RESUMEN
Although studies on non-tuberculous mycobacteria have increased in recent years because they cause a considerable proportion of infections, their cellulolytic system is still poorly studied. This study presents a characterization of the cellulolytic activities of environmental mycobacterial isolates derived from soil and water samples from the central region of Argentina, aimed to evaluate the conservation of the mechanism for the degradation of cellulose in this group of bacteria. The molecular and genomic identification revealed identity with Mycolicibacterium septicum. The endoglucanase and total cellulase activities were assessed both qualitatively and quantitatively and the optimal enzymatic conditions were characterized. A specific protein of around 56 kDa with cellulolytic activity was detected in a zymogram. Protein sequences possibly arising from a cellulase were identified by mass spectrometry-based shotgun proteomics. Results showed that M. septicum encodes for cellulose- and hemicellulose-related degrading enzymes, including at least an active ß-1,4 endoglucanase enzyme that could be useful to improve its survival in the environment. Given the important health issues related to mycobacteria, the results of the present study may contribute to the knowledge of their cellulolytic system, which could be important for their ability to survive in many different types of environments.
Asunto(s)
Proteínas Bacterianas , Celulasa , Celulosa , Microbiología del Suelo , Celulosa/metabolismo , Celulasa/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Argentina , Microbiología del Agua , Proteómica/métodos , Mycobacteriaceae/genética , Mycobacteriaceae/enzimologíaRESUMEN
Mycobacterium neoaurum DSM 1381 originated from Mycobacterium neoaurum ATCC 25790 by mutagenesis screening is a strain of degrading phytosterols and accumulating important C22 steroid intermediates, including 22-hydroxy-23, 24-bisnorchola-4-en-3-one (4-HP) and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (HPD). However, the metabolic mechanism of these C22 products in M. neoaurum DSM 1381 remains unknown. Therefore, the whole-genome sequencing and comparative genomics analysis of M. neoaurum DSM 1381 and its parent strain M. neoaurum ATCC 25790 were performed to figure out the mechanism. As a result, 28 nonsynonymous single nucleotide variants (SNVs), 17 coding region Indels, and eight non-coding region Indels were found between the genomes of the two strains. When the wild-type 3-ketosteroid-9α-hydroxylase subunit A1 (KshA1) and ß-hydroxyacyl-CoA dehydrogenase (Hsd4A) were overexpressed in M. neoaurum DSM 1381, the steroids were transformed into the 4-androstene-3, 17- dione (AD) and 1,4-androstadiene-3,17-dione (ADD) instead of C22 intermediates. This result indicated that 173N of KshA1 and 171K of Hsd4A are indispensable to maintaining their activity, respectively. Amino acid sequence alignment analysis show that both N173D in KshA1 and K171E in Hsd4A are conservative sites. The 3D models of these two enzymes were predicted by SWISS-MODEL and AlphaFold2 to understand the inactivation of the two key enzymes. These results indicate that K171E in Hsd4A may destroy the inaction between the NAD+ with the NH3+ and N173D in KshA1 and may disrupt the binding of the catalytic domain to the substrate. A C22 steroid intermediates-accumulating mechanism in M. neoaurum DSM 1381 is proposed, in which the K171E in Hsd4A leads to the enzyme's inactivation, which intercepts the C19 sub-pathways and accelerates the C22 sub-pathways, and the N173D in KshA1 leads to the enzyme's inactivation, which blocks the degradation of C22 intermediates. In conclusion, this study explained the reasons for the accumulation of C22 intermediates in M. neoaurum DSM 1381 by exploring the inactivation mechanism of the two key enzymes.
