Functions encoded by the most widely distributed plasmid (pTRACA22) were found to be enriched in the human gut microbiome. Homologues of the RelE toxin were associated with all major bacterial divisions.
Functions encoded by the most widely distributed plasmid (pTRACA22) were found to be enriched in the human gut microbiome. Homologues of the RelE toxin were associated with all major bacterial divisions.
Functions encoded by the most widely distributed plasmid (pTRACA22) were found to be enriched in the human gut microbiome. Homologues of the RelE toxin were associated with all major bacterial divisions.
Gut Microbes 1:6, 415-431; November/December 2010; 2010 Landes Bioscience ARTICLE ADDENDUM ARTICLE ADDENDUM Addendum to: Jones BV, Sun F, Marchesi JR. Comparative metagenomic analysis of plasmid encoded functions in the human gut microbi- ome. BMC Genomics 2010; 11:46; PMID:20085629; DOI: 10.1186/1471-2164-11-46. Key words: mobile genetic elements, mobile metagenome, gut microbiota, horizontal gene transfer, toxin-antitoxin addiction module, metagenomics, hologenome, holobiont Submitted: 07/22/10 Revised: 10/21/10 Accepted: 11/02/10 Previously published online: www.landesbioscience.com/journals/ gutmicrobes/article/14108 DOI: 10.4161/gmic.1.6.14087 Correspondence to: Brian V. Jones; Email: B.V.Jones@Brighton.ac.uk U sing the culture independent TRACA system in conjunction with a comparative metagenomic approach, we have recently explored the pool of plasmids associated with the human gut mobile metagenome. This revealed that some plasmids or plasmid families are present in the gut microbiomes of geo- graphically isolated human hosts with a broad global distribution (America, Japan and Europe), and are potentially unique to the human gut microbiome. Functions encoded by the most widely distributed plasmid (pTRACA22) were found to be enriched in the human gut microbiome when compared to micro- bial communities from other environ- ments, and of particular interest was the increased prevalence of a putative RelBE toxin-antitoxin (TA) addiction module. Subsequent analysis revealed that this was most closely related to putative TA modules from gut associated bacteria belonging to the Firmicutes, but homo- logues of the RelE toxin were associated with all major bacterial divisions com- prising the human gut microbiota. In this addendum, functions of the gut mobile metagenome are considered from the per- spective of the human host, and within the context of the hologenome theory of human evolution. In doing so, our origi- nal analysis is also extended to include the gut metagenomes of a further 124 individuals comprising the METAHIT dataset. Differences in the incidence and relative abundance of pTRACA22 and associated TA modules between healthy individuals and those with inammatory bowel diseases are explored, and poten- tial functions of pTRACA22 type RelBE The human gut mobile metagenome A metazoan perspective Brian V. Jones Centre for Biomedical and Health Science Research; School of Pharmacy and Biomolecular Sciences; University of Brighton; Brighton UK modules in the human gut microbiome are discussed. Introduction Humans share the planet with an esti- mated 10 30 prokaryotic cells and interac- tion with these microbes has shaped the course of our development. 1-8 Modern humans have co-evolved with microbial communities that have colonized various habitats offered by the human body, and now maintain commensal or symbiotic relationships with their metazoan hosts. Of the numerous microbial communities harbored by the human body, the gastro- intestinal tract (GIT) is home to the larg- est. The microbiota resident in the distal colon of an individual adult consists of an estimated 10 13 10 14 individual pro- karyotic cells belonging to an estimated 150800 species, which are derived from ~1,3804,018 distinct operational taxo- nomic units observed in collective gut microbiomes analyzed. 8-11 The numerous benecial functions undertaken by this microbial community, and its capacity to direct development of host physiology, epitomises the co-evolution of the gut microbiota with its human host. 3,7,11-17 This inuence may even extend to aspects of human behavior, and the benets gained by acquisition of benecial microbes may have partly contributed to the evolution of social behavior and group living, which facilitates the transmission of microbial symbionts to new hosts. 18 Investigation of host-microbe interac- tion in the GIT has begun to reveal the mechanisms underlying these benecial functions, and emphasised the intricate 416 Gut Microbes Volume 1 Issue 6 For example, the recent genome sequence of the human gut commensal E. coli strain SE11 included six plasmids, which carried genes involved in adherence to epithelial cells, tetracycline resistance and bacterio- cine production. 41 Conversely, plasmid encoded functions may also contribute to virulence in patho- genic bacteria, and gut associated patho- gens harbor some of the best characterized examples. 52-59 These virulence plasmids have been found to carry genes required for the production of specic virulence factors such as toxins, or factors that facilitate colonization and survival in this environment. 52-59 In the latter case, there is potential for pathogens to acquire these functions from commensal or symbiotic members of this community through HGT, while conjugative plasmids in gen- eral may contribute to colonization of the human GI tract by both pathogens and commensals, since conjugation machinery has been found to facilitate adherence of bacteria to various surfaces including epi- thelial cells. 40,53,60 On the whole, genes and functions encoded by MGE that are long term members of the gut mobile metagenome will also reect the co-evolution of resi- dent microbes and their human host. 22,24,49
Recent studies indicate that plasmids har- bored by gut bacteria also display the sig- natures of long-term association with the human host evident in the core genomes of their bacterial hosts, with the observed patterns of long-term gene convergence and conservation evident in chromosomes of gut bacteria, even more pronounced for plasmids. 49 These observations sup- port the hypothesis for a role of the mobile metagenome in broader aspects of com- munity development and are likely to be reected in the gut mobile metagenome in general. 24 The functions encoded by this ex- ible gene pool, along with the capacity for MGE to mediate gene ow between dis- tinct bacterial species, undoubtedly gen- erates an additional sphere of complexity and variability in terms of how this com- munity develops, functions, evolves and interacts with the human host. Despite this, the majority of the human gut mobile metagenome remains unexplored and undened, both in terms of its signicance DNA anking these genes, 32 and evidence for an evolutionarily recent transfer of tetQ from animal to human gut bacterial species has been presented. 25 Overall, the gut mobile metagenome is considered a reservoir for antibiotic resistance genes. 31,38 This view has been strengthened by recent function-driven metagenomic studies highlighting the frequent association of resistance deter- minants with genes and sequences related to MGE in the mammalian gut micro- biota, 27,38 and the observation that MGE encoding resistance to antibiotics such as tetracycline were prevalent in gut isolates of Bacteriodes sp. collected prior to the widespread clinical use of these drugs. 28,31
However, there is growing interest in other functions that may be encoded by MGE resident in the gut microbiota, and the role of the mobile metagenome in the develop- ment and functioning of this community. Characterization of plasmids from cultivatable commensal and pathogenic species found in the human GI tract have illuminated some of the functions encoded by this sphere of the gut microbiome. 24,39-59
These include functions relevant to sur- vival in the GIT and interaction with the human host, such as utilization of car- bohydrates and other nutrients, bacterio- cine production, adhesion to host cells, resistance to bile acids and virulence fac- tors. 24,41-59 A particularly interesting exam- ple of MGE encoding such functions are the large megaplasmids that appear to be widespread in Lactobacillus salivarius, and in other Lactobacilli sp. resident in the human GIT. 41,44 In the case of the probiotic L. salivarius UCC118, this organism harbors three plasmids, including a megaplasmid encod- ing genes related to bile tolerance, redox balance, cell wall biosynthesis and bacte- riocine production. 44 Bacteriocine produc- tion has also been identied as a plasmid encoded function in other gut associated species, 44 while genes providing the ability to resist the antimicrobial effects of bile in the small intestine appear to be encoded by plasmids and MGE associated with a diverse range of gut bacterial species. 24,44,51
Functions involved in gut colonization and survival have also been characterized on plasmids infecting the Enterobacterial population of the human gut microbiota. and intimate relationship between eukary- otic host and prokaryotic symbiont. 12-17
More recently, metagenomic approaches which permit access to the greater uncul- tivated fraction of the gut microbiota have revealed functions enriched in the gut microbiome, and highlighted gene fami- lies shared among the disparate micro- bial lineages in this ecosystem. 10,14,19-22 In parallel to these studies, there is currently much effort focused on dening the bac- terial and archaeal species comprising the common phylogenetic core of the human gut microbiome. 10,11 Together, such studies will ultimately lead to the resolution of the fundamental structure and metabolic out- puts of this community, which will allow us to properly dene its impact on human health and development. However, just as the human gut har- bors a complex microbial ecosystem, bac- teria comprising the gut microbiota in turn play host to their own hitchhikers in the form of mobile genetic elements (MGE). These include plasmids, transpo- sons and integrons and collectively the dif- ferent elements associated with the human gut microbiota may be referred to as its mobile metagenome. 22-24 Bacteriophage also share many of the properties associ- ated with these elements, and although are not MGE in the same sense as plasmids, transposons or integrons, they are capa- ble of mediating horizontal gene transfer (HGT) between bacteria, and for the pur- poses of this article they are considered to be part of the gut mobile metagenome. Investigation of MGE infecting key cultivatable species comprising the human gut microbiota have highlighted the role of elements such as plasmids and conju- gative transposons (CTn) in the mainte- nance and spread of antibiotic resistance genes within this community. 25-39
