f(x) = x R² = RPKM (M8.MXB) f(x) = x E-014 R² = 1 RPKM (M31.
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1 14 12 f(x) = x R² = RPKM (M8.MXA) RPKM (M8.MXB) f(x) = x E-14 R² = 1 RPKM (M31.XA) RPKM (M31.XB) Figure S1. Reproducibility between the biological replicates (MXA and MXB) for mixed cultures. Correlations for RPKM values were plotted once assigned to M8 (upper panel), with a R =.9956, or M31 (lower panel) with R =1.
2 Monitorización: Monitoring: ODDO 6 6,9.9 OD6,8.8,7.7,6.6,5.5,4.4 M8PureOD M31PureOD MXOD,3.3,2.2, Time (h) Monitorización: recuentos por DAPI y qpcr MM8 Pure (cells/ml) MM31 Pure (cells/ml) MMX (cells/ml) 9, ,5 8.5 MM8 Pure qpcr MM31Pure qpcr MM8X (qpcr) MM31X (qpcr) log cells/ml 7, , , , Time (h) Figure S2. Growth curves for pure cultures of M8 (blue) and M31 (red). In the upper figure lines represent OD reads for pure and mixed cultures, as indicated in the insert. The bottom figure show log cells/ml values for M8 and M31 cultures measured by quantitative PCR (black and red dots for mixed and triangles for pure cultures) and DAPI (continuous lines). Circled the point selected for sequencing.) and M31
3 Cumulative frequency curve. S. ruber M8, pure cultures Expression level (RPKM) Cumulative percentage (%) <2 RPKM 2-1 RPKM >1 RPKM (VHEG) Cumulative frequency curve. S. ruber M31, pure cultures Expression level (RPKM) Cumulative percentage (%) <2 RPKM 2-1 RPKM >1 RPKM (VHEG) Figure S3. RPKM transcription levels for M8 (upper panel) and M31 (lower panel) genes in pure cultures. Cumulative curves represents the percentage of genes with transcription levels between -2 RPKM (blue), 2-1 RPKM (orange) and above 1 RPKM (yellow).
4 S. ruber M31 replicons. Normalized expression frequency histogram Normalized frequency level S.ruber M31 replicons. Expression frequency histogram normalized Expression level RPKM (1bin=2 RPKM) S.ruber M31. plasmid psru S.ruber M31. plasmid psru S.ruber M31. Chromosome S. ruber M8 replicons. Normalized expression frequency histogram Normalized frequency level S.ruber M8 replicons. Expression frecuency histogram normalized S.ruber M8 plasmid psr11 S.ruber M8 plasmid psr11 psr56 plasmid psr56 S.ruber S.ruber M8 M8 plasmid plasmid psr61 psr61 psr84 S.ruber M8 plasmid psr84 S ruber M8 Chromosome S ruber M8 Chromosome Expression level RPKM (1bin=2 RPKM) Figure S4. Normalized gene distribution for M31 (upper figure) and M8 (bottom figure) based on their expression level (RPKM). All the replicons were represented in both cases.
5 M8 Pure culture. Relation between %GC and expression values A Expression level (RPKM) %GC Normalized frequency level %GC M31 Pure culture. Relation between %GC and expression values Expression level (RPKM) B %GC Normalized frequency level %GC Figure S5. Normalized gene distribution for M8 (A) and M31 (B) based on their % GC and RPKM expression levels. For each strain, genes with expression values below 3 RPKM are shown in bottom panels.
6 (<-.25) (-.25;-.5) (-.5;.5) (.5,.25) (>.25)5) Figure S6. M31 citrate cycle (TCA cycle) KEGG map (sru:2) showing the differential expression changes (log 2 fold change) of S.ruber M31 mean a percentage color gradient. The green boxes represent genes that increase their expression and the reds that decrease it. In red letters the genes that change its expression significantly (SRU_828 = ; (SRU_1969 = ). The circles show the increases (green) or decreases (red) of intermediate metabolites based on the genes expression levels that encode the enzymes that produce them.
