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The Anabaena sp. PCC 7120 Exoproteome: Taking a Peek outside the Box.

Oliveira P, Martins NM, Santos M, Couto NA, Wright PC, Tamagnini P - Life (Basel) (2015)

Bottom Line: The evidence presented here shows that Anabaena sp.Furthermore, the activity of selected exoproteins associated with oxidative stress has been assessed, suggesting their involvement in redox homeostasis mechanisms in the extracellular space.Finally, we discuss our results in light of other cyanobacterial exoproteome studies and focus on the potential of exploring cyanobacteria as cell factories to produce and secrete selected proteins.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto 4150-180, Portugal. paulo.oliveira@ibmc.up.pt.

ABSTRACT
The interest in examining the subset of proteins present in the extracellular milieu, the exoproteome, has been growing due to novel insights highlighting their role on extracellular matrix organization and biofilm formation, but also on homeostasis and development. The cyanobacterial exoproteome is poorly studied, and the role of cyanobacterial exoproteins on cell wall biogenesis, morphology and even physiology is largely unknown. Here, we present a comprehensive examination of the Anabaena sp. PCC 7120 exoproteome under various growth conditions. Altogether, 139 proteins belonging to 16 different functional categories have been identified. A large fraction (48%) of the identified proteins is classified as "hypothetical", falls into the "other categories" set or presents no similarity to other proteins. The evidence presented here shows that Anabaena sp. PCC 7120 is capable of outer membrane vesicle formation and that these vesicles are likely to contribute to the exoproteome profile. Furthermore, the activity of selected exoproteins associated with oxidative stress has been assessed, suggesting their involvement in redox homeostasis mechanisms in the extracellular space. Finally, we discuss our results in light of other cyanobacterial exoproteome studies and focus on the potential of exploring cyanobacteria as cell factories to produce and secrete selected proteins.

No MeSH data available.


Related in: MedlinePlus

Negative staining electron micrographs of Anabaena sp. PCC 7120 concentrated exoproteome samples collected from cultures grown in nitrogen-fixing conditions (BG110), or in medium supplemented with nitrate (BG11) or ammonia (BG110 + NH4Cl). (Top) The presence and abundance of outer membrane vesicles (some highlighted by white arrowheads) in each sample (size bar, 200 nm); (bottom) the details of selected vesicles (size bar, 100 nm).
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life-05-00130-f003: Negative staining electron micrographs of Anabaena sp. PCC 7120 concentrated exoproteome samples collected from cultures grown in nitrogen-fixing conditions (BG110), or in medium supplemented with nitrate (BG11) or ammonia (BG110 + NH4Cl). (Top) The presence and abundance of outer membrane vesicles (some highlighted by white arrowheads) in each sample (size bar, 200 nm); (bottom) the details of selected vesicles (size bar, 100 nm).

Mentions: A good example to demonstrate how much is unknown in terms of extracellular processes is outer membrane vesicles (OMV) formation. A large number of heterotrophic Gram-negative bacteria naturally produce OMV, spherical bilayered vesicles released from the outer membrane, ranging in size from 50 to 250 nm in diameter [59]. Their suggested functions include toxin trafficking, DNA transfer and uptake, protein delivery and communication (reviewed by [59,60]). OMV formation by photoautotrophs was not described until earlier in 2014, when Biller and co-workers [61] were able to show it in cyanobacteria of the genera Prochlorococcus and Synechococcus. In that work, not only cultures of Prochlorococcus were shown to continuously release lipid vesicles containing proteins, DNA and RNA, but also that these vesicles could be found abundantly in coastal and open-ocean sea water samples. Most interestingly, Prochlorococcus vesicles were demonstrated to support the growth of heterotrophic bacteria, as well as to have the capacity of being recognized and infected by cyanophages [61]. Thus, as recognized by the authors, the ability to form vesicles by marine photoautotrophs adds another layer of complexity to the flow of information, energy and biomolecules in marine microbial ecosystems [61]. However, this may not be restricted to marine ecosystems, if OMV formation proves to be a common mechanism widespread to other cyanobacteria occupying different ecological niches. Proteomic analyses of Prochlorococcus MED4 vesicles identified a diverse set of proteins, including membrane nutrient transporters and porins, but also, predictably, soluble proteases and hydrolases and several proteins of unknown function [61]. Interestingly, from the 40 Prochlorococcus MED4 reported vesicle proteins, eight homologues could be found in the exoproteome of Anabaena sp. PCC 7120, including the porin TolC-like HgdD (Alr2887), the membrane transporters All4575 (phosphate), All1951 (ABC-type) and All4388 (putative polysaccharide exporter), the phosphoribosylglycinamide formyltransferase 2 (Alr1299), the ATP synthase α-subunit (All0005), the RuBisCO large subunit RbcL (Alr1524) and the hypothetical protein, All0268. Furthermore, it is also noteworthy that even though it was not possible to find the respective homologues for some of the Prochlorococcus MED4 vesicles’ proteins, related counterparts could be found for proteases (Alr1381, Alr0996), aminotransferases (All1683, Alr1004, Alr1080, Alr4853, Alr5103) and even ribosomal proteins (All4214) (see the Supplementary Information). Until now, there have been no reports describing the ability of Anabaena sp. PCC 7120 to form and release vesicles. Therefore, the different Anabaena sp. PCC 7120 concentrated exoproteome samples were subjected to negative staining transmission electron microscopy, in an attempt to find OMVs. Remarkably, the three samples presented numerous small spherical structures (Figure 3) that strongly resemble the membrane vesicles reported for Prochlorococcus [61].


