메뉴 건너뛰기




Volumn 287, Issue 5458, 2000, Pages 1652-1655

An ultrasensitive bacterial motor revealed by monitoring signaling proteins in single cells

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL PROTEIN; CHEMOTACTIC PEPTIDE; GREEN FLUORESCENT PROTEIN;

EID: 0034009901     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5458.1652     Document Type: Article
Times cited : (519)

References (37)
  • 1
    • 0032496358 scopus 로고    scopus 로고
    • A rare example of single-cell study of biochemical networks is provided by a recent analysis of Xenopus oocytes, which revealed the mitogen-activated protein kinase cascade as a sharp epigenetic switch [J. E. Ferrell Jr. and E. Machleder, Science 280, 895 (1998)].
    • (1998) Science , vol.280 , pp. 895
    • Ferrell J.E., Jr.1    Machleder, E.2
  • 4
    • 0032480007 scopus 로고    scopus 로고
    • U. Alon et al., EMBO J. 17, 4238 (1998).
    • (1998) EMBO J. , vol.17 , pp. 4238
    • Alon, U.1
  • 8
    • 0002574321 scopus 로고
    • J. A. Hoch and T. J. Silhavy, Eds. American Society for Microbiology, Washington, DC
    • R. M. Macnab, in Two Component Signal Transduction, J. A. Hoch and T. J. Silhavy, Eds. (American Society for Microbiology, Washington, DC, 1995), pp. 181-199.
    • (1995) Two Component Signal Transduction , pp. 181-199
    • Macnab, R.M.1
  • 12
    • 0026572085 scopus 로고
    • M. Welch, K. Oosawa, S. Aizawa, M. Eisenbach, Proc. Natl. Acad. Sci. U.S.A. 90, 8787 (1993); R. Barak and M. Eisenbach, Biochemistry 31, 1821 (1992).
    • (1992) Biochemistry , vol.31 , pp. 1821
    • Barak, R.1    Eisenbach, M.2
  • 13
    • 0342333747 scopus 로고    scopus 로고
    • note
    • In addition to crieV, this strain also lacks two other genes coding for CheB, which demethylates the receptors, and CheZ, which dephosphorylates CheY-P. Several lines of evidence from in vitro data and (4) indicate that, in this strain, essentially all of the CheY present in the cytoplasm is phosphorylated. To verify if the presence of the GFP modified the phosphorylation of CheY-GFP, we performed in vitro kinase assays (27). There is no noticeable difference between phosphorylation rates for CheY and CheY-GFP.
  • 14
    • 0029973636 scopus 로고    scopus 로고
    • CheY-GFP was expressed under control of lacOP with the low-copy plasmid pMGS98 (CmR). This plasmid was engineered by replacing the cheY gene in pLC576 (4) with an in-frame cheY-gfp fusion constructed by polymerase chain reaction amplification of the cheY gene (including the ribosome binding site) and gfp#2 [B. P. Cormack, R. H. Valdivia, S. Falkow, Gene 173, 33 (1996)].
    • (1996) Gene , vol.173 , pp. 33
    • Cormack, B.P.1    Valdivia, R.H.2    Falkow, S.3
  • 15
    • 0343203316 scopus 로고    scopus 로고
    • note
    • The glass slide was coated with amino-silanes and rabbit antibodies to flagellin that were covalently cross-linked to amino-silanes with glutaraldehyde. Transitions from CW to CCW and from CCW to CW states were manually scored from slowed-down (to 1/3 speed) video recordings and recorded on charts with LabView software (National Instrument, Austin, TX). Errors on the timing of transitions were less than 0.2 s. The incident excitation light was provided by the 488-nm line of an argon laser (air-cooled ion laser 160, Spectra Physics, Mountain View, CA). The laser beam was expanded to a diameter of 4 mm, attenuated with a neutral density filter down to 0.1 mW, and fed to an oil immersion objective X 100 (numerical aperture = 1.3, Olympus Uplan Fl, Melville, NY). We used a dichroic mirror (Q505LP, Chroma, Brattleboro, VT) to set up an epifluorescence illumination. The green fluorescence emission was rejected from the side of the microscope with a cold mirror (CP-SM-550, CVI, Livermore, CA). The residual blue component was filtered out from a long-pass green filter (06-515, CVI). A convergent lens focused the outcoming beam onto the core (50 μm) of a multimode optical fiber (Spectran, Sturbridge, MA). The optical fiber was connected to an avalanche photodiode (SPCM, EG&G, Quebec, Canada) that delivered a TTL pulse for each detected photon.
