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Microbiology and Molecular Biology Reviews, September 2007, p. 495-548, Vol. 71, No. 3
1092-2172/07/$08.00+0 doi:10.1128/MMBR.00005-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum
Marco Ventura,1*
Carlos Canchaya,2
Andreas Tauch,3
Govind Chandra,4
Gerald F. Fitzgerald,2
Keith F. Chater,4 and
Douwe van Sinderen2*
Department of Genetics, Biology of Microorganisms, Anthropology, Evolution, University of Parma, Parma, Italy,1 Alimentary Pharmabiotic Centre and Department of Microbiology, National University of Ireland, Cork, Ireland,2 Institute for Genome Research and Systems Biology Center for Biotechnology, Bielefeld University, Universitaetsstrasse 25, D-33615 Bielefeld, Germany,3 Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich, United Kingdom4
SUMMARY INTRODUCTION General Features of Actinobacteria Evolution and Dynamics of Bacterial Genomes Gene duplications. HGT. Gene decay. Genome rearrangements. Taxonomy of Actinobacteria Actinobacterial Genome Sequencing Projects GENOMICS OF BIFIDOBACTERIUM General Features Comparative Bifidobacterial Genome Analysis DNA Regions Acquired by HGT in Bifidobacterial Genomes Prophage-Like Elements in Bifidobacteria Extrachromosomal DNA Elements Bifidobacteria and Carbohydrate Metabolism Bifidobacteria and Prebiotic Properties Interaction of Bifidobacteria with the GIT GENOMICS OF TROPHERYMA General Features Tropheryma Comparative Genome Analysis DNA Region Acquired by HGT in T. whipplei Genomes Tropheryma Genome and Biological Lifestyle Interaction of Tropheryma with the Environment GENOMICS OF PROPIONIBACTERIUM General Features Extrachromosomal DNA Elements in Propionibacterium DNA Region Acquired by HGT Prophage-Like Elements in Propionibacterium P. acnes Genome and Biological Lifestyle Interaction of P. acnes with Its Environment GENOMICS OF MYCOBACTERIUM General Features Genomics of M. tuberculosis M. tuberculosis genome architecture. M. tuberculosis genome and biological lifestyle. Comparative genomics within the M. tuberculosis complex. Prophage-like elements in M. tuberculosis. Genomics of M. bovis M. bovis genome architecture. M. bovis genome and biological lifestyle. Comparative Genomics of M. bovis and M. tuberculosis Genomics of M. leprae Genomics of M. avium subsp. paratuberculosis Extrachromosomal DNA Elements in Mycobacterium GENOMICS OF NOCARDIA General Features Nocardia Comparative Genome Analysis Extrachromosomal DNA Elements in Nocardia N. farcinica Genome and Biological Lifestyle GENOMICS OF CORYNEBACTERIUM General Features Corynebacterium Genome Architecture Corynebacterium Comparative Genome Analysis DNA Regions in C. glutamicum Acquired by HGT Prophage-Like Elements in the C. glutamicum Genome DNA Acquired by HGT in the C. efficiens Genome Prophage-Like Element in the Genome of C. diphtheriae DNA Regions in the C. diphtheriae Genome Acquired by HGT DNA Regions in the C. jeikeium Genome Acquired by HGT Extrachromosomal DNA Elements Corynebacterial Genomes and Biological Lifestyle Adherence to pharyngeal epithelial cells by C. diphtheriae. Adaptation to amino acid production by C. glutamicum and C. efficiens. Adaptation to elevated temperatures by C. efficiens. Adaptation to the lipophilic lifestyle by C. jeikeium. GENOMICS OF LEIFSONIA General Features Extrachromosomal DNA Elements in Leifsonia DNA Regions Acquired by HGT in Leifsonia Prophage-Like Elements in Leifsonia L. xyli subsp. xyli Genome and Biological Lifestyle GENOMICS OF THE MYCELIAL ACTINOBACTERIA: STREPTOMYCES, FRANKIA, AND THERMOBIFIDA General Features Architecture of Mycelial Actinobacterial Genomes Comparative Genomics of Mycelial Actinobacterial Genomes Multiply represented metabolic genes. Genes unexpectedly missing from mycelial Actinobacteria. Conservons and transposons. DNA Regions in Mycelial Actinobacterial Genomes Acquired by HGT Streptomyces Extrachromosomal Elements Prophage-Like Elements in Streptomyces Mycelial Actinobacterial Genomes and Biological Lifestyle Ecology. Secondary metabolism. P450 cytochromes (CYPs). Development. Specialized use of the rare UUA leucine codon. COMPARATIVE GENOMICS OF ACTINOBACTERIA Synteny of Actinobacterial Genomes Actinobacterial Core Genome Sequences: Phylogenomics IMPACT OF ACTINOBACTERIAL GENOMICS ON TAXONOMY New Approaches to Investigate Taxonomic Relationships in Actinobacteria Based on Whole-Genome Sequences Actinobacterial Taxonomy Based on Multilocus Approach CONCLUSIONS ACKNOWLEDGMENTS REFERENCES
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Summary: Actinobacteria constitute one of the largest phyla among Bacteria and represent gram-positive bacteria with a high G+C content in their DNA. This bacterial group includes microorganisms exhibiting a wide spectrum of morphologies, from coccoid to fragmenting hyphal forms, as well as possessing highly variable physiological and metabolic properties. Furthermore, Actinobacteria members have adopted different lifestyles, and can be pathogens (e.g., Corynebacterium, Mycobacterium, Nocardia, Tropheryma, and Propionibacterium), soil inhabitants (Streptomyces), plant commensals (Leifsonia), or gastrointestinal commensals (Bifidobacterium). The divergence of Actinobacteria from other bacteria is ancient, making it impossible to identify the phylogenetically closest bacterial group to Actinobacteria. Genome sequence analysis has revolutionized every aspect of bacterial biology by enhancing the understanding of the genetics, physiology, and evolutionary development of bacteria. Various actinobacterial genomes have been sequenced, revealing a wide genomic heterogeneity probably as a reflection of their biodiversity. This review provides an account of the recent explosion of actinobacterial genomics data and an attempt to place this in a biological and evolutionary context.
