Environmental Microbiology Microbiology (2016) (2015) 18(2), 401–413 Environmental

doi:10.1111/1462-2920.12958 doi:10.1111/1462-2920.12958

Specific global responses to N and Fe nutrition in toxic and non-toxic Microcystis aeruginosa Ralitza Alexova,1 The Cuong Dang,2 Manabu Fujii,2,3 Mark J. Raftery,4 T. David Waite,2 Belinda C. Ferrari1,5 and Brett A. Neilan1,5* Schools of 1Biotechnology and Biomolecular Sciences and 2 Civil and Environmental Engineering, 4Bioanalytical Mass Spectrometry Facility and 5 Australian Centre for Astrobiology, University of New South Wales, Sydney, NSW 2052, Australia. 3 Department of Civil Engineering, Tokyo Institute of Technology, 2-12-1-M1-4 Ookayama, Tokyo 152-8552, Japan. Summary The bloom-forming cyanobacteria species Microcystis aeruginosa includes toxic and non-toxic (microcystin-producing) strains. Certain stress conditions stimulate synthesis of microcystin (MCYST) and enhance the binding of the MCYST molecule to proteins. In this quantitative proteomic study, we compared the response of a wild-type toxic strain PCC 7806, an mcyH− knockout non-toxic strain, and a naturally occurring non-toxic strain, PCC 7005, after 8 days in low iron (Fe) and nitrogen (N) starvation in order to assess the benefit of MCYST synthesis in non-optimal conditions. Fe limitation increased MCYST synthesis and caused an accumulation of phycobilisome proteins and the ferric iron transporter FutA only in the toxic PCC 7806 but not the non-toxic strains. In N starvation, photosynthetic, C and N metabolism proteins were more abundant in the non-toxic strains, as were chaperones and proteases. Significant interaction between nutrient availability and toxicity existed for thioredoxin peroxidase and several thioredoxinregulated proteins. We propose a competition of MCYST for binding sites in thioredoxin-regulated proteins during oxidative stress (low Fe) but not in growth-limiting conditions (low N). This then leads to differences in the regulation of C:N metabolism in toxic and non-toxic M. aeruginosa in nutrient-replete and nutrient-limited conditions. Received 11 March, 2015; revised 5 June, 2015; accepted 15 June, 2015. *For correspondence. E-mail [email protected]; Tel. (+612) 9385 3235; Fax (+612) 9385 1591.

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Introduction Microcystis aeruginosa is a species of cyanobacteria that occurs in algal bloom events worldwide. Some M. aeruginosa strains are capable of synthesizing microcystin (MCYST), a hepatotoxic non-ribosomal peptide, encoded by a conserved mcy gene cluster. Toxic and non-toxic strains often dominate different seasons or stages of bloom formation in the same water body, and such successions appear to be driven by nutrient and light availability (Van de Waal et al., 2011; Briand et al., 2012). The capacity of toxic cyanobacterial strains to synthesize MCYST appears to correlate with their increased fitness during nutrient limitation or in high light (Tonk et al., 2005; Zilliges et al., 2011). The postulated linear relationship between growth rate and cellular toxin quota (Vezie et al., 2002; Downing et al., 2005; Dai et al., 2008; Van de Waal et al., 2009) is not a straightforward one, as MCYST levels have also been shown to increase during growth-limiting low Fe stress (Utkilen and Gjolme, 1995; Dittmann et al., 2001; Kardinaal et al., 2007). The changes that are observed at the transcript level for mcy genes are not necessarily reflected in methanol-extractable microcystin content, such as in the case of N and high light stress (Kaebernick et al., 2000; Sevilla et al., 2010). A direct comparison of toxin content across different environmental conditions could also be an unreliable measure of toxicity as MCYST may accumulate in the media as a result of cell lysis, and the toxin has recently been shown to bind to proteins in the producing cells (Pearson et al., 2004; Zilliges et al., 2011; Meissner et al., 2013), thereby potentially underestimating toxin levels. Proteome and transcriptome analyses allow for a global overview of the changes that occur in cyanobacterial cells during their adaptation to a new environment, and recently have been applied to comparative investigations of toxic and non-toxic M. aeruginosa (Alexova et al., 2011b; Zilliges et al., 2011; Tonietto et al., 2012; Makower et al., 2015). Comparative proteomic studies of nutrientreplete naturally occurring toxic and non-toxic strains of M. aeruginosa show that the two groups differ in the expression of proteins involved in the maintenance of cellular C:N balance and redox sensing, including carboxysome components, nutrient transporters and nitrogen sensing proteins (Alexova et al., 2011b; Tonietto et al., 2012). These findings suggest that metabolic

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Fig. 1. Growth curves of Microcystis aeruginosa in nutrient-replete BG11 media (A) and N-free BG110 media (B). Batch cultures of three strains, PCC 7806 (toxic wild-type), PCC 7806 mcyH− (non-toxic PCC 7806 mcyH knockout) and PCC 7005, were maintained for 8 days, after which cells for proteomics were harvested. Mean values and standard deviation (n = 3) are shown for each growth curve.