  • 16
    • 0016366591 scopus 로고
    • D. Magde, E. Elson, W. W. Webb, Biopolymers 13, 29 (1974); E. Elson and D. Magde, Biopolymers 13, 1 (1974); R. Rigler, in Fluorescence Correlation Spectroscopy, O. S. Wolfbeis, Ed. (Springer-Verlag, Berlin, 1992), pp. 13-24.
    • (1974) Biopolymers , vol.13 , pp. 29
    • Magde, D.1    Elson, E.2    Webb, W.W.3
  • 17
    • 0016379116 scopus 로고
    • D. Magde, E. Elson, W. W. Webb, Biopolymers 13, 29 (1974); E. Elson and D. Magde, Biopolymers 13, 1 (1974); R. Rigler, in Fluorescence Correlation Spectroscopy, O. S. Wolfbeis, Ed. (Springer-Verlag, Berlin, 1992), pp. 13-24.
    • (1974) Biopolymers , vol.13 , pp. 1
    • Elson, E.1    Magde, D.2
  • 18
    • 0016366591 scopus 로고
    • O. S. Wolfbeis, Ed. Springer-Verlag, Berlin
    • D. Magde, E. Elson, W. W. Webb, Biopolymers 13, 29 (1974); E. Elson and D. Magde, Biopolymers 13, 1 (1974); R. Rigler, in Fluorescence Correlation Spectroscopy, O. S. Wolfbeis, Ed. (Springer-Verlag, Berlin, 1992), pp. 13-24.
    • (1992) Fluorescence Correlation Spectroscopy , pp. 13-24
    • Rigler, R.1
  • 20
    • 0343203314 scopus 로고    scopus 로고
    • note
    • This method allowed us fast and consecutive concentration measurements of cells of interest without bleaching irreversibly (less 5%) the GFP. The lower data point (0 μM) gives the amplitude of the background noise. The limit of the detection ranged from ∼0.15 to 50 μM, corresponding to a background noise of three molecules and an upper limit of ∼1000 molecules in the detection volume, respectively. Because of the errors in focusing and alignment of the laser spot with respect to the cell body, concentration could not be determined with precision better than 15%.
  • 23
    • 0342768692 scopus 로고    scopus 로고
    • note
    • To check whether the presence of a GFP molecule in the CheY-GFP fusion could modify the cooperativity of the binding with the motors, we analyzed, on video recordings, the swimming behavior of individual cells. Two populations of cells carrying either wild-type CheY or CheY-GFP (both expressed from the same low-copy plasmid vector) were grown under the same conditions (see Fig. 2A) and preinduced with 5 μM of IPTG. From these two populations, we reported, on two histograms, the number of cells (out of 60) having either a "smooth," "intermediate," or "tumbly" behavior. We defined these three types of behavior as "smooth" for cells having a tumbling frequency (TF) inferior to 0.1, "intermediate" when 0.1 < TF < 1, and "tumbly" when TF > 1. We followed individual cells and manually scored the number of tumbles. We computed TF, for single cells, as the ratio of the number of tumbles to the duration of the recording. The two histograms were remarkably similar (27). This rather arduous analysis of the cell behavior provides a strong indication that the presence of a GFP molecule in the CheY-GFP fusion should not affect substantially the cooperativity of the binding to the motors and CheY phosphorylation.
  • 24
    • 0031006611 scopus 로고    scopus 로고
    • note
    • 2-terminal fusions of proteins to GFP, where the folding of the chromophore was observed to be tightly coupled to the folding of the fused protein (28).
  • 25
    • 0020535125 scopus 로고
    • The top curve of figure 5 in the Ishihara et al. article shows a high degree of correlation in the bias between two neighboring motors within the same cell. This result strongly suggests that motors are responding identically to the same signal, implying a uniformity of their functioning
    • A. Ishahara, J. S. Segall, S. M. Block, H. C. Berg, J. Bacteriol. 155, 228 (1983). The top curve of figure 5 in the Ishihara et al. article shows a high degree of correlation in the bias between two neighboring motors within the same cell. This result strongly suggests that motors are responding identically to the same signal, implying a uniformity of their functioning.
    • (1983) J. Bacteriol. , vol.155 , pp. 228
    • Ishahara, A.1    Segall, J.S.2    Block, S.M.3    Berg, H.C.4
  • 26
    • 0343638931 scopus 로고    scopus 로고
    • note
    • We calculated the Hill coefficient from the Hill plot in a standard fashion by retaining all data points with the CW bias between 0.1 and 0.9 (these limits do not necessarily correspond here to the physiologically relevant range of bacterial behavior).