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In terms of number and variety of identified species, the phylum Actinobacteria represents one of the largest taxonomic units among the 18 major lineages currently recognized within the domain Bacteria (406), including 5 subclasses and 14 suborders (404). It comprises gram-positive bacteria with a high G+C content in their DNA, ranging from 51% in some corynebacteria to more than 70% in Streptomyces and Frankia. An exception to this is the genome of the obligate pathogen Tropheryma whipplei, with less than 50% G+C.
Actinobacteria exhibit a wide variety of morphologies, from coccoid (Micrococcus) or rod-coccoid (e.g., Arthrobacter) to fragmenting hyphal forms (e.g., Nocardia spp.) or permanent and highly differentiated branched mycelium (e.g., Streptomyces spp.) (15). They also exhibit diverse physiological and metabolic properties, such as the production of extracellular enzymes and the formation of a wide variety of secondary metabolites (389). Notably, many such secondary metabolites are potent antibiotics (255), a trait that has turned Streptomyces species into the primary antibiotic-producing organisms exploited by the pharmaceutical industry (29). Furthermore, various different lifestyles are encountered among Actinobacteria, and the phylum includes pathogens (e.g., Mycobacterium spp., Nocardia spp., Tropheryma spp., Corynebacterium spp., and Propionibacterium spp.), soil inhabitants (Streptomyces spp.), plant commensals (Leifsonia spp.), nitrogen-fixing symbionts (Frankia), and gastrointestinal tract (GIT) inhabitants (Bifidobacterium spp.). Unusual developmental features are displayed by many actinobacterial genera, such as formation of sporulating aerial mycelium in Streptomyces species or the persistent nonreplicating state exhibited by certain mycobacteria. Actinobacteria are widely distributed in both terrestrial and aquatic (including marine) ecosystems, especially in soil, where they play a crucial role in the recycling of refractory biomaterials by decomposition and humus formation (152, 403). Furthermore, many bifidobacteria are used as active ingredients in a variety of so-called functional foods due to their perceived health-promoting or probiotic properties, such as protection against pathogens mediated through the process of competitive exclusion, bile salt hydrolase activity, immune modulation, and the ability to adhere to mucus or the intestinal epithelium (273, 329, 407).
The actinobacterial genomes sequenced so far belong to organisms relevant to human and veterinary medicine, biotechnology, and ecology, and the observed genomic heterogeneity is assumed to be a reflection of their biodiversity. This review will give an account of the recent explosion of actinobacterial genomics data and will place this in a biological and evolutionary context.
The principal genetic events that determine genome shape and structure are believed to be gene duplication, horizontal gene transfer (HGT), gene loss, and chromosomal rearrangements. Despite efforts to quantify the relative contribution of each of these processes, no reliable model can yet explain and trace the evolutionary development of bacteria based on their current genome structure (8, 183, 243, 398).
It was previously thought that bacterial genomes have evolved from a much smaller ancestral genome through numerous gene duplication events and the consequent generation of paralogs (244). However, an analysis based on the currently available bacterial genome data does not support this theory and shows that gene duplications contribute only modestly to genome evolution (79). Despite this, it has been noted that genes involved in a specific adaptation have been preserved after duplications, suggesting that gene duplication does have an evolutionary role (79). This is nicely illustrated by the mycobacterial paranome, which largely corresponds to a functional class of genes involved in fatty acid metabolism, in agreement with the complex nature of the mycobacterial cell wall and probably reflecting adaptive evolution of this cellular structure (79, 432).
The introduction of novel or alien genes by HGT allows for rapid niche-specific adaptation, which in turn may lead to bacterial diversification and speciation (80). Bacterial genome evolution is based on the combined outcome of genes acquired through cell division, i.e., vertically inherited, and by HGT (482). Taking this concept to its extreme, one can claim that two bacterial taxa are more related to each other than to a third one not because they share a more recent ancestor but because they exchange genes more frequently (151). HGT is held responsible for enhancing the competitiveness of bacteria in their natural environments. For example, in some pathogenic bacteria, segments of DNA containing many virulence genes and gene clusters, called pathogenicity islands, appear to have been acquired by HGT (321). Actinobacterial examples of transmission of virulence genes through HGT are rare (376). Of these, the following three cases appear to represent obvious HGT events: (i) phages of Corynebacterium diphtheriae carry the major diphtheria toxin gene, (ii) a linear plasmid carries the genes for the macrolide toxin responsible for the ulceration that gives Mycobacterium ulcerans its name (411), and (iii) a large segment of the chromosome of Streptomyces turgidiscabies concerned with causing potato scab can be transferred by conjugation (280). In addition, it has been argued that the Mycobacterium tuberculosis Rv0986-8 virulence operon, which plays an important role in parasitism of host phagocytic cells by increasing the ecological fitness of the infecting mycobacterium (339), was acquired horizontally by the ancestor of M. tuberculosis, Mycobacterium prototuberculosis. Other genetic studies of the ancestral M. prototuberculosis species have indicated that various HGT events occurred before the evolutionary bottleneck that led to the emergence of the M. tuberculosis complex (167), probably from the Indian subcontinent (124).
Bioinformatic methods to identify HGT events are based principally on the analysis of divergence in the G+C content (GC deviation), dinucleotide differences, four-letter genomic signatures, and/or codon usage, though geneticists would often be satisfied with HGT as the explanation for genes found in only one organism. If the latter is correct, it would mean that HGT frequency is rather low (below 10% of the total gene complement) (243, 398). Interestingly, a recent analysis showed that many of the proteins that appeared to be specific for actinobacteria are also encoded by the genome of an alphaproteobacterium, Magnetospirillum magnetotacticum, but not by any other sequenced alphaproteobacterial genome, leading to the proposal that M. magnetotacticum acquired these genes by HGT from actinobacterial species (137).
Two other interesting cases of HGT between Chlamydia and a subset of Actinobacteria (e.g., Streptomyces, Tropheryma, Bifidobacterium, Leifsonia, Arthrobacter, and Brevibacterium) have recently been described (158). In the enzyme serine hydroxymethyltransferase (GlyA protein), two conserved inserts of 3 and 31 amino acids (aa) are present in various chlamydiae as well as the above-mentioned subset of Actinobacteria. Similarly, these bacteria contain a conserved 16-amino-acid insert in the peptidoglycan biosynthesis enzyme UDP-N-acetylglucosamine enolpyruvyl transferases (MurA). The functional and physiological significance of these apparent HGT events between chlamydiae and Actinobacteria is presently unclear.
