Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Summary

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Abstract

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Introduction

Cyanobacteria are widely recognized as prolific sources of natural products with promising biotechnological/biomedical applications. Therefore, understanding cyanobacterial secretion mechanisms and optimization of the extraction/recovery methods are essential to implement cyanobacteria as efficient microbial cell factories.

Many cyanobacterial strains are able to produce extracellular polymeric substances (EPS), mainly formed by heteropolysaccharides, that remain associated to the cell surface or are released into the medium¹. These released carbohydrate polymers have distinct features compared to those from other bacteria, which make them suitable for a wide range of applications (e.g., antivirals², immunostimulatory³, antioxidant⁴, metal-chelating⁵, emulsifying⁶, and drug delivery agents^7,8). Methodology for the isolation of these polymers largely contributes not only to improved yield but also to increased purity and the specific physical properties of the polymer obtained⁹. A vast majority of these methods for isolation of the polymers rely on precipitation strategies from the culture medium that are easily accomplished due to the polymer’s strong anionic nature^9,10. In addition, removal of the solvents used in the precipitation step can be rapidly achieved by evaporation and/or lyophilization. Depending on the foreseen application, different steps can be coupled either after or before polymer precipitation in order to tailor the final product, which include trichloroacetic acid (TCA) treatment, filtration, or size exclusion chromatography (SEC) column purification¹⁰.

Cyanobacteria are also able to secrete a wide range of proteins through pathways dependent on membrane transporters (classical)¹¹ or mediated by vesicles (non-classical)¹². Therefore, analysis of the cyanobacterial exoproteome constitutes an essential tool, both to understand/manipulate cyanobacterial protein secretion mechanisms and understand the specific extracellular function of these proteins. Reliable isolation and analysis of exoproteomes require the concentration of the extracellular milieu, since the abundance of secreted proteins is relatively low. In addition, other physical or chemical steps (e.g., centrifugation, filtration, or protein precipitation) may optimize the quality of the exoproteome obtained, enriching the protein content¹³, and avoiding the presence of contaminants (e.g., pigments, carbohydrates, etc.)^14,15 or the predominance of intracellular proteins in the samples. However, some of these steps may also restrict the set of proteins that can be detected, leading to a biased analysis.

This work describes efficient protocols for the isolation of released carbohydrate polymers and exoproteomes from cyanobacteria culture media. These protocols can be easily adapted to the study’s specific objectives and user needs, while maintaining the basic steps presented here.

Protocol

1. Cyanobacterial released carbohydrate polymer isolation

1. Polymer isolation and removal of contaminants
   1. Cultivate the cyanobacterial strain under standard conditions [e.g., 30 °C under a 12 h light (50 μE m⁻²s⁻¹)/12 h dark regimen, with orbital shaking at 150 rpm]. Measure growth using standard protocols [e.g., optical density at 730 nm (OD_730nm), chlorophyll a, dry-weight, etc.], then measure the production of released polysaccharides according to the phenol-sulfuric acid method¹⁶.
   2. Transfer the culture into dialysis membranes (12-14 kDa of molecular weight cut-off) and dialyze against a minimum of 10 volumes of deionized water for 24 h with continuous stirring.
      NOTE: Depending on the volume of culture to dialyze and medium composition, it may be necessary to change the dialysis water.
   3. Centrifuge the culture at 15,000 x g for 15 min at 4 °C. Transfer the supernatant to a new vial and discard the pellet (cells).
   4. Centrifuge again at 20,000 x g for 15 min at 4 °C to remove contaminants such as cell wall debris or lipopolysaccharides (LPS).
   5. Transfer the supernatant to a glass beaker and discard the pellet.
2. Precipitation of the polymer
   1. Add 2 volumes of 96% ethanol to the supernatant.
   2. Incubate the suspension at 4 °C, at least overnight.
   3. Polymer recovery
      1. For small or not visible amounts of precipitated polymer: centrifuge the suspension at 13,000 x g for 25 min at 4 °C. Discard the supernatant and resuspend the pellet in 1 mL or 2 mL of autoclaved deionized water. Transfer the aqueous suspension to a vial.
         CAUTION: The supernatant should be discarded gently, as it can become easily resuspended.
      2. For visible/large amounts of precipitated polymer: collect the precipitated polymer with sterile metal forceps to a vial. Squeeze the polymer and discard the excess ethanol.
   4. Optional: depending on the degree of polymer purification required, repeat the precipitation step with 96% ethanol after resuspension of the polymers in deionized water.
3. Lyophilization of the polymer
   1. Keep the vials with the precipitated polymer at -80 °C, at least overnight.
   2. Freeze-dry (lyophilize) the polymer for at least 48 h (do not let the suspension defrost before freeze-drying).
   3. Store the dried polymer at room temperature (RT) until further use.
      NOTE: Storing the polymer in a desiccator is advisable, as it can absorb water over time.