  • 27
    • 0031029852 scopus 로고    scopus 로고
    • Standard techniques of immunoblotting ignored the inherent diversity of bacteria, which is especially acute when proteins in the cells are expressed from plasmids. Variability of protein distribution within a population of cells depends on precise experimental conditions and the choice of strains, plasmids, or promoters [see, e.g., H. H. McAdams and A. Arkin, Proc. Natl. Acad. Sci. U.S.A. 94, 814 (1997)]. Therefore, in all of these methods, the true output characteristic of flagellar motors was effectively convoluted with [CheY-P] distributions, leading typically to smooth-out characteristics with lower Hill coefficients. To mimic the use of immunoblots, we simulated on a computer the convolution of our data (sigmoid motors characteristic curve, Fig. 2A) with a Gaussian distribution of proteins (σ = 24% of the mean) [M. D. Levin, C. J. Morton-Firth, W. N. Abouhamad, R. B. Bourret, D. Bray, Biophys J. 74, 175 (1998)]. In good agreement with our data and (4-6), we found that Hill coefficients ranging from 8 to 20 were respectively smoothed out from 3.9 to 4.2 when convoluted with such distribution. Generally, immunoblots may thus be inadequate tools for defining sharp slopes of biological switches. To check the validity of these computer simulations, we performed the same experiment as in (4) with wild-type CheY and CheY-GFP fusion. For several concentrations of IPTG (0, 1, 2.5, and 5 μM), the two populations of cells showed the same swimming behavior [thanks to U. Alon, these data were provided with the same setup as in (4)]. In both cases, wild-type CheY and CheY-GFP fusion reproduced identical results to those reported in figure 5 of (4) (27).
    • (1997) Proc. Natl. Acad. Sci. U.S.A. , vol.94 , pp. 814
    • McAdams, H.H.1    Arkin, A.2
  • 28
    • 0031982512 scopus 로고    scopus 로고
    • In good agreement with our data and (4-6), we found that Hill coefficients ranging from 8 to 20 were respectively smoothed out from 3.9 to 4.2 when convoluted with such distribution. Generally, immunoblots may thus be inadequate tools for defining sharp slopes of biological switches. To check the validity of these computer simulations, we performed the same experiment as in (4) with wild-type CheY and CheY-GFP fusion. For several concentrations of IPTG (0, 1, 2.5, and 5 μM), the two populations of cells showed the same swimming behavior [thanks to U. Alon, these data were provided with the same setup as in (4)]. In both cases, wild-type CheY and CheY-GFP fusion reproduced identical results to those reported in figure 5 of (4) (27)
    • Standard techniques of immunoblotting ignored the inherent diversity of bacteria, which is especially acute when proteins in the cells are expressed from plasmids. Variability of protein distribution within a population of cells depends on precise experimental conditions and the choice of strains, plasmids, or promoters [see, e.g., H. H. McAdams and A. Arkin, Proc. Natl. Acad. Sci. U.S.A. 94, 814 (1997)]. Therefore, in all of these methods, the true output characteristic of flagellar motors was effectively convoluted with [CheY-P] distributions, leading typically to smooth-out characteristics with lower Hill coefficients. To mimic the use of immunoblots, we simulated on a computer the convolution of our data (sigmoid motors characteristic curve, Fig. 2A) with a Gaussian distribution of proteins (σ = 24% of the mean) [M. D. Levin, C. J. Morton-Firth, W. N. Abouhamad, R. B. Bourret, D. Bray, Biophys J. 74, 175 (1998)]. In good agreement with our data and (4-6), we found that Hill coefficients ranging from 8 to 20 were respectively smoothed out from 3.9 to 4.2 when convoluted with such distribution. Generally, immunoblots may thus be inadequate tools for defining sharp slopes of biological switches. To check the validity of these computer simulations, we performed the same experiment as in (4) with wild-type CheY and CheY-GFP fusion. For several concentrations of IPTG (0, 1, 2.5, and 5 μM), the two populations of cells showed the same swimming behavior [thanks to U. Alon, these data were provided with the same setup as in (4)]. In both cases, wild-type CheY and CheY-GFP fusion reproduced identical results to those reported in figure 5 of (4) (27).
