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Laboratory evolution of synthetic electron transport system variants reveals a larger metabolic respiratory system and its plasticity

ABSTRACT: Laboratory evolution of synthetic electron transport system variants reveals a larger metabolic respiratory system and its plasticity

PROVIDER: PRJNA834787 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19066548_1.fastq.gz	Fastqsanger.gz
	SRR19066548_2.fastq.gz	Fastqsanger.gz
	SRR19066549_1.fastq.gz	Fastqsanger.gz
	SRR19066549_2.fastq.gz	Fastqsanger.gz
	SRR19066550_1.fastq.gz	Fastqsanger.gz

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Project description:The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions and its induction is controlled by two histidine kinases, DosS and DosT. Recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. This report demonstrates that DosS responds to a reduced electron transport system (ETS), although this does not rule out a role for oxygen in silencing signaling. Under aerobic conditions induction of the DosR regulon by DosS but not DosT was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating the menaquinone pool is a limiting factor in regulon induction. Taken together these data indicate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS, rescue inhibition of menaquinone synthesis, and is less toxic. The menaquinone pool is central to the ETS and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Aerobically growing logarithmic phase DosS and DosT mutant strains were analyzed after treatment with the cytochrome c reductant ascorbate to examine the effect on DosR signaling caused by reducing the electron transport system. Experiments were repeated in triplicate (dosT mutant) or duplicate (dosS mutant).

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Laboratory evolution of synthetic electron transport system variants reveals a larger metabolic respiratory system and its plasticity

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