Comparative genomics for mycobacterial peptidoglycan remodelling enzymes reveals extensive genetic multiplicity
© Machowski et al.; licensee BioMed Central Ltd. 2014
Received: 27 December 2013
Accepted: 12 March 2014
Published: 24 March 2014
Mycobacteria comprise diverse species including non-pathogenic, environmental organisms, animal disease agents and human pathogens, notably Mycobacterium tuberculosis. Considering that the mycobacterial cell wall constitutes a significant barrier to drug penetration, the aim of this study was to conduct a comparative genomics analysis of the repertoire of enzymes involved in peptidoglycan (PG) remodelling to determine the potential of exploiting this area of bacterial metabolism for the discovery of new drug targets.
We conducted an in silico analysis of 19 mycobacterial species/clinical strains for the presence of genes encoding resuscitation promoting factors (Rpfs), penicillin binding proteins, endopeptidases, L,D-transpeptidases and N-acetylmuramoyl-L-alanine amidases. Our analysis reveals extensive genetic multiplicity, allowing for classification of mycobacterial species into three main categories, primarily based on their rpf gene complement. These include the M. tuberculosis Complex (MTBC), other pathogenic mycobacteria and environmental species. The complement of these genes within the MTBC and other mycobacterial pathogens is highly conserved. In contrast, environmental strains display significant genetic expansion in most of these gene families. Mycobacterium leprae retains more than one functional gene from each enzyme family, underscoring the importance of genetic multiplicity for PG remodelling. Notably, the highest degree of conservation is observed for N-acetylmuramoyl-L-alanine amidases suggesting that these enzymes are essential for growth and survival.
PG remodelling enzymes in a range of mycobacterial species are associated with extensive genetic multiplicity, suggesting functional diversification within these families of enzymes to allow organisms to adapt.
KeywordsPeptidoglycan Transglycosylases Amidases D,D-carboxypeptidases Transpeptidases Endopeptidases
Bacteria inhabit every environment on earth with a resilience that is central to their survival and consequently, they continue to serve as a major source of human disease. A critical factor, which has been central to the success of these organisms, is the diversity entrenched within their cell walls, which serves as a major barrier to drug treatment. The mycobacterial cell wall is an incredibly complex structure, with multiple layers that collectively constitute a waxy, durable coat around the cell, which serves as the major permeability barrier to drug action [1–4]. Considering this, the cell wall and related components are attractive for the mining of new drug targets, and remain relatively unexploited for drug discovery in the case of certain bacterial pathogens [2, 5, 6]. Peptidoglycan (PG or the murein sacculus) is a rigid layer that constricts the cell membrane and the cell within, providing mechanical stability to counteract imbalances of cytoplasmic turgour pressure, and plays an important role in determining cell size and shape [7–10]. Mycobacteria possess a highly complex additional lipid rich outer membrane, with different constituents anchored either directly to the cell membrane or to the PG [6, 11]. Arabinoglactan (AG), a structure unique to actinomycetes, is bound externally to an N-acetyl muramic acid (NAM) moiety of the PG [3, 12]. In mycobacteria, a certain proportion of the muramic acid is N-glycolylated  through the activity of NamH, a UDP-N-acetylmuramic acid hydroxylase . This modification results in altered tumour necrosis factor α production [15, 16] however, abrogation of NamH activity does not lead to decreased virulence in mice .
The PG in bacterial cell walls is an incredibly dynamic structure that requires constant expansion and remodelling during growth to accommodate the insertion of new PG subunits, secretion apparatus, flagellae etc. [9, 10]. During cell division, pre-septal PG synthesis and subsequent degradation of the septum is critical to daughter cell separation; consequently these processes are carefully regulated . In this regard, there is a diversity of enzymes involved in cross-linking, degradation and remodelling of PG, which are illustrated in Figure 1. A ubiquitous feature in bacteria is the genetic multiplicity associated with these functions, which presumably contributes to the ability of different organisms to adapt under varying environmental conditions [7, 9, 10]. In the case of Mycobacterium tuberculosis, the causative agent of tuberculosis, there is a dire need for new drugs with novel modes of action. The increased prevalence of drug resistant strains has raised concerns regarding the sustainability of the current treatment regimen. To address this, several aspects of mycobacterial metabolism are being assessed for potential new drug targets . The genetic redundancy associated with PG biosynthesis together with the reliance on robust bacterial growth to achieve significant drug target vulnerability, has hampered drug development initiatives that target the cell wall . For other bacterial pathogens, PG has been successfully used as an antibiotic target in the past, as evidenced by the widespread use of β-lactam antibiotics among others, the biosynthesis and degradation of this macromolecule in mycobacteria is meritorious of further investigation.
In this study, we undertake a comprehensive analysis of the genomic repertoire of PG remodelling enzymes in various pathogenic and environmental mycobacteria to determine the level of genetic multiplicity/redundancy and degree of conservation. We focus on those enzymes involved in cross-linking and remodelling of the PG in the periplasmic compartment, including: resuscitation promoting factors (Rpfs), penicillin binding proteins (PBPs), transpeptidases, endopeptidases, and N-acetylmuramoyl-L-alanine amidases. Our data reveal extensive genetic multiplicity for the 19 strains analysed in this study, which allowed grouping of strains into three families based on their complement of PG remodelling enzymes, including the MTBC, other pathogenic mycobacteria and non-pathogenic environmental organisms.
Results and Discussion
Genetic complement for PG remodelling enzymes in 19 mycobacterial species
Other mycobacterial pathogens
Environmental mycobacterial species
M. tuberculosis H37Rv
M. tuberculosis H37Ra
M. tuberculosis CDC1551
M. africanum GM041182
M. bovis AF2122/97
M. bovisBCG Pasteur 1173P2
M. avium 104
M. avium subsp. paraTB K-10
M. intracellulare ATCC 13950
M. ulcerans AGY99
M. marinum M
M. abscessus ATCC19977
M. vanbaalenii PYR–1
M. gilvum PYR–GCK
M. leprae TN
Resuscitation promoting factors
Mjls_ 4643 Mjls_1689
Penicillin binding proteins
Mkms_5716 Mkms_1458 Mkms_2496
Mjls_ 2488 Mjls_4564 Mjls_4520
Mflv_5292 Mflv_0895 Mflv_2839 Mflv_3663
Mkms_2497 Mkms_1465 Mkms_5687 Mkms_5720
Mjls_ 2489 Mjls_ 4472 Mjls_ 4557 Mjls_ 4529
Mflv_5324 Mflv_5288 Mflv_0902 Mflv_2843
Mkms_3349 Mkms_5661 Mkms_1453
Mjls_3298 Mjls_4528 Mjls_4570
Mflv_2959 Mflv_5385 Mflv_5350 Mflv_2808 Mflv_0888
Mvan_4102 Mvan_3651 Mvan_5854
Mkms_5721 Mkms_3714 Mkms_1466
Mjls_3646 Mjls_4532 Mjls_4556
Mflv_2542 Mflv_5287 Mflv_0904
Mflv_5330 Mflv_2824 Mflv_5369 Mflv_0479
Resuscitation promoting factors (lytic transglycosylases)
Rpfs are classified as lytic transglycosylases (LTs) based on sequence conservation and three-dimensional protein structure [29, 39–41]. LTs cleave the ß-1,4-glycosidic bonds between the NAG-NA/GM sugar subunits, Figure 1, and their activity is required for insertion of new PG units and expansion of the glycan backbone . In mycobacteria RpfB contains a lysozyme-like, transglycosylase-like PFAM domain, and consequently this group of enzymes are predicted to cleave the glycan backbone of PG [39–41]. Direct evidence for this is lacking and moreover, the mechanism through which Rpf-mediated cleavage of PG results in growth stimulation remains unknown. The repertoire of rpf genes is highly conserved in the MTBC; in contrast, other pathogenic mycobacteria lack rpfD, including M. leprae, Table 1. Based on the distribution of rpfC and rpfD, we categorize the 19 strains analysed in this study into the MTBC (which retains all five rpf homologues present in M. tuberculosis), other pathogenic mycobacteria (which lack rpfD) and environmental strains (which lack both rpfC and rpfD). This classification is supported by phylogenetics analysis which confirms these clusters and duplication/loss of genes, Additional file 1: Figure S1. Recently, it has been shown that the Rpfs can serve as potent antigens  and Rpf-directed host immune responses allow for detection of TB in latently infected individuals . It is noteworthy that strains lacking different combinations of rpf genes confer significant protective efficacy when used as vaccine strains in mice . Hence, any variation in rpf gene complement between pathogenic mycobacteria may have significant consequences for broadly protective effects of future Rpf-based vaccines.
