Supplementary Material

Supplement to Jiang & Liu (2020) – Aquat Microb Ecol 85:47-58 – https://doi.org/10.3354/ame01939

TEXT S1: ANALYSIS OF THE SPECIFIC GROWTH RATE

The specific growth rate was calculated according to Equation S1, in which D0 and D1 were

the cell density at the beginning (t0) and the end (t1) of the selected time interval during the exponential growth phase, respectively.

µ(/day) = (LnD1 – LnD0)/(t1 – t0) (S1)

TEXT S2: ANALYSIS OF MICROCYSTINS CONTENT

On the last day of culture (stationary phase), 40 mL of the culture medium was sampled from the low nutrient group, the high nitrogen group and the high phosphorus group. The extracellular and intracellular fractions of MCs were separated from each other by low-vacuum filtration through a Whatman GF/B filtration membrane (1µm pore size). The cyanobacterial cells intercepted by the filtration membrane were freeze-dried, extracted with 10 mL of 40% (v/v) aqueous methanol under sonication and centrifuged at 4000×g at 4°C for 20 min to remove the cell debris. The extract was further cleaned up by solid phase extraction with an Oasis HLB glass cartridge (200 mg, 5 mL) to obtain the intracellular fraction of MCs. MCs in the filtrated culture medium were directly extracted through an Oasis HLB glass cartridge (200 mg, 5 mL) to obtain the extracellular fraction of MCs. The intracellular/extracellular extract was blown down to dryness under a gentle flow of nitrogen, spiked with 100 ng of enkephalin as an internal standard, dissolved with 500 µL of methanol, and subjected to the Agilent 1200 series liquid chromatograph

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coupled with an Agilent series 6410B triple quadrupole mass spectrometer (LC-MS/MS). The operation parameters of the LC-MS/MS and the detection limits of target MCs were described in detail in a previous study of our group (Liu et al. 2012).

TEXT S3: ANALYSIS OF PROTEOMIC RESPONSES

Protein Extraction and Digestion

The cyanobacterial cells were mixed with 900 µL of lysis buffer (4% SDS, 100 mM Tris-HCl,

1 mM DTT, pH 7.6) and Mpbio FastPrep Lysing Matrix A (MP Biomedicals, China), and then homogenized in a FastPrep-24™ system (MP Biomedicals, China). The mixture was incubated at

100 for 15 min, subjected to an ultrasonic cell pulverizer at 100 W for 4 min (ultrasonic time: 10 s; rest time: 10 s), and then incubated at 100 for another 15 min. After that, 180 µL of the supernatant was mixed with 1.6 mL of ice-cold acetone and incubated at -20 for 12 h. The protein pellet was collected by centrifugation (14,000×g, 4, 30 min), washed twice by ice-cold acetone, and finally dissolved in 90 µL of lysis buffer. The protein content was determined with a

DCTM Protein Assay kit (Bio-Rad, USA) according to the protocol manuals. During the determination of the protein content for each sample (low nutrient group/high nutrient group), only when the relative standard deviations of both the biological replicates and the technical replicates were below 10% could the sample be defined as qualified. The purified protein (400 µg) was reduced with 100 mM DTT in a boiling water bath for 5 min. After cooling, the protein sample was mixed with 200 µL of UA buffer (8 M Urea, 150 mM Tris-HCl, pH 8.0), transferred to a 10 KDa ultra-filtration centrifuge tube, and then centrifuged at 14,000×g for 15 min. The reduced protein was mixed with 100 µL of 50 mM iodoacetamide in UA buffer to block reduced

Supplement to Jiang & Liu (2020) – Aquat Microb Ecol 85:47-58 – https://doi.org/10.3354/ame01939

cysteine residues, and incubated at room temperature in the dark for 30 min. After that, the tube was centrifuged at 14,000×g for 10 min and consequently washed with 200 µL of UA buffer and

200 µL DS buffer (50 mM triethylammoniumbicarbonate, pH 8.5). The protein sample on the ultra-filtration membrane was digested with 8 µg trypsin in 40 µL DS buffer at 37 for 18 h, and the filtrate was collected by centrifugation at 14,000×g for 10 min. The digested peptides were dried under vacuum.

Peptide Labeling

The peptide samples were labeled with an iTRAQ Reagent 8-plex kit (AB SCIEX, China) according to the manufacturer’s recommendations. Both high nitrogen and phosphorus groups were labeled with iTRAQs 113, 114 and 115, and the low nutrient group was labeled with iTRAQs 116, 117 and 118. The labeling was independently repeated 3 times (each time for one biological replicate of the test groups). Six replicates (3 biological replicates × 2 technical replicates) were prepared for each test group for the quantitative analysis of proteome.