Bacterial genome size is determined by the outcome of several opposing forces. Deletion bias and genetic drift cause genomes to contract, while selection on gene function promotes genomes to preserve DNA. Genome increments depend on both gene duplications and acquisition of alien DNA, coupled to adaptive benefits (293). DNA loss may range from large deletions that span multiple loci to deletions of one or a few nucleotides (7). The influence of these different routes is variable among bacterial lineages (293). Inactivating and deleterious mutations in genes with little contribution to fitness can be transmitted to progeny and accumulate in populations, eventually leading to gene loss; whereas such mutations in genes that are critical will prevent the production of progeny and so will be eliminated from populations, resulting in the preservation of the functional gene (320).
Gene inactivation and loss are particularly apparent in several bacterial groups with a host-associated lifestyle, in which the host supplies many of the metabolic intermediates, thereby obviating the need to maintain many biosynthetic genes. In endosymbiotic bacteria, such as Buchnera and Rickettsia, loss of individual loci or operons is the only source of divergence in the gene inventories between species (289, 419). A clear example of genome degradation is provided by Mycobacterium leprae, which has discarded more than 1,000 genes compared with M. tuberculosis (84). Moreover, the presence of an even larger set of nonfunctional genes, i.e., pseudogenes, in M. leprae indicates that this genome contraction is still in progress. Although the criteria for identifying pseudogenes differ among studies, the overall rationale is identical: the predicted protein must be altered to a degree that abolishes its function. The thresholds applied for pseudogene identification are based on the known size and organization of functional domains within proteins, the observed length variation within individual gene families, and available information on experimentally disrupted proteins (320). Generally, pseudogenes include cases in which a stop codon or deletion has resulted in an encoded protein that is less than 80% of the length of its functional counterpart in the contrasted genome and cases in which a frameshift or insertion has altered more than 20% of the amino acid sequence (263). Most of the pseudogenes so far annotated in bacterial genomes are among the open reading frames (ORFs) whose functions are unknown. The lack of pseudogenes shared among multiple strains of the same species suggests that pseudogenes are generated continually, are eliminated rapidly, and thus only rarely persist in bacterial genomes (320). Other bacteria show a lower level of gene loss: in the obligate intracellular pathogen Rickettsia prowazekii only 76% of the potential coding capacity is used, while just 12 pseudogenes were identified (9); and a recent genome analysis of two Streptococcus thermophilus strains (33) found that 10% of the genes were pseudogenes, perhaps reflecting adaptation of S. thermophilus to its specialized environment, milk (33).
When all bacterial genomes are compared with each other, a set of only 50 to 100 genes, which are called the core genome sequences, appear to be maintained universally (for a review, see reference 147).
Apart from the events described in the previous sections that affect gene content, the organization of a genome is subject to change through chromosome rearrangements. Synteny, a term used here to indicate the conservation of gene order between genomes, can be applied as a phylogenetic tool to investigate relationships between species, since the degree of genome rearrangements increases linearly in relation to the time of divergence of bacterial taxa (236, 484).
Chromosomal rearrangements are largely dependent on the activity of repeated and mobile elements such as insertion sequences (ISs), transposons, prophage sequences, and plasmids (233). Bacterial genomes containing a higher repeat density have higher rates of rearrangements, leading to an accelerated loss of gene order (371). Homologous recombination events between such repeat sequences catalyze both gene rearrangement and gene loss in the genome, thus leading to diversification of taxa. Such recombination events may have promoted speciation in the T. whipplei taxon (357). Furthermore, chromosome evolution is influenced by large chromosomal rearrangements, e.g., large inversions, roughly symmetrically centered around the replication origin, which lead to the occurrence of X-shaped patterns in the alignments of whole genomes (117).
Actinobacteria include many organisms that exhibit, or have a tendency towards, mycelial growth. 16S rRNA gene sequencing has led to the recognition of 39 families and 130 genera, which also include high-G+C gram-positive bacteria with simpler morphology, such as bifidobacteria and micrococci (Fig. 1) (119). The deepest branch separates bifidobacteria from all other known families. The divergence of actinobacteria from other bacteria is so ancient that it is not possible to identify the phylogenetically closest bacterial group to Actinobacteria with confidence (119).
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FIG. 1. Phylogenetic tree of Actinobacteria based on 1,500 nucleotides of 16S rRNA. Scale bar, 5 nucleotides. Families containing members subjected to complete genome sequencing at the time of this writing are depicted in bold. Orders are indicated.
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The first actinobacterial genome to be sequenced was that of the paradigm strain of the human tuberculosis agent, M. tuberculosis H37Rv (83). In the last few years, genomes of 20 different Actinobacteria (in some cases multiple strains of the same species) have been sequenced to completion (Table 1), while sequencing of genomes from representatives of 43 other high-G+C bacteria are still in progress (Table 1) (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi).
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TABLE 1. Published data for actinobacterial genomes
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Below we examine relevant genomic information from some of the best-known actinobacterial taxa (Bifidobacterium, Mycobacterium, Streptomyces, Corynebacterium, Thermobifida, Leifsonia, Frankia, Nocardia, Propionibacterium, and Tropheryma), partially in the light of what is known for Escherichia coli or Bacillus subtilis, as paradigms of gram-negative proteobacteria and gram-positive low-G+C-content bacteria, respectively. We discuss how genomic information can be used to gain insights into the physiology, genetics, and evolution of Actinobacteria.
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The Bifidobacteriaceae family comprises four genera, Bifidobacterium, Gardnerella, Scardovia, and Parascardovia (404), of which only the first contains more than one species. Bifidobacteria form a deep-branching lineage within the Actinobacteria (136, 137). The Bifidobacterium genus contains six phylogenetic clusters, named B. boum, B. asteroides, B. adolescentis, B. longum, B. pullorum, and B. pseudolongum (448).
Bifidobacteria are nonmotile, nonsporulating, non-gas-producing, anaerobic, and saccharoclastic bacteria. They have been isolated from five different, though somewhat connected, ecological niches: the intestine, the oral cavity, food, the insect gut, and sewage. Those that inhabit the GIT (e.g., B. breve, B. longum biotype longum, and B. longum biotype infantis) have been the subject of growing interest due to their probiotic properties. Bifidobacteria ferment a large variety of oligosaccharides in the GIT, some of which, in particular those that are not digested by their host, are commercially exploited to enhance bifidobacterial numbers (as well as other probiotic bacteria) in situ, a practice that is referred to as the prebiotic concept (146).