adaptations of naturally occurring strains to nutrient availability in a particular eco niche may affect their response to seasonal nutrient fluctuations. Alternatively, knockout strains in which an mcy gene has been insertionally inactivated can be used to explore the effect of MCYST synthesis in the absence of background adaptation processes to a particular environmental niche that are likely to occur in natural isolates. Such strains are typically maintained in nutrient-rich media, and despite this exhibit impaired growth, pigmentation and disorganized thylakoid membranes (Schatz et al., 2007), suggesting that recent loss of MCYST biosynthetic ability affects multiple metabolic processes. A proteome analysis of the model toxic strain PCC 7806 and a mcyB− knockout established that microcystin binds to intracellular proteins, particularly in an oxidative stress-inducing environment, such as high light and low Fe, and may prevent protein processing by proteases (Zilliges et al., 2011). It remains unclear how and whether this enhanced synthesis of MCYST and its binding to proteins during stress benefits the producing strains in suboptimal nutrient conditions. To gain a greater understanding of the links between the capacity for MCYST synthesis and the nutrient stress response in M. aeruginosa, we quantified protein expression in Fe- or N-limited batch cultures. For this analysis, the response of the model toxic strain PCC 7806, the non-toxic wild-type strain PCC 7005 and a PCC 7806 mcyH− strain (hereafter referred to as mcyH−) were compared. This experimental design allowed a reconstruction of metabolic adaptations that occur after 8 days of Fe limitation and N starvation in the presence or absence of MCYST synthesis in this bloom-forming cyanobacterium.

Results Growth, pigmentation and free microcystin content We have previously reported that, in the presence of 10 nmol l−1 (Fe-stressed) but not 100 nmol l−1 Fe (Felimited), the toxic strain PCC 7806 responds with a significant increase in microcystin content and does not bleach, unlike the non-toxic PCC 7005 and mcyH−, which become severely chlorotic (Alexova et al., 2011a). Earlier studies have also shown that the difference in cell density does not extend to a difference in cell size of Fe-replete and Fe-limited cells (Alexova et al., 2011a; Cuong et al., 2012). Despite these differences in pigmentation at 10 nmol l−1 Fe, the three strains have similar growth rates (for growth curves, refer to Alexova et al., 2011a). The lack of bleaching in Fe-stressed PCC 7806 was also observed in the present study. Not only was the phycobiliprotein content not reduced in this strain, but growth in 10 nmol l−1 Fe in fact led to an accumulation of phycobilisome proteins, evident in the 1D SDS-PAGE gel (Fig. S1A) and was further confirmed by the mass spectrometric quantitation of differentially expressed proteins. In BG11, the three strains grew at the same exponential rate for the duration of the experiment (Fig. 1A). Exponential growth in BG110 for all strains was supported only for 2 days after transfer in the new media (Fig. 1B), after which growth was stationary. After 3 days in this media, chlorosis became pronounced in all strains. A secondary increase in cell density occurred for all strains between days 10 and 14. There was no significant difference in MCYST content (0.019 versus 0.017 nmol l−1 MCYST cell−1, for N-replete versus N-limited cells, respectively, t-test, n = 3).

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Nutrient Nutrient stress proteome of Microcystis aeruginosa 4033 stress proteome of Microcystis aeruginosa Fig. 2. Principal component analysis (PCA) of the proteomes of Microcystis aeruginosa grown for 8 days in 10 and 1000 nmol l−1 Fe in Fraquil media (A) and in BG11 or BG110 media (B). The first two components (PC1 and PC2) contributing to the variance in the data are shown. Nutrient-stressed samples are bound by dotted lines, and samples from control conditions are bound by unbroken lines; numbers represent individual replicates. Red – PCC 7806 (toxic wild-type); blue – PCC 7005 (non-toxic wild type); and green – PCC 7806 mcyH− (non-toxic PCC 7806 mcyH knockout).

Differential expression of proteins in nutrient-stressed and nutrient-replete strains of M. aeruginosa For M. aeruginosa grown in Fraquil media, 512 unique proteins were identified across the three strains and two Fe concentrations with high confidence. For the same strains grown in BG11 or BG110, 463 proteins could be quantified. When the proteomes of the three strains of M. aeruginosa were visualized by principal component analysis (PCA) (Fig. 2), it was evident that, despite their similar growth rates, the three strains that were chosen for analysis had distinct proteomes under nutrient-replete conditions in both Fraquil (Fig. 2A) and BG11 (Fig. 2B) media. Significantly different protein abundance between PCC 7806 and both non-toxic strains is listed in Tables S1–S8. Both Fe and N limitation caused changes in the proteome of the toxic strain PCC 7806, which separated the control and treatment groups clearly along the first two principal components (PC1 and PC2). In contrast, the proteomes of nutrient-replete and nutrient-stressed non-toxic strains (PCC 7005 and mcyH−) were less distinct. This applied in particular for the Fe-stressed knockout mutant mcyH−, which seemed to have a very restricted response to Fe limitation, but responded similarly to PCC 7005 when grown in BG110 (Fig. 2). Next, we performed multiple t-tests to determine which proteins were significantly changing between the toxic PCC 7806 and each of the non-toxic strains in control and nutrient-limited conditions, respectively (Figs 3–6 and Supporting information), as well as a two-way analysis of variance (ANOVA) to highlight proteins that showed significant interaction between nutrient availability (replete or limited) and microcystin production (toxic or non-toxic) (Tables S5 and S6). Such comparisons revealed proteins with similar abundance differences relative to PCC 7806 in both the naturally occurring non-toxic PCC 7005, which would have been subject to natural adaptation processes before it was isolated, and in the recently generated mutant mcyH− for which no other genomic differences to the wild-type PCC 7806 have been documented. Components of protein complexes, such as the phycobilisome, or proteins functioning in the same metabolic pathway were affected in a similar manner. This led us to focus our

