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The small membrane protein YohP induces membrane depolarization and ppGpp accumulation in Escherichia coli

ABSTRACT: Small membrane proteins represent an abundant and ubiquitous class of proteins that are often up-regulated when cells encounter unfavorable conditions, yet details about their exact function are largely missing. In bacteria, these proteins consist of typically less than 50 amino acids and contain a single transmembrane domain but lack any detectable catalytic activity. Thus, the benefit of producing these proteins during stress conditions is unknown. In the current study we used a multidisciplinary approach to determine the function of the 27 amino acid long protein YohP in E. coli. Our proteomics approach revealed that YohP production leads to an up-regulation of proteins involved in membrane protection and to a down-regulation of many enzymes involved in key metabolic processes, such as nucleotide biosynthesis. Further biochemical characterizations revealed increased cardiolipin content in the membrane, a partial dissipation of the membrane potential and reduced membrane fluidity in YohP-containing membranes. Finally, our data show that YohP production induces the stringent response and leads to elevated levels of (p)ppGpp. Overall, our data indicate that the YohP-induced proteome and membrane changes initiate a state of metabolic silencing that protects E. coli against stress and helps to conserve cellular resources.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Julian Langer

LAB HEAD: Julian Langer

PROVIDER: PXD063229 | Pride | 2025-10-20

REPOSITORIES: Pride

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			Action	DRS
	230310_2318_EcUM2_Pepsep50_90min_DIA_1uL_1_20230313225257.raw	Raw
	230310_2318_EcUM2_Pepsep50_90min_DIA_1uL_2_20230314023813.raw	Raw
	230310_2318_EcUM2_Pepsep50_90min_DIA_1uL_3_20230314062325.raw	Raw
	230310_2318_EcUM2_Pepsep50_90min_DIA_1uL_4.raw	Raw
	230310_2318_EcUM2_Pepsep50_90min_DIA_1uL_5.raw	Raw

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Project description:Guanosine 3 ,5 -bispyrophosphate (ppGpp), also known as ‘‘magic spot,’’ has been shown to bind prokaryotic RNA polymerase to down-regulate ribosome production and increase transcription of amino acid biosynthesis genes during the stringent response to amino acid starvation. Because many environmental growth perturbations cause ppGpp to accumulate, we hypothesize ppGpp to have an overarching role in regulating the genetic program that coordinates transitions between logarithmic growth (feast) and growth arrest (famine). We used the classic glucose-lactose diauxie as an experimental system to investigate the temporal changes in transcription that accompany growth arrest and recovery in wildtype Escherichia coli and in mutants that lack RelA (ppGpp synthetase) and other global regulators, i.e., RpoS and Crp. When cultured on a mixture of glucose and lactose, E. coli grows preferentially on glucose until it is exhausted, resulting in growth arrest while the cells adjust to growth on lactose, i.e., diauxie. Diauxie was delayed in the relA mutant and was accompanied by a 15% decrease in the number of carbon sources used and a 3-fold overall decrease in the induction of RpoS and Crp regulon genes. Thus the data significantly expand the previously known role of ppGpp and support a model wherein the ppGpp-dependent redistribution of RNA polymerase across the genome is the driving force behind control of the stringent response, general stress response, and starvation-induced carbon scavenging. Our conceptual model of diauxie describes these global control circuits as dynamic, interconnected, and dependent upon ppGpp for the efficient temporal coordination of gene expression that programs the cell for transitions between feast and famine. E. coli MG1655 and isogenic mutants were cultured in a 2 l Biostat B fermentor (B. Braun Biotech International) containing 1 liter of Morpholinepropanesulfonic acid (MOPS) minimal medium with 0.5 g/l of glucose and 1.5 g/l of lactose. The temperature was maintained at 37 ºC and pH was kept constant at 7.2 by the addition of 2 M NaOH. The dissolved oxygen level was maintained above 20% of saturation by adjusting the agitation speeds in the range of 270-500 rpm with fixed 1 l/min air flow.

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The small membrane protein YohP induces membrane depolarization and ppGpp accumulation in Escherichia coli

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