Strong Cation Exchange (SCX) Fractionation

The labeled peptide mixture was vacuum-dried, reconstituted in buffer A (10 mM KH2PO4,

25% acetonitrile, pH 3.0) and loaded into an AKTA Purifier 100 SCX chromatography (GE

Healthcare, China) equipped with a PolySULFOETHYL 4.6 × 100 mm column (5 µm, 200 Å)

(PolyLC Inc, USA). The peptide mixture was fractionated at a flow rate of 1 mL/min using an

increasing gradient of buffer B (10 mM KH2PO4, 500 mM KCl, 25% acetonitrile, pH 3.0) in a gradient elution protocol for a total of 60 min (100% buffer A for the initial 25 min, 0%-45% buffer B for 22 min, 45%-100% buffer B for 5 min and 100% buffer B for 8 min). A total of 30

SCX fractions were collected along the gradient, desalted with Sep-Pak C18 cartridges (Waters

Supplement to Jiang & Liu (2020) – Aquat Microb Ecol 85:47-58 – https://doi.org/10.3354/ame01939

Corporation, USA) and vacuum-dried.

Protein Identification and Quantification

The peptide mixture was re-suspended in solvent A (2% acetonitrile, 0.1% formic acid). 5 µL of the re-suspended sample was loaded on to a 150 µm × 100mm RP-C18 Thermo EASY column

(Thermo Fisher Scientific, USA). Separation was performed at a flow rate of 300 nL/min using a

Thermo Scientific EASY-nLC 1000 UHPLC system (Thermo Fisher Scientific, USA). Peptides were eluted from the column by a linear gradient from 0% solvent B (84% acetonitrile, 0.1% formic acid) to 45% solvent B in 100min, followed by ramping up to 100% solvent B in 8 min, and then held on 100% for 12 min. Peptides separated by UHPLC were analyzed by a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA). Survey full-scan MS spectra (350-1800m/z) was acquired with a mass resolution of 70000 at m/z 200. Twenty most abundant precursor ions from the survey scan were selected for high-energy collisional dissociation (HCD) fragmentation.

Determination of the target value was based on Automatic Gain Control (AGC). A dynamic exclusion of 10 s was used to remove redundant MS/MS scan. The resolution of MS/MS scan set to 17500 at m/z 200. Normalized collision energy was 30 eV and the underfill ratio was defined as

0.1%.

The .raw files were converted to .mgf files using Proteome Discoverer 1.4 (Thermo Fisher

Scientific, USA), submitted to MASCOT 2.2 (Matrix Science, UK), and searched against the database of M. aeruginosa from UniProt (updated on 09/15/2019).The enzyme specificity of trypsin was used and two missed cleavages were allowed. Mascots were searched with a parent ion tolerance of 20 ppm and a fragment ion mass tolerance of 0.1 Da. Carbamidomethylation of cysteine, as well as N-term and lysine labeled by iTRAQ 8-plex were set as fixed modifications.

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Oxidation of methionine and tyrosine labeled by iTRAQ 8-plex were specified as variable modifications. False discovery rate (FDR) correction was adopted with a threshold of 0.01 to reduce the false identification of peptide. The minimum protein identification probability and the minimum peptide identification probability were set as 99%, and the minimum number of peptides was identified as 1.

Relative quantification of identified proteins was performed with Proteome Discoverer 1.4.

The integration window tolerance was set as 20 ppm, and most confident centroid was used as the integration method. The protein ratios were calculated as the median of only unique peptides of the protein. Final ratios were then normalized by the median protein ratio for all six labeled samples. This correction is based on the assumption that the expression of most proteins does not change. A protein was considered to be differentially expressed if the iTRAQ ratio was > 1.50 or

< 0.67 in each high nutrient group compared with the low nutrient group (p < 0.05 according to

Student’s t-test) (Ralston-Hooper et al. 2013).

REFERENCES

Liu Y, Gao BY, Yue QY, Guan YT, Wang Y, Huang LH (2012) Influences of two antibiotic

contaminants on the production, release and toxicity of microcystins. Ecotoxicol Environ Saf

77:79−87

Ralston-Hooper KJ, Turner ME, Soderblom EJ, Villeneuve D, Ankley GT, Moseley MA, Hoke

RA, Ferguson PL (2013) Application of a label-free, gel-free quantitative proteomics method

for ecotoxicological studies of small fish species. Environ Sci Technol 47:1091−1100

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Table S1 The identified results of differentially expressed proteins in high nitrogen group compared with the low nutrient group.

