Second messenger signaling

 

Second Messenger Signaling in Bacteria

We use Forster Resonance Energy Transfer based imaging to visualize dynamics of c-di-GMP signaling in live bacteria.

The Research group:Hemantha Kulasekara, Bridget Kulasekara, Erik Peterson, Erez Mills, Kathleen Barry and Dr. Toni Kline.

Research description:

1. Biological basis of asymmetrical distribution of c-di-GMP upon cell division

2. Discovery of inhibitors of c-di-GMP metabolism

3. Second messenger based signaling cascades that induce rapid physiological changes in bacterial cells.

Collaborators:

Dr. Matthias Christen PhD, Basel, Switzerland.

Dr. Paul Wiggins, Dept of Physics, University of Washington.

Equipments:

1) Nikon/Andor Spinning Disc confocal microscope: with Yokogawa CSU-X1, Nikon Ti-E inverted scope, Andor Revolution 6 color laser Launcher, Andor iXon Ultra 897 EMCCD (Single Photon Sensitive ultra high speed back thinned EX2 CCD chip), Andor Mosaic photo conversion arm, Nikon TIRF.

2) Nikon Ti-E Inverted Microscope with Cascade II 1024 EMCCD, Sutter Xenon light source, Sutter Lambda Filter wheels.

3) Nikon TE2000E with Andor iXON 987 EMCCD camera.

Relevant Publications:

The response threshold of Salmonella PilZ domain proteins is determined by their binding affinities for c-di-GMP. Pultz IS, Christen M, Kulasekara HD, Kennard A, Kulasekara B, Miller SI.  Mol Microbiol. 2012 Dec;86(6):1424-40.

Christen M, Kulasekara HD, Christen B, Kulasekara BR, Hoffman LR, Miller SI. Asymmetrical distribution of the second messenger c-di-GMP upon bacterial cell division. Science. 2010 Jun 4;328(5983):1295-7. PubMed PMID: 20522779. Christen et al

Hoffman LR, D’Argenio DA, MacCoss MJ, Zhang Z, Jones RA, Miller SI. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature. 2005 Aug 25;436(7054):1171-5. PubMed PMID: 16121184. Hoffman et al

Mills E, Pultz IS, Kulasekara HD and Miller SI. Bacterial Second Messenger Signaling, Trends in Microbiology.

D’Argenio DA, Miller SI.Cyclic di-GMP as a bacterial second messenger. Microbiology. 2004 Aug;150(Pt 8):2497-502. Review. D’Argenio et al

Background:

c-di-GMP specific Biosensor

What is a biosensor: Biosensors is a devise that measure instance changes in electromagnatic radiation, similar to electromagnatic sensors such as photo resistors, CCD and CMOS chips. Therefore, in vivo gene expression reporters are not biosensors.

Biosensor is consisted of three parts: (i) biological receptor element sensitive to any form of energy, (ii) a measuring device that quantify electromagnetic radiation and (iii) a transducer that acts as a bridge between the biological receptor and the that measuring device by converting the initial form of  energy to a different form of electromagnatic radiation that can be read by the measuring device.

c-di-GMP specific biosensor  is made of evolutionary optimized CFP and YFP varients, mCYpet and mYpet that flanks a PilZ domain proteins. Of all the PilZ based biosensors we have made in the lab, the Salmonella flagella brake PilZ domain protein produced the ideal biosensor to detect c-di-GMP both in vitro and an in vivo.

c-di-GMP specific YcgR biosensor schematics

c-di-GMP specific YcgR biosensor schematics

 

 

 

 

 

 

 

 

c-di-GMP specific PilZ domain based biosensor

See An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins; Tomoo Ohashi, Stephane D. Galiacy, Gina Briscoe, and Harold P. Erickson