Of the currently recognized 29 Bifidobacterium species, three strains that belong to the B. longum and B. adolescentis phylogenetic groups have been sequenced to completion (Table 2), while the sequences of others, e.g., B. dentium Bd1, are at various stages of completion: detailed sequence information for some of these genomes is expected to become publicly available in the near future. Furthermore, genome sequencing of B. breve M-16V, B. breve Yacult, B. animalis subsp. lactis, B. longum biotype longum, and B. longum biotype infantis (276) is under way. These genomes range in size from 1.9 to 2.9 Mb and generally display architectural features of a typical bacterial chromosome. Some of these are the co-orientation of gene transcription and DNA replication (288); a G-rich, C-poor bias in the nucleotide composition of the leading DNA strand (129); and a typical presumptive origin-of-replication region (350), including a gene constellation near the origin (comprising rpmH, dnaA, dnaN, and recF), a particular GC nucleotide skew ([G-C]/[G+C]), and the presence of multiple DnaA boxes and AT-rich sequences immediately upstream of the dnaA gene (77).
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TABLE 2. General features of bifidobacterial genomes
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Dot plot comparisons (at the nucleotide level) of the fully sequenced bifidobacterial genomes revealed a high degree of conservation and synteny across the entire genomes., i.e., those of B. longum biotype longum NCC2705, B. longum biotype longum DJO10A, B. breve UCC2003, and B. adolescentis ACC15703. Preliminary analysis against the draft genome sequences of B. dentium Bd1 confirmed and extended this result. However, there are also several breakpoint regions that seem to represent inversions or DNA insertion/deletion points (S. Leahy and D. Van Sinderen, unpublished data).
Recently, a B. longum biotype longum NCC2705-based spotted DNA microarray was employed to compare the genomes of 10 bifidobacterial strains, including other B. longum biotype longum strains as well as the closely related B. longum biotype infantis and B. longum biotype suis taxa (232). Results revealed seven large genome regions of variability, the majority of which encompass DNA with a deviating G+C content. These regions correspond to a prophage remnant; a cluster of genes for enzymes involved in sugar metabolism, such as an 
-mannosidase; and a capsular polysaccharide biosynthesis gene cluster, which could play a role in host-bacterium interactions (see Fig. S1 in the supplemental material). Though very useful, microarray-based comparative genome analyses suffer from some limitations. It is not possible to identify regions present in the test strains but absent from the strain that was used to construct the array, and it will generally not allow synteny studies.
It has been suggested that selected genes involved in sugar metabolism as well as in the production of exopolysaccharides in B. longum biotype longum NCC2705 have been acquired via HGT, as part of the adaptation of this organism to a specific ecological niche. For example, a region encoding rhamnosyl transferases seems to have been acquired from streptococci (384), while two other regions that contain genes encoding restriction-modification systems also appear to have been acquired through HGT. Overall, about 5% of the B. longum biotype longum NCC2705 genome content seems to have been recently acquired by this mechanism (384).
Until recently bifidobacteria were not considered to be suitable targets for phage infection. However, prophage-like elements, designated Bbr-1, Bl-1, and Blj-1, are present in the genomes of B. breve UCC2003, B. longum biotype longum NCC2705, and B. longum biotype longum DJO10A (455). These prophage-like elements display homology to genes of double-stranded DNA phages that infect a broad phylogenetic range of bacteria. Surprisingly, using the proteomic tree method to investigate the evolution of these phages (373), it became clear that the Bbr-1, Bl-1, and Blj-1 prophage-like elements exhibit a close phylogenetic relationship with phages infecting low-G+C bacteria (e.g., lactococcal and staphylococcal phages) (455), perhaps because these bacteria and their phages have shared the same ecological niche (i.e., the animal GIT) during their evolution, thereby allowing DNA exchange. This may therefore point to DNA transfer events between low- and high-G+C bacteria. Perhaps such phages originally infected the ancestor of high-G+C gram-positive bacteria, in line with the concept that high-G+C gram-positive bacteria originated from low-G+C ancestors (455). The unfinished B. dentium Bd1 genome contains at least two prophage-like elements, one of which resembled that of the NCC2705 Bl-1 prophage (Fig. 2). Notably, all three published bifidobacterial prophage-like elements are integrated in a tRNAMet gene, which is the first case of this tRNA gene as a target for phage integration in any gram-positive or gram-negative bacterium (56). Analysis of the distribution of this integration site revealed that the attB sites are well conserved in many bifidobacterial species and in a phylogenetically unrelated bacterium, Thermosynechococcus elongatus BP-1 (306), but surprisingly not in other sequenced Actinobacteria.
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FIG. 2. Comparative genome maps of the prophage-like elements detected in Bifidobacterium genomes. Genes sharing similarity are linked. Probable functions of encoded proteins identified by bioinformatic analysis are indicated. The modular structure is color coded: red, lysogeny; green, DNA packaging and head; blue, tail; mauve, tail fiber; violet, lysis module; yellow, transcriptional regulator; orange, DNA replication; gray, unknown genes; black, genes similar to other functionally unknown bacteriophage genes. Vertical blue lines, tRNA genes.
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The Bbr-1 and Bl-1 prophage-like elements appear to be defective prophages, although they may constitute functional satellite phages, whose mobility depends on helper phages in a manner similar to that described for the cryptic mycophages Rv1 and Rv2 (175, 455).
Plasmids are not ubiquitous in bifidobacteria (332, 392), and when present they are small, i.e., ranging from 1.5 kb to 15 kb. Completely sequenced plasmids from different B. longum biotype longum strains include pMB1 (378); pKJ36 and pKJ50 (332, 333); pBLO1 (384); pNAC1, pNAC2, and pNAC3 (95); pDOJH10S and pDOJH10L (260); pTB6 (421); pB44 (GenBank accession number NC004443); and pNAL8 (162). In addition, six plasmids from other bifidobacterial species have been sequenced: pVS809 from Bifidobacterium globosum (285), pCIBb1 from B. breve (327), pNBb1 from B. breve (GenBank accession number E17316), pAP1 from B. asteroides (GenBank accession number Y11549), pBC1 from Bifidobacterium catenulatum (5), and p4M from Bifidobacterium pseudocatenulatum (GenBank accession number NC003527). These plasmids do not encode any obvious phenotypic trait, except for the plasmid isolated from B. bifidum NCFB 1454 (492), which was proposed to encode a bacteriocin, bifidocin B.
