Fire.biol.wwu.edu
Genome-scale analyses of health-
promoting bacteria: probiogenomics
Marco Ventura*, Sarah O'Flaherty‡, Marcus J. Claesson§, Francesca Turroni*, Todd R. Klaenhammer‡, Douwe van Sinderen§ and Paul W. O'Toole§
Abstract The human body is colonized by an enormous population of bacteria (microbiota) that provides the host with coding capacity and metabolic activities. Among the human gut microbiota are health-promoting indigenous species (probiotic bacteria) that are commonly consumed as live dietary supplements. Recent genomics-based studies (probiogenomics) are starting to provide insights into how probiotic bacteria sense and adapt to the gastrointestinal tract environment. In this Review, we discuss the application of probiogenomics in the elucidation of the molecular basis of probiosis using the well-recognized model probiotic bacteria genera
Bifidobacterium and
Lactobacillus as examples.
The availability of the human genome sequence has proliferation and differentiation, pH, the development of
The collective microbial
enabled us to better understand the genetic basis of the immune system and innate and acquired responses
community or population that
many aspects of human health and disease. However, to pathogens1,9,10.
resides in a particular locale at
to fully understand the human genotype and its rela-
Alterations in the composition of the intestinal
a given time.
tionship with susceptibility to disease we need better microbiota have recently been linked to various con-
information on how environmental and developmental ditions, including inflammatory bowel disease, al ergy
Groups of bacteria that are
factors interact with the genome to influence health. and obesity6,11–14. Among the variable constituents of
defined by percentage identity
Human beings are colonized by, or transiently harbour, the microbiota are health-promoting indigenous spe-
in their 16S rRNA gene
a diverse, complex and dynamic collection of bacteria cies (or mucosa-adherent microbiota). According to
that outnumber the human somatic and germ cel s and that the Food and Agriculture Organization (FAO)/WHO
collectively represent significantly more genetic variety criteria, probiotics are defined as "live microorganisms
than the genomes of their hosts1. However, the com- which when administered in adequate amounts confer
ponents of the human microbiota remain poorly char- a health benefit on the host"15.
acterized. Recent culture-independent studies of the
The mechanisms by which probiotic microorgan-
microbiota of the human gastrointestinal tract (GIT) isms benefit human health (reviewed in REFS 16,17)
have identified more than 1,000 phylotypes, which rep- are typically divided into several general categories,
*Department of Genetics, Biology of Microorganisms,
resent more than 7,000 strains and belong to 8 major including strengthening of the intestinal barrier,
Anthropology and Evolution,
phyla1–4 (reviewed in REF. 5).
modulation of the immune response and antagonism
University of Parma
It has been suggested that the composition of the of pathogens, either by the production of antimicrobial
43100, Italy. ‡
gut microbiota is the result of selective pressures that compounds or through competition for mucosal bind-
Department of Food,
Bioprocessing and Nutrition
are imposed by the host, and is further modulated by ing sites16,18. Although there is some evidence for each
Sciences, North Carolina
competition between constituent bacterial members6. of these functional claims, the molecular mechanisms
State University, Raleigh,
The interactions between bacteria and the human host by which these activities are achieved remain largely
North Carolina 27695, USA.
can be categorized as a continuum that ranges from sym- unknown.
§Alimentary Pharmabiotic
biosis and commensalism (mutualism) to pathogenesis.
Genomics could accelerate research into probiotic
Centre and Department of Microbiology, University
In the human gut, adaptive co-evolution of humans and bacteria. In recent years, genome sequencing of gut
College Cork, Western Road,
bacteria has resulted in the establishment of commensal commensals and symbionts has come to the fore, cur-
Cork, Ireland.
relationships in which neither partner is disadvantaged rently represented by the development of a new disci-
Correspondence to P.W.O.
and in symbiotic relationships in which both partners pline called probiogenomics19, which aims to provide
benefit, be it from unique metabolic activities or from insights into the diversity and evolution of commensal
Published online 24 November
other benefits. The intestinal microbiota contributes and probiotic bacteria and to reveal the molecular basis
to host nutrition1,7,8 and impacts on intestinal cell for their health-promoting activities. The integration of
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macidophilum
subsp.
