This work was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia/Minist&#233;rio da Ci&#234;ncia, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-028779 and the grant SFRH/BD/99715/2014 (CF).

1. Cyanobacterial released carbohydrate polymer isolation
<ol>
	<li>Polymer isolation and removal of contaminants 
	<ol>
		<li>Cultivate the cyanobacterial strain under standard conditions [e.g., 30 &#176;C under a 12 h light (50 &#956;E m&#8722;2s&#8722;1)/12 h dark regimen, with orbital shaking at 150 rpm]. Measure growth using standard protocols [e.g., optical density at 730 nm (OD730nm), chlorophyll a, dry-weight, etc.], then measure the production of relea...

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emulsifying,+flocculating,+and+physicochemical+properties+of+exopolysaccharide+produced+by+cyanobacterium+Nostoc+flagelliforme.">Emulsifying, flocculating, and physicochemical properties of exopolysaccharide produced by cyanobacterium Nostoc flagelliforme.</a> Applied Biochemistry and Biotechnology. 172 (1), 36-49 (2014).</li><li>Leite, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanobacterium&#8208;Derived+Extracellular+Carbohydrate+Polymer+for+the+Controlled+Delivery+of+Functional+Proteins.">Cyanobacterium&#8208;Derived Extracellular Carbohydrate Polymer for the Controlled Delivery of Functional Proteins.</a> Macromolecular Bioscience. 17 (2), 1600206 (2017).</li><li>Estevinho, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+cyanobacterial+extracellular+polymeric+substance+in+the+microencapsulation+of+vitamin+B12.">Application of a cyanobacterial extracellular polymeric substance in the microencapsulation of vitamin B12.</a> Powder Technology. 343, 644-651 (2019).</li><li>Klock, J. H., Wieland, A., Seifert, R., Michaelis, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+polymeric+substances+(EPS)+from+cyanobacterial+mats:+characterisation+and+isolation+method+optimisation.">Extracellular polymeric substances (EPS) from cyanobacterial mats: characterisation and isolation method optimisation.</a> Marine Biology. 152 (5), 1077-1085 (2007).</li><li>Delattre, C., Pierre, G., Laroche, C., Michaud, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+extraction+and+characterization+of+microalgal+and+cyanobacterial+exopolysaccharides.">Production, extraction and characterization of microalgal and cyanobacterial exopolysaccharides.</a> Biotechnology Advances. 34 (7), 1159-1179 (2016).</li><li>Costa, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+systems+in+gram-negative+bacteria:+structural+and+mechanistic+insights.">Secretion systems in gram-negative bacteria: structural and mechanistic insights.</a> Nature Reviews Microbiology. 13 (6), 343-359 (2015).</li><li>Roier, S., Zingl, F. G., Cakar, F., Schild, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+outer+membrane+vesicle+biogenesis:+a+new+mechanism+and+its+implications.">Bacterial outer membrane vesicle biogenesis: a new mechanism and its implications.</a> Microbial Cell. 3 (6), 257-259 (2016).</li><li>Sergeyenko, T. V., Los, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+secreted+proteins+of+the+cyanobacterium+Synechocystis+sp.+strain+PCC+6803.">Identification of secreted proteins of the cyanobacterium Synechocystis sp. strain PCC 6803.</a> FEMS Microbiology Letters. 193 (2), 213-216 (2000).</li><li>Oliveira, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatile+TolC-like+Slr1270+in+the+cyanobacterium+Synechocystis+sp.+PCC+6803.">The versatile TolC-like Slr1270 in the cyanobacterium Synechocystis sp. PCC 6803.</a> Environmental Microbiology. 18 (2), 486-502 (2016).</li><li>Flores, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alternative+sigma+factor+SigF+is+a+key+player+in+the+control+of+secretion+mechanisms+in+Synechocystis+sp.+PCC+6803.">The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803.</a> Environmental Microbiology. 21 (1), 343-359 (2018).</li><li>Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., Smith, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+method+for+determination+of+sugars+and+related+substances.">Colorimetric method for determination of sugars and related substances.</a> Analytical Chemistry. 28 (3), 350-356 (1956).</li><li>Parikh, A., Madamwar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+characterization+of+extracellular+polysaccharides+from+cyanobacteria.">Partial characterization of extracellular polysaccharides from cyanobacteria.</a> Bioresource Technology. 97 (15), 1822-1827 (2006).</li><li>R&#252;hmann, B., Schmid, J., Sieber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+identify+the+unexplored+diversity+of+microbial+exopolysaccharides.">Methods to identify the unexplored diversity of microbial exopolysaccharides.</a> Frontiers in Microbiology. 6, 565 (2015).</li><li>Pathak, J., Rajneesh, R., Sonker, A. S., Kannaujiya, V. K., Sinha, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanobacterial+extracellular+polysaccharide+sheath+pigment,+scytonemin:+A+novel+multipurpose+pharmacophore.">Cyanobacterial extracellular polysaccharide sheath pigment, scytonemin: A novel multipurpose pharmacophore.</a> Marine Glycobiology. , 343-358 (2016).</li><li>Nguyen, A. T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performances+of+different+protocols+for+exocellular+polysaccharides+extraction+from+milk+acid+gels:+Application+to+yogurt.">Performances of different protocols for exocellular polysaccharides extraction from milk acid gels: Application to yogurt.</a> Food Chemistry. 239, 742-750 (2018).</li><li>Jamshidian, H., Shojaosadati, S. A., Mousavi, S. M., Soudi, M. R., Vilaplana, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+recovery+procedures+on+structural+and+rheological+properties+of+schizophyllan+produced+from+date+syrup.">Implications of recovery procedures on structural and rheological properties of schizophyllan produced from date syrup.</a> International Journal of Biological Macromolecules. 105, 36-44 (2017).</li><li>Couto, N., Schooling, S. R., Dutcher, J. R., Barber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+profiles+of+outer+membrane+vesicles+and+extracellular+matrix+of+Pseudomonas+aeruginosa+biofilms.">Proteome profiles of outer membrane vesicles and extracellular matrix of Pseudomonas aeruginosa biofilms.</a> Journal of Proteome Research. 14 (10), 4207-4222 (2015).</li></ol>

