RESUMO
The epidemiological importance of mycobacterial species is indisputable, and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important bacterial metabolites, represents a set of targets for inhibitors of Mycobacterium tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene disruption, gene knockdown, point mutations (D61W, R134A, E321N), and kinetic analysis to evaluate aroA gene essentiality and vulnerability of its protein product, EPSPS, from Mycolicibacterium (Mycobacterium) smegmatis (MsEPSPS). We demonstrate that aroA-deficient cells are auxotrophic for aromatic amino acids (AroAAs) and that the growth impairment observed for aroA-knockdown cells grown on defined medium can be rescued by AroAA supplementation. We also evaluated the essentiality of selected MsEPSPS residues in bacterial cells grown without AroAA supplementation. We found that the catalytic residues R134 and E321 are essential, while D61, presumably important for protein dynamics and suggested to have an indirect role in catalysis, is not essential under the growth conditions evaluated. We have also determined the catalytic efficiencies (Kcat/Km) of recombinant wild-type (WT) and mutated versions of MsEPSPS (D61W, R134A, E321N). Our results suggest that drug development efforts toward EPSPS inhibition may be ineffective if bacilli have access to external sources of AroAAs in the context of infection, which should be evaluated further. In the absence of AroAA supplementation, aroA from M. smegmatis is essential, its essentiality is dependent on MsEPSPS activity, and MsEPSPS is vulnerable. IMPORTANCE We found that cells from Mycobacterium smegmatis, a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l-tryptophan, l-phenylalanine, and l-tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs. The depleted enzyme, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes the sixth step of shikimate pathway. Depletion of this enzyme inside cells was performed by disrupting or silencing the EPSPS-encoding aroA gene. Finally, we evaluated the essentiality of specific residues from EPSPS that are important for its catalytic activity, determined with experiments of enzyme kinetics using recombinant EPSPS mutants.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/metabolismo , Aminoácidos Aromáticos/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/enzimologia , 3-Fosfoshikimato 1-Carboxiviniltransferase/química , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Cinética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Alinhamento de SequênciaRESUMO
Macrophages are important in host defense and can differentiate into functionally distinct subsets named classically (M1) or alternatively (M2) activated. In several inflammatory disorders, macrophages become tolerized to prevent deleterious consequences. This tolerization reduces the ability of macrophages to respond to bacterial components (e.g., LPS) maintaining a low level of inflammation but compromising the ability of macrophages to mount an effective immune response during subsequent pathogen encounters. In this study, we aimed to reactivate human monocyte-derived macrophages chronically exposed to LPS by re-stimulation with muramyl dipeptide (MDP). We observed an undefined profile of cell surface marker expression during endotoxin tolerance and absence of TNFα production. Stimulating macrophages chronically exposed to LPS with LPS+MDP restored TNFα, production together with an increased production of IL1, IL6, IFNγ, IL4, IL5 and IL10. These results suggest that macrophages chronically exposed to LPS possess a mixed M1-M2 phenotype with sufficient antimicrobial and homeostatic potential.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunofenotipagem , Macrófagos/classificação , Macrófagos/metabolismo , Macrófagos/microbiologia , Monócitos/citologia , Mycobacterium smegmatis/crescimento & desenvolvimento , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.
Assuntos
Ácido Graxo Sintases/genética , Metabolismo dos Lipídeos , Mycobacterium smegmatis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Regulação Bacteriana da Expressão Gênica , Mutação , Mycobacterium smegmatis/genética , ÓperonRESUMO
Upon Mycobacterium tuberculosis infection, macrophages may undergo apoptosis, which has been considered an innate immune response. The pathways underlying the removal of dead cells in homeostatic apoptosis have been extensively studied, but little is known regarding how cells that undergo apoptotic death during mycobacterial infection are removed. This study shows that macrophages induced to undergo apoptosis with mycobacteria cell wall proteins are engulfed by J-774A.1 monocytic cells through the mannose receptor. This demonstration was achieved through assays in which phagocytosis was inhibited with a blocking anti-mannose receptor antibody and with mannose receptor competitor sugars. Moreover, elimination of the mannose receptor by a specific siRNA significantly diminished the expression of the mannose receptor and the phagocytosis of apoptotic cells. As shown by immunofluorescence, engulfed apoptotic bodies are initially located in Rab5-positive phagosomes, which mature to express the phagolysosome marker LAMP1. The phagocytosis of dead cells triggered an anti-inflammatory response with the production of TGF-ß and IL-10 but not of the proinflammatory cytokines IL-12 and TNF-α. This study documents the previously unreported participation of the mannose receptor in the removal of apoptotic cells in the setting of tuberculosis (TB) infection. The results challenge the idea that apoptotic cell phagocytosis in TB has an immunogenic effect.