Most of the plasmids contain characteristic genetic features for plasmid replication via a rolling-circle replication system, i.e., repB, traA, and mob genes. In contrast, pDOJH10S from B. longum biotype longum DJO10A and pBC1 from B. catenulatum contain sequences homologous to replication functions of theta-type replicating plasmids (5, 260).
Rep proteins from different bifidobacterial plasmids do not cluster together phylogenetically (110) but resemble replication proteins from different hosts, including gram-negative bacteria such as E. coli (5, 260) (Fig. 3). Horizontal transfer is also indicated for pDOJH10S, which may have been acquired from another Actinobacteria member, possibly Rhodococcus rhodochrous (260).
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FIG. 3. Phylogenetic relationships of Rep proteins from actinobacterial plasmids and several prototype plasmids of different plasmid families from gram-positive and gram-negative bacteria. The phylogenetic tree was calculated by the sequence distance method using the neighbor-joining algorithm.
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Mammalian (including human) biology is partially shaped by the vast community of commensal bacteria that colonize the GIT. Plant-based foods that are commonly consumed by mammals are rich in complex polysaccharides that contain, among others, glucose, fructose, xylan, pectin, and arabinose moieties. Mammalian genomes do not appear to encode the enzymes necessary for degrading most of these glycans (CAZy database; see below), which are supplied instead by the distal GIT microbiome (16). The human GIT microbiome is enriched in genes involved in metabolism of sugars, including glucose, galactose, fructose, arabinose, mannose, and xylose, as well as other sugars that escape digestion by the host's enzymes, including many prebiotic compounds, such as fructooligosaccharides, galactooligosaccharides, glucooligosaccharides, xylooligosaccharides, lactulose, and raffinose (for reviews, see references 148, 161). Gill et al. (148) describe more than 81 different glycoside hydrolase families distributed in a mixture of anaerobic bacteria, i.e., the GIT microbiome, which includes Bifidobacteriales, Clostridiales, Bacteroidales, Enterobacteriales, Fusobacterales, Thermoanaerobacteriales, and Methanobacteria (148, 447. Many of these enzymes are not represented in the human glycobiome. Moreover, GIT mucus provides an abundant reservoir of glycans for microbiota, which serve to reduce the effects of marked changes in the availability of dietary polysaccharides (16).
The type of sugar available is likely to influence the species composition and abundance of the microbiota along the GIT (447). In this context, bacteria such as Lactobacillus are particularly prevalent in the upper GIT, where they mainly ferment relatively simple mono-, di-, and trisaccharides (447). In contrast, bacteria active in the lower parts of the colon, such as bifidobacteria, probably owe their specific ecological success to their capacity to metabolize complex carbohydrates. It therefore comes as no surprise that genes for complex sugar metabolism abound in the genomes of B. breve and B. longum biotype longum. According to the sequence-based classification of carbohydrate-active enzymes (CAZy), over 8% of the annotated genes of these bifidobacterial genomes may encode enzymes involved in the metabolism of carbohydrates, including various glycosyl hydrolases for utilization of diverse, but in most cases not identified, plant-derived dietary fiber or complex carbohydrate structures. Relatively few of these glycosyl hydrolases are predicted to be secreted, including those that are thought to hydrolyze arabinogalactans and arabinoxylans (384). Instead, most of the bifidobacterial glycosyl hydrolases are predicted to be intracellular, and the genes that encode them are almost without exception associated with genes predicted to encode systems for the uptake of structurally diverse carbohydrate substrates (see below). Moreover, carbohydrate-modifying enzymes may also shape the overall metabolic state of the colon to sustain a microbiota that indirectly provides the host with about 10 to 15% of its calories from the degradation of complex carbohydrates through short-chain fatty acids (447).
Bifidobacteria can also utilize sialic acid-containing complex carbohydrates in mucin, glycosphingolipids, and human milk (187, 465). Thus, the mammalian host supplies substrates for intestinal commensals such as bifidobacteria and lactobacilli, in a remarkable symbiotic (or altruistic) relationship (94, 308). Starch and amylopectin are other examples of polysaccharides which may escape digestion in the upper human GIT and which are plant-derived high-molecular-weight carbohydrates. The ability to degrade these sugars appears to be restricted to certain species or to certain strains of a particular species, including B. breve and B. adolescentis (379).
Nearly 10% of the total bifidobacterial gene content is dedicated to sugar internalization, via ABC transporters, permeases, and proton symporters rather than phosphoenolpyruvate-phosphotransferase systems (PEP-PTSs) (384), though a PEP-PTS has been experimentally demonstrated in B. breve to be active for the internalization of glucose (108). The PTS acts through the concomitant internalization and phosphorylation of carbohydrates, in which the transfer of phosphate from PEP to the incoming sugar is mediated via a phosphorylation chain involving enzyme I (EI), histidine-containing protein (HPr), and EII. The B. longum biotype longum NCC2705 genome has a single EII-encoding locus, and that of B. breve UCC2003 has four (287). The latter system was shown to transport fructose, but it appears to transport glucose as well (287). This difference in the number of PEP-PTSs may indicate that B. breve more frequently encounters less complex sugars in its preferred niche, the GITs of infants, than B. longum biotype longum encounters in the GITs of adults, where it is prevalent. Thus, the different diets of infants and adults may affect the compositions of their GIT microbiomes.
Prebiotics, such as fructo- and galactooligosaccharides, are indigestible food ingredients that beneficially affect the host by selectively stimulating growth of commensal bacteria (36, 370). Bifidobacterial genomes have a rich arsenal of genes for such prebiotic metabolism (116, 176, 218, 259). For example, B. breve UCC2003 has a fos operon encompassing a putative permease-encoding gene, a gene specifying an unknown protein, and a ß-fructofuranosidase gene, which has been shown to be involved in fructooligosaccharide degradation (380). Prebiotic oligosaccharides are also provided in human milk (467). These include galactooligosaccharides (325) ranging in degree of polymerization from 3 to over 32 galactose moieties (247). Certain bifidobacteria can also hydrolyze high-molecular-weight prebiotic carbon sources, such as trans-galactooligosaccharides, the latter through an extracellular enzyme encoded by galA (176).