The environmental species retain three rpf genes [rpfA, rpfB (duplicated in Mycobacterium sp. JLS, Mycobacterium sp. KMS, Mycobacterium sp. MCS) and rpfE], Table 1 and Additional file 1: Figure S1. Although rpfC (Rv1884c in M. tuberculosis) homologues have been annotated as present in all mycobacteria , our analysis shows that the M. tuberculosis rpfC homologue is absent from environmental species. Artemis Comparison Tool (ACT) whole genome alignment reveals that the region encoding rpfC in M. tuberculosis is absent in M. smegmatis and all other environmental mycobacteria (data not shown). Thus, based on gene synteny, there is no direct rpfC homologue in these strains. However, there is a local duplication of rpfE in all the environmental strains (annotated as MSMEG_4643 in M. smegmatis), Table 1, Additional file 1: Figure S1. Consequently, we re-annotate MSMEG_4640 to rpfE2, as a homologue of MSMEG_4643, rather than a homologue of Rv1884c. As RpfE interacts with the Rpf Interacting Protein A (RipA) , there may be some functional consequence to the presence of multiple copies in M. smegmatis and other environmental bacteria.
The restriction of rpfC and rpfD homologues to pathogenic and MTBC strains, along with the duplication of rpfB in some environmental species, raises interesting questions regarding the nature of growth stimulation in these organisms. These differences suggest that the latter require fewer secreted Rpfs and are more reliant on the membrane bound RpfB homologue. This could be related to the fact that environmental organisms are required to grow in diverse niches of varying size and complexity making them more dependent on localised growth stimulatory activity through a membrane bound Rpf rather than paracrine signalling from diffusible Rpfs produced by neighbouring organisms. It is noteworthy that of all five homologues in M. tuberculosis, deletion of rpfB individually or in combination with rpfA results in colony forming defects and prolonged time to reactivation from chronic infection in mice [21, 34, 35].
The role of Rpfs in TB disease in humans remains enigmatic. It has been demonstrated that sputum from patients with active TB disease, before the initiation of treatment, is characterised by a population of dormant bacteria that require Rpfs for growth . These data provide tantalizing preliminary evidence that Rpfs play an important role in determining bacterial population dynamics in TB infected patients and moreover are critical for disease transmission. Within the granulomatous environment, it may be preferable for the bacterial population as a whole to facilitate emergence of fitter clones which are able to exit from arrested growth. This could explain clonal emergence in clinical samples if few strains are able to expand sufficiently to cause tubercular lung disease.
Penicillin binding proteins
Penicillin Binding proteins (PBPs) are a large family of evolutionarily related cell wall associated enzymes, that bind β-lactam antibiotics [48, 49]. PBPs are classified according to their molecular weight as either high molecular mass (HMM) or low molecular mass (LMM) and are broken down into Class A, Class B and Class C . In mycobacteria, Class A PBPs constitute bi-functional enzymes designated as ponA1 (PBP1, Rv0050, ); and ponA2 (PBP1A, Rv3682 ), Figure 2. They contain separate domains for transpeptidase and transglycosylase activities. Both these genes are present in all mycobacteria and, as previously reported for M. smegmatis and other environmental strains, there is a duplication of ponA2 which was annotated as ponA3 , Table 1 and Additional file 1: Figure S2.
Class B PBP proteins PbpA (pbpA; Rv0016c, ), PbpB (pbpB; Rv2163c, ) and PBP-lipo (Rv2864c, ) are predicted to contain only transpeptidase domains and possibly additional dimerisation domains, but lack transglycosylase activities, Figure 2. Both PbpA and PbpB (FtsI) are involved in progression to cell division in M. smegmatis where gene deletion or depletion manifests in altered cell morphology and antibiotic resistance profiles . In this family of PBPs – as exemplified by ponA2 - there is a distal duplication of PBP-lipo in the environmental strains, Table 1 and Additional file 1: Figure S3. No experimental data on this are currently available, but the lipophilic domain is speculated to allow for cell wall association.
D,D-carboxypeptidases (DD-CPases) are designated as Class C PBPs and are generally present in high abundance . DD-CPases remove the D-Ala residue at position 5 of pentapeptides  and through this activity prevent cross linking of the stem peptide into 4 → 3 bridges, Figure 1. In mycobacteria, the dacB2-encoded DD-CPase is not affected by penicillin – though it does bind the antibiotic . Inhibition of DacB through treatment with meropenem results in the accumulation of pentapeptides in M. tuberculosis . In this context, DD-CPases have been implicated in regulating the amount of cross-linking that can occur within the PG sacculus . Our analysis shows that M. tuberculosis H37Rv encodes three distinct DD-CPase homologues: dacB1 (Rv3330), dacB2 (Rv2911) and Rv3627c, Table 1, Figure 2 and Additional file 1: Figure S4. Rv3627c carries two PF02113 domains, one of which is contracted. In the environmental species there is a local duplication of the dacB2 (Rv2911) homologue, leading to consecutive numbering of the resulting duplicated genes for example, MSMEG_2432 and MSMEG_2433 in M. smegmatis. In addition, a distant DD-CPase homologue (annotated as MSMEG_1900 in M. smegmatis) was identified in the environmental strains, as well as in M. abscessus but not in the other pathogenic mycobacteria and MTBC, Table 1. Two additional loci - Rv0907 and Rv1367c – were identified in M. tuberculosis by in silico analysis through their predicted ß-lactamase domains and are grouped among Class C PBPs . Analysis of these proteins revealed that they retain a β-lactamase binding domain (of the AmpH family) but further classification into the functional classes studied herein proved difficult. Consequently, we have not analysed these genes further.
