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<title>Proteomic comparison of the cytosolic proteins of three Bifidobacterium longum human isolates and B. longum NCC2705</title>
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Aires et al. BMC Microbiology 2010, 10:29
http://www.biomedcentral.com/1471-2180/10/29

RESEARCH ARTICLE

Open Access

Proteomic comparison of the cytosolic proteins of
three Bifidobacterium longum human isolates and
B. longum NCC2705
Julio Aires1*, Patricia Anglade2, Fabienne Baraige2, Monique Zagorec2, Marie-Christine Champomier-Vergès2,
Marie-José Butel1

Abstract
Background: Bifidobacteria are natural inhabitants of the human gastrointestinal tract. In full-term newborns, these
bacteria are acquired from the mother during delivery and rapidly become the predominant organisms in the
intestinal microbiota. Bifidobacteria contribute to the establishment of healthy intestinal ecology and can confer
health benefits to their host. Consequently, there is growing interest in bifidobacteria, and various strains are
currently used as probiotic components in functional food products. However, the probiotic effects have been
reported to be strain-specific. There is thus a need to better understand the determinants of the observed benefits
provided by these probiotics. Our objective was to compare three human B. longum isolates with the sequenced
model strain B. longum NCC2705 at the chromosome and proteome levels.
Results: Pulsed field electrophoresis genotyping revealed genetic heterogeneity with low intraspecies strain
relatedness among the four strains tested. Using two-dimensional gel electrophoresis, we analyzed qualitative
differences in the cytosolic protein patterns. There were 45 spots that were present in some strains and absent in
others. Spots were excised from the gels and subjected to peptide mass fingerprint analysis for identification. The
45 spots represented 37 proteins, most of which were involved in carbohydrate metabolism and cell wall or cell
membrane synthesis. Notably, the protein patterns were correlated with differences in cell membrane properties
like surface hydrophobicity and cell agglutination.
Conclusion: These results showed that proteomic analysis can be valuable for investigating differences in
bifidobacterial species and may provide a better understanding of the diversity of bifidobacteria and their potential
use as probiotics.

Background
Bifidobacteria are anaerobic high G + C Gram-positive
bacteria that belong to the Bifidobacterium genus, which
contains more than 30 species. Bifidobacterium is a prevalent bacterial genus in the human colon that represents
up to 90% of all bacteria in fecal samples of breast-fed
infants and 3 to 5% of adult fecal microbiota [1,2]. In
full-term breast-fed infants, the intestinal microbiota is
rapidly dominated by bifidobacteria that are acquired
from mothers’ microbiota during birth. These bacteria
contribute to the establishment of healthy intestinal
* Correspondence: julio.aires@univ-paris5.fr
1
Université Paris Descartes, EA 4065, Faculté des Sciences Pharmaceutiques
et Biologiques, Paris, France

ecology and can confer health benefits to their host.
Indeed, impairment of bifidobacterial colonization is a
risk factor for allergic diseases [3] and for necrotizing
enterocolitis in preterm infants [4]. Consequently, bifidobacteria are the subject of growing interest due to their
assumed contribution to the maintenance of gastrointestinal health [5-12]. For these reasons, some bifidobacterial
strains are used as health-promoting or probiotic components in functional food products [13].
Although bifidobacteria have been reported to exert a
number of positive biological effects, there has been limited research into the molecular mechanisms underlying
these effects. This may be due in part to reports that
some of the positive biological activities of bifidobacteria
are strain-dependent [14] and to the limited number of

