Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs.

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is hypothesized to play a major role in mRNA degradation because of its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the nonpathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in degradation of the transcripts encoded by at least 89% of protein-coding genes, with leadered transcripts often being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in M. smegmatis and Mycobacterium tuberculosis and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negative bacteria. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming that RNase E has a broad impact on the M. tuberculosis transcriptome.

Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system.

BACKGROUND: Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. RESULTS: Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. CONCLUSION: The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Expression of Synthetic cyp102A1-LG23 Gene and Functional Analysis of Recombinant Cytochrome P450 BM3-LG23 in the Actinobacterium Mycolicibacterium smegmatis.

Cytochrome CYP102A1 (P450 BM3) of Priestia megaterium (bas. Bacillus megaterium) has several unique functional features and thus provides an ideal object for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme part was obtained that hydroxylates several androstanes at C7ß with the formation of products with the anti-inflammatory and neuroprotective activities. In this study, synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant was expressed as a component of either monocistronic operon or bicistronic operon containing the gdh (glucose dehydrogenase, GDH) or zwf2 (glucose 6-phosphate dehydrogenase, G6PD) gene in Mycolicibacterium smegmatis BD cells. The recombinant bacteria were able hydroxylate androst-4-ene-3,17-dione (AD) into 7ß-OH-AD. Their biocatalytic activity was increased twice by increasing the solubility of CYP102A1-LG23 protein in the cells and supplementing the cells with the additional cofactor regeneration system by introducing GDH and G6PD. The maximum 7ß-OH-AD yield (37.68 mol%) was achieved by co-expression of cyp102A1-LG23 and gdh genes in M. smegmatis. These results demonstrate the possibility of using synthetic genes to obtain recombinant enzymes and expand our understanding of the processes involved in steroid hydroxylation by bacterial cytochromes. The data obtained can be used to develop new approaches for microbiological production of 7ß-hydroxylated steroids in genetically modified Mycolicibacterium species.

A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation.

Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN-FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.

Structure of subunit I of the anthranilate synthase complex of Mycolicibacterium smegmatis.

The tryptophan biosynthesis pathway, which does not exist in mammals, is highly conserved in Mycobacterium. Anthranilate synthase (AS) catalyzes the initial reactions in the tryptophan biosynthesis pathway in many microorganisms, catalyzing the conversion of glutamine and chorismate to form pyruvate and anthranilate. Here, the crystal structure of anthranilate synthase component I (AS I) from Mycolicibacterium smegmatis (MsTrpE) has been determined to 1.7 Å resolution. MsTrpE crystallizes in the space group P1 with two monomers in the asymmetric unit, which is consistent with the oligomeric state in solution as confirmed by analytical ultracentrifugation. Inspection of the active site shows that it is in the active form with a bound Mg2+ ion and a ligand that is modelled as benzoate. The position of benzoate mimics the position of the anthranilate product in the active site. The structure of MsTrpE will provide a starting point for the investigation of latent biotechnology and pharmaceutical applications of anthranilate synthase component I.

Metabolic profiling of dormant Mycolicibacterium smegmatis cells' reactivation reveals a gradual assembly of metabolic processes.