analysis on the comparative responses of proteins within the same functional category. In all cases, aside from the heterogeneous ‘hypothetical’ and ‘other’ functional categories, many proteins involved in photosynthesis and respiration, and in energy metabolism, changed in abundance (Tables S1–S8). Proteins in the ‘amino acid’ and ‘translation’ functional categories, as well as proteins involved in redox sensing and reactive oxygen species (ROS) detoxification, nutrient transport, and the cell envelope, also contributed to a large percentage of the differentially expressed proteins. These functional categories are known to be important in the adaptation of cyanobacteria to nutrient stress and showed distinct responses in the toxic PCC 7806 and the non-toxic strains in all growth conditions (Figs 3–6). The proteomes with the greatest distinction between PCC 7806 and the non-toxic strains were found in the N-starved cells (Fig. 6). Photosynthesis and respiration proteins As is evident from the PCA in Fig. 2, the three strains had very distinct proteomes in nutrient-replete conditions, which could affect their response when transferred to nutrient-limited media. The proteins with significant difference between PCC 7806 and PCC 7005 and mcyH− during growth in Fraquil or BG11 media are presented in Fig. 3 and Fig. 5 respectively. With respect to proteins involved in photosynthesis and respiration, the non-toxic strains exhibited a lower content of photosystem I subunit III relative to PCC 7806, but higher abundance of ATP synthase F1 beta subunit (Fig. 3). In BG11, photosystem I subunit III was also less abundant in the non-toxic strains, but the photosystem I core protein PsaB was more abundant in the non-toxic strains relative to PCC 7806 (Fig. 5). The phycobilisome content was also lower in BG11-grown non-toxic M. aeruginosa. Two proteins involved in C-fixation, CcmM and the large subunit of RuBisCo were also differentially expressed in toxic and non-toxic strains (Fig. 5). The lack of chlorosis is the most characteristic response of the toxic PCC 7806 grown in 10 nmol l−1 Fe Fraquil media. The quantitative analysis of the proteomes of PCC

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Fig. 3. Proteins with significant difference in abundance between Microcystis aeruginosa PCC 7806, PCC 7806 mcyH − and PCC 7005 after 8 days of growth in Fraquil media supplemented with 1000 nmol l−1 Fe. The cell diagram shows proteins in six functional groups, which were upregulated (red) or downregulated (blue) (t-test P-value < 0.05, false discovery rate 5%) in both non-toxic strains relative to the toxic PCC 7806. The right panel shows the log10 fold change in individual proteins and their assignment to the functional groups. Functional categories according to cyanobase, gi accession numbers and normalized quantitative values are provided in the Supporting information.

7806 revealed that rather than the photosynthetic machinery being degraded, which is typical of Fe-starved cyanobacteria, several of the phycobiliproteins, and the protective orange carotenoid protein, were more abundant in low Fe growth conditions (Fig. 4, Table S7). In the nontoxic strains, allophycocyanin alpha and beta subunits, and phycocyanin alpha subunit, were maintained at a lower level (1.4–4-fold) relative to PCC 7806, and the differences were more pronounced in PCC 7005 (Fig. 4, Table S2). In N-starved PCC 7806, components of the photosynthetic machinery were strongly downregulated relative to the non-toxic strains (Fig. 6, Table S4). This included not only phycobilisome proteins, but also core subunits of photosystem I and II and ATP synthase subunits, and enzymes involved in the Calvin-Benson cycle (Fig. 6). C and N metabolism Given that the phycobilisome, photosystem and respiration protein abundance was extensively remodelled at

different nutrient availability in all strains, it is likely that these responses would be accompanied by abundance changes in energy pathways as cells attempt to balance their C:N metabolism under stress. Non-toxic cells harvested from Fraquil media at both Fe concentrations contained less glutamate-ammonia ligase than PCC 7806 (Figs 3 and 4). After 8 days of N starvation, the non-toxic strains matched the higher abundance of photosynthetic proteins with a higher abundance of enzymes involved in N and C metabolic pathways (glutamate-ammonia ligase, glutamine synthetase, fructose-1,6-bisphosphate aldolase, phosphoglucokinase and transketolase) (Fig. 6). Redox sensitive proteins and protein processing Cells achieve fine control over the response to nutrient stress with a network of regulatory proteins that control enzymatic activity and gene expression. In the present analysis, thioredoxin peroxidase (TPx) showed significant

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Nutrient Nutrient stress proteome of Microcystis aeruginosa 4055 stress proteome of Microcystis aeruginosa

Fig. 4. Proteins with significant difference in abundance between Microcystis aeruginosa PCC 7806, PCC 7806 mcyH − and PCC 7005 after 8 days of growth in Fraquil media supplemented with 10 nmol l−1 Fe. The cell diagram shows proteins in six functional groups, which were upregulated (red) or downregulated (blue) (t-test P-value < 0.05, false discovery rate 5%) in both non-toxic strains relative to the toxic PCC 7806. The right panel shows the log10 fold change in individual proteins and their assignment to the functional groups. Functional categories according to cyanobase, gi accession numbers and normalized quantitative values are provided in the Supporting information.

