RESUMEN
The concerted action of DNA replication and cell division has been extensively investigated in eukaryotes. Well demarcated checkpoints have been identified in the cell cycle, which provides the correct DNA stoichiometry and appropriate growth in the progeny. In bacteria, which grow faster and less concerted than eukaryotes, the linkages between cell elongation and DNA synthesis are unclear. dTTP, one of the canonical nucleotide-building blocks of DNA, is also used for cell wall biosynthesis in mycobacteria. We hypothesize that the interconnection between DNA and cell wall biosynthesis through dTTP may require synchronization of these processes by regulating dTTP availability. We investigated growth, morphology, cellular dNTP pool, and possible signs of stress in Mycobacterium smegmatis upon perturbation of rhamnose biosynthesis by the overexpression of RmlA. RmlA is a cell wall synthetic enzyme that uses dTTP as the precursor for cross-linking the peptidoglycan with the arabinogalactan layers by a phosphodiester bond in the mycobacterial cell wall. We found that RmlA overexpression results in changes in cell morphology, causing cell elongation and disruption of the cylindrical cell shape. We also found that the cellular dTTP pool is reduced by half in RmlA overexpressing cells and that this reduced dTTP availability does not restrict cell growth. We observed 2-6-fold increases in the gene expression of replication and cell wall biosynthesis stress factors upon RmlA overexpression. Using super-resolution microscopy, we found that RmlA, acting to crosslink the nascent layers of the cell wall, localizes throughout the whole cell length in a helical pattern in addition to the cellular pole.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium smegmatis/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleótidos de Timina/metabolismo , Pared Celular/metabolismo , Replicación del ADN , ADN Bacteriano , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Ramnosa/metabolismo , Estrés FisiológicoRESUMEN
Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an emerging area with established protocols and promising results. Traditional proteomic studies, especially involving bacteria, have been limited to suspension cultures with large protein yields. Such studies, however, remain population centered with the uniqueness of individual responses to environmental challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony from 7H10 media, with growth supplements for optimal growth. Following protein purification and digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer. Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative power while increasing reproducibility, of "very limiting samples."
Asunto(s)
Proteínas Bacterianas/análisis , Mycobacterium/química , Proteómica/métodos , Cromatografía Liquida/métodos , Humanos , Mycobacterium/citología , Infecciones por Mycobacterium/microbiología , Mycobacterium smegmatis/química , Mycobacterium smegmatis/citología , Proteoma/análisis , Espectrometría de Masas en Tándem/métodosRESUMEN
Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that can infect and replicate in macrophages. Peptidoglycan (PGN) is a major component of the mycobacterial cell wall and is recognized by host pattern recognition receptors (PRRs). Many bacteria modulate and evade the immune defenses of their hosts through PGN deacetylation. Rv1096 was previously characterized as a PGN N-deacetylase gene in Mtb. However, the underlying mechanism by which Rv1096 regulates host immune defenses during macrophage infection remains unclear. In the present study, we investigated the role of Rv1096 in evading host immunity using a recombinant M. smegmatis expressing exogenous Rv1096 and Rv1096-deleted Mtb strain H37Rv mutant. We found that Rv1096 promoted intracellular bacillary survival and inhibited the inflammatory response in M. smegmatis- or Mtb-infected macrophages. The inhibition of mycobacteria-induced inflammatory response in macrophages was at least partially due to NF-κB and MAPK activation downstream of TLR and NOD signaling pathways. Furthermore, we found that Rv1096 inhibitory effect on inflammatory response was associated with TLR2, TLR4 and NOD2. Finally, we demonstrated the PGN deacetylase activity of Rv1096 by Fourier transform IR and Rv1096 NODB deficient mutant. Our findings suggest that Rv1096 may deacetylate PGNs to evade PRRs recognition, thus protecting Mtb from host immune surveillance and clearance in macrophages.
