Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Summary

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Abstract

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.

We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms¹. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

Introduction

c-di-GMP is a key second messenger used by most bacteria to control various aspects of their growth and behavior. For instance, c-di-GMP regulates cell cycle progression, motility and the expression of exopolysaccharides and surface adhesins^2-4. Through the coordination of such processes c-di-GMP promotes biofilm formation, a process which is associated with chronic infections of a range of pathogenic bacteria⁵. c-di-GMP is synthetized by enzymes called diguanylate cyclases (DGCs) that harbor a catalytic GGDEF domain⁴. Some DGCs possess an inhibitory site that down regulates the cyclase activity upon c-di-GMP binding. The degradation o....

Protocol

1. Lysate Preparation

1. Grow P. aeruginosa cells in LB to the desired OD.
   NOTE: For guidance: use ≈ 100 ml culture/sample for stationary phase cultures and ≈ 500 ml cultur /sample for log phase cultures (OD_600nm = 0.5).
2. Pellet by centrifugation for 20 min at 5,000 x g.
3. Resuspend 0.5-1 g of pellet in 1 ml lysis buffer (6.7 mM MES, 6.7 mM HEPES, 200 mM NaCl, 6.7 mM KAc, DDT 1 mM, pH 7.5) and add protease inhibitor (complete mini, EDTA-free) as well as DNaseI.
4. Lyse the cells by 3 passages through a French pressure cell, at 20,000 psi (see Materials List).
5. Ultra-centrifu....

Results

To identify novel c-di-GMP effectors in P. aeruginosa we systematically used CCMS to analyze the soluble and membrane fractions of P. aeruginosa strain PAO1 from a log phase culture (OD₆₀₀ = 0.5). Here we summarize and discuss representative results of this fishing expedition. Four independent biological replicas were used. For each experiment two different cdG-CC concentrations were used (5 µM and 10 µM). To probe for specificity, experiments were carried out in the presence or absence of 1 .......

Discussion

Special care should be taken at several steps of the protocol. The protein concentration is a critical parameter with a concentration of 10 mg/ml being difficult to reach when cells are grown under specific growth conditions (e.g. biofilms or small colony variants). Thus, the pellet resuspension should be performed in a low volume of lysis buffer. Protein concentrations can be decreased to 8 mg/ml. Compared to the method published by Nesper et al.¹, we added various nucleotides to the capture.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
caproBox	caprotec bioanalytics	1-5010-001 (220 V)	UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation
caproMag	caprotec bioanalytics	included in the CCMS Starter Kit	For easy handling of magnetic particles without pipetting
c-di-GMP caproKit	caprotec bioanalytics	upon request	The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer
Disposable PD-10 Desalting Columns	GE Healthcare	17-0851-01
12-tube PCR strips	Thermo Scientific	AB-1114
UIS250v sonicator with VialTweeter	Hielscher ultrasound technology	UIS250v and VialTweeter
Miniature French Pressure Cell	Thermo Electron Corporation	FA-003