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 ...

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 agents7,8). Methodology for the isolation of these polymers largely contributes&#160;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&#8217;s strong anionic nature9,10. 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&#8217;s specific objectives and user needs, while maintaining the basic steps presented here.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Dialysis membranes</td>
			<td style="width:191px;">Medicell Membranes Ltd&#160;</td>
			<td style="width:119px;">DTV.12000.07</td>
			<td style="width:777px;">Visking Tubing Size 7, Dia 23.8 mm, Width 39-41 mm 30m Roll&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Ethanol 96%</td>
			<td style="width:191px;">AGA - &#193;lcool e G&#233;neros Alimentares, S.A.</td>
			<td style="width:119px;">4.000.02.02.00</td>
			<td>Fermentation ethyl alcohol 96% AGA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">PES Filter 0.2 &#956;m</td>
			<td style="width:191px;">Fisher Scientific, Lda</td>
			<td style="width:119px;">15206869</td>
			<td>Syringe filter polystyrene 33MM 0.2&#181;M STR&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Amicon Ultra-15, Ultracel-3K</td>
			<td style="width:191px;">Merck Millipore Ltd.</td>
			<td style="width:119px;">UFC900324</td>
			<td>Centrifugal filters with a nominal molecular weight cut-off of 3 kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Thermo Scientific&#160;Pierce BCA Protein Assay</td>
			<td style="width:191px;">Fisher Scientific, Lda</td>
			<td style="width:119px;">10741395</td>
			<td>Green-to-blue, precise, detergent-compatible assay reagent to measure total protein concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brillant Blue G Colloidal Concentrate&#160;</td>
			<td style="width:191px;">Sigma Aldrich Qu&#237;mica SL</td>
			<td style="width:119px;">B2025-1EA</td>
			<td>Coomassie blue&#160;</td>
		</tr>
	</tbody>
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looking outwards: isolation of cyanobacterial released carbohydrate polymers and proteins

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.

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 ...

Watch this Scientific Journal Video about Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins at JoVE.com

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

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.

