Edwards A, Pachebat JA, Blackmore TM, et al. (2012) A
comparison of the microbiome and the metabolome of different regions of the
equine hindgut. FEMS Microbiol Ecol 82: 642652.
7. Belanche A, Doreau M, Edwards JE, Moorby JM, Pinloche E, et al. (2012) Shifts
in the rumen microbiota due to the type of carbohydrate and level of protein
ingested by dairy cattle are associated with changes in rumen fermentation.
J Nutr 142: 16841692.
8. Tajima K, Aminov RI, Nagamine T, Matsui H, Nakamura M, et al. (2001) Diet-
dependent shifts in the bacterial population of the rumen revealed with real-time
PCR. Appl Environ Microbiol 67: 27662774.
9. Van Soest PJ, Robertson JB, Lewis BA (1991) Methods for dietary fiber, neutral
detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
J Dairy Sci 74: 35833597.
10. Li LL, McCorkle SR, Monchy S, Taghavi S, van der Lelie D (2009)
Bioprospecting metagenomes: glycosyl hydrolases for converting biomass.
Biotechnol Biofuels 18: 210.
11. Flint HJ (1997) The rumen microbial ecosystem some recent developments.
Trends Microbiol 5: 483488.
12. Flint HJ, Bayer EA, Lamed R, White BA (2008) Polysaccharide utilization by
gut bacteria: potential for new insights from genomic analysis. Nature Rev
Microbiol 6: 121131.
13. Wolin MJ, Miller TL (1983) Interactions of microbial populations in cellulose
fermentation. Federation proceedings 42: 109113.
14. Bayer EA, Kenig R, Lamed R (1983) Adherence of Clostridium thermocellum to
cellulose. J Bacteriol 156: 818827.
15. Lamed R, Setter E, Bayer EA (1983) Characterization of a cellulose-binding,
cellulase-containing complex in Clostridium thermocellum. J Bacteriol 156: 828836.
16. Lamed R, Setter E, Kenig R, Bayer EA (1983) The cellulosome a discrete cell
surface organelle of Clostridium thermocellum which exhibits separate antigenic,
cellulose-binding and various cellulolytic activities. Biotechnol Bioeng Symp 13:
163181.
17. Lamed R, Bayer EA (1988) The cellulosome of Clostridium thermocellum. Adv Appl
Microbiol 33: 146.
18. Bayer EA, Shimon LJW, Lamed R, Shoham Y (1998) Cellulosomes: structure
and ultrastructure. J Struct Biol 124: 221234.
19. Shoham Y, Lamed R, Bayer EA (1999) The cellulosome concept as an efficient
microbial strategy for the degradation of insoluble polysaccharides. Trends
Microbiol 7: 275281.
20. Bayer EA, Shoham Y, Lamed R (2006) Cellulose-decomposing prokaryotes and
their enzyme systems. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H,
Stackebrandt E, The Prokaryotes, Third Edition. New York: Springer-Verlag.
pp. 578617.
21. Raman B, Pan C, Hurst GB, Rodriguez M, McKeown CK, et al. (2009) Impact
of pretreated switchgrass and biomass carbohydrates on Clostridium thermocellum
ATCC 27405 cellulosome composition: a quantitative proteomic analysis. PLoS
ONE 4: e5271.
22. Schwarz WH, Zverlov VV, Bahl H (2004) Extracellular glycosyl hydrolases from
Clostridia. Adv Appl Microbio 56: 215261.
23. Bayer EA, Morag E, Lamed R (1994) The cellulosome A treasure-trove for
biotechnology. Trends Biotechnol 12: 379386.
24. Salamitou S, Raynaud O, Lemaire M, Coughlan M, Beguin P, et al. (1994)
Recognition specificity of the duplicated segments present in Clostridium
thermocellum endoglucanase CelD and in the cellulosome-integrating protein
CipA. J Bacteriol 176: 28222827.
25. Salamitou S, Tokatlidis K, Beguin P, Aubert J-P (1992) Involvement of separate
domains of the cellulosomal protein S1 of Clostridium thermocellum in binding to
cellulose and in anchoring of catalytic subunits to the cellulosome. FEBS Lett
304: 8992.
26. Tokatlidis K, Dhurjati P, Beguin P (1993) Properties conferred on Clostridium
thermocellum endoglucanase CelC by grafting the duplicated segment of
endoglucanase CelD. Protein Eng 6: 947952.
27. Tokatlidis K, Salamitou S, Beguin P, Dhurjati P, Aubert J-P (1991) Interaction
of the duplicated segment carried by Clostridium thermocellum cellulases with
cellulosome components. FEBS Lett 291: 185188.
28. Shoseyov O, Takagi M, Goldstein MA, Doi RH (1992) Primary sequence
analysis of Clostridium cellulovorans cellulose binding protein A. Proc Natl Acad Sci
USA 89: 34833487.
