2.4.1 Origin and Evolution of the copper storage system
Figure 2.6 - Evolution of the Ccsp, SLI_RS17245 (3624) and SLI_RS17250 (3625) in the Tree of Life.
The occupancy and phylogenetic patterns point to multiple transfer events (indicated with arrows) from Bacteria to the other domains of the Tree of Life. Eukaryota has been circled in red due to issues in validating sample taxonomy (see Discussion).
43 It has been possible to determine the evolutionary history and origins of Ccsp, SLI_RS17245 and SLI_RS17250 by constructing the phylogenetic trees and their taxonomic occupation presented in this chapter. It has been hypothesised that all three genes derive from a bacterial last common ancestor (LCA) due to their wide distribution among the main Bacteria groups and were subjected to several gene losses in independent bacterial lineages. Although, it cannot be disregarded that possible LGT events occurred as there is a shortfall in resolution in parts of the phylogenetic trees. On the other hand, Ccsp, SLI_RS17245 and SLI_RS17250 are lacking in various members of Archaea and Eukaryota, along with inadequate evolutionary relationships between their sequences. Lest a great number of secondary gene losses occurred to explain their scarcity, this overall suggests an absence of these genes in the LCA of each of those domains. The likely explanation for this is due to multiple LGT events but with more genetic sampling this theory may change.
Figure 2.6 has helped by illustrating possible transfer patterns across all three phylogenetic trees as high levels of conservation in these genes have yielded some surprising phylogenetic patterns. Figure 2.6 initially shows the bacterial Ccsp gene jumped twice to Euryarcheota, twice to the TACK, and twice to eukaryotes (fungi and plants). As for SLI_RS17245, it is possible that at least five independent LGT events occurred towards Archaea and three towards Eukaryota. The SLI_RS17250 gene displayed the least amount of movement showing only two transfer events to various Archaea and two others to eukaryotic fungi. From this data alone, it is not possible to suggest any coincidence in the timing of the multiple transfers of the three genes. Interestingly, it was found that three species of TACK archaeans (related to the genus Nitrososphaera, an ammonia oxidizing archaean) possess all three genes in their genomes and suggests that, Ccsp, SLI_RS17245 and SLI_RS17250 have been transferred together simultaneously. For this to be validated, more information into these three genes structure and function within Nitrososphaera must be obtained to support this theory. Also, it has been observed that all three genes have jumped multiple times towards multicellular groups such as plants, slime molds, and fungi, as well as some unicellular algae.
Despite fascinating observations made from these phylogenetic analyses especially regarding transfer events of all three genes occurring in Eukaryota, it is essential to note that these eukaryotic targets could have been mistaken for bacterial species. It has been mentioned for Ccsp in particular, that due to a high sequence similarity between eukaryotic homologues and Ccsp raises this suspicion as stated by Dennison et al. in their review of Csps (75). As suggested by Dennison et al. this error is not surprising as many bacterial species are
44 soil dwelling or widespread in nature (75). Thus, contamination of samples with bacterial DNA is highly likely and gives this probable error in the phylogenetic analyses (75). The same issue could also be applied for SLI_RS17245 and SLI_RS17250 eukaryotic targets. The Eukaryota branch in Figure 2.6 has also been highlighted for this purpose, see also Appendix 1.1.
It was widely considered that the bacterial cytosol did not possess the machinery to store copper due to the absence of metabolic requirement and the toxicity of copper. This is unlike mammals, which possess various metallothioneins which are able to bind cadmium(II), copper(I) , zinc(II) or two of these metal species simultaneously (75, 140). The unexpected discovery and characterisation of cytosolic copper storage proteins known as Csp3s overturned the previous ideas about cytosolic copper in bacteria. This has now led to the discovery of Ccsp in S. lividans and has revealed its extensive taxonomic distribution across the Tree of Life (Figs. 2.3-2.5). Additionally, the phylogenetic distribution of SLI_RS17245 (Na+/H+ antiporter) and SLI_RS17250 (DUF4396) could suggest the existence of a copper cytosolic storage and regulation system that have not yet been characterised in other bacteria. It is well established that S. lividans requires copper as part of its development (19, 118). Stemming from this, previous studies in the extensive characterisation of S. lividans copper efflux and trafficking system, CsoR/CopZ/P1-type ATPase, have yielded some interesting transcriptional responses to copper (17-19, 138, 139). Many genes other than CsoR/CopZ/ATPase efflux system responded to copper stress by becoming up- or down regulated (18). As mentioned previously in this chapter, these genes included the upregulation of Ccsp, SLI_RS17245 and SLI_RS17250, not under control of CsoR (Fig. 2.2) (18, 19) and thus appear to be under the control of an unknown regulator. As suggested in the study by Dwarakanath et al., it is possible that multiple copper homeostatic mechanisms are simultaneously involved in regulating copper other than the CsoR regulon in S. lividans such as that described in redox homeostasis (18). Overall, these results present a potential new model for cytosolic copper storage and transport in S. lividans that requires further testing.
45
3 Chapter Three
Characterisation of a
cytosolic copper storage
protein from Streptomyces
lividans
Results from this Chapter have been published in:
Straw, Megan L., Chaplin, Amanda K., Hough, Michael A., Paps, Jordi, Bavro, Vassiliy N., Wilson, Michael T., Vijgenboom, Erik, Worrall, Jonathan A. R. “A cytosolic copper storage protein provides a second level of copper tolerance in Streptomyces lividans” 2018 Metallomics, 10, 180-193
46
3.1 Introduction
Streptomyces lividans shows a distinct dependence on copper (Cu) for initiating a
morphological switch from vegetative to aerial growth that simultaneously produces secondary metabolites (12, 44, 103, 141, 142). From a biotechnology perspective there is interest in using S. lividans as an industrial cell factory for the heterologous production of high value proteins and enzymes for processing biomass waste, diagnostic, therapeutic and agricultural uses (143). Indeed, the Streptomyces genus contains some important antibiotics and industrially useful enzymes that would prove beneficial if exploited correctly. The bioavailability of metal ions in microbial growth cultures is known to be important for optimizing batch processes, and to a certain extent this has been shown for Cu bioavailability in S. lividans and as such can impact on growth morphology in submerged (liquid) cultures (103). Thus a thorough understanding of how Cu is utilized in the host, i.e. in ‘correctly’ metalating secreted nascent apo-enzymes or proteins (37, 144, 145) in Cu resistance mechanisms (146) and in Cu trafficking pathways is important for creating re-engineered strains for optimized and improved growth.
Linked to understanding Cu handling in S. lividans is the discovery of a cytosolic copper storage (Ccsp) protein (Fig. 3.1) that has been described in Chapter 2. This Chapter describes initial structural and biochemical characterization of Ccsp and its ability to bind Cu(I). In addition, a ccsp null-mutant in S. livdians has been constructed by collaborators at Leiden University, The Netherlands, and its effect on growth and morphology investigated under increasing exogenous Cu concentrations. Cu(I) trafficking has also been investigated, and using size-exclusion chromatography evidence for a S. lividans Cu(I) metallochaperone, CopZ, being able to traffic Cu(I) to Ccsp is presented.