B. pseudolongum
B. ther subsp
. thermophilum
pseudolongum
B. pseudolongum
t s a orynef
B. thermophilum
B. indicum
B. thermacidophilum
B. bifidum
B. animalis
subsp.
animalis
B. choerinum
B. minimum
B. gallicum
cardia farcinic
B. cuniculi
B. tsurumiense
B. dentium
B. pseudoc
B. ruminantium
B. gallinarum
a B. merycicua
B. saecular
B. pullorum
subsp
. suis
B. longum
f L aginal
ennini edioc p
L. reuteri
L. acidipis
L. coleohominis
L. ingluviei
L. ruminis
L. gastricus
L. saerimneri
cis L iarius L. animalis s
Bacillus subtili
L. rossii L. siligionis
L. algidus
L. lindneri
L. suebicus
L. homohiochii
L. harbinensis
L. fructivi
L. spicher
L. delbrueckii su
L. hammesii
L. hilgardii
L. buchneri
bsp. bulgaricu
L L. parakefir
L. delbrueckii
subsp
. lactis
L. delbrueckii subsp.
delbrueckii
L. fornicalis
L. jensenii
L. pantheris
L. hamster
L. plantaru
L. manihotiv
aciens ovorus
L. nantensis
L. graminis
L. curvatu
L. acidophilu
. a aliment ch
. fuchuensi
L. kitasat
rciminis e
L. crispatus
L. intestinalis
L. kalixensis
L. helveticus
L. acetotoler
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Table 1
General features of sequenced Bifidobacterium and Lactobacillus genomes
Species
genome size %
genes Proteins Source
Accession
Bifidobacterium longum subsp.
longum
NCC2705
Bifidobacterium longum subsp.
longum DJ010A
Bifidobacterium breve UCC2003
Bifidobacterium adolescentis ATCC15703
Bifidobacterium adolescentis L2-32
Bifidobacterium animalis subsp.
lactis HN019
Lactobacillus acidophilus NCFM
Lactobacillus casei ATCC334
Lactobacillus gasseri ATCC33323
Lactobacillus johnsonii NCC533
Lactobacillus plantarum WCFS1
Lactobacillus reuteri F275
Lactobacillus fermentum IFO 3956
Lactobacillus salivarius subsp.
salivarius
UCC118GIT, gastrointestinal tract.
Neighbour-joining tree
probiogenomics and functional genomic information of mammals, birds and insects19. Those bifidobacte-
A tree that reconstructs the
with data on host gene expression in the human gut wil rial species that have been isolated from the human
evolutionary development of
expand our understanding of the roles of (probiotic) intestine have attracted the interest of genomic
organisms on the basis of
microbiota, microbe–microbe and host–microbe inter- researchers owing to their probiotic properties.
distances between pairs of
actions. These omics approaches allow the simultane- However, of the bifidobacterial taxa described so far,
ous analysis of huge numbers of genes and proteins20. genomes of only three species, which belong to the
Probiogenomics is thus just one strand of gut systems and
The integration of genomics
microbiology. significantly, when studied in combina- groups, have been sequenced to completion (TABLE 1).
methodology and data with
tion with host genome variation, probiogenomics offers The availability of six genome sequences provides
functional genomic analyses involving transcriptomics,
a comprehensive systems model, even at the individual genetic evidence that bifidobacteria are prototrophic
proteomics, metabolomics and
subject level.
and therefore well adapted to growth in an environ-
Here we address current developments in analysing ment such as the human colon, which contains low
the genome sequences of probiotic bacteria and how concentrations of some growth substrates (for example,
these data can be integrated into a global view using vitamins, amino acids and nucleotides)23. These bifi-
omics approaches to elucidate genome evolution and dobacterial genome sequences harbour genes for the
genetic adaptation of these bacteria to the human gut synthesis of at least 19 amino acids and they encode
niche. We have focused on the model probiotic bacteria all of the enzymes that are needed for the biosynthesis
Bifidobacterium spp. and
Lactobacillus spp., which of pyrimidine and purine nucleotides, as well as those
are phylogenetically distant relatives (FIG. 1) that have that are required for the synthesis of the B vitamins,
different features from one another.
folic acid, thiamine and nicotinate24 (s. leahy and
D.v.s., unpublished observations). Annotation and
Genomics of the genus Bifidobacterium
pathway prediction revealed that bifidobacterial spe-
The genus
Bifidobacterium is small, with 30 char- cies possess the genetic information that is required to
acterized species and a low level of phylogenetic shunt many monosaccharides or disaccharides into the
and genomic diversity21 (FIG. 1a). Bifidobacteria were fructose-6-phosphate pathway23.
originally isolated from a breast-fed infant22 and 30
species have since been isolated from the GIT contents
Adaptation to the human gut. The amount and types
of ‘non-digestible' saccharides in the diet (some of
which are referred to as prebiotics) have major influ-
Figure 1
Evolutionary relationships between the main gastrointestinal tract
commensal bacterial groups. Bifidobacteria are shown in panel
a and lactobacilli are
ences on the numbers and metabolic activities of dif-
shown in panel
b. Both panels are based on a neighbour-joining tree of 16S rRNA gene
ferent groups of bacteria in the enteric microbiota25.
sequences. Bacterial taxa for which the whole-genome sequences are available are
The range of polysaccharide substrates that arrive in
shaded in pink. Bootstrap values above 600 are indicated. The outgroups are shaded in
the intestine is extremely broad26. This diversity of
green. Scale bars indicate 0.1 nucleotide substitutions per site.
carbon substrates potentially generates a vast array
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content is dedicated to sugar internalization, through
ATP-binding cassette (ABC) transporters, permeases
and proton symporters rather than through phos-
phoenolpyruvate phosphotransferase systems24,31,32.