7 Table S1. M8 (Gene ID SRM_) and M31 (Gene ID SRU_) genes with transcription below the detection threshold in pure culture. In blue, strain specific genes. In green, genes untranscribed both in M8 and M31.I: Island; CR: conserved region; HP: Hypothetical protein; CHP: Conserved hypothetical protein. GeneID Location Function GeneID Location Function SRM_4 HP SRM_87 I2 HP SRM_141 HP SRM_88 I2 HP SRM_142 CHP SRM_1218 HP SRM_143 HP SRM_145 HP SRM_152 HP SRM_1571 CHP SRM_22 HP SRM_1654 HP SRM_24 HP SRM_1683 HP SRM_239 I1 HP SRM_176 CHP SRM_318 I1 HP SRM_1835 HP SRM_328 I1 HP SRM_1949 HP SRM_332 I1 HP SRM_211 HP SRM_376 HP SRM_2231 HP SRM_41 HP SRM_232 CR HP SRM_479 HP SRM_2491 CR SRM_536 HP SRM_2677 CHP SRM_67 HP SRM_2688 HP SRM_686 I2 transposase SRM_2978 HP SRM_756 I2 HP PSR_5634 PSR56 HP SRM_765 I2 HP PSR_6115 PSR61 HP SRM_775 I2 HP PSR_6126 PSR61 HP SRM_784 I2 HP PSR_6131 PSR61 HP SRM_799 I2 transposase PSR_847 PSR84 HP SRM_875 HP PSR_8447 PSR84 HP SRM_116 HP PSR_8458 PSR84 HP SRM_147 CHP PSR_8461 PSR84 HP SRM_174 HP PSR_8463 PSR84 HP SRM_113 HP PSR_8467 PSR84 HP SRM_1182 HP SRU_13 HP SRU_863 I3 CHP SRU_125 CHP SRU_114 I3 HP SRU_181 I1 HP SRU_117 I3 HP SRU_186 I1 HP SRU_1376 CHP SRU_187 I1 Transcription regulator SRU_146 SRU_212 I1 CHP SRU_1562 CHP SRU_214 I1 CHP SRU_1834 HP SRU_215 I1 CHP SRU_221 CR HP SRU_217 I1 HP SRU_2339 HP SRU_25 I1 HP SRU_2394 HP sensor kinase protein rcsc
8 SRU_263 I1 HP SRU_2457 CHP SRU_284 HP SRU_2465 HP SRU_578 HP SRU_2634 HP SRU_593 I2 HP SRU_2862 SRU_625 I2 HP SRU_p8 tyrosine recombinase XerD
9 Table S2. Genes with expression above 1 RPKM (VHEG: Very highly expressed genes ). Pink: M8 VHEG. Blue: M31 VHEG. Gene ID RPKM Annotation Location a Ortholog. SRM_ hypothetical protein M8 specific SRM_ S ribosomal protein L13 SRU_38 SRM_ conserved hypothetical protein SRU_81 SRM_ hypothetical protein I1 M8 specific SRM_ hypothetical protein M8 specific SRM_ cytochrome c oxidase subunit II SRU_313 SRM_ hypothetical protein SRU_375 SRM_ AsnC family transcriptional regulator SRU_384 SRM_ glutamine synthetase SRU_385 SRM_ hypothetical protein SRU_435 SRM_ TonB protein SRU_51 SRM_ Chain length determinant protein SRU_594 SRM_ Phage integrase family protein I2 SRU_595 SRM_ UDP-glucose 6-dehydrogenase I2 M8 specific SRM_ hypothetical protein I2 M8 specific SRM_7 162 acyl-[acyl-carrier-protein]--udp-n- I2 SRU_66 SRM_ succinyl-coa synthetase, alpha subunit SRU_67 SRM_ PKD repeat protein SRU_96 SRM_ ATP synthase subunit C SRU_912 SRM_ cold-shock DNA-binding protein SRU_966 SRM_ hypothetical protein M8 specific SRM_ S ribosomal protein S12 SRU_13 SRM_ S ribosomal protein S17 SRU_144 SRM_ S ribosomal protein L17 SRU_161 SRM_ hypothetical protein M8 specific SRM_ adenosylhomocysteinase SRU_1139 SRM_ S