Functional specificity of extracellular nuclease EndA
81
Discussion
are likely required for disulfide bond formation. Proper folding and activation of EndA might therefore be restricted to oxidizing milieus such as the extracellular space to protect the cells from deleterious effects of nuclease activity in the cytoplasm [250, 401]. Accordingly, the nucleolytic activity of purified EndA was completely inhibited upon addition of DTT to generate a reducing environment. Interestingly, endA (SO_0833) resides in an operon with gene SO_0831, encoding a putative ATP-dependent glutathione synthetase (GshB). Cotranscription of gshB with endA and GshB-mediated synthesis of glutathione might provide reducing conditions in the cytoplasm that prevent folding of EndA prior to cotranslational transport into the periplasm. Strikingly, gshB genes seem to be closely associated with endA genes in other species as well, including dns in V. cholerae and endA in E. coli, further indicating linked functions of both proteins (data not shown).
1.8.2 Regulation of EndA-mediated extracellular nucleolytic activity
Quantitative real-time RT-PCR analysis performed by M. Heun indicated that endA is not differentially regulated in response to phosphorus starvation. This was a rather surprising finding since EndA had also been shown to enable S. oneidensis MR-1 to exploit DNA as sole source of phosphorus under starvation conditions [292]. However, the results of this study demonstrated that deletion of endA results in extensive accumulation of eDNA in stationary phase cultures, both in minimal and rich medium. This clearly shows that even under non-starvation conditions, ΔendA deletion mutants exhibit abnormal phenotypes that might impact S. oneidensis MR-1 physiology and long-term survival. On the one hand, eDNA might be an important alternative energy and nutrient source in addition to other common sources such as N-acetylglucosamine, small organic acids, or amino acids. On the other hand, extensive accumulation of eDNA resulted in a highly viscous environment that might inhibit motility or reach toxic concentrations in late stationary phase [198].
Accordingly, the results of this study demonstrate that endA transcription is regulated by growth phase and not in response to phosphorus starvation. In planktonic cultures of wild-type S. oneidensis MR-1, native endA transcription was induced during exponential phase and decreased in stationary phase, indicating that EndA is required already in early phases of planktonic growth or that EndA might be stable in the supernatant over longer periods. Notably, episomal overexpression of endA in planktonic cultures resulted in strongly decreased eDNA levels and elevated nucleolytic activity in culture supernatants, indicating that transcription is the limiting factor under the conditions tested. However, factors involved in transcriptional regulation of endA remain to be identified. The fact that endA is induced even under optimal growth conditions during exponential phase, indicates that it is of general importance. Extracellular DNA has been shown to occur in high amounts in marine sediments and to play important roles in microbial ecosystems in the deep-sea [172]. Natural habitats of S. oneidensis and other members of the Shewanellaceae family comprise marine sediments. Thus, EndA may be a crucial factor for growth and long-term survival of Shewanella species in their natural environment. Finally, the control of DNA uptake and horizontal gene transfer might be another role of EndA that could be of particular importance in eDNA-rich environments such as marine sediments. However, conclusive evidence for the occurrence of natural transformation in Shewanella species is still missing.
EndA resides in an operon with phoA, encoding extracellular alkaline phosphatase PhoA. PhoA contributes significantly to extracellular phosphatase activity in culture supernatants but is not essential for growth on eDNA. In the absence of PhoA, residual phosphatase activity was observed, indicating the presence of additional enzymes that exhibit redundant functions in
Functional specificity of extracellular nuclease EndA
83 S. oneidensis MR-1. Indeed, several other extracellular phosphatases can be identified in S. oneidensis MR-1, and the resulting activity is likely sufficient for growth on eDNA in the absence of other phosphorus sources [173].
EndA is homologous to other bacterial nucleases, such as V. vulnificus Vvn, E. coli EndA, V. cholerae Dns, and A. hydrophila Dns. All of these enzymes contain signal peptides for Sec-dependent secretion and remain either in the periplasm, like Vvn and E. coli EndA, or are exported into the extracellular space, like Dns in V. cholerae and A. hydrophila [250, 295, 402]. In vivo DNA degradation assays in planktonic cultures demonstrated nucleolytic activity in cell-free supernatants but not in suspensions with washed cells, suggesting that Shewanella EndA is an extracellular enzyme similar to Dns and does not remain in the periplasm like Vvn or E. coli EndA. However, release through vesiculation or cell lysis cannot be ruled out, but this is rather unlikely to occur under the conditions tested (short incubation time, logarithmic phase cultures) [38, 403].