UniProt Protein Protein Coverage Unique Peptides PSMb Theoretical Fold AsNoa name symbol percentage (%) peptides pI/Mw change Biological process: Biosynthetic process B0JV60 Acetylornithine aminotransferase ArgD 22.12 1 6 11 5.00/46808 1.54 B0JMS6 Aminotransferase AspC 36.69 11 11 54 5.60/42173 1.59 B0JWV0 ATP synthase subunit delta AtpH 82.42 1 11 72 6.34/19629 1.55 B0JKM3 GDP-L-fucose synthase Fcl 45.83 1 10 32 4.96/34940 0.61 B0JQ90 Glutathione synthetase GshB 10.09 3 3 6 5.20/36309 0.58 B0JW58 GMP synthase [glutamine-hydrolyzing] GuaA 5.20 1 1 1 5.73/60816 1.53 B0JVF7 Histidinol dehydrogenase HisD 12.20 1 3 4 5.53/46016 0.59 B0JRP2 Ketol-acid reductoisomerase (NADP(+)) IlvC 72.73 2 19 192 5.39/35871 0.63 B0JM73 Precorrin-6y C5,15-methyltransferase (Decarboxylating) MAE_02680 3.41 1 1 2 6.14/44248 1.87 B0JMU9 7,8-dihydroneopterin aldolase MAE_02910 32.20 1 4 10 5.56/13343 0.56 B0JUD0 3-oxoacyl-[acyl-carrier-protein] synthase 2 MAE_14960 28.85 1 8 41 5.80/43646 0.62 B0JW35 Aspartate kinase MAE_49560 32.11 1 15 31 5.20/63644 1.59 B0JHR2 Non-ribosomal peptide synthetase MAE_56520 14.38 12 13 27 5.24/124456 1.56 B0JJW8 McnE protein McnE 18.41 1 20 38 5.14/159501 1.55 B0JPV3 McyC protein McyC 18.15 4 16 29 5.26/147846 1.68 B0JPW0 McyF protein McyF 56.97 9 10 47 5.72/28043 1.52 B0JPW2 McyH protein McyH 6.88 2 2 2 5.51/67042 1.57 B0JPT8 Methylthioribose-1-phosphate isomerase MtnA 33.42 1 9 22 5.11/38321 1.57 B0JPI3 NAD kinase NadK 17.05 4 4 4 5.66/33392 1.58 B0JGU8 3-methyl-2-oxobutanoate hydroxymethyltransferase PanB 15.29 3 3 4 5.77/27251 0.43 B0JPG5 Phycocyanobilin:ferredoxin oxidoreductase PcyA 11.43 1 3 3 5.29/27685 0.57 B0JVN8 Flavin prenyltransferase UbiX UbiX 18.05 3 3 4 6.51/22652 1.56 Biological process: Carbohydrate metabolic process B0JMP6 Enolase Eno 56.71 1 16 135 4.92/46543 0.63 B0JUD1 Transketolase MAE_14970 55.54 1 25 142 5.45/72232 0.59 B0JWB9 Glycosyl transferase group 1 MAE_17770 7.16 2 2 5 6.14/42663 1.57

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B0JHH3 Glyceraldehyde-3-phosphate dehydrogenase MAE_25030 76.92 1 25 362 6.19/36436 0.61 B0JMM4 Probable phosphoketolase MAE_34870 41.69 4 30 121 6.00/91842 0.56 B0JVZ9 Glycosyl transferase family 2 MAE_49200 3.59 1 1 3 6.59/38169 1.63 Biological process: Catabolic process B0JJ08 Aminomethyltransferase GcvT 23.91 1 8 27 5.48/40123 0.58 B0JXF9 Dipeptide epimerase MAE_19640 13.43 4 4 6 5.55/38850 1.58 B0JU62 Phycocyanin alpha phycocyanobilin lyase related protein NblB 43.89 1 7 39 4.36/24007 1.56 Biological process: Cell communication B0JT21 Uncharacterized protein MAE_12430 0.77 1 1 1 3.95/171948 1.98 Biological process: Cell division B0JIG6 Cell division protein FtsZ FtsZ 29.40 1 8 27 5.01/43364 1.74 Biological process: Cellular component organization B0JPL0 Gas vesicle structural protein GvpC 48.04 1 12 89 9.37/25092 1.74 B0JPL8 Gas vesicle protein GvpW 20.37 3 3 7 5.10/26159 1.56 Biological process: Gene expression B0JVN3 50S ribosomal protein L33 RpmG 17.19 1 1 5 10.11/7401 1.64 B0JTB0 30S ribosomal protein S1 Rps1 51.19 1 12 41 4.95/37057 1.59 B0JGK4 30S ribosomal protein S20 RpsT 30.93 1 1 2 10.75/10550 1.67 Biological process: Methylation B0JLC9 Putative type IIS restriction/modification enzyme MAE_63060 2.67 1 1 2 6.53/138827 0.57 Biological process: Nitrogen compound metabolic process B0JRH7 Glutamate--ammonia ligase GlnN 36.10 6 23 82 5.60/79050 1.64 B0JGF2 Ferredoxin-nitrate reductase NarB 1.62 1 1 1 7.03/80194 1.69 B0JH03 Anthranilate synthase component II TrpG 4.10 1 1 4 5.25/21662 0.53 Biological process: Not defined B0JFF9 Antitoxin MAE_00990 11.46 1 1 1 5.70/10853 0.60 B0JMV8 Cell envelope-related transcriptional attenuator MAE_03000 10.43 1 2 2 8.64/51512 1.53 B0JVM2 Carbon dioxide concentrating mechanism protein CcmK1 67.26 1 7 157 6.74/12118 1.79 B0JVM0 Carbon dioxide concentrating mechanism protein CcmM 65.80 1 43 243 8.77/70344 1.71 Biological process: Oxidation-reduction process B0JIQ8 Hydrogenase subunit of the bidirectional hydrogenase HoxY 21.43 3 3 5 4.54/20025 0.61