INTRODUCTION: Under gradual acidification of the culture medium mycobacterial cells transit into a specific state characterized by low level of metabolic activity and morphological alterations. This state of non-replicative persistence (dormancy) is directly linked to physiological drug resistance, which complicates the efforts to eradicate the latent forms of TB. In order to find new anti-latent TB compounds, the metabolic processes which may occur in the state of dormancy and during the transition into the active state (reactivation) should be characterized. OBJECTIVES: In the current study we analyzed the untargeted metabolomic profiles of dormant and reactivating Mycolicibacterium smegmatis cells (a model microorganism, bearing many common physiological traits of MTB), on the global scale level, since the characterization and analysis of the metabolites' dynamics would provide a comprehensive overview on global biochemical responses of the bacteria to stress conditions. METHODS: The reactivation process was tracked by measuring the value of membrane potential, applying a ratio-metric approach, by the method of flow-cytometry. The crucial timepoints were selected and the bacteria were sampled to LC-MS metabolic profiling. RESULTS: Reactivation of these cells after 60 days of storage revealed that this process proceeds in two stages: (I) a period, which lasts for 10 h and is characterized by a constant CFU number, unchangeable cell size, a minuscule increase of respiratory activity and a noticeable increase in membrane potential value, indicating the onset of the first metabolic processes during this time interval; the second phase (10-26 h) is characterized by acceleration of endogenous respiration, changes in the size of the cells and it finishes with the beginning of cells division. Analysis of the changes in the relative abundances of KEGG-annotated metabolites revealed that a significant number of metabolites, such as stearic acid, glycerol, D-glucose, trehalose-6-phosphate decrease their concentrations over the reactivation time, whereas in contrast, such metabolites as dodecanoic acid, mycobactin S, and other compounds of PG/AG biosynthesis are synthesized during reactivation. Differential analysis of metabolic profiles disclosed the activation of a number of metabolic pathways at the early reactivation stage: biosynthesis of secondary metabolites, purine and pyrimidine metabolism, glycerophospholipid and fatty acids metabolism etc. CONCLUSION: The data obtained indicate, despite the long-term storage of dormant cells in a state of minimal metabolic activity, according to metabolic profiling, they still retained a large number of metabolites. In the process of reactivation, the incremental stochastic assembly of the complete metabolic pathways occurs.

Short communication: Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, causing pyogranulomatous mastitis and its cross-reactivity in bovine (para)tuberculosis testing.

Different mycobacterial species are encountered in bovine medicine. The fastidiously growing mycobacteria (Mycobacterium bovis as the cause of bovine tuberculosis, and Mycobacterium avium ssp. paratuberculosis, MAP, as the cause of paratuberculosis) are well known and targeted in eradication/control or monitoring programs in different countries, whereas the rapidly growing species is only rarely identified from bovine disease. The latter have occasionally been reported as the cause of bovine clinical mastitis, but recent reports are scarce. In this study, Mycolicibacterium smegmatis (basonym Mycobacterium smegmatis) was identified as cause of granulomatous, relapsing clinical mastitis in 2 cows from one Belgian dairy herd. Milk, blood, and fecal samples were collected, as well as tissue samples after the cows were culled. Serological analysis conducted on milk and serum samples resulted in positive reactions for MAP, but negative for Mycobacterium bovis. Production of IFN-γ showed sensitization with mycobacteria or similar organisms, other than M. bovis, in one cow. Detection of MAP by bacteriological culture and IS900-based quantitative PCR on milk and feces remained negative. In conclusion, this paper describes M. smegmatis as a cause of bovine clinical mastitis in Belgium and suggests cross-reactivity of the intramammary M. smegmatis infection with routinely used serological tests for MAP.

Impact of MSMEG5257 Deletion on Mycolicibacterium smegmatis Growth.

Mycobacterial membrane proteins play a pivotal role in the bacterial invasion of host cells; however, the precise mechanisms underlying certain membrane proteins remain elusive. Mycolicibacterium smegmatis (Ms) msmeg5257 is a hemolysin III family protein that is homologous to Mycobacterium tuberculosis (Mtb) Rv1085c, but it has an unclear function in growth. To address this issue, we utilized the CRISPR/Cas9 gene editor to construct Δmsmeg5257 strains and combined RNA transcription and LC-MS/MS protein profiling to determine the functional role of msmeg5257 in Ms growth. The correlative analysis showed that the deletion of msmeg5257 inhibits ABC transporters in the cytomembrane and inhibits the biosynthesis of amino acids in the cell wall. Corresponding to these results, we confirmed that MSMEG5257 localizes in the cytomembrane via subcellular fractionation and also plays a role in facilitating the transport of iron ions in environments with low iron levels. Our data provide insights that msmeg5257 plays a role in maintaining Ms metabolic homeostasis, and the deletion of msmeg5257 significantly impacts the growth rate of Ms. Furthermore, msmeg5257, a promising drug target, offers a direction for the development of novel therapeutic strategies against mycobacterial diseases.

Structural and functional characterization of FabG4 from Mycolicibacterium smegmatis.

The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID-19, with multi-drug-resistant strains of TB having an ∼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD+ and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.

Ergothioneine production by Corynebacterium glutamicum harboring heterologous biosynthesis pathways.

