Bacteria use population heterogeneity, the presence of more than one phenotypic variant in a clonal population, to endure diverse environmental challenges - a 'bet-hedging' strategy. Phenotypic variants have been described in many bacteria, but the phenomenon is not well-understood in mycobacteria, including the environmental factors that influence heterogeneity. Here, we describe three reproducible morphological variants in M. smegmatis - smooth, rough, and an intermediate morphotype that predominated under typical laboratory conditions. M. abscessus has two recognized morphotypes, smooth and rough. Interestingly, M. tuberculosis exists in only a rough form. The shift from smooth to rough in both M. smegmatis and M. abscessus was observed over time in extended static culture, however the frequency of the rough morphotype was high in pellicle preparations compared to planktonic culture, suggesting a role for an aggregated microenvironment in the shift to the rough form. Differences in growth rate, biofilm formation, cell wall composition, and drug tolerance were noted among M. smegmatis and M. abscessus variants. Deletion of the global regulator lsr2 shifted the M. smegmatis intermediate morphotype to a smooth form but did not fully phenocopy the naturally generated smooth morphotype, indicating Lsr2 is likely downstream of the initiating regulatory cascade that controls these morphotypes. Rough forms typically correlate with higher invasiveness and worse outcomes during infection and our findings indicate the shift to this rough form is promoted by aggregation. Our findings suggest that mycobacterial population heterogeneity, reflected in colony morphotypes, is a reproducible, programmed phenomenon that plays a role in adaptation to unique environments and this heterogeneity may influence infection progression and response to treatment.

The non-pathogenic bacterium Mycobacterium smegmatis mc2155 has been widely used as a model organism in mycobacterial research, yet a detailed study about its transcription landscape remains to be established. Here we report the transcriptome, expression profiles and transcriptional structures through growth-phase-dependent RNA sequencing (RNA-seq) as well as other related experiments. We found: (1) 2,139 transcriptional start sites (TSSs) in the genome-wide scale, of which eight samples were randomly selected and further verified by 5'-RACE; (2) 2,233 independent monocistronic or polycistronic mRNAs in the transcriptome within the operon/sub-operon structures which are classified into five groups; (3) 47.50% (1016/2139) genes were transcribed into leaderless mRNAs, with the TSSs of 41.3% (883/2139) mRNAs overlapping with the first base of the annotated start codon. Initial amino acids of MSMEG_4921 and MSMEG_6422 proteins were identified by Edman degradation, indicating the presence of distinctive widespread leaderless features in M. smegmatis mc2155. (4) 150 genes with potentially wrong structural annotation, of which 124 proposed genes have been corrected; (5) eight highly active promoters, with their activities further determined by β-galactosidase assays. These data integrated the transcriptional landscape to genome information of model organism mc2155 and lay a solid foundation for further works in Mycobacterium.

A new biotechnological process for the production of testosterone (TS) has been developed to turn the model strain Mycobacterium smegmatis suitable for TS production to compete with the current chemical synthesis procedures. We have cloned and overexpressed two genes encoding microbial 17β-hydroxysteroid: NADP 17-oxidoreductase, from the bacterium Comamonas testosteroni and from the fungus Cochliobolus lunatus. The host strains were M. smegmatis wild type and a genetic engineered androst-4-ene-3,17-dione (AD) producing mutant. The performances of the four recombinant bacterial strains have been tested both in growing and resting-cell conditions using natural sterols and AD as substrates respectively. These strains were able to produce TS from sterols or AD with high yields. This work represents a proof of concept of the possibilities that offers this model bacterium for the production of pharmaceutical steroids using metabolic engineering approaches.

Non-tuberculous mycobacteria can cause catheter associated blood stream infections. The causative agents are generally rapid growers that belong to the Mycobacterium fortuitum and Mycobacterium mucogenicum groups. A 65 year hospitalized patient with temporary central venous catheter who developed Mycobacterium smegmatis bacteremia. Bacteremia cleared after removal of the catheter. Patient was treated initially with 4 weeks of intravenous amikacin, intravenous meropenem, oral doxycycline and oral ethambutol and then deescalated to oral doxycycline and oral ciprofloxacin for 8 weeks. He improved clinically and remained stable. A literature search identified total of 22 articles that reported 47 unique cases of Mycobacterium smegmatis infection. To our knowledge, this is the first case of Mycobacterium smegmatis central venous catheter associated bacteremia in an immunocompetent host.

Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis.Methods: Recombinant wild type and mutant Rv2629 strains were constructed. Rv2629 expression was evaluated by real-time PCR and western blot. Microarray and interaction network analyses were used to identify the gene interactions associated with wild type and mutant Rv2629. Bacterial growth was assessed in Balb/c mice infected with wild type and mutant Rv2629 strains using CFU assay and histological analysis of the organs. Results: Overexpression of Rv2629 could delay the entry of the Mycobacterium tuberculosis cells into the log-phase, while Rv2629 decreased the number of ribosomes and the expression of uridylate kinase in Mycobacterium smegmatis. The Gene Ontology (GO) and pathway analysis indicated that 122 genes correlated with wild type Rv2629, whereas the Rv2629 mutation led to decrease in the ribosome production, oxidative phosphorylation, and virulence in Mycobacterium tuberculosis. Overexpression of Rv2629 slightly enhanced the drug resistance of Mycobacterium smegmatis to antibiotics, and increased its survival and pathogenicity in Balb/c mice. Conclusion: It is suggested that Rv2629 is involved in the survival of the clinical drug-resistant strain via bacterial growth repression and bacterial persistence induction.

The disaccharide d-N-acetylglucosamine-l-rhamnose plays an important role in the mycobacterial cell wall as a linker connecting arabinogalactan and peptidoglycan via a phosphodiester linkage. The first step of the disaccharide linker is the formation of decaprenyl phosphate-GlcNAc, which is catalyzed by GlcNAc-1-phosphate transferase. In Gram-negative bacteria, the wecA gene specifies the UDP-GlcNAc: undecaprenyl phosphate GlcNAc-1-phosphate transferase (WecA), which catalyzes the first step in the biosynthesis of lipopolysaccharide O-antigen. Mycobacterium tuberculosis Rv1302 and Mycobacterium smegmatis MSMEG_4947 show homology to Escherichia coli WecA protein. We cloned Rv1302 and MSMEG_4947 and introduced plasmids pYJ-1 (carrying Rv1302) and pYJ-2 (carrying MSMEG_4947) into a wecA-defective strain of E. coli MV501, respectively. Lipopolysaccharide analysis demonstrated that lipopolysaccharide synthesis in MV501 (pYJ-1) and MV501 (pYJ-2) was restored upon complementation with Rv1302 and MSMEG_4947, respectively. This provides the first evidence that Rv1302 and MSMEG_4947 have the same function as E. coli WecA. We also generated an M. smegmatis MSMEG_4947 knockout mutant using a homologous recombination strategy. The disruption of MSMEG_4947 in the M. smegmatis genome resulted in the loss of viability at a nonpermissive temperature. Scanning electron microscopy and transmission electron microscopy results showed that the lack of the MSMEG_4947 protein causes drastic morphological changes in M. smegmatis.

MSMEG_4305 is a two-domain protein of Mycolicibacterium smegmatis (Mycobacterium smegmatis) (Mycolicibacterium smegmatis). The N-terminal domain of MSMEG_4305 encodes an RNase H type I. The C-terminal domain is a presumed CobC, predicted to be involved in the aerobic synthesis of vitamin B12. Both domains reach their maximum at distinct pH, approximately 8.5 and 4.5, respectively. The presence of the CobC domain influenced RNase activity in vitro in homolog Rv2228c. Here, we analyzed the role of MSMEG_4305 in vitamin B12 synthesis and the functional association between both domains in vivo in M. smegmatis. We used knock-out mutant of M. smegmatis, deficient in MSMEG_4305. Whole-cell lysates of the mutants strain contained a lower concentration of vitamin B12, as it determined with immunoenzimatic assay. We observed growth deficits, related to vitamin B12 production, on media containing sulfamethazine and propionate. Removal of the CobC domain of MSMEG_4305 in ΔrnhA background hardly affected the growth rate of M. smegmatis in vivo. The strain carrying truncation showed no fitness deficit in the competitive assay and it did not show increased level of RNA/DNA hybrids in its genome. We show that homologs of MSMEG_4305 are present only in the Actinomycetales phylogenetic branch (according to the old classification system). The domains of MSMEG_4305 homologs accumulate mutations at a different rate, while the linker region is highly variable. We conclude that MSMEG_4305 is a multidomain protein that most probably was fixed in the phylogenetic tree of life due to genetic drift.

