Comparative genome analysis of mycobacteria focusing on tRNA and non-coding RNA.

BACKGROUND: The Mycobacterium genus encompasses at least 192 named species, many of which cause severe diseases such as tuberculosis. Non-tuberculosis mycobacteria (NTM) can also infect humans and animals. Some are of emerging concern because they show high resistance to commonly used antibiotics while others are used and evaluated in bioremediation or included in anticancer vaccines. RESULTS: We provide the genome sequences for 114 mycobacterial type strains and together with 130 available mycobacterial genomes we generated a phylogenetic tree based on 387 core genes and supported by average nucleotide identity (ANI) data. The 244 genome sequences cover most of the species constituting the Mycobacterium genus. The genome sizes ranged from 3.2 to 8.1 Mb with an average of 5.7 Mb, and we identified 14 new plasmids. Moreover, mycobacterial genomes consisted of phage-like sequences ranging between 0 and 4.64% dependent on mycobacteria while the number of IS elements varied between 1 and 290. Our data also revealed that, depending on the mycobacteria, the number of tRNA and non-coding (nc) RNA genes differ and that their positions on the chromosome varied. We identified a conserved core set of 12 ncRNAs, 43 tRNAs and 18 aminoacyl-tRNA synthetases among mycobacteria. CONCLUSIONS: Phages, IS elements, tRNA and ncRNAs appear to have contributed to the evolution of the Mycobacterium genus where several tRNA and ncRNA genes have been horizontally transferred. On the basis of our phylogenetic analysis, we identified several isolates of unnamed species as new mycobacterial species or strains of known mycobacteria. The predicted number of coding sequences correlates with genome size while the number of tRNA, rRNA and ncRNA genes does not. Together these findings expand our insight into the evolution of the Mycobacterium genus and as such they establish a platform to understand mycobacterial pathogenicity, their evolution, antibiotic resistance/tolerance as well as the function and evolution of ncRNA among mycobacteria.

Intracellular localization of the mycobacterial stressosome complex.

Microorganisms survive stresses by alternating the expression of genes suitable for surviving the immediate and present danger and eventually adapt to new conditions. Many bacteria have evolved a multiprotein "molecular machinery" designated the "Stressosome" that integrates different stress signals and activates alternative sigma factors for appropriate downstream responses. We and others have identified orthologs of some of the Bacillus subtilis stressosome components, RsbR, RsbS, RsbT and RsbUVW in several mycobacteria and we have previously reported mutual interactions among the stressosome components RsbR, RsbS, RsbT and RsbUVW from Mycobacterium marinum. Here we provide evidence that "STAS" domains of both RsbR and RsbS are important for establishing the interaction and thus critical for stressosome assembly. Fluorescence microscopy further suggested co-localization of RsbR and RsbS in multiprotein complexes visible as co-localized fluorescent foci distributed at scattered locations in the M. marinum cytoplasm; the number, intensity and distribution of such foci changed in cells under stressed conditions. Finally, we provide bioinformatics data that 17 (of 244) mycobacteria, which lack the RsbRST genes, carry homologs of Bacillus cereus genes rsbK and rsbM indicating the existence of alternative σF activation pathways among mycobacteria.

Author Correction: Extensive genomic diversity among Mycobacterium marinum strains revealed by whole genome sequencing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Insight into the biology of Mycobacterium mucogenicum and Mycobacterium neoaurum clade members.

