9 research outputs found
Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. Smegmatis mutants
The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor (s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.Fil: Suárez, Cristian Alejandro. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Laboratorio de MicrobiologĂa Molecular; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; ArgentinaFil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Laboratorio de MicrobiologĂa Molecular; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; ArgentinaFil: Tasselli, Sabrina Emilse. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Laboratorio de MicrobiologĂa Molecular; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; ArgentinaFil: Morbidoni, HĂ©ctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Laboratorio de MicrobiologĂa Molecular; Argentin
Analysis of Novel Mycobacteriophages Indicates the Existence of Different Strategies for Phage Inheritance in Mycobacteria
Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100?400 nm, genome size in the 50?70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30uC, eight of them failed to propagate at 37uC. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences) allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.Fil: Stella, Emma Julieta. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Tasselli, Sabrina Emilse. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Morbidoni, HĂ©ctor Ricardo. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia
Complete genome sequences of nine mycobacteriophages
Genome analyses of a large number of mycobacteriophages, bacterial viruses that infect members of the genus Mycobacterium, yielded novel enzymes and tools for the genetic manipulation of mycobacteria. We report here the complete genome sequences of nine mycobacteriophages, including a new singleton, isolated using Mycobacterium smegmatis mc(2)155 as a host strain.Fil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Cs.mĂ©dicas. Escuela de Cs.mĂ©dicas. Cátedra de MicrobiologĂa,parasitologĂa y Virologia; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Suárez, Cristian Alejandro. Universidad Nacional de Rosario. Facultad de Cs.mĂ©dicas. Escuela de Cs.mĂ©dicas. Cátedra de MicrobiologĂa,parasitologĂa y Virologia; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Teran, Lucrecia Cecilia. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Raya, Raul Ricardo. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Morbidoni, HĂ©ctor Ricardo. Universidad Nacional de Rosario. Facultad de Cs.mĂ©dicas. Escuela de Cs.mĂ©dicas. Cátedra de MicrobiologĂa,parasitologĂa y Virologia; Argentin
A katG S315T or an ahpC promoter mutation mediate Mycobacterium tuberculosis resistance to 2-thiophen carboxylic acid hydrazide, an inhibitor resembling the anti-tubercular drugs Isoniazid and Ethionamide
Clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis are differentially susceptible to 2-Thiophen Hydrazide (TCH); however its mechanism of action or the reasons for that difference are unknown. We report herein that under our experimental conditions, TCH inhibits M. tuberculosis in solid but not in liquid medium, and that in spite of resembling Isoniazid and Ethionamide, it does not affect mycolic acid synthesis. To understand the mechanisms of action of TCH we isolated M. tuberculosis TCH resistant mutants which fell into two groups; one resistant to TCH and Isoniazid but not to Ethionamide or Triclosan, and the other resistant only to TCH with no, or marginal, cross resistance to Isoniazid. A S315T katG mutation conferred resistance to TCH while katG expression from a plasmid reduced M. tuberculosis MIC to this drug, suggesting a possible involvement of KatG in TCH activation. Whole genome sequencing of mutants from this second group revealed a single mutation in the alkylhydroperoxide reductase ahpC promoter locus in half of the mutants, while the remaining contained mutations in dispensable genes. This is the first report of the genetics underlying the action of TCH and of the involvement of ahpC as the sole basis for resistance to an anti-tubercular compound.Fil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; ArgentinaFil: Belardinelli, Juan Manuel. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. State University of Colorado - Fort Collins; Estados UnidosFil: Tong, Ping. University College Dublin; Irlanda. University of Edinburgh; Reino UnidoFil: Loftus, Brendan. University College Dublin; IrlandaFil: Recio Balsells, Alejandro Iván. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de QuĂmica Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de QuĂmica Rosario; ArgentinaFil: Labadie, Guillermo Roberto. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de QuĂmica Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de QuĂmica Rosario; ArgentinaFil: Gordon, Stephen V.. University College Dublin; IrlandaFil: Morbidoni, HĂ©ctor Ricardo. Universidad Nacional de Rosario; Argentin
Aislamiento y caracterizaciĂłn molecular de micobacteriofagos: sus aplicaciones al estudio y diagnĂłstico de micobacterias patĂłgenas
En las Ăşltimas dĂ©cadas el estudio de los bacteriofagos especĂficos de micobacterias-micobacteriofagos- se ha constituido en una pieza clave para los avances alcanzados en el estudio de la fisiologĂa y genĂ©tica de este gĂ©nero bacteriano, que comprende numerosas especias entre ellas patĂłgenos que suponen una amenaza en salud pĂşblica como M. tuberculosis y M.leprae, agentes causales de las tuberculosis y la lepra respectivamente.
Durante el desarrollo de la presente Tesis Doctoral se han aislado siete micobacteriofagos usando M. smegmatis como bacteria indicadora; los cuales junto a otros once micobacteriofagos aislados previamente en el mismo laboratorio de trabajo, han sido caracterizados fenotĂpicamente, en cuanto a su naturaleza lĂtica o temperada, su rango de huĂ©sped y sus caracterĂsticas morfomĂ©tricas. Algunos de ellos se han integrado con facilidad al cromosoma de M. smegmatis mientras que seis de los dieciocho micobacteriofagos en estudio fueron capaces de infectar M. tuberculosis. TambiĂ©n, gracias a la secuenciaciĂłn genĂłmica de estos especĂmenes los mismos pudieron agruparse en distintas categorĂas definidas en la literatura, se determinaron el nĂşmero de ORFs en cada caso, y la funciĂłn probable para los mismos, cuando esta pudo ser asignada; este análisis permitiĂł finalizar la secuenciagenimica para diez de ellos, las cuales han sido depositadas en Genbank. Inesperadamente en una alta proporciĂłn, estos aislamientos presentaron loci de particiĂłn, constituĂdos por los elementos ParA, ParB y ParS. Mediante un análisis filogenĂ©tico de los mismos, y considerando la naturaleza temperada de los micobacteriofagos que poseen estos sistemas, es probable que dada la reticencia del gĂ©nero Mycobacterium al intercambio gĂ©nico, sus virus hayan adquirido estos loci de particiĂłn como una alternativa para perpetuarse junto con las micobacterias sin “perturbar” su genoma.