interaction between nutrition and toxicity in both low Fe and N stress conditions (Tables S5 and S6). This enzyme was significantly lower in both non-toxic strains during growth in 1000 nmol l−1 Fe Fraquil than in the toxic strain (Fig. 3, Table S1). This trend was conserved, but not significant, during growth in complete BG11 where thioredoxin A was downregulated in both non-toxic strains relative to PCC 7806 (Fig. 5). The TPx was also differentially expressed in N-starved non-toxic and toxic M. aeruginosa, whereas it was less abundant in PCC 7005 and mcyH− (Fig. 6). Protein abundance and function in a stress-inducing environment can also be affected by the efficiency of the protein processing machinery (translation, protein folding, refolding and degradation). Several enzymes with such function were changed in abundance between the different proteomes presented here. In particular, N starvation led to accumulation of several chaperones and proteases of the FtsH class in the non-toxic strains (Fig. 6). The

chaperone GroL was one protein with different abundance in all strains and media (Figs 3–6). Proteins involved in solute binding and nutrient transport Numerous proteins responsible for the transport of nutrients, including nitrate, phosphate, sulfate, bicarbonate, iron and amino acids, contributed to differences in the proteomes of M. aeruginosa strains in all growth conditions studied (Figs 3–6). The general trend was for the non-toxic strains to express less nutrient transporter and cell envelope proteins relative to PCC 7806 in all growth conditions tested (Figs 3–6), despite the similar cell numbers between the three strains at the time of harvest (Alexova et al., 2011a; Fig. 1). FutA1 and a ferrous iron transporter FeoA showed significant interaction between Fe availability and toxicity (Table S5). Importantly, in limited Fe, PCC 7806 upregulated the ferric transporter FutA1, while the

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Fig. 5. Proteins with significant difference in abundance between Microcystis aeruginosa PCC 7806, PCC 7806 mcyH − and PCC 7005 after 8 days of growth in nutrient-replete BG11 media. The cell diagram shows proteins in six functional groups, which were upregulated (red) or downregulated (blue) (t-test P-value < 0.05, false discovery rate 5%) in both non-toxic strains relative to the toxic PCC 7806. The right panel shows the log10 fold change in individual proteins and their assignment to the functional groups. Functional categories according to cyanobase, gi accession numbers and normalized quantitative values are provided in the Supporting information.

non-toxic strains did not change the expression of this iron transporter significantly (Table S7, Fig. 4). In BG11 and BG110, but not in Fraquil media, MrpA, a protein known to be associated with MCYST expression, had significantly lower expression in the non-toxic strains (Figs 5 and 6). In these media, irrespective of N availability, transporters for nitrate, phosphate and sulfate represented some of the largest differences between PCC 7806 and the non-toxic strains. Finally, a hypothetical protein homologous to MAE_ 47530 in the M. aeruginosa NIES-843 genome was significantly different in both non-toxic strains relative to PCC 7806, being more abundant in Fraquil media and less abundant in BG11, irrespective of Fe or N availability (Figs 3–6). This protein has no conserved domains and its function has not been predicted, but its expression profile

points to differential responsiveness to environmental conditions based on strain toxicity. Discussion Previously, it has been shown that naturally occurring toxic and non-toxic strains of M. aeruginosa grown in optimal conditions have distinct protein expression profiles that differ particularly in the levels of proteins involved in nitrogen and carbon metabolism and nutrient transport (Alexova et al., 2011b; Tonietto et al., 2012). Such preexisting proteome differences in a nutrient-rich environment will subsequently determine the response of individual strains to fluctuations in nutrient concentrations and could account for the different distribution of toxic and non-toxic strains during the course of bloom formation.

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Nutrient Nutrient stress proteome of Microcystis aeruginosa 4077 stress proteome of Microcystis aeruginosa

Fig. 6. Proteins with significant difference in abundance between Microcystis aeruginosa PCC 7806, PCC 7806 mcyH − and PCC 7005 after 8 days of growth in nutrient-replete BG110 media. The cell diagram shows proteins in six functional groups, which were upregulated (red) or downregulated (blue) (t-test P-value < 0.05, false discovery rate 5%) in both non-toxic strains relative to the toxic PCC 7806. The right panel shows the log10 fold change in individual proteins and their assignment to the functional groups. Functional categories according to cyanobase, gi accession numbers and normalized quantitative values are provided in the Supporting information.