Endopeptidases are enzymes that cleave within the stem peptides in PG. In this study, we focus on the Nlp/P60 class of endopeptidases, which cleave within the stem peptides between positions 2 and 3 as exemplified by RipA, Figure 1. RipA is an essential PG hydrolytic enzyme that synergistically interacts with RpfB and RpfE [46, 57] to form a complex that is able to degrade PG. The RipA-RpfB hydrolytic complex is negatively regulated by PonA2  suggesting a dynamic interplay between PG hydrolases, one that would be significantly nuanced with the presence of multiple RipA and Rpf homologues. In this regard, our analysis reveals four endopeptidases in M. tuberculosis that display strong homology to ripA, Table 1, Figure 2, Additional file 1: Figure S5. With the exception of Mycobacterium abscessus and M. leprae, pathogenic mycobacteria retain all five of these homologues. Environmental strains display enhanced expansion of endopeptidases, with the exception of the ripD homologue (Rv1566c). The functional consequence of this remains unknown but it is noteworthy that these strains have also expanded their rpfE and rpfB gene repertoire, suggesting that the multiplicity in this case allows for a greater number of RipA-RpfB/E protein complexes, as well as for protein complexes with different subunit composition. Dysregulated expression of RipA leads to dramatic alterations in cellular morphology and growth  suggesting that careful regulation of this protein, both at the expression level as well as by post-translational level is essential. Genetic expansion of RipA homologues along with two copies of RpfB and RpfE, both of which interact with RipA implies a functional consequence of this expansion. In addition, strong regulation of these multiple copies would be required to prevent any detrimental effects on cell growth.
RipB displays strong sequence homology RipA in M. tuberculosis (100% amino acid identity over 58% coverage) and similar domain organization , but lacks the N-terminal motif, Figure 2, that has been implicated in auto inhibition by blocking the active site in the three-dimensional crystal structure . More recently, high resolution crystal structures of RipB and the C-terminal module of RipA (designated as RipAc) revealed striking differences in the structure of these proteins, specifically in the N-terminal fragments that cross the active site . Both RipB and RipAc are able to bind high molecular weight PG and retain the ability to cleave PG with variable substrate specificity, which is not regulated by the presence of the N-terminal domain . This suggests that the N-terminus does not regulate PG degrading activity and in this context, the physiological consequences of the reduced size of RipB and RipD, Figure 2, remain unknown. The high degree of conservation of RipB across all pathogenic mycobacteria including M. leprae, Table 1, Additional file 1: Figure S5 indicates that variable substrate specificity in PG hydrolases in essential for pathogenesis. The Mycobacterium marinum homologues of Rv1477 and Rv1478, iipA and iipB (MMAR_2284 and MMAR_2285 respectively), Table 1, Additional file 1: Figure S5, have been implicated in macrophage invasion, antibiotic susceptibility and cell division . As with the other enzymes assessed in this study, environmental mycobacteria display greater genetic multiplicity for these homologues, Table 1.
Structural analysis of RipD reveals alterations in the catalytic domain, consistent with the inability of this protein to hydrolyse PG . Nevertheless the core domain of RipD is able to bind mycobacterial PG and this binding is negatively regulated by the C-terminal region . However, RipD homologues in the environmental mycobacteria lack the 63C-terminal amino acids, Table 1 (shown in parentheses), possibly allowing for stronger binding of this enzyme to PG.
Rv2190c encodes another NlpC/P60-type PG hydrolase in mycobacteria. Deletion of this gene in M. tuberculosis results in altered colony morphology, attenuated growth in vitro, defective PDIM production and reduced colonisation of mouse lungs in the murine model of TB infection . Consistent with this, homologues of Rv2190c are found in all pathogenic mycobacteria, Table 1, with notable genetic expansion in some environmental species. In contrast, the Rv0024 is absent from environmental species, suggesting that it could be required for intracellular growth or some other component of the pathogenic process, Table 1, Additional file 1: Figure S5.
L,D - Transpeptidases
L,D-transpeptidases (Ldt) are a group of carbapenem sensitive enzymes in M. tuberculosis  that contribute to the formation of a 3 → 3 link between the two adjacent mDAP (mDap → mDap bridges) residues in PG, distinct from the classic 4 → 3 link (D-Ala → mDAP), Figure 1. M. abscessus  and M. tuberculosis  exhibit increased ratios of the 3 → 3 cross-link in stationary axenic culture, indicating that mycobacteria are capable of modulating their PG at the level of transpeptidation in response to growth stage and the availability of nutrients. Both LdtMt1 and LdtMt2 (Rv0116c and Rv2518c respectively) were experimentally shown to affect M. tuberculosis H37Rv morphology, growth characteristics and antibiotic susceptibility in vivo . The crystal structure of LdtMt2 places the extramembrane domain 80–100 Å from the membrane surface and indicates that this enzyme is able to remodel PG within this spatial region of the PG sacculus . More recently, it has been demonstrated that the combinatorial loss of both LdtMt1 and LdtMt2 in M. tuberculosis resulted in morphological defects and altered virulence in the murine model of TB infection . A notable variability of L,D-transpeptidase genes is found in mycobacteria, Table 1, Figure 2 and Additional file 1: Figure S6. Five homologues are present in all but one pathogenic strain, while multiple homologues are evident in most environmental strains. The exception is ldt Mt3 (Rv1433), which is absent from the pathogen Mycobacterium ulcerans and from the environmental species Mycobacterium vanbaalenii, M sp. MCS and M. sp. KMS, yet its presence in M. leprae suggests functional importance. As with RipA, M. gilvum shows the greatest expansion of the ldt genes. Biochemical characterisation of all five M. tuberculosis H37Rv homologues, LdtMt1 - LdtMt5, confirms PG cross-linking and/or ß-lactam acylating enzyme activities in all of these enzymes . This activity can be abolished by treatment with imipenem and cephalosporins, indicating that this group of enzymes holds great promise for TB drug development [70, 71]. Moreover, the functionality of all the Ldt homologues present in M. tuberculosis raises interesting questions with respect to the functional consequences of the expansion of this protein family in environmental strains, which may require greater flexibility in Ldt function.
While endopeptidases and transpeptidases are responsible for cleavage within or between peptide stems, amidases act to remove the entire peptide stem from the glycan strands, cleaving between the NA/GM moiety and the L-Ala in the first position of the stem peptide, Figure 1. The amidases have been implicated in PG degradation, antibiotic resistance/tolerance and cell separation in Escherichia coli and other organisms, and can be organised into 2 main families containing either an amidase_2 or amidase_3 – type domain [8, 9, 72]. The amidases of E. coli (which retains 5 amidases designated AmiA, AmiB, AmiC, AmiD and AmpD) have specific substrate requirements governed by the structural confirmation of the NAM carbohydrate moiety. Knockout of these amidases results in chaining phenotypes, abnormal cell morphologies and/or increased susceptibility to certain antibiotics [72–74]. Amidases have also been implicated in spore formation, germination and cell communication in Bacillus subtilis [75, 76]. The role of amidases in mycobacterial growth, virulence and resuscitation from dormancy is unknown and any impact of these on mycobacterial morphology and antibiotic resistance remains to be demonstrated. Analysis of the amidase gene complement in mycobacteria reveals the presence of four homologues in M. tuberculosis, two containing the amidase_2 domain (ami3; Rv3811 and ami4; Rv3594) and two the amidase_3 domain (ami1; Rv3717 and ami2; Rv3915), Table 1, Figure 2 and Additional file 1: Figure S7. The crystal structure of Rv3717 from M. tuberculosis confirms that this enzyme is able to bind and cleave muramyl dipeptide . The amidase family distinguishes itself from all other enzyme families by absence of a homologue (ami4) from non-MTBC pathogens and its presence in the MTBC and environmental strains. M. leprae retains only the ami1 and ami2 genes – both containing the amidase_3 domain. This suggests that amidase_2 domain amidase activity is dispensable specifically in this species, but required for peptidoglycan remodelling in the other pathogenic mycobacteria.