Assuntos
Apoptose , Parede Celular/imunologia , Lectinas Tipo C/fisiologia , Macrófagos/imunologia , Lectinas de Ligação a Manose/fisiologia , Monócitos/imunologia , Mycobacterium smegmatis/imunologia , Fagocitose , Receptores de Superfície Celular/fisiologia , Animais , Linhagem Celular Tumoral , Vesículas Extracelulares/ultraestrutura , Imunofluorescência , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Lectinas Tipo C/genética , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Macrófagos/citologia , Macrófagos/microbiologia , Receptor de Manose , Lectinas de Ligação a Manose/genética , Camundongos , Mycobacterium smegmatis/crescimento & desenvolvimento , Fagossomos/imunologia , Fagossomos/ultraestrutura , RNA Interferente Pequeno , Receptores de Superfície Celular/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas rab5 de Ligação ao GTP/análiseRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Tuberculosis (TB) is an infectious disease mainly caused by Mycobacterium tuberculosis (Mtb), which generates 9 million new cases worldwide each year. The Mayo ethnicity of southern Sonora, Mexico is more than 2000 years old, and the Mayos possess extensive knowledge of traditional medicine. AIMS OF THE STUDY: To evaluate the antimycobacterial activity levels of extracts of medicinal plants used by the Mayos against Mtb and Mycobacterium smegmatis (Msm) in the treatment of TB, respiratory diseases and related symptoms. MATERIALS AND METHODS: A total of 34 plant species were collected, and 191 extracts were created with n-hexane, dichloromethane, ethyl acetate (EtOAc), methanol and water. Their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined against Mtb H37Rv using the microplate alamar blue assay (MABA) and against Msm using the resazurin microplate assay (REMA) at 6 and 2 days of exposure, respectively, and at concentrations of 250-1.9µg/mL (n-hexane extracts) and 1000-7.81µg/mL (extracts obtained with dichloromethane, EtOAc, methanol and water). RESULTS: Rhynchosia precatoria (Willd.) DC. (n-hexane root extract), Euphorbia albomarginata Torr. and A. Gray. (EtOAc shoot extract) and Helianthus annuus L. (n-hexane stem extract) were the most active plants against Mtb H37Rv, with MICs of 15.6, 250, 250µg/mL and MBCs of 31.25, 250, 250µg/mL, respectively. R. precatoria (root) was the only active plant against Msm, with MIC and MBC values of ≥250µg/mL. None of the aqueous extracts were active. CONCLUSIONS: This study validates the medicinal use of certain plants used by the Mayo people in the treatment of TB and related symptoms. R. precatoria, E. albomarginata and H. annuus are promising plant sources of active compounds that act against Mtb H37Rv. To our knowledge, this is the first time that their antimycobacterial activity has been reported. Crude extracts obtained with n-hexane, EtOAc and dichloromethane were the most active against Mtb H37Rv.
