We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 4

1.
Fig. 3.

Fig. 3. From: High stimulus unmasks positive feedback in an autoregulated bacterial signaling circuit.

Autoregulation amplifies PhoP-regulated transcription under conditions of high stimulus associated with growth-limiting [Mg2+]. (A) Transcription of the PhoP-regulated mgrB promoter (YFP/CFP) for autoregulated (P1+) and non-autoregulated (P1) strains (TIM44 and TIM45, respectively) cultured in growth-limiting Mg2+ concentrations. Fluorescence ratios were determined from the average of ≈200 cells. Points indicate the mean and range for two cultures. (B) Transcription of the mgrB promoter (YFP/CFP) for cells (TIM219) in growth-limiting Mg2+ concentrations with the expression of phoPphoQ under IPTG inducible control. The expression of phoPphoQ was determined by western blotting against PhoP and normalizing by the level of PhoP in wild-type cells (TIM210) grown under the same condition. Fluorescence ratios were determined from the average of ≈200 cells and normalized by the corresponding ratio for wild-type cells grown under the same condition. Points indicate the mean and range for two cultures.

Tim Miyashiro, et al. Proc Natl Acad Sci U S A. 2008 November 11;105(45):17457-17462.
2.
Fig. 1.

Fig. 1. From: High stimulus unmasks positive feedback in an autoregulated bacterial signaling circuit.

Positive autoregulation does not affect steady-state PhoQ/PhoP signaling over a wide range of stimuli. (A) Positive autoregulation in the PhoQ/PhoP system. The membrane protein PhoQ autophosphorylates and subsequently transfers the phosphoryl group to PhoP. Phosphorylated PhoP activates transcription of phoPphoQ from the P1 promoter. The P2 promoter is constitutively active. (B) PhoP western blot of autoregulated (P1+), non-autoregulated (P1), or phoPphoQ strains (TIM68, TIM69, and TIM13, respectively) grown in the indicated Mg2+ concentrations. (C) Steady-state PhoP-regulated transcription levels (YFP/CFP) for autoregulated (P1+) or non-autoregulated (P1) strains (TIM44 and TIM45, respectively) grown in the indicated Mg2+ concentrations. Transcription was determined from YFP expressed from a chromosomal operon fusion of yfp to the PhoP-regulated gene mgrB. The strains also contain a chromosomal copy of cfp under control of a constitutive promoter. CFP fluorescence was used as a normalization. (Above) Fluorescence ratios were determined from the average of ≈200 cells. Points and bars indicate mean and range for two cultures. (Below) Corresponding single-cell distributions for the indicated magnesium concentrations.

Tim Miyashiro, et al. Proc Natl Acad Sci U S A. 2008 November 11;105(45):17457-17462.
3.
Fig. 2.

Fig. 2. From: High stimulus unmasks positive feedback in an autoregulated bacterial signaling circuit.

A model of the PhoQ/PhoP circuit predicts autoregulation will amplify the steady-state output at sufficiently high stimulus. (A, Left) The autoregulation module determines the total levels of PhoP ([PhoP]total) and PhoQ ([PhoQ]total) as a function of phosphorylated PhoP ([PhoP-P]). The ratio ([PhoP]total/[PhoQ]total) is assumed to be constant and much greater than 1. (Right) Steady-state [PhoP]total is taken to be a saturating function of [PhoP-P]. The basal expression level is due to the constitutive promoter P2; the dotted line represents [PhoP]total in a P1 (non-autoregulated) strain. (B, Left) The phosphorylation cycle determines [PhoP-P] as a function of stimulus, [PhoP]total, and [PhoQ]total. The autophosphorylation, phosphotransfer, and phosphatase steps are indicated by the arrows within the box. (Right) Steady-state [PhoP-P] saturates as a function of [PhoP]total to a level set by the input stimulus (see SI for details). (C, Left) the complete circuit consists of the interconnected autoregulation and phosphorylation cycle modules. (Right) The steady-state circuit output ([PhoP-P]) is governed by the intersections of the curves in (A) and (B). Arrows illustrate the amplification in [PhoP-P] due to autoregulation in P1+ strains (closed symbols) vs. P1 strains (open symbols) for different stimuli. For high-stimulus conditions, the increase in [PhoP]total from autoregulation causes a strong increase in [PhoP-P]. For low-stimulus conditions, the corresponding increase in [PhoP]total does not result in a significant change in [PhoP-P].

Tim Miyashiro, et al. Proc Natl Acad Sci U S A. 2008 November 11;105(45):17457-17462.
4.
Fig. 4.

Fig. 4. From: High stimulus unmasks positive feedback in an autoregulated bacterial signaling circuit.

A phosphatase-defective PhoQ eliminates saturation associated with phoPphoQ induction and shows strong positive feedback in an autoregulated strain. (A) PhoQ autophosphorylation and phosphotransfer to PhoP for cytoplasmic fragments of wild-type PhoQ and a T281R mutant. PhoP was added after ≈25 min. (B) In vitro PhoP-P dephosphorylation by cytoplasmic fragments of wild type and a T281R mutant in the presence of ADP. (C) Transcription from the mgrB promoter (YFP/CFP) for cells grown in 1 mM [Mg2+] with the expression of phoPphoQ (blue) or phoPphoQ(T281R) (red) under inducible control (strains TIM220 and TIM270, respectively). The induction level of phoPphoQ was determined by western blotting against PhoP and normalizing by the level of PhoP in wild-type cells (TIM210) grown under the same condition. Fluorescence ratios were determined from the average of ≈200 cells and normalized by the corresponding ratio for wild-type cells grown under the same condition. Points indicate the mean and range for two cultures. (D) Transcription from the mgrB promoter (YFP/CFP) for autoregulated (P1+) and non-autoregulated (P1) strains expressing wild-type phoPphoQ or phoPphoQ(T281R) grown in 1 mM Mg2+. The strains are TIM284 (solid blue), TIM285 (striped blue), TIM286 (solid red), TIM287 (striped red), and TIM215 (white). (Left) Average fluorescence ratios from ≈200 cells (error bars denote the ranges from two cultures). (Right) Corresponding histograms of single-cell fluorescence.

Tim Miyashiro, et al. Proc Natl Acad Sci U S A. 2008 November 11;105(45):17457-17462.

Supplemental Content

Recent activity

Your browsing activity is temporarily unavailable.

Write to the Help Desk