Very little is known about in vitro growth and division of M. leprae, as it can only be grown in animal models. From our analysis, it is apparent that M. leprae habours notable genetic redundancy for PG remodelling enzymes (Table 1) in contrast to its minimal gene set for other areas of metabolism . Considering that PG subunits or precursors cannot be scavenged from the host, it is expected that pathogenic bacteria would retain complete pathways for biosynthesis and remodelling of PG. However, the presence in M. leprae of multiple homologues within each class of PG remodelling enzyme assessed in this study, suggests that some level of multiplicity is required to ensure substrate flexibility. Further work in this regard is difficult due to the limited tractability of M. leprae for in vitro manipulation.
Mycobacteria represent a wide range of species with a great variety of phenotypes. Exposure to stresses which they encounter at various stages of their life cycles demands the ability to adapt. Consistent with this, many mycobacteria encode a multiplicity of genes for numerous important pathways such as respiration and cofactor biosynthesis [79, 80], which allows for a more nuanced regulation of physiology. The analysis performed herein summarises the general distribution of PG remodelling genes in diverse strains and reveals an emerging trend towards gene multiplicity in environmental mycobacteria. There is great conservation within the MTBC and other pathogenic mycobacteria. Of all strains, M. gilvum displays the greatest degree of gene expansion, containing a total 44 PG remodelling genes, Table 1. This organism has not been studied extensively but may represent a potential model system for understanding how the genetic multiplicity for PG remodelling enzymes contributes to bacterial physiology. As expected M. leprae shows a reduction in the number of genes that encode the enzymes assessed in this study but still retains more than one representative of each functional class. This, together with the striking degree of conservation in some families of PG remodelling enzymes in pathogenic mycobacteria, suggests that PG biosynthesis, remodelling and possibly recycling are all potential vulnerable pathways for drug development. The extracellular nature of these enzymes provides an added advantage for drug screening since small molecules need not enter the cell for biological activity. Entry of compounds into mycobacterial cells remains the major confounding factor in current drug development initiatives. Moreover, the lack of human counterparts would ensure a high degree of specificity. In conclusion, the gene complements for PG remodelling revealed in this study most likely reflect the differential requirements of various mycobacteria for murein expansion/turnover during colonisation of and proliferation within host organisms or environmental niches.
The 19 mycobacterial strain sequences used in this study were all complete and either published [24, 78, 81–90] or directly submitted to GenBank  (Additional file 2: Table S1). The following sites were utilized for analysis of the genomes (Additional file 2: Table S2): The comparative genomic profile for the enzymes of interest were initiated by homology searches of known M. tuberculosis H37Rv genes at TubercuList , GenoList  or TBDB . Where necessary for further analysis direct BLAST analysis was performed at NCBI , utilising protein sequence for BLASTp or DNA sequence for BLASTn particularly for the analysis of Mycobacterium sp. JLS, M. africanum and M. intracellulare which are not or only partially annotated at TBDB. To confirm the absence of genes, protein sequence was used for tBLASTn analysis. Additional homologues that are absent from M. tuberculosis H37Rv were identified by advanced search at SmegmaList (Mycobrowser) . Where information was required for sequence level analysis, the Sanger Artemis Comparison Tool (ACT)  was utilized on annotated sequences obtained from the Integrated Microbial Genomes (IMG) site at the DOE Joint Genome Institute . Phylogeny was established from FASTA files from all genes in Table 1 at EMBL-EBI by ClustalO  alignment and ClustalW2  analysis and visualized using FigTree V1.4 software (http://tree.bio.ed.ac.uk/software/figtree). Functional annotation of each of the M. tuberculosis proteins was identified at InterScanPro , for PFAM domains , signal sequences (SignalP)  and membrane anchoring domains (TMHMM) .
This work was supported by grants from the South African National Research Foundation (NRF), the Medical Research Council, the Department of Science and Technology. BK was supported by an Early Career Scientist award from the Howard Hughes Medical Institute. C.S.E was supported by postdoctoral fellowships from the NRF and the Centre of Aids Programme Research in South Africa (CAPRISA).
- Nikaido H, Jarlier V: Permeability of the mycobacterial cell wall. Res Microbiol. 1991, 142 (4): 437-443. 10.1016/0923-2508(91)90117-S.PubMedGoogle Scholar
- Chatterjee D: The mycobacterial cell wall: structure, biosynthesis and sites of drug action. Curr Opin Chem Biol. 1997, 1 (4): 579-588. 10.1016/S1367-5931(97)80055-5.PubMedGoogle Scholar
- Brennan PJ, Besra GS: Structure, function and biogenesis of the mycobacterial cell wall. Biochem Soc Trans. 1997, 25 (1): 188-194.PubMedGoogle Scholar
- Jarlier V, Nikaido H: Mycobacterial cell wall: structure and role in natural resistance to antibiotics. FEMS Microbiol Lett. 1994, 123 (1–2): 11-18.PubMedGoogle Scholar
- Barry CE, Mdluli K: Drug sensitivity and environmental adaptation of mycobacterial cell wall components. Trends Microbiol. 1996, 4 (7): 275-281. 10.1016/0966-842X(96)10031-7.PubMedGoogle Scholar
- Favrot L, Ronning DR: Targeting the mycobacterial envelope for tuberculosis drug development. Expert Rev Anti Infect Ther. 2012, 10 (9): 1023-1036. 10.1586/eri.12.91.PubMed CentralPubMedGoogle Scholar
- Typas A, Banzhaf M, Gross CA, Vollmer W: From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol. 2012, 10 (2): 123-136.Google Scholar