29. Gerngross UT, Romaniec MPM, Kobayashi T, Huskisson NS, Demain AL
(1993) Sequencing of a Clostridium thermocellum gene (cipA) encoding the
cellulosomal SL-protein reveals an unusual degree of internal homology. Mol
Microbiol 8: 325334.
30. Poole DM, Morag E, Lamed R, Bayer EA, Hazlewood GP, et al. (1992)
Identification of the cellulose binding domain of the cellulosome subunit S1 from
Clostridium thermocellum. FEMS Microbiol Lett 99: 181186.
31. Morag E, Lapidot A, Govorko D, Lamed R, Wilchek M, et al. (1995)
Expression, purification and characterization of the cellulose-binding domain of
the scaffoldin subunit from the cellulosome of Clostridium thermocellum. Appl
Environ Microbiol 61: 19801986.
32. Lemaire M, Miras I, Gounon P, Beguin P (1998) Identification of a region
responsible for binding to the cell wall within the S-layer protein of Clostridium
thermocellum. Microbiology 144: 211217.
33. Berg Miller ME, Antonopoulos DA, Rincon MT, Band M, Bari A, et al. (2009)
Diversity and strain specificity of plant cell wall degrading enzymes revealed by
the draft genome of Ruminococcus flavefaciens FD-1. PLoS ONE 4: e6650.
Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 13 July 2014 | Volume 9 | Issue 7 | e99221
34. Rincon MT, Dassa B, Flint HJ, Travis AR, Jindou S, et al. (2010) Abundance
and diversity of dockerin-containing proteins in the fiber-degrading rumen
bacterium, Ruminococcus flavefaciens FD1. PLoS ONE 5: e12476.
35. Rincon MT, Martin JC, Aurilia V, McCrae SI, Rucklidge G, et al. (2004) ScaC,
an adaptor protein carrying a novel cohesin that expands the dockerin-binding
repertoire of the Ruminococcus flavefaciens 17 cellulosome. J Bacteriol 186: 2576
2585.
36. Rincon MT, Cepeljnik T, Martin JC, Lamed R, Barak Y, et al. (2005)
Unconventional mode of attachment of the Ruminococcus flavefaciens cellulosome to
the cell surface. J Bacteriol 187: 75697578.
37. Morrison M, Daugherty SC, Nelson WC, Davidsen T, Nelson KE (2010) The
FibRumBa database: A resource for biologists with interests in gastrointestinal
microbial ecology, plant biomass degradation, and anaerobic microbiology.
Microb Ecol 59: 212213.
38. Ohara H, Karita S, Kimura T, Sakka K, Ohmiya K (2000) Characterization of
the cellulolytic complex (cellulosome) from Ruminococcus albus. Biosci Biotechnol
Biochem 64: 254260.
39. Ohara H, Noguchi J, Karita S, Kimura T, Sakka K, et al. (2000) Sequence of
egV and properties of EgV, a Ruminococcus albus endoglucanase containing a
dockerin domain. Biosci Biotechnol Biochem 64: 8088.
40. Morrison M, Miron J (2000) Adhesion to cellulose by Ruminococcus albus: a
combination of cellulosomes and Pil-proteins? FEMS Microbiol Lett 185: 109
115.
41. Pegden RS, Larson MA, Grant RJ, Morrison M (1998) Adherence of the gram-
positive bacterium Ruminococcus albus to cellulose and identification of a novel
form of cellulose-binding protein which belongs to the Pil family of proteins.
J Bacteriol 180: 59215927.
42. Rakotoarivonina H, Jubelin G, Hebraud M, Gaillard-Martinie B, Forano E, et
al. (2002) Adhesion to cellulose of the Gram-positive bacterium Ruminococcus albus
involves type IV pili. Microbiology 148: 18711880.
43. Rakotoarivonina H, Larson MA, Morrison M, Girardeau JP, Gaillard-Martinie
B, et al. (2005) The Ruminococcus albus pilA1-pilA2 locus: expression and putative
role of two adjacent pil genes in pilus formation and bacterial adhesion to
cellulose. Microbiology 151: 12911299.
44. Miron J, Morag E, Bayer EA, Lamed R, Ben-Ghedalia D (1998) An adhesion-
defective mutant of Ruminococcus albus SY3 is impaired in its capability to degrade
cellulose. J Appl Microbiol 84: 249254.
45. Miron J, Jacobovitch J, Bayer EA, Lamed R, Morrison M, et al. (2001)
Subcellular distribution of glycanases and related components in Ruminococcus
albus SY3 and their role in cell adhesion to cellulose. J Appl Microbiol 91: 677
685.