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B0JPF1 3-hydroxyacid dehydrogenase MAE_06400 39.02 1 8 15 5.29/31821 0.45 B0JNP5 Ferredoxin I PetF1 16.00 2 2 12 3.88/10606 1.85 B0JSD0 Ferredoxin I PetF2 20.39 1 1 2 4.33/11287 1.57 B0JHU8 Thioredoxin TrxA 64.81 1 6 13 8.43/12162 0.60 Biological process: Phosphorus metabolic process B0JQJ1 Dihydroorotase PyrC 9.59 3 3 6 5.96/38398 1.64 B0JX00 5'-nucleotidase SurE SurE 24.07 4 4 6 5.41/29383 1.58 Biological process: Photosynthesis B0JXA2 Phycocyanin alpha subunit CpcA2 13.50 1 2 48 5.41/17758 1.74 B0JHB5 Phycocyanin beta subunit CpcB1 82.35 1 1 5 5.12/18164 1.58 B0JXA3 Phycocyanin beta subunit CpcB2 92.41 1 8 444 5.14/18059 1.59 B0JXB7 Cytochrome b6-f complex iron-sulfur subunit PetC 70.95 1 8 56 5.23/19065 1.74 B0JHN7 Plastocyanin PetE 52.80 1 4 51 6.82/13280 1.83 B0JR68 Photosystem II CP43 reaction center protein PsbC 24.85 9 9 61 6.11/50234 1.67 Biological process: Protein folding B0JN66 Protein GrpE GrpE 39.58 1 9 51 4.60/26992 0.64 Biological process: Protein metabolic process B0JKT4 Serine protease HtrA 30.75 7 7 22 5.48/44728 0.56 B0JI96 Deoxyribodipyrimidine photolyase-related protein MAE_25720 7.75 3 3 5 6.41/59238 1.58 B0JIG4 Periplasmic carboxyl-terminal protease MAE_26400 51.63 1 18 36 6.13/47445 1.64 B0JNH9 Peptidylprolyl isomerase MAE_35870 40.00 1 11 81 4.98/27831 0.58 B0JS62 Serine/threonine protein kinase MAE_41980 2.35 1 1 1 7.02/40031 1.65 B0JXY8 HesB/YadR/YfhF family protein MAE_52040 8.47 1 1 3 6.04/12860 0.51 B0JWG8 Methionine aminopeptidase Map 21.65 2 3 15 6.05/27531 1.55 Biological process: Regulation of cell shape B0JQ06 Rod shape-determining protein MreC 4.82 1 1 1 9.52/27727 1.52 Biological process: Regulation of gene expression B0JU72 Two-component response regulator OmpR subfamily MAE_14380 4.84 1 1 3 8.99/29658 0.59 B0JJF3 Transcriptional regulator MAE_27730 3.73 1 1 1 8.32/34528 1.57 B0JRG0 Global nitrogen regulator Ycf28 NtcA 56.30 1 5 18 6.92/25206 1.84 Biological process: Response to stimulus