Assuntos
Antituberculosos/farmacologia , Euphorbia/química , Fabaceae/química , Helianthus/química , Medicina Tradicional , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Extratos Vegetais/farmacologia , Tuberculose/tratamento farmacológico , Acetatos/química , Antituberculosos/isolamento & purificação , Etnobotânica , Etnofarmacologia , Hexanos/química , Humanos , Cloreto de Metileno/química , México , Testes de Sensibilidade Microbiana , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fitoterapia , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Solventes/química , Tuberculose/microbiologiaRESUMO
The main purpose of our study is to understand how mycobacteria exert control over the biosynthesis of their membrane lipids and find out the key components of the regulatory network that control fatty acid biosynthesis at the transcriptional level. In this article we describe the identification and purification of FasR, a transcriptional regulator from Mycobacterium sp. that controls the expression of the fatty acid synthase (fas) and the 4-phosphopantetheinyl transferase (acpS) encoding genes, whose products are involved in the fatty acid and mycolic acid biosynthesis pathways. In vitro studies demonstrated that fas and acpS genes are part of the same transcriptional unit and that FasR specifically binds to three conserved operator sequences present in the fas-acpS promoter region (Pfas). The construction and further characterization of a fasR conditional mutant confirmed that FasR is a transcriptional activator of the fas-acpS operon and that this protein is essential for mycobacteria viability. Furthermore, the combined used of Pfas-lacZ fusions in different fasR backgrounds and electrophoretic mobility shift assays experiments, strongly suggested that long-chain acyl-CoAs are the effector molecules that modulate the affinity of FasR for its DNA binding sequences and therefore the expression of the essential fas-acpS operon.
Assuntos
Proteínas de Bactérias/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Ácido Graxo Sintases/genética , Genes Reguladores , Metabolismo dos Lipídeos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Óperon , Regiões Promotoras Genéticas , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
The study of the in vitro cell growth of mycobacteria still remains a fastidious, difficult, and time-consuming procedure. In addition, assessing mycobacterial growth in the laboratory is often complicated by cell aggregation and slow growth-rate. We now report that the use of a stainless steel spring in the culture led to an absence of large cell clumps, to a decrease of dead cells in the exponential phase and to growth of a more homogeneous population of large cells. We also report that flow cytometry is a rapid, simple and reliable approach to monitor mycobacterial cell growth and viability. Here, we monitored Mycobacterium smegmatis cellular growth by optical density, dry cell mass, and colony forming units; in addition, viability, cell size and granularity profiles were analyzed by flow cytometry, and cell morphology by electron microscopy. Cultures monitored by flow cytometry may lead to a better understanding of the physiology of mycobacteria. Moreover, this methodology may aid in characterizing the cell growth of other fastidious species of microorganisms.
Assuntos
Técnicas Bacteriológicas/métodos , Meios de Cultura/química , Mycobacterium smegmatis/crescimento & desenvolvimento , Citometria de Fluxo , Viabilidade Microbiana , Aço InoxidávelRESUMO
This study evaluated the antimicrobial and hemolytic activities and phytochemical constituents of hydroalcoholic extract and its fractions from Buchenavia tetraphylla leaves. Cyclohexane (BTCF), ethyl acetate (BTEF), and n-butanol-soluble (BTSBF) and non-soluble (BTNBF) fractions were obtained from a liquid-liquid partition of hydroalcoholic extract (BTHE) from B. tetraphylla leaves. The hemolytic activity of active fractions was checked. The BTHE inhibited the growth of Micrococcus luteus (MIC: 0.10 mg/mL), Pseudomonas aeruginosa (MIC: 0.20 mg/mL), Mycobacterium smegmatis (MIC: 0.39 mg/mL), Proteus vulgaris, and Staphylococcus aureus (MIC: 0.78 mg/mL for both). The more active fractions were BTCF and BTBSF. BTCF showed better potential to inhibit M. luteus (0.10 mg/mL), P. aeruginosa (0.20 mg/mL), S. enteritidis (0.39 mg/mL), and S. aureus (1.56 mg/mL). BTBSF showed the best results for M. luteus (0.10 mg/mL), M. smegmatis, B. subtilis (0.39 mg/mL for both), and P. vulgaris (0.10 mg/mL). The HC50 were greater than observed MIC: 20.30, 4.70 and 2.53 mg/mL, respectively, to BTBF, BTHE and BTCF, which. The phytochemical analysis detected the presence of flavanoids, triterpene, carbohydrate, and tannin. Our work showed for the first time the broad-spread antimicrobial activity of B. tetraphylla, which has nonhemolytic action, creating a new perspective on the interesting association of traditional and scientific knowledge.