Asunto(s)
Mycobacteriaceae , Mycobacterium , Fitosteroles , Mycobacterium/genética , Mycobacterium/metabolismo , Esteroides/metabolismo , Mycobacteriaceae/genética , Mycobacteriaceae/metabolismo , Oxigenasas de Función Mixta/metabolismo , Fitosteroles/metabolismoRESUMEN
Conjugation is considered the main horizontal gene transfer mechanism in bacterial adaptation and evolution. In the Mycobacteriaceae family, Mycolicibacterium smegmatis has been used as the model organism for the conjugative transfer of hybrid plasmids. However, the natural conjugation process in any bacteria would involve the transfer of naturally occurring plasmids. Currently, there is a gap in this regard about this abundant environmental genus of Mycobacteriaceae. Here, we performed conjugation experiments between wild Mycolicibacterium sp. strains involving naturally occurring plasmids, and interestingly, evidence of conjugative transfer was obtained. Thus, it is likely that conjugation occurs in Mycolicibacterium in the natural environment, representing a source of diversification and evolution in this genus of bacteria.
Asunto(s)
Conjugación Genética , Mycobacteriaceae , Bacterias/genética , Transferencia de Gen Horizontal , Mycobacteriaceae/genética , Plásmidos/genéticaRESUMEN
Introduction: Infections caused by non-tuberculosis mycobacteria are significantly worsening across the globe. M. fortuitum complex is a rapidly growing pathogenic species that is of clinical relevance to both humans and animals. This pathogen has the potential to create adverse effects on human healthcare. Methods: The MF GZ001 clinical strain was collected from the sputum of a 45-year-old male patient with a pulmonary infection. The morphological studies, comparative genomic analysis, and drug resistance profiles along with variants detection were performed in this study. In addition, comparative analysis of virulence genes led us to understand the pathogenicity of this organism. Results: Bacterial growth kinetics and morphology confirmed that MF GZ001 is a rapidly growing species with a rough morphotype. The MF GZ001 contains 6413573 bp genome size with 66.18 % high G+C content. MF GZ001 possesses a larger genome than other related mycobacteria and included 6156 protein-coding genes. Molecular phylogenetic tree, collinearity, and comparative genomic analysis suggested that MF GZ001 is a novel member of the M. fortuitum complex. We carried out the drug resistance profile analysis and found single nucleotide polymorphism (SNP) mutations in key drug resistance genes such as rpoB, katG, AAC(2')-Ib, gyrA, gyrB, embB, pncA, blaF, thyA, embC, embR, and iniA. In addition, the MF GZ001strain contains mutations in iniA, iniC, pncA, and ribD which conferred resistance to isoniazid, ethambutol, pyrazinamide, and para-aminosalicylic acid respectively, which are not frequently observed in rapidly growing mycobacteria. A wide variety of predicted putative potential virulence genes were found in MF GZ001, most of which are shared with well-recognized mycobacterial species with high pathogenic profiles such as M. tuberculosis and M. abscessus. Discussion: Our identified novel features of a pathogenic member of the M. fortuitum complex will provide the foundation for further investigation of mycobacterial pathogenicity and effective treatment.
Asunto(s)
Farmacorresistencia Bacteriana , Mycobacteriaceae , Animales , Humanos , Persona de Mediana Edad , Pruebas de Sensibilidad Microbiana , Filogenia , Farmacorresistencia Bacteriana/genética , Mycobacteriaceae/efectos de los fármacos , Mycobacteriaceae/genéticaRESUMEN
Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum, including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Mycobacteriaceae/genética , Sistemas CRISPR-Cas/genética , Reparación del ADN por Unión de Extremidades , PlásmidosRESUMEN
BACKGROUND: Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. RESULTS: The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a ß-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. CONCLUSION: KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.