- Vollmer W, Blanot D, de Pedro MA: Peptidoglycan structure and architecture. FEMS Microbiol Rev. 2008, 32 (2): 149-167. 10.1111/j.1574-6976.2007.00094.x.PubMedGoogle Scholar
- Vollmer W, Joris B, Charlier P, Foster S: Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol Rev. 2008, 32 (2): 259-286. 10.1111/j.1574-6976.2007.00099.x.PubMedGoogle Scholar
- Boneca IG: The role of peptidoglycan in pathogenesis. Curr Opin Microbiol. 2005, 8 (1): 46-53. 10.1016/j.mib.2004.12.008.PubMedGoogle Scholar
- Kaur D, Guerin ME, Skovierova H, Brennan PJ, Jackson M: Chapter 2: Biogenesis of the cell wall and other glycoconjugates of Mycobacterium tuberculosis. Adv Appl Microbiol. 2009, 69: 23-78.PubMed CentralPubMedGoogle Scholar
- Besra GS, Brennan PJ: The mycobacterial cell wall: biosynthesis of arabinogalactan and lipoarabinomannan. Biochem Soc Trans. 1997, 25 (3): 845-850.PubMedGoogle Scholar
- Mahapatra S, Crick DC, McNeil MR, Brennan PJ: Unique structural features of the peptidoglycan of Mycobacterium leprae. J Bacteriol. 2008, 190 (2): 655-661. 10.1128/JB.00982-07.PubMed CentralPubMedGoogle Scholar
- Raymond JB, Mahapatra S, Crick DC, Pavelka MS: Identification of the namH gene, encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan. J Biol Chem. 2005, 280 (1): 326-333.PubMedGoogle Scholar
- Coulombe F, Divangahi M, Veyrier F, de Leseleuc L, Gleason JL, Yang Y, Kelliher MA, Pandey AK, Sassetti CM, Reed MB, Behr MA: Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide. J Exp Med. 2009, 206 (8): 1709-1716. 10.1084/jem.20081779.PubMed CentralPubMedGoogle Scholar
- Hansen JM, Golchin SA, Veyrier FJ, Domenech P, Boneca IG, Azad AK, Rajaram MV, Schlesinger LS, Divangahi M, Reed MB, Behr MA: N-Glycolylated Peptidoglycan Contributes to the Immunogenicity but Not Pathogenicity of Mycobacterium tuberculosis. J Infect Dis. 2013, 209 (7): 1045-1054.PubMedGoogle Scholar
- Desmarais SM, De Pedro MA, Cava F, Huang KC: Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly. Mol Microbiol. 2013, 89 (1): 1-13. 10.1111/mmi.12266.PubMed CentralPubMedGoogle Scholar
- Zumla A, Nahid P, Cole ST: Advances in the development of new tuberculosis drugs and treatment regimens. Nat Rev Drug Discov. 2013, 12 (5): 388-404. 10.1038/nrd4001.PubMedGoogle Scholar
- Boshoff HI, Barry CE: Is the mycobacterial cell wall a hopeless drug target for latent tuberculosis?. Drug Discovery Today: Disease Mechanisms. 2006, 3 (2): 237-245. 10.1016/j.ddmec.2006.06.002.Google Scholar
- Sassetti CM, Boyd DH, Rubin EJ: Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol. 2003, 48 (1): 77-84. 10.1046/j.1365-2958.2003.03425.x.PubMedGoogle Scholar
- Griffin JE, Gawronski JD, Dejesus MA, Ioerger TR, Akerley BJ, Sassetti CM: High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. PLoS Pathog. 2011, 7 (9): e1002251-10.1371/journal.ppat.1002251.PubMed CentralPubMedGoogle Scholar
- Mukamolova GV, Kaprelyants AS, Young DI, Young M, Kell DB: A bacterial cytokine. Proc Natl Acad Sci U S A. 1998, 95 (15): 8916-8921. 10.1073/pnas.95.15.8916.PubMed CentralPubMedGoogle Scholar
- Mukamolova GV, Murzin AG, Salina EG, Demina GR, Kell DB, Kaprelyants AS, Young M: Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation. Mol Microbiol. 2006, 59 (1): 84-98. 10.1111/j.1365-2958.2005.04930.x.PubMedGoogle Scholar
- Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, et al: Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 1998, 393 (6685): 537-544. 10.1038/31159.PubMedGoogle Scholar
- Kana BD, Mizrahi V: Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling. FEMS Immunol Med Microbiol. 2009, 58 (1): 39-50.PubMedGoogle Scholar
- Mukamolova GV, Turapov OA, Young DI, Kaprelyants AS, Kell DB, Young M: A family of autocrine growth factors in Mycobacterium tuberculosis. Mol Microbiol. 2002, 46 (3): 623-635. 10.1046/j.1365-2958.2002.03184.x.PubMedGoogle Scholar
- Kana BD, Mizrahi V: Resuscitation promoting factors in bacterial population dynamics during TB infection. Drug Discovery Today: Disease Mechanisms. 2010, 7: e13-e18.Google Scholar
- Keep NH, Ward JM, Cohen-Gonsaud M, Henderson B: Wake up! Peptidoglycan lysis and bacterial non-growth states. Trends Microbiol. 2006, 14 (6): 271-276. 10.1016/j.tim.2006.04.003.PubMedGoogle Scholar
- Keep NH, Ward JM, Robertson G, Cohen-Gonsaud M, Henderson B: Bacterial resuscitation factors: revival of viable but non-culturable bacteria. Cell Mol Life Sci. 2006, 63 (22): 2555-2559. 10.1007/s00018-006-6188-2.PubMedGoogle Scholar
- Kell DB, Young M: Bacterial dormancy and culturability: the role of autocrine growth factors. Curr Opin Microbiol. 2000, 3 (3): 238-243. 10.1016/S1369-5274(00)00082-5.PubMedGoogle Scholar
- Tufariello JM, Chan J, Flynn JL: Latent tuberculosis: mechanisms of host and bacillus that contribute to persistent infection. Lancet Infect Dis. 2003, 3 (9): 578-590. 10.1016/S1473-3099(03)00741-2.PubMedGoogle Scholar
- Downing KJ, Mischenko VV, Shleeva MO, Young DI, Young M, Kaprelyants AS, Apt AS, Mizrahi V: Mutants of Mycobacterium tuberculosis lacking three of the five rpf-like genes are defective for growth in vivo and for resuscitation in vitro. Infect Immun. 2005, 73 (5): 3038-3043. 10.1128/IAI.73.5.3038-3043.2005.PubMed CentralPubMedGoogle Scholar
- Kana BD, Gordhan BG, Downing KJ, Sung N, Vostroktunova G, Machowski EE, Tsenova L, Young M, Kaprelyants A, Kaplan G, Mizrahi V: The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro. Mol Microbiol. 2008, 67 (3): 672-684. 10.1111/j.1365-2958.2007.06078.x.PubMed CentralPubMedGoogle Scholar
- Russell-Goldman E, Xu J, Wang X, Chan J, Tufariello JM: A Mycobacterium tuberculosis Rpf double-knockout strain exhibits profound defects in reactivation from chronic tuberculosis and innate immunity phenotypes. Infect Immun. 2008, 76 (9): 4269-4281. 10.1128/IAI.01735-07.PubMed CentralPubMedGoogle Scholar
- Tufariello JM, Mi K, Xu J, Manabe YC, Kesavan AK, Drumm J, Tanaka K, Jacobs WR, Chan J: Deletion of the Mycobacterium tuberculosis resuscitation-promoting factor Rv1009 gene results in delayed reactivation from chronic tuberculosis. Infect Immun. 2006, 74 (5): 2985-2995. 10.1128/IAI.74.5.2985-2995.2006.PubMed CentralPubMedGoogle Scholar