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8 R. R. Alexova et al. 408 Alexova et al. Therefore, we sought to determine whether the dominance of either MCYST-producing or non-toxic strains in nutrient-limited environments could be explained by their proteome profile. A proposed role for microcystin in cellular redox control The biological significance and potential benefit of increased MCYST synthesis that has been reported to occur in ROS-producing conditions, such as high light or low Fe, have been the subject of much debate. Recent metabolome studies have indicated that an mcyB− experiences more stress than PCC 7806 wild-type in high light (Meissner et al., 2015). The toxin molecule itself is not likely to be directly involved in the scavenging of oxidative species during conditions of high light and low iron, as microcystin seems to be readily attacked by ROS (Song et al., 2009). The possibility remains that ROS modifies the toxin molecule, and this product in turn serves a role in the oxidative stress response. Results of immunogoldlabelling indicated that the majority of intracellular microcystin is associated with the thylakoid region and carboxysomes (Young et al., 2005; Gerbersdorf, 2006; Zilliges et al., 2011). Recent proteome analysis has shown that the toxin molecule is able to bind covalently to Cys residues of proteins, a process that is stimulated in high light, low Fe and chemically induced oxidative stress (Zilliges et al., 2011). MCYST binds to the phycobiliproteins and has been suggested to function as a toxin-storage mechanism or in regulating proteolysis by preventing access of proteases to their targets (Juttner and Luthi, 2008; Zilliges et al., 2011). The results that we report here for Fe-stressed M. aeruginosa and those by Zilliges and colleagues (2011) for high light and low Fe raise the possibility that MCYST could inhibit the disassembly of the photosynthetic machinery under nutrient-replete and ROSgenerating conditions by blocking access of ROS to reactive Cys in proteins. Given the predicted membrane localization of the ABC-transporter-like McyH protein and the increased disorganization of thylakoid membranes in the mcyH− strain (Pearson et al., 2004), as well as its limited response to Fe stress that we report here, it is possible that McyH is also involved in maintaining the stability of thylakoid membrane protein complexes, or in facilitating MCYST binding to photosynthetic proteins during oxidative stress in toxic M. aeruginosa. Protein Cys is a target site for thioredoxin binding, and many of the differentially expressed proteins that we observed in low Fe and N starvation, including PGK, FBA, GAPDH, ApcE, CpcB, CcmM, AtpA, AtpD and NrtA, are predicted thioredoxin targets and redox-sensitive proteins (Mata-Cabana et al., 2007; Lindahl and Kieselbach, 2009; Perez-Perez et al., 2009; Ansong et al., 2014). Thus,

microcystin and thioredoxin may be competing for reactive Cys in proteins of toxic M. aeruginosa. We have previously reported a difference in the abundance of thioredoxin M between naturally occurring toxic and non-toxic strains of M. aeruginosa (Alexova et al., 2011b). The abundance of thioredoxin reductase, which reactivates oxidized thioredoxin, was significantly different between PCC 7806 and mcyB− (Zilliges et al., 2011). Here, we identified TPx abundance, and in some instances thioredoxin A, as being significantly regulated in opposite directions in both N and Fe stress, and between toxic and non-toxic M. aeruginosa. This process is likely to occur synergistically with the action of chaperones and proteases, which act to repair or remove misfolded proteins and allow metabolic processes to proceed in conditions of cellular stress. In the presence of microcystin, the toxin itself may be interacting with proteases and redox-sensitive proteins as proposed by Zilliges and colleagues (2011). As microcystin concentration and GroL abundance increase in Fe-stressed PCC 7806, they may prevent oxidative damage to the phycobilisome and facilitate refolding of ROS-damaged proteins. In our study, free microcystin does not accumulate in N-starved PCC 7806 cells that have entered stationary growth phase and have become chlorotic. Instead, the response of this toxic strain to N starvation seems to be driven by changes in TPx abundance and leads to reduced expression of photosynthetic proteins and enzymes involved in C and N metabolism. In contrast, in the N-starved non-toxic M. aeruginosa, higher abundance of chaperones and proteases could maintain metabolic activity and protein function by refolding damaged proteins or recycling proteinogenic amino acids. Phycobilisome responses during Fe and N stress The increase in phycobilisome abundance in PCC 7806 grown in low Fe does not simply equal insensitivity to iron stress. We have previously reported decreased growth rates, and increased transcript levels of isiA and futA in PCC 7806 grown in the presence of 10 nmol l−1 Fe (Alexova et al., 2011a), as expected under iron limitation (Michel and Pistorius, 2004). In the proteome analysis presented here, PCC 7806 had higher abundance of the ferric transporter FutA, suggesting that this strain was able to sense external Fe and respond with an attempt to increase scavenging of this nutrient. On the other hand, we were unable to quantify changes in abundance in the photosystem I protective IsiA (CP43′). This is possibly due to the availability of only a truncated version of the C-terminal portion of IsiA in the M. aeruginosa PCC 7806 genome database, thus making it less likely to identify tryptic peptides derived from this protein. During N stress, protein synthesis is limited by the low availability of amino acids, and this can affect the

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stress proteome of Microcystis aeruginosa Nutrient Nutrient stress proteome of Microcystis aeruginosa 4099 photosystems as well as the phycobiliproteins, which account for up to a third of the soluble protein in cyanobacteria (Barrios-Llerena et al., 2006), and therefore represent a significant sink for N. In an N-starvation scenario, increased phycobilisome protein abundance, as was observed for Fe-stressed PCC 7806, may not be economical for the cell as amino acid pools become depleted and could not support continued renewal of highturnover proteins such as D1 and the high-abundance phycobiliproteins. Nutrient transport proteins in toxic and non-toxic Microcystis In the aquatic environment, complex interactions between nutrients determine their bioavailability, and often nutrient stress causes changes in the expression of transporters for nutrients other than the limiting factor (Hernandez-Prieto et al., 2012; Harke and Gobler, 2013). This was illustrated in the present study via the coordinated changes in abundance in multiple transporter proteins. In particular, FutA, the main transporter in nonsiderophore-mediated Fe uptake (Katoh et al., 2001), was significantly affected by nutrient availability and toxicity in both media. This confirms our previous physiological and transcript-level observations that PCC 7806 is particularly efficient in the uptake of ferric iron from the media (Alexova et al., 2011a) and appears to be capable of inducing the expression of this transporter more strongly in Fe stress than other strains. We, and others, have shown that toxic and non-toxic strains have different proteome profiles in nutrient-replete conditions, and some of these differences involve nutrient transporter subunits, such as NrtA and CmpA (Alexova et al., 2011b; Tonietto et al., 2012). This was also the case in the present study in which nitrate, sulfate, phosphate and bicarbonate transporters were differentially expressed in PCC 7806 and the non-toxic wild-type and mutant. These results point to differences in nutrient acquisition and assimilation in M. aeruginosa strains. Nevertheless, the growth rates of the different strains were similar in each media tested, suggesting that the capacity of non-toxic strains to take up nutrients may be regulated by a different subset of nutrient transporters, such as has been shown for the bicarbonate transport system in Microcystis strains (Sandrini et al., 2014). Additionally, a lower uptake of nutrients in the non-toxic strains may be offset by more efficient metabolic use of the same nutrients. Conclusions and future perspectives In summary, quantitative label-free proteomic analysis of a toxic and a non-toxic M. aeruginosa strain revealed

