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Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies.Protein Science : a Publication of the... Jan 2015Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional... (Review)
Review
Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.
Topics: Bacterial Proteins; Cloning, Molecular; Genetic Vectors; Models, Molecular; Mycobacterium; Mycobacterium smegmatis; Protein Processing, Post-Translational; Recombinant Proteins
PubMed: 25303009
DOI: 10.1002/pro.2584 -
Nucleic Acids Research Jul 2022Bacteria and bacteriophages co-evolve in a constant arms race, wherein one tries and finds newer ways to overcome the other. Phage resistance poses a great threat to the...
Bacteria and bacteriophages co-evolve in a constant arms race, wherein one tries and finds newer ways to overcome the other. Phage resistance poses a great threat to the development of phage therapy. Hence, it is both essential and important to understand the mechanism of phage resistance in bacteria. First identified in Mycobacterium smegmatis, the gene mpr, upon overexpression, confers resistance against D29 mycobacteriophage. Presently, the mechanism behind phage resistance by mpr is poorly understood. Here we show that Mpr is a membrane-bound DNA exonuclease, which digests DNA in a non-specific manner independent of the sequence, and shares no sequence or structural similarity with any known nuclease. Exonuclease activity of mpr provides resistance against phage infection, but the role of mpr may very well go beyond just phage resistance. Our experiments show that mpr plays a crucial role in the appearance of mutant colonies (phage resistant strains). However, the molecular mechanism behind the emergence of these mutant/resistant colonies is yet to be understood. Nevertheless, it appears that mpr is involved in the survival and evolution of M. smegmatis against phage. A similar mechanism may be present in other organisms, which requires further exploration.
Topics: Bacteriophages; Mycobacterium smegmatis
PubMed: 35713559
DOI: 10.1093/nar/gkac505 -
Journal of Bacteriology Jan 2020Stringent response is a conserved stress response mechanism in which bacteria employ the second messengers guanosine tetraphosphate and guanosine pentaphosphate...
Stringent response is a conserved stress response mechanism in which bacteria employ the second messengers guanosine tetraphosphate and guanosine pentaphosphate [collectively termed (p)ppGpp] to reprogram their cellular processes under stress. In mycobacteria, these alarmones govern a multitude of cellular phenotypes, such as cell division, biofilm formation, antibiotic tolerance, and long-term survival. possesses the bifunctional Rel as a (p)ppGpp synthetase and hydrolase. In addition, it contains a short alarmone synthetase MS_RHII-RSD (renamed RelZ), which contains an RNase H domain in tandem with the (p)ppGpp synthetase domain. The physiological functions of Rel have been well documented, but there is no clear picture about the cellular functions of RelZ in RelZ has been implicated in R-loop induced stress response due to its unique domain architecture. In this study, we elucidate the differential substrate utilization pattern of RelZ compared to that of Rel We unveil the ability of RelZ to use GMP as a substrate to synthesize pGpp, thereby expanding the repertoire of second messengers known in mycobacteria. We have demonstrated that the pGpp synthesis activity of RelZ is negatively regulated by RNA and pppGpp. Furthermore, we investigated its role in biofilm formation and antibiotic tolerance. Our findings highlight the complex role played by the RelZ in cellular physiology of and sheds light upon its functions distinct from those of Rel Bacteria utilize nucleotide messengers to survive the hostile environmental conditions and the onslaught of attacks within the host. The second messengers guanosine tetraphosphate and pentaphosphate [(p)ppGpp] have a profound impact on the long-term survival, biofilm formation, antibiotic tolerance, virulence, and pathogenesis of bacteria. Therefore, understanding the stress response mechanism regulated by (p)ppGpp is essential for discovering inhibitors of stress response and potential drug targets. contains two (p)ppGpp synthetases: Rel and RelZ. Our study unravels the novel regulatory mechanisms of RelZ activity and its role in mediating antibiotic tolerance. We further reveal its ability to synthesize novel second messenger pGpp, which may have regulatory roles in mycobacteria.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Gene Expression Regulation, Bacterial; Guanine Nucleotides; Guanosine Tetraphosphate; Mycobacterium smegmatis; Promoter Regions, Genetic
PubMed: 31659009
DOI: 10.1128/JB.00444-19 -
Microbiology (Reading, England) Mar 2008The growth and nutritional requirements of mycobacteria have been intensively studied since the discovery of Mycobacterium tuberculosis more than a century ago. However,... (Comparative Study)
Comparative Study Review
The growth and nutritional requirements of mycobacteria have been intensively studied since the discovery of Mycobacterium tuberculosis more than a century ago. However, the identity of many transporters for essential nutrients of M. tuberculosis and other mycobacteria is still unknown despite a wealth of genomic data and the availability of sophisticated genetic tools. Recently, considerable progress has been made in recognizing that two lipid permeability barriers have to be overcome in order for a nutrient molecule to reach the cytoplasm of mycobacteria. Uptake processes are discussed by comparing M. tuberculosis with Mycobacterium smegmatis. For example, M. tuberculosis has only five recognizable carbohydrate transporters in the inner membrane, while M. smegmatis has 28 such transporters at its disposal. The specificities of inner-membrane transporters for sulfate, phosphate and some amino acids have been determined. Outer-membrane channel proteins in both organisms are thought to contribute to nutrient uptake. In particular, the Msp porins have been shown to be required for uptake of carbohydrates, amino acids and phosphate by M. smegmatis. The set of porins also appears to be different for M. tuberculosis and M. smegmatis. These differences likely reflect the lifestyles of these mycobacteria and the availability of nutrients in their natural habitats: the soil and the human body. The comprehensive identification and the biochemical and structural characterization of the nutrient transporters of M. tuberculosis will not only promote our understanding of the physiology of this important human pathogen, but might also be exploited to improve tuberculosis chemotherapy.