Asunto(s)
Amidohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Sistema de Señalización de MAP Quinasas , Viabilidad Microbiana , Mycobacterium smegmatis/citología , Mycobacterium tuberculosis/enzimología , FN-kappa B/metabolismo , Peptidoglicano/metabolismo , Animales , Proteínas Bacterianas/química , Citocinas/metabolismo , Femenino , Inflamación/patología , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium tuberculosis/crecimiento & desarrollo , Dominios Proteicos , Células RAW 264.7RESUMEN
Mycobacterial cells elongate via polar deposition of cell wall material, similar to the filamentous Streptomyces species, which contain a tip-organizing centre. Coiled-coiled proteins such as DivIVA play an important role in this process. The genome of Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes many coiled-coil proteins that are homologous to DivIVA with a potential role in mycobacterial cell elongation. Here we describe studies on Mycobacterium smegmatis MSMEG_2416, a homologue of M. tuberculosis Rv2927c. Two previous independent studies showed that MSMEG_2416 was involved in septation (subsequently referred to as sepIVA). Contrary to these previous reports, we found sepIVA to be dispensable for growth in laboratory media by generating a viable null mutant. The mutant strain did, however, show a number of differences, including a change in colony morphology and biofilm formation that could be reversed on complementation with sepIVA as well as Rv2927c, the sepIVA homologue from M. tuberculosis. However, analysis of cell wall lipids did not reveal any alterations in lipid profiles of the mutant strain. Microscopic examination of the mutant revealed longer cells with more septa, which occurred at irregular intervals, often generating mini-compartments, a profile similar to that observed in the previous studies following conditional depletion, highlighting a role for sepIVA in mycobacterial growth.
Asunto(s)
Proteínas Bacterianas/metabolismo , División Celular , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Pared Celular/química , Eliminación de Gen , Genes Bacterianos , Lípidos/análisis , Mutación , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Dominios ProteicosRESUMEN
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.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Liposomas/metabolismo , Modelos Biológicos , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/ultraestructuraRESUMEN
The study was designed to assess whether plant extracts / phytochemical (D-Pinitol) synergistically combine with antituberculosis drugs and act on Mycobacterium smegmatis (M. smegmatis) as well as assess their mode of action on Mycobacterium tuberculosis (M.tb) Filamenting temperature sensitive mutant Z (FtsZ) protein. Resazurin microtitre plate assay (Checker board) was performed to analyze the activity of plant extracts against M. smegmatis. Synergistic behaviour of plant extracts / D-Pinitol with Isoniazid (INH) and Rifampicin (RIF) were determined by time-kill and checker board assays. Elongation of M. smegmatis cells due to this treatment was determined by light microscopy. The effect of Hexane methanol extract (HXM) plant extracts on cell viability was determined using PI/SYTO9 dual dye reporter Live/Dead assay. Action of HXM plant extracts / D-Pinitol on inhibition of FtsZ protein was done using Guanosine triphosphatase (GTPase) light scattering assay and quantitative Polymerase Chain Reaction (qPCR). The Hexane-methanolic plant extract of Acacia nilotica, Aegle marmelos and Glycyrrhiza glabra showed antimycobacterial activity at 1.56 ± 0.03, 1.32 ± 0.02 and 1.25 ± 0.03 mg/mL respectively and that of INH and RIF were 4.00 ± 0.06 µg/mL and 2.00 ± 0.04 µg/mL respectively. These plant extracts and major phytochemical exudate D-Pinitol was found to act synergistically with antimycobacterial drugs INH and RIF with an FIC index ~ 0.20. Time-Kill kinetics studies indicate that, these plant extracts were bacteriostatic in nature. D-Pinitol in conjunction with INH and RIF exhibited a 2 Log reduction in the growth of viable cells compared to untreated. Attempt to elucidate their mode of action through phenotypic analysis indicated that these plant extracts and D-Pinitol was found to interfere in cell division there by leading to an abnormal elongated cellular morphology. HXM extracts and D-Pinitol synergistically combined with the first line tuberculosis drugs, INH and RIF, to act on M. smegmatis. The increase in the length of M. smegmatis cells on treatment with D-Pinitol and HXM extract of the plants indicated that they hinder the cell division mechanism thereby leading to a filamentous phenotype, and finally leading to cell death. In addition, the integrity of the bacterial cell membrane is also altered causing cell death. Further gene expression analysis showed that these plant extracts and D-Pinitol hampers with function of FtsZ protein which was confirmed through in vitro inhibition of FtsZ-GTPase enzymatic activity.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas del Citoesqueleto/genética , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Plantas Medicinales , Antituberculosos/administración & dosificación , Proteínas Bacterianas/antagonistas & inhibidores , División Celular/efectos de los fármacos , Proteínas del Citoesqueleto/antagonistas & inhibidores , Sinergismo Farmacológico , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/efectos de los fármacos , Humanos , Técnicas In Vitro , Inositol/administración & dosificación , Inositol/análogos & derivados , Isoniazida/administración & dosificación , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium smegmatis/citología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Extractos Vegetales/administración & dosificación , Rifampin/administración & dosificación , TemperaturaRESUMEN
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.