    • (1998) Biophys J. , vol.74 , pp. 175
    • Levin, M.D.1    Morton-Firth, C.J.2    Abouhamad, W.N.3    Bourret, R.B.4    Bray, D.5
  • 31
    • 0019996706 scopus 로고
    • D. E. Koshland, A. Goldbeter, J. B. Stock, Science 217, 220 (1982); A. Goldbeter and D. E. Koshland Jr., Proc. Natl. Acad. Sci. U.S.A. 78, 6840 (1981).
    • (1982) Science , vol.217 , pp. 220
    • Koshland, D.E.1    Goldbeter, A.2    Stock, J.B.3
  • 33
    • 18144446008 scopus 로고    scopus 로고
    • CheZ is a specific phosphatase interacting with CheY-P. It has recently been discovered in vitro [Y. Blat, B. Gillepsie, A. Bren, F. W. Dahlquist, M. Eisenbach, J. Mol. Biol. 284, 1191 (1998)] that the CheZ activity is cooperative with respect to CheY-P and that the phosphatase activity is regulated at the level of the ratio CheY-P/CheZ interaction [B. E. Scharf, K. A. Fahrner, H. C. Berg, J. Bacteriol. 180, 5123 (1998)]. It might be tempting to think that CheZ acts as a CheY-P concentration regulator. CheZ would contribute to the adjustment of the CheY-P concentration in the functioning range of the motors (3 μM). As noted by Scharf et al., "the swarming abilities of both strains RP437 and AW405 suggested that the absolute values of CheY and CheZ are not as crucial for chemotaxis as their ratios" (p. 5125). Conversely, one could argue that the natural level of expression of the CheZ gene is sufficient, without any further regulation, to dephosphorylate the right amount of CheY-P to adjust the system to the operational range of the motors.
    • (1998) J. Mol. Biol. , vol.284 , pp. 1191
    • Blat, Y.1    Gillepsie, B.2    Bren, A.3    Dahlquist, F.W.4    Eisenbach, M.5
  • 34
    • 0031709168 scopus 로고    scopus 로고
    • It might be tempting to think that CheZ acts as a CheY-P concentration regulator. CheZ would contribute to the adjustment of the CheY-P concentration in the functioning range of the motors (3 μM). As noted by Scharf et al., "the swarming abilities of both strains RP437 and AW405 suggested that the absolute values of CheY and CheZ are not as crucial for chemotaxis as their ratios" (p. 5125). Conversely, one could argue that the natural level of expression of the CheZ gene is sufficient, without any further regulation, to dephosphorylate the right amount of CheY-P to adjust the system to the operational range of the motors
    • CheZ is a specific phosphatase interacting with CheY-P. It has recently been discovered in vitro [Y. Blat, B. Gillepsie, A. Bren, F. W. Dahlquist, M. Eisenbach, J. Mol. Biol. 284, 1191 (1998)] that the CheZ activity is cooperative with respect to CheY-P and that the phosphatase activity is regulated at the level of the ratio CheY-P/CheZ interaction [B. E. Scharf, K. A. Fahrner, H. C. Berg, J. Bacteriol. 180, 5123 (1998)]. It might be tempting to think that CheZ acts as a CheY-P concentration regulator. CheZ would contribute to the adjustment of the CheY-P concentration in the functioning range of the motors (3 μM). As noted by Scharf et al., "the swarming abilities of both strains RP437 and AW405 suggested that the absolute values of CheY and CheZ are not as crucial for chemotaxis as their ratios" (p. 5125). Conversely, one could argue that the natural level of expression of the CheZ gene is sufficient, without any further regulation, to dephosphorylate the right amount of CheY-P to adjust the system to the operational range of the motors.
    • (1998) J. Bacteriol. , vol.180 , pp. 5123
    • Scharf, B.E.1    Fahrner, K.A.2    Berg, H.C.3
  • 35
    • 0342768689 scopus 로고    scopus 로고
    • P. Cluzel, M. Surette, S. Leibler, data not shown
    • P. Cluzel, M. Surette, S. Leibler, data not shown.
  • 37
    • 0343203311 scopus 로고    scopus 로고
    • note
    • We thank U. Alon, N. Barkai, C. Bonnet, M. Elowitz, T. Griggs, C. Guet, T. Silhavy, J. Stock, J. Vilar, and E. Winfree for many helpful discussions and comments on the manuscript. This work was partially sponsored by the NIH. P.C. acknowledges support by a fellowship from the Program in Mathematics and Molecular Biology at the Florida State University, with funding from the NSF under grant DMS-9406348.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.