The molecular basis of interactions with the host epithelium has been investigated in detail for several pathogens such as Listeria monocytogenes and Salmonella spp. (256, 291), but little is known about this for commensal bacteria such as bifidobacteria. Bifidobacterial genome analyses did not reveal clear candidate genes for GIT-bifidobacterial interaction. However, bifidobacteria are predicted to encode cell envelope-associated structures which may play a role in host association. All sequenced bifidobacteria appear to encode an extracellular polysaccharide (EPS) or capsular polysaccharide, and such an extracellular structure may be important in bacterial adherence to host cells, while it could also contribute to resistance to stomach acids and bile salts (338).
The various different EPS clusters present in the commensal microorganism Bacteroides tetaiotamicron help to avoid immune recognition by the host (240). The B. longum biotype longum NCC2705 genome has two regions related to polysaccharide biosynthesis that, like the cps/eps cluster of B. breve UCC2003, are flanked by IS elements and show a strong divergence in G+C content relative to the remainder of the genome. These appear to be a genetic hallmark of cps/eps loci examined thus far (127) and may facilitate inter- and intraspecies transfer of such gene clusters.
Genes predicted to encode glycoprotein-binding fimbria-like structures, which have been identified in the genome sequences of both B. longum biotype longum NCC2705 and B. longum biotype longum DJO10A, may mediate another interaction with the host (232, 384). In addition, B. longum biotype longum NCC2705 encodes a serpin-like protease inhibitor that has been demonstrated to contribute to host interaction in the GIT (199). The NCC2705 serpin is an efficient inhibitor of human neutrophil and pancreatic elastases, whose release by activated neutrophils at the sites of intestinal inflammation represents an interesting mechanism of innate immunity (199).
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The only sequenced member of the genus Tropheryma is T. whipplei, the causative agent of Whipple's disease, which is characterized by intestinal malabsorption leading to cachexia and death. T. whipplei isolates are typically found in human intracellular niches, such as inside intestinal macrophages and circulating monocytes (355, 356). However, extracellular and metabolically active T. whipplei cells have been found in the intestinal lumen (130). An environmental reservoir of T. whipplei is also suspected, as PCR experiments revealed its presence in sewage water (283).
Phylogenetic analyses based on the 16S rRNA, 5S rRNA, 23S rRNA, groEL, and rpoB genes placed T. whipplei within the phylum Actinobacteria (282, 479). T. whipplei was difficult to propagate until relatively recently, when cultivation methods using human fibroblasts were established (354). Two T. whipplei strains, TW08/27 and Twist, have been fully sequenced (27, 357). Both strains have a small genome (less than 1 Mb) (Table 1) bearing the traits of strictly host-adapted microorganisms, which include pronounced deficiencies in energy metabolism, dependence on external amino acids, and a lower G+C content (i.e., 46%) than free-living relatives (302).
A large amount of coding capacity is devoted to the biosynthesis of surface-associated features that may sustain the intricate interaction with eukaryotic cells. These surface features include a prominent family of predicted surface proteins termed WiSP (Wnt-induced secreted protein), ranging in size from 103 to 2,308 aa residues. Only a few WiSP family members contain a predicted transmembrane motif near the C terminus that can anchor such proteins to the bacterial membrane. Alignment of all the WiSP members revealed the presence of a single ß-strand motif (27).
The two T. whipplei genomes contain many noncoding repetitive DNA regions, which may promote recombination events that allow the bacteria to expose different sets of proteins at their surface, possibly in response to host defense actions and/or specific environmental conditions (27, 357). All these genome characteristics are discussed in detail below. It is noteworthy that the genomes of T. whipplei Twist and TW08/27 contain 808 and 784 coding sequences (CDSs), respectively, with only a small number of pseudogenes. This apparent low degree of gene decay, which contrasts with the conspicuous gene decay of M. leprae (see below), may be related to the complete absence of mobile genetic elements within the genomes, or it may mean that most redundant DNA has already been removed.
The two available T. whipplei genomic sequences are >99% identical but differ by a large chromosomal inversion (Fig. 4). The extremities of this inversion include two identical nucleotide sequences corresponding to the WND domain of the WiSPs. Consequently, the inversion event caused differences in the WiSPs in the two strains: TW08/27 has eight copies of WND domain sequences that are identical across an 800-bp nucleotide span, whereas the rest of these WiSP genes do not display any DNA similarity. This suggests that WND motifs act both as coding regions and as DNA repeats to promote genome recombination (357).
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FIG. 4. Circular map of genome diversity found in Tropheryma. From inside to outside: ring 1, GC deviation; ring 2, G+C content; ring 3, atlas of T. whipplei strain TW08/27; ring 4, comparison to the genome sequences of T. whipplei Twist. Green indicates homologies of >95%. The synteny plot comparing the order of homologous genes in sequenced genomes of Tropheryma is depicted in the panel inside the circular map.
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HGT events appear to be less frequent for intracellular bacteria with small genomes than for free-living bacteria (27, 40, 321, 357). In T. whipplei Twist, only nine genes, about 1% of the entire genome, appear to have been acquired by HGT. These encompass aminoacyl-tRNA synthetase-encoding genes, genes involved in nucleotide metabolism (purB and pyrB), and genes specifying hypothetical proteins (357).
Possibly, the comparatively nonpromiscuous lifestyles of intracellular bacteria do not offer extensive opportunities to exchange DNA with other bacteria. The absence of mobile elements is also consistent with the notion that T. whipplei resides in an isolated niche, being sheltered from foreign bacterial DNA.
Metabolic reconstruction of T. whipplei from genome data indicates the absence of biosynthetic pathways for arginine, tryptophan, and histidine biosynthesis and incomplete pathways for the synthesis of glycine, serine, leucine, and cysteine. There are also deficiencies in cofactor biosynthesis, energy metabolism, and carbohydrate metabolism. The absence of genes required for prototrophic growth is another indicator of the reliance of these microorganisms on their host for various compounds. Nevertheless, among intracellular microorganisms with reduced genomes, T. whipplei has the most complete biosynthetic pathways for purine and pyrimidine nucleotides, fatty acids, several cofactors, and other small biomolecules. Like Buchnera, T. whipplei genomes have maintained about half of the amino acid biosynthetic pathways. In Buchnera the retained metabolic pathways correspond to those necessary for the synthesis of amino acids essential for their insect hosts. However, since no such symbiotic association is known for T. whipplei, its retained biosynthetic capacity is probably a reflection of the amino acids that are in limited supply in its natural environment or host. All this genome-acquired knowledge allowed the formulation, through computer modeling of the T. whipplei metabolic networks, of a comprehensive culture medium that supported axenic growth of this organism (366). Acquisition of iron is crucially important for bacterial pathogens in the iron-depleted host environment. The genome survey of T. whipplei identified a gene cluster predicted to be involved in ferri-siderophore uptake. However, apart from the presence of a homolog of the gene for the mycobacterial iron dependent regulatory protein IdeR, no genes with clear homology to known siderophore genes have been identified, indicating that T. whipplei might only be able to scavenge xenosiderophores (27).