46. Weimer PJ, Price NP, Kroukamp O, Joubert LM, Wolfaardt GM, et al. (2006)
Studies of the extracellular glycocalyx of the anaerobic cellulolytic bacterium
Ruminococcus albus 7. Appl Environ Microbiol 72: 75597566.
47. Mosoni P, Gaillard-Martinie B (2001) Characterization of a spontaneous
adhesion-defective mutant of Ruminococcus albus strain 20. Archives of
microbiology 176: 5261.
48. Devillard E, Goodheart DE, Karnati SK, Bayer EA, Lamed R, et al. (2004)
Ruminococcus albus 8 mutants defective in cellulose degradation are deficient in
two processive endocellulases, Cel48A and Cel9B, both of which possess a novel
modular architecture. J Bacteriol 186: 136145.
49. Xu Q, Morrison M, Bayer EA, Atamna N, Lamed R (2004) A novel family of
carbohydrate-binding modules identified with Ruminococcus albus proteins. FEBS
Lett 566: 1116.
50. Rakotoarivonina H, Terrie C, Chambon C, Forano E, Mosoni P (2009)
Proteomic identification of CBM37-containing cellulases produced by the rumen
cellulolytic bacterium Ruminococcus albus 20 and their putative involvement in
bacterial adhesion to cellulose. Arch Microbiol 191: 379388.
51. Ezer A, Matalon E, Jindou S, Borovok I, Atamna N, et al. (2008) Cell-surface
enzyme attachment is mediated by a family-37 carbohydrate-binding module,
unique to Ruminococcus albus. J Bacteriol 190: 82208222.
52. Brulc JM, Wilson MK, Yeoman CJ, Berg Miller ME, Jeraldo P, et al. (2011)
Cellulosomics, a gene-centric approach to investigating the intraspecific diversity
and adaptation of Ruminococcus flavefaciens within the rumen. PLoS ONE 6:
e25329.
53. Jindou S, Levy-Assaraf M, Rincon MT, Flint HJ, Berg ME, et al. (2008)
Cellulosome gene cluster analysis for gauging the diversity of the ruminal
cellulolytic bacterium Ruminococcus flavefaciens. FEMS Microbiol Lett 285: 188
194.
54. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, et al. (2009)
The Carbohydrate-Active Enzymes database (CAZy): an expert resource for
glycogenomics. Nucleic Acids Res 37: D233238.
55. Ding S-Y, Rincon MT, Lamed R, Martin JC, McCrae SI, et al. (2001)
Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens. J Bacteriol
183: 19451953.
56. Rincon MT, Ding S-Y, McCrae SI, Martin JC, Aurilia V, et al. (2003) Novel
organization and divergent dockerin specificities in the cellulosome system of
Ruminococcus flavefaciens. J Bacteriol 185: 703713.
57. Jindou S, Borovok I, Rincon MT, Flint HJ, Antonopoulos DA, et al. (2006)
Conservation and divergence in cellulosome architecture between two strains of
Ruminococcus flavefaciens. J Bacteriol 188: 79717976.
58. Rincon MT, Cepeljnik T, Martin JC, Barak Y, Lamed R, et al. (2007) A novel
cell surface-anchored cellulose-binding protein encoded by the sca gene cluster of
Ruminococcus flavefaciens. J Bacteriol 189: 47747283.
59. Brulc JM, Antonopoulos DA, Berg Miller ME, Wilson MK, Yannarell AC, et al.
(2009) Gene-centric metagenomics of the fiber-adherent bovine rumen
microbiome reveals forage specific glycoside hydrolases. Proc Natl Acad Sci
USA 106: 19481953.
60. Hess M, Sczyrba A, Egan R, Kim TW, Chokhawala H, et al. (2011)
Metagenomic discovery of biomass-degrading genes and genomes from cow
rumen. Science 331: 463467.
61. Kelly WJ, Leahy SC, Altermann E, Yeoman CJ, Dunne JC, et al. (2010) The
glycobiome of the rumen bacterium Butyrivibrio proteoclasticus B316(T) highlights
adaptation to a polysaccharide-rich environment. PLoS One 5: e11942.
62. Purushe J, Fouts DE, Morrison M, White BA, Mackie RI, et al. (2010)
Comparative genome analysis of Prevotella ruminicola and Prevotella bryantii: insights
into their environmental niche. Microb Ecol 60: 721729.
63. Ferrer M, Ghazi A, Beloqui A, Vieites JM, Lopez-Cortes N, et al. (2012)
Functional metagenomics unveils a multifunctional glycosyl hydrolase from the
family 43 catalysing the breakdown of plant polymers in the calf rumen. PLoS
One 7: e38134.
64. Dassa B, Borovok I, Lamed R, Henrissat B, Coutinho P, et al. (2012) Genome-
wide analysis of Acetivibrio cellulolyticus provides a blueprint of an elaborate
cellulosome system. BMC Genomics 13: 210.