Bifidobacteria use a ‘docking station' to sequester
and capture high-molecular-weight carbohydrate
molecules such as xylose- and arabinose-containing
polysaccharides (FIG. 2) and bind these to their cell
surface29,32, presumably to avoid losing them to
nearby competitors. This is reminiscent of a putative
carbohydrate utilization system that was identified
in the genome of
and in a
system used bor starch
utilization34. enteric bifidobacteria can also use sialic-
acid-containing complex carbohydrates in mucin,
glycosphingolipids and human milk35,36. Thus, these
bifidobacteria have acquired adaptations to allow them
to exploit a rich repertoire of otherwise indigestible
components of the human or animal diet.
Figure 2 Acquisition of sugars by bifidobacteria. The figure shows a strategy that
Whole bacterial genome sequencing efforts have
might be adopted by bifidobacteria to acquire sugar nutrients. Bifidobacteria use a
also provided general indications about the genetic
‘docking station' to capture complex sugars, such as
xylan- tur
ar views Micr
adaptation of some organisms to specific ecologi-
and bind these to the bacterial cell surface to prevent loss of the sugars to competitors.
cal niches. In the case of bifidobacteria, although
The docking station is a complex of modular glycanases, which are anchored at the cell
genomic information is still currently limited to a
surface by a transmembrane domain. The enzymatic activities degrade the arabino- or
few genomes, it was possible to identify an operon
xylan-based molecules to oligosaccharides that are subsequently transported across the bacterial membrane by a transporter protein; the presence of the bacterial cell-wall
that encodes for enzymes that are involved in the
material might prohibit diffusion of nutrients away from the transporters.
breakdown of complex sugars such as starch, amy-
lopectin and pullulan, which is present only in the
genomes of . As B. breve is one
of ecological niches that can be exploited by gut bac- of the dominant bacteria in the infant microbiota37,
teria. Although some members of the gut microbiota this enzyme might be important during weaning
can switch rapidly between using different substrates when non-milk foods are supplemented in the diet
(for example, derived from diet or from host origin), and when infants are, for the first time, exposed to
others (for example, those bacteria associated with complex carbohydrates that are different from those
insoluble substrates) are far more specialized27. In present in mother's milk.
this context, bifidobacteria have been presumed to
Characterization of the metabolism of prebi-
have an ecological advantage owing to their capacity otic compounds by bifidobacteria has led to the
to metabolize complex sugars that are derived from identification of specific transporters and hydro-
the diet as well as from the host28. Genome annota- lases for oligosaccharides29,38,39. These studies indi-
tion has confirmed that genes that are required for cated that bifidobacteria ferment different types
the breakdown of complex sugars are abundant in of fructo-oligosaccharides; accordingly, the respec-
sequenced bifidobacterial genomes19. more than 8% of tive fructo-oligosaccharide metabolism operons
annotated bifidobacterial genes encode enzymes that have different genetic architectures40, suggesting that
are involved in carbohydrate metabolism. This is 30% these genes were acquired following evolutionary
higher than GIT-resident bacteria such adivergence of the species. Prebiotic oligosaccharides
or and than non-GIT residents (such as galacto-oligosaccharides) are also contained
such as 19. However, the level of in human milk and these are hydrolysed by bifido-
sugar-fermentative coding capacity in bifidobacteria bacteria through the action of extracellular enzymes
is similar to that of one other intestinal commensal that are encoded by the galA gene29,41. In addition to
genus, Bacteroides19. Bifidobacterial enzymes that are galacto-oligosaccharides, human milk provides large
involved in sugar metabolism include various glycosyl amounts of small peptides, which are derived from
hydrolases (GH), which are used on diverse, but in the digestion of milk proteins by the gastric protease
most cases unidentified, plant-derived dietary fibres or pepsin42. Bifidobacterium genomes encode several
complex carbohydrate structures.
enzymes, such as dipeptidyl aminopeptidases and
Growth substrates that are preferential y (or ideal y,
most of the bifidobacterial GHs are predicted to oligopeptide uptake systems, that are involved in the
exclusively) metabolized by a
be intracellular, including those that are predicted breakdown and internalization of peptides (m.v. and
single genus or species and
to hydrolyse arabinogalactans and arabinoxylans, D.v.s. unpublished observations).
that may thus be used as
starch and related polysaccharides24,29,30. The genes
dietary supplements to
for these GHs are associated with genetic loci for Interaction with the host. Bacterium–host interactions
promote growth of a targeted health-promoting
the uptake of structurally diverse sugar substrates. that benefit the host can be elucidated by identifica-
Altogether, about 5% of the total bifidobacterial gene tion and molecular analysis of the bacterial proteins
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B. adolesc
B. dentiumBd1
Figure 3 comparative analysis of Bifidobacterium genomes. a Circular plot of genome diversity in
bifidobacteria. The white and green colouring in the three outer rings indicates
genome r ture Re
egions vie
pr ws Micr
absent, respectively, in the bifidobacterial genomes, relative to the Bifidobacterium dentium Bd1 genome map.