- Gupta RK, Srivastava R: Resuscitation promoting factors: a family of microbial proteins in survival and resuscitation of dormant mycobacteria. Indian J Microbiol. 2012, 52 (2): 114-121. 10.1007/s12088-011-0202-6.PubMed CentralPubMedGoogle Scholar
- Haiser HJ, Yousef MR, Elliot MA: Cell wall hydrolases affect germination, vegetative growth, and sporulation in Streptomyces coelicolor. J Bacteriol. 2009, 191 (21): 6501-6512. 10.1128/JB.00767-09.PubMed CentralPubMedGoogle Scholar
- Hett EC, Rubin EJ: Bacterial growth and cell division: a mycobacterial perspective. Microbiol Mol Biol Rev. 2008, 72 (1): 126-156. 10.1128/MMBR.00028-07. table of contentsPubMed CentralPubMedGoogle Scholar
- Cohen-Gonsaud M, Barthe P, Bagneris C, Henderson B, Ward J, Roumestand C, Keep NH: The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes. Nat Struct Mol Biol. 2005, 12 (3): 270-273. 10.1038/nsmb905.PubMedGoogle Scholar
- Cohen-Gonsaud M, Keep NH, Davies AP, Ward J, Henderson B, Labesse G: Resuscitation-promoting factors possess a lysozyme-like domain. Trends Biochem Sci. 2004, 29 (1): 7-10. 10.1016/j.tibs.2003.10.009.PubMedGoogle Scholar
- Ruggiero A, Tizzano B, Pedone E, Pedone C, Wilmanns M, Berisio R: Crystal structure of the resuscitation-promoting factor ΔDUFRpfB from M. tuberculosis. J Mol Biol. 2009, 385 (1): 153-162. 10.1016/j.jmb.2008.10.042.PubMedGoogle Scholar
- Romano M, Aryan E, Korf H, Bruffaerts N, Franken CL, Ottenhoff TH, Huygen K: Potential of Mycobacterium tuberculosis resuscitation-promoting factors as antigens in novel tuberculosis sub-unit vaccines. Microbes Infect. 2011, 14 (1): 86-95.PubMedGoogle Scholar
- Riano F, Arroyo L, Paris S, Rojas M, Friggen AH, van Meijgaarden KE, Franken KL, Ottenhoff TH, Garcia LF, Barrera LF: T cell responses to DosR and Rpf proteins in actively and latently infected individuals from Colombia. Tuberculosis (Edinb). 2012, 92 (2): 148-159. 10.1016/j.tube.2011.12.005.Google Scholar
- Kondratieva T, Rubakova E, Kana BD, Biketov S, Potapov V, Kaprelyants A, Apt A: Mycobacterium tuberculosis attenuated by multiple deletions of rpf genes effectively protects mice against TB infection. Tuberculosis (Edinb). 2011, 91 (3): 219-223. 10.1016/j.tube.2011.01.005.Google Scholar
- Ravagnani A, Finan CL, Young M: A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement. BMC Genomics. 2005, 6 (1): 39-10.1186/1471-2164-6-39.PubMed CentralPubMedGoogle Scholar
- Hett EC, Chao MC, Deng LL, Rubin EJ: A mycobacterial enzyme essential for cell division synergizes with resuscitation-promoting factor. PLoS Pathog. 2008, 4 (2): e1000001-10.1371/journal.ppat.1000001.PubMed CentralPubMedGoogle Scholar
- Mukamolova GV, Turapov O, Malkin J, Woltmann G, Barer MR: Resuscitation-promoting factors reveal an occult population of tubercle bacilli in sputum. Am J Respir Crit Care Med. 2009, 181 (2): 174-180.PubMed CentralPubMedGoogle Scholar
- Goffin C, Ghuysen JM: Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs. Microbiol Mol Biol Rev. 1998, 62 (4): 1079-1093.PubMed CentralPubMedGoogle Scholar
- Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P: The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev. 2008, 32 (2): 234-258. 10.1111/j.1574-6976.2008.00105.x.PubMedGoogle Scholar
- Billman-Jacobe H, Haites RE, Coppel RL: Characterization of a Mycobacterium smegmatis mutant lacking penicillin binding protein 1. Antimicrob Agents Chemother. 1999, 43 (12): 3011-3013.PubMed CentralPubMedGoogle Scholar
- Patru MM, Pavelka MS: A role for the class A penicillin-binding protein PonA2 in the survival of Mycobacterium smegmatis under conditions of nonreplication. J Bacteriol. 2010, 192 (12): 3043-3054. 10.1128/JB.00025-10.PubMed CentralPubMedGoogle Scholar
- Dasgupta A, Datta P, Kundu M, Basu J: The serine/threonine kinase PknB of Mycobacterium tuberculosis phosphorylates PBPA, a penicillin-binding protein required for cell division. Microbiology. 2006, 152 (Pt 2): 493-504.PubMedGoogle Scholar
- Plocinski P, Ziolkiewicz M, Kiran M, Vadrevu SI, Nguyen HB, Hugonnet J, Veckerle C, Arthur M, Dziadek J, Cross TA, Madiraju M, Rajagopalan M: Characterization of CrgA, a new partner of the Mycobacterium tuberculosis peptidoglycan polymerization complexes. J Bacteriol. 2011, 193 (13): 3246-3256. 10.1128/JB.00188-11.PubMed CentralPubMedGoogle Scholar
- Ghosh SS, Dakoji S, Tanaka Y, Cho YJ, Mobashery S: Properties of analogues of an intermediate in the process of mechanism-based inactivation of carboxypeptidase A. Bioorg Med Chem. 1996, 4 (9): 1487-1492. 10.1016/0968-0896(96)00142-3.PubMedGoogle Scholar
- Bourai N, Jacobs WR, Narayanan S: Deletion and overexpression studies on DacB2, a putative low molecular mass penicillin binding protein from Mycobacterium tuberculosis H37Rv. Microb Pathog. 2012, 52 (2): 109-116. 10.1016/j.micpath.2011.11.003.PubMedGoogle Scholar
- Kumar P, Arora K, Lloyd JR, Lee IY, Nair V, Fischer E, Boshoff HI, Barry CE: Meropenem inhibits D, D-carboxypeptidase activity in Mycobacterium tuberculosis. Mol Microbiol. 2012, 86 (2): 367-381. 10.1111/j.1365-2958.2012.08199.x.PubMed CentralPubMedGoogle Scholar
- Hett EC, Chao MC, Steyn AJ, Fortune SM, Deng LL, Rubin EJ: A partner for the resuscitation-promoting factors of Mycobacterium tuberculosis. Mol Microbiol. 2007, 66 (3): 658-668. 10.1111/j.1365-2958.2007.05945.x.PubMedGoogle Scholar
- Hett EC, Chao MC, Rubin EJ: Interaction and modulation of two antagonistic cell wall enzymes of mycobacteria. PLoS Pathog. 2010, 6 (7): e1001020-10.1371/journal.ppat.1001020.PubMed CentralPubMedGoogle Scholar
- Chao MC, Kieser KJ, Minami S, Mavrici D, Aldridge BB, Fortune SM, Alber T, Rubin EJ: Protein complexes and proteolytic activation of the cell wall hydrolase RipA regulate septal resolution in mycobacteria. PLoS Pathog. 2013, 9 (2): e1003197-10.1371/journal.ppat.1003197.PubMed CentralPubMedGoogle Scholar
- Both D, Schneider G, Schnell R: Peptidoglycan remodeling in Mycobacterium tuberculosis: comparison of structures and catalytic activities of RipA and RipB. J Mol Biol. 2013, 413 (1): 247-260.Google Scholar