Asunto(s)
Redes y Vías Metabólicas , Mycobacteriaceae/genética , Mycobacteriaceae/metabolismo , Fitosteroles/metabolismo , Profármacos/metabolismo , Esteroides/metabolismo , Proteínas Bacterianas/genética , Coenzima A Transferasas/genética , Edición Génica , Técnicas de Inactivación de Genes , Genoma Bacteriano , Hidroliasas/genética , Oxidorreductasas/genéticaRESUMEN
In the present work, we tried to identify the mechanism why by which the steroid alcohols accumulated when hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was present to enhance the sterol conversion rate. Compared with the bioconversion system without HP-ß-CD, the reaction rate was greatly improved in presence of HP-ß-CD, but the steroid alcohols largely accumulated concurrently. In a reaction system with an enhanced reaction rate, the higher intracellular NADH/NAD+ level was detected, and the production of steroid alcohols increased also. Mycobacterium neoaurum mutants with higher KshA activity (3-ketosteroid 9α-hydrolase, a monooxygenase hydroxylating the nucleus at C-9 at the expense of NAD(P)H consumption) reduced the steroid alcohol production, and in the meantime, the NADH/NAD+ level was decreased consequently. Further research found that oxygen availability was seriously inhibited by the cyclodextrin in a reaction system. These results indicated that NADH formed in the bioconversion was not properly regenerated via the respiratory chain because of the poor oxygen bioavailability. The inhibitory effect of cyclodextrin on oxygen bioavailability is a key factor for the metabolic flux redistribution toward steroid alcohols in phytosterol resting cells bioconversion.
Asunto(s)
2-Hidroxipropil-beta-Ciclodextrina/farmacología , Proteínas Bacterianas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Mutación , Mycobacteriaceae/metabolismo , Oxígeno/metabolismo , Fitosteroles/biosíntesis , Proteínas Bacterianas/genética , Oxigenasas de Función Mixta/genética , Mycobacteriaceae/genética , Fitosteroles/genéticaRESUMEN
The mobilome plays a crucial role in bacterial adaptation and is therefore a starting point to understand and establish the gene flow occurring in the process of bacterial evolution. This is even more so if we consider that the mobilome of environmental bacteria can be the reservoir of genes that may later appear in the clinic. Recently, new genera have been proposed in the family Mycobacteriaceae, including the genus Mycolicibacterium, which encompasses dozens of species of agricultural, biotechnological, clinical and ecological importance, being ubiquitous in several environments. The current scenario in the Mycobacteriaceae mobilome has some bias because most of the characterized mycobacteriophages were isolated using a single host strain, and the few plasmids reported mainly relate to the genus Mycobacterium. To fill in the gaps in these issues, we performed a systematic in silico study of these mobile elements based on 242 available genomes of the genus Mycolicibacterium. The analyses identified 156 putative plasmids (19 conjugative, 45 mobilizable and 92 non-mobilizable) and 566 prophages in 86 and 229 genomes, respectively. Moreover, a contig was characterized by resembling an actinomycete integrative and conjugative element (AICE). Within this diversity of mobile genetic elements, there is a pool of genes associated with several canonical functions, in addition to adaptive traits, such as virulence and resistance to antibiotics and metals (mercury and arsenic). The type-VII secretion system was a common feature in the predicted plasmids, being associated with genes encoding virulent proteins (EsxA, EsxB, PE and PPE). In addition to the characterization of plasmids and prophages of the family Mycobacteriaceae, this study showed an abundance of these genetic elements in a dozen species of the genus Mycolicibacterium.
Asunto(s)
Variación Genética , Mycobacteriaceae/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Simulación por Computador , Microbiología Ambiental , Genoma Bacteriano , Secuencias Repetitivas Esparcidas , Microbiota , Mycobacteriaceae/clasificación , Mycobacteriaceae/aislamiento & purificación , Mycobacteriaceae/virología , Filogenia , Plásmidos/genética , Plásmidos/metabolismo , Profagos/clasificación , Profagos/genética , Profagos/aislamiento & purificaciónRESUMEN
To date, only 26 cases of Mycobacterium wolinskyi infections have been reported in humans. We herein report a first case of prosthetic valve endocarditis due to this organism after cardiovascular surgery. An 82-year-old man presented with repeat episodes of syncope and fever after aortic valve replacement, mitral valve replacement, left atrial appendage closure, and pulmonary vein isolation. Blood cultures maintained in aerobic bottles were repeatedly positive after 90-100 hours, and Gallium scan revealed abnormal accumulations in the sternum and left testis. While colonies formed by culturing the fluid of the parasternal area and blood cultures revealed gram-positive rods, we could not analyze the colony using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF). M. wolinskyi was finally identified on 16S rRNA, hsp65, and rpoB gene sequencing. We treated the patient with multiple antimycobacterial drugs, i.e., amikacin, imipenem, and clarithromycin for 6 weeks, which was changed to oral ciprofloxacin and minocycline for 12 months. This case highlights the need to consider rapidly growing mycobacteria, including M. wolinskyi, if chronic fever persists from weeks to months after surgery, the blood culture is positive, and the organism is not identified. In addition, sequencing the 16S rRNA, hsp65, and rpoB genes is essential for diagnosis.