From outside to the outside: ring 1 shows a comparison with the genome sequence of Bifidobacterium longum
subsp. longum NCC2705; ring 2 shows a comparison with the genome sequence of B. longum subsp. longum
DJO10A; ring 3 shows a comparison with the genome sequence of Bifidobacterium adolescentis ATCC15703; ring 4
shows the GC content; ring 5 shows the GC deviation. Deviations from the average GC content are shown in either
green (high GC spike) or violet (low GC spike). b Comparison of gene-order conservation between two genome
pairs, illustrating different forms of bifidobacterial genome evolution. The x and y axes represent the linearized
chromosomes of B. dentium Bd1 and B. adolescentis ATCC15703, respectively. Blue dots indicate pairs of
homologous genes that are in the same orientation in both genomes, whereas red dots indicate pairs that are
in an inverted orientation in one relative to the other.
or macromolecules involved. For example, a potential phenotypes among community members has already
probiotic effector molecule that is a homologue of been described in other microbial communities that
the eukaryotic-type serine protease inhibitor (serpin) degrade cellulose46. Alternatively, shifts in transcrip-
was identified in the genome of B. longum subsp. tion patterns could represent responses to competition
longum24,43. members of the serpin family regulate (see below).
various signalling pathways in eukaryotes and some
The elucidation of the molecular impact of the human
are recognized for their ability to suppress inflam- microbiota on the human host was analysed by study-
matory responses by inhibiting elastase activity44. ing the host epithelium response to co-colonization
Recent findings showed that the bifidobacterial by B. longum subsp. longum and B. thetaiotaomicron45.
serpin-like protein performs an immunomodulatory Remarkably, the host response to these two bacte-
role in a murine model of colitis by reducing intestinal rial species was different. The host response to
B. thetaiotaomicron was focused on tumour necrosis
Transcriptomic approaches have been useful factor-α and lipopolysaccharide-responsive cytokine
for studying how individual organisms in bacterial produced by natural killer and T macrophages,
communities affect one another's transcriptomes. whereas B. longum subsp. longum promoted the acti-
Transcriptomic analyses were performed on bacteria vation of T-cell-produced cytokine interferon-γ and
from germ-free mice that had been mono-associated reduced host production of antibacterial proteins
with B. thetaiotaomicron — one of the dominant such as regenerating islet-derived-3γ (Reg3γ) and
components of the human gut microbiota — and sub- pancreatitis-associated protein (Pap). Thus, the host
sequently challenged with B. longum subsp. longum. response to enteric bifidobacteria may not only pro-
The presence of B. longum subsp. longum provoked mote bifidobacterial survival in the human intestine,
an expansion in the diversity of polysaccharides that but may also affect the composition of the overall
are targeted for breakdown by B. thetaiotaomicron, human gut microbiota.
such as mannose- and xylose-containing glycans45.
The changes in the transcriptional profiles of Comparative genomics of bifidobacteria
The subset of genes that are transcribed in an organism. It
polysaccharide-utilization-related genes by B. longum Comparisons at the nucleotide level of the fully
represents dynamic links
subsp. longum and B. thetaiotaomicron might imply sequenced bifidobacterial genomes revealed a high
between a genome, proteins
the existence of symbiosis between these microbial degree of conservation and synteny across the entire
and cellular phenotypes.
species, where each species possesses a complement genomes19. However, several breakpoint regions were
of GH activities, which when combined allow both also reported, apparently representing inversions or
SyntenyGenetic linkage or conservation
to participate in a synergic harvest of xylose- and DnA deletion/insertion points. DnA regions uniquely
of gene order.