In this study, Corynebacterium glutamicum was engineered to produce ergothioneine, an amino acid derivative with high antioxidant activity. The ergothioneine biosynthesis genes, egtABCDE, from Mycolicibacterium smegmatis were introduced into wild-type and l-cysteine-producing strains of C. glutamicum to evaluate their ergothioneine production. In the l-cysteine-producing strain, ergothioneine production reached approximately 40 mg L-1 after 2 weeks, and the amount was higher than that in the wild-type strain. As C. glutamicum possesses an ortholog of M. smegmatis egtA, which encodes an enzyme responsible for γ-glutamyl-l-cysteine synthesis, the effect of introducing egtBCDE genes on ergothioneine production in the l-cysteine-producing strain was evaluated, revealing that a further increase to more than 70 mg L-1 was achieved. As EgtBs from Methylobacterium bacteria are reported to use l-cysteine as a sulfur donor in ergothioneine biosynthesis, egtB from Methylobacterium was expressed with M. smegmatis egtDE in the l-cysteine-producing strain. As a result, ergothioneine production was further improved to approximately 100 mg L-1. These results indicate that utilization of the l-cysteine-producing strain and introduction of heterologous biosynthesis pathways from M. smegmatis and Methylobacterium bacteria are effective for improved ergothioneine production by C. glutamicum.

Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in Mycolicibacterium smegmatis MC2 155 by proteogenomic analysis.

Accurate identification of novel peptides remains challenging because of the lack of evaluation criteria in large-scale proteogenomic studies. Mirror proteases of trypsin and lysargiNase can generate complementary b/y ion series, providing the opportunity to efficiently assess authentic novel peptides in experiments other than filter potential targets by different false discovery rates (FDRs) ranking. In this study, a pair of in-house developed acetylated mirror proteases, Ac-Trypsin and Ac-LysargiNase, were used in Mycolicibacterium smegmatis MC2 155 for proteogenomic analysis. The mirror proteases accurately identified 368 novel peptides, exhibiting 75-80% b and y ion coverages against 65-68% y or b ion coverages of Ac-Trypsin (38.9% b and 68.3% y) or Ac-LysargiNase (65.5% b and 39.6% y) as annotated peptides from M. smegmatis MC2 155. The complementary b and y ion series largely increased the reliability of overlapped sequences derived from novel peptides. Among these novel peptides, 311 peptides were annotated in other public M. smegmatis strains, and 57 novel peptides with more continuous b and y pairs were obtained for further analysis after spectral quality assessment. This enabled mirror proteases to successfully correct six annotated proteins' N-termini and detect 17 new coding open reading frames (ORFs). We believe that mirror proteases will be an effective strategy for novel peptide detection in both prokaryotic and eukaryotic proteogenomics.

H2O2 concentration-dependent kinetics of gene expression: linking the intensity of oxidative stress and mycobacterial physiological adaptation.

Defence against oxidative stress is crucial for Mycobacterium tuberculosis to survive and replicate within macrophages. Mycobacteria have evolved multilayer antioxidant systems, including scavenging enzymes, iron homeostasis, repair pathways, and metabolic adaptation, for coping with oxidative stress. How these systems are coordinated to enable the physiological adaptation to different intensities of oxidative stress, however, remains unclear. To address this, we investigated the expression kinetics of the well-characterized antioxidant genes at bacteriostatic H2O2 concentrations ranging from 1 mM to 10 mM employing Mycolicibacterium smegmatis as a model. Our results showed that most of the selected genes were expressed in a H2O2 concentration-dependent manner, whereas a subset exhibited sustained induction or repression without dose-effect, reflecting H2O2 concentration-dependent physiological adaptations. Through analyzing the dynamics of the coordinated gene expression, we demonstrated that the expressions of the H2O2 scavenging enzymes, DNA damage response, and Fe-S cluster repair function were strikingly correlated to the intensity of oxidative stress. The sustained induction of mbtB, irtA, and dnaE2 indicated that mycobacteria might deploy increased iron acquisition and error-prone lesion bypass function as fundamental strategies to counteract oxidative damages, which are distinct from the defence tactics of Escherichia coli characterized by shrinking the iron pool and delaying the DNA repair. Moreover, the distinct gene expression kinetics among the tricarboxylic acid cycle, glyoxylate shunt, and methylcitrate cycle suggested that mycobacteria could dynamically redirect its metabolic fluxes according to the intensity of oxidative stress. This work defines the H2O2 concentration-dependent gene expression kinetics and provides unique insights into mycobacterial antioxidant defence strategies.

