Benzoic Acid-Inducible Gene Expression in Mycobacteria

Dragset, Marte Singsås; Barczak, Amy K.; Kannan, Nisha; Flo, Trude Helen; Valla, Svein; Rubín, Eric J.; Steigedal, Magnus

doi:10.1371/journal.pone.0134544

Cited by 7 publications

(7 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The step-response simulations show that the use of an sRNA to tune the effective feedback strength of an autorepressor allows a decoupling of response time from steady-state output. This contrasts with previously-described feedback circuits, including a recent transcriptional negative autoregulation feedback circuit, where the use of an RNA transcriptional attenuator did not allow decoupling of output and response time ( 105 ). The dynamic simulations with a temporal disturbance in external input concentrations suggest that other disturbances such as temperature and nutrient changes or depletion of shared cellular machinery would be rejected well by the closed-loop sRNA feedback circuit.…”

Section: Discussioncontrasting

confidence: 66%

Synthetic negative feedback circuits using engineered small RNAs

Kelly

Harris

Steel

et al. 2018

Nucleic Acids Research

View full text Add to dashboard Cite

Negative feedback is known to enable biological and man-made systems to perform reliably in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have primarily relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules that can inhibit translation of target messenger RNAs (mRNAs). In this work, we modelled, built and validated two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised tet-based autorepressor, incorporating an externally-inducible sRNA to tune the effective feedback strength. This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal, steep input–output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA, which inhibits translation of the mRNA encoding the output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.

show abstract

Section: Discussioncontrasting

confidence: 66%

Synthetic negative feedback circuits using engineered small RNAs

Kelly

Harris

Steel

et al. 2018

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…Msmeg mutant strains were constructed by recombineering, replacing the gene in question with an antibiotic resistance marker, using strains expressing the mycobacteriophage recombinases gp60 and gp61 on a nitril-inducible, counter-selectable plasmid or on the acetamide-inducible plasmid pJV53 58 . Details for construct preparations and selection processes are contained in Supplementary SI Methods 59,60 .…”

Section: Methodsmentioning

confidence: 99%

Genome-wide Phenotypic Profiling Identifies and Categorizes Genes Required for Mycobacterial Low Iron Fitness

Dragset

Ioerger

Zhang

et al. 2019

Sci Rep

Self Cite

View full text Add to dashboard Cite

Iron is vital for nearly all living organisms, but during infection, not readily available to pathogens. Infectious bacteria therefore depend on specialized mechanisms to survive when iron is limited. These mechanisms make attractive targets for new drugs. Here, by genome-wide phenotypic profiling, we identify and categorize mycobacterial genes required for low iron fitness. Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis (TB), can scavenge host-sequestered iron by high-affinity iron chelators called siderophores. We take advantage of siderophore redundancy within the non-pathogenic mycobacterial model organism M . smegmatis ( Msmeg ), to identify genes required for siderophore dependent and independent fitness when iron is low. In addition to genes with a potential function in recognition, transport or utilization of mycobacterial siderophores, we identify novel putative low iron survival strategies that are separate from siderophore systems. We also identify the Msmeg in vitro essential gene set, and find that 96% of all growth-required Msmeg genes have a mutual ortholog in Mtb . Of these again, nearly 90% are defined as required for growth in Mtb as well. Finally, we show that a novel, putative ferric iron ABC transporter contributes to low iron fitness in Msmeg , in a siderophore independent manner.

show abstract

“…The TetR variant used, TetR#28, functions as a repressor for P myc1 with anhydrotetracycline as corepressor (Klotzsche et al 2009 ). We also tested a double control system developed for Mycobacterium tuberculosis (Dragset et al 2015 ) in which the TetR#28 repressor variant is used to repress expression of xylS from the P myc 1 /TetO promoter/operator. In this plasmid (pRMG4), the luciferase gene is expressed from the P m promoter, which depends on XylS together with its coactivator m-toluate for efficient expression.…”

Section: Resultsmentioning

confidence: 99%

Improved site-specific mutagenesis in Rhodococcus opacus using a novel conditional suicide plasmid

Jain

Ertesvåg

2022

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus. The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales, the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis, but not for Corynebacterium glutamicum, and the new vector was used to study if mycolic acid is essential for R. opacus. We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication. Key points • Improved vector for homologous recombination in R. opacus. • Mycolic acid is important for survival of R. opacus like it is for Mycobacterium. • Similar conditional suicide plasmids may be constructed for other bacteria. Graphical abstract

show abstract

Benzoic Acid-Inducible Gene Expression in Mycobacteria

Cited by 7 publications

References 53 publications

Synthetic negative feedback circuits using engineered small RNAs

Synthetic negative feedback circuits using engineered small RNAs

Genome-wide Phenotypic Profiling Identifies and Categorizes Genes Required for Mycobacterial Low Iron Fitness

Improved site-specific mutagenesis in Rhodococcus opacus using a novel conditional suicide plasmid

Contact Info

Product

Resources

About