mannose-containing sugars. Complementation of present in one genome and absent in others were also
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identified. most of these, including prophage-like ele- Adaptation to the human gut. The metabolic diver-
Proteinaceous substances that
ments, restriction modification systems, integrative sity of the Lactobacillus genome sequences that are
are produced by one
plasmids and genes that are involved in the biosyn- available so far is illustrated in FIG. 4. Taking the
bacterium to kil another
thesis of extracellular structures such as exopolysac- L. plantarum WCFs1 genome as a reference, it is
bacterium, usual y by inducing
charides, correspond to genetic elements that were clear that there is considerable variation in the COG
leakage or lysis. Bacteriocins are composed of one or two
presumably acquired by horizontal gene transfer assignments of the gene sets that are harboured by the
short peptides that can be
(HGT) events (FIG. 3). Another set of genes that dis- respective genomes. Intestinal lactobacilli compensate
seminated via HGT in bifidobacteria is the CRIsPR- for their auxotrophy by encoding multiple genes for
related system (CAss), which is implicated in defence transporters. Their genomes also contain genes that
against phages and plasmids47 and which has been encode acid and bile resistance, capacity for uptake
Clusters of orthologous groups are delineated by comparing
identified in the genome of of macromolecules, metabolism of complex carbo-
protein sequences that are
Bd1 as well as in the genome of B. breve uCC2003 hydrates and cell-surface proteins that interact with
encoded in complete genomes,
(m.v. and D.v.s., unpublished observations; s. leahy the intestinal mucosa52. more strikingly than is evi-
representing major
and D.v.s., unpublished observations). notably, these dent for bifidobacteria, the adaptation to life in the
phylogenetic lineages. Each COG consists of individual
in silico analyses were also confirmed by comparative GIT becomes evident when the genome sequences of
proteins or groups of
genome hybridization analyses48.
intestinal isolates are compared with food-adapted
paralogues from at least 3
There is little phylogenetic diversity in the genus lactobacilli such as Lactobacillus bulgaricus and
lineages and thus corresponds
Bifidobacterium compared with Lactobacillus (see . L. bulgaricus is widely used
to an ancient conserved
below). This is underlined at the whole-genome level as a starter culture in yoghurt fermentations and has
when one compares the oral species (B. dentium), undergone genome decay to adapt to the milk envi-
which is frequently identified as a component of the ronment53. Thus, it harbours numerous degraded or
Members of the microbiota
microbiota that is associated with dental caries49, with partial carbohydrate pathways and bile salt hydrolase
that are growing where they
the probiotic species B. adolescentis (FIG. 3). Despite the pseudogenes52,53. In addition, L. bulgaricus has a pref-
are found, as distinct from transient species that are only
large phenotypic differences, there is a remarkable erence for growth on lactose, further emphasizing
passing through the
degree of overall synteny. This reductionist model of its niche adaptation to milk. The genome sequence
genome evolution may be useful for identifying niche- of L. helveticus, a widely used cheese starter culture,
specific genes and genes that are related to specialized has been reported recently54. Compared to the closely
related L. acidophilus, L. helveticus has additional genes
for fatty acid biosynthesis and specific amino-acid
Genomics of the genus Lactobacillus
metabolism, but notably fewer cell-surface proteins
The genus Lactobacillus has more than 100 cultured and phospho enolpyruvate phosphotransferase systems
species (and probably more that are poorly culturable for sugar utilization54,55. Additionally, no functional
or non-culturable) and is noteworthy for its extreme mucus-binding proteins or transporters for complex
phylogenetic, phenotypic and ecological diversity50 carbohydrates, such as raffinose and fructo-oligosac-(FIG. 1b). However, the real extent of Lactobacillus charides, are encoded by the L. helveticus genome,
diversity is not ful y known and culture-independent reflecting the degree of adaptation of L. helveticus to a
16s rRnA gene surveys of complex ecosystems (for milk environment.
example, the human gut microbiota) are expected to
By contrast, L. acidophilus has adapted to the gut
uncover novel phylotypes that belong to the genus ecological niche by retaining the functional gene sets
Lactobacillus. The microbiological characteriza- that are absent from L. helveticus, emphasizing the
tion of lactobacilli is historically better developed importance of these gene sets for probiotic functional-
than that of bifidobacteria, but the genomic analy- ity and niche adaptation by autochthonous lactobacilli
sis is recent. Of the 14 sequenced and published that natural y reside in the GIT.
Lactobacillus genomes, 8 (
several studies have examined commensal
Lactobacillus casei, Lactobacillus gene expression in animal model sys-
, tems. using a stringent lincomycin-resistance-based
and selection, Walter and colleagues identified just three
L. plantarum) are from cultures or species that are genes that were differential y expressed in vivo56. Bron
considered to be probiotic (TABLE 1). Interestingly, et al.57 used a modified in vivo expression technology
11% of the overall coding capacity of the L. salivarius to identify 72 genes that are expressed by L. plantarum
genome is present on pmP118, the first megaplasmid in the mouse GIT, most of which were associated with
described in lactic acid bacteria51. This megaplasmid carbon metabolism, amino-acid metabolism and
encodes biologically important features such as a stress resistance57. notably, many of these functions
locus for bacteriocin production, a bile salt hydrolase in pathogens were associated with survival or adap-
and two genes that complete the phosphoketolase tation. L. casei actively transcribes metabolic genes
pathway, officially reclassifying this organism as a in the murine intestine and initiates de novo protein
facultative heterofermenter51. Plasmids account for synthesis58. L. johnsonii nCC533 expresses different
15% of the genome of L. salivarius, which is not the sets of genes depending on its location in the GIT59,
case with other sequenced probiotic lactobacilli, even and surprisingly, 44% of the genome remains untran-
though members of this genus are considered to be scribed both in vitro and in vivo59. Interestingly, the
replete with plasmids9.
