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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

In-gel analysis of the SOD activities of Anabaena sp. PCC 7120. Cells were grown in nitrogen-fixing conditions (N2) or in medium with nitrate (NO3−) or ammonia (NH4+). Cell-free extracts were obtained from the collected cells, while the exoproteome was concentrated from the respective cell-free medium. Three hundred micrograms of total protein (total protein) and the protein content present in approximately 20 mL of culture (exoproteome) were separated by native-polyacrylamide gel electrophoresis. (A) Zymogram depicting total SOD activity. The Fe-SOD and Fe-SOD/Mn-SOD complexes’ activity bands are highlighted; (B) Zymogram showing the SOD activity bands, as a result of the specific inhibition of Fe-SOD with 5 mM H2O2 [41]. The Mn-SOD activity bands/smear are highlighted.
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life-05-00130-f004: In-gel analysis of the SOD activities of Anabaena sp. PCC 7120. Cells were grown in nitrogen-fixing conditions (N2) or in medium with nitrate (NO3−) or ammonia (NH4+). Cell-free extracts were obtained from the collected cells, while the exoproteome was concentrated from the respective cell-free medium. Three hundred micrograms of total protein (total protein) and the protein content present in approximately 20 mL of culture (exoproteome) were separated by native-polyacrylamide gel electrophoresis. (A) Zymogram depicting total SOD activity. The Fe-SOD and Fe-SOD/Mn-SOD complexes’ activity bands are highlighted; (B) Zymogram showing the SOD activity bands, as a result of the specific inhibition of Fe-SOD with 5 mM H2O2 [41]. The Mn-SOD activity bands/smear are highlighted.

Mentions: The ability of prokaryotes to export ROS protein scavengers to the extracellular space is well documented in the literature, particularly for pathogenic bacteria, such as mycobacteria [63,64], streptococci [65], Campylobacter [66] and Corynebacterium [58]. Since phagocytic cells produce reactive oxygen intermediates to kill invading bacteria, it is not surprising that these enzymes are important for virulence [63,64]. In contrast, reports describing the presence and activity of ROS detoxifying proteins in the extracellular space of cyanobacteria are limited to the work of Shirkey et al. [67]. In this work, the exudates (supernatant fractions) of desiccated colonies of N. commune ENG/1996 and cultures of N. commune DRH1 were shown to have high amounts of SOD, as well as high levels of enzyme activity [67]. It was also demonstrated that the extensive extracellular polysaccharide matrix of N. commune DRH1 generated superoxide radicals upon exposure to ultra-violet irradiation [67]. Hence, it was proposed that the SOD released by N. commune is crucial for the cyanobacterium to deal with the oxidative stress imposed by multiple cycles of desiccation and rehydration of the extracellular matrix during ultra-violet irradiation in situ [67]. Anabaena sp. PCC 7120 does not form a complex extracellular polysaccharide matrix similar to that described for N. commune strains, and still, SOD could be identified accumulating in the extracellular space. Fe-SOD was detected in the exoproteomes of cells grown under all tested conditions (Table 1) and not just in the exoproteome of cells grown under conditions eliciting the synthesis and secretion of large amounts of polysaccharides, as in nitrogen-fixing conditions. Therefore, we have decided to evaluate whether SOD activity could be detected in the three isolated exoproteomes. Our in-gel activity results clearly show the presence of SOD activity in all three isolated exoproteomes (Figure 4); in addition to the clear SOD activity band, other fainter bands/smear could also be observed (Figure 4A). We hypothesized that the additional fainter bands could be resulting from the activity of Fe-SOD (Alr2938) and Mn-containing SOD (All0070; not identified in this work) complexes. To test that hypothesis, the Fe-SOD activity was inhibited with the presence of H2O2; in such conditions, the intensity of the clearest SOD activity band completely disappeared, as well as part of the signal from the other fainter bands/smear, remaining however as part of the signal (Figure 4B). This result supports our initial suggestion that Fe-SOD and the Mn-containing SOD may indeed form complexes in the extracellular milieu.