Almost 15% of the T. whipplei predicted proteins and 74% of the hypothetical proteins are expected to be exported from the cell or associated with the cell envelope. The assignment of so many extracellular proteins may be a reflection of the importance of host interactions to the organism. The T. whipplei WiSPs (see above) resemble proteins known to be involved in pathogenesis and host-immune evasion, such as the Staphylococcus aureus Bap protein, a biofilm-associated protein that projects from the cell surface, allowing interactions with adjacent surfaces (100).
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Currently, only one complete propionibacterial genome sequence, that of Propionibacterium acnes strain KPA171202, is publicly available (46). P. acnes is a non-spore-forming, anaerobic, pleomorphic rod whose end products of fermentation include propionic acid. The organism belongs to the human cutaneous propionibacteria, along with P. avidum, P. granulosum, P. innocuum, and P. propionibacterium. P. acnes is ubiquitous on human skin, preferably within sebaceous follicles, where it is generally a harmless commensal. Nevertheless, P. acnes is an opportunistic pathogen (180, 197). It has been isolated from sites of infection and inflammation ranging from acne to diverse other conditions such as corneal ulcers, endocarditis, synovitis, pulmonary angitis, hyperostosis, endophthalmitis, and osteitis (SAPHO) syndrome (99, 193, 203). P. acnes grows slowly and can resist phagocytosis and persist intracellularly within macrophages (472). This resistance to phagocytosis may be conferred by a complex cell wall structure, which also includes a surface fibrillar layer (301).
The circular 2.5-Mb chromosome of P. acnes KPA171202 (DSM16379) contains 2,333 predicted genes and 35 pseudogenes (Table 1). A function has been assigned for around 70% of the identified genes.
Data concerning the relatedness of the skin isolate KPA171202 to other P. acnes isolates are limited to just a few genes, including the 16S rRNA, gehA, groEL, and dnaK genes. Such analyses are not very informative, since only limited variability exists between homologs of these genes at the DNA level. Comparative genome analyses with closely related genera, such as Mycobacterium, Streptomyces, and Corynebacterium, revealed that the closest related genome is that of Streptomyces avermitilis. However, genomic synteny between P. acnes and S. avermitilis is limited to just a few gene clusters (46).
Endogenous small plasmids, of 6 to 10 kb, have been identified in a few Propionibacterium species, i.e., P. acidipropionici, P. jensenii, P. granulosum, and P. freudenreichii (363). Only four have been sequenced: pRGO1 from P. acidipropionici (224), p545 from P. freudenreichii (211), the cryptic plasmid pPGO1 from P. granulosum (NCBI source NC_004526), and pLME106 from P. jensenii (NCBI source NC_005705). Two genes, repA and repB, encoding putative replication proteins similar to those of ColE theta-type replicating plasmids, were found in all but pPGO1, indicating that a similar replication mechanism is used by many Propionibacterium plasmids (Fig. 3).
A survey of the P. acnes genome for DNA regions that show an uncommon codon usage as well as a deviation in G+C content highlighted 10 regions that may have been acquired by HGT (45) (Fig. 5a). These include a cryptic prophage (see below), a cluster of genes predicted to be involved in conjugal DNA transfer, and a putative lanthionine biosynthesis cluster. Various other suspected HGT-acquired DNA regions, apart from those that specify predicted PTS-mediated substrate uptake systems, are assumed to express virulence traits. These include genes specifying factors for iron acquisition and adhesion, as well as hemolysin/cytotoxin factors. Another interesting alien DNA region is a 43-gene cluster predicted to encode a nonribosomal peptide synthetase similar to a nonribosomal peptide synthetase system of a Streptomyces species that enhances the biological fitness of the bacterium (62).
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FIG. 5. (a) Genome map of Propionibacterium acnes KPA171202. The genome variability regions are indicated by black boxes. (b) Genome map of the P. acnes KPA171202 Pro-1 prophage. Probable functions of encoded proteins indicated by bioinformatics analysis are noted.
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P. acnes KPA171202 contains a single, apparently defective, prophage-like element, named Pro-1 (57). The small number of phage-related genes represent functions (e.g., an antirepressor, holin, helicase, DNA polymerase, and primase) found in bacteriophages infecting low-G+C bacteria (Enterococcus, Streptococcus, Clostridium, and Lactobacillus) (Fig. 5b). These phage-related genes are not organized in the modular structure typical of lambdoid phages (35), but are dispersed among genes displaying classical bacterial origins, such as genes encoding an NADH oxidoreductase and a peptidase. Interestingly, Pro-1 carries a gene (abiF) encoding a protein resembling a Lactococcus lactis phage defense system that interferes with intracellular phage multiplication (for a review, see reference 72). The Pro-1 element is flanked on one site by a glycine tRNA gene, although no clear attachment sites can be distinguished in the flanking phage sequences. Based on its genetic structure, it seems that Pro-1 has been subject to intensive genome reshuffling and decay, indicating that the integration of this prophage was not a recent event.
The genome sequence of P. acnes reflects its presence and activity as a ubiquitous commensal on human skin. Metabolic reconstruction revealed the capacity of P. acnes to cope with varying oxygen availability, in accordance with its growth under microaerobic as well as anaerobic conditions (96, 156). The genome encodes all key components of oxidative phosphorylation, employing two terminal oxidases, a cytochrome aa3 oxidase, a cytochrome d oxidase whose E. coli homolog predominates when cells are grown at low aeration, and an F0F1-type ATP synthase (46). All genes of the Embden-Meyerhof and pentose phosphate pathways are present. Under anaerobic conditions P. acnes grows on different carbon sources, including glucose, ribose, fructose, mannitol, trehalose, mannose, N-acetylglucosamine, and glycerol (46). Fermentation products are short-chain fatty acids, especially propionic acid. P. acnes can also mobilize metabolic capabilities such as nitrate reductase, dimethyl sulfoxide reductase, and fumarate reductase to allow anaerobic respiration.