Asunto(s)
Endocarditis Bacteriana , Endocarditis , Prótesis Valvulares Cardíacas , Anciano de 80 o más Años , Endocarditis Bacteriana/diagnóstico , Endocarditis Bacteriana/tratamiento farmacológico , Prótesis Valvulares Cardíacas/efectos adversos , Humanos , Masculino , Mycobacteriaceae/genética , ARN Ribosómico 16S/genéticaRESUMEN
4-Androstenedione (4-AD) and progesterone (PG) are two of the most important precursors for synthesis of steroid drugs, however their current manufacturing processes suffer from low efficiency and severe environmental issues. In this study, we decipher a dual-role reductase (mnOpccR) in the phytosterols catabolism, which engages in two different metabolic branches to produce the key intermediate 20-hydroxymethyl pregn-4-ene-3-one (4-HBC) through a 4-e reduction of 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA) and 2-e reduction of 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA), respectively. Inactivation or overexpression of mnOpccR in the Mycobacterium neoaurum can achieve exclusive production of either 4-AD or 4-HBC from phytosterols. By utilizing a two-step synthesis, 4-HBC can be efficiently converted into PG in a scalable manner (100 gram scale). This study deciphers a pivotal biosynthetic mechanism of phytosterol catabolism and provides very efficient production routes of 4-AD and PG.
Asunto(s)
Proteínas Bacterianas/metabolismo , Oxidorreductasas/metabolismo , Fitosteroles/metabolismo , Pregnenos/metabolismo , Androstenodiona/síntesis química , Proteínas Bacterianas/genética , Biocatálisis , Mycobacteriaceae/enzimología , Mycobacteriaceae/genética , Oxidorreductasas/genética , Pregnenos/química , Progesterona/síntesis químicaRESUMEN
A 57-year-old Caucasian woman suffered from dyspnea on exertion. One year following a supposed pulmonary embolism event, a chronic thromboembolic vasculopathy was diagnosed and a pulmonary thromboendarterectomy was performed. However, a granulomatous pulmonary arterial vasculitis was identified upon examination. DNA of Mycobacterium goodii was detected as the most likely causative agent. Anti-inflammatory and anti-mycobacterial therapy was initiated for more than 12 months. Regular PET-CT scans revealed improvement under therapy. The last PET-CT did not show any tracer uptake following 10 months of therapy.
Asunto(s)
Infecciones por Bacterias Grampositivas/microbiología , Enfermedades Pulmonares/microbiología , Mycobacteriaceae/aislamiento & purificación , Vasculitis/microbiología , Femenino , Infecciones por Bacterias Grampositivas/diagnóstico por imagen , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Humanos , Enfermedades Pulmonares/diagnóstico por imagen , Enfermedades Pulmonares/tratamiento farmacológico , Persona de Mediana Edad , Mycobacteriaceae/genética , Tomografía Computarizada por Tomografía de Emisión de Positrones , Vasculitis/diagnóstico por imagen , Vasculitis/tratamiento farmacológicoRESUMEN
Mycobacteroides immunogenum is an emerging opportunistic pathogen implicated in nosocomial infections. Comparative genome analyses may provide better insights into its genomic structure, functions and evolution. The present analysis showed that M. immunogenum has an open pan-genome. Approximately 36.8% of putative virulence genes were identified in the accessory regions of M. immunogenum. Phylogenetic analyses revealed two potential novel subspecies of M. immunogenum, supported by evidence from ANIb (average nucleotide identity using blast) and GGDC (Genome to Genome Distance Calculator) analyses. We identified 74 genomic islands (GIs) in Subspecies 1 and 23 GIs in Subspecies 2. All Subspecies 2-harboured GIs were not found in Subspecies 1, indicating that they might have been acquired by Subspecies 2 after their divergence. Subspecies 2 has more defence genes than Subspecies 1, suggesting that it might be more resistant to the insertion of foreign DNA and probably explaining why Subspecies 2 has fewer GIs. Positive selection analysis suggest that M. immunogenum has a lower selection pressure compared to non-pathogenic mycobacteria. Thirteen genes were positively selected and many were involved in virulence.