Thymidine biosynthesis is essential in all cells. Inhibitors of the enzymes involved in this pathway (e.g. methotrexate) are thus frequently used as cytostatics. Due to its pivotal role in mycobacterial thymidylate synthesis dUTPase, which hydrolyzes dUTP into the dTTP precursor dUMP, has been suggested as a target for new antitubercular agents. All mycobacterial genomes encode dUTPase with a mycobacteria-specific surface loop absent in the human dUTPase. Using Mycobacterium smegmatis as a fast growing model for Mycobacterium tuberculosis, we demonstrate that dUTPase knock-out results in lethality that can be reverted by complementation with wild-type dUTPase. Interestingly, a mutant dUTPase gene lacking the genus-specific loop was unable to complement the knock-out phenotype. We also show that deletion of the mycobacteria-specific loop has no major effect on dUTPase enzymatic properties in vitro and thus a yet to be identified loop-specific function seems to be essential within the bacterial cell context. In addition, here we demonstrated that Mycobacterium tuberculosis dUTPase is fully functional in Mycobacterium smegmatis as it rescues the lethal knock-out phenotype. Our results indicate the potential of dUTPase as a target for antitubercular drugs and identify a genus-specific surface loop on the enzyme as a selective target.

Subcellular organization of the bacterial nucleoid and spatiotemporal dynamics of DNA replication and segregation have been studied intensively, but the functional link between these processes remains poorly understood. Here we use quantitative time-lapse fluorescence microscopy for single-cell analysis of chromosome organization and DNA replisome dynamics in Mycobacterium smegmatis. We report that DNA replication takes place near midcell, where, following assembly of the replisome on the replication origin, the left and right replication forks colocalize throughout the replication cycle. From its initial position near the cell pole, a fluorescently tagged chromosomal locus (attB, 245° from the origin) moves rapidly to the replisome complex just before it is replicated. The newly duplicated attB loci then segregate to mirror-symmetric positions relative to midcell. Genetic ablation of ParB, a component of the ParABS chromosome segregation system, causes marked defects in chromosome organization, condensation, and segregation. ParB deficiency also results in mislocalization of the DNA replication machinery and SMC (structural maintenance of chromosome) protein. These observations suggest that ParB and SMC play important and overlapping roles in chromosome organization and replisome dynamics in mycobacteria.

Peptidoglycan (PG), a polymer cross-linked by d-amino acid-containing peptides, is an essential component of the bacterial cell wall. We found that a fluorescent d-alanine analog (FDAA) incorporates chiefly at one of the two poles in Mycobacterium smegmatis but that polar dominance varies as a function of the cell cycle in Mycobacterium tuberculosis: immediately after cytokinesis, FDAAs are incorporated chiefly at one of the two poles, but just before cytokinesis, FDAAs are incorporated comparably at both. These observations suggest that mycobacterial PG-synthesizing enzymes are localized in functional compartments at the poles and septum and that the capacity for PG synthesis matures at the new pole in M. tuberculosis Deeper knowledge of the biology of mycobacterial PG synthesis may help in discovering drugs that disable previously unappreciated steps in the process.IMPORTANCE People are dying all over the world because of the rise of antimicrobial resistance to medicines that could previously treat bacterial infections, including tuberculosis. Here, we used fluorescent d-alanine analogs (FDAAs) that incorporate into peptidoglycan (PG)-the synthesis of which is an attractive drug target-combined with high- and super-resolution microscopy to investigate the spatiotemporal dynamics of PG synthesis in M. smegmatis and M. tuberculosis FDAA incorporation predominates at one of the two poles in M. smegmatis In contrast, while FDAA incorporation into M. tuberculosis is also polar, there are striking variations in polar dominance as a function of the cell cycle. This suggests that enzymes involved in PG synthesis are localized in functional compartments in mycobacteria and that M. tuberculosis possesses a mechanism for maturation of the capacity for PG synthesis at the new pole. This may help in discovering drugs that cripple previously unappreciated steps in the process.

