Disruption of trehalose periplasmic recycling dysregulates cAMP-CRP signaling in Escherichia coli during stationary phase

Abstract

Survival during starvation hinges on the ability to manage intracellular energy reserves and to initiate appropriate metabolic responses to perturbations of such reserves. How Escherichia coli manage carbon storage systems under starvation stress, as well as transpose changes in intracellular metabolite levels into regulatory signals, is not well understood. Endogenous trehalose metabolism may be at the center of these processes, coupling carbon storage with carbon starvation responses. The coupled transport to the periplasm and subsequent hydrolysis of trehalose back to glucose for transport to the cytoplasm may function as a crucial metabolic signaling pathway. Although trehalose has been characterized as a stress protectant in E. coli, the disaccharide also functions as both an energy storage compound and a regulator of carbohydrate metabolism in fungi, plants, and other bacteria. Our research explores the metabolic regulatory properties of trehalose in E. coli and a potential mechanism by which the intracellular carbon pool is interconnected with regulatory circuits, enabling long-term survival.

Keywords: carbohydrate metabolism; long-term survival; stationary phase; trehalose.

Fig 1

Model for trehalose metabolism in E. coli during stationary phase. Trehalose can be derived from the environment or synthesized endogenously during stationary phase (23). The biosynthetic enzyme OtsA condenses glucose-6-phosphate with UDP-glucose to form trehalose-6-phosphate, and the phosphate is subsequently cleaved by OtsB. Trehalose can then be hydrolyzed into two glucose molecules by the cytoplasmic trehalase TreF. Alternatively, trehalose can be excreted through stretch-activated porins (SAP) into the periplasm, where it is converted into glucose by the periplasmic trehalase TreA. Environmental trehalose that enters the cell through the LamB outer membrane porin is also degraded by TreA. Liberated glucose returns to the cytoplasm via the glucose-specific PTS. Dashed arrows indicate exponential phase conditions; solid arrows indicate under stationary phase conditions.

Fig 2

Mutation of periplasmic trehalase TreA impairs survival in monoculture yet increases fitness in competition during LTSP. (A) Viable cell counts of monocultures of strains lacking either the cytoplasmic trehalase TreF, the periplasmic trehalase TreA, or both enzymes were measured for 10 days. Coculture competition between (B) the treA mutant strain and the WT strain, (C) the treF mutant strain and the WT strain, and (D) the treA-treF double mutant strain and the WT strain. Data are mean ± s.d. (n = 3 cultures per strain).

Fig 3

Mutation of trehalases results in altered trehalose accumulation during stationary phase. Enzymatic assay to determine intracellular concentrations of trehalose for WT or mutant strains at 16 h post-inoculation (stationary phase). Data are reported as µg trehalose per 10⁸ cells. Statistical significance (unpaired t-test, two-sided) is represented relative to the WT strain. Data are mean ± s.d.; *, P < 0.05; **, P < 0.01 (n = 3 cultures per strain).

Fig 4

The treA strain exhibits enhanced resource scavenging and elevated ATP levels. (A) Stationary phase cell yields in conditioned medium experiments. WT, treA, or treF mutant cells are inoculated into conditioned medium prepared from cultures of each of the three strains; n = 3 cultures per strain (data are mean ± s.d.; *, P < 0.05; ***, P < 0.001; ns, P > 0.05; unpaired t-test; two-sided). (B) ATP assay of stationary phase cultures of the WT, treA, or treF mutant strains. Luminescence measurements were normalized to 1.0 for WT; n = 3 cultures per strain (data are mean ± s.d.; *, P < 0.05; ***, P < 0.001; unpaired t-test; two-sided).

Fig 5

The treA strain displays upregulation of the CAP and the Cra regulons. RT-PCR of mRNA from 16 hour cultures, with fold change in gene expression relative to the WT strain; n = 3 cultures per strain (data are mean ± s.d.; *, P < 0.05; **, P < 0.01; unpaired t-test; two-sided).

Fig 6

Supplementation of the treA strain with a nonmetabolizable glucose analog partially rescues viability during LTSP. Supplementation of (A) WT or (B) treA cultures with 0.5% α-MGlc or an equal volume of water (solvent control). Data are mean ± s.d. (n = 3 cultures per strain, with or without supplementation).

Fig 7

Codeletion of the cyaA gene with the treA mutation reduces competitive fitness but partially restores viability during LTSP. (A) Monoculture survival of the WT strain and cyaA. (B) Monocultures of the treA and the treA-cyaA double knockout mutant strain. (C) Coculture competition between the WT strain and the cyaA mutant strains. (D) Competition coculture of the WT strain and the treA-cyaA double knockout mutant strain. Data are mean ± s.d. (n = 3 cultures per strain). Asterisks indicate that cfu mL⁻¹ was below the limit of detection (<10³ cfu mL⁻¹).

Fig 8

A model of trehalose periplasmic recycling facilitating metabolic fine-tuning during stationary phase. (A) During stationary phase, RpoS-induced expression of the biosynthetic genes otsA and otsB leads to the accumulation of intracellular trehalose, which is then transported into the periplasm via stretch-activated porins (SAP). Once in the periplasm, trehalose is hydrolyzed by TreA into glucose. Flux of this liberated glucose through the PTS sugar transporter EIIC leads to the dephosphorylation of EIIA and EIIB, the phosphorylation state of which is important for modulating adenylate cyclase (AC) activity. Phosphorylated glucose can be converted back into trehalose or enter other metabolic pathways. (B) Mutation of the periplasmic trehalase treA results in the accumulation of trehalose in the periplasm and reduced glucose flux through the PTS, leading to the phosphorylated EIIA-mediated activation of AC, which converts ATP into cAMP. Higher cAMP levels generate increased activation of the cAMP receptor protein (CRP), upregulating the expression of high- and moderate-affinity transporters for carbon substrates, such as sugars, as well as enzymes for alternative carbon metabolism. Thus, suggesting that trehalose metabolism is a crucial modulator of carbon starvation responses during stationary phase.