Asunto(s)
Redes y Vías Metabólicas , Metabolómica , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/metabolismoRESUMEN
The metabolic activity of bacterial cells largely differentiates even within a clonal population. Such metabolic divergence among cells is thought to play an important role for phenotypic adaptation to ever-changing environmental conditions, such as antibiotic persistence. It has long been thought that persisters are in a state called dormancy, in which cells are metabolically inactive and do not grow. However, recent studies suggest that some types of persisters are not necessarily dormant, triggering a debate about the mechanisms of persisters. Here, we combined single-cell Raman imaging spectroscopy and D2O labeling to analyze metabolic activities of bacterial persister cells. Metabolically active cells uptake deuterium through metabolic processes and give distinct C-D Raman bands, which are direct indicators of metabolic activity. Using this imaging method, we characterized the metabolic activity of Mycobacterium smegmatis, a fast-growing model for Mycobacterium tuberculosis. We found that persister cells of M. smegmatis show certain metabolic activity and active cell growth in the presence of the antibiotic rifampicin. Interestingly, persistence is not correlated with growth rate prior to antibiotic exposure. These results show that dormancy is not responsible for the persistence of M. smegmatis cells against rifampicin, suggesting that the mechanism of persistence largely varies depending on the type of antibiotics and bacteria. Our results successfully demonstrate the potential of our perfusion-based single-cell D2O Raman imaging system for the analysis of the metabolic activity and growth of bacterial persister cells.
Asunto(s)
Óxido de Deuterio/metabolismo , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/metabolismo , Análisis de la Célula Individual , Antibacterianos/farmacología , Óxido de Deuterio/química , Pruebas de Sensibilidad Microbiana , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Espectrometría RamanRESUMEN
The low intensity ultrasound has been adopted by researchers to enhance the bactericidal effect against bacteria in vitro and in vivo. Although the mechanism is not completely understood, one dominant opinion is that the permeability increases because of acoustic cavitation. However, the relationship between ultrasonic exposure parameters and cavitation effects is not definitely addressed. In this paper, by establishing a modified artificial neural network (ANN) model between ultrasonic parameters and cavitation effects, the cavitation effects can be predicted and inversely the direction for choosing parameters can be given despite of different ultrasonic systems. Compared with the generic model, the computational results obtained by modified model are more close to experimental results with low calculation cost. It means that as an efficient solution, the validity of the new model has been proved. Although the research is of preliminary stage, the new method may have great value and significance because of reducing the experimental expense. The next step of this research is to explore an optimization method to obtain the most suitable parameters based on this identification model. We hope it can give a guideline for future applications in ultrasonic therapy.
Asunto(s)
Biología Computacional/métodos , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/metabolismo , Redes Neurales de la Computación , Ondas Ultrasónicas , PermeabilidadRESUMEN
Functionally uncharacterized gene Rv3627c is predicted to encode a carboxypeptidase in the pathogen of Mycobacterium tuberculosis (M. tuberculosis), which remains a major threat to human health. Here, we sought to reveal the function of Rv3627c and to elucidate its effects on mycobacterial growth. Rv3627c was purified from E. coli using Ni2+-NTA affinity chromatography, and its identity was confirmed with a monoclonal anti-polyhistidine antibody. An enzyme activity assay involving a d-amino acid oxidase-peroxidase coupled colorimetric reaction and high-performance thin layer chromatography was performed. A pull-down assay and MS-MS were also employed to identify putative interaction partners of Rv3627c. Scanning electron microscopy and transmission electron microscopy were performed to observe any morphological alterations to Mycobacterium smegmatis (M. smegmatis). We successfully obtained soluble expressed Rv3627c and identified it as carboxypeptidase using prepared peptidoglycan. Four proteins were identified as potential interaction partners with Rv3627c based on results obtained from both a pull-down assay and MS/MS analysis. Rv3627c over-expression induced M. smegmatis cells to become elongated, and promoted the formation of increased numbers of Z-rings. Rv3627c, a novel carboxypeptidase in M. tuberculosis identified in this study, exerts important effects on mycobacterial cell morphology and cell division. This functional information provides a promising insight into anti-mycobacterial target designs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carboxipeptidasas/metabolismo , División Celular , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Carboxipeptidasas/genética , Clonación Molecular , Dipéptidos/metabolismo , Humanos , Mapas de Interacción de Proteínas , Homología de SecuenciaRESUMEN