Various P. acnes gene products can degrade and use host-derived substances. Before the genome decipherment, knowledge of the capacity of P. acnes to use skin tissue was limited to a secreted extracellular triacylglycerol lipase, GehA, isolated from P. acnes P-37 (294). This enzyme degrades skin lipids, such as sebum, which may be a crucial activity for skin colonization. Furthermore, it was proposed that free fatty acids, released by P. acnes lipase activity on sebum, assist bacterial adherence and colonization of the sebaceous follicle (155). As expected, the genome of P. acnes KPA171202 contains a gehA homolog, while it also contains other genes that encode predicted extracellular (secreted and cell wall-bound) lipases.
The degradation of host tissues is also facilitated by hyaluronate lyase, which acts on a key constituent of the extracellular matrix of connective tissues (408). The P. acnes genome encodes such an enzyme, as well as numerous additional enzymatic activities with suspected roles in host tissue degradation, such as two endoglycoceramidases and four sialidases, a putative endo-ß-N-acetylglucosaminidase, and various extracellular peptidases. There are also genes specifying homologs of CAMP factors, which are typically found in pathogenic staphylococci (140). The CAMP reaction causes synergistic lysis of erythrocytes due to the interaction of the CAMP factor with the Staphylococcus aureus sphingomyelinase C. CAMP factors can bind to the Fc fragment of immunoglobulins of the immunoglobulin G and immunoglobulin M classes (140).
The capacity of P. acnes to modulate the immune system has been thoroughly studied. Increased cellular and humoral immunity to P. acnes has been detected in patients with severe acne (209, 237). The P. acnes genome encodes various cell surface or surface-exposed proteins that may exhibit cell-adherent properties. Many of these possess a C-terminal LPXTG-type cell wall-sorting signal required for binding of surface proteins to the cell wall through the action of a so-called sortase (92). Three genes within the P. acnes genome possess contiguous stretches of 12 to 16 guanine or cytosine residues, distributed either in the promoter or in the coding region. The sequences within these regions were ambiguous with respect to the length of the poly(C/G) stretch (46). Such variable homopolymeric C or G stretches, which are generated by slipped-strand mispairing during replication, have been reported to be involved in phase variation, an adaptive strategy commonly noticed in bacterial pathogens (46). P. acnes has a lipoglycan-based cell wall envelope (474), which may well play a role in adherence to skin and in the formation of a biofilm matrix (53). Furthermore, the genome of P. acnes contains three clusters involved in EPS biosynthesis. All these extracellular structures may modulate immunogenicity towards the microorganism and/or may constitute a barrier against antimicrobial compounds.
P. acnes abundantly produces porphyrins, which might contribute to skin damage (156). The interaction of porphyrins with oxygen is thought to contribute to keratinocyte damage and consequently to have implications regarding the pathogenesis of progressive macular hypomelanosis (473). However, it is known that P. acnes can be eradicated by illumination with intense blue light, which induces photoexcitation of bacterial porphyrins, singlet oxygen production, and thus bacterial destruction (14). The P. acnes genome contains two clusters of 26 and 8 genes, respectively, which are involved in vitamin B12 biosynthesis (46).
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The genera Corynebacterium, Mycobacterium, and Nocardia form a monophyletic taxon, the so-called CMN group, within the Actinobacteria (119). These bacteria share an unusual cell envelope composition, characterized by the presence of a waxy cell envelope containing mycolic acids, conferring alcohol and acid-fast staining properties on these bacteria which distinguish them from other bacteria.
The genus Mycobacterium is highly diverse and comprises 85 different species, which have been identified since the isolation of M. leprae in 1873 (358). In addition, there are a number of Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine substrains, which are derived from an attenuated M. bovis strain obtained in 1921 (24). Finally, individual Mycobacterium species, such as M. tuberculosis, display great diversity (219). Generally, mycobacteria are free-living saprophytes (121) and are well adapted to different habitats, such as soil (485) and aquatic environments (87). A few species, such as M. bovis and M. tuberculosis, first identified in infected animals, have never been isolated from other environments, suggesting that they are obligate parasites of humans and animals (86). Nevertheless, caution should be taken in drawing such a conclusion: the pathogenic M. ulcerans has also been isolated as a soil inhabitant in symbiosis with roots of certain plants present in tropical rain forests or similar environments (174).
Mycobacteria are the causative agents of a broad epidemiological, clinical, and pathological spectrum of diseases in humans. Mycobacterial diseases are very often associated with immunocompromised patients, especially AIDS patients. M. tuberculosis and related species, such as M. bovis, cause tuberculosis, surviving within macrophages. M. tuberculosis may primarily cause pulmonary disease, although organs other than lungs may be affected. M. leprae causes leprosy, living within Schwann cells and macrophages to give rise to a chronic granulomatous disease of the skin and peripheral nerves (201). M. ulcerans is the third most common mycobacterial disease (443). However, in contrast to the other mycobacteria, M. ulcerans grows outside of its host cells, and its pathogenicity is attributed to the secretion of a toxin. The M. ulcerans-mediated chronic disease results in painless, expanding skin ulcers. Many other environmental mycobacteria (e.g., M. avium) may on occasion cause localized or disseminated clinical illness such as lymphadenitis (121).
Due to their clinical importance, genome sequences of several mycobacterial species have been determined (Table 1). These include M. tuberculosis and M. leprae (83, 84, 126); M. bovis, which causes bovine tuberculosis (139); and M. avium subsp. paratuberculosis, the agent of Johne's disease in cattle (271). Additional ongoing mycobacterial genome sequencing projects include those for three undetermined mycobacterial species (NCBI sources NC_008146, NZ_AAQC00000000, and NZ_AAQD00000000), M. flavescens PYR-GCK (NCBI source NZ_AAQ00000000), M. tuberculosis C (NCBI source NZ_AAKR00000000), M. tuberculosis F11 (NCBI source NZ_AAIX00000000), M. tuberculosis strain Haarlem (NCBI source NZ_AASN00000000), and M. vanbaalenii PYR-1 (NCBI source NZ_AAPF00000000). Here we focus on the published and completely sequenced mycobacterial genomes (84, 126, 139, 271).