Dissemination of erythromycin resis- tance between Bacteriodes species in the human gut has been largely attributed to horizontal transfer of the relevant genes by conjugative transposons, 26,28-31 and the role of MGE in the acquisition and spread of tetracycline resistance in the human gut microbiota is also well docu- mented. 25-27,32-34 Signicant similarity has been identied between tetracycline resis- tance genes present in human and animal gut commensals, including regions of www.landesbioscience.com Gut Microbes 417 gut microbiota to date, which identied around 1,0001,150 prevalent bacterial species present in the combined gut com- munities of the 124 individuals investi- gated. 10 However, only 18 of these species were common to all individuals, and it was estimated that only around 38% of the bacterial genes present in an individu- als gut metagenome are shared with other individuals. 10 This is in stark contrast to our primary eukaryotic genome, which in comparison varies little between indi- viduals (~0.1% by single nucleotide poly- morphisms, 0.3% by inversions, 1.2% by copy number variations/insertions or dele- tions), 65 or even between Homo sapiens and its closest living relative, Pan troglo- dytes (~4%). 66 In addition, our primary eukaryotic genomes are essentially xed in terms of content throughout the life time of the host, and lack the plasticity inherent in our secondary prokaryotic metagenomes. Prokaryotes frequently exchange genetic information, and the mobile metage- nome associated with the human micro- biota may represent an important fraction of the human hologenome. This highly exible tertiary gene pool may benet the human host indirectly via effects on constituent species, or encode functions that impact directly on tness of the holo- biont. 24 Studies of MGE associated with microbial communities from plants and invertebrates demonstrate the potential for the gut mobile metagenome to encode genes involved in symbiosis of human and microbe, as well as specic functions of direct benet to the human host. 67-71 Therefore, while the primary human segment of our genome evolves relatively slowly due to a long generation time and its relatively static nature, our secondary prokaryotic segment has the capacity to rapidly adapt to new environmental con- ditions by virtue of short generation times and recruitment of new genetic material. 4
A major advantage of this arrangement for the human host is the retention of a genetically exible secondary genome which may facilitate adaptation to new environmental conditions such as changes in available food supply. This model also affords the possibility that horizontal gene transfer (HGT) among human-associated microbes is of direct benet to the human prokaryotic and eukaryotic cells, and fre- quently referred to as superorganisms. 2-8
In this context the gut microbiota has been described as a virtual organ that undertakes a wealth of functions from which we benet, but which our eukary- otic cells have not evolved for themselves. 3
These include a barrier function against colonization by intestinal pathogens, sal- vage of energy from dietary components exigent to host digestive mechanisms, development of the immune system, as well as aspects of intestinal physiology and control of epithelial cell proliferation in the GIT. 7,12-16,24,61,62 If we accept the paradigm of humans as gestalt entities consisting of both pro- karyotic and eukaryotic cells, with the gut microbiota as a virtual organ, then we must also acknowledge the human genome to be composed of both eukary- otic and prokaryotic components. This has resulted in the development of eco- logical and evolutionary models which integrate both components of the human genome to explain the co-evolution of host and microbe. 4-6,8,18 Recently, Zilber- Rosenberg & Rosenberg (2008) described humans and other complex metazoans as holobionts and presented the hologenome theory of evolution, in which the overall unit of selection in evolution is composed of the total genetic content of the eukary- otic host organism, plus that of its sym- biotic microbial partners. 4 In this model, our eukaryotic cells carry the primary core of indispensible, human dening genetic information, while our prokaryotic sym- bionts collectively house a secondary, vari- able portion of the human genome, which directs benecial accessory functions not encoded for by the primary eukaryotic segment. The content of the principal prokary- otic segment may be dened as the core genome content of the collective microbial species comprising communities such as the gut microbiota, and is itself a metage- nome. Consequently this secondary pro- karyotic metagenome has the potential to vary in terms of genetic content (and therefore its functional output and impact on host tness) not only between indi- viduals but also over the lifetime of the host. 8-11,63,64 This is highlighted by the most in depth analysis of the human to the human host and human evolution, as well as total genetic content. Overall, the mobile metagenome is a facet of the gut microbiota not readily captured by current efforts to characterize this com- munity, but one that will be important in attaining a complete understanding of this virtual organ. Utilizing a comparative metagenomic approach we have recently revealed the existence of plasmids potentially unique to the human gut microbiome, which appear to be present in the gut communities of geographically isolated hosts distributed across the globe. 22 Of particular interest was the unexpected nding that some of these plasmids encoded genes which are enriched in the gut microbial community when compared to other microbial eco- systems, 22 reinforcing the hypothesis of the human gut mobile metagenome as a reservoir of genetic information involved in key aspects of community function. This addendum attempts to place the gut mobile metagenome within the framework of recent theories of host-microbe co-evo- lution, considering functions of this mal- leable genetic resource from the perspective of the human host. In doing so, our origi- nal analysis of plasmids in the human gut mobile metagenome is extended to encom- pass an additional 124 human gut micro- biomes, 10 and a preliminary exploration of differences in plasmid encoded functions in health and disease is presented. An Integrated View of the Human Genome: A Place for the Mobile Metagenome? The understanding that our prokaryotic passengers are more than simply oppor- tunistic hitchhikers, and play an intimate role in our development and wellbeing, has resulted in a revision to the view of humans (and many other complex organ- isms) as entities composed exclusively of eukaryotic cells. In light of our increasing knowledge of host-microbe (prokaryote- eukaryote) interaction in the gastrointesti- nal tract and other body sites, the concept of humans and other animals as organisms has evolved to encompass our symbiotic and commensal prokaryotes. 2-8 Mammals, including humans, are now generally accepted to be amalgams of both 418 Gut Microbes Volume 1 Issue 6 environmental stresses arise (exposure to relevant antibiotics). Perhaps of greater importance is the potential for pathogens to acquire new functions from well adapted gut symbi- onts, which may include traits that facili- tate colonization of the gut or inhibit treatment of infections. In this context the transfer of antibiotic resistance genes from members of the normal gut microbiota to transiently colonizing pathogens is cause for concern. Species naturally present in the human gut that survive in the external environment and cause disease at other body sites, such as uropathogenic E. coli, may also serve to disseminate antibiotic resistance and other genes from the gut microbiome. Host tness and rapid adaptation. On the scale of the human holobiont, a major function of the mobile metagenome may be as a conduit for gene ow between the secondary prokaryotic portion of the human genome (in this case the gut metagenome) and the wider, external pool of prokaryotic genetic information. The mobile metagenome facilitates adapta- tion and evolution of the secondary pro- karyotic fraction of the human genome by mediating the introduction of new traits into this community. 71-74 These directly or indirectly affect the tness of the human host, and in so doing, the human holobi- ont as a whole. Adding functional capacity to the gut microbiota through HGT should be a faster route to acquiring new activities and adapting to new environmental factors, 4,6
while entailing far less risk than recruiting new member species. Although attaining new traits by recruitment of new species is undoubtedly an important mechanism underpinning the evolution of the gut microbiome, and the development of this community in new hosts, in the case of an established community this entails the adaptation of an entire organism to the gut environment and its integration into a complex and pre-existing metabolic net- work, without compromising host tness. In contrast the acquisition of new genetic material, and ultimately new functional capacity through HGT is more likely to maintain the status quo and essentially permits the upgrade of an already func- tioning and well adapted ecosystem. members of this community may also be of benet to the human host. HGT is a driving force in bacterial evo- lution and facilitates rapid adaptation to new environments. This may benet the metazoan host either directly by facilitat- ing development of benecial functions of the gut microbiota, or indirectly by facilitating the adaptation of community members to the gut environment, and development of a stable gut microbiota with associated benets to the host. 24
While the levels of HGT extant in the gut microbiota may be primarily a conse- quence of the environmental parameters in the mammalian gut, such as a high population density in close spatial proxim- ity, it is possible that this facet of the com- munity has been of benet to the human host and is important in the evolution of the human holobiont as a whole. This theory is supported by advantages offered directly to the human host though HGT between autochthonous species of the gut microbiota, as well as between indigenous and transiently colonizing allochthonous species: (1) Introduction of new benecial traits and rapid adaptation to environmen- tal change, possibly within the life time of the host. (2) Generation of functional redundancy, increasing functional stabil- ity of the gut microbiota. Both proposed advantages of HGT to the human holo- biont are supported by the hologenome theory of evolution. 4 In contrast, there is also potential for such widespread HGT to introduce functions which may ultimately prove deleterious to the human host, such as antibiotic resistance. 28-39 However, it would be expected that the introduction of overtly harmful traits into the human hologenome, including those that may destabilize important functions under- taken by our prokaryotic components (such as the gut microbiota), would be countered by holobiont level selection, and ultimately reduced in or eliminated from the gut microbiome. Traits such as antibiotic resistance may be exceptions, since carriage of antibiotic resistance genes does not appear to be directly harm- ful to the human holobiont, and results in detrimental effects only when harbored by community members able to cause disease, and then only when particular host, and raises the possibility that HGT in the gut microbiota (as well as other human associated microbial communi- ties) is in itself an important process in the evolution of the human holobiont as a whole. The nding that certain MGE and the functions they encode are enriched and generally conserved in the human gut microbiome, 10,20-24,72 underscores the potential contribution of mobile genetic elements to the development and perhaps overall functioning of this community, and therefore also to host-microbe co- evolution. For example, the genes under- lying many of the core functions of the gut microbiota appear broadly distributed among members of this community, and HGT seems to have underwritten the expansion of these benecial gene sets ensuring functional stability of the gut ecosystem. 