[Knockout of ribosomal genes bS22 and bL37 increases the sensitivity of mycobacteria to antibiotics].

In recent years, two novel proteins in the ribosomes of mycobacteria have been discovered by cryo-electron microscopy. The protein bS22 is located near the decoding center of the 30S subunit, and the protein bL37 is located near the peptidyl transferase center of the 50S subunit. Since these two proteins bind to conserved regions of the ribosome targeted by antibiotics, it is speculated that they might affect the binding of related drugs to these targets. Therefore, we knocked out the genes encoding these two proteins in wild-type Mycolicibacterium smegmatis mc2155 through homologous recombination, and then determined the growth curves of these mutants and their sensitivity to related antibiotics. The results showed that compared with the wild-type strain, the growth rate of these two mutants did not change significantly. However, mutant ΔbS22 showed increased sensitivity to capreomycin, kanamycin, amikacin, streptomycin, gentamicin, paromomycin, and hygromycin B, while mutant ΔbL37 showed increased sensitivity to linezolid. These changes in antibiotics sensitivity were restored by gene complementation. This study hints at the possibility of using ribosomal proteins bS22 and bL37 as targets for drug design.

A research program-linked, course-based undergraduate research experience that allows undergraduates to participate in current research on mycobacterial gene regulation.

Undergraduate instructional biology laboratories are typically taught within two paradigms. Some labs focus on protocols and techniques delivered in "cookbook" format with defined experimental outcomes. There is increasing momentum to alternatively employ student-driven, open-ended, and discovery-based strategies, often via course-based undergraduate research experiences (CUREs) using crowd-sourcing initiatives. A fraction of students also participate in funded research in faculty research labs, where they have opportunities to work on projects designed to expand the frontiers of human knowledge. These experiences are widely recognized as valuable but are not scalable, as most institutions have many more undergraduates than research lab positions. We sought to address this gap through our department's curriculum by creating an opportunity for students to participate in the real-world research process within a laboratory course. We conceived, developed, and delivered an authentic, guided research experience to students in an upper-level molecular biology laboratory course. We refer to this model as a "research program-linked CURE." The research questions come directly from a faculty member's research lab and evolve along with that research program. Students study post-transcriptional regulation in mycobacteria. We use current molecular biology methodologies to test hypotheses like "UTRs affect RNA and protein expression levels," "there is functional redundancy among RNA helicases," and "carbon starvation alters mRNA 5' end chemistries." We conducted standard assessments and developed a customized "Skills and Concepts Inventory" survey to gauge how well the course met our student learning outcomes. We report the results of our assessments and describe challenges addressed during development and execution of the course, including organizing activities to fit within an instructional lab, balancing breadth with depth, and maintaining authenticity while giving students the experience of obtaining interpretable and novel results. Our data suggest student learning was enhanced through this truly authentic research approach. Further, students were able to perceive they were participants and contributors within an active research paradigm. Students reported increases in their self-identification as scientists, and a positive impact on their career trajectories. An additional benefit was reciprocation back to the funded research laboratory, by funneling course alumni, results, materials, and protocols.

Ac-LysargiNase efficiently helps genome reannotation of Mycolicibacterium smegmatis MC2 155.