Nontuberculous mycobacteria, NTM, are of growing concern and among these members of the Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clades can cause infections in humans and they are resistant to first-line anti-tuberculosis drugs. They can be isolated from different ecological niches such as soil, tap water and ground water. Mycobacteria, such as Mmuc and Mneo, are classified as rapid growing mycobacteria, RGM, while the most familiar, Mycobacterium tuberculosis, belongs to the slow growing mycobacteria, SGM. Modern "omics" approaches have provided new insights into our understanding of the biology and evolution of this group of bacteria. Here we present comparative genomics data for seventeen NTM of which sixteen belong to the Mmuc- and Mneo-clades. Focusing on virulence genes, including genes encoding sigma/anti-sigma factors, serine threonine protein kinases (STPK), type VII (ESX genes) secretion systems and mammalian cell entry (Mce) factors we provide insight into their presence as well as phylogenetic relationship in the case of the sigma/anti-sigma factors and STPKs. Our data further suggest that these NTM lack ESX-5 and Mce2 genes, which are known to affect virulence. In this context, Mmuc- and Mneo-clade members lack several of the genes in the glycopeptidolipid (GLP) locus, which have roles in colony morphotype appearance and virulence. For the M. mucogenicum type strain, MmucT, we provide RNASeq data focusing on mRNA levels for sigma factors, STPK, ESX proteins and Mce proteins. These data are discussed and compared to in particular the SGM and fish pathogen Mycobacterium marinum. Finally, we provide insight into as to why members of the Mmuc- and Mneo-clades show resistance to rifampin and isoniazid, and why MmucT forms a rough colony morphotype.

Comparative genomics of Mycobacterium mucogenicum and Mycobacterium neoaurum clade members emphasizing tRNA and non-coding RNA.

BACKGROUND: Mycobacteria occupy various ecological niches and can be isolated from soil, tap water and ground water. Several cause diseases in humans and animals. To get deeper insight into our understanding of mycobacterial evolution focusing on tRNA and non-coding (nc)RNA, we conducted a comparative genome analysis of Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clade members. RESULTS: Genome sizes for Mmuc- and Mneo-clade members vary between 5.4 and 6.5 Mbps with the complete MmucT (type strain) genome encompassing 6.1 Mbp. The number of tRNA genes range between 46 and 79 (including one pseudo tRNA gene) with 39 tRNA genes common among the members of these clades, while additional tRNA genes were probably acquired through horizontal gene transfer. Selected tRNAs and ncRNAs (RNase P RNA, tmRNA, 4.5S RNA, Ms1 RNA and 6C RNA) are expressed, and the levels for several of these are higher in stationary phase compared to exponentially growing cells. The rare tRNAIleTAT isoacceptor and two for mycobacteria novel ncRNAs: the Lactobacillales-derived GOLLD RNA and a homolog to the antisense Salmonella typhimurium phage Sar RNA, were shown to be present and expressed in certain Mmuc-clade members. CONCLUSIONS: Phages, IS elements, horizontally transferred tRNA gene clusters, and phage-derived ncRNAs appears to have influenced the evolution of the Mmuc- and Mneo-clades. While the number of predicted coding sequences correlates with genome size, the number of tRNA coding genes does not. The majority of the tRNA genes in mycobacteria are transcribed mainly from single genes and the levels of certain ncRNAs, including RNase P RNA (essential for the processing of tRNAs), are higher at stationary phase compared to exponentially growing cells. We provide supporting evidence that Ms1 RNA represents a mycobacterial 6S RNA variant. The evolutionary routes for the ncRNAs RNase P RNA, tmRNA and Ms1 RNA are different from that of the core genes.

Extended insight into the Mycobacterium chelonae-abscessus complex through whole genome sequencing of Mycobacterium salmoniphilum outbreak and Mycobacterium salmoniphilum-like strains.

Members of the Mycobacterium chelonae-abscessus complex (MCAC) are close to the mycobacterial ancestor and includes both human, animal and fish pathogens. We present the genomes of 14 members of this complex: the complete genomes of Mycobacterium salmoniphilum and Mycobacterium chelonae type strains, seven M. salmoniphilum isolates, and five M. salmoniphilum-like strains including strains isolated during an outbreak in an animal facility at Uppsala University. Average nucleotide identity (ANI) analysis and core gene phylogeny revealed that the M. salmoniphilum-like strains are variants of the human pathogen Mycobacterium franklinii and phylogenetically close to Mycobacterium abscessus. Our data further suggested that M. salmoniphilum separates into three branches named group I, II and III with the M. salmoniphilum type strain belonging to group II. Among predicted virulence factors, the presence of phospholipase C (plcC), which is a major virulence factor that makes M. abscessus highly cytotoxic to mouse macrophages, and that M. franklinii originally was isolated from infected humans make it plausible that the outbreak in the animal facility was caused by a M. salmoniphilum-like strain. Interestingly, M. salmoniphilum-like was isolated from tap water suggesting that it can be present in the environment. Moreover, we predicted the presence of mutational hotspots in the M. salmoniphilum isolates and 26% of these hotspots overlap with genes categorized as having roles in virulence, disease and defense. We also provide data about key genes involved in transcription and translation such as sigma factor, ribosomal protein and tRNA genes.