- Ruggiero A, Marasco D, Squeglia F, Soldini S, Pedone E, Pedone C, Berisio R: Structure and functional regulation of RipA, a mycobacterial enzyme essential for daughter cell separation. Structure. 2010, 18 (9): 1184-1190. 10.1016/j.str.2010.06.007.PubMedGoogle Scholar
- Gao LY, Pak M, Kish R, Kajihara K, Brown EJ: A mycobacterial operon essential for virulence in vivo and invasion and intracellular persistence in macrophages. Infect Immun. 2006, 74 (3): 1757-1767. 10.1128/IAI.74.3.1757-1767.2006.PubMed CentralPubMedGoogle Scholar
- Both D, Steiner EM, Izumi A, Schneider G, Schnell R: RipD (Rv1566c) from Mycobacterium tuberculosis: adaptation of an NlpC/p60 domain to a non-catalytic peptidoglycan-binding function. Biochem J. 2013, 457 (1): 33-41.Google Scholar
- Parthasarathy G, Lun S, Guo H, Ammerman NC, Geiman DE, Bishai WR: Rv2190c, an NlpC/P60 family protein, is required for full virulence of Mycobacterium tuberculosis. PLoS One. 2012, 7 (8): e43429-10.1371/journal.pone.0043429.PubMed CentralPubMedGoogle Scholar
- Lavollay M, Fourgeaud M, Herrmann JL, Dubost L, Marie A, Gutmann L, Arthur M, Mainardi JL: The peptidoglycan of Mycobacterium abscessus is predominantly cross-linked by L, D-transpeptidases. J Bacteriol. 2011, 193 (3): 778-782. 10.1128/JB.00606-10.PubMed CentralPubMedGoogle Scholar
- Lavollay M, Arthur M, Fourgeaud M, Dubost L, Marie A, Veziris N, Blanot D, Gutmann L, Mainardi JL: The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by L, D-transpeptidation. J Bacteriol. 2008, 190 (12): 4360-4366. 10.1128/JB.00239-08.PubMed CentralPubMedGoogle Scholar
- Gupta R, Lavollay M, Mainardi JL, Arthur M, Bishai WR, Lamichhane G: The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin. Nat Med. 2010, 16 (4): 466-469. 10.1038/nm.2120.PubMed CentralPubMedGoogle Scholar
- Both D, Steiner EM, Stadler D, Lindqvist Y, Schnell R, Schneider G: Structure of LdtMt2, an L, D-transpeptidase from Mycobacterium tuberculosis. Acta Crystallogr D Biol Crystallogr. 2013, 69 (Pt 3): 432-441.PubMed CentralPubMedGoogle Scholar
- Schoonmaker MK, Bishai WR, Lamichhane G: Non-classical transpeptidases of Mycobacterium tuberculosis alter cell size, morphology, cytosolic matrix, protein localization, virulence and resistance to beta-lactams. J Bacteriol. 2014, 196 (7): 1394-1402. 10.1128/JB.01396-13.PubMed CentralPubMedGoogle Scholar
- Cordillot M, Dubee V, Triboulet S, Dubost L, Marie A, Hugonnet JE, Arthur M, Mainardi JL: In vitro cross-linking of peptidoglycan by Mycobacterium tuberculosis L,D-transpeptidases and inactivation of these enzymes by carbapenems. Antimicrob Agents Chemother. 2013, 57 (12): 5940-5945. 10.1128/AAC.01663-13.PubMed CentralPubMedGoogle Scholar
- Dubee V, Triboulet S, Mainardi JL, Etheve-Quelquejeu M, Gutmann L, Marie A, Dubost L, Hugonnet JE, Arthur M: Inactivation of Mycobacterium tuberculosis L, D-transpeptidase LdtMt1 by carbapenems and cephalosporins. Antimicrob Agents Chemother. 2012, 56 (8): 4189-4195. 10.1128/AAC.00665-12.PubMed CentralPubMedGoogle Scholar
- Heidrich C, Templin MF, Ursinus A, Merdanovic M, Berger J, Schwarz H, de Pedro MA, Holtje JV: Involvement of N-acetylmuramyl-L-alanine amidases in cell separation and antibiotic-induced autolysis of Escherichia coli. Mol Microbiol. 2001, 41 (1): 167-178. 10.1046/j.1365-2958.2001.02499.x.PubMedGoogle Scholar
- Korsak D, Liebscher S, Vollmer W: Susceptibility to antibiotics and beta-lactamase induction in murein hydrolase mutants of Escherichia coli. Antimicrob Agents Chemother. 2005, 49 (4): 1404-1409. 10.1128/AAC.49.4.1404-1409.2005.PubMed CentralPubMedGoogle Scholar
- Jacobs C, Joris B, Jamin M, Klarsov K, Van Beeumen J, Mengin-Lecreulx D, van Heijenoort J, Park JT, Normark S, Frere JM: AmpD, essential for both beta-lactamase regulation and cell wall recycling, is a novel cytosolic N-acetylmuramyl-L-alanine amidase. Mol Microbiol. 1995, 15 (3): 553-559. 10.1111/j.1365-2958.1995.tb02268.x.PubMedGoogle Scholar
- Popham DL, Helin J, Costello CE, Setlow P: Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proc Natl Acad Sci U S A. 1996, 93 (26): 15405-15410. 10.1073/pnas.93.26.15405.PubMed CentralPubMedGoogle Scholar
- Smith TJ, Blackman SA, Foster SJ: Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. Microbiology. 2000, 146 (Pt 2): 249-262.PubMedGoogle Scholar
- Prigozhin DM, Mavrici D, Huizar JP, Vansell HJ, Alber T: Structural and Biochemical Analyses of Mycobacterium tuberculosis N-Acetylmuramyl-L-alanine Amidase Rv3717 Point to a Role in Peptidoglycan Fragment Recycling. J Biol Chem. 2013, 288 (44): 31549-31555. 10.1074/jbc.M113.510792.PubMed CentralPubMedGoogle Scholar
- Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson NR, Wheeler PR, Honoré N, Garnier T, Churcher C, Harris D, Mungall K, Basham D, Brown D, Chillingworth T, Connor R, Davies RM, Devlin K, Duthoy S, Feltwell T, Fraser A, Hamlin N, Holroyd S, Hornsby T, Jagels K, Lacroix C, Maclean J, Moule S, Murphy L, Oliver K, Quail MA, et al: Massive gene decay in the leprosy bacillus. Nature. 2001, 409 (6823): 1007-1011. 10.1038/35059006.PubMedGoogle Scholar
- Kana D, Machowski E, Schechter N, Shin JT, Rubin H, Mizrahi V: Electron transport and respiration. Mycobacterium: Genomics and Molecular Biology. 2009, Caister Academic Press: Norfolk, UK, 35-64.Google Scholar
- Williams MJ, Kana BD, Mizrahi V: Functional analysis of molybdopterin biosynthesis in mycobacteria identifies a fused molybdopterin synthase in Mycobacterium tuberculosis. J Bacteriol. 2011, 193 (1): 98-106. 10.1128/JB.00774-10.PubMed CentralPubMedGoogle Scholar
- Bentley SD, Comas I, Bryant JM, Walker D, Smith NH, Harris SR, Thurston S, Gagneux S, Wood J, Antonio M, Quail MA, Gehre F, Adegbola RA, Parkhill J, de Jong BC: The genome of Mycobacterium africanum West African 2 reveals a lineage-specific locus and genome erosion common to the M. tuberculosis complex. PLoS Negl Trop Dis. 2012, 6 (2): e1552-10.1371/journal.pntd.0001552.PubMed CentralPubMedGoogle Scholar
