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.
Fire.biol.wwu.eduGenome-scale analyses of health- promoting bacteria: probiogenomicsMarco 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 nATuRe RevIeWs microbiology
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2009 Macmillan Publishers Limited. All rights reserved 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 62 jAnuARy 2009 vOlume 7
2009 Macmillan Publishers Limited. All rights reserved Table 1 General features of sequenced Bifidobacterium and Lactobacillus genomes
genome size %
genes Proteins Source
Bifidobacterium longum subsp. longum NCC2705Bifidobacterium 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 nATuRe RevIeWs microbiology
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2009 Macmillan Publishers Limited. All rights reserved 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 64 jAnuARy 2009 vOlume 7
2009 Macmillan Publishers Limited. All rights reserved 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
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 nATuRe RevIeWs microbiology
vOlume 7 jAnuARy 2009 65
2009 Macmillan Publishers Limited. All rights reserved 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 66 jAnuARy 2009 vOlume 7
2009 Macmillan Publishers Limited. All rights reserved (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 nATuRe RevIeWs microbiology
vOlume 7 jAnuARy 2009 67
2009 Macmillan Publishers Limited. All rights reserved 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.
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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