Extensive genomic diversity among Mycobacterium marinum strains revealed by whole genome sequencing.

Mycobacterium marinum is the causative agent for the tuberculosis-like disease mycobacteriosis in fish and skin lesions in humans. Ubiquitous in its geographical distribution, M. marinum is known to occupy diverse fish as hosts. However, information about its genomic diversity is limited. Here, we provide the genome sequences for 15 M. marinum strains isolated from infected humans and fish. Comparative genomic analysis of these and four available genomes of the M. marinum strains M, E11, MB2 and Europe reveal high genomic diversity among the strains, leading to the conclusion that M. marinum should be divided into two different clusters, the "M"- and the "Aronson"-type. We suggest that these two clusters should be considered to represent two M. marinum subspecies. Our data also show that the M. marinum pan-genome for both groups is open and expanding and we provide data showing high number of mutational hotspots in M. marinum relative to other mycobacteria such as Mycobacterium tuberculosis. This high genomic diversity might be related to the ability of M. marinum to occupy different ecological niches.

The Mycobacterium phlei Genome: Expectations and Surprises.

Mycobacterium phlei, a nontuberculosis mycobacterial species, was first described in 1898-1899. We present the complete genome sequence for theM. phlei CCUG21000(T)type strain and the draft genomes for four additional strains. The genome size for all five is 5.3 Mb with 69.4% Guanine-Cytosine content. This is ≈0.35 Mbp smaller than the previously reported M. phlei RIVM draft genome. The size difference is attributed partly to large bacteriophage sequence fragments in theM. phlei RIVM genome. Comparative analysis revealed the following: 1) A CRISPR system similar to Type 1E (cas3) in M. phlei RIVM; 2) genes involved in polyamine metabolism and transport (potAD,potF) that are absent in other mycobacteria, and 3) strain-specific variations in the number of σ-factor genes. Moreover,M. phlei has as many as 82 mce(mammalian cell entry) homologs and many of the horizontally acquired genes in M. phlei are present in other environmental bacteria including mycobacteria that share similar habitat. Phylogenetic analysis based on 693 Mycobacterium core genes present in all complete mycobacterial genomes suggested that its closest neighbor is Mycobacterium smegmatis JS623 and Mycobacterium rhodesiae NBB3, while it is more distant toM. smegmatis mc2 155.

Comparative Sigma Factor-mRNA Levels in Mycobacterium marinum under Stress Conditions and during Host Infection.

We have used RNASeq and qRT-PCR to study mRNA levels for all σ-factors in different Mycobacterium marinum strains under various growth and stress conditions. We also studied their levels in M. marinum from infected fish and mosquito larvae. The annotated σ-factors were expressed and transcripts varied in relation to growth and stress conditions. Some were highly abundant such as sigA, sigB, sigC, sigD, sigE and sigH while others were not. The σ-factor mRNA profiles were similar after heat stress, during infection of fish and mosquito larvae. The similarity also applies to some of the known heat shock genes such as the α-crystallin gene. Therefore, it seems probable that the physiological state of M. marinum is similar when exposed to these different conditions. Moreover, the mosquito larvae data suggest that this is the state that the fish encounter when infected, at least with respect to σ-factor mRNA levels. Comparative genomic analysis of σ-factor gene localizations in three M. marinum strains and Mycobacterium tuberculosis H37Rv revealed chromosomal rearrangements that changed the localization of especially sigA, sigB, sigD, sigE, sigF and sigJ after the divergence of these two species. This may explain the variation in species-specific expression upon exposure to different growth conditions.