The Anabaena sp. PCC 7120 Exoproteome: Taking a Peek outside the Box.

Oliveira P, Martins NM, Santos M, Couto NA, Wright PC, Tamagnini P - Life (Basel) (2015)

Negative staining electron micrographs of Anabaena sp. PCC 7120 concentrated exoproteome samples collected from cultures grown in nitrogen-fixing conditions (BG110), or in medium supplemented with nitrate (BG11) or ammonia (BG110 + NH4Cl). (Top) The presence and abundance of outer membrane vesicles (some highlighted by white arrowheads) in each sample (size bar, 200 nm); (bottom) the details of selected vesicles (size bar, 100 nm).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390845&req=5

life-05-00130-f003: Negative staining electron micrographs of Anabaena sp. PCC 7120 concentrated exoproteome samples collected from cultures grown in nitrogen-fixing conditions (BG110), or in medium supplemented with nitrate (BG11) or ammonia (BG110 + NH4Cl). (Top) The presence and abundance of outer membrane vesicles (some highlighted by white arrowheads) in each sample (size bar, 200 nm); (bottom) the details of selected vesicles (size bar, 100 nm).
Mentions: A good example to demonstrate how much is unknown in terms of extracellular processes is outer membrane vesicles (OMV) formation. A large number of heterotrophic Gram-negative bacteria naturally produce OMV, spherical bilayered vesicles released from the outer membrane, ranging in size from 50 to 250 nm in diameter [59]. Their suggested functions include toxin trafficking, DNA transfer and uptake, protein delivery and communication (reviewed by [59,60]). OMV formation by photoautotrophs was not described until earlier in 2014, when Biller and co-workers [61] were able to show it in cyanobacteria of the genera Prochlorococcus and Synechococcus. In that work, not only cultures of Prochlorococcus were shown to continuously release lipid vesicles containing proteins, DNA and RNA, but also that these vesicles could be found abundantly in coastal and open-ocean sea water samples. Most interestingly, Prochlorococcus vesicles were demonstrated to support the growth of heterotrophic bacteria, as well as to have the capacity of being recognized and infected by cyanophages [61]. Thus, as recognized by the authors, the ability to form vesicles by marine photoautotrophs adds another layer of complexity to the flow of information, energy and biomolecules in marine microbial ecosystems [61]. However, this may not be restricted to marine ecosystems, if OMV formation proves to be a common mechanism widespread to other cyanobacteria occupying different ecological niches. Proteomic analyses of Prochlorococcus MED4 vesicles identified a diverse set of proteins, including membrane nutrient transporters and porins, but also, predictably, soluble proteases and hydrolases and several proteins of unknown function [61]. Interestingly, from the 40 Prochlorococcus MED4 reported vesicle proteins, eight homologues could be found in the exoproteome of Anabaena sp. PCC 7120, including the porin TolC-like HgdD (Alr2887), the membrane transporters All4575 (phosphate), All1951 (ABC-type) and All4388 (putative polysaccharide exporter), the phosphoribosylglycinamide formyltransferase 2 (Alr1299), the ATP synthase α-subunit (All0005), the RuBisCO large subunit RbcL (Alr1524) and the hypothetical protein, All0268. Furthermore, it is also noteworthy that even though it was not possible to find the respective homologues for some of the Prochlorococcus MED4 vesicles’ proteins, related counterparts could be found for proteases (Alr1381, Alr0996), aminotransferases (All1683, Alr1004, Alr1080, Alr4853, Alr5103) and even ribosomal proteins (All4214) (see the Supplementary Information). Until now, there have been no reports describing the ability of Anabaena sp. PCC 7120 to form and release vesicles. Therefore, the different Anabaena sp. PCC 7120 concentrated exoproteome samples were subjected to negative staining transmission electron microscopy, in an attempt to find OMVs. Remarkably, the three samples presented numerous small spherical structures (Figure 3) that strongly resemble the membrane vesicles reported for Prochlorococcus [61].

Bottom Line: The evidence presented here shows that Anabaena sp.Furthermore, the activity of selected exoproteins associated with oxidative stress has been assessed, suggesting their involvement in redox homeostasis mechanisms in the extracellular space.Finally, we discuss our results in light of other cyanobacterial exoproteome studies and focus on the potential of exploring cyanobacteria as cell factories to produce and secrete selected proteins.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto 4150-180, Portugal. paulo.oliveira@ibmc.up.pt.

ABSTRACT
The interest in examining the subset of proteins present in the extracellular milieu, the exoproteome, has been growing due to novel insights highlighting their role on extracellular matrix organization and biofilm formation, but also on homeostasis and development. The cyanobacterial exoproteome is poorly studied, and the role of cyanobacterial exoproteins on cell wall biogenesis, morphology and even physiology is largely unknown. Here, we present a comprehensive examination of the Anabaena sp. PCC 7120 exoproteome under various growth conditions. Altogether, 139 proteins belonging to 16 different functional categories have been identified. A large fraction (48%) of the identified proteins is classified as "hypothetical", falls into the "other categories" set or presents no similarity to other proteins. The evidence presented here shows that Anabaena sp. PCC 7120 is capable of outer membrane vesicle formation and that these vesicles are likely to contribute to the exoproteome profile. Furthermore, the activity of selected exoproteins associated with oxidative stress has been assessed, suggesting their involvement in redox homeostasis mechanisms in the extracellular space. Finally, we discuss our results in light of other cyanobacterial exoproteome studies and focus on the potential of exploring cyanobacteria as cell factories to produce and secrete selected proteins.

No MeSH data available.


Related in: MedlinePlus