Accurate genome annotation, the foundation of life science research in the genome era, is hampered by limited known gene models, nonstandard start codons, and the limited homology of annotated genes in other organisms. LysargiNase mirrors trypsin at the cleavage sites, providing the opportunity to identify peptides other than tryptic peptides. In this study, we used an in-house developed acetylated LysargiNase (Ac-LysargiNase) with higher activity and stability in non-pathogenic Mycolicibacterium smegmatis MC2 155 to supplement the widely used trypsin in proteomic studies. We identified 27,582 peptides from 3844 annotated proteins and 332 novel genome search-specific peptides (GSSPs). Among these GSSPs, 88 peptides were annotated in another M.smegmatis genome database, and 41 were verified as novel peptides by predicted theoretical spectra and their corresponding 15N-labeling spectra. Further analysis revealed that 17 verified GSSPs corrected the N-terminus of the 13 annotated genes. The other 24 verified GSSPs helped identify 17 novel open reading frames (ORFs) missed in previously annotated M. smegmatis genomes. Among these novel ORFs, four relatively small proteins with amino acid residues less than 100 and three were precisely identified with C-terminal peptides. Ac-LysargiNase helps with genome reannotation by identifying new genes and events in proteogenomic studies. SIGNIFICANCE: Correct genomic annotation is vital in the field of life sciences. The nonstandard start codons seriously affect the confirmation of the translation initiation sites (TISs) of an open reading frame (ORF), and unknown structural genes are easily missed in automated gene prediction. Although proteogenomics presents new avenues for validating gene expression and gene structure refinement based on conventional tryptic peptides, determining the TISs and potential encoding genes is complicated. Thus, validation of TISs and encoding ORFs is crucial and urgent. Therefore, we recommend Ac-LysargiNase, a mirror enzyme of trypsin that can identify additional novel peptides for N-terminal correction and ORF identification.

In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract.

Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the nitrate assimilation pathway. Here, we present a detailed protocol for measuring in vitro nitrate reductase (NaR) activity of NAS enzymes from Mycolicibacterium smegmatis crude extract using both physiological and non-physiological electron donors.

Mycolicibacterium smegmatis possesses operational agmatinase but contains no detectable polyamines.

Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.

Transcriptomic dataset of Mycolicibacterium smegmatis exposed to an imidazo[1,2-b][1,2,4,5]tetrazine.

Deciphering the mechanism of action of novel anti-tuberculosis compounds is a key step in the drug development process. We have previously described a number of imidazo[1,2-b][1,2,4,5]tetrazines with a promising activity on Mycobacterium tuberculosis[1]. These compounds had predicted activity as serine­threonine protein kinase inhibitors, however spontaneous drug resistant Mycolicibacterium smegmatis mc 2 155 (formerly Mycobacterium smegmatis) revealed only the mycobacterial mechanism of resistance to imidazo[1,2-b][1,2,4,5]tetrazines: mutations in MSMEG_1380 gene lead to overexpression of the mmpS5-mmpL5 operon in M. smegmatis, thus providing resistance to imidazo[1,2-b][1,2,4,5]tetrazines via enhanced efflux [2]. Here we report the RNA sequencing data of M. smegmatis mc 2  155 culture treated with one of the imidazo[1,2-b][1,2,4,5]tetrazines for 1.5 h and the untreated culture as a control. The mapped reads showed that a total of 1386 genes are differentially expressed in this experiment. A further analysis of these data can shed light of the mechanism of action of imidazo[1,2-b][1,2,4,5]tetrazines. The data generated by RNA-seq (raw reads) have been deposited to NCBI sequence read archive (SRA) and have been assigned a BioProject accession number PRJNA615922.

Mycolicibacterium smegmatis, Basonym Mycobacterium smegmatis, Expresses Morphological Phenotypes Much More Similar to Escherichia coli Than Mycobacterium tuberculosis in Quantitative Structome Analysis and CryoTEM Examination.

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.

Knockout of ribosomal genes bS22 and bL37 increases the sensitivity of mycobacteria to antibiotics / 生物工程学报

In recent years, two novel proteins in the ribosomes of mycobacteria have been discovered by cryo-electron microscopy. The protein bS22 is located near the decoding center of the 30S subunit, and the protein bL37 is located near the peptidyl transferase center of the 50S subunit. Since these two proteins bind to conserved regions of the ribosome targeted by antibiotics, it is speculated that they might affect the binding of related drugs to these targets. Therefore, we knocked out the genes encoding these two proteins in wild-type Mycolicibacterium smegmatis mc2155 through homologous recombination, and then determined the growth curves of these mutants and their sensitivity to related antibiotics. The results showed that compared with the wild-type strain, the growth rate of these two mutants did not change significantly. However, mutant ΔbS22 showed increased sensitivity to capreomycin, kanamycin, amikacin, streptomycin, gentamicin, paromomycin, and hygromycin B, while mutant ΔbL37 showed increased sensitivity to linezolid. These changes in antibiotics sensitivity were restored by gene complementation. This study hints at the possibility of using ribosomal proteins bS22 and bL37 as targets for drug design.