Characterization of Three Mycobacterium spp. with Potential Use in Bioremediation by Genome Sequencing and Comparative Genomics.

We provide the genome sequences of the type strains of the polychlorophenol-degrading Mycobacterium chlorophenolicum (DSM43826), the degrader of chlorinated aliphatics Mycobacterium chubuense (DSM44219) and Mycobacterium obuense (DSM44075) that has been tested for use in cancer immunotherapy. The genome sizes of M. chlorophenolicum, M. chubuense, and M. obuense are 6.93, 5.95, and 5.58 Mb with GC-contents of 68.4%, 69.2%, and 67.9%, respectively. Comparative genomic analysis revealed that 3,254 genes are common and we predicted approximately 250 genes acquired through horizontal gene transfer from different sources including proteobacteria. The data also showed that the biodegrading Mycobacterium spp. NBB4, also referred to as M. chubuense NBB4, is distantly related to the M. chubuense type strain and should be considered as a separate species, we suggest it to be named Mycobacterium ethylenense NBB4. Among different categories we identified genes with potential roles in: biodegradation of aromatic compounds and copper homeostasis. These are the first nonpathogenic Mycobacterium spp. found harboring genes involved in copper homeostasis. These findings would therefore provide insight into the role of this group of Mycobacterium spp. in bioremediation as well as the evolution of copper homeostasis within the Mycobacterium genus.

Draft Genome Sequence of Saccharopolyspora rectivirgula.

We have sequenced the genome of Saccharopolyspora rectivirgula, the causative agent of farmer's lung disease. The draft genome consists of 182 contigs totaling 3,977,051 bp, with a GC content of 68.9%.

Uterine cervix cancer treatment at Radiumhemmet: 90 years' experience. Time trends of age, stage, and histopathology distribution.

Since the introduction of screening programs for cervical cancer (CC) the incidence has decreased and CC is discovered at an earlier stage. The purpose of this study was to analyze time trends in age, stage, and histopathology over a 90-year period and to our knowledge this is the largest single institutional series in the literature of invasive cervical carcinoma (CC) cases. This is a retrospective study comprising 18,472 women treated for CC from 1914 until 2004 at Radiumhemmet, Stockholm. The material is part of the international CC statistics published since 1937 in the League of Nations' Annual Reports, and since 1958 under the patronage of International Federation of Gynecology and Obstetrics (FIGO). During the 90-year study period, the annual number of cases treated increased to over 400 up until 1965, after which there was a gradual drop to less than 100 cases in 2004. A pronounced shift toward earlier stages at diagnosis was noted. The mean age at diagnosis increased in all stages, predominantly in advanced stages. A reduction in squamous cell carcinoma (SCC) cases and a sixfold increase in the proportion of adenocarcinoma (AC) cases were observed. The mean age at diagnosis for squamous and AC cases shifted after 1970, when the SCC cases ultimately became 3 years older than the AC cases in contrast to around 1950 when they were 3 years younger than the AC cases. The changes in the distribution by age, stage, and histopathology during this 90-year period are probably associated with: improved social conditions and increased access to health care, the introduction of screening programs for CC in the 1960s, and a change in the risk factors for CC (changed sexual behavior, introduction of contraceptive pills, and changed smoking habits).

Identification and expression of stressosomal proteins in Mycobacterium marinum under various growth and stress conditions.

Like other bacteria, Mycobacterium spp. have developed different strategies in response to environmental changes such as nutrient limitations and other different stress situations. We have identified candidate genes (rsb genes) from Mycobacterium marinum involved in the regulation of the activity of the alternative sigma factor, σ(F) . This is a homolog of the master regulator of general stress response, σ(B) , and the sporulation-specific sigma factor, σ(F) , in Bacillus subtilis. The organization of these genes in M. marinum and B. subtilis is similar. Transcriptome and qRT-PCR data show that these genes are indeed expressed in M. marinum and that the levels of expression vary with growth phase and exposure to stress. In particular, cold stress caused a significant rise in the expression of all identified rsb and sigF genes. We discuss these data in relation to what is currently known for other Mycobacterium spp.