prolonged murine gut persistence of nCC533, but not
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(sugar transport and metabolism)
Figure 4 comparative analysis of Lactobacillus genomes. Circular genome atlas of Lactobacillus plantarum WCFS1
Nature Reviews Microbiology
with mapped orthologues (defined as reciprocal best FastA hits with more than 30% identity over at least 80% of both
protein lengths) from 13 publicly available Lactobacillus genomes. The outer circle shows L. plantarum WCFS1
followed, inwards, by Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus reuteri F275, L. reuteri F275 (Japanese),
Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus gasseri,
Lactobacillus bulgaricus ATCC 11842, L. bulgaricus ATCC BAA-365, Lactobacillus casei, Lactobacillus sakei, GC
percentage, and GC skew (green shows high GC spikes whereas violet shows low GC spikes; window-sizes 10,000
basepairs). COG categories in metabolism are shown in red, information storage and processing are shown in green,
cellular processes and signalling are shown in blue, and poorly or not categorized COGs are shown in grey. Rings on
yellow backgrounds indicate genomes from species that are considered to be resident in the gastrointestinal tract. EPS,
exopolysaccharides; NpsA, non-ribosomal peptide synthetase.
of L. johnsonii, was recently shown to induce expres- context of the extremely complex intestinal ecosys-
sion of exopolysaccharide synthesis genes, mannose- tem61. lactobacillaceae account for approximately 36
uptake genes and a gene for a putative protease in this phylotypes out of the >1,000 phylotypes in the human
strain60. In summary, although there are tantalizing GIT microbiota5. In the short term, intervention
glimpses of commensal Lactobacillus gene expression studies in animal models and human subjects should
in vivo, these are as yet limited to animal models; data provide key insights into our current understanding of
from human volunteer studies is keenly awaited.
interaction with other intestinal commensals.
some lactobacilli have subtle effects on the micro-
Interaction with other commensal bacteria. Although biota. Consumption of DR20
the biology of commensal bacteria can be investigated transiently alters the proportions of lactobacilli,
in isolation, it must ultimately be understood in the bifidobacteria, enterococci and Bacteroidetes, but the
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variations were general y smal 62 and mechanisms were revealed that selective pressure from niche-specific
not investigated. The development of genomic tools adaptation has impacted on the genome evolution of
facilitated a study that examined the molecular basis of these species53,54,69.
interactions between the different components of the gut
In addition to gene duplication, HGT is also evi-
microbiota45. such analyses were performed by the dent in probiotic lactobacilli. For example, the meta-
colonization of germ-free mice with B. thetaiotaomi- bolic diversity of L. plantarum is underpinned by the
cron and B. longum as wel as with L. casei, or combina- expanded coding capacity that is afforded by its larger
tions of these organisms45. Presence of L. casei resulted 3 mb genome and by a low-GC-content region coding
in an expanded capacity of B. thetaiotaomicron to for sugar transport and metabolism genes that is likely
metabolize polysaccharides and increased expression to have been acquired by HGT70. Genes encoding
of genes for inorganic ion transport and metabolism45. cell-surface factors in L. johnsonii and the exopoly-
The L. casei-induced changes in the B. thetaiotaomi- saccharide cluster in the L. acidophilus complex are
cron transcriptome were functional y similar to those further examples of HGT in probiotic lactobacilli55,71.
caused by B. longum, but distinct from those induced moreover, production of reuterin (3-hydroxypropi-
by administration of Bifidobacterium animalis to the onaldehyde), a potent broad-spectrum antimicrobial
mice. Administration of Lactobacillus paracasei or compound72, is encoded by a genomic island that is
L. rhamnosus to germ-free mice colonized with human present in some L. reuteri strains73–75 and that is absent
infant microbiota caused modest changes in levels of from the sequenced genome of a mouse L. reuteri
a limited number of species monitored by culture isolate74 and the closely related L. fermentum75.
techniques, but major changes to levels of diverse With genomes of 12 of the 147 recognized species76
metabolites, including amino acids, methylamines now ful y sequenced, Lactobacillus spp. have been tar-
and short-chain fatty acids63. The metabolism of the geted for several comparative whole-genome analyses.
administered probiotics, coupled with competition for starting with the report of extreme diversity between
substrates and small molecules, are the likely reasons the first two available genomes77, genome sequencing
for the transcriptional and metabolic alterations that of L. acidophilus, L. gasseri,
are described in these studies.