- Brosch R, Gordon SV, Garnier T, Eiglmeier K, Frigui W, Valenti P, Dos Santos S, Duthoy S, Lacroix C, Garcia-Pelayo C, Inwald JK, Golby P, Garcia JN, Hewinson RG, Behr MA, Quail MA, Churcher C, Barrell BG, Parkhill J, Cole ST: Genome plasticity of BCG and impact on vaccine efficacy. Proc Natl Acad Sci U S A. 2007, 104 (13): 5596-5601. 10.1073/pnas.0700869104.PubMed CentralPubMedGoogle Scholar
- Fleischmann RD, Alland D, Eisen JA, Carpenter L, White O, Peterson J, DeBoy R, Dodson R, Gwinn M, Haft D, Hickey E, Kolonay JF, Nelson WC, Umayam LA, Ermolaeva M, Salzberg SL, Delcher A, Utterback T, Weidman J, Khouri H, Gill J, Mikula A, Bishai W, Jacobs Jr WR, Venter JC, Fraser CM: Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J Bacteriol. 2002, 184 (19): 5479-5490. 10.1128/JB.184.19.5479-5490.2002.PubMed CentralPubMedGoogle Scholar
- Garnier T, Eiglmeier K, Camus JC, Medina N, Mansoor H, Pryor M, Duthoy S, Grondin S, Lacroix C, Monsempe C, Simon S, Harris B, Atkin R, Doggett J, Mayes R, Keating L, Wheeler PR, Parkhill J, Barrell BG, Cole ST, Gordon SV, Hewinson RG: The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci U S A. 2003, 100 (13): 7877-7882. 10.1073/pnas.1130426100.PubMed CentralPubMedGoogle Scholar
- Kim BJ, Choi BS, Lim JS, Choi IY, Lee JH, Chun J, Kook YH, Kim BJ: Complete genome sequence of Mycobacterium intracellulare strain ATCC 13950(T). J Bacteriol. 2012, 194 (10): 2750-10.1128/JB.00295-12.PubMed CentralPubMedGoogle Scholar
- Li L, Bannantine JP, Zhang Q, Amonsin A, May BJ, Alt D, Banerji N, Kanjilal S, Kapur V: The complete genome sequence of Mycobacterium avium subspecies paratuberculosis. Proc Natl Acad Sci U S A. 2005, 102 (35): 12344-12349. 10.1073/pnas.0505662102.PubMed CentralPubMedGoogle Scholar
- Shallom SJ, Gardina PJ, Myers TG, Sebastian Y, Conville P, Calhoun LB, Tettelin H, Olivier KN, Uzel G, Sampaio EP, Holland SM, Zelazny AM: New Rapid Scheme for Distinguishing the Subspecies of the Mycobacterium abscessus Group and Identifying Mycobacterium massiliense Isolates with Inducible Clarithromycin Resistance. J Clin Microbiol. 2013, 51 (9): 2943-2949. 10.1128/JCM.01132-13.PubMed CentralPubMedGoogle Scholar
- Stinear TP, Seemann T, Harrison PF, Jenkin GA, Davies JK, Johnson PD, Abdellah Z, Arrowsmith C, Chillingworth T, Churcher C, Clarke K, Cronin A, Davis P, Goodhead I, Holroyd N, Jagels K, Lord A, Moule S, Mungall K, Norbertczak H, Quail MA, Rabbinowitsch E, Walker D, White B, Whitehead S, Small PL, Brosch R, Ramakrishnan L, Fischbach MA, Parkhill J, Cole ST: Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Res. 2008, 18 (5): 729-741. 10.1101/gr.075069.107.PubMed CentralPubMedGoogle Scholar
- Stinear TP, Seemann T, Pidot S, Frigui W, Reysset G, Garnier T, Meurice G, Simon D, Bouchier C, Ma L, Tichit M, Porter JL, Ryan J, Johnson PD, Davies JK, Jenkin GA, Small PL, Jones LM, Tekaia F, Laval F, Daffé M, Parkhill J, Cole ST: Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer. Genome Res. 2007, 17 (2): 192-200. 10.1101/gr.5942807.PubMed CentralPubMedGoogle Scholar
- Zheng H, Lu L, Wang B, Pu S, Zhang X, Zhu G, Shi W, Zhang L, Wang H, Wang S, Zhao G, Zhang Y: Genetic basis of virulence attenuation revealed by comparative genomic analysis of Mycobacterium tuberculosis strain H37Ra versus H37Rv. PLoS One. 2008, 3 (6): e2375-10.1371/journal.pone.0002375.PubMed CentralPubMedGoogle Scholar
- Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW: GenBank. Nucleic Acids Res. 2013, 41 (Database issue): D36-D42.PubMed CentralPubMedGoogle Scholar
- Cole ST: Learning from the genome sequence of Mycobacterium tuberculosis H37Rv. FEBS Lett. 1999, 452 (1–2): 7-10.PubMedGoogle Scholar
- Lechat P, Hummel L, Rousseau S, Moszer I: GenoList: an integrated environment for comparative analysis of microbial genomes. Nucleic Acids Res. 2008, 36 (Database issue): D469-D474.PubMed CentralPubMedGoogle Scholar
- Reddy TB, Riley R, Wymore F, Montgomery P, DeCaprio D, Engels R, Gellesch M, Hubble J, Jen D, Jin H, Koehrsen M, Larson L, Mao M, Nitzberg M, Sisk P, Stolte C, Weiner B, White J, Zachariah ZK, Sherlock G, Galagan JE, Ball CA, Schoolnik GK, et al: TB database: an integrated platform for tuberculosis research. Nucleic Acids Res. 2009, 37 (Database issue): D499-D508.PubMed CentralPubMedGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215 (3): 403-410.PubMedGoogle Scholar
- Kapopoulou A, Lew JM, Cole ST: The MycoBrowser portal: a comprehensive and manually annotated resource for mycobacterial genomes. Tuberculosis (Edinb). 2011, 91 (1): 8-13. 10.1016/j.tube.2010.09.006.Google Scholar
- Carver TJ, Rutherford KM, Berriman M, Rajandream MA, Barrell BG, Parkhill J: ACT: the Artemis Comparison Tool. Bioinformatics. 2005, 21 (16): 3422-3423. 10.1093/bioinformatics/bti553.PubMedGoogle Scholar
- Markowitz VM, Chen IM, Palaniappan K, Chu K, Szeto E, Grechkin Y, Ratner A, Jacob B, Huang J, Williams P, Huntemann M, Anderson I, Mavromatis K, Ivanova NN, Kyrpides NC: IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 2012, 40 (Database issue): D115-D122.PubMed CentralPubMedGoogle Scholar
- Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG: Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011, 7: 539-PubMed CentralPubMedGoogle Scholar
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal X version 2.0. Bioinformatics. 2007, 23 (21): 2947-2948. 10.1093/bioinformatics/btm404.PubMedGoogle Scholar
- Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R: InterProScan: protein domains identifier. Nucleic Acids Res. 2005, 33 (Web Server issue): W116-W120.PubMed CentralPubMedGoogle Scholar
- Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer EL, Eddy SR, Bateman A, Finn RD, et al: The Pfam protein families database. Nucleic Acids Res. 2012, 40 (Database issue): D290-D301.PubMed CentralPubMedGoogle Scholar
- Petersen TN, Brunak S, von Heijne G, Nielsen H: SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011, 8 (10): 785-786. 10.1038/nmeth.1701.PubMedGoogle Scholar
- Moller S, Croning MD, Apweiler R: Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics. 2001, 17 (7): 646-653. 10.1093/bioinformatics/17.7.646.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.