Inositol monophosphatase (IMPase) is a crucial enzyme for the biosynthesis of phosphatidylinositol, an essential component in mycobacterial cell walls. IMPase A (ImpA) from Mycobacterium smegmatis is a bifunctional enzyme that also functions as a fructose-1,6-bisphosphatase (FBPase). To better understand the bifunctional nature of this enzyme, point mutagenesis was conducted on several key residues and their enzyme activity was tested. Our results along with active site models support the fact that ImpA is a bifunctional enzyme with residues Gly94, Thr95 hypothesized to be contributing to the FBPase activity and residues Trp220, Asp221 hypothesized to be contributing to the IMPase activity. Double mutants, W220A + D221A reduced both FBPase and IMPase activity drastically while the double mutant G94A + T95A surprisingly partially restored the IMPase activity compared to the single mutants. This study establishes the foundation toward obtaining a better understanding of the bifunctional nature of this enzyme.

A series of structome analyses, that is, quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level, have already been achieved individually in Exophiala dermatitidis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Myojin spiral bacteria, and Escherichia coli. In these analyses, sample cells were processed through cryo-fixation and rapid freeze-substitution, resulting in the exquisite preservation of ultrastructures on the serial ultrathin sections examined by transmission electron microscopy. In this paper, structome analysis of non pathogenic Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, was performed. As M. smegmatis has often been used in molecular biological experiments and experimental tuberculosis as a substitute of highly pathogenic M. tuberculosis, it has been a task to compare two species in the same genus, Mycobacterium, by structome analysis. Seven M. smegmatis cells cut into serial ultrathin sections, and, totally, 220 serial ultrathin sections were examined by transmission electron microscopy. Cell profiles were measured, including cell length, diameter of cell and cytoplasm, surface area of outer membrane and plasma membrane, volume of whole cell, periplasm, and cytoplasm, and total ribosome number and density per 0.1 fl cytoplasm. These data are based on direct measurement and enumeration of exquisitely preserved single cell structures in the transmission electron microscopy images, and are not based on the calculation or assumptions from biochemical or molecular biological indirect data. All measurements in M. smegmatis, except cell length, are significantly higher than those of M. tuberculosis. In addition, these data may explain the more rapid growth of M. smegmatis than M. tuberculosis and contribute to the understanding of their structural properties, which are substantially different from M. tuberculosis, relating to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance in relation to the ratio of the targets to the corresponding drugs. In addition, data obtained from cryo-transmission electron microscopy examination were used to support the validity of structome analysis. Finally, our data strongly support the most recent establishment of the novel genus Mycolicibacterium, into which basonym Mycobacterium smegmatis has been classified.

RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleotides are present. Here we investigated the importance of RNase H type I encoding genes for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis genome without affecting the growth rate of the mutant. Further, we demonstrate that deletion of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Finally, we question the possibility of existence of RNase HI related alternative mode of initiation of DNA replication in M. smegmatis, the process initially discovered in Escherichia coli. We suspect that synthetic lethality of double mutant lacking RNases H type I is caused by formation of R-loops leading to collapse of replication forks. We report Mycobacterium smegmatis as the first bacterial species, where function of RNase H type I has been found essential.

Small non-coding RNAs play a key role in bacterial adaptation to various stresses. Mycobacterium tuberculosis small RNA MTS1338 is upregulated during mycobacteria infection of macrophages, suggesting its involvement in the interaction of the pathogen with the host. In this study, we explored the functional effects of MTS1338 by expressing it in non-pathogenic Mycobacterium smegmatis that lacks the MTS1338 gene. The results indicated that MTS1338 slowed the growth of the recombinant mycobacteria in culture and increased their survival in RAW 264.7 macrophages, where the MTS1338-expressing strain significantly (p < 0.05) reduced the number of mature phagolysosomes and changed the production of cytokines IL-1β, IL-6, IL-10, IL-12, TGF-β, and TNF-α compared to those of the control strain. Proteomic and secretomic profiling of recombinant and control strains revealed differential expression of proteins involved in the synthesis of main cell wall components and in the regulation of iron metabolism (ESX-3 secretion system) and response to hypoxia (furA, whiB4, phoP). These effects of MTS1338 expression are characteristic for M. tuberculosis during infection, suggesting that in pathogenic mycobacteria MTS1338 plays the role of a virulence factor supporting the residence of M. tuberculosis in the host.