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)

In-gel analysis of the SOD activities of Anabaena sp. PCC 7120. Cells were grown in nitrogen-fixing conditions (N2) or in medium with nitrate (NO3−) or ammonia (NH4+). Cell-free extracts were obtained from the collected cells, while the exoproteome was concentrated from the respective cell-free medium. Three hundred micrograms of total protein (total protein) and the protein content present in approximately 20 mL of culture (exoproteome) were separated by native-polyacrylamide gel electrophoresis. (A) Zymogram depicting total SOD activity. The Fe-SOD and Fe-SOD/Mn-SOD complexes’ activity bands are highlighted; (B) Zymogram showing the SOD activity bands, as a result of the specific inhibition of Fe-SOD with 5 mM H2O2 [41]. The Mn-SOD activity bands/smear are highlighted.
© Copyright Policy
Related In: Results  -  Collection

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

life-05-00130-f004: In-gel analysis of the SOD activities of Anabaena sp. PCC 7120. Cells were grown in nitrogen-fixing conditions (N2) or in medium with nitrate (NO3−) or ammonia (NH4+). Cell-free extracts were obtained from the collected cells, while the exoproteome was concentrated from the respective cell-free medium. Three hundred micrograms of total protein (total protein) and the protein content present in approximately 20 mL of culture (exoproteome) were separated by native-polyacrylamide gel electrophoresis. (A) Zymogram depicting total SOD activity. The Fe-SOD and Fe-SOD/Mn-SOD complexes’ activity bands are highlighted; (B) Zymogram showing the SOD activity bands, as a result of the specific inhibition of Fe-SOD with 5 mM H2O2 [41]. The Mn-SOD activity bands/smear are highlighted.
Mentions: The ability of prokaryotes to export ROS protein scavengers to the extracellular space is well documented in the literature, particularly for pathogenic bacteria, such as mycobacteria [63,64], streptococci [65], Campylobacter [66] and Corynebacterium [58]. Since phagocytic cells produce reactive oxygen intermediates to kill invading bacteria, it is not surprising that these enzymes are important for virulence [63,64]. In contrast, reports describing the presence and activity of ROS detoxifying proteins in the extracellular space of cyanobacteria are limited to the work of Shirkey et al. [67]. In this work, the exudates (supernatant fractions) of desiccated colonies of N. commune ENG/1996 and cultures of N. commune DRH1 were shown to have high amounts of SOD, as well as high levels of enzyme activity [67]. It was also demonstrated that the extensive extracellular polysaccharide matrix of N. commune DRH1 generated superoxide radicals upon exposure to ultra-violet irradiation [67]. Hence, it was proposed that the SOD released by N. commune is crucial for the cyanobacterium to deal with the oxidative stress imposed by multiple cycles of desiccation and rehydration of the extracellular matrix during ultra-violet irradiation in situ [67]. Anabaena sp. PCC 7120 does not form a complex extracellular polysaccharide matrix similar to that described for N. commune strains, and still, SOD could be identified accumulating in the extracellular space. Fe-SOD was detected in the exoproteomes of cells grown under all tested conditions (Table 1) and not just in the exoproteome of cells grown under conditions eliciting the synthesis and secretion of large amounts of polysaccharides, as in nitrogen-fixing conditions. Therefore, we have decided to evaluate whether SOD activity could be detected in the three isolated exoproteomes. Our in-gel activity results clearly show the presence of SOD activity in all three isolated exoproteomes (Figure 4); in addition to the clear SOD activity band, other fainter bands/smear could also be observed (Figure 4A). We hypothesized that the additional fainter bands could be resulting from the activity of Fe-SOD (Alr2938) and Mn-containing SOD (All0070; not identified in this work) complexes. To test that hypothesis, the Fe-SOD activity was inhibited with the presence of H2O2; in such conditions, the intensity of the clearest SOD activity band completely disappeared, as well as part of the signal from the other fainter bands/smear, remaining however as part of the signal (Figure 4B). This result supports our initial suggestion that Fe-SOD and the Mn-containing SOD may indeed form complexes in the extracellular milieu.

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