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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.
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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| INTRODUCTION |
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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.
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.
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).
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).
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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.
| GENOMICS OF BIFIDOBACTERIUM |
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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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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.
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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).
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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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.
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).
| GENOMICS OF TROPHERYMA |
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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.
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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.
| GENOMICS OF PROPIONIBACTERIUM |
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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).
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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).
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).
| GENOMICS OF MYCOBACTERIUM |
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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).
In contrast to the situation described for fast-growing bacteria such as B. subtilis, the orientation of genes with respect to the direction of replication is less biased (59% of them are transcribed with the same polarity as the replication forks, instead of 75% in B. subtilis [246]). It is believed that higher expression levels can be achieved by coordinating directions of transcription and replication, and it is therefore assumed
