A two-step strategy for the complementation of M: tuberculosis mutants

Abstract

The sequence of Mycobacterium tuberculosis, completed in 1998, facilitated both the development of genomic tools, and the creation of a number of mycobacterial mutants. These mutants have a wide range of phenotypes, from attenuated to hypervirulent strains. These phenotypes must be confirmed, to rule out possible secondary mutations that may arise during the generation of mutant strains. This may occur during the amplification of target genes or during the generation of the mutation, thus constructing a complementation strain, which expresses the wild-type copy of the gene in the mutant strain, becomes necessary. In this study we have introduced a two-step strategy to construct complementation strains using the Ag85 promoter. We have constitutively expressed dosR and have shown dosR expression is restored to wild-type level.

New antituberculosis drugs, regimens, and adjunct therapies: needs, advances, and future prospects

“…Por ende, se hace indispensable emplear otro sistema de complementación para evitar efectos adversos por la sobreexpresión de la proteína recombinante, como consecuencia de una aumentada actividad promotora y la multicopia del plásmido extracromosomal. De este modo, se recomienda el uso de vectores integrativos (única copia) para prevenir estos inconvenientes, dado que expresan de manera estable la proteína recombinante y generan menor carga metabólica en el huésped (Movahedzadeh, Frita and Gutka, 2011;Kilpeläinen et al, 2018).…”

“…Por lo tanto, la búsqueda de sistemas óptimos de complementación se convierte en una prioridad para futuros ensayos, debido a que este procedimiento permite comprobar que el fenotipo mutante se debe a la pérdida del gen de interés, y no a mutaciones secundarias que podrían surgir durante la construcción del mutante (Movahedzadeh, Frita and Gutka, 2011). La implementación de plásmidos integrativos (pMV361 y pMH94) para la complementación de cepas mutantes de Mtb, está siendo considerada en nuestro grupo de investigación, no obstante, esta fuera de los objetivos del presente trabajo.…”

Mycobacterium tuberculosis and M. bovis BCG Moreau Fumarate Reductase Operons Produce Different Polypeptides That May Be Related to Non-canonical Functions

“…Complementation was performed using the integrative vector pMV361H, which carries integration signals from mycobacteriophage L5 ( Stover et al, 1991 ), substituting the vector’s original promoter by the frd operon and its own, native promoter. Some studies report that a disadvantage of the L5 integrative vector could be related to the integration locus, which might not favor the transcription of the integrated genes under control of their own, native promoters ( Murry et al, 2005 ; Movahedzadeh et al, 2011 ). Additionally, the frd promoter region included in our complementation constructs may lack other transcriptional regulatory sequences, resulting in a different expression pattern for the frd operon.…”

Rv0100: An essential acyl carrier protein from M. tuberculosis important in dormancy

“…In order to complement the Rv0100 mutant, this gene was cloned into pFM209 [29] under the Ag85 promoter, to produce pFM225. Then the HindIII cassette of pUC-Gm-int, which has…”