Topics: Bacterial Proteins; Biological Transport; Carrier Proteins; Mycobacterium smegmatis; Mycobacterium tuberculosis
PubMed: 18310015
DOI: 10.1099/mic.0.2007/012872-0 -
Proceedings of the National Academy of... Apr 2021Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is...
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 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.
Topics: Bacterial Proteins; Cryoelectron Microscopy; Mycobacterium smegmatis; Organelles; Peroxidases
PubMed: 33853951
DOI: 10.1073/pnas.2025658118 -
Journal of Infection and Public Health Sep 2020An alternate host for mycobacteria is Mycobacterium smegmatis which is used frequently. It is a directly budding eco-friendly organism not emulated as human infection.... (Review)
Review
An alternate host for mycobacteria is Mycobacterium smegmatis which is used frequently. It is a directly budding eco-friendly organism not emulated as human infection. It is mainly useful for the investigation of various microorganisms in the sort of Mycobacteria in cell culture laboratories. Some Mycobacterium species groups that is normal, unsafe ailments, likely to Mycobacterium leprae, Mycobacterium tuberculosis and Mycobacterium bovis. At present, various laboratories are clean and culture this type of species to make an opinion that fascinating route of harmful Mycobacteria. This publication provides aggregate data on cell shape, genome studies, ecology, pathology and utilization of M. smegmatis.
Topics: Bacterial Proteins; Humans; Liposomes; Models, Biological; Mycobacterium Infections, Nontuberculous; Mycobacterium smegmatis; Mycobacterium tuberculosis
PubMed: 32674978
DOI: 10.1016/j.jiph.2020.06.023 -
Acta Crystallographica. Section F,... Jul 2022The unintended crystallization of proteins which generally originate from the expression host instead of the target recombinant proteins is periodically reported....
The unintended crystallization of proteins which generally originate from the expression host instead of the target recombinant proteins is periodically reported. Despite the massive technological advances in the field, assigning a structural model to the corresponding diffraction data is not a trivial task. Here, the structure of acyl-carrier protein synthase (AcpS) from Mycobacterium smegmatis (msAcpS), which crystallized inadvertently in an experimental setup to grow crystals of a Mycobacterium tuberculosis protein using M. smegmatis as an expression system, is reported. After numerous unsuccessful attempts to solve the structure of the target protein by the molecular-replacement method no convincing solutions were obtained, indicating that the diffraction data may correspond to a crystal of an artifactual protein, which was finally identified by the Sequence-Independent Molecular replacement Based on Available Databases (SIMBAD) server. The msAcpS structure was solved at 2.27 Å resolution and structural analysis showed an overall conserved fold. msAcpS formed a trimeric structure similar to those of other reported structures of AcpS from various organisms; however, the residues involved in trimer formation are not strictly conserved. An unrelated metal ion (Ni), which was possibly incorporated during protein purification, was observed in the proximity of His49 and His116. Structural and sequence differences were observed in the loop connecting the α3 and α4 helices that is responsible for the open and closed conformations of the enzyme. Moreover, the structural analysis of msAcpS augments the current understanding of this enzyme, which plays a crucial role in the functional activation of acyl-carrier proteins in the fatty-acid biosynthesis pathway.
Topics: Acyl Carrier Protein; Bacterial Proteins; Crystallization; Crystallography, X-Ray; Mycobacterium smegmatis; Transferases
PubMed: 35787552
DOI: 10.1107/S2053230X22005738 -
MBio Apr 2022Mycobacteria mediate horizontal gene transfer (HGT) by a process called distributive conjugal transfer (DCT) that is mechanistically distinct from -mediated plasmid...