and L. helveticus allowed attention to be focused on
numerous studies have reported that consumption the ‘acidophilus complex'54,55,78–80. large regions of syn-
of probiotics provides benefits for a range of GIT con- teny were observed between these species55,78. multi-
ditions and infections64,65,66,67, but mechanistic insights locus sequence analysis of five housekeeping genes,
are generally lacking. A reduction in the levels of comparative-genome hybridizations and DnA-typing
vaginal Lactobacil us spp., which results in vaginosis, revealed consistent and stepwise-decreasing levels of
has been linked to the production of a bacteriocin-like similarity in the group, indicating a strong role for
substance by commensal enterococci66. Also, the abil- vertical evolution78. Conversely, differences between
ity of L. salivarius to eliminate Listeria monocytogenes trees from 16s rRnA genes and 401 core genes
from a mouse model was dependent on the produc- from L. acidophilus, L. johnsonii and L. delbrueckii
tion of the broad spectrum bacteriocin Abp118 (also indicated a high level (40%) of HGT79.
known as salivaricin)67, and bacteriocin-producing
To infer robust phylogenetic relationships with
lactobacilli become dominant among strains in a minimal incongruence, or to elucidate functional
cocktail that reduces Salmonel a shedding in pigs68. differences between species, a set of careful y selected
Thus, bacteriocin production is probably an impor- single-copy ubiquitously-present genes is necessary.
tant mechanism in the interaction of many lactobacilli A comparison of 354 core genes from 5 lactobacilli
with other commensals.
underscored the substantial diversification of the
genus and suggested that these lactobacilli could be
Comparative genomics of Lactobacillus
subdivided into 3 groups81. Furthermore, 2 overlap-
sequencing of the genomes of 20 lactic acid bacteria ping comparative studies, which included 9 additional
has demonstrated that loss and decay of ancestral genes lactobacillales genomes, expanded the core genome
has played a key role in the evolution of lactobacil ales. to 567 order-specific genes50,82. The finer granularity
lactobacillales diverged from their Bacillus ancestor provided by laCOGs (lactobacil ales-specific COGs)
with an estimated loss of 600–1,200 genes from a total allowed detection of two genes, the gene-contexts of
gene repertoire of 2,100–2,200 (REF. 50). many of these which suggest housekeeping and protein-modification
genes encoded biosynthetic enzymes or functioned functions. Recently, we extracted 141 core genes from
in sporulation50. However, in addition to major gene 12 Lactobacillus spp. genomes to investigate the case
losses, gene gains also occurred that seem to reflect the for a single congruent genus phylogeny51,83. These were
nutrient-rich niches, such as milk and the GIT, that are operationally characterized by absent genes rather
occupied by lactic acid bacteria. For example, genes than by gained or retained genes, consistent with the
encoding peptidases and amino-acid transport pro- findings of an earlier study82.
teins as well as genes involved in the metabolism and
transport of carbohydrates have been duplicated50. In Evolutionary trends in probiotic genomes
addition, comparative analysis between GIT-associated Collective analyses of probiotic genome sequences have
species L. acidophilus, L. gasseri and L. johnsonii revealed some conserved genetic traits24,51,55,70,71,75,82,
and the dairy species L. bulgaricus and L. helveticus which might reflect adaptation to the intestinal niche1.
68 jAnuARy 2009 vOlume 7
2009 Macmillan Publishers Limited. All rights reserved
However, as probiotic bacteria are diverse and taxo- screening was used to correlate comparative genomic
nomically heterogeneous groups of microorganisms, hybridization patterns with a particular phenotype
the analysis of phyletic (phylogenetic) patterns, that (mannose-sensitive adhesin) to successfully identify
is, patterns of gene presence/absence in a particular this gene from the genomic background87. Thus,
set of genomes, may be overwhelmingly influenced comparative genomic analysis of probiotic strains
by the evolutionary distance between distant phyla. with well-defined phenotypic characteristics can be
nevertheless, common trends in the evolution of the a fruitful approach to identify strain-specific effector
genomes of both Bifidobacterium and Lactobacil us molecules/mechanisms that can then be functionally
species can be discerned. These include gene loss (for validated. However, other effector mechanisms that are
example, of genes encoding biosynthetic enzymes), probably involved in probiosis, such as the modulation
gene duplication and HGT. The adaptation of probiotic of cytokine production by the composition of lipotei-
bacteria to successful y exist and compete in the human choic acid88, were not identified by a comparative
gut must have been driven by the occurrence of DnA genomics approach at all, so conserved components
duplications and genetic acquisitions. many genes that must not be overlooked.