Tuberculosis, caused by Mycobacterium tuberculosis has been one of the primal afflictions to human, and owing to the current scenario of drug resistance, newer drugs, and alternate targets are required to mitigate the disease. FtsZ is a GTP hydrolyzing protein, conserved in prokaryotes that plays a central role in Z-ring formation during cell division cytokinesis stage. This study employs the use of pharmacophore models derived from two different datasets based on Mtb-FtsZ GTPase inhibition and whole cell antibacterial activity, to virtually screen and shortlist novel compounds from In-house small molecule library as Mtb-FtsZ inhibitors and evaluate their in-vitro and ex-vivo activity. The results revealed Piperine (IC50â¯=â¯21.2⯱â¯0.7⯵M), 4-Bromo di-methoxy coumarin (IC50â¯=â¯13.0⯱â¯1.6⯵M) and Di-ethyl amino methyl coumarin (IC50â¯=â¯19.4⯱â¯1.1) as potent Mtb-FtsZ GTPase inhibitors which showed considerable antibacterial activity (84.0⯱â¯2.6⯵M, 56.0⯱â¯4.3⯵M and 108⯱â¯7.1⯵M respectively) against M. smegmatis. They appear to be bacteriostatic, as well as treatment with these compounds led to a 3× increase in cell length of M. smegmatis. Further these molecules also altered the FtsZ gene expression by 3-fold when compared to untreated. In addition compound Aloin, an Aloe exudate showed potent Mtb-FtsZ inhibition (IC50â¯=â¯16.7⯱â¯0.4⯵M) but exhibited poor anti-mycobacterial activity (>500⯵M).
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Mycobacterium smegmatis/efectos de los fármacos , Alcaloides/farmacología , Proteínas Bacterianas/genética , Benzodioxoles/farmacología , División Celular/efectos de los fármacos , Cumarinas/farmacología , Citocinesis , Proteínas del Citoesqueleto/genética , Bases de Datos de Compuestos Químicos , Evaluación Preclínica de Medicamentos/métodos , GTP Fosfohidrolasas/antagonistas & inhibidores , Humanos , Simulación del Acoplamiento Molecular , Mycobacterium smegmatis/citología , Mycobacterium tuberculosis/metabolismo , Piperidinas/farmacología , Alcamidas Poliinsaturadas/farmacología , Relación Estructura-ActividadRESUMEN
Mycobacterium tuberculosis (MTB) infections rely on continued growth and division. Despite the substantial global burden of tuberculosis, the underlying mechanism governing growth is incompletely understood. Bifunctional penicillin-binding protein (PBP1), encoded by Rv0050 (ponA1) of MTB, is a key peptidoglycan synthase and plays a central role in mycobacterial growth and division by its interaction with Rpf-interacting protein A (RipA, peptidoglycan endopeptidase). Our previous work suggested that the hyper-variable proline repeats are located at the N end of PBP1. In this study, we prove that altered secondary structure resulting from polymorphic proline repeats modulates the interaction between PBP1 and RipA. Without proper coordination of peptidoglycan synthase and hydrolase, cell elongation and division is also altered resulting in phenotypic changes in the population as indicated by altered dispersion, slowed growth, or shortened cell length. Together, our data reveal that polymorphisms in Rv0050 induce mycobacterial growth and morphologic changes, and hence are responsible for giving bacteria their shape.
Asunto(s)
Mycobacterium smegmatis/genética , Mycobacterium/crecimiento & desarrollo , Proteínas de Unión a las Penicilinas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Mycobacterium/citología , Mycobacterium/genética , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/crecimiento & desarrollo , Proteínas de Unión a las Penicilinas/química , Dominios Proteicos Ricos en ProlinaRESUMEN
Although mycobacteria are rod shaped and divide by simple binary fission, their cell cycle exhibits unusual features: unequal cell division producing daughter cells that elongate with different velocities, as well as asymmetric chromosome segregation and positioning throughout the cell cycle. As in other bacteria, mycobacterial chromosomes are segregated by pair of proteins, ParA and ParB. ParA is an ATPase that interacts with nucleoprotein ParB complexes - segrosomes and non-specifically binds the nucleoid. Uniquely in mycobacteria, ParA interacts with a polar protein DivIVA (Wag31), responsible for asymmetric cell elongation, however the biological role of this interaction remained unknown. We hypothesised that this interaction plays a critical role in coordinating chromosome segregation with cell elongation. Using a set of ParA mutants, we determined that disruption of ParA-DNA binding enhanced the interaction between ParA and DivIVA, indicating a competition between the nucleoid and DivIVA for ParA binding. Having identified the ParA mutation that disrupts its recruitment to DivIVA, we found that it led to inefficient segrosomes separation and increased the cell elongation rate. Our results suggest that ParA modulates DivIVA activity. Thus, we demonstrate that the ParA-DivIVA interaction facilitates chromosome segregation and modulates cell elongation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , ADN Bacteriano/metabolismo , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/enzimología , Proteínas Bacterianas/genética , Segregación Cromosómica , Análisis Mutacional de ADN , Mycobacterium smegmatis/crecimiento & desarrolloRESUMEN
In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III2IV2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Qo and Qi sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c1 and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.