Assuntos
Antibacterianos/farmacologia , Combretaceae/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , 1-Butanol/química , Acetatos/química , Antibacterianos/isolamento & purificação , Carboidratos/análise , Cicloexanos/química , Relação Dose-Resposta a Droga , Flavonoides/análise , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Micrococcus luteus/efeitos dos fármacos , Micrococcus luteus/crescimento & desenvolvimento , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/crescimento & desenvolvimento , Proteus vulgaris/efeitos dos fármacos , Proteus vulgaris/crescimento & desenvolvimento , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Solubilidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Taninos/análise , Triterpenos/análiseRESUMO
BACKGROUND: Cationic bilayers based on the inexpensive synthetic lipid dioctadecyldimethylammonium bromide (DODAB) have been useful as carriers for drug delivery, immunoadjuvants for vaccines and active antimicrobial agents. METHODS: Rifampicin (RIF) or isoniazid (ISO) interacted with DODAB bilayer fragments (BF) or large vesicles (LV). Dispersions were evaluated by dynamic light-scattering for zeta-average diameter (Dz) and zeta-potential (ζ) analysis; dialysis for determination of drug entrapment efficiency; plating and CFU counting for determination of cell viability of Mycobacterium smegmatis or tuberculosis, minimal bactericidal concentration (MBC) and synergism index for DODAB/drug combinations. RESULTS: DODAB alone killed micobacteria over a range of micromolar concentrations. RIF aggregates in water solution were solubilised by DODAB BF. RIF was incorporated in DODAB bilayers at high percentiles in contrast to the leaky behavior of ISO. Combination DODAB/RIF yielded MBCs of 2/2 and 4/0.007 µg/mL against Mycobacterium smegmatis or Mycobacterium tuberculosis, respectively. Synergism indexes equal to 0.5 or 1.0, indicated synergism against the former and independent action, against the latter species. CONCLUSIONS: In vitro, DODAB acted effectively both as micobactericidal agent and carrier for rifampicin. The novel assemblies at reduced doses may become valuable against tuberculosis.
Assuntos
Antibacterianos/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Rifampina/farmacologia , Antibacterianos/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antibióticos Antituberculose/química , Antibióticos Antituberculose/farmacologia , Portadores de Fármacos , Composição de Medicamentos/métodos , Sinergismo Farmacológico , Bicamadas Lipídicas/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Compostos de Amônio Quaternário/química , Rifampina/química , Tecnologia Farmacêutica/métodosRESUMO
Endothelial cells are susceptible to infection by several pathogens, but little is known about mycobacterial infection. We analyzed some features of mycobacteria-endothelial cell interactions and the innate response to the infection. Intracellular growth in human umbilical vein endothelial cells (HUVECs) of three Mycobacterium species: M. tuberculosis (MTB), M. abscessus (MAB) and M. smegmatis (MSM) was analyzed. M. smegmatis was eliminated; M. abscessus had an accelerate intracellular replication and M. tuberculosis did not replicate or was eliminated. M. abscessus infection induced profound cytoskeleton rearrangements, with M. tuberculosis infection changes were less marked, and with MSM were slight. Nitric oxide (NO) production was induced differentially: M. abscessus induced the highest levels followed by M. tuberculosis and M. smegmatis; the contrary was true for reactive oxygen species (ROS) production. Only M. tuberculosis infection caused beta-1 defensin over-expression. As a whole, our results describe some aspects of the innate response of HUVEC infected by mycobacteria with different virulence and suggest that a strong cytoskeleton mobilization triggers a high NO production in these cells.