2. Cyanobacterial exoproteome isolation

1. Medium concentration
   1. Cultivate cyanobacteria under standard conditions [e.g., 30 °C under a 12 h light (50 μE m⁻²s⁻¹)/12 h dark regimen, with orbital shaking at 150 rpm]. Monitor the cyanobacterium growth using standard procedures (e.g., OD_730nm, chlorophyll a, dry-weight, etc.).
   2. Centrifuge the cultures at 4,000 x g, for 10 min at RT.
   3. Transfer the supernatant to a flask and discard the cell pellet.
   4. Filter the decanted medium through a 0.2 μm pore size filter.
      NOTE: The protocol can be paused here for a short time period, if the medium is kept at 4 °C.
   5. Concentrate the medium approximately 500x (considering the initial volume of filtered medium), using centrifugal concentrators with a nominal molecular weight cut-off of 3 kDa. Centrifugation should be operated at 4,000 x g (maximum 1 h per centrifugation round) at 15 °C.
      CAUTION: For the majority of concentrator brands, the filter device needs to be rinsed by centrifugation with ultrapure water before use. Once the filter is wet, do not let it dry out. Leave enough fluid on the reservoir when the device is not being used.
      NOTE: Reducing the centrifugation temperature to 4 °C may be helpful if the aim is to study protein activity, though it will increase the time necessary for sample concentration. The protocol can be paused between centrifugation steps for short time periods if the medium is kept at 4 °C.
   6. Rinse the walls of the filter device sample reservoir with the concentrated sample and transfer the content to a microcentrifuge tube.
   7. Perform an additional washing step of the filter device sample reservoir with autoclaved culture medium to ensure maximal exoproteome recovery.
      NOTE: To quantify the percentage of recovery, follow the specific manufacturer’s instructions.
   8. Store the exoproteome samples at -20 °C until further use.
      NOTE: Addition of protease inhibitors is recommended for long-term storage.
2. Analysis of the exoproteome
   1. Quantify the protein content by BCA protein assay in 96-well plate according to the manufacturer’s instructions.
   2. Separate the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using standard staining protocols (e.g., Coomassie blue, silver staining).
   3. Cut out the bands/gel regions of interest and collect them in different microcentrifuge tubes containing appropriate volumes of ultra-pure water.
   4. Proceed with identification and analysis of the proteins by mass spectrometry.

Results

A schematic representation of the method described to extract released carbohydrate polymers from cyanobacterial cultures is depicted in Figure 1. Precipitated polymers from the moderate EPS producer cyanobacterium Synechocystis sp. PCC 6803 and the efficient EPS producer Cyanothece sp. CCY 0110 are shown in Figure 2. In Figure 3, lyophilized polymers with different degrees of contamination are shown, highlighting ...

Discussion

To better understand bacterial secretion mechanisms and study the released products, it is of extreme importance to demonstrate the efficient isolation and analysis of the biomolecules present in the extracellular bacterial environment (such as released carbohydrate polymers and proteins).

Cyanobacterial extracellular carbohydrate polymers are extremely complex, mainly due to the number and proportion of different monosaccharides that constitute their composition¹. The ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dialysis membranes	Medicell Membranes Ltd	DTV.12000.07	Visking Tubing Size 7, Dia 23.8 mm, Width 39-41 mm 30m Roll
Ethanol 96%	AGA - Álcool e Géneros Alimentares, S.A.	4.000.02.02.00	Fermentation ethyl alcohol 96% AGA
PES Filter 0.2 μm	Fisher Scientific, Lda	15206869	Syringe filter polystyrene 33MM 0.2µM STR
Amicon Ultra-15, Ultracel-3K	Merck Millipore Ltd.	UFC900324	Centrifugal filters with a nominal molecular weight cut-off of 3 kDa
Thermo Scientific Pierce BCA Protein Assay	Fisher Scientific, Lda	10741395	Green-to-blue, precise, detergent-compatible assay reagent to measure total protein concentration
Brillant Blue G Colloidal Concentrate	Sigma Aldrich Química SL	B2025-1EA	Coomassie blue