© 2010 Aires et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Aires et al. BMC Microbiology 2010, 10:29
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sequenced genomes. Applying genomics to bifidobacteria
is essential for a better understanding of their effects.
Indeed, comparative genomic studies of the few
sequenced genomes of bifidobacteria has contributed to
a better understanding of the stress response [15,16],
bacterial phylogeny and ecological adaptation [16,17],
and genetic variability [16,18]. Within the Bifidobacterium genus, the first completed genome sequence was
that of the probiotic strain B. longum NCC2705, which
became available in 2002 [16] and was revised in
2005 (GenBank database accession no. AE014295).
Recently, the assembled genome of B. longum DJO10A
becameavailable in the NCBI database (NCBI source
NZ_AABM00000000), allowing this genetic information
to be used for comparisons and functional analyses
such as proteomic comparisons.
Unlike genome studies, investigations at the proteomic
level provide insights into protein abundance and/or
post-transcriptional modifications. Proteomic studies of
the Bifidobacterium genus have established reference
maps [19,20]; comparisons of differentially expressed
proteins have shed light on bacterial adaptations to gastrointestinal tract factors such as bile [21,22] and acidic
pH [23]. Although two-dimensional electrophoresis (2Delectrophoresis) has been used to analyze bacterial protein polymorphisms and to distinguish between closely
related pathogenic organisms [24-26], 2D-electrophoresis has not been used to compare bifidobacteria.
In this study, our objective was to compare three
human B. longum isolates with the model sequenced
strain B. longum NCC2705 at the chromosome and proteome levels. Pulse-field gel electrophoresis (PFGE)
revealed a high degree of heterogeneity. Moreover, the
isolates showed different patterns in terms of their cytoplasmic proteins that may reveal correlations with specific phenotypic differences of the B. longum strains. Our
results show that this approach is a valuable tool for
exploring the natural diversity and the various capabilities of bifidobacteria strains.

Results and Discussion
In the present study, we chose B. longum NCC2705 as
the reference strain because (i) B. longum is one of
three species used as probiotics; (ii) the entire genome
sequence is available, allowing protein identification
using a public database [16]; (iii) a proteome reference
map had been established for this strain [19]. Three
B. longum human isolates with known biological effects
were compared to this reference strain. In an animal
model, B. longum BS89 has a protective role against
necrotizing enterocolitis via a sharp decrease of clostridia [27]. The two other isolates show differences in
their abilities to stimulate the intestinal immune system
in gnotobiotic mice by inducing either T-helper 2

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(B. longum BS64) or T-helper 1 cytokines (B. longum
BS49) [28].
Genotype comparison using PFGE

We first compared the four strains at the genome level
using PFGE [29]. XbaI macro-restriction analysis of
genomic DNA from B. longum strains NCC2705, BS49,
BS64 and BS89 generated clear and easy-to-interpret
PFGE patterns (Figure 1). The four strains exhibited a
high degree of genomic heterogeneity and low intraspecies relatedness: BS89, BS49 and BS64 shared 57.9, 29.3
and 20.9% identity, respectively, with NCC2705 macrorestriction patterns. Such genetic variability is consistent
with the comparative genomic analysis of B. longum
strains NCC2705 and DJO10A, which showed substantial loss of genome regions, probably due to multiple
phage insertion sites [18,30]. Considering the various
biological effects and genomic heterogeneity of the isolates, one might speculate that this heterogeneity could
be related to functional differences that could be identified using proteomic analysis.
Comparison of cytosolic protein patterns of the B. longum
strains

We next used 2D-electrophoresis to analyze the cytosolic
protein content of these four strains. Spot differences
between the three human isolates, BS89, BS49 and BS64,
and B. longum NCC2075 are summarized in Table S1
(Additional file 1). A total of 45 spots (Additional file 2),
representing 37 different proteins, were present in some
strains and absent in others. The 38 proteins fell mainly
into the following functional categories: (i) metabolismrelated proteins, especially proteins related to cell wall/
membrane/envelope biogenesis; (ii) proteins involved in
nucleotide or amino acid transport and metabolism; (iii)
proteins involved in energy production and conversion;
(iv) proteins related to transcription and translation. No
Cluster of Orthologous Group (COG) proteins, involved
in cell control or cell division, showed differences among
the four strains; these proteins are over-represented in B.
longum NCC2705 [16]. This was not surprising because
the bacteria were grown in a rich medium so that stress
was minimal. In addition, the proteins in the bifidobacterial shunt pathway, which is a characteristic pathway of
the Bifidobacterium genus, were well conserved among
all strains.
Differences in cell wall, membrane and envelope
biogenesis proteins in the B. longum strains