significant flexibility in the proteome response to iron and nitrogen depletion. This is based on underlying strainspecific differences in photosynthesis, energy metabolism and nutrient transport in nutrient-replete media. These differences highlight the possibility of misinterpreting -omic results when only two strains (toxic versus non-toxic natural isolates, or toxic versus knockout) are compared, and emphasize the importance of choice of starting control growth conditions as a potential source of variability, which could lead to significantly different experimental findings in comparative proteomic experiments. Metaproteomics is an emerging tool in studying the complex relationships between bacterial populations and their environment. With burgeoning genomic information of cyanobacterial cultures, both metagenomic and metaproteomic studies of a bloom community hold promise in answering some of these real-world questions regarding the ecophysiology of toxin production in cyanobacteria. It is well documented that laboratory-engineered mcy− mutants of PCC 7806 have altered pigmentation, exhibit disorganized thylakoid membranes, and are more sensitive to nutrient and light stress than the wild-type strain (Schatz et al., 2005). The present study complements these findings with data for the naturally occurring strain PCC 7005, and supports the proposal that the putative binding of microcystin to cellular proteins has a physiological role and is not simply a toxin storage mechanism. We propose that microcystin is able to bind phycobilisomes and carbon metabolism proteins and alter their regulation by thioredoxin under N- and Fe-replete conditions and in ROS-generating conditions, such as iron stress, when the photosynthetic machinery is particularly susceptible to oxidative damage. Experimental procedures Culturing and protein extraction Stock cultures of M. aeruginosa PCC 7806 (toxic), PCC 7806 mcyH− (non-toxic) and PCC 7005 (non-toxic) were grown in BG11 mineral medium (Rippka et al., 1979). For nitrogenstress studies, 50 ml of stock culture cells were washed with 10 volumes of BG11 containing no nitrate and supplemented with ferric citrate, instead of ferric ammonium citrate (referred to as BG110 in the main text), and 7 × 105 cells ml−1 were used to inoculate triplicate 100 ml batch cultures in BG11 (nitrogen-replete) and BG110 (nitrogen-limited). The growth experiment was replicated independently three times. All cultures were maintained in 250 ml Erlenmeyer flasks for 8 days at 28oC under a 14:10 light/dark cycle (25 μmol photons m−2 s−1) supplied by cool white fluorescent lamps. Cultures were gently agitated by hand twice daily. Samples were taken daily for 14 days from a parallel set of cultures for measurement of cell numbers in a Neubauer hemocytometer. For iron stress studies, cells were cultured in 200 ml modified Fraquil media (Andersen, 2005) in 250 ml Nalgene

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10 R.R.Alexova et et al. al. 410 Alexova Biotainer polycarbonate bottles (Thermo Scientific, MA, USA) that were acid-washed prior to use to avoid iron contamination. Strains in the BG11 stock cultures were washed in equal volume of Fraquil containing 100 nmol l−1 Fe and acclimated in this media for 2 weeks. Experiments were then initiated by washing and re-suspending 5 × 104 cells ml−1 in Fraquil supplemented with 10 nmol l−1 or 1000 nmol l−1 Fe. These conditions were chosen as the Fe transformation, kinetics and bioavailability have been well defined in the literature (Alexova et al., 2011a; Fujii et al., 2011; 2014). In addition, these growth conditions induce the most dramatic changes in toxicity and iron-regulated gene transcription (Alexova et al., 2011a). Triplicate batch cultures of M. aeruginosa PCC 7806 and PCC 7005 were maintained for 8 days at 27oC under a 14:10 light/dark cycle (90 μmol photons m−2 s−1) supplied by cool white fluorescent lamps and gently agitated daily. The higher light intensity compared with growth in BG11 media was required as the bioavailability of Fe in Fraquil media is determined by its interaction with EDTA and light-mediated Fe reduction (unlike BG11 which uses citrate and thermal dissociation of ferric citrate is the dominant process). Fe-replete Fraquil (1000 nmol l−1 Fe) is sufficient to support exponential growth of the three M. aeruginosa strains studied here for at least 10 days with no evidence of high light stress on the cells. For protein extraction, 100 ml of cells from triplicate cultures after 8 days of either Fe or N stress or control conditions were collected in the middle of the light period. The triplicate cultures were pooled to collect enough material for proteomics, and the experiment was performed three times. Cells were collected by centrifugation and partially lysed by three cycles of alternating freezing in liquid nitrogen and incubation at 37oC. The partial lysate was sonicated for 3 × 15 s in acid extraction buffer (Herbert et al., 2006; Alexova et al., 2011b) before pelleting insoluble cellular debris by centrifugation at 20 000× g for 5 min at 4oC. The supernatant was buffer-exchanged with phosphate-buffered saline at pH 8 supplied with 1 mM PMSF (Sigma, St Louis, MO, USA) and treated with Benzonase nuclease (Sigma). Protein concentration was determined using a Bradford assay (Bio-Rad, Hercules, CA, USA).