Receptor domain Emission ratio 527/480 nma KDb ∆S° c ∆H° d
free bound % Change nM ± s.d. kJ mol-1K-1 ± s.d. kJ mol-1 ± s.d.
DgrA 2.04 1.78 -12.7 26  ± 5.5 -0.101 ± 0.023 -72.07 ± 6.81
YcgR 2.31 1.4 -60.6 195  ± 6.1 -0.158 ± 0.007 -84.29 ± 2.19
PA0012 3.04 4.07 33.8 402  ± 39 -0.166 ± 0.014 -84.88 ± 4.17
PA2799 2.87 2.67 -6.8 1998  ± 448 -0.233 ± 0.028 -102.08 ± 8.56
PA4608 2.66 2.09 -21.6 6126  ± 525 -0.191 ± 0.024 -86.09 ± 7.23
PA2960 (PilZ) 2.07 2.08 0.2 NDe
DgrARRAA 1.96 1.93 -1.5 NDe
DgrA aa1-24 2.7 2.62 -3 NDe

 Table: binding constance and entropy and enthalpy changes upon c-di-GMP binding to variety of PilZ domain biosensors.

YcgR-biosensor-cdgmp-titration

 

 

 

 

 

 

 

 

Measurment of binding constance of c-diGMP to YcgR biosensor. Left: titration with c-di-GMP. Right: with GTP.

YcgR-biosensor-temperature-dependency-of-binding

 

 

 

 

 

 

 

 

Above:Temperature dependency of c-diGMP binding to YcgR biosensor. The changes to entropy and enthalpy are in the table above.

Below: The c-di-GMP biosensor responds to c-di-GMP fluctuations within seconds.

temporal-changes-to-FRET-upon-cdgmp-addtion

 

 

 

 

 

 

Asymmetrical distribution of cdgmp in Pseudomonas

Time Lapse images of c-di-GMP dynamics in Pseudomonas upon cell division. Upper panel: FRET images. Lower panel: polar flagella (and the surface) labeled by cy5 to monitor flagellation status of the bacteria (images taken by Bridget Kulasekara)

Biosensor Protocols:

Purifying mYPet-YcgR-mCYPet biosensor for in vitro binding assays.

Please refer to the materials and methods in Christen et al.

Use BL21 DE3 or BLR (recA negative). BLR minimizes recombination between YPet and CYPet.
Always use freshly transformed bacteria with the pET15B:: biosensor plasmid (resistance is Ampicillin)
Innoculate 4L of buffered LB from the freshly transformed overnight grown bacteria and grow the 4L to OD of 0.5 at 30C, induce with 500ug of IPTG, grow for 2 hours and then cool down the temperature to 18C, grow overnight.
Next day, pellet the culture, and purify the biosensor using standard Histag affinity purification methods. Use 25mM Tris, pH 8, 500mM NaCl, 10% glycerol as the buffer during purification.
Always use a Hitrap column that has been stripped using the guanidium HCL method. (see the protocol section). Sometimes, majority of the biosensor may elute at 50mM immidazol. So save all the fractions.
After the His tag purification step, don’t dialyze the protein. Instead, use a sephadex gel filtration column to remove the contaminating cleavage product which is mostly YFP. The YFP band could be substantial and will affect FRET calculations.
Once the biosensor is purified via gel filtration, dilute it to less than 0.3mg/ml and freeze it in 20% glycerol 25mM Tris, pH 8, 500mM NaCl buffer with 5mM BME.

Test the biosensor with the ligand in a spectrofluorometer. Note that if you are using aplate reader, a dual detector is superior to that of using a single detector. Therefore, try to find an instrument such as Envision dual detector plate reader. Otherwise, try to find a Horiba Jobin Yvon Fluoromax 4 fluorometer.

Protocol for using Envision Dual detector plate reader- coming soon

Protocol for using in vivo biosensor for single cell FRET microscopy- coming soon

Instrument needed.

Widefield systems with sutter wheels and EMCCD camera are superior to laser scanning confocal microscopy for FRET measurements in bacteria.