ribosomal protein L31 SRU_1162 SRM_ S ribosomal protein S21 M8 specific SRM_ ATP-dependent Clp protease, ATPase subunit SRU_1317 SRM_ iron-sulfur cluster assembly accessory protein SRU_1318 SRM_ S ribosomal protein L27 SRU_1419 SRM_ S ribosomal protein L21 SRU_142 SRM_ Aconitate hydratase SRU_1477 SRM_ Xanthorhodopsin SRU_15 SRM_ hypothetical protein SRU_1536 SRM_ Azurin SRU_159 SRM_ hypothetical protein M8 specific SRM_ MotA/TolQ/ExbB proton channel SRU_1681 SRM_ superoxide dismutase SRU_1724 SRM_ cold-shock protein SRU_1727 SRM_ hypothetical protein SRU_1731 SRM_ DNA-directed RNA polymerase subunit beta' SRU_1757 SRM_ S ribosomal protein L7/L12 SRU_1759 SRM_ S ribosomal protein S15 SRU_1774 SRM_ conserved hypothetical protein SRU_1783 SRM_ S ribosomal protein S1 SRU_1787 SRM_ hypothetical protein SRU_1919 SRM_ kda chaperonin CR SRU_2121
10 SRM_ kda chaperonin CR SRU_2122 SRM_ TonB-dependent receptor CR SRU_2178 SRM_ hypothetical protein CR M8 specific SRM_ outer membrane protein CR SRU_2181 SRM_ TonB-dependent receptor CR SRU_2182 SRM_ peptidyl-prolyl cis-trans isomerase CR SRU_228 SRM_ conserved hypothetical protein SRU_2378 SRM_ ATP synthase beta chain SRU_2429 SRM_ ATP synthase subunit epsilon SRU_243 SRM_ cold-shock protein SRU_2449 SRM_ acyl carrier protein SRU_2754 SRM_ S ribosomal protein L35 SRU_284 SRM_ S ribosomal protein L2 SRU_285 SRM_ hypothetical protein M8 specific SRM_ conserved hypothetical protein SRU_287 PSR_ hypothetical protein containing Lambda repressor-like, DNA-binding domain PSR11 M8 specific M8ncRNA SAM (RF162)SAM riboswitch (S box leader) pb pb M31ncRNA1 M8ncRNA TmRNA (RF23) transfer-messenger RNA pb pb M31ncRNA2 M8ncRNA TPP (RF59) TPP riboswitch (THI element) pb pb M31ncRNA3 SRU_ ribosomal protein L28 SRM_22 SRU_ conserved hypothetical protein SRM_88 SRU_ chaperonin GroEL I1 SRM_297 SRU_ cytochrome c oxidase subunit II SRM_393 SRU_ hypothetical protein SRM_452 SRU_ putative AsnC-family transcriptional regulator SRM_46 SRU_ glutamine synthetase, type I SRM_461 SRU_ NADH-quinone oxidoreductase chain b SRM_47 SRU_ conserved hypothetical protein SRM_512 SRU_ TonB SRM_59 SRU_ Chain length determinant protein I2 SRM_689 SRU_ Phage integrase family protein I2 SRM_69 SRU_ UDP-glucose dehydrogenase I2 M31 specific SRU_ ABC transporter, multidrug efflux family I2 M31 specific SRU_ NDP-sugar dehydrogenase, putative I2 SRM_718 SRU_6 195 nucleoside-diphosphate-sugar epimerase I2 M31 specific SRU_ perosamine synthetase, putative I2 SRM_695 SRU_ succinyl-coa synthetase, alpha subunit SRM_846 SRU_ bioy family protein SRM_97 SRU_ hypothetical protein SRM_151 SRU_ conserved protein SRM_198 SRU_ ATP synthase F, C subunit SRM_115 SRU_ 'Cold-shock' DNA-binding domain, putative SRM_1159 SRU_ ribosomal protein L3 SRM_1236 SRU_ S ribosomal protein S17 SRM_1245 SRU_ ribosomal protein L24 SRM_1247 SRU_ ribosomal protein S13p/S18e SRM_1258 SRU_ ribosomal protein L17 SRM_1262 SRU_ adenosylhomocysteinase SRM_1319 SRU_ ribosomal protein L31 SRM_1343 SRU_ osmotically inducible protein C SRM_1497