In-gel proteolysis and nanoLC-MS/MS analysis For proteomic analysis, 60 μg of protein extract was separated by 1D SDS-PAGE in a precast 4–20% Criterion Tris-Acetate gel (Bio-Rad) and stained with Coomassie G-250. For each experimental condition, technical triplicates of pooled biological triplicate samples were used (Fig. S1). Each lane was divided into 14 fractions, and in-gel digestion was performed as described in Alexova and colleagues (2011b). Finally, tryptic peptides were extracted in 50% ACN/1% formic acid, dried completely and re-suspended in 10 μl 1% formic acid/ 0.05% v/v heptafluorobutyric acid (HFBA) solution. Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex). Samples (1 μl) were concentrated and desalted onto a micro C18 precolumn (500 μm × 2 mm, Michrom Bioresources) with H2O:ACN (98:2, 0.05% HFBA) at 20 μl min−1. After a 4 min wash, the pre-column was switched (Valco 10 port valve, Dionex) into line with a fritless nano column

(75 μm × ∼ 10 cm) containing C18 media (5 μm, 200 Å Magic, Michrom) manufactured according to Gatlin and colleagues (1998). Peptides were eluted using a linear gradient of H2O:ACN (98:2, 0.1% formic acid) to H2O:ACN (64:36, 0.1% formic acid) at 350 nl min−1 over 30 min. High voltage (1800 V) was applied to a low volume tee (Upchurch Scientific), and the column tip positioned ∼ 0.5 cm from the heated capillary (T = 250°C) of an LTQ FT Ultra (Thermo Electron) mass spectrometer. Positive ions were generated by electrospray and the LTQ FT Ultra operated in datadependent acquisition mode. A survey scan m/z 350–1750 was acquired in the FT ICR cell (resolution = 100 000 at m/z 400 with an accumulation target value of 1 × 106 ions). The six most abundant ions (> 3000 counts) with charge states > +2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30 000 ions. M/z ratios selected for MS/MS were dynamically excluded for 30 s.

Data analysis Peak lists were generated using Mascot Daemon/ extract_msn (Matrix Science) using the default parameters, and submitted to the database search programme Mascot (version 2.1, Matrix Science). The following were the search parameters: precursor tolerance 4 p.p.m. and product ion tolerances ± 0.4 Da; Met(O), Acryl(C) and Carbamidomethyl(C) were specified as variable modification, enzyme specificity was trypsin, one missed cleavage was possible, and the M. aeruginosa PCC 7806 protein database (containing 5161 entries downloaded from National Center for Biotechnology Information (NCBI), version 01/11/07) was searched. A decoy database was searched to calculate the false discovery rate. SCAFFOLD 2 (Proteome Software) was used to validate MS/MS-based peptide and protein identifications. All MS/MS samples were analysed using Mascot (Matrix Science) and X! TANDEM (http://www.thegpm.org; version 2007.01.01.1). X! TANDEM and Mascot were set up to search the custom M. aeruginosa PCC 7806 proteome database, assuming tryptic digestion. Protein identifications were compared with the M. aeruginosa NIES-843 protein database, and functional categories were assigned according to annotation in Cyanobase (http://genome.microbedb.jp/cyanobase/ Microcystis). Mascot and X! TANDEM were searched with a fragment ion mass tolerance of 0.40 Da and a parent ion tolerance of 4.0 p.p.m. Oxidation of methionine and iodoacetamide derivative of cysteine were specified in X! TANDEM as variable modifications. Oxidation of methionine, iodoacetamide derivative of cysteine and the acrylamide adduct of cysteine were specified in Mascot as variable modifications. Protein identifications were accepted if they could be established at greater than 99% probability and contained at least two identified peptides. Peptide identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet algorithm (Keller et al., 2002). Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be

© 2015 Society for Applied and Microbiology and & Sons Ltd, Environmental Microbiology C 2015 Society V for Applied Microbiology John Wiley & John Sons Wiley Ltd, Environmental Microbiology, 18, 401–413

proteome of Microcystis aeruginosa411 11 NutrientNutrient stress stress proteome of Microcystis aeruginosa differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Differentially displayed proteins were determined from normalized spectral counts obtained in SCAFFOLD (quantitative values), which were used for further statistical analysis. Multiple t-tests with false discovery rate set at 5% and two-way ANOVA with Tukey posthoc test were performed in GRAPHPAD PRISM (v 6.01), and PCA was performed in R (R version 3.1.2; RStudio version 0.98.1091). Mass spectra were exported from SCAFFOLD as .mgf files and submitted to the PRIDE database (www.ebi.ac.uk/pride) using PRIDE converter 2.4.9 (Barsnes et al., 2009). The data are available under accession numbers 16401–16436 (username: review39418, password: mm7rNBg-).

Microcystin analysis Concurrently with protein extraction, free microcystins were extracted by diluting 1 ml of culture to a final concentration of 70% methanol. Toxin content normalized to cell number was determined by the protein phosphatase 2A inhibition assay as described previously (Carmichael and An, 1999).