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Genómica/métodos , Mycobacteriaceae/clasificación , Factores de Virulencia/genética , Genoma Bacteriano , Islas Genómicas , Tipificación de Secuencias Multilocus , Mycobacteriaceae/genética , Mycobacteriaceae/patogenicidad , Filogenia , ARN Ribosómico 16S/genética , Selección Genética , Especificidad de la EspecieRESUMEN
BACKGROUND: Mycobacterium houstonense is rapidly growing mycobacteria (RGM) that belongs to M. fortuitum group. So far, there have been few associated reports of human diseases induced by M. houstonense worldwide. CASE PRESENTATION: We present a delayed-onset postoperative endophthalmitis caused by M. houstonense after glaucoma drainage implant (GDI) surgery. The ocular infection lasted for 2 months without appropriate treatment that developed into endophthalmitis and the patient underwent an emergency enucleation. CONCLUSION: Implant erosion and a delay in diagnosis of ocular infection could lead to irreversible damage as observed in our case. Ophthalmologists should be alert for ocular RGM infection, and prompt laboratory diagnosis with initiation of effective multidrug therapy might prevent loss of vision.
Asunto(s)
Endoftalmitis/diagnóstico , Endoftalmitis/etiología , Implantes de Drenaje de Glaucoma/efectos adversos , Mycobacteriaceae/genética , Complicaciones Posoperatorias/diagnóstico , Amicacina/uso terapéutico , Antibacterianos/uso terapéutico , Quimioterapia Combinada , Endoftalmitis/tratamiento farmacológico , Endoftalmitis/cirugía , Enucleación del Ojo , Estudios de Seguimiento , Humanos , Levofloxacino/uso terapéutico , Masculino , Persona de Mediana Edad , Mycobacteriaceae/aislamiento & purificación , Complicaciones Posoperatorias/tratamiento farmacológico , Complicaciones Posoperatorias/microbiología , Complicaciones Posoperatorias/cirugía , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Resultado del TratamientoRESUMEN
Mycobacterium neoaurum strains can transform phytosterols to 4-androstene-3,17-dione (4-AD), a key intermediate for the synthesis of advanced steroidal medicines. In this work, we presented the complete genome sequence of the M. neoaurum strain HGMS2, which transforms ß-sitosterol to 4-AD. Through genome annotation, a phytosterol-degrading pathway in HGMS2 was predicted and further shown to form a 9,10-secosteroid intermediate by five groups of enzymes. These five groups of enzymes included three cholesterol oxidases (ChoM; group 1: ChoM1, ChoM2 and Hsd), two monooxygenases (Mon; group 2: Mon164 and Mon197), a set of enzymes for side-chain degradation (group 3), one 3-ketosteroid-1,2-dehydrogenase (KstD; group 4: KstD211) and three 3-ketosteroid-9a-hydroxylases (Ksh; group 5: KshA226, KshA395 and KshB122). A gene cluster encoding Mon164, KstD211, KshA226, KshB122 and fatty acid ß-oxidoreductases constituted one integrated metabolic pathway, while genes encoding other key enzymes were sporadically distributed. All key enzymes except those from group 3 were prepared as recombinant proteins and their activities were evaluated, and the proteins exhibited distinct activities compared with enzymes identified from other bacterial species. Importantly, we found that the KstD211 and KshA395 enzymes in the HGMS2 strain retained weak activities and caused the occurrence of two major impurities, i.e., 1,4-androstene-3,17-dione (ADD) and 9-hydroxyl-4-androstene-3,17-dione (9OH-AD) during ß-sitosterol fermentation. The concurrence of these two 4-AD analogs not only lowered 4-AD production yield but also hampered 4-AD purification. HGMS2 has the least number of genes encoding KstD and Ksh enzymes compared with current industrial strains. Therefore, HGMS2 could be a potent strain by which the 4-AD production yield could be enhanced by disabling the KstD211 and KshA395 enzymes. Our work also provides new insight into the engineering of the HGMS2 strain to produce ADD and 9OH-AD for industrial application.