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B0JSS2 16.6 kDa small heat shock protein HspA 47.79 2 4 17 4.76/15205 0.59 Biological process: Transport B0JM22 Amino acid transport system permease protein MAE_02170 3.48 1 1 1 9.77/30710 1.68 B0JPC9 Urea transport system ATP-binding protein MAE_06180 28.45 5 5 10 7.85/25624 1.63 B0JPD1 Urea transport system permease protein MAE_06200 2.86 1 1 2 8.98/42462 0.57 B0JU64 ABC-transporter ATP-binding protein MAE_14300 10.09 2 2 3 5.67/35554 1.54 B0JXP2 ABC_transp_aux domain-containing protein MAE_20470 19.69 4 8 12 9.12/56329 0.51 B0JXP4 ABC-transporter ATP-binding protein MAE_20490 16.23 4 4 7 6.13/34496 1.60 B0JJH9 Porin type major outer membrane protein MAE_27990 21.99 2 13 232 5.40/60274 0.59 B0JQV4 Ammonium transporter MAE_40010 4.60 1 1 1 5.70/50340 1.85 B0JR43 Putative HlyD family secretion protein MAE_40900 41.86 1 16 34 9.80/51996 0.54 B0JR91 Oligopeptides ABC-transporter permease protein MAE_41380 3.73 1 1 1 9.09/41272 1.56 B0JS84 Amino acid transport system ATP-binding protein MAE_42200 25.32 5 5 9 9.59/25900 1.59 B0JUG5 Extracellular solute-binding protein MAE_45910 3.47 1 1 2 7.53/60079 0.47 B0JHQ8 ABC-transporter ATP-binding protein MAE_56480 3.77 1 1 1 5.45/76838 1.72 B0JLC0 Amino acid transport system ATP-binding protein MAE_62970 25.49 5 5 11 5.85/27643 1.57 B0JUB1 Nitrate/nitrite transport system ATP-binding protein NtcD 17.17 5 5 10 5.16/36882 0.63 B0JL11 Copper-transporting P-type ATPase PacS 4.12 2 2 2 8.75/80254 0.56 B0JRJ7 Phosphate import ATP-binding protein PstB PstB 35.38 7 7 16 8.52/29880 0.53 B0JWH6 Phosphate import ATP-binding protein PstB PstB2 22.44 4 4 10 8.22/29390 0.40 B0JWI0 Phosphate-binding protein PstS 31.64 3 8 38 5.28/39891 0.58 a: Accession number; b: Peptide spectrum match

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Table S2 The identified results of differentially expressed proteins in high phosphorus group compared with the low nutrient group.

UniProt Protein name Protein Coverage Unique Peptides PSMb Theoretical Fold AsNoa symbol percentage (%) peptides pI/Mw change Biological process: Biosynthetic process B0JXE8 N-acetyl-gamma-glutamyl-phosphate reductase ArgC 47.88 1 12 57 5.99/38195 1.55 B0JWU7 ATP synthase subunit c AtpE 8.64 1 1 3 4.94/7979 0.53 B0JWV0 ATP synthase subunit delta AtpH 91.21 2 12 77 6.34/19629 1.51 B0JFP4 Biotin synthase BioB 4.86 2 2 2 5.57/39354 0.62 B0JU89 Geranylgeranyl pyrophosphate synthase CrtE 34.30 3 8 25 4.92/33249 0.64 B0JL16 Adenylyl-sulfate kinase CysC 64.67 3 9 37 5.56/20110 1.58 B0JJZ9 Cysteine synthase CysK 65.20 2 13 48 5.31/34371 1.55 B0JWW6 1-deoxy-D-xylulose 5-phosphate reductoisomerase Dxr 48.21 12 12 22 5.23/42189 1.56 B0JL88 1-deoxy-D-xylulose-5-phosphate synthase Dxs 23.43 11 11 51 6.12/69114 0.59 B0JJI5 Glucose-1-phosphate adenylyltransferase GlgC 64.57 1 31 179 6.57/48418 1.59 B0JIM9 Glutamine--fructose-6-phosphate aminotransferase [isomerizing] GlmS 19.59 10 10 39 5.27/70310 1.53 B0JJJ7 Histidinol-phosphate aminotransferase HisC 25.29 6 6 12 5.36/39189 0.64 B0JFQ9 Imidazole glycerol phosphate synthase subunit HisH HisH 40.09 2 7 27 6.34/23011 1.58 B0JUF7 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD 30.80 2 6 6 5.71/24856 1.58 B0JLJ1 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF 50.91 4 4 23 7.02/17221 0.58 B0JJJ8 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ferredoxin) IspG 45.57 5 16 60 5.67/44480 0.59 B0JVA7 4-hydroxy-3-methylbut-2-enyl diphosphate reductase IspH 18.16 6 7 13 5.29/45595 0.60 B0JM73 Precorrin-6y C5,15-methyltransferase (Decarboxylating) MAE_02680 3.41 1 1 2 6.14/44248 1.82 B0JMR1 1-acyl-sn-glycerol-3-phosphate acyltransferase MAE_35240 42.52 8 8 17 9.71/23394 0.63 B0JS63 Dolichyl-phosphate-mannose synthase MAE_41990 9.52 3 3 11 9.22/48181 1.54 B0JUV1 Uroporphyrin-III C-methyltransferase MAE_47270 5.64 1 1 3 6.16/28255 1.58 B0JW04 Sulfolipid biosynthesis protein MAE_49250 60.57 2 17 69 5.86/43141 1.64 B0JPT8 Methylthioribose-1-phosphate isomerase MtnA 33.42 1 9 22 5.11/38321 1.59 B0JTU1 2,3-diketo-5-methylthiopentyl-1-phosphate enolase MtnW 26.68 7 7 15 5.76/41549 1.54 B0JPI3 NAD kinase NadK 17.05 4 4 4 5.66/33392 1.64 B0JHT4 Nucleoside diphosphate kinase Ndk 49.65 1 7 44 5.6/16472 0.64