65. Selinger LB, Forsberg CW, Cheng KJ (1996) The rumen: a unique source of
enzymes for enhancing livestock production. Anaerobe 2: 263284.
66. Peer A, Smith SP, Bayer EA, Lamed R, Borovok I (2009) Non-cellulosomal
cohesin and dockerin-like modules in the three domains of life. FEMS Microbiol
Lett 291: 116.
67. Mizrahi I (2012) The rumen plasmidome: A genetic communication hub for the
rumen microbiome. Mob Genet Elements 2: 152153.
68. Halliwell GA, Bryant MP (1963) The cellulolytic activity of pure strains of
bacteria from the rumen of cattle. J Gen Microbiol 32: 441448.
69. Mizrahi I (2013) Rumen symbiosis. In: Rosenberg E, The Prokaryotes, Fourth
Edition. Berlin: Springer-Verlag. pp. 533544.
70. Shi Y, Weimer PJ (1996) Utilization of individual cellodextrins by three
predominant ruminal cellulolytic bacteria. Appl Environ Microbiol 62: 1084
1088.
71. Brumm P, Mead D, Boyum J, Drinkwater C, Deneke J, et al. (2011) Functional
annotation of Fibrobacter succinogenes S85 carbohydrate active enzymes. Appl
Biochem Biotechnol 163: 649657.
72. Flint HJ, McPherson CA, Bisset J (1989) Molecular cloning of genes from
Ruminococcus flavefaciens encoding xylanase and b(1-3,1-4)glucanase activities.
Appl Environ Microbiol 55: 12301233.
73. Stewart CS, Duncan SH, Flint HJ (1990) The properties of forms of Ruminococcus
flavefaciens which differ in their ability to degrade cotton cellulose. FEMS
Microbiol Lett 72: 4750.
74. Wood TM, Wilson CA, Stewart CS (1982) Preparation of the cellulase from the
cellulolytic anaerobic rumen bacterium Ruminococcus albus and its release from the
bacterial cell wall. Biochem J 205: 129137.
75. Hungate RE, Stack RJ (1982) Phenylpropanoic acid: growth factor for
Ruminococcus albus. Appl Environ Microbiol 44: 7983.
76. Stewart CS, Paniagua C, Dinsdale D, Cheng KJ, Garrow SH (1981) Selective
isolation and characteristics of Bacteriodes succinogenes from the rumen of a cow.
Appl Environ Microbiol 41: 504510.
77. Vodovnik M, Duncan SH, Reid MD, Cantlay L, Turner K, et al. (2013)
Expression of cellulosome components and type IV pili within the extracellular
proteome of Ruminococcus flavefaciens 007. PLoS ONE 8: e65333.
78. Altschul SF, Madden TL, Scha ffer AA, Zhang J, Zhang Z, et al. (1997) Gapped
BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucl Acids Res 25: 33893402.
79. Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, et al. (1994)
CLUSTAL W: improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Res 22: 46734680.
80. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, et al. (2010) New
algorithms and methods to estimate maximum-likelihood phylogenies: Assessing
the performance of PhyML 3.0. Syst Biol 59: 307321.
81. Page RD (1996) TreeView: an application to display phylogenetic trees on
personal computers. Comput Appl Biosci 12: 357358.
82. Flint HJ, Martin J, McPherson CA, Daniel AS, Zhang JX (1993) A bifunctional
enzyme, with separate xylanase and b(1,3-1,4)-glucanase domains, encoded by
the xynD gene of Ruminococcus flavefaciens. J Bacteriol 175: 29432951.
83. Zhang JX, Flint HJ (1992) A bifunctional xylanase encoded by the xynA gene of
the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 comprises two
dissimilar domains linked by an asparagine/glutamine-rich sequence. Mol
Microbiol 6: 10131023; (Erratum p. 3627).
84. Aurilia V, Martin JC, McCrae SI, Scott KP, Rincon MT, et al. (2000) Three
multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus
flavefaciens 17 that carry divergent dockerin sequences. Microbiology 146:
13911397.
85. Berg Miller ME, Yeoman CJ, Tringe SG, Edwards RA, Flint HJ, et al. (2012)
Phage-bacteria relationships and CRISPR elements revealed by a metagenomic
survey of the rumen microbiome. Environ Microbiol 14: 207227.
86. Suen G, Stevenson DM, Bruce DC, Chertkov O, Copeland A, et al. (2011)
Complete genome of the cellulolytic ruminal bacterium Ruminococcus albus 7.
J Bacteriol 193: 55745575.
Ruminococcal Cellulosomics
PLOS ONE | www.plosone.org 14 July 2014 | Volume 9 | Issue 7 | e99221