20,21,72-74 MGE are also capable of introducing new genetic material into an existing microbial community, facili- tating the development of new functional pathways within an established ecosystem, which is almost certainly of signicance to community function past, present and future. 24,72-74 In broader terms, the abil- ity of MGE to transfer genetic material between disparate bacterial species may in itself be an attribute of the mobile metage- nome as a whole, that has, and continues to benet both host and microbe, and HGT may constitute a key mechanism underlying the proposed adaptive role of the prokaryotic fraction of the human hologenome. The Gut Mobile Metagenome: A Role for Microbial HGT in Human Evolution? Activities of MGE are generally studied in terms of their impact on tness of the bacterial host. However, activities of bac- teria belonging to communities associated with higher host organisms, such as the gut microbiota, are ultimately selected for at the level of the metazoan host through effects on host tness. 2,8,24 It is therefore of note that the human gut microbiome is considered to be a hotspot for genetic exchange and there is increasing evidence to support this claim. 20,71-73,75 If so, then it follows that a high level of HGT among www.landesbioscience.com Gut Microbes 419 Thus, it appears that this activity was acquired by members of the Japanese gut microbiota through conjugal trans- fer of plasmids encoding this ability from marine bacteria to gut symbionts. Subsequently its utility resulted in integra- tion and xation in the genomes of human gut symbionts, enhancing functionality of the Japanese gut microbiome. Notably, this appears to be an evolutionarily recent gain in function, and this activity could not be detected in any of the gut metage- nomic datasets derived from American individuals analyzed in this study, 71 indi- cating that the Japanese gut microbiota has evolved this trait as a specic adap- tation to host diet. These ndings rein- force the proposed adaptive role of the human prokaryotic genome segment in the human holobiont, and illustrate how the gut mobile metagenome can facilitate this process. Evidence for a role of HGT in the evo- lution of other key functions of the gut microbiota relating to dietary energy sal- vage and maintenance of gut health, have also recently emerged. Carbohydrates that escape host digestive processes are fer- mented by the colonic microbiota result- ing in the production of short chain fatty acids (SCFA). 81,82 This process not only liberates additional energy from the diet that would not normally be accessible to the host, but certain SCFA are considered pivotal to gut health. 81-83 Butyrate in par- ticular has been associated with a range of benecial effects, 81-83 and not only is this SCFA a primary energy source for colonic epithelial cells, but has also been impli- cated in the regulation of epithelial cell proliferation. 81-83 Recent characterization of butyrate synthesizing bacteria from the human colon has implicated HGT in the development of this pathway in intestinal bacteria. 74 It seems likely that this origi- nally arose as a general adaptation to the gut environment, 74 and subsequently, this pathway may have been selected for at host-level due to its benecial effects. Overall, these studies demonstrate the capacity for the mobile metagenome to facilitate adaptation of the gut microbiota to new environmental conditions, such as composition of the host diet or envi- ronmental conditions in the GIT itself, and in doing so facilitate adaptation of bacteriophage is notable in this context, and may in part result from selective pres- sure exerted by the aphid-bacteria-phage holobiont to provide a functionally stable gut microbiota, and minimize the disrup- tion to key prokaryotic components of the holobiont that could arise from infection by lytic phage. Overall, this example dem- onstrates how the mobile metagenome can add functional capacity to the prokaryotic fraction of an organisms hologenome, and in so doing facilitate adaptation and inu- ence evolution of the holobiont as a whole. Recent comparisons of functional dif- ferences between the gut microbiomes of American and Japanese individuals have also provided evidence for HGT mediated adaptation of the human holobiont, and identied a role of the human gut mobile metagenome in this process. In this case a gain in function of direct benet to the human host was identied in the Japanese gut microbiome, and its acquisi- tion through HGT revealed. 71 This func- tional gain relates to a primary activity of the human gut microbiome: the salvage of energy from the diet. 2,14,80 It has been esti- mated that up to 10% of our daily calories are derived from microbial fermentation of plant polysaccharides resistant to host digestive mechanisms, 80 which recovers energy from the diet that is otherwise inaccessible to the human host. Seaweeds, and in particular Nori, are major components of the Japanese diet and marine bacteria colonizing these algae produce a range of porphyranse and aga- rase enzymes in order to utilize the poly- saccharides they generate. 71 Hehemann and co workers demonstrated the acquisi- tion of porphyranse and agarase degrading ability by the gut microbiota of Japanese individuals, through horizontal transfer of these genes from marine bacteria natu- rally colonizing dietary seaweed (which are consumed without cooking), to mem- bers of the Japanese gut microbiota. 71 In this case genes associated with conjugal transfer machinery were identied in regions of DNA surrounding the acquired porphyranase genes in the gut symbi- ont Bacteriodes plebius. 71 These showed homology to plasmid gene sequences which encode the putative ancestral por- phyranase genes in the candidate donor species of marine bacteria. 71 Examples of holobiont adaptation mediated by microbial HGT, and the mobile metagenome of host associ- ated microbial communities have been described previously. 67-70,74,76-79 For instance, the symbiotic relationship between Rhizobium sp. and the roots of leguminous plants is heavily inuenced by MGE encoded genes, with the majority of functions essential for symbiosis carried by plasmids, and frequently requiring the acquisition of several distinct MGE for successful host-microbe interaction. 67,68
Here, Rhizobial plasmids have been found to encode essential functions which facili- tate adaptation not only to plant-microbe symbiosis, but also to a free living lifestyle, such as tolerance of low pH, heat, drought or starvation. 67,68,77-79 This strategy pro- vides Rhizobium with a highly mobile gene pool that facilitates rapid adaptation to diverse and often transient ecological niches, and subsequently allows modula- tion of core community functions under- taken by the symbiotic microbiota to be adapted to the host legume. 24,69 As such the Rhizobial mobile metagenome likely facilitates rapid adaptation of the plant- microbe holobiont as a whole. A further example of how the mobile metagenome may encompass functions of direct benet to the metazoan host comes from recent studies of the aphid symbiont Hamiltonia defensa. 70 H. defensa protects its aphid host against attack from parasitoid wasps by killing wasp larvae before they can fully develop and bring about the demise of the aphid. 70 Notably, the production of toxins necessary to kill wasp larvae is not directed by the prin- cipal genome of H. defensa, but acquired through infection of the microbial symbi- ont with a lysogenic bacteriophage carry- ing the necessary genes. 70 In this instance, the phage encodes functions which essen- tially ensure that the habitat of its bacterial host (the aphid) is protected and therefore that suitable host bacteria will be avail- able for replication. However, since aphids with a microbiota lacking this ability will be vulnerable to attack from parasitoid wasps, the benet to the metazoan host will also result in selection for this attri- bute from the level of the aphid host, and therefore for the phage providing these functions. The lysogenic life cycle of this 420 Gut Microbes Volume 1 Issue 6 in the gut environment must be overcome without adverse effects on host tness, and the resulting host-level selection could drive the dissemination of relevant genes to many diverse members of this commu- nity, generating functional redundancy in the process. The salvage of energy from the host diet is a prime example of a core function of the gut microbiota that exhibits a high level of redundancy and stability in this commu- nity. 2,9,20,61,71-74 The ability of gut microbes to utilize carbohydrates and release energy to the host in the form of SCFA, appears to be distributed among a diverse array of species spanning the main bacterial divisions of the human gut microbiota (Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria). 2,9,20,61,71-74 While it is likely that some of this redundancy is the result of early events in the development of the gut microbiota, reecting the recruit- ment of species capable of colonizing and utilizing available nutrients without harming the host, recent studies have also highlighted the pivotal role of the mobile metagenome in the convergence and expansion of the required gene sets. 71-74 Comparative genomic and metage- nomic analyses of Bacteroides species from the normal human gut microbiota with non-gut Bacteroides sp. have highlighted the importance of HGT in the adaptation of gut-associated species to the human GI tract. 73 This included the acquisi- tion of genes related to the interaction of these organisms with the host immune system, as well as utilization of carbohy- drates indigestible to the human host. 73
This feature of the analyzed genomes also demonstrates that in these species MGE mediate access to a diverse genetic resource, through which adaption to new ecological niches and environmental con- ditions may be rapidly achieved. In a more global analysis, Lozupone et al. (2008) investigated the distribution of glycoside hydrolase and glycoside transferase encod- ing genes among 36 genomes representing the dominant bacterial divisions in the gut microbiota, as well as archaeal species. 72