Acknowledgements Ralitza Alexova was supported by a postgraduate scholarship from the Environmental Biotechnology Cooperative Research Council. Funding for this project was provided by the Australian Research Council and the Japanese Society for the Promotion of Science. We thank Cristián Holzmann for his help with statistical analysis in R. Mass spectrometric results were obtained at the Bioanalytical Mass Spectrometry Facility within the Analytical Centre of the University of New South Wales. BAN is a fellow of the Australian Research Council. The authors declare no conflict of interest.

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proteome of Microcystis aeruginosa413 13 NutrientNutrient stress stress proteome of Microcystis aeruginosa Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Representative 1D SDS-PAGE gel of Microcystis aeruginosa proteins used for the following mass spectrometric analysis. (A) 10 nM and 1000 nM Fe Fraquil and (B) BG11 or BG110 media. For each strain, 15 μg of protein was separated by electrophoresis on a 4–20% Criterion gel (Bio-Rad). MWM: Precision Plus molecular weight markers (Bio-Rad); N: nitrogen-replete; N0: nitrogen stress. Table S1. Proteins with significantly different abundance between M. aeruginosa PCC 7806 (toxic) and both non-toxic strains PCC 7806 mcyH− and PCC 7005 in Fraquil media containing 1000 nmol l−1 Fe. Significance was determined after t-test (P-value < 0.05, n = 3, with a false discovery rate set at 5%). The normalized quantitative values are calculated by the proteome analysis software SCAFFOLD for each protein and functional categories are from CyanoBase. Proteins with lower abundance in the non-toxic strains relative to PCC 7806 are shown in blue; proteins with higher abundance in the non-toxic strains are in red. Table S2. Proteins with significantly different abundance between M. aeruginosa PCC 7806 (toxic) and both non-toxic strains PCC 7806 mcyH− and PCC 7005 in Fraquil media containing 10 nmol l−1 Fe. Significance was determined after t-test (P-value < 0.05, n = 3, with a false discovery rate set at 5%). The normalized quantitative values are calculated by the proteome analysis software SCAFFOLD for each protein and functional categories are from CyanoBase. Proteins with lower abundance in the non-toxic strains relative to PCC 7806 are shown in blue; proteins with higher abundance in the non-toxic strains are in red. Table S3. Proteins with significantly different abundance between M. aeruginosa PCC 7806 (toxic) and both non-toxic strains PCC 7806 mcyH− and PCC 7005 in nutrient-replete BG11 media. Significance was determined after t-test (P-value < 0.05, n = 3, with a false discovery rate set at 5%). The normalized quantitative values are calculated by the proteome analysis software SCAFFOLD for each protein and functional categories are from CyanoBase. Proteins with lower abundance in the non-toxic strains relative to PCC 7806 are shown in blue; proteins with higher abundance in the non-toxic strains are in red. TO: detected in the toxic strain only. Table S4. Proteins with significantly different abundance between M. aeruginosa PCC 7806 (toxic) and both non-toxic

strains PCC 7806 mcyH− and PCC 7005 in nitrogen-free BG110 media. Significance was determined after t-test (P-value < 0.05, n = 3, with a false discovery rate set at 5%). The normalized quantitative values are calculated by the proteome analysis software SCAFFOLD for each protein and functional categories are from CyanoBase. Proteins with lower abundance in the non-toxic strains relative to PCC 7806 are shown in blue; proteins with higher abundance in the non-toxic strains are in red. TO: detected in the toxic strain only; NTO: not detected in the toxic strain. Table S5. Proteins with significant interaction between Fe content and toxicity in Fraquil media. Significance was determined after two-way ANOVA with Tukey as a post-hoc test. The normalized quantitative values are calculated by the proteome analysis software SCAFFOLD for each protein (colour-coded from blue to red in order of increasing magnitude) and functional categories are from CyanoBase. Table S6. Proteins with significant interaction between N content and toxicity in BG11 media. Significance was determined after two-way ANOVA with Tukey as a post-hoc test. The normalized quantitative values are calculated by the proteome analysis software SCAFFOLD for each protein (colour-coded from blue to red in order of increasing magnitude) and functional categories are from CyanoBase. Table S7. Proteins with significantly different abundance between M. aeruginosa in Fraquil media containing 1000 or 10 nmol l−1 Fe. Significance was determined after t-test (P-value < 0.05, n = 3), with a false discovery rate set at 5%. The normalized quantitative values are calculated by the proteome analysis software SCAFFOLD for each protein and functional categories are from CyanoBase. Proteins with lower abundance in the low Fe stress relative to control are shown in blue; proteins with higher abundance in the stressed cells are in red. Table S8. Proteins with significantly different abundance between M. aeruginosa in BG11 or BG110 media. Significance was determined after t-test (P-value < 0.05, n = 3), with a false discovery rate set at 5%. The normalized quantitative values are calculated by the proteome analysis software SCAFFOLD for each protein and functional categories are from CyanoBase. Proteins with lower abundance in the low Fe stress relative to control are shown in blue; proteins with higher abundance in the stressed cells are in red. BG11: protein identified only in nutrient-replete conditions; BG110: protein identified only in N-free media.

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Specific global responses to N and Fe nutrition in toxic and non-toxic Microcystis aeruginosa.

The bloom-forming cyanobacteria species Microcystis aeruginosa includes toxic and non-toxic (microcystin-producing) strains. Certain stress conditions...
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