are involved in sugar metabolism and transport were
duplicated or acquired early in the evolution of pro- Conclusions
biotic bacteria, including those that encode enolase, most of the probiotic bacteria marketed today were
β-galactosidase and many other GHs50. In addition, originally selected on the basis of technological sta-
expansion of peptidases and amino-acid transporters bility or by various easily measurable phenotypes
has occurred in several lineages of lactobacil ales and such as ability to tolerate bile salts or survive GIT
bifidobacteria. Furthermore, several expanded fami- passage, but not necessarily for their ability to confer
lies include proteins, such as β-lactamases, that are health benefits. It is crucial to identify the precise
involved in antibiotic resistance in other bacteria84.
mechanisms by which such probiotic microorganisms
extensive evidence of HGT by bacteriophages or affect human health. such studies should be acceler-
conjugation has been documented in lactobacillales ated by omics approaches, including genomics and
and seems to be important for niche-specific adapta- functional analyses. molecular interaction models
tion in probiotic bacteria. In probiotic lactobacilli, are currently being developed, although more are
HGT played an important role in shaping the com- required, to monitor the activation of cellular and
mon ancestor, in which 84 genes were inferred to systemic responses in vivo in animal models and in
be acquired by horizontal transfer from different feeding trial participants through the measurement of
sources50. In some cases the ancestor acquired an addi- previously validated biomarkers. The combination of
tional pseudoparalogous copy of a gene by HGT (for validated molecular models with functional and com-
example, enolase in lactobacil ales), whereas in other parative genomics-based approaches should enable
cases xenologous displacement, that is, acquisition of selection of the most appropriate probiotic strain for
genes by HGT followed by the loss of the ancestral a particular health benefit or should enable improve-
orthologous gene85, seems to have occurred.
ment of strain processing and administration regimes
With the imminent availability of an even greater that optimize established health effects. This might
number of whole-genome sequences from probi- allow the selection of specific probiotics for a par-
otic bacteria, a future challenge is the identification ticular human genotype, by analogy with personalized
of the core probiogenome, which would comprise genomic medicine efforts.
the core genome functions of probiotic bacteria.
several issues regarding the sequences of complete
However, only seven genes present in bifidobacteria, probiotic bacterial genomes remain unresolved. so far,
but absent from the genomes of the other mem- only a limited number of completed probiotic bacterial
bers of the Actinobacteria phylum, are shared with genome sequences are available, and these only partial y
lactobacillales. Only one of these genes, which represent the total biodiversity of probiotic bacteria
encodes a functionally uncharacterized membrane residing in the human gut. In this context, understanding
protein, is present in all of the lactobacil ales genomes the human gut microbiome will be an important challenge
that have been sequenced so far50.
for the future89. Furthermore, sequencing the genomes
notably, many current claims of health-promoting of environmental organisms and carrying out metage-
properties in commercially available products that nomic surveys of diverse gut environments (human
include probiotic agents are based on strain-specific versus animal GITs, for example) will provide not only
properties. Thus, another intriguing goal of probiog- an improved understanding of microbial biodiversity
enomics is to provide the molecular basis for such but also insights into the evolution of bacterial factors
strain-specific genes and gene products. large-scale that may be crucial for the establishment of commensals
An extra copy of a gene that is already present in a genome
parallel sequencing of multiple strains of single species (probiotics) in these different gut niches90.
that was acquired by lateral
wil resolve issues such as conserved and variable gene
The first decade of bacterial genomics has afforded
gene transfer rather than by
families at inter- and intra-specific levels. The power unprecedented insights into the evolution of bacterial
gene duplication.
of this approach has been demonstrated by a recent pathogens (bacterial pathogenomics)81. The next dec-
pathogenomic study that narrowed 10-fold the focus ade holds the promise of being even more rewarding, as
The collective genome of
of a follow-up investigative phase of effector mol- the new discoveries about probiotic bacteria provided
ecules86. In the case of L. plantarum, biodiversity-based by probiogenomic efforts can be exploited.
nATuRe RevIeWs microbiology
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for Outstanding Young Researcher scheme "Incentivazione
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five species. Microbiology 152, 3185–3196
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SJIF Impact Factor 2.026 ejpmr, 2015,2(6), 141-146 Research Article EUROPEAN JOURNAL OF PHARMACEUTICAL Gopalakrishnan et al. European Journal of Pharmaceutical and Medical Resea N 3 294-3211 AND MEDICAL RESEARCH HEPATOPROTECTIVE ACTIVITY STUDIES OF CUCUMIS TRIGONUS ROXB.
TRINIDAD & TOBAGO Third national report CONTENTS A. REPORTING PARTY . 2 Information on the preparation of the report. 3 B. PRIORITY SETTING, TARGETS AND OBSTACLES. 4 Priority Setting. 6 Challenges and Obstacles to Implementation. 7 2010 Target. 10 Global Strategy for Plant Conservation (GSPC). 36 Ecosystem Approach . 49 C. ARTICLES OF THE CONVENTION. 50
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