Asunto(s)
Androstenodiona/biosíntesis , Mycobacteriaceae/enzimología , Mycobacteriaceae/genética , Fitosteroles/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Redes y Vías Metabólicas , Secuenciación Completa del GenomaRESUMEN
The conversion of low value-added phytosterols into 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is an important step in the steroid pharmaceutical industry. However, the highly dense cell envelope with extremely low permeability largely affects the overall transformation efficiency. Here, we preliminarily located the key gene embC required for the synthesis of lipoarabinomannan from lipomannan in Mycobacterium neoaurum. The genetic manipulation of embC indicated that it might be the only functional enzyme catalyzing the above synthesis process. The deficiency of lipoarabinomannan led to a significantly increased cell permeability, which in turn caused the enhanced uptake capacity of cells. The sterol substrate conversion efficiency of mycobacterial cells was increased by about 52.4 % after 72-h conversion. Ultimately, the absence of embC increased the productivity from 0.0927â¯g/L/h to 0.1031â¯g/L/h, as confirmed by a resting cell system. This study verified the feasibility of improving the efficiency of the microbial conversion system through the cell envelope engineering strategy.
Asunto(s)
Androstenodiona/metabolismo , Biotransformación , Membrana Celular/metabolismo , Pared Celular/metabolismo , Lipopolisacáridos/biosíntesis , Mycobacteriaceae/genética , Mycobacteriaceae/metabolismo , Fitosteroles/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico , Eliminación de Gen , Genes Bacterianos/genética , Lipopolisacáridos/genética , Ingeniería Metabólica , Permeabilidad , Esteroles/metabolismoRESUMEN
Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5-9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity.
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Proteínas Bacterianas/metabolismo , Enzimas Inmovilizadas/metabolismo , Enzimas/metabolismo , Mycobacteriaceae/enzimología , Proteínas Recombinantes/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Biocatálisis , Microscopía por Crioelectrón , Cristalografía por Rayos X , Estabilidad de Enzimas , Enzimas/genética , Microscopía Electrónica de Transmisión , Mycobacteriaceae/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestructura , Especificidad por Sustrato , TemperaturaRESUMEN
Large-scale -omics data now allows for investigating genome-wide functional elements. Using RNA-Seq data, we tested the expression of 134 pseudogenes which do not show duplicated protein coding genes in the M. smegmatis genome. We observe significant expression and translation of 28 pseudogenes. Further examination using RNA-Seq reads suggested the sequencing errors in many pseudogenes. These include some of the functionally relevant genes such as recN and manB. We propose that the analysis of transcriptional and translational landscape using multi-dimensional -omics data could shed light on the current annotations of the bacterial pseudogenes.