Of the 38 identified proteins, nine were directly or
indirectly linked to cell wall/membrane/envelope biogenesis (Figure 2). Five proteins (BL0228, BL0229,
BL1175, BL1245 and BL1267) were directly involved in
cell wall/membrane/envelope biogenesis and include the
following: dTDP-4-keto-L-rhamnose reductase/dTDP-4keto-6-deoxyglucose-3,5-epimerase (BL0228), a dTDPglucose 4,6-dehydratase (RmlB1) (BL0229), a glutamine

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% similarity
10

50

100

NCC2705
BS89
BS64
BS49
Dendrogram

PFGE pattern

Figure 1 Comparison of B. longum genomic DNA XbaI macrorestriction patterns using pulsed field gel electrophoresis (PFGE)
genotyping.

fructose-6-phosphate transaminase (GlmS) (BL1175), a
UDP-galactopyranose mutase (Glf) (BL1245) and a carboxyvinyltransferase (MurA) (BL1267). In addition, two
of the identified proteins were involved in carbohydrate
metabolism, which is important for cell wall biogenesis:
a b-galactosidase (LacZ) (BL0978) and a galactose-1phosphate uridyltransferase (GalT) (BL1211). Finally,
two spots corresponded to proteins indirectly linked to
cell wall structure: cyclopropane fatty acid (CFA)
synthase (BL1672) and bile salt hydrolase (BSH)
(BL0796).
Two of these proteins, BL0229 and BL0228, were
detected only in the NCC2705 proteome pattern (Additional file 1 and 2). These proteins play a role in peptidoglycan biogenesis by producing rhamnose, a
polysaccharide component of the Bifidobacterium peptidoglycan [31]. Rhamnose is synthesized by a de novo
biosynthetic pathway that starts with dTDP-glucose and
leads to the formation of dTDP-L-rhamnose via dehydration and epimerase/reductase reactions mediated by
RmlB1 dTDP-glucose 4,6-dehydratase and BL0228
dTDP-4-keto-6-deoxyglucose-3,5-epimerase/dTDP-4keto-L-rhamnose reductase, respectively [31] (Figure 2).
These two enzymes are encoded by genes belonging to
the same operon, which is located just downstream of a
gene coding for a hypothetical transmembrane protein
that may be involved in polysaccharide biosynthesis
(BL0230). Interestingly, glutamine fructose-6-phosphate
transaminase GlmS (BL1175) was detected in NCC2705
as well as in BS49. GlmS links the D-fructose-6-phosphate shunt of bifidobacteria to the early steps of the de
novo amino acid sugar biosynthetic pathway, a pathway
that is important for the synthesis of cell wall peptidoglycan precursors.
The proteins MurA (BL1267) and Glf (BL1245) were
not detected in the BS64 cytosolic proteome. Both

proteins are involved in peptidoglycan biosynthesis.
MurA is directly linked to the transformation of N-acetylglucosamine in that MurA catalyses the first committed step of its incorporation into the peptidoglycan
(Figure 2). Meanwhile, Glf catalyzes the ring contraction
of UDP-galactopyranose to UDP-galactofuranose, which
is then used to form the galactofuran structures that are
incorporated into the peptidoglycan (Figure 2).
The spot corresponding to b-galactosidase (lacZ,
BL0978) was present in B. longum NCC2705 and BS89,
but not in strains B. longum BS49 and BS64. When
grown on LB agar medium supplemented with X-gal,
b-galactosidase activity was observed not only in
NCC2705 and BS89, but also in the BS49 strain (data
not shown). This suggests that b-galactosidase activity
might be repressed in BS64 and that BS49 may use an
enzyme other than BL0978 to metabolize X-gal. The latter is consistent with the observation that several
b-galactosidase-encoding genes are predicted in the
B. longum NCC2705 genome (BL1168 and BL0259). It
is noteworthy that the b-galactosidase LacZ is a saccharolytic enzyme, explaining the adaptation of Bifidobacterium to its ecological niche, e.g., digestion of complex
carbohydrates that escape digestion in the human gastrointestinal tract. In fact, Bifidobacterium b-galactosidases show transgalactosylation activity resulting in the
production of galacto-oligosaccharides, which are considered prebiotics [32]. The protein differences observed
between the four strains may thus reflect different sugar
utilization mechanisms that might confer different beneficial properties for the host in terms of probiotic and/
or prebiotic activity.
The Leloir pathway enzyme GalT (BL1211) was
observed in BS89 and BS49. This enzyme is involved in
the UDP-glucose and galactose metabolism that links
the anabolic pathway of carbohydrate synthesis to cell