Induced by the pathogen Mycobacterium tuberculosis, tuberculosis remains one of the most dangerous infectious diseases in the world. As a special virus, prophage is domesticated by its host and are major contributors to virulence factors for bacterial pathogenicity. The function of prophages and their genes in M. tuberculosis is still unknown.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a global burden, responsible for over 1 million deaths annually. The emergence and spread of drug-resistant M. tuberculosis strains (MDR-, XDR- and TDR-TB) is the main challenge in global TB-control, requiring the development of novel drugs acting on new biotargets, thus able to overcome the drug-resistance. Tryptanthrin is a natural alkaloid, with great therapeutic potential due to its simple way of synthesis and wide spectrum of biological activities including high bactericidal activity on both drug-susceptible and MDR M. tuberculosis strains. InhA was suggested as the target of tryptanthrins by in silico modeling, making it a promising alternative to isoniazid, able to overcome drug resistance provided by katG mutations. However, neither the mechanism of action of tryptanthrin nor the mechanism of resistance to tryptanthrins was ever confirmed in vitro. We show that the MmpS5-MmpL5 efflux system is able to provide resistance to tryptanthrins using an in-house test-system. Comparative genomic analysis of spontaneous tryptanthrin-resistant M. smegmatis mutants showed that mutations in MSMEG_1963 (EmbR transcriptional regulator) lead to a high-level resistance, while those in MSMEG_5597 (TetR transcriptional regulator) to a low-level one. Mutations in an MFS transporter gene (MSMEG_4427) were also observed, which might be involved in providing a basal level of tryptanthrins-resistance.

Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single-particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding.

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.

Mycobacteria, along with exospore forming Streptomyces, belong to the phylum actinobacteria. Mycobacteria are generally believed to be non-differentiating. Recently however, we showed that the mycobacterial model organism M. smegmatis is capable of forming different types of morphologically distinct resting cells. When subjected to starvation conditions, cells of M. smegmatis exit from the canonical cell division cycle, segregate and compact their chromosomes, and become septated and multi-nucleoided. Under zero nutrient conditions the differentiation process terminates at this stage with the formation of Large Resting Cells (LARCs). In the presence of traces of carbon sources this multi-nucleoided cell stage completes cell division and separates into Small Resting Cells (SMRCs). Here, we carried out RNA-seq profiling of SMRC and LARC development to characterize the transcriptional program underlying these starvation-induced differentiation processes.

Cofactor F(420) is a unique electron carrier in a number of microorganisms including Archaea and Mycobacteria. It has been shown that F(420) has a direct and important role in archaeal energy metabolism whereas the role of F(420) in mycobacterial metabolism has only begun to be uncovered in the last few years. It has been suggested that cofactor F(420) has a role in the pathogenesis of M. tuberculosis, the causative agent of tuberculosis. In the absence of a commercial source for F(420), M. smegmatis has previously been used to provide this cofactor for studies of the F(420)-dependent proteins from mycobacterial species. Three proteins have been shown to be involved in the F(420) biosynthesis in Mycobacteria and three other proteins have been demonstrated to be involved in F(420) metabolism. Here we report the over-expression of all of these proteins in M. smegmatis and testing of their importance for F(420) production. The results indicate that co-expression of the F(420) biosynthetic proteins can give rise to a much higher F(420) production level. This was achieved by designing and preparing a new T7 promoter-based co-expression shuttle vector. A combination of co-expression of the F(420) biosynthetic proteins and fine-tuning of the culture media has enabled us to achieve F(420) production levels of up to 10 times higher compared with the wild type M. smegmatis strain. The high levels of the F(420) produced in this study provide a suitable source of this cofactor for studies of F(420)-dependent proteins from other microorganisms and for possible biotechnological applications.