Asymmetric growth and division in Mycobacterium spp.: compensatory mechanisms for non-medial septa.

Mycobacterium spp., rod-shaped cells belonging to the phylum Actinomycetes, lack the Min- and Noc/Slm systems responsible for preventing the placement of division sites at the poles or over the nucleoids to ensure septal assembly at mid-cell. We show that the position for establishment of the FtsZ-ring in exponentially growing Mycobacterium marinum and Mycobacterium smegmatis cells is nearly random, and that the cells often divide non-medially, producing two unequal but viable daughters. Septal sites and cellular growth disclosed by staining with the membrane-specific dye FM4-64 and fluorescent antibiotic vancomycin (FL-Vanco), respectively, showed that many division sites were off-centre, often over the nucleoids, and that apical cell growth was frequently unequal at the two poles. DNA transfer through the division septum was detected, and translocation activity was supported by the presence of a putative mycobacterial DNA translocase (MSMEG2690) at the majority of the division sites. Time-lapse imaging of single live cells through several generations confirmed both acentric division site placement and unequal polar growth in mycobacteria. Our evidence suggests that post-septal DNA transport and unequal polar growth may compensate for the non-medial division site placement in Mycobacterium spp.

Cervical cancer in the screening era: who fell victim in spite of successful screening programs?

OBJECTIVE: To compare profiles of a prescreening and screening cohort of women with cervical cancer regarding histopathology and clinical variables in order to identify those remaining at risk despite successful screening programs. By analyzing these profiles we hope to improve future screening methods. METHODS: The prescreening and screening cohorts consisted of 5,046 and 1,174 women, respectively, treated for cervical cancer at the Department of Gynecological Oncology at Radiumhemmet, Karolinska University Hospital, during the periods 1944-1957 and 1990-2004. RESULTS: Mean age increased from 48.9 years to 55.3 years in the cohorts treated 1944-1957 and 1990-2004, respectively. The percentage of patients older than 69 years was 5.4% and 27.3% in the prescreening and screening period, respectively. A shift towards earlier stages at diagnosis, a reduction of squamous cervical cancer and an increase of adenocarcinoma were observed in the screening cohort. The percentage of adenocarcinoma was about 6 times higher among younger patients. Cases of stump cancer and cervical cancer associated with pregnancy have declined. Eighty-seven women in the screening cohort had a history of treatment for in situ carcinoma by conization; 28% of these cases developed cervical cancer within one year after conization. CONCLUSION: The profile changed in the screening era indicating a need to refine screening for improved detection of in older women. This study, one of the largest clinical series of cervical cancer, provides an important baseline with which later studies can be compared to evaluate the effects of human papillomavirus vaccine and other important changes in this field.

Carcinoma of the cervical stump: fifty years of experience.

A series of patients with carcinoma of the cervical stump in relation to age, clinical stage, histopathology, changes in relative incidence, treatment outcome and long-term survival, were studied and the findings were compared with matched controls that have an intact uterus and cancer of the cervix. Of 8,028 women treated for invasive cervical carcinoma between 1959-2004, 161 were diagnosed with stump cancer, accounting for 2.0% of all cervical cancers. The mean time interval between subtotal hysterectomy and stump cancer diagnosis was 17.6 years, with a range of 1-46 years. In 80% of cases, symptoms drove the patient to seek medical attention and postcoital, intermenstrual or postmenopausal bleeding was the main reason. Among 161 stump cancer cases 89% were squamous cell carcinoma (SCC) and the remaining 17 cases were adenocarcinomas (AC). Cumulative cause-specific survival rate was significantly worse for adenocarcinoma than for squamous cell carcinoma (SCC) of the cervical stump (Log-rank p = 0.027, Cox-Mantel p = 0.015, Cox F-test p = 0.01). The stump cancer cases show a worse stage profile compared with the cancer cases in intact uterus. We conclude that the total effect of stump cancers following subtotal hysterectomies is not to be neglected.

tRNA accumulation and suppression of the bldA phenotype during development in Streptomyces coelicolor.