11 SRU_ ATP-dependent Clp protease, ATPase subunit SRM_151 SRU_ iron-sulfur cluster assembly accessory protein, SRM_1511 SRU_ ribosomal protein L9 SRM_1562 SRU_ ribosomal protein L27 SRM_1611 SRU_ aconitate hydratase, putative SRM_167 SRU_ xanthorhodopsin SRM_1698 SRU_ putative cold shock-like protein cspg M31 specific SRU_ auracyanin A precursor SRM_1789 SRU_ MotA/TolQ/ExbB proton channel family protein SRM_1885 SRU_ Mn-superoxide dismutase SRM_1935 SRU_ conserved domain protein SRM_1939 SRU_ histone H1-like protein SRM_1942 SRU_ DNA-directed RNA polymerase, beta' subunit SRM_197 SRU_ ribosomal protein L7/L12 SRM_1972 SRU_ ribosomal protein S15 SRM_1986 SRU_ conserved hypothetical protein SRM_1995 SRU_ ribosomal protein S1, putative SRM_1999 SRU_ putative regulatory protein, FmdB family SRM_218 SRU_ conserved hypothetical protein SRM_213 SRU_ chaperonin, 1 kda CR SRM_2344 SRU_ chaperonin GroEL CR SRM_2345 SRU_ fasciclin domain protein CR SRM_2399 SRU_ putative outer membrane protein, probably CR SRM_241 SRU_ putative outer membrane protein probably CR SRM_245 SRU_ putative outer membrane protein probably CR SRM_246 SRU_ peptidyl-prolyl cis-trans isomerase, FKBP-type CR SRM_2434 SRU_ tetratricopeptide repeat domain protein SRM_2581 SRU_ conserved hypothetical protein SRM_2598 SRU_ conserved domain protein SRM_2671 SRU_ acyl carrier protein SRM_2965 SRU_ oxidoreductase, putative SRM_2995 SRU_ hypothetical HIT-like protein slr1234 SRM_39 SRU_ ribosomal protein L2 SRM_321 SRU_ conserved hypothetical protein SRM_386 M31ncRNA SAM (RF162)SAM riboswitch (S box leader) pb pb M8ncRNA1 M31ncRNA TmRNA (RF23) transfer-messenger RNA pb pb M8ncRNA2 M31ncRNA3 595 TPP (RF59) TPP riboswitch (THI element) pb pb M8ncRNA3 a I: Island; CR: Conserved region
12 Table S3. Expression level differences between paralogous genes. 25 gene duplication events, 182 pre-m8/m31 divergence (1) and 23 post-m8/m31 divergence and detected in M8 strain (), and genes involved are shown in the table. An average of Log2 fold change value was obtained in order to estimate expression level differences, 1.31±1.26 and 2.5±2.13 for duplication events accounted after (; orange) and before (1; yellow) M8 and M31 strains divergence respectively. 1= Paralogs M8+M31 Gene ID Absolute value = Paralogs M8 Strain specific (log2foldchange) 1 SRM_ SRM_261; SRM_ SRM_ SRM_2262; SRM_ SRM_213; SRM_ SRM_27; SRM_ SRM_ SRM_ SRM_ SRM_ SRM_221;SRM_ SRM_789; SRM_ SRM_ SRM_232; SRM_ SRM_1359; SRM_ SRM_231; SRM_ SRM_129; SRM_ SRM_ SRM_1838; SRM_ SRM_1954; SRM_ SRM_685; SRM_ SRM_49; SRM_ SRM_135; SRM_ SRM_468; SRM_ SRM_88; SRM_ SRM_627; SRM_ SRM_167; SRM_961; SRM_ SRM_31; SRM_ SRM_ SRM_ SRM_322; SRM_1443; SRM_ SRM_73; SRM_173; SRM_ SRM_173; SRM_73; SRM_ SRM_ SRM_972; SRM_ SRM_ SRM_12; SRM_ SRM_546; SRM_ SRM_1932; SRM_ SRM_349; SRM_1219; SRM_