Assuntos
Defensinas/biossíntese , Células Endoteliais/imunologia , Células Endoteliais/microbiologia , Imunidade Inata , Infecções por Mycobacterium/imunologia , Mycobacterium smegmatis/crescimento & desenvolvimento , Carga Bacteriana , Citoesqueleto/ultraestrutura , Defensinas/análise , Células Endoteliais/citologia , Células Endoteliais/ultraestrutura , Feminino , Feto , Interações Hospedeiro-Parasita , Humanos , Microscopia Eletrônica de Varredura , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/patogenicidade , Infecções por Mycobacterium/microbiologia , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/patogenicidade , Óxido Nítrico/análise , Gravidez , Cultura Primária de Células , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Veias Umbilicais/citologia , Virulência/imunologiaRESUMO
BACKGROUND: The ParA/Soj and ParB/Spo0J proteins, and the cis-acting parS site, participate actively in chromosome segregation and cell cycle progression. Genes homologous to parA and parB, and two putative parS copies, have been identified in the Mycobacterium bovis BCG and Mycobacterium smegmatis chromosomes. As in Mycobacterium tuberculosis, the parA and parB genes in these two non-pathogenic mycobacteria are located near the chromosomal origin of replication. The present work focused on the determination of the transcriptional organisation of the ~6 Kb orf60K-parB region of M. bovis BCG and M. smegmatis by primer extension, transcriptional fusions to the green fluorescence protein (GFP) and quantitative RT-PCR. RESULTS: The parAB genes were arranged in an operon. However, we also found promoters upstream of each one of these genes. Seven putative promoter sequences were identified in the orf60K-parB region of M. bovis BCG, whilst four were identified in the homologous region of M. smegmatis, one upstream of each open reading frame (ORF).Real-time PCR assays showed that in M. smegmatis, mRNA-parA and mRNA-parB levels decreased between the exponential and stationary phases. In M. bovis BCG, mRNA-parA levels also decreased between the exponential and stationary phases. However, parB expression was higher than parA expression and remained almost unchanged along the growth curve. CONCLUSION: The majority of the proposed promoter regions had features characteristic of Mycobacterium promoters previously denoted as Group D. The -10 hexamer of a strong E. coli sigma70-like promoter, located upstream of gidB of M. bovis BCG, overlapped with a putative parS sequence, suggesting that the transcription from this promoter might be regulated by the binding of ParB to parS.
Assuntos
Proteínas de Bactérias/genética , Mycobacterium bovis/genética , Mycobacterium smegmatis/genética , Óperon , Regiões Promotoras Genéticas , Transcrição Gênica , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Cromossomos Bacterianos/genética , Genes Bacterianos , Dados de Sequência Molecular , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium smegmatis/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Origem de Replicação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Sítio de Iniciação de TranscriçãoRESUMO
All mycobacteria studied to date have an rRNA operon, designated rrnA, located downstream from a single copy of the murA gene, which encodes an enzyme (EC 2.5.1.7) important for peptidoglycan synthesis. The rrnA operon has a promoter, P1(A), located within the coding region of murA, near the 3' end. Samples of RNA were isolated from Mycobacterium tuberculosis at different stages of the growth cycle and from Mycobacterium smegmatis grown under different conditions. RNase protection assays were used to investigate transcripts of both murA and rrnA. Transcription of murA was found to continue into the 16S rRNA gene, as if murA and rrnA form a hybrid (protein coding-rRNA coding) operon. During the growth of M. tuberculosis, the hybrid operon contributed approximately 2% to total pre-rRNA. Analysis of M. smegmatis RNA revealed that the level of murA RNA depended on the growth rate and that the patterns of expression during the growth cycle were different for murA and rrnA. M. smegmatis has a second rRNA operon, rrnB, located downstream from a single copy of the tyrS gene, encoding tyrosyl-tRNA synthetase. Transcription of tyrS was found to continue into the 16S rRNA gene rrnB. The hybrid tyrS-rrnB operon contributed 0.2 to 0.6% to rrnB transcripts. The pattern of tyrS expression during the growth cycle matched the pattern of rrnB expression, reflecting the essential role of TyrS and rRNA in protein biosynthesis.