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Respiración de la Célula/fisiología , Proteínas del Complejo de Cadena de Transporte de Electrón/fisiología , Complejo III de Transporte de Electrones/fisiología , Modelos Moleculares , Mycobacterium smegmatis/metabolismo , Microscopía por Crioelectrón , Transporte de Electrón , Proteínas del Complejo de Cadena de Transporte de Electrón/química , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Complejo III de Transporte de Electrones/química , Mycobacterium smegmatis/citología , Oxidación-Reducción , Oxígeno , Estructura Terciaria de ProteínaRESUMEN
In many model organisms, diffuse patterning of cell wall peptidoglycan synthesis by the actin homolog MreB enables the bacteria to maintain their characteristic rod shape. In Caulobacter crescentus and Escherichia coli, MreB is also required to sculpt this morphology de novo. Mycobacteria are rod-shaped but expand their cell wall from discrete polar or subpolar zones. In this genus, the tropomyosin-like protein DivIVA is required for the maintenance of cell morphology. DivIVA has also been proposed to direct peptidoglycan synthesis to the tips of the mycobacterial cell. The precise nature of this regulation is unclear, as is its role in creating rod shape from scratch. We find that DivIVA localizes nascent cell wall and covalently associated mycomembrane but is dispensable for the assembly process itself. Mycobacterium smegmatis rendered spherical by peptidoglycan digestion or by DivIVA depletion are able to regain rod shape at the population level in the presence of DivIVA. At the single cell level, there is a close spatiotemporal correlation between DivIVA foci, rod extrusion and concentrated cell wall synthesis. Thus, although the precise mechanistic details differ from other organisms, M. smegmatis also establish and propagate rod shape by cytoskeleton-controlled patterning of peptidoglycan. Our data further support the emerging notion that morphology is a hardwired trait of bacterial cells.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular/fisiología , Esqueleto de la Pared Celular/biosíntesis , Mycobacterium smegmatis , Peptidoglicano/metabolismo , Esferoplastos/crecimiento & desarrollo , Esferoplastos/metabolismo , Proteínas de Microfilamentos/metabolismo , Microscopía , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/crecimiento & desarrollo , Esferoplastos/citologíaRESUMEN
OBJECTIVE: To determine how Csm4 protein expression affects intracellular survival of Mycobacterium smegmatis(MS). METHODS: Csm 4 gene was amplified by PCR to construct pMV261-Csm4 shuttle expression plasmid. The Csm4 protein expression in MS_Csm4 was detected by Western blot after electroporation of the recombinant plasmid into MS. The growth kinetics of MS_Csm4 in vitro and the influence of reactive N,O species on the growth of MS_Csm4were observed. The intracellular survival of MS_Csm4 and expressions of inducible nitric oxide synthase gene (iNOS) and nitric oxide production (NO) were detected after infection with THP-1 macrophages. RESULTS: Csm4 protein was successfully expressed in MS_Csm4,which did not affect the growth of the recombinant MS. Reactive N,O species decreased MS_Csm4 colony forming unit (CFU) in vitro. THP-1 increased the expression of iNOS and NO production and decreased intracellular survival of MS_Csm4. CONCLUSION: Recombinant MS_Csm4 is susceptible to reactive N,O species in vitro. THP-1 promotes NO release and thus discourages intracellular survival of MS.
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Proteínas Bacterianas/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Macrófagos/enzimología , Mycobacterium smegmatis/citología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Citoplasma/microbiología , Humanos , Macrófagos/microbiología , Mycobacterium tuberculosis , Óxido Nítrico/metabolismo , Células THP-1RESUMEN
Time-resolved atomic force microscopy (AFM) offers countless new modes by which to study bacterial cell physiology on relevant time scales, from mere milliseconds to hours and days on end. In addition, time-lapse AFM acts as a complementary tool to optical fluorescence microscopy (OFM), for which the combination offers a correlative link between the physical manifestation of bacterial phenotypes and molecular mechanisms obeying those principles. Herein we describe the essential materials and methods necessary for conducting time-resolved AFM and dual AFM/OFM experiments on bacteria.