This illuminated the convergence of key carbohydrate active gene families among diverse members of the gut microbiota, and again indicated HGT as a major pro- cess facilitating the dissemination and the gut microbiota to uctuate through- out the lifetime of the host, there is scope for considerable variation in the prokary- otic section of the human genome, and therefore the functional output of this community. 2,3,8,63, 64 Despite this, in the adult human holobiont, the human gut microbiota is considered to exhibit a high level of stability in terms of the major core functions of this community, such as energy salvage. 2,8 How this functional output is stabilized and maintained in spite of the variable nature of the prokaryotic metagenome has not yet been fully elucidated, but the gen- eration of functional redundancy within this community is considered an impor- tant mechanism. 8,20,21,72 Redundancy may be achieved through the wide spread dis- tribution of genes underlying community level functions to diverse and disparate members of the gut community. 8,20,21,72
This should guard against the loss of key activities and pathways from the commu- nity and allow the gut microbiota to main- tain its overall functional output despite shifts in community composition. Although redundancy of core func- tions may be achieved via the recruit- ment of diverse species with overlapping metabolic capabilities, evidence for a key role of HGT in generating functional redundancy in this community has also emerged. 20,21,72-74 The movement of MGE between disparate species belonging to the mammalian gut microbiota has been demonstrated both in vitro, in vivo, and in silico, 21,22,24-27,86-88 and HGT has already been linked with development of several primary activities of the gut microbiota which exhibit a high level of redundancy in this community. 21,72,73,88,89 Therefore, the mobile metagenome has likely facili- tated the dissemination of key traits involved in core community functions to a wide range of community members, play- ing a major role in developing a function- ally stable ecosystem. 21,24,72 In particular, it would be expected that challenges faced by most or all of the microbes colonizing a particular environ- ment, would promote the acquisition of genes providing solutions from other spe- cies colonizing the same habitat. 8,21,72 For bacteria comprising the human gut micro- biota, the barriers to survival encountered the human holobiont as a whole. In par- ticular, the capacity to rapidly adapt to new dietary components would serve the human holobiont by allowing new food sources to be exploited as they become available or when access to the normal food supply is restricted. It is easy to see how such dietary exibility, and the ability to obtain the maximum possible energy yield from a wide range of food sources would benet the ancestors of modern humans, for whom the source of each meal would almost certainly vary signicantly. 84,85 This may be reected in the division of the genetic information encoding these important pathways of dietary energy sal- vage, which despite the obvious impact on host tness, are not present in the primary eukaryotic portion of our genomes, but remain in the secondary, exible prokary- otic segment. The reason for this, at least in part, may be the advantage of rapid adaptation offered by the prokaryotic metagenome, which through the mobile metagenome has access to a vast genetic resource. In the examples discussed here, HGT rather than changes in species com- position, has facilitated the adaptation of the holobiont, 67,69-71,74,76-79 supporting the hypothesis that HGT in the gut microbi- ota is of direct benet to the human host. This also affords the intriguing pos- sibility that the observed degree of gene exchange extant in the gut microbial com- munity may not only be due to the physi- cal characteristics of the gut environment and intrinsic properties of species com- prising this ecosystem, but also the result of host-level selection for activities of the gut microbiota which impact favorably on host tness. However, more in depth stud- ies of the relative levels of HGT between various host associated microbial com- munities and those not subject to host level selection are required to validate this theory. The mobile metagenome and func- tional stability of the gut microbiota. Core functions of the gut microbiota that impact on host health are ultimately selected for at the level of the human host, and there is pressure to ensure they are stable over time and consistently delivered by this community. 8 However, in light of the plasticity of bacterial genomes and the potential for the species composition of www.landesbioscience.com Gut Microbes 421 Therefore, the identication and charac- terization of MGE enriched in the gut has the potential to greatly increase our under- standing of the gut microbiota, and its interaction with the human host. However, cataloguing the MGE comprising the human gut mobile metagenome, and iden- tication of gut enriched elements is a chal- lenging task. In light of the enormous and predominantly uncharacterized diversity of the microbial world, the inherent pro- miscuity of MGE, and the methodological challenges associated with studying these elements, identication of MGE poten- tially unique to or enriched in the gut will not be straightforward. 24 Furthermore, membership of the gut mobile metage- nome, in accordance with the nature of MGE, is likely to be much less exclusive or committed than for the core commen- sal or symbiotic bacterial species compris- ing this community. As such this section of the human hologenome is likely to be a much less clearly denable gene-space than the principal prokaryotic gut metagenome. Nevertheless, several recent studies includ- ing our own, have provided evidence sup- porting the existence of gut specic MGE, and offered good candidate elements. In our recent study, plasmids were iso- lated from the gut microbiome using the culture-independent TRACA system, which is capable of capturing plasmids from a wide range of bacterial species comprising the gut microbiota, and facili- tating their maintenance in surrogate host species. 22-24 The complete nucleotide sequences of six plasmids captured from the human gut microbiome were used to search a range of metagenomic data sets derived from the human gut, 19,20 murine gut, 14 marine 90 and terrestrial environ- ments. 91 In this comparative metage- nomic analysis, sequences homologous to two of our plasmids (pTRACA10 and pTRACA22) were detected in mul- tiple human gut metagenomes, indicat- ing a broad distribution. 22 In contrast no sequences with homology to any of the six plasmids analyzed were identied in any of the non-human metagenomes studied. 22 While none of the metagenomic data sets currently available afford complete coverage of their representative com- munities (particularly for environmental ecosystems), this nonetheless indicates MGE Enriched in the Gut Mobile Metagenome The proposed integration of the prokary- otic mobile metagenome into the human hologenome proper, leads to certain pre- dictions about the composition of the pool of MGE comprising the gut mobile metagenome that may be investigated in more detail. In particular, if the gut mobile metagenome constitutes a compo- nent of the human hologenome as a whole, then the co-evolution of host and microbe should also be reected in the MGE asso- ciated with the gut microbiota, and the functions encoded by constituent MGE will also be subject to selective pressure at the level of the human host. 22-24 This hypothesis is supported by recent studies of gene convergence in gut associ- ated bacterial species compared to non-gut species, in which plasmids carried by gut bacteria were found to reect the overall signatures of increased specialization at short-phylogenetic distances and long-term gene convergence at greater phylogenetic distances, that were observed for bacterial chromosomes. 49 In addition, the long-term convergence of gene content identied in gut associated plasmids also suggest the general functional conservation exhibited by the human gut microbiome of indi- vidual human hosts, may also extend to aspects of the gut mobile metagenome. 49 As such it would be expected that the most successful and well adapted MGE in this community, which satisfy the demands of both bacterial and human hosts, will exhibit the greatest prevalence in the gut mobile metagenome, and be common in the gut microbiomes of a board range of individuals. These gut specic elements would be more likely to encode factors that provide bacterial hosts with an advantage in this environment, such as colonization of and survival in the gut, as well as to carry genes involved in host-microbe interaction and functions of the community inuencing host health. MGE mediated host-microbe interactions have already been described in bacterial communities associated with plant and insect hosts, and in particular the example of bacteriophage encoded protection of aphid hosts from parasitoid wasps would appear to support this theory. 67-70,76,79 expansion of gene sets involved in core community functions. 72 Microbes inhabiting the human gut also face a number of direct barriers to colonization which must be overcome without reducing host tness. 21 Among these are the toxic effects of conjugated bile acids (CBA) encountered in the small intestine. HGT has also been implicated in the dissemination of genes which facili- tate resistance to CBA (bile salt hydrolase: BSH) among a broad range of microbes in the gut community. 21,24,88,89 The real- ization that bile acids also function as key signalling molecules regulating important aspects of host metabolism and immune function, means that bile acid modica- tion by gut microbes also has the potential to greatly inuence host physiology and development. 21 Although our understanding of how this aspect of the gut microbiota relates to host health is currently limited, the very broad distribution of BSH activity and the resulting stability of this function in the community indicates host-level selection for this activity, and therefore a benet to the human holobiont as a whole. 21 In keep- ing with this, the general enrichment and excellent functional redundancy of BSH activity means that although overall capac- ity for this activity may uctuate over the lifetime of the host in response to changes in population structure of the gut micro- biota, it is highly unlikely that this func- tion will be lost from an established gut community. 21 It seems likely that much of the functional redundancy for BSH activ- ity in this community has been achieved through HGT, perhaps even transcending barriers between distinct domains of life (Bacteria and Archaea). 21,24,51,88,89 In our recent comparative metagenomic study of plasmids from the gut mobile metagenome, we also provided evidence for a high level of redundancy and gen- eral enrichment of certain toxin-antitoxin (TA) modules in this ecosystem. 22 This was indicated by the broad phylogenetic distribution of the pTRACA22 type RelE toxin sequences among all major bacterial divisions in the gut microbiota. However, the role of these TA modules, if any, in the overall functioning of the gut microbiota remains to be established, and is discussed in subsequent sections of this article. 422 Gut Microbes Volume 1 Issue 6 level of inter-individual variation in car- riage of phage GB124 remains to be estab- lished. 