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Figure 2 Schematic representation of peptidoglycan and exopolysaccharide production. Proteins present or absent in the B. longum
strains are indicated using B. longum NCC2705 identification code.

wall components and to exopolysaccharide synthesis;
galactosides are frequently used as building blocks for
exopolysaccharides. Indeed, UDP-galactose is one biosynthetic donor of the galactopyranosyl unit to the
galactoconjugates that make up the surface constituents
of bacteria, e.g., peptidoglycan (Figure 2) [33,34].
Cyclopropane fatty acid (CFA) synthase (BL1672) was
detected only in the NCC2705 strain. Interestingly, CFA
synthase is directly linked to modifications in the bacterial membrane fatty acid composition that reduce membrane fluidity and helps cells adapt to their environment
[35].
Proteins with changes in mobility

Mass spectrometry analysis revealed that 12 spots,
representing 6 proteins, showed changes in mobility due
to charge changes (Additional file 1 and 2). These proteins included a hypothetical protein of unknown function (BL1050), a probable UDP-galactopyranose mutase
(Glf) (BL1245), elongation factor Ts (BL1504), a transcription elongation factor (NusA) (BL1615), an UDPgalactopyranose mutase (GalE) (BL1644) and the

adenylosuccinate lyase (PurB, BL1800). All had pIs that
clearly differed from corresponding proteins in B.
longum NCC2705. In addition, four spots were identified
as different isoforms of the BSH. However, the posttranscriptional modifications leading to the mobility differences are unknown.
Biological variability among B. longum strains

Among the 29 spots that differed (present/absent)
between the NCC2705 and BS64 proteomes, only 11
proteins from BS64 had an orthologous gene in
NCC2705. Comparison of the BS49 and BS89 proteomes
to the NCC2705 proteome showed 23 and 26 differences, of which 22 and 14 proteins, respectively, could
be identified by comparison to the NCC2705 genome
database. Moreover, in BS64, missing spots were identified as enzymes directly or indirectly involved in cell
wall/membrane/envelope biogenesis, as noted above.
This suggested that BS64 and NCC2705 might show
some biological differences in terms of the cell wall
properties. To investigate this hypothesis, we compared
the surface hydrophobicity of the four strains and their

Aires et al. BMC Microbiology 2010, 10:29
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ability to aggregate; these traits reflect the cell surface
properties of the strains [36]. Interestingly, BS64 showed
three times more autoaggregation than NCC2705 (Figure 3a) and the surface hydrophobicity of BS64 was
three times higher than that of NCC2705 (Figure 3b).
Because autoaggregation and surface hydrophobicity
may impact intestinal colonization, these observations
suggest that BS64 and NCC2705 may have different
adhesion capabilities. It also suggests possible differences
in peptidoglycan between the strains, since peptidolycan
is the principal constituent of the bacterial outer membrane that directly contacts the surrounding environment. Adhesion of bifidobacteria to the gastrointestinal
epithelium plays an important role in colonization of
the gastrointestinal tract and provides a competitive
advantage in the ecosystem against pathogens.