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Mycobacterium smegmatis/genética , Seudogenes/genética , Análisis de Secuencia de ARN/métodos , Cromatografía Liquida/métodos , Biología Computacional/métodos , Expresión Génica/genética , Mycobacteriaceae/genética , ARN/genética , Espectrometría de Masas en Tándem/métodos , Transcripción Genética/genética , Transcriptoma/genéticaRESUMEN
Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) infection remains a serious public threat despite decades of creative endeavors. There are few reports on the roles of M. tuberculosis enzymes involved in cell envelope biosynthesis in pathogen survival and persistence. M. tuberculosis Rv3717 encodes N-acetylmuramoyl-l-alanine amidase, a cell-wall hydrolase that hydrolyzes the bond between N-acetylmuramic acid and l-alanine in cell-wall peptidoglycan. In this paper, we demonstrated the Rv3717 promoted the survival of Mycolicibacterium smegmatis(M. smegmatis) within macrophages. More importantly, we demonstrated that this effect is because MS_Rv3717 reduces the release of host pro-inflammatory cytokines such as IL-1ß, IL-6, IL-12 p40, TNF-α, and increased transcription of anti-inflammatory cytokine IL-10. At the same time, MS_Rv3717 inhibits apoptosis by inhibiting the activation of Caspase-3/9, reducing the host's elimination of M. smegmatis. Finally, from a bacterial perspective, we found Rv3717 decreased the survival of M. smegmatis under stresses such as SDS and low pH. This is the first report of the involvement of Mycobacterium cell envelope biosynthetic enzyme in host-pathogen interaction.
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Apoptosis/inmunología , Proteínas Bacterianas/metabolismo , Caspasas/metabolismo , Mycobacteriaceae/genética , Mycobacteriaceae/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Caspasas/genética , Supervivencia Celular , Biología Computacional , Secuencia Conservada , Regulación Bacteriana de la Expresión Génica/fisiología , Humanos , Inmunidad Innata , Ratones , Monocitos/microbiología , Monocitos/fisiología , Células RAW 264.7 , Estrés Fisiológico , Células THP-1RESUMEN
Mobile genetic elements (MGEs) are agents of bacterial evolution and adaptation. Genome sequencing provides an unbiased approach that has revealed an abundance of MGEs in prokaryotes, mainly plasmids and integrative conjugative elements. Nevertheless, many mobilomes, particularly those from environmental bacteria, remain underexplored despite their representing a reservoir of genes that can later emerge in the clinic. Here, we explored the mobilome of the Mycobacteriaceae family, focusing on strains from Brazilian Atlantic Forest soil. Novel Mycolicibacterium and Mycobacteroides strains were identified, with the former ones harbouring linear and circular plasmids encoding the specialized type-VII secretion system (T7SS) and mobility-associated genes. In addition, we also identified a T4SS-mediated integrative conjugative element (ICEMyc226) encoding two T7SSs and a number of xenobiotic degrading genes. Our study uncovers the diversity of the Mycobacteriaceae mobilome, providing the evidence of an ICE in this bacterial family. Moreover, the presence of T7SS genes in an ICE, as well as plasmids, highlights the role of these mobile genetic elements in the dispersion of T7SS.
Asunto(s)
Secuencias Repetitivas Esparcidas , Mycobacteriaceae/clasificación , Plásmidos/genética , Análisis de Secuencia de ADN/métodos , Brasil , Conjugación Genética , Bosques , Transferencia de Gen Horizontal , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Mycobacteriaceae/genética , Filogenia , Microbiología del SueloRESUMEN
Household water purifiers are increasingly used to treat drinking water at the household level, but their influence on the microbiological safety of drinking water has rarely been assessed. In this study, representative purifiers, based on different filtering processes, were analyzed for their impact on effluent water quality. The results showed that purifiers reduced chemical qualities such as turbidity and free chlorine. However, a high level of bacteria (102-106 CFU/g) was detected at each stage of filtration using a traditional culture-dependent method, whereas quantitative PCR with propidium monoazide (PMA) treatment showed 106-108 copies/L of total viable bacteria in effluent water, indicating elevated microbial contaminants after purifiers. In addition, high-throughput sequencing revealed a diverse microbial community in effluents and membranes. Proteobacteria (22.06-97.42%) was the dominant phylum found in all samples, except for purifier B, in which Melainabacteria was most abundant (65.79%). For waterborne pathogens, Escherichia coli (100-106 copies/g) and Pseudomonas aeruginosa (100-105 copies/g) were frequently detected by qPCR. Sequencing also demonstrated the presence of E. coli (0-6.26%), Mycobacterium mucogenicum (0.01-3.46%), and P. aeruginosa (0-0.16%) in purifiers. These finding suggest that water from commonly used household purifiers still impose microbial risks to human health.