93 Currently, our understanding of how bacteriophage may inuence human health is limited, but phage not only have the potential to alter microbial community dynamics and metabolic output by selec- tive elimination of species within the gut microbiota, but may themselves interact directly with the host via the immune system. 94 However, coverage of the gut com- munity offered by the datasets utilized in our original analysis of plasmids resident in the human gut mobile metagenome is generally low, and it was not possible to retrieve the complete sequence of the most widely distributed plasmid, pTRACA22, from either individual or combined data sets. 22 In total we found that 81.9% of the pTRACA22 nucleotide sequence was represented in these combined datasets at an identity of 90% or greater, and likely indicates the general enrichment and con- servation of a closely related family of plas- mids of which pTRACA22 is a member. 22 This suggests that metagenomic data sets with greatly increased coverage of the gut microbiota will afford a concor- dant increase in the degree of coverage mobile metagenome, may be a manifesta- tion of community adaptation to modern environmental stresses such as the use of antibiotics. 20 Metagenomic studies have also revealed the high abundance of bacteriophage associated with all microbial communi- ties including the human gut microbi- ome, where it is estimated a minimum of ~1,000 distinct viruses exist (although this is likely to be a substantial underesti- mate). 92 Because the host range for bacte- riophage typically varies from a few closely related species down to individual strains, the existence of gut specic bacteriophage seems a likely prospect, and candidate phage apparently unique to the human gut microbiome have already been described in reference 93. Phage infecting a common gut commensal, Bacteroides sp. GB124 which has been tentatively identied as a strain of Bacteroides ovatus (Ebdon J, personal com- munication), were isolated from human feces. 92 However, this phage was found to be absent from fecal samples derived from a wide range of common domestic and wild animals, and was not present in the general environment. 93 Furthermore, widespread carriage among the human population was also indicated by this study, but the actual the capture of human gut specic MGE. The presence of sequences homologous to pTRACA22 in particular, in multiple human gut metagenomes derived from individuals with broad geographic origins (America, Japan and Europe), suggests a gut specic family of mobile elements which has a deep co-evolutionary rela- tionship with this community and global distribution. 22 A similar broad global distribution and general enrichment for a family of conjugative transposons in the human gut microbiome has also been described in reference 20. Designated CTnRINT (Conjugative transposons rich in intes- tine), these elements are related to the Tn-1549 family CTns found in a range of gut associated pathogenic microbes includ- ing Enterococcus faecalis and Clostridium difcile. 20 CTnRINT elements display similar gene architecture to Tn-1549 ele- ments from other bacteria, but differ in an accessory gene region. It is possible that the genes encoded by this region of the CTnRINT elements relate to activi- ties advantageous to life as a human gut commensal or symbiont. 20 Alternatively, the current enrichment of CTnRINT, as well as other MGE in the human gut Figure 1 (See opposite page). Incidence and relative abundance of plasmids pTRACA10, pTRACA17, pTRACA18, pTRACA20, pTRACA22 and pTRACA30 in the METAHIT gut metagenome data set. The complete nucleotide sequences of each plasmid were used to search the METAHIT 10 dataset using Blastn, and calculate incidence and relative abundance according to methods described previously in reference 21 and 22. For this analysis, only hits matching the following criteria were considered signifcant: An identity of 90% or greater over 100 nucleotides or more, and an e-value of 1 e -10
or lower. In addition to the complete METAHIT data set, incidence and relative abundance of plasmids in sub sets of individuals represented in the METAHIT dataset was also explored based on nationality (Danish or Spanish), gender, and disease status (healthy, Crohn disease or ulcerative colitis). For the purpose of this analysis individuals were designated as healthy unless indicated as being diagnosed with ulcerative colitis (UC) or Crohn disease (CD), regardless of age or body mass index. The statistical signifcance of observed incidence and relative abundance data was explored using the 2 distribution. 22 Where signifcant diferences in relative abundance of plasmids between metagenomic datasets were identifed, the statistical power aforded by the available sample sizes in each group was calculated using the Piface program. 111 This indicates the probability that the observed diferences are truly refective of the wider population, with a probability of 0.8 considered to indicate adequate power. Symbols above bars indicate the level of signifcance in 2 analysis, *p 0.05; **p 0.01; ***p 0.001. Statistical power is shown in adjacent parentheses, and in all cases has been computed to p = 0.05 using variation (standard deviation) observed within the total METAHIT dataset. Danish HEALTHY, Spanish HEALTHY = Combined gut metagenomes from individuals of each nationality in the METAHIT data set, excluding those diagnosed with IBD; Male HEALTHY, Female HEALTHY = Combined gut metagenomes from individuals of each gender in the METAHIT data set, excluding those diagnosed with IBD; Healthy TOTAL = Com- bined gut metagenomes from all healthy individuals in the METAHIT dataset; CD, UC = Combined gut metagenomes from individuals diagnosed with respective IBDs; TOTAL METAHIT = Combined gut metagenomes of all 124 individuals regardless of nationality, gender or disease status. (A) Shows the incidence of each plasmid (and closely related elements) among the gut metagenomes comprising the METAHIT data set, when grouped by national- ity, gender and disease statues, as well in the METAHIT cohort as a whole. Incidence is expressed as the percentage of metagenomes in which at least one signifcant hit to each plasmid was detected, (according to criteria outlined above). In this analysis, only diferences found to be supported by ad- equate statistical power (0.8 at p = 0.05) were considered signifcant overall. (B) Shows the relative abundance of each plasmid, expressed as hits/Mb, among the gut metagenomes comprising the METAHIT data set, as grouped in (A). 21,22 For this analysis relative abundance was calculated based on the total number of signifcant hits (according to criteria outlined above) in each group (gender, nationality, disease status), and the average relative abun- dance in each cohort displayed. INSET: Shows the overall inter-individual variation for each plasmid among all 124 individuals in the METHIT dataset, with yellow bars representing lowest observed abundance, red bars highest observed abundance, and green bars showing the average abundance over all 124 individuals. (C) Shows the percentage of each plasmid nucleotide sequence that could be recovered from the total METHIT dataset. Colors of segments represent % identity of recovered METAHIT sequences corresponding to respective regions of each plasmid, and numbers below pie charts provide the coordinates in the complete plasmid sequence for each segment. Yellow segments = 9294% identity; orange segments = 9496% identity; red segments = 9698% identity; black segments = 98100% identity. www.landesbioscience.com Gut Microbes 423 homology could be retrieved (pTRACA17, pTRACA20, pTRACA30; Fig. 1). 22
or lower) in these human gut microbiomes compared to the other plasmids, and the greatest incidence of the six plasmids with sequences homologous to pTRACA22 detected in 73.3% of the 124 individual metagenomes comprising the METAHIT data set (Fig. 1). However, it is not clear if this is solely due to the greater cover- age afforded by the METAHIT data set, or is a result of its European origin and therefore a reection of variation in the gut mobile metagenomes of different eth- nic groups, but is likely a combination of both factors. 124 European individuals. 10 As predicted, using this data set permitted a much greater proportion of the pTRACA22 nucleotide sequence to be retrieved, and in general homologous sequences with much higher identity were recovered for all plasmids analyzed in our original study (Fig. 1C). In total we were able to recover 99.4% of the pTRACA22 nucleotide sequence at an average identity of 97.7% or greater using the METAHIT data set (Fig. 1C). The METAHIT data set also permit- ted the retrieval of sequences homologous to several other plasmids characterized in our recent study, which were either poorly represented in the American and Japanese data sets originally utilized (pTRACA18), or for which no sequences with signicant of the plasmids analyzed in our study. Furthermore, since the general conserva- tion of plasmid pTRACA22 in the gut mobile metagenomes of geographically isolated individuals suggests their asso- ciation with the human gut microbiota is ancient, some divergence in plasmids resident in the gut microbiomes of vari- ous ethnic groups is to be expected. As such, use of larger metagenomic data sets that are also derived from the same broad ethnic group (European) as the original pTRACA22 plasmid should also increase coverage of these plasmids. Therefore, the recently released METAHIT data set, which affords the greatest coverage of the gut metagenome to date, was utilized to extend our original analysis to a further Figure 1. For fgure legend, see page 423. 424 Gut Microbes Volume 1 Issue 6 bacterial or human host, or is the result of the addictive nature of these modules alone. 96 Perhaps the simplest explanation for the enrichment of the pTRACA22 type RelBE modules identied in our study, stems from the latter possibility, and relates to the potential for these gene systems to exist purely as selsh entities. 96 It is conceiv- able that these pTRACA22 type TA mod- ules present no appreciable impact on host tness, and in terms of the gut microbiota and the human holobiont as a whole, are evolutionarily neutral. This explanation does not appear to account for the differential enrichment of the pTRACA22 RelBE family of TA modules over other TA systems, and per- haps even over other RelBE variants. 22
However, the observed trend in relative abundance of the TA systems we exam- ined ts comfortably with this selsh DNA hypothesis, since there is no evi- dence at present which excludes the pos- sibility of selection against the other TA systems, rather than explicit selection for the pTRACA22 sub-family. Therefore, the prevalence of the pTRACA22 type RelBE modules we identied may simply result from their addictive nature, coupled with a neutral or insignicant impact on tness of the bacterial and ultimately the human host. If this is so, then TA modules expanded in microbial communities such as the human gut may be under pressure to minimize any adverse effects on bacte- rial or human hosts. In this context it is notable that among the pTRACA22 RelE homologues we retrieved from the human gut metagenomes analyzed, cer- tain amino acid residues were found to be completely or predominantly conserved specically among gut associated RelE. 22