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B0JGU8 3-methyl-2-oxobutanoate hydroxymethyltransferase PanB 15.29 3 3 4 5.77/27251 1.54 B0JI57 4-hydroxythreonine-4-phosphate dehydrogenase PdxA 17.52 5 5 19 5.78/37537 0.58 B3DF93 Pyridoxine 5'-phosphate synthase PdxJ 17.92 2 2 3 5.53/26549 1.69 B0JVX4 Prephenate dehydratase PheA 3.87 1 1 2 4.97/31496 1.62 B0JXE3 Phosphate acyltransferase PlsX 31.34 7 7 26 5.79/36182 1.74 B0JFJ0 Polyphosphate kinase Ppk 40.72 24 24 70 5.79/82027 0.57 B0JWW5 Gamma-glutamyl phosphate reductase ProA 22.97 8 8 17 5.60/47184 0.61 B0JSC4 Thiamine-phosphate synthase ThiE 37.87 1 12 29 5.91/37947 0.56 B0JYF1 Thiamine-monophosphate kinase ThiL 10.25 2 2 2 5.08/34733 1.52 B0JTM2 Indole-3-glycerol phosphate synthase TrpC 53.04 12 12 33 5.92/32652 0.58 B0JVN8 Flavin prenyltransferase UbiX UbiX 18.05 3 3 4 6.51/22652 1.58 Biological process: Carbohydrate metabolic process B0JSC7 Anhydro-N-acetylmuramic acid kinase AnmK 11.11 2 2 3 6.00/41291 1.54 B0JLP5 Fructose-1,6-bisphosphate aldolase FbaA 56.92 1 14 169 5.40/38952 0.56 B0JUD1 Transketolase MAE_14970 55.54 1 25 142 5.45/72232 0.56 B0JHH3 Glyceraldehyde-3-phosphate dehydrogenase MAE_25030 76.92 1 25 361 6.19/36436 1.54 B0JMM4 Probable phosphoketolase MAE_34870 41.69 4 30 121 6.00/91842 0.58 B0JNS9 Mannose-6-phosphate isomerase MAE_36870 12.03 2 2 3 5.44/14479 0.59 B0JR93 Transketolase, central region MAE_41400 52.19 28 28 122 5.64/78581 0.64 B0JQG3 N-acetylmuramic acid 6-phosphate etherase MurQ 23.20 2 6 10 5.84/32607 0.59 B0JUS7 Putative N-acetylmannosamine-6-phosphate 2-epimerase NanE 20.26 1 3 7 5.08/24129 0.64 B0JVE7 Glucose-6-phosphate isomerase Pgi 58.40 25 25 100 5.81/58113 1.57 B0JKD7 Ribulose-phosphate 3-epimerase Rpe 31.91 7 7 37 5.44/25314 0.58 B0JM55 Ribose-5-phosphate isomerase A RpiA 75.85 1 11 115 4.81/24870 1.55 B0JSQ7 Triosephosphate isomerase Tpi 62.17 2 10 40 5.11/25339 0.56 B0JT83 Glucose-6-phosphate 1-dehydrogenase Zwf 45.89 20 20 57 7.7/58013 0.59 Biological process: Catabolic process B0JFI6 Alpha/beta fold family hydrolase MAE_01260 5.74 1 1 5 5.80/32832 1.71 B0JXF9 Muconate cycloisomerase MAE_19640 13.43 4 4 6 5.55/38850 1.62 B0JQV7 Putative hydrolase MAE_40040 7.99 2 2 2 5.86/33037 1.55 B0JU62 Phycocyanin alpha phycocyanobilin lyase related protein NblB 43.89 1 7 39 4.36/24007 1.63