13 1 SRM_235; SRM_ SRM_8; SRM_113; SRM_ SRM_322; SRM_1284; SRM_ SRM_35; SRM_ SRM_1666; SRM_ SRM_1159; SRM_ SRM_221; SRM_1378; SRM_147; SRM_ SRM_221; SRM_686; SRM_1378; SRM_147; SRM_ SRM_118; SRM_ SRM_286; SRM_ SRM_145; SRM_ SRM_145; SRM_ SRM_136; SRM_ SRM_2; SRM_ SRM_ SRM_ SRM_ SRM_1585; SRM_ SRM_72; SRM_ SRM_197; SRM_ SRM_783; SRM_736; SRM_ SRM_23; SRM_243; SRM_ SRM_1276; SRM_ SRM_1276; SRM_ SRM_ SRM_383; SRM_ SRM_37; SRM_ SRM_181; SRM_ SRM_1264; SRM_ SRM_736; SRM_747; SRM_ SRM_ SRM_134; SRM_ SRM_1228; SRM_ SRM_891; SRM_ SRM_293; SRM_ SRM_ SRM_156; SRM_221; SRM_226; SRM_ SRM_ SRM_ SRM_164; SRM_ SRM_224; SRM_2476; SRM_864; SRM_ SRM_432; SRM_ SRM_216; SRM_ SRM_751; SRM_ SRM_ SRM_128; SRM_ SRM_159; SRM_ SRM_ SRM_434; SRM_ SRM_ SRM_185; SRM_ SRM_ SRM_478; SRM_
14 1 SRM_885; SRM_ SRM_ SRM_296; SRM_627; SRM_832; SRM_ SRM_296; SRM_ SRM_216; SRM_685; SRM_1377; SRM_148; SRM_ SRM_222; SRM_ SRM_217; SRM_794; SRM_787; SRM_ SRM_229; SRM_ SRM_926; SRM_ SRM_291; SRM_ SRM_292; SRM_ SRM_2145; SRM_ SRM_ SRM_ SRM_ SRM_2; SRM_ SRM_1338; SRM_ SRM_ SRM_ SRM_1667; SRM_ SRM_798; SRM_ SRM_435; SRM_ SRM_ SRM_ SRM_351; SRM_ SRM_351; SRM_ SRM_792; SRM_937; SRM_2135; SRM_285; SRM_44; SRM_ SRM_793; SRM_ SRM_475; SRM_ SRM_ SRM_ SRM_247; SRM_ SRM_31; SRM_929; SRM_ SRM_ SRM_94; SRM_15; SRM_ SRM_ SRM_75; SRM_ SRM_ SRM_2171; SRM_2555; SRM_ SRM_ SRM_ SRM_251; SRM_ SRM_241; SRM_ SRM_745; SRM_ SRM_383; SRM_ SRM_819; SRM_ SRM_223; SRM_236; SRM_ SRM_ SRM_ SRM_1464; SRM_ SRM_1464; SRM_ SRM_547; SRM_ SRM_356; SRM_
15 1 SRM_ SRM_435; SRM_ SRM_228; SRM_ SRM_93; SRM_157; SRM_2263; SRM_ SRM_549; SRM_115; SRM_ SRM_114; SRM_181; SRM_191; SRM_ SRM_691; SRM_ SRM_167; SRM_961; SRM_1947; SRM_853; SRM_98; SRM_157; SRM_158; SRM_1948; SRM_ SRM_ SRM_715; SRM_82; SRM_ SRM_561; SRM_ SRM_317; SRM_ SRM_316; SRM_129; SRM_2127; SRM_ SRM_34; SRM_ SRM_69; SRM_735; SRM_833; SRM_69; SRM_735; SRM_ SRM_186; SRM_ SRM_69; SRM_735; SRM_ SRM_11; SRM_412; SRM_433; SRM_549; SRM_115; SRM_117; SRM_ SRM_ SRM_338; SRM_ SRM_ SRM_ SRM_ SRM_161; SRM_ SRM_161; SRM_ SRM_315; SRM_ SRM_5; SRM_285; SRM_198; SRM_ SRM_88; SRM_ SRM_ SRM_ SRM_381 1 SRM_489 1 SRM_53; SRM_51; SRM_511 1 SRM_221; SRM_686; SRM_1378; SRM_147 1 SRM_765 1 SRM_78 1 SRM_781 1 SRM_86 1 SRM_89 1 SRM_115 1 SRM_ SRM_ SRM_156 1 SRM_ SRM_ SRM_ SRM_292 1 SRM_2141 1
16 1 1 SRM_ SRM_ SRM_271 1 SRM_2916 SRM_686; SRM_147 SRM_784; SRM_735
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