This observation is more signicant in light of the broad phylogenetic distribu- tion identied for the pTRACA22 type RelE toxin sequences, and while the majority of these were afliated with members of the Firmicutes division, rep- resentatives also grouped with members of the Bacteroidetes, Actinobacteria and Proteobacteria. 22 Together, a broad phylo- genetic distribution, and a conserved gut associated sequence motif, indicates the development and expansion of a gut spe- cic RelE sub type. However, the selsh in the dissemination and ultimate enrich- ment of genes involved in core commu- nity level outputs, the overall abundance of genes and functions encoded by gut specic MGE is of considerable interest. It would be expected that the genes and functions encoded by MGE enriched in this community exhibit an increased prev- alence in the gut microbiome as a whole. This is indeed the case for genes encoded by the CTnRINT elements described by Kurowkawa et al. (2006), 20 as well as certain genes encoded by the pTRACA10 and pTRACA22 plasmids analyzed in our study. 22 For CTnRINT, genes encoded by this gut associated family of MGE were found to account for around 0.8% of the 5,325 predicted ORFs identied in the combined metagenomic sequence data from 15 Japanese gut microbiomes. 20 These genes were also found to be enriched in gut metagenomes of American individuals, highlighting CTnRINT as a long standing member of the gut mobile metagenome. 20 Utilizing these same metagenomic data sets, we also observed the enrich- ment of several ORFs encoded by plas- mids pTRACA10 and pTRACA22 in the human gut metagenome. 22 These enriched ORFs cover a wide range of functions and include genes for phosphoesterases or phophohydrolases, replication proteins, a TA addiction module, as well as genes of unknown function. 22 Of particular inter- est was the increased prevalence of a puta- tive RelBE TA addiction module encoded by pTRACA22, which was unexpected in this community. 22 While TA modules are generally highly abundant in free liv- ing bacteria and archaea (i.e., those not forming obligate intracellular relation- ships with eukaryotic host cells), 95 further analysis revealed that this enrichment in the gut was conned to homologues of the pTRACA22 type RelBE module, and was not observed for MazEF, ParDE or HigBA modules. 22 This naturally leads to the fol- lowing questions: why is this particular family of TA modules enriched in the human gut microbiota? What role, if any, do these genes play in the overall opera- tion of the gut microbial community? In addressing the rst question, it is important to consider whether the observed enrichment results from selection for these modules due to benets conferred on the Furthermore, the plasmids isolated in our original analysis range in size from 3.7 to 10.8 kb. It is possible that this size range reects a restriction of the TRACA system used to isolate them, the dominance of smaller plasmids in the gut microbiome, or a combination of these factors. 22-24
While the smaller size of these plasmids do not alter the observation that some plasmid families are generally enriched and broadly distributed in the human gut microbiome, 22 similar studies of larger plasmids would be useful in understand- ing how plasmid size may relate to cover- age in metagenomic datasets. Overall, MGE potentially enriched in the human gut mobile metagenome such as pTRACA22 family plasmids, CTnRINT type transposons and phage GB124, along with numerous as yet uncharacterized gut specic or gut enriched MGE, could conceivably constitute the mobile metage- nome equivalent of a conserved phyloge- netic core. It also seems most probable that MGE enriched in the human gut mobile metagenome will be associated with numerically dominant members of this community. Given the potential for a bacterial host to support a range of MGE, those elements that are most frequently associated with abundant, and presum- ably successful and well adapted species, in the gut microbiota are likely to confer selective advantages. While enrichment of these elements or association with numeri- cally dominant members of the commu- nity does not necessarily indicate a role in community function or survival in the gut environment, characterization of such elements is most likely to provide insights into the role of MGE in the development and functioning of the gut microbiota, particularly given that plasmids (and most likely other MGE comprising the mobile metagenome) also appear to be susceptible to long-term habitat associ- ated convergence of gene content in this environment. 49 Plasmid Encoded Functions Enriched in the Human Gut Microbiome In light of the prevalence of certain MGE within the human gut mobile metage- nome, and the prominent role of HGT www.landesbioscience.com Gut Microbes 425 question of what role these gene systems may play in the human gut microbiota or for the human holobiont as a whole, and there are a range of possible answers. TA modules have been associated with a wide variety of activities which may be relevant to microbes inhabiting the human gut. These include contribution to tness of the bacterial host through modulation of gene expression, 95,100,101
the establishment of new regulatory gene networks, 102 formation of persister cells and resistance to environmental stresses such as nutrient limitation, 95,103,104 as well as the potential for direct host-microbe interaction through toxic effects of RelE on eukaryotic cells. 105-107 Known or pro- posed functions of TA modules that may be relevant to the human gut microbi- ome are summarized in Table 1 along with the potential utility of these attri- butes to gut microbes and implications for both the human host and microbiota as a whole. Importantly, in the case of mecha- nisms underlying bacterial colonization and persistence in the gut, these must not only permit bacteria to overcome particu- lar environmental stresses, but must do so without any negative effect on tness of the human host, and TA modules would appear to full this remit. It is also con- ceivable that discrete functions mediated by TA modules are not only differentially selected for in various host bacteria, (with different host species beneting from dis- tinct functions of the same TA module), but are also of most benet at particu- lar stages in the development of the gut microbiota or under specic environmen- tal parameters which are not a constant feature of the gut environment. If so, the observed prevalence of these TA modules may result from the wide range of func- tions potentially mediated by TA (Table 1), which make them of benet to a broad range of community members under a wide range of conditions. The addictive properties of these gene systems would only serve to reinforce any positive selec- tion for these modules and stabilize them within the community. When considered from the perspective of the human host and within the con- text of the hologenome theory of human evolution, there also exists the possibility selective effects, rather than a general cloning bias. This is reinforced by the well established presence and functionality of the TA systems we examined in E. coli (RelBE, MazEF, ParDE, HigBA). 95,99 However, to investigate this issue more denitively, the METAHIT data set was again utilized, 10 and the relative abun- dance of the pTRACA22 RelBE family of TA modules in the combined gut metage- nomes of the 124 individuals represented was assessed (Fig. 2). This illuminated the large inter-individual variation in the rela- tive abundance of pTRACA22 like RelBE modules between the individuals in this dataset (Fig. 2A), and demonstrated a pervading trend of a higher abundance of RelB anti-toxin in the majority of indi- viduals (Fig. 2B). The incidence of these pTRACA22 like RelBE TA modules was high, and we were able to detect homolo- gous TA modules in 93% of individuals represented in the METAHIT data set (Fig. 2B). Notably the METHIT data set was generated using distinctly different methods for DNA extraction, sequenc- ing, and library construction than the previously analyzed data sets, including the use of small and very small insert libraries unlikely to harbor intact TA modules. 10 Regardless of these differ- ences in methodology, no signicant differences were observed in the relative abundance of RelBE modules between our previous analysis (based on the combined American and Japanese data- sets) 19,20 and that calculated from the METAHIT data (Fig. 2C). 10 The varia- tion in pTRACA22 type TA module abundance between the data sets utilized in our original study and healthy indi- viduals in the METAHIT dataset was found to be 0.004 and 0.012 hits/Mb for RelE and RelB respectively. Thus it appears highly unlikely that the observed enrichment of the pTRACA22 family of RelBE TA modules is an artefact of the metagenomic approach. Apart from the selsh DNA hypoth- esis, there also remains the possibility that the pTRACA22 family RelBE mod- ules have been selected due to a benecial function or attribute they confer upon members of the gut community or even the human host. This leads back to the DNA hypothesis for the observed enrich- ment of pTRACA22 type RelBE modules in the human gut microbiome is neither supported, nor refuted by our ndings, and further investigation of the role of these systems in the gut microbiota is required to test this hypothesis. Alternatively, the addictive proper- ties of TA modules could account for the observed enrichment through more nefarious mechanisms which lead to perturbations in metagenomic data sets. The contribution of TA modules to gen- erational stability of plasmids is well established, and plasmid encoded TA modules ensure that daughter cells retain a copy of the plasmid through a post seg- regation killing mechanism (PSK). 95,97,98
Loss of the plasmid effectively results in loss of the unstable antitoxin, leading to cell death or growth arrest through the continued activity of the stable toxin component which persists in plasmid free cells. 95,97,98 PSK may account for the enrichment of certain RelBE TA mod- ules by stabilizing metagenomic clones in the surrogate E. coli hosts used to construct metagenomic libraries. This could lead to the articial enrichment of TA module harboring clones in the nal metagenomic data set. In addition, the issue of biases generated by use of differ- ent extraction procedures and sequencing strategies used to generate metagenomic data sets, may also be an underlying fac- tor in the observed trends. Despite this, there is much evidence indicating that the prevalence of pTRACA22 type TA modules in the human gut microbiome is not the result of bias in these data sets, and reinforces the utility of available metagenomic data. The existence of orphan toxin genes identied in metagenomic sequences argues against bias induced by TA medi- ated stabilization of metagenomic clones, and no signicant differences in terms of relative gene abundance were identi- ed between the Japanese and American data sets utilized in our previous study. 22
