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Elucidating the regulation and function of metabolic stress in Escherichia coli

Award Number: R15GM114710
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 07/01/2016
PERIOD OF PERFORMANCE END DATE: 08/31/2022

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Elucidating the regulation and function of metabolic stress in Escherichia coli - DESCRIPTION (provided by applicant): In all domains of life, small RNAs (sRNAs) are vital regulators of normal cell function and responses to a variety of stresses. In bacteria, the mechanisms by which sRNAs regulate gene expression have been well- characterized, while related regulation and sRNA roles in stress are often poorly understood. The sRNA SgrS is a model for studying stress in Escherichia coli and other bacteria. SgrS and the transcriptional regulator SgrR are both essential for regulating the response to glucose-phosphate stress, which occurs when glucose- phosphate accrues in the cell and impedes growth. SgrR activates expression of SgrS, which helps rescue cells from stress largely by inhibiting translation of mRNAs that encode sugar transporters. While regulation by SgrS is well-described, little is known about regulation by SgrR or other associated regulatory networks. The long-term objectives of this proposal are to characterize the regulation and physiological functions of the glucose-phosphate stress response. In particular, Aim 1 will characterize novel genes regulated by SgrR and their role in glucose-phosphate stress, while Aim 2 will elucidate the regulatory relationship of SgrR-SgrS to the stringent response to nutrient starvation. For Aim 1, preliminary experiments utilizing high-throughput RNA sequencing (RNA-seq) analysis have identified dozens of novel gene targets potentially regulated by SgrR. We will verify these SgrR-mediated changes in expression using quantitative PCR (qPCR), and assess SgrR binding to target gene promoters using in vivo titration assays. To examine the functions of target genes in glucose-phosphate stress, we will delete and overexpress the genes and assess the effects on induction of the stress response and growth during stress. For Aim 2, previous findings strongly support the notion that glucose-phosphate stress is due to glycolytic or related nutrient depletion similar to stringent nutrient starvation. In addition, preliminary data support likely roles for stringent response regulators DksA and the "alarmone" ppGpp (synthesized by RelA and SpoT) in glucose-phosphate stress, as mutations in dksA or relA spoT result in growth defects during stress. We will further examine the role of dksA and ppGpp null mutations in GP stress response regulation as measured by expression of a PsgrS-lacZ transcriptional fusion. To determine whether the glucose-phosphate stress response affects stringent induction, we will examine the effects of mutating sgrR and sgrS on stringent response induction and growth under stringent conditions. Finally, to identify individual stringent response genes with functions in glucose-phosphate stress, we will use RNA-seq to monitor gene expression of dksA and ppGpp null mutants grown under glucose-phosphate stress. As with potential SgrR targets, we will then employ genetic analyses to assess stress-related phenotypes of stringent gene mutations. This work will aid in our understanding of how cells use sRNAs related regulators of their physiology in order to adapt to stress. Both sRNAs and sugar-phosphate stress play a role in virulence of certain bacteria, so this research also could ultimately contribute to finding new targets for treating disease.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2023 ( Subtotal = -$2,269 )
2023	2016	UNIVERSITY OF WISCONSIN PARKSIDE	900 WOOD RD	KENOSHA	WI	53144	KENOSHA	USA	Biomedical Research and Research Training	000	1	10/1/2022	NEW	-$2,269
														Subtotal = -$2,269

Issue Date FY: 2022 ( Subtotal = $0 )
2022	2016	UNIVERSITY OF WISCONSIN SYSTEM	900 WOOD RD	KENOSHA	WI	53144	KENOSHA	USA	Biomedical Research and Research Training	000	1	11/16/2021	NEW	$0
														Subtotal = $0

Issue Date FY: 2021 ( Subtotal = $0 )
2021	2016	UNIVERSITY OF WISCONSIN SYSTEM	900 WOOD RD	KENOSHA	WI	53144	KENOSHA	USA	Biomedical Research and Research Training	000	1	3/8/2021	NEW	$0
														Subtotal = $0

Issue Date FY: 2020 ( Subtotal = $0 )
2020	2016	UNIVERSITY OF WISCONSIN SYSTEM	900 WOOD RD	KENOSHA	WI	53144	KENOSHA	USA	Biomedical Research and Research Training	000	1	5/7/2020	NEW	$0
														Subtotal = $0

Issue Date FY: 2016 ( Subtotal = $381,039 )
2016	2016	UNIVERSITY OF WISCONSIN PARKSIDE	900 WOOD ROAD BOX 2000	KENOSHA	WI	53144	KENOSHA	USA	Biomedical Research and Research Training	000	1	5/20/2016	NEW	$381,039
														Subtotal = $381,039

Grand Total All Awards = $378,770

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Elucidating the regulation and function of metabolic stress in Escherichia coli

Award Number: R15GM114710

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 07/01/2016

PERIOD OF PERFORMANCE END DATE: 08/31/2022

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