Streptomyces coelicolor undergoes distinct morphological changes as it grows on solid media where spores differentiate into vegetative and aerial mycelium that is followed by the production of spores. Deletion of bldA, encoding the rare tRNA(Leu) UAA, blocks development at the stage of vegetative mycelium formation. From previous data it appears that tRNA(Leu) UAA accumulates relatively late during growth while two other tRNAs do not. Here, we studied the expression of 17 different tRNAs including bldA tRNA, and the RNA subunit of the tRNA processing endoribonuclease RNase P. Our results showed that all selected tRNAs and RNase P RNA increased with time during development. However, accumulation of bldA tRNA and another rare tRNA(Leu) isoacceptor started at an earlier stage compared with the other tRNAs. We also introduced the bldA tRNA anticodon (UAA) into other tRNAs and introduced these into a bldA deletion strain. In particular, one such mutant tRNA derived from the tRNA(Leu) CAA isoacceptor suppressed the bldA phenotype. Thus, the bldA tRNA scaffold is not critical for function as a regulator of S. coelicolor cell differentiation. Further substitution experiments, in which the 5'- and 3'-flanking regions of the suppressor tRNA were changed, indicated that these regions were important for the suppression.

Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.

RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.

Invasive carcinoma of the uterine cervix associated with pregnancy: 90 years of experience.

BACKGROUND: This study is a representation of 90 years of experience with carcinoma of the uterine cervix in pregnancy. The objective was to retrospectively study changes in the distribution of cervical carcinoma (CC) by age, disease stage, histopathology, survival, and the development of second primary cancers. METHODS: Altogether, 18,474 women with newly diagnosed CC were examined and treated at the Radiumhemmet between 1914 and 2004, including 9247 women who were of a childbearing age (<50 years) and 219 women who were pregnant. RESULTS: The mean patient age declined from 35 years (during 1914-1943) to 32.2 years (during 1960-2004). Similarly, the age range changed from ages 23 to 51 years (during 1914-1943) to ages 21 to 47 years (during 1960-2004). The relative incidence for all women aged <50 years who were treated for CC dropped considerably from 4.2% (during 1914-1943) to 1.2% (during 1960-2004), which translated into a reduction of by approximately 66%. At the time of diagnosis, stage I CC was observed in 75.6% of patients during 1960 to 2004 compared with 24.8% of patients during 1914 to 1943. The 10-year actuarial survival rate improved significantly during the study period from 27% (1914-2004) to 79% (1960-2004). The 10-year cause-specific cumulative actuarial survival rate for 41 women who were treated during 1960 to 2004 did not differ statistically from the rate for an age-matched, stage-matched, and histopathology-matched control series from the total cohort of women with CC who were treated at the Radiumhemmet during the same period (log-rank test; P = .85). CONCLUSIONS: During the study period, the incidence of CC during pregnancy declined, the cases were discovered at earlier stages, and survival improved. Furthermore, there was no increase in second primary cancers, and pregnancy did not appear to influence prognosis.

Sporulation in mycobacteria.

Mycobacteria owe their success as pathogens to their ability to persist for long periods within host cells in asymptomatic, latent forms before they opportunistically switch to the virulent state. The molecular mechanisms underlying the transition into dormancy and emergence from it are not clear. Here we show that old cultures of Mycobacterium marinum contained spores that, upon exposure to fresh medium, germinated into vegetative cells and reappeared again in stationary phase via endospore formation. They showed many of the usual characteristics of well-known endospores. Homologues of well-known sporulation genes of Bacillus subtilis and Streptomyces coelicolor were detected in mycobacteria genomes, some of which were verified to be transcribed during appropriate life-cycle stages. We also provide data indicating that it is likely that old Mycobacterium bovis bacillus Calmette-Guérin cultures form spores. Together, our data show sporulation as a lifestyle adapted by mycobacteria under stress and tempt us to suggest this as a possible mechanism for dormancy and/or persistent infection. If so, this might lead to new prophylactic strategies.