Conclusion
This study used proteomics to analyze cytosolic proteins
extracted from four strains of bifidobacteria grown in a
rich laboratory medium. The results validated proteomics as a tool for exploring the natural diversity and
biological effects of bifidobacteria. Specifically, proteomics allowed identification of phenotype differences in
B. longum strains that have different in vitro properties.
Interestingly, by comparing 2D-electrophoresis patterns
and by identifying proteins that were present in some
strains but not others, we found that the protein diversity observed between the strains was related to differences in cell wall/membrane biogenesis. In one of the
strains (BS64), it was associated with better autoaggregation and greater surface hydrophobicity. This strain has
been reported to be an inducer of T-helper 2 cytokines;
in contrast, NCC2705 had the lowest surface hydrophobicity of the four strains and has been reported to

Page 5 of 7

induce T-helper 1 cytokines [28]. This study showed
that proteomic approach may help researchers understand the differential effects of bifidobacteria and be
useful for identifying bifidobacteria with probiotic
potential.

Methods
Strains, media and growth conditions

B. longum NCC2705 was kindly provided by the Nestlé
Research Center (Lausanne, Switzerland). B. longum
CUETM 89-215 (BS89), BS49 and BS64 were isolated
from the dominant fecal flora of healthy infants [28].
Strains were cultured on Wilkins-Chalgren anaerobe
agar (Oxoid) supplemented with 1% (w/v) D-glucose,
0.05% (w/v) L-cysteine, 0.5% (v/v) Tween 80 (WCB) and
incubated for 48 hrs at 37°C in a chamber under anaerobic conditions (CO2:H2:N2, 10:10:80). After genomic
DNA extraction, Bifidobacterium strains were identified
by multiplex PCR and amplification and sequencing of
the 16S rRNA, as previously described [37].
TGYH broth (tryptone peptone, 30 g l-1; glucose, 5 g
-1
l ; yeast extract, 20 g l-1; haemin, 5 g l-1) was used for
cell growth prior to protein extraction. Three independent growth experiments were performed for each strain
to extract cytosolic proteins. b-galactosidase activity was
visualized on Luria-Bertani (LB) (Oxoid) agar plates supplemented with X-gal (40 mg l-1).
Genotyping using PFGE

PFGE was performed as previously described using the
XbaI restriction enzyme [29]. Gels were run using a
clamped homogeneous electric-field apparatus (CHEFDRIII, Bio-Rad), and Staphylococcus aureus NCTC 8325
DNA was used as a reference. GelCompar software
(Bio-Rad) was used for cluster analysis (Applied Maths)
with the Dice correlation coefficient, and a dendrogram

Figure 3 Aggregation (a) and cell surface hydrophobicity (b) of B. longum NCC2705 (black circle), BS64 (black diamond), BS89 (black
triangle) and BS49 (black square).

Aires et al. BMC Microbiology 2010, 10:29
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was produced with the unweighted pair-group method
using the arithmetic averages clustering algorithm.
Cytosolic protein extraction and 2D-electrophoresis

Cytosolic cell extracts were obtained from 300 ml of
culture in TGYH medium that was collected at the midlog exponential growth phase (OD600 of 0.8-0.9). Cytosolic protein extraction and 2D-electrophoresis were
performed as previously described [21]. The protein
concentration of each bacterial extract was measured
using the Coomassie Protein Assay Reagent kit (Pierce
Biotechnology) according to the manufacturer’s instructions. For electrophoresis, proteins from bifidobacterial
extracts (350 μg) were loaded onto strips (17 cm) with a
pH range of 4 to 7 (Bio-Rad), focused for 60,000 V·h,
and the second dimension was carried out using a
12.5% SDS-polyacrylamide gel. The gels were stained
with Bio-Safe Coomassie (Bio-Rad). Spot (present in all
replicates) detection was carried out using Progenesis
SameSpots software (Nonlinear Dynamics) and a master
gel image was produced. The reproducibility of spot differences was confirmed by analyzing three gels for each
strain, each obtained using an independent culture.
Spots of interest were subjected to tryptic in-gel digestion and identified by matrix-assisted laser desorption
ionization-time of flight mass spectrometry (MALDITOF/MS) using a Voyager DE STR Instrument (Applied
Biosystems), as previously described [38]. The a-cyano4-hydroxycinnamic acid matrix was prepared at 4 g l-1
in 0.1% TFA, 50% acetonitrile. An equal volume (1 μl)
of matrix and sample were spotted onto the MALDITOF target plate. Spectra were acquired in the reflector
mode with the following parameters: 2250 laser intensity, 20 kV accelerating voltage, 62% grid voltage, 135 ns
delay. The mass gates used were 700-4000 Da. Internal
calibration was performed by using the trypsin peptides
at 842.5 and 2211.1 Da. Spots mass accuracy varied
between 15-30 ppm. The carbamidomethylation of
cysteines, methionine oxidation and one miscleavage
were considered during the search. A minimum of four
matching peptides and a sequence coverage above 25%
were required before considering this a result of the
database search. Additional parameters were used to
assume a correct identification: theoretical molecular
weight and isoelectric point in good agreement with
experimental values.
Proteins were identified using MS-Fit software (University of California San Francisco Mass Spectrometry
Facility; http://prospector.ucsf.edu and Mascot software
(Matrix Science Inc., Boston, MA; http://www.
matrixscience.com). The genome database entries of the
chromosome of B. longum NCC2705 (GenBank database
accession no. AE014295) were used to assign putative
genes encoding the cytosolic proteins of interest from
the four B. longum extracts using peptide mass