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Biological process: Cellular component organization B0JPL0 Gas vesicle structural protein GvpC 48.04 1 12 89 9.37/25092 1.53 B0JPL8 Gas vesicle protein GvpW 20.37 3 3 7 5.10/26159 0.58 Biological process: Gene expression B0JQ56 Putative pre-16S rRNA nuclease MAE_06930 21.54 2 2 2 9.33/14732 1.59 B0JNQ5 50S ribosomal protein L1 RplA 52.29 1 13 90 9.32/25823 1.66 B0JTA6 50S ribosomal protein L10 RplJ 77.47 3 12 56 8.92/19645 0.64 B0JSJ8 50S ribosomal protein L20 RplT 43.48 5 5 23 12.24/13346 0.64 B0JVN3 50S ribosomal protein L33 RpmG 17.19 1 1 5 10.11/7401 1.66 B0JSN9 DNA-directed RNA polymerase subunit gamma RpoC1 39.46 1 21 61 8.65/71097 0.64 B0JYC2 30S ribosomal protein S14 RpsN 30.75 3 3 13 10.61/11967 0.53 B0JGK4 30S ribosomal protein S20 RpsT 30.93 1 1 2 10.75/10550 1.57 B0JJJ4 tRNA N6-adenosine threonylcarbamoyltransferase TsaD 9.59 2 2 4 6.04/36967 1.59 Biological process: Methylation B0JP12 Type I restriction-modification system DNA methylase MAE_05010 2.68 1 1 2 4.94/40932 0.62 B0JRP0 Putative methyltransferase MAE_09680 52.99 6 10 22 5.32/29282 0.59 B0JLC9 Putative type IIS restriction/modification enzyme MAE_63060 2.67 1 1 2 6.53/138827 0.59 Biological process: Nitrogen compound metabolic process B0JY82 Ribonuclease H like MAE_52980 17.31 5 5 7 6.98/31128 0.50 B0JGC3 Cysteine desulfurase NifS 5.95 3 3 3 5.54/46195 1.54 B0JH03 Anthranilate synthase component II TrpG 4.10 1 1 4 5.25/21662 0.58 Biological process: Not defined B0JVL9 Carbon dioxide concentrating mechanism protein CcmN 12.27 2 2 8 4.55/22858 0.61 Biological process: Oxidation-reduction process B0JIQ8 Hydrogenase subunit of the bidirectional hydrogenase HoxY 21.43 3 3 5 4.54/20025 0.57 B0JV94 Aldo/keto reductase MAE_16060 41.64 3 8 21 8.86/34812 0.58 B0JG74 Cytochrome P450 MAE_22570 7.38 3 3 4 8.25/52298 1.57 B0JTQ0 Ferredoxin MAE_45310 52.53 1 3 13 8.87/10730 1.52 B0JPG4 NAD(P)H-quinone oxidoreductase chain 4 1 NdhD1 1.71 1 1 2 7.72/57976 0.61 B0JV02 NADH dehydrogenase subunit 4 NdhD3 5.54 2 2 2 9.44/56048 1.62 B0JWI3 Ferredoxin--nitrite reductase NirA 54.97 5 24 86 5.46/57077 1.53