1.8.3 EndA is a planktonic growth-specific nuclease
Deletion of endA was shown to result in cellular aggregation during planktonic growth of S. oneidensis MR-1 [292]. Although this might suggest a possible role in community behavior of S. oneidensis MR-1, deletion of endA did not result in abnormal biofilm formation under hydrodynamic conditions and did not exhibit uncontrolled eDNA accumulation, as observed in ΔexeM mutants [241]. Overexpression of endA in static biofilms did not result in biofilm dispersal, although expression levels of endA were shown to correlate with nucleolytic activity in supernatants of planktonic cultures. Moreover, external addition of MBP-EndA did not release biomass from biofilms while addition of DNaseI readily induced biofilm dispersal, both under hydrodynamic and static conditions.
These data pose intriguing questions about structural and functional differences of extracellular nucleases that might determine whether a nuclease is functional under biofilm conditions or under planktonic growth conditions. Since purified MBP-EndA was shown to exhibit significant nucleolytic activity in vitro, it is rather unlikely that it lacks access to eDNA in biofilms due to the MBP fusion. Possibly, biofilm conditions render EndA inactive, e.g. as a result of reducing conditions in anaerobic biofilm areas, loss of the metal cofactor, or presence of other inhibitory cofactors. Indeed, slight inhibition of MBP-EndA nucleolytic activity was observed in vitro in the presence of Ca2+ (data not shown). However, it is unclear whether locally increased Ca2+ levels occur in biofilm environments, and if so, whether this would suffice to inactivate the nucleolytic activity of EndA. Furthermore, at least a residual loss of biomass from the upper layers of the biofilms would be expected in any of the mentioned cases. Recently, it has been demonstrated that eDNA physically interacts with other matrix components in biofilms formed by M. xanthus and P. aeruginosa [131, 146]. In starvation biofilms and fruiting bodies of M. xanthus, eDNA was bound to an unknown exopolysaccharide, resulting in greater physical strength and stress resistance compared to DNase I treated matrices [131]. Similarly, exopolysaccharide Psl in P. aeruginosa biofilms was shown to interact with eDNA to form a web of Psl-eDNA fibers in the center of pellicles to give structural support [146]. Possibly, binding of exopolysaccharides or other matrix components might mask eDNA and protect it from unspecific cleavage by extracellular nucleases under biofilm conditions. It would be interesting to determine whether similar interactions occur in S. oneidensis MR-1 biofilms and whether removal of such matrix components would make eDNA
Discussion
succeptible to cleavage by EndA. Another possibility is that biofilm-specific eDNA is modified as protection from degradation by EndA, as has been suggested for methylated eDNA in N. gonorrhoeae biofilms that was resistant to degradation by extracellular nuclease Nuc [184].
However, this study reveals that a significant fraction of eDNA that is required for structural biofilm formation, is released by prophage-induced cell lysis and therefore represents genomic DNA that is most likely identical to eDNA in planktonic stationary phase cultures. Moreover, in vitro degradation assays with biofilm eDNA indicated that most of the eDNA was readily degraded by the MBP-EndA fusion protein. However, only a small fraction of the eDNA might exhibit structural functions in biofilms. Under static conditions, deletion of endA resulted in increased accumulation of biofilm biomass, indicating that native EndA plays at least a minor role in biofilm formation. However, it is possible that improved cell aggregation in ΔendA mutants supports initial attachment or early biofilm formation but does not affect the structural integrity of the eDNA matrix in later phases. The phenotype of the ΔendA deletion mutant would therefore be rather indirect. Notably, extracellular nuclease Dns in V. cholerae has been shown to affect biofilm formation [242]. To better understand why homologous enzymes such as EndA and Dns exhibit different functions, it would be interesting to determine whether addition of purified Dns to biofilms formed either by V. cholerae or by S. oneidensis MR-1, would result in biofilm dispersal.
In conclusion, the role of EndA in biofilm formation requires further elucidation, mainly regarding biofilm-specific conditions or eDNA modifications that protect biofilms from dissolution by EndA. The results of this study strongly suggest functional specificity of extracellular nucleases in S. oneidensis MR-1. The fact that many bacteria possess various structurally distinct extracellular nucleases indicates that functional specificity and ‘division of labor‘ by extracellular nucleases might represent a common phenomenon and likely an adaptation to variable lifestyle and environments, including planktonic growth and biofilm formation.
Molecular characterization of ExeM
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