This is in spite of the reported paucity of sequences from Bacteroides sp. in the lat- ter data set. 19 Furthermore, the differential enrichment of pTRACA22 family RelBE modules compared to other TA systems in itself points to enrichment though specic 426 Gut Microbes Volume 1 Issue 6 metagenome may be an auxiliary, ex- ible gene pool that introduces additional variation at a level distinct from that of the principal gut metagenome (which is based on the core genome content of con- stituent species). Changes in the structure and metabolic output of the human gut microbiota are being increasingly linked to human disease, and there is potential for the mobile metagenome to contribute to such changes. Bacteriophage in particular may be involved in the pathogenesis of disorders such as inammatory bowel diseases (IBD). As well as mediating HGT and MGE, Community Output and Intestinal Disease: A Preliminary Investigation Our original comparative metagenomic analysis also hinted at the inter-individual variation of the gut mobile metagenome, 22
and this has been further supported by the analysis of the METAHIT dataset pre- sented in this article (Fig. 1). Variation in this sphere of the human hologenome, as with other sections, has the potential to contribute to differences in the metabolic output of gut communities between indi- viduals. 22 In this respect the gut mobile that the primary reason for the observed enrichment may be selection for these pTRACA22 type RelBE TA modules at the level of the human host. In this case, carriage of these modules may impose a tness cost on the bacterial host which is ultimately offset by increased tness of the holobiont as a whole. However, further study is required to determine if these modules simply persist as selsh entities in the human hologenome, with no tan- gible impact on the human holobiont or contribute to functions undertaken by the gut microbiota and its interaction with the human host. Figure 2. For fgure legend, see page 427. www.landesbioscience.com Gut Microbes 427 pTRACA10 in healthy Spanish compared to healthy Danish individuals, although power calculations indicated this is almost certainly not a true representation of the wider population (Fig. 1B). The incidence of all plasmids was found to vary between different groups, particularly healthy indi- viduals and those diagnosed with CD, but no signicant differences were observed (Fig. 1A). However, signicant differences in relative abundance of pTRACA10, pTRACA22 and pTRACA30 were identi- ed in individuals with CD or ulcerative colitis (UC), compared to healthy indi- viduals, but in most cases available sample sizes do not provide adequate statistical power to infer an association in the wider population (Fig. 1B). Interestingly, the relative abundance of pTRACA22 type TA modules was also altered in both CD and UC disease states (Fig. 2C). In the CD cohort the pervad- ing trend for higher abundance of RelB antitoxin over the RelE toxin in healthy individuals was reversed, with RelE toxin elevated and RelB antitoxin reduced in comparison to healthy individuals and those with UC, although this was not sta- tistically signicant (Fig. 2C), despite the distinct separation of the gut microbiomes of healthy individuals and those with CD and UC reported by Qin et al. (2010). 10 In the case of UC, a statistically signicant In the case of the human gut specic phage infecting Bacteroides GB124, the observation that the host species for these phage may be a strain of Bacteroides ova- tus also has implications for their involve- ment in CD. The titre of serum antibodies against Bacteroides ovatus is signicantly elevated in patients with CD, in contrast to that of other common gut associated Bacteriodes sp. (B. fragilis and B. vulga- tus). 110 While it is presently unclear if this is indicative of a role for B. ovatus in the pathogenesis of CD, the immunogenicity of this organism, and the increased levels of both IgG and IgA anti-ovatus antibod- ies in CD patients, suggests a possible pro- tective role for bacteriophage capable of killing this organism. The METAHIT data set also allowed us to explore more fully the incidence and relative abundance of plasmids cap- tured in our original study (pTRACA10, pTRACA17, pTRACA18, pTRACA20, pTRACA22 and pTRACA30) among individuals of different nationalities, genders or disease status (Fig. 1). For all plasmids a large inter-individual varia- tion in incidence and relative abundance was observed (Fig. 1B inset), but when group data was analyzed, no signicant difference between healthy individuals by nationality or gender was identied, with the exception of relative abundance of harboring genes involved in important activities of the gut microbiota, bacterio- phage may also be a major force behind changes in the species composition of the gut community within an individual. Selective attack on dominant species in the gut microbiota opens new niches for less abundant members, and phage driven population shifts are well documented in other bacterial ecosystems. 108 This aspect of phage-bacteria interaction is of particu- lar signicance in light of recent obser- vations by Lepage et al. (2008), 109 who described distinct differences in abun- dance of bacteriophage between healthy individuals and those suffering from Crohn disease (CD). This raises the pos- sibility that bacteriophage drive the dys- biosis of the gut microbiota observed in CD patients, and that phage driven shifts in community structure may be involved in disease initiation or progression. 109
Alternatively, the observed potential for bacteriophage to translocate from the gut environment and themselves act as anti- gens raises the possibility that phage play a direct role in the pathogenesis of CD, 94
and also ts with their increased preva- lence in CD patients. 109 In both hypoth- eses, the relapsing and remitting nature of CD may correspond to predator-prey population dynamics of relevant phage and their bacterial hosts. Figure 2 (See opposite page). Relative abundance of pTRACA22 type RelBE modules in the METAHIT dataset and variation in infammatory bowel disease. The relative abundance of pTRACA22 type RelBE modules in the gut microbiomes of individuals represented in the METHIT 10 dataset was cal- culated exactly as described previously in reference 22. METHIT data was searched with pTRACA22 RelB and RelE amino acid sequences using tBlastn to identify homologous sequences, and only hits generating e-values of 1 e -5 or lower with a length of 30 aa or over were considered signifcant, and used to calculate hits/Mb. 22 For the purpose of this analysis individuals were designated as healthy unless indicated as being diagnosed with ulcerative colitis (UC) or Crohn disease (CD), regardless of age or body mass index. Statistically signifcant diferences in relative abundance or incidence of RelBE modules within and between metagenomic datasets were explored using the 2 distribution. 22 Where signifcant diferences in relative abundance of RelBE homologues were identifed within or between groups comprised of metagenomes from the METAHIT dataset, the statistical power aforded by the available sample sizes in each group was calculated using the Piface program. 111 This provides the probability that the observed diferences refect the wider population, with a probability of 0.8 generally considered to indicate adequate power. Symbols above bars indicate the level of signifcance in 2 analysis, *p 0.05; **p 0.01; ***p 0.001. Statistical power is shown in adjacent parentheses, and in all cases has been computed to p = 0.05 using variation (standard deviation) observed within the total METAHIT dataset. (A) Shows the relative abundance of pTRACA22 type RelBE homo- logues in the individual gut microbiomes of all 124 individuals represented in the METAHIT dataset. Each bar represents the relative abundance data (Hits/Mb) for RelB (blue) overlaid with that of RelE (red). Purple regions show the least abundant ORF in each individual metagenome, while the color of upper regions indicates the abundance of the most prevalent ORF in each individual metagenome. Individuals in which the RelB anti-toxin is most abundant are indicated by blue upper regions of bars, while those in which the RelE toxin is most abundant are indicated by red upper regions of bars. Individuals in which the level of RelB and RelE are equal are indicated by exclusively purple bars. (B) Shows the percentage of individuals represented in the entire METAHIT dataset with varying ratios of RelB:RelE abundance. Blue = % of individuals in which RelB dominates, red = % of individuals in which RelE dominates, purple = % of individuals with equal relative abundance of both RelB and RelE; white = % of individuals in which neither RelB nor RelE could be detected within the criteria employed in this study. (C) Comparison of overall relative abundance in combined human gut metage- nomes of Japanese and American individuals (utilized in our original analysis), and the METAHIT dataset. 10,19,20 Red bars show relative abundance of RelE homologues and Blue bars show relative abundance of RelB homologues. Am&Jp = American and Japanese gut metagenomes utilized in our original study; 19,20 METAHIT Healthy = Combined gut metagenomes of individuals from the METAHIT dataset without IBD; METAHIT CD = Combined gut metagenomes of individuals from the METAHIT dataset diagnosed with Crohn disease; METAHIT UC = Combined gut metagenomes of individuals from the METAHIT dataset diagnosed with ulcerative colitis; METAHIT TOTAL = Combined gut metagenomes of all 124 individuals represented in the METAHIT dataset. 428 Gut Microbes Volume 1 Issue 6 and disease risk, not only between indi- viduals but also within an individual over time. The gut mobile metagenome is a partic- ularly enigmatic and little explored aspect of the human holobiont. MGE encoding genes required for bacteria to establish symbiotic relationships with metazoan host organisms, as well as MGE conferring a direct benet to the metazoan host, have already been described among the mobile metagenomes of microbial communi- ties colonizing plants, invertebrates and also humans. 