Page 6 of 7

fingerprinting. Based on comparison against the master
gel, we identified spots that were not present in all
strains, i.e. pattern differences. The presence or absence
of a spot (protein) can reflect whether the gene encoding the protein is present, is expressed or repressed, or
may reflect a change in the location of the spot on the
gel. Our approach resulted in identification of spots
(proteins) corresponding to genes in the NCC2705
genome.
Aggregation and cell surface hydrophobicity assays

The aggregation assay was performed using bacteria
grown at 37°C for 48 hrs in TGYH broth that was harvested and resuspended in TGYH at an OD600 of 0.5.
During incubation at 37°C, the OD600 of the suspension
was monitored at 30, 60, 120 and 180 min, and aggregation was expressed as [1-(OD 600 upper suspension/
OD600 total bacterial suspension)] × 100 [36]. To assay
cell surface hydrophobicity, bacteria were grown in
TGYH as described above, washed twice in 10 ml phosphate buffer (pH 6.5, 50 mM) and diluted in the same
buffer to OD600 = 1. This bacterial suspension (2 ml)
was added to an equal volume of xylene and mixed for
2 min by vortexing. The OD600 was measured. Cell surface hydrophobicity (H) was calculated as follows: [(1ODaqueous phase)/ODinitial] × 100 [39].
Additional file 1: Table S1- Identification of selected protein spots
that showed variation (presence/absence) among the B. longum
NCC2705, BS49, BS89 and BS64 strains. Additional file 1 contains
Table S1 where are presented spot identification and characteristics.
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1471-2180-1029-S1.XLS ]
Additional file 2: 2D-electrophoretic gel of B. longum NCC2705,
BS49, BS89 and BS64 cytosolic proteins. Spots that are present in
some strains and absent in others are highlighted. Spot
characteristics are listed in Table S1. Additional file 2 contains 2Delectrophoretic gel pictures of B. longum NCC2705, BS49, BS89 and BS64
cytosolic proteins.
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1471-2180-1029-S2.PPT ]

Acknowledgements
We thank the PAPPSO (Plateforme d’Analyse Protéomique de Paris Sud
Ouest) at the INRA Center at Jouy en Josas for performing the MALDI-TOF/
MS experiments.
Author details
1
Université Paris Descartes, EA 4065, Faculté des Sciences Pharmaceutiques
et Biologiques, Paris, France. 2INRA, FLEC, UR309, Domaine de Vilvert, 78350
Jouy en Josas, France.
Authors’ contributions
JA performed the PFGE, proteomic and phenotype experiments. PA helped
design the study and performed protein spot detection using Progenesis
SameSpot software. FB prepared samples for MALDI-TOF/MS and identified
proteins using protein identification software. JA, MZ, MCCV and MJB
conceived the study, participated in the study design process, and helped
write the manuscript. All authors read and approved the final manuscript.

Aires et al. BMC Microbiology 2010, 10:29
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Received: 1 October 2009
Accepted: 29 January 2010 Published: 29 January 2010
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