Asunto(s)
Membrana Celular/ultraestructura , Microscopía de Fuerza Atómica/métodos , Mycobacterium smegmatis/ultraestructura , Imagen de Lapso de Tiempo/métodos , Ciclo Celular , Células Inmovilizadas/citología , Células Inmovilizadas/efectos de los fármacos , Dimetilpolisiloxanos/química , Microscopía Fluorescente , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/crecimiento & desarrollo , Polilisina/farmacologíaRESUMEN
The distinctive cell walls of mycobacteria are characteristic features of these bacteria. Individual cell wall components influence diverse mycobacterial phenotypes, such as colony morphology, virulence and stress resistance. To investigate the role of the hypothetical protein Rv2387, we constructed a Mycobacterium smegmatis strain that heterologously expressed this ORF, and we observed that the M. smegmatis strain expressing Rv2387 exhibited altered colony morphology and cell wall lipid composition, leading to a marked decrease in the resistance against acidic conditions. This study demonstrates that due to its impact on cell wall remodeling, Rv2387 might play an important role in mycobacterial physiology.
Asunto(s)
Pared Celular/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculosis/microbiología , Pared Celular/genética , Expresión Génica , Humanos , Viabilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrolloRESUMEN
Diadenosine polyphosphates, Ap(2-7)A, which contain two adenosines in a 5',5' linkage through phosphodiester bonds involving 2-7 phosphates, regulate diverse cellular functions in all organisms, from bacteria to humans, under normal and stress conditions. We had earlier reported consistent occurrence of asymmetric constriction during division (ACD) in 20-30% of dividing mycobacterial cells in culture, irrespective of different growth media, implying exogenous action of some factor of mycobacterial origin. Consistent with this premise, concentrated culture supernatant (CCS), but not the equivalent volume-wise concentrated unused medium, dramatically enhanced the ACD proportion to 70-90%. Mass spectrometry and biochemical analyses of the bioactive fraction from CCS revealed the ACD-effecting factor to be Ap6A. Synthetic Ap6A showed a mass spectrometry profile, biochemical characteristics, and bioactivity identical to native Ap6A in the CCS. Thus, the present work reveals a novel role for Ap6A in generating cell-length asymmetry during mycobacterial cell-division and thereby cell-length heterogeneity in the population.
Asunto(s)
División Celular/efectos de los fármacos , Fosfatos de Dinucleósidos/metabolismo , Mycobacterium/citología , Mycobacterium/metabolismo , Fosfatos de Dinucleósidos/aislamiento & purificación , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/metabolismoRESUMEN
Mycobacterium tuberculosis Rv3717 has been identified as a zinc-dependent amidase which can hydrolyze peptidoglycan (PG). To demonstrate the relationship of Rv3717 and cell division, in this study, Rv3717 gene was first amplified and expressed and the resulting protein was purified by using a His-tagged approach. M. smegmatis mc2155, a fast-growing and nonpathogenic mycobacterium was used to evaluate the effect of Rv3717 on cell division. Scan electron microscope (SEM) results indicated that M. smegmatis with division site was more exhibited and some of the cells turned larger in size after Rv3717 treatment. Transmission electron microscope (TEM) results revealed that MSMEG_6281 gene knockout strain named M sm-ΔM_6281 (MSMEG_6281 in M. smegmatis mc2155 is the homologous gene of Rv3717) tended to have a division defect with a severely abnormal morphology, and division septa were distorted. Gene expression analysis indicated also that the gene involved in cell division such as M. smegmatis ftsZ was significantly up-regulated with treatment time. The findings demonstrated that physiological role of Rv3717 was related to cell division and regulated possibly division septum formation. Further, fibronectin (Fn) binding ability of Rv3717 was evaluated by protein binding experiment, and the results confirmed the interaction of Rv3717 with Fn in a dose dependent manner. We found also that the invasion rate of M. sm-ΔM_6281 to A549â¯cells was reduced by 59% compared to the control strain, and the invasion defect could be rescued by Rv3717 addition. RT-PCR results showed that M. smegmatis fbpC were up-regulated after Rv3717 addition. These clues may be significant to explore roles of Rv3717 in growth and colonization of mycobacteria.