24,67-69,71,76-79 Therefore genes important for life in the gastrointestinal tract, as well as activities that impact on host-microbe or microbe-microbe interac- tions, are likely to be encoded by the gut mobile metagenome. Perhaps of more signicance is the over- all capacity of the mobile metagenome to move information both into and out of the gut community. This has profound impli- cations for the human holobiont, in that the prokaryotic mobile metagenome effec- tively links the human hologenome with the wider prokaryotic world. In this view, the hologenome of an individual human may be likened to an island in an ocean of prokaryotic genetic information. The primary eukaryotic core of the human hologenome constitutes the rocky heart of the island which changes only slowly. Our principal prokaryotic metagenome sits at the interface between the island and the ocean forming a beach of relatively stable but ever shifting sands. The ter- tiary mobile metagenome forms the shal- low waters of the surrounding reef which connects to the wider ocean beyond, and through which new genetic material enters the lagoon, washes onto the prokaryotic beach and is distributed along the shore. Acknowledgements I would like to thank Dr. Caroline Jones for valuable discussion and constructive criticism of the manuscript, and Elizabeth Cheek for expert advice regarding sta- tistical analyses of the data presented. Research in the laboratory of Dr. B.V. Jones is currently supported by funding from the Medical Research Council, The Hospital Infection Society, The Society for Applied Microbiology, The Royal Society and the Centre for Biomedical (power = 0.796; Fig. 1C). Although, for functions analyzed here, even when suf- cient samples are available to provide the required statistical power, it is not yet clear if a statistically signicant difference between gut metagenomes from distinct groups equates to any meaningful biologi- cal effect and vice versa. Nonetheless, although far from proven a role for the mobile metagenome in intes- tinal disease cannot yet be precluded, par- ticularly as MGE may also be enriched or reduced by the microbial dysbiosis char- acteristic of IBD (Fig. 1B). These initial ndings are in agreement with numer- ous other studies, including the original METHIT study, 10 showing differences in structure and function of the gut micro- biome in IBD patients and potentially extends the dysbiosis of the gut microbiota observed in IBD to the mobile metage- nome and specically the plasmid popu- lation (Fig. 1B). Such disturbances could lead to alterations in the abundance of MGE and the genes they encode, includ- ing pTRACA22 type RelBE modules, in the gut microbiomes of individuals with IBD and therefore perturb or amplify any effects they may have on the human host. Further research will be required to substantiate or refute these preliminary ndings, establish their relevance to ini- tiation, progression or diagnosis of IBD, and answer the question: Do attributes of, or functions encoded by, the human gut mobile metagenome play any role in the pathogenesis of IBD or other intestinal disorders? Summary If we accept the paradigm of humans as chimeras of both eukaryotic and prokary- otic cells, with the gut microbiota consti- tuting a virtual organ, and forming an integral part of the human (holo)genome, then we must also accept that our knowl- edge of how our bodies function, and the mechanisms of many prevalent diseases will remain obscure until we have also gained an in-depth understanding of our prokaryotic components. The gut micro- biota especially is far more versatile than any of our own tissues (both metaboli- cally and genetically), and undoubtedly contributes greatly to differences in health reduction in both RelB and RelE homo- logues was observed, and analysis of indi- vidual gut metagenomes showed that in 33.33% of this cohort no pTRACA22 type RelBE modules could be detected, compared to only 2.02% of individuals in the healthy group (Fig. 2A). These observations potentially associ- ate pTRACA22 type TA modules with aspects of these diseases (Fig. 2). While several suggested functions of TA mod- ules, particularly their potential toxicity towards eukaryotic cells (Table 1), 105-107
appear to t with an association with IBD, the actual role of these gene systems in the human gut remains unknown, and toxic- ity of RelE has not yet been demonstrated outside articial delivery to, and expres- sion in mammalian cells via specialized vectors. 105-107 Overall, the most probable explanation remains that the observed changes are the result of the well estab- lished population shifts characteristic of CD, rather than a direct involvement in the disease, and it is perhaps not surprising that the mobile metagenome also appears disrupted in individuals diagnosed with these diseases. While these initial ndings are intrigu- ing and indicate that this aspect of the gut metagenome should be subjected to further study, it should also be noted that only a small number of individuals with CD or UC (n = 4, and n = 21 respec- tively) are present in the METAHIT data set. Although signicant differences were observed between the metagenomes analyzed here, power calculations clearly demonstrate that in most cases the small numbers of gut metagenomes from indi- viduals with CD or UC do not provide adequate power (generally accepted to be 0.8 at p = 0.05). Where statistical power is low, the probability that the observed differences are truly reective of the wider population is correspondingly low, and as such any apparently signicant differences should be treated with caution. However, adequate statistical power appears to be available to demonstrate the greater number of individuals in which pTRACA22 type RelBE modules were undetected among the UC group (Fig. 2A); and possibly the greater relative abundance of pTRACA10 in individuals with UC compared to healthy individuals www.landesbioscience.com Gut Microbes 429 Table 1. Proposed or confirmed functions of TA addiction modules and relevance to the human gut microbiota Confirmed or proposed function of TA modules Significance/relevance to human gut microbiome 1 Plasmid stabilization Stabilize vertical transmission of neighbouring DNA segments The stabilization of plasmids though post-segregational killing (PSK) by TA modules should allow the retention of plasmids within the gut microbiome in the absence of direct selective pressure for functions they encode. 24 Chromosomally encoded TA modules may stabilize neigh- bouring gene regions, and ensure vertical transmission of associated genes is maintained even in the absence of direct selective pressure for functions they encode. 95 Together, these stabili- zation effects may serve to retain important functions within the gut microbiome that are not under continual selection. These may be functions required only intermittently, or during key stages of holobiont development, such as transmission of the microbiota to new hosts, or initial formation of the community. Plasmid anti-addiction modules Bacteriophage countermeasure Chromosomally encoded TA modules may function as anti-addiction modules, preventing PSK by plasmid encoded TA systems and destabilising the plasmid within the host bacterial popula- tion. 112 Both plasmid and chromosomally encoded TA systems may also function as phage exclu- sion mechanisms, restricting the capacity of bacteriophage to infect a bacterial population. 113
These attributes could constitute a mechanism which controls the rate of HGT in the gut micro- biome by restricting the range of MGE that may participate in this process. TA modules may also limit the impact of bacteriophage on the gut microbiota, stabilising the community against bacteriophage attacks. This attribute fits well with the observed enrich- ment of pTRACA22 type RelBE modules in the gut microbiome, 22 and is of particular interest in light of the potential role of bacteriophage in dysbiosis of the gut microbiota in CD. 109 Modulation of host gene expression Formation of new regulatory gene networks Specific mechanisms through which plasmids and other MGE alter global gene expression of the bacterial host have recently been described. 45,114 Modulation of gene expression and the for- mation of new regulatory gene networks in bacterial cells have also been linked with TA mod- ules. 95,100-102 These potential functions of TA systems could serve to facilitate rapid adaptation of gut associated bacterial species to the GIT, or changing conditions within this environment. This may be of significance during colonization of pristine gut environments, as well as survival of bacterial cells in the external environment during transmission to new hosts. This feature of TA systems may also allow members of an established gut microbiota to deal with shifts in envi- ronmental conditions, such as changes in host diet. Formation of persister cells and survival of exposure to environmental stresses TA modules have been consistently associated with the formation of persister cells, and survival of environmental stresses. 96,104,105 These include nutrient limitation and exposure to antimicro- bial agents, both of which are likely to be encountered during colonization of the human gut. In addition, the ability to survive under nutrient limiting conditions may also be of benefit to members of an established community. For example the ability to survive under nutrient limit- ing conditions may be important during changes in host diet which reduce the levels of usable nutrients to particular members of the gut community, or during periods of host starvation. In particular, wide variation in diet and periods of starvation were undoubtedly more common in the ancestors of modern humans, and access to a consistent food supply is still a problem for a significant fraction of the human population alive today. Alternatively, the association of per- sister cells with survival of exposure to antimicrobial agents, may indicate a potential role for TA systems in the ability of the gut microbiota to recover following exposure to antibiotics. Biofilm dispersal The recent finding that TA modules may play a role in regulating aspects of biofilm formation is also of relevance to the human gut microbiome. 115 The formation of biofilm communities associ- ated with the gut mucosa, or the surface of indigestible food particles, is generally considered to be an important and consistent behaviour in the gut microbial community, which may be involved in the persistence of microbes in the gut, and also in the pathogenesis of intestinal diseases. 116,117 Control of epithelial cell proliferation Perhaps the most contentious of the proposed functions for RelBE TA modules is the potential for a direct role of these gene systems in host-microbe interaction. 22 This theory stems from the observation that RelE toxins are active in eukaryotic cells via the same mechanism employed to inhibit growth of or kill bacterial cells. 105-107 Importantly, in cultured human cells expression of bacterial RelE leads to cell death by induction of apoptosis. As such these systems may contrib- ute to homeostasis of the intestinal epithelium, as has been documented for other activities of the gut microbiota. However, this activity has to date only been observed using artificial mam- malian gene expression systems, 105-107 and it is not yet known if RelE expressed by microbial cells in the gut microbiota, or in even in model co-culture systems, will recreate these effects. The observed enrichment of the pTRACA22 type RelBE in the human gut microbiome argues for further study of this potential activity of RelBE TA modules. 22 Previously presented functions or activities of TA modules which may be relevant to the human gut microbiota are described, and the significance of these functions to the gut microbiota is considered. All functions of TA that are discussed are based on previous studies which have provided evi- dence for particular roles of TA modules, and relevant literature cited. 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