Por otro lado, en el marco de una colaboraciĂłn establecida con el grupo del Dr. C. MartĂn (Universidad de Zaragoza, España), se ha construido una mutante en el ORF MSMEG_4724, codificante para una oligorribonucleasa hipotĂ©tica (ORN) con actividad 3´-5´exonucleasa. La ausencia de la misma ha generado una reducciĂłn en la eficiencia de formaciĂłn de placas de lisis para más de la mitad de los micobacteriofagos ensayados, bloqueando parcialmente el ciclo de replicaciĂłn de los mismos en la etapa de inyecciĂłn del material genĂ©tico, y en algĂşn otro punto que aĂşn no pudo ser elucidado. Además, mediante el análisis de mutantes espontáneas resistentes a distintos micobacteriofagos, se han podido dividir a los aislamientos locales en dos grupos definidos, aquellos que usarĂan GPLs como molĂ©culas receptoras, y un grupo mayoritario que fue capaz de replicarse en mutantes deficientes en estas molĂ©culas, y por lo tanto tendrĂan preferencia por otro tipo de molĂ©culas para iniciar la infecciĂłn en M. smegmatis.Fil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; Argentina
Mycobacteriophage CRB2 defines a new subcluster in mycobacteriophage classification
Mycobacteriophages are viruses -mostly temperates- that infect Mycobacterium smegmatis and sometimes Mycobacterium tuberculosis. Mycobacteriophages are grouped in clusters on the basis of the overall nucleotide sequence homology, being further divided in subclusters as more mycobacteriophage genomes are sequenced and annotated. As part of our on-going screening for novel isolates, we herein report the bioinformatics analysis of CRB2, a mycobacteriophage belonging into the Siphoviridae family that propagates at 30°C. CRB2 has a 72,217 bp genome with a 69.78% GC content that belongs to Cluster B; nucleotide comparison with other B cluster members positions CRB2 as the sole member of a new subcluster, B9, being mycobacteriophage Saguaro (belonging into subcluster B7) its closest relative. Sequencing and annotation of 14 mycobacteriophages isolated by our group has yielded six cluster A members, a singleton, four of the five members of subcluster B6, one of the three reported members of subcluster G4, and CRB2 which defines subcluster B9. Considering the massive mycobacteriophage search performed in USA and the relatively rarity of our phages, we propose that factors other than size of the sampling determine the variability of mycobacteriophage distribution, and thus a world-wide concerted mining would most likely bring extremely rare and yet undiscovered mycobacteriophages.Fil: Suarez, Cristian Alejandro. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Laboratorio de MicrobiologĂa Molecular; ArgentinaFil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Laboratorio de MicrobiologĂa Molecular; ArgentinaFil: Morbidoni, HĂ©ctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Laboratorio de MicrobiologĂa Molecular; Argentin
Deletion of msmeg_1350 in mycobacterium smegmatis causes loss of epoxy-mycolic acids, fitness alteration at low temperature and resistance to a set of mycobacteriophages
Mycobacterium smegmatis is intrinsically resistant to thiacetazone, an anti-tubercular thiourea; however we report here that it causes a mild inhibition in growth in liquid medium. Since mycolic acid biosynthesis was affected, we cloned and expressed Mycobacterium smegmatis mycolic acid methyltransferases, postulated as targets for thiacetazone in other mycobacterial species. During this analysis we identified MSMEG_1350 as the methyltransferase involved in epoxy mycolic acid synthesis since its deletion led to their total loss. Phenotypic characterization of the mutant strain showed colony morphology alterations at all temperatures, reduced growth and a slightly increased susceptibility to SDS, lipophilic and large hydrophilic drugs at 20°C with little effect at 37°C. No changes were detected between parental and mutant strains in biofilm formation, sliding motility or sedimentation rate. Intriguingly, we found that several mycobacteriophages severely decreased their ability to form plaques in the mutant strain. Taken together our results prove that, in spite of being a minor component of the mycolic acid pool, epoxy-mycolates are required for a proper assembly and functioning of the cell envelope. Further studies are warranted to decipher the role of epoxy-mycolates in the M. smegmatis cell envelope.Fil: Di Capua, Cecilia Beatriz. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Escuela de Ciencias MĂ©dicas. Cátedra de MicrobiologĂa, ParasitologĂa y VirologĂa; ArgentinaFil: Belardinelli, Juan Manuel. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Escuela de Ciencias MĂ©dicas. Cátedra de MicrobiologĂa, ParasitologĂa y VirologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; ArgentinaFil: Buchieri, MarĂa Virginia. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Escuela de Ciencias MĂ©dicas. Cátedra de MicrobiologĂa, ParasitologĂa y VirologĂa; ArgentinaFil: Bortolotti, Ana. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Escuela de Ciencias MĂ©dicas. Cátedra de MicrobiologĂa, ParasitologĂa y VirologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; ArgentinaFil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Escuela de Ciencias MĂ©dicas. Cátedra de MicrobiologĂa, ParasitologĂa y VirologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; ArgentinaFil: Morbidoni, HĂ©ctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias MĂ©dicas. Escuela de Ciencias MĂ©dicas. Cátedra de MicrobiologĂa, ParasitologĂa y VirologĂa; Argentin