Mycobacteria mediate horizontal gene transfer (HGT) by a process called distributive conjugal transfer (DCT) that is mechanistically distinct from -mediated plasmid transfer. The transfer of multiple, independent donor chromosome segments generates transconjugants with genomes that are mosaic blends of their parents. Previously, we had characterized contact-dependent conjugation between two independent isolates of Mycobacterium smegmatis. Here, we expand our analyses to include five independent isolates of M. smegmatis and establish that DCT is both active and prevalent among natural isolates of M. smegmatis. Two of these five strains were recipients but exhibited distinct conjugal compatibilities with donor strains, suggesting an ability to distinguish between potential donor partners. We determined that a single gene, , was responsible for conferring mating compatibility using a combination of comparative DNA sequence analysis, bacterial genome-wide association studies (GWAS), and targeted mutagenesis. maps within the secretion locus, and we establish that it confers mycobacterial self-identity with parallels to kin recognition. Similar to other kin model systems, orthologs of Msmeg0070 are highly polymorphic. The identification of a kin recognition system in M. smegmatis reinforces the concept that communication between cells is an important checkpoint prior to DCT commitment and implies that there are likely to be other, unanticipated forms of social behaviors in mycobacteria. Conjugation, unlike other forms of HGT, requires direct interaction between two viable bacteria, which must be capable of distinguishing between mating types to allow successful DNA transfer from donor to recipient. We show that the conjugal compatibility of Mycobacterium smegmatis isolates is determined by a single, polymorphic gene located within the conserved secretion locus. This gene confers self-identity; the expression of identical Msmeg0070 proteins in both donor-recipient partners prevents DNA transfer. The presence of this polymorphic locus in many environmental mycobacteria suggests that kin identification is important in promoting beneficial gene flow between nonkin mycobacteria. Cell-cell communication, mediated by kin recognition and ESX secretion, is a key checkpoint in mycobacterial conjugation and likely plays a more global role in mycobacterial biology.
Topics: Conjugation, Genetic; DNA; Genome-Wide Association Study; Mycobacterium; Mycobacterium smegmatis
PubMed: 35297678
DOI: 10.1128/mbio.00213-22 -
BMC Microbiology Aug 2020The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in...
BACKGROUND
The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in cell wall metabolism and identified an aceE gene mutant (aceE-mut) which presented a deficient colony morphology on 7H10 agar by screening transposon mutagenesis in Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis (M. smegmatis). This study aimed to identify the functional role of aceE gene in cell wall biosynthesis in M. smegmatis.
RESULTS
We observed that the colony morphology of aceE-mut was quite different, smaller and smoother on the solid culture medium than the wild-type (WT) strain during the transposon library screening of M. smegmatis. Notably, in contrast with the WT, which aggregates and forms biofilm, the aceE-mut lost its ability of growing aggregately and biofilm formation, which are two very important features of mycobacteria. The morphological changes in the aceE-mut strain were further confirmed by electron microscopy which indicated smoother and thinner cell envelope images in contrast with the rough morphology of WT strains. Additionally, the aceE-mut was more fragile to acidic stress and exhibited a pronounced defects in entering the macrophages as compared to the WT. The analysis of mycolic acid (MA) using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE-mut strain whereas complementation of the aceE-mut with a wild-type aceE gene restored the composition of MA.
CONCLUSIONS
Over all, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology, biofilm formation of M. smegmatis and bacteria invasion of macrophage.
Topics: Bacterial Proteins; Biofilms; Chromatography, Liquid; Genetic Complementation Test; Mass Spectrometry; Membrane Proteins; Microbial Sensitivity Tests; Microscopy, Electron; Mutagenesis, Site-Directed; Mutation; Mycobacterium smegmatis; Mycolic Acids
PubMed: 32811434
DOI: 10.1186/s12866-020-01940-2 -
MBio Feb 2015Subcellular organization of the bacterial nucleoid and spatiotemporal dynamics of DNA replication and segregation have been studied intensively, but the functional link...
UNLABELLED
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.
IMPORTANCE
We studied the spatiotemporal organization of the chromosome and DNA replication machinery in Mycobacterium smegmatis, a fast-growing relative of the human pathogen Mycobacterium tuberculosis. We show that genetic ablation of the DNA-binding proteins ParB and SMC disturbs the organization of the chromosome and causes a severe defect in subcellular localization and movement of the DNA replication complexes. These observations suggest that ParB and SMC provide a functional link between chromosome organization and DNA replication dynamics. This work also reveals important differences in the biological roles of the ParABS and SMC systems in mycobacteria versus better-characterized model organisms, such as Escherichia coli and Bacillus subtilis.
Topics: Cell Cycle Proteins; Chromosome Segregation; Chromosomes, Bacterial; DNA Replication; DNA, Bacterial; Gene Deletion; Microscopy, Fluorescence; Mycobacterium smegmatis; Single-Cell Analysis; Spatio-Temporal Analysis; Time-Lapse Imaging
PubMed: 25691587
DOI: 10.1128/mBio.01999-14