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

These protocols that we developed are important to study the secretion mechanisms in cyanobacteria and their released products, namely the extracellular carbohydrate polymers and proteins. The knowledge generated will allow us to customize these products for several applications, namely biotechnological and biomedical applications. These protocols are very straightforward.They can be tailored according to the producing organism, the user needs, and the final application of the product. The main advantage of these protocols is that they can easily be adapted to other living systems, in particular to other bacteria. These protocols are easy.However, one may need to introduce some changes depending on the bacterial strain or the degree of purity required. To begin, cultivate the cyanobacterial strain under standard conditions. Measure growth using standard protocols.Next, transfer the culture into dialysis membranes, and dialyze against a minimum of 10 volumes of deionized water for 24 hours with continuous stirring. Centrifuge the culture at 15, 000 times g at four degrees Celsius for 15 minutes. Transfer the supernatant to a new vial, and discard the pellet.Centrifuge again at 20, 000 times g at four degrees Celsius for 15 minutes to remove contaminants, such as cell wall debris or lipopolysaccharides. After the centrifugation, transfer the supernatant to a glass beaker, and discard the pellet. To precipitate the polymer, add two volumes of 96%ethanol to the supernatant, and incubate the suspension at four degrees Celsius overnight.For small or not visible amounts of precipitated polymer, centrifuge the suspension at 13, 000 times g at four degrees Celsius for 25 minutes. Gently discard the supernatant, and resuspend the pellet in one to two milliliters of autoclaved deionized water. Transfer the aqueous suspension to a vial.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. To perform the lyophilization process otherwise known as freeze-drying, keep the vials with the precipitated polymer at minus 80 degrees Celsius overnight.Lyophilize the polymer for at least 48 hours. Then, store the dried polymer in a desiccator at room temperature until further use. To concentrate the medium, first cultivate cyanobacteria under standard conditions.Monitor the cyanobacterium growth using standard procedures. Then, centrifuge the cultures at 4, 000 times g at room temperature for 10 minutes. Transfer the supernatant to a flask, and discard the cell pellet.Next, filter the decanted medium through a 0.2-micron pore size filter. To concentrate the medium approximately 500 times, use centrifugal concentrators with a nominal molecular weight cut-off of three kilodaltons to centrifuge 4, 000 times g and 15 degrees Celsius for a maximum of one hour per centrifugation round. After centrifugation, rinse the walls of the filter device sample reservoir with the concentrated sample, and transfer the content to a microcentrifuge tube.Perform an additional washing step of the filter device sample reservoir with the autoclaved culture medium to ensure maximal exoproteome recovery. Then, store the exoproteome samples at minus 20 degrees Celsius until further use. The ethanol must be added vigorously to optimize the polymer precipitation and yield.After centrifuging the precipitated polymer, the discard of the supernatant should be made carefully in order to avoid resuspension of the pellet that may be loosely attached on the wall of flask. If manual filtration is performed, it should be gentle to avoid breaking the filter membrane. Some precipitated polymers can only be visible after centrifugation, mainly on the walls of the flasks, such as the case of EPS from Synechocystis.In other cases, such as Cyanothece polymer, it is possible to see the polymer clumps floating in the glass beaker just after precipitation. Some contaminations can be easily detected macroscopically after polymer lyophilization since they will alter polymer pigmentation, usually white or light brown. Orange polymers are contaminated with carotenoids or structures containing these pigments.For example, green polymers are contaminated with cell debris and chlorophyll. Exoproteomes can vary significantly depending on the bacterial strain. See, for example, the exoproteome from a unicellular cyanobacterium and the one from a filamentous strain.The high amount of polysaccharides in exoproteome-concentrated samples can hinder the exoproteome analysis. For example, it can delay protein separation and mask less abundant proteins. The user can characterize the polymer in terms of composition, physical/chemical properties, test polymer biological activity, or modified polymer components.For protein identification, separation of proteins in gel and mass spectrometry can be performed. And for separation of vesicles, ultracentrifugation of the exoproteome should be performed. In our group, these protocols have already paved the way to unravel cyanobacterial secretion mechanisms or applications of the extracellular carbohydrate polymers in different fields, such as in bioremediation or as antitumor or drug delivery agents.

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6.4K visualizações.  Universidade do Porto. Esses protocolos que desenvolvemos são importantes para estudar os mecanismos de secreção em cianobactérias e seus produtos liberados, ou seja, os polímeros e proteínas de carboidratos extracelulares. O conhecimento gerado nos permitirá personalizar esses produtos para diversas aplicações, ou seja, aplicações biotecnológicas e biomédicas. Esses protocolos são muito simples.Eles podem ser adaptados de acordo com o organismo produtor, as necessidades do usuário e a aplica?...