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B0JNP5 Ferredoxin I PetF1 16.00 2 2 12 3.88/10606 1.85 Biological process: Phosphorus metabolic process B0JJM3 Carbamoyl-phosphate synthase small chain CarA 32.72 7 7 31 6.01/41653 0.59 B0JUS6 Inorganic pyrophosphatase Ppa 52.07 2 13 75 4.77/18846 1.58 B0JH30 Ribose-phosphate pyrophosphokinase Prs 28.79 7 7 33 7.08/35358 1.51 B0JQJ1 Dihydroorotase PyrC 9.59 3 3 6 5.96/38398 1.58 B0JHQ6 Orotate phosphoribosyltransferase PyrE 36.92 2 5 8 5.17/20660 1.63 B0JJL8 Orotate phosphoribosyltransferase PyrE 27.88 7 7 17 5.26/53055 1.82 Biological process: Photosynthesis B0JFY1 Phycobilisome core component ApcF 88.17 2 14 138 5.22/18632 1.51 B0JVL8 Ribulose bisphosphate carboxylase large chain CbbL 59.64 2 15 239 6.04/52544 1.56 B0JHB6 Phycocyanin alpha subunit CpcA1 73.12 2 8 350 5.82/17516 0.53 B0JHB5 Phycocyanin beta subunit CpcB1 93.33 2 11 898 5.12/18164 1.71 B0JXA3 Phycocyanin beta subunit CpcB2 93.02 1 10 859 5.14/18059 1.71 B0JHK5 NAD(P)H-quinone oxidoreductase subunit 2 NdhB 1.95 1 1 4 6.74/54738 0.64 B0JXB7 Cytochrome b6-f complex iron-sulfur subunit PetC 70.95 1 8 56 5.23/19065 1.58 B0JHN8 Cytochrome c6 PetJ 43.18 3 3 13 7.76/9132 0.58 B0JT88 Photosystem I reaction center subunit XI PsaL 45.63 4 4 48 5.53/16723 1.64 Biological process: Protein folding B0JQV2 Chaperone protein HtpG HtpG 30.00 1 20 76 5.01/75081 0.61 Biological process: Protein metabolic process B0JRV1 Zinc metalloprotease MAE_10290 23.86 1 7 20 5.28/40045 0.59 B0JI96 Deoxyribodipyrimidine photolyase-related protein MAE_25720 7.75 3 3 5 6.41/59238 1.52 B0JXY8 HesB/YadR/YfhF family protein MAE_52040 8.47 1 1 3 6.04/12860 0.63 B0JYD9 Serine/threonine-protein kinase B MAE_53550 3.03 2 2 2 9.37/58279 1.62 B0JHT0 TPR repeat protein MAE_56700 5.26 3 4 5 5.34/102374 1.61 B0JGJ4 Peptidyl-prolyl cis-trans isomerase YtfC 48.26 2 6 43 5.16/17842 1.60 Biological process: Regulation of gene expression B0JUZ9 Transcriptional regulator MAE_15110 29.94 1 8 16 6.72/35111 1.54 Biological process: Response to stimulus B0JH22 Two-component response regulator OmpR subfamily MAE_23520 14.03 2 2 5 5.99/24556 1.51

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B0JVX6 Universal stress protein MAE_48970 33.33 1 5 14 5.48/18192 1.61 Biological process: Transport B0JU64 ABC-transporter ATP-binding protein MAE_14300 10.09 2 2 3 5.67/35554 1.53 B0JXP2 ABC_transp_aux domain-containing protein MAE_20470 19.69 4 8 12 9.12/56329 0.64 B0JJH9 Porin type major outer membrane protein MAE_27990 21.99 2 13 232 5.40/60274 1.76 B0JHS8 Iron transport system substrate-binding protein MAE_56680 46.15 2 14 66 9.67/38743 0.56 B0JRJ7 Phosphate import ATP-binding protein PstB PstB 35.38 7 7 16 8.52/29880 0.59 B0JWH6 Phosphate import ATP-binding protein PstB PstB2 22.44 4 4 10 8.22/29390 0.57 B0JWI0 Phosphate-binding protein PstS 31.64 3 8 38 5.28/39891 0.58 B0JP76 Protein-export membrane protein SecF SecF 5.99 2 2 2 5.50/34253 0.64 B0JWI1 Phosphate transport system substrate-binding protein SphX 14.53 2 4 7 5.33/38267 1.59 a: Accession number; b: Peptide spectrum match

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Fig. S1 A Venn diagram showing the overlap of the differentially expressed proteins in M. aeruginosa exposed to high nitrogen level and high phosphorus level. The numbers in the overlapping regions represent proteins common to the two treatments (AtpH, CpcB1, CpcB2,

GvpC, GvpW, HoxY, MAE_02680, MAE_14300, MAE_14970, MAE_19640, MAE_20470,

MAE_25030, MAE_25720, MAE_27990, MAE_34870, MAE_52040, MAE_63060, MtnA,

NadK, NblB, PanB, PetC, PetF1, PstB, PstB2, PstS, PyrC, RpmG, RpsT, TrpG, UbiX), and the numbers in the non-overlapping regions represent proteins unique to the indicated treatment. The diagram was plotted using VENNY (http://bioinfogp.cnb.csic.es/tools/venny/index.html).

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Fig. S2 (a) The interaction network of the differentially expressed proteins in high nitrogen group and (b) the significantly enriched (p < 0.05) PPI module in this network, constructed according to the STRING database. The protein symbols are shown in Table S1 in the Supplementary material. The connection degree of each protein is indicated by color.

Supplement to Jiang & Liu (2020) – Aquat Microb Ecol 85:47-58 – https://doi.org/10.3354/ame01939

Fig. S3 (a) The interaction network of the differentially expressed proteins in high phosphorus group and (b) the significantly enriched (p < 0.05) PPI module in this network, constructed according to the STRING database. The protein symbols are shown in Table S2 in the Supplementary material. The connection degree of each protein is indicated by color.