Photosynthesis Research 75: 211–221, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
211
Regular paper
Cyanobacterial signature genes Kirt A. Martin1 , Janet L. Siefert2 , Sailaja Yerrapragada1 , Yue Lu1 , Thomas Z. McNeill1,3 , Pedro A. Moreno1 , George M. Weinstock3 , William R. Widger1 & George E. Fox1,∗ 1 Department of Biology and Biochemistry, University
of Houston, Houston, TX 77204-5001, USA; 2 Department of Statistics, Rice University, Houston, TX 77251-1892, USA; 3 Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA; ∗ Author for correspondence (e-mail:
[email protected]; fax: +1-713-743-8351)
Received 28 June 2002; accepted in revised form 7 November 2002
Key words: comparative genomics, cyanobacteria, signature genes
Abstract A comparison of 8 cyanobacterial genomes reveals that there are 181 shared genes that do not have obvious orthologs in other bacteria. These signature genes define aspects of the genotype that are uniquely cyanobacterial. Approximately 25% of these genes have been associated with some function. These signature genes may or may not be involved in photosynthesis but likely they will be in many cases. In addition, several examples of widely conserved gene order involving two or more signature genes were observed. This suggests there may be regulatory processes that have been preserved throughout the long history of the cyanobacterial phenotype. The results presented here will be especially useful because they identify which of the many genes of unassigned function are likely to be of the greatest interest.
Introduction Although claims for the earliest fossilized cyanobacteria at 3.5 Ga (Schopf and Packer 1987) have been seriously questioned (Brasier et al. 2002) there is strong agreement that membrane biomarkers in wellpreserved sediments reveal the presence of cyanobacteria at 2.7 Ga (Brocks et al. 1999). It is therefore of interest to studies of the early Earth to understand what shared properties these early cyanobacteria likely had. The obvious example is the ability to carry out oxygenic photosynthesis, which is widely regarded as the cause the rise of oxygen in the Archaean atmosphere at 2.2 Ga(Knoll 1999; Catling et al. 2001). There may, however, be other shared properties of the cyanobacteria that would have had more subtle impact on the early Earth and which may persist even to this day. The ability to sequence whole genomes makes it possible to examine the distribution of genes in a very detailed way. The completion of the Synechocystis 6803 genome (Kaneko et al. 2001) made the full complement of genes carried by a cyanobacterium
available for the first time. In order to determine which of these genes, if any, might contribute to a unique shared cyanobacterial genotype we compared this initial cyanobacterial genome to seven other genomes that are now available. Based on rRNA comparisons, these eight genomes represent five major lineages at the crown of the cyanobacterial tree (Turner et al. 1999) (Figure 1). Their common ancestor would predate the rise of the heterocyst, which has been dated at 2.1 Ga by geologic evidence (Amard and BertrandSarfati 1997). An inter-comparison of the genomic content of these eight genomes, defines a working set of signature genes, Table 1. This signature set contains 181 genes that were initially among the almost 1000 putative open reading frames found in the Synechocystis 6803 genome (Kaneko et al. 2001).
Materials and methods The gene content of eight completely sequenced cyanobacterial genomes was examined. The genomes
212 Table 1. The 181 cyanobacterial signature gene set derived from the set of core genes. Column headings indicate the cyanobacterial species used in this study and the numbers in each column refer to the gene name of the likely ortholog detected by the analysis reported here. The gene designators are those used by the individual genome projects as of October of 2002. In some cases, e.g. Nostoc the name consists of a contig followed by the gene number on that contig. In some cases the designator, e.g. sll0558 indicates a relative direction of transcription too. The signature genes are tabulated in the physical order in which they occur in Synechocystis sp. PCC 6803. This allows the identification of putative operons containing two or more signature genes by simply scanning the Table. Signature genes included in these putative operons are tabulated in bold. Synechosystis sp. PCC6803
Anabaena sp. PCC7120
N. punctiforme
P. marinus MED4
P. marinus MIT9313
Synechococcus sp. WH8102
Trichodesmium erythraeum
T. elongatus
sll0558 sll1399 sll1398 slr1495 slr1122 slr0728 slr0730 slr0731 slr0732 slr0734 slr0737 sll0226 slr0250 ssr0390 sll1321 slr1780 ssr2998 slr1796 slr1800 sll1656 sll1654 sll1194 slr1306 sll0933 ssl1972 ssr1789 slr1596 slr1599 slr1600 sll1507 sll1752 slr2032 slr2034 ssr3451 smr0006 slr2049 sll1934 slr1908 slr1915 slr1918 ssr1698 slr1034 slr1384 slr1699 ssr2843
all1826 alr0301 all0801 alr4075 alr1700 alr5367 alr4016 alr4017 alr0613 all4252 all0329 all4289 alr4067 asr4775 all0011 all0216 asl4482 all2716 alr5279 all3977/all4113 alr4132 alr1216 alr4172 all0748 asl4547 asl2354 all1673 alr3954 alr3603 alr4178 all4871 all3013 alr3844 asr3845 asr3846 alr0617 alr1356 alr2231 alr2980 all3259 asl4369 all4779 all4892 alr1085 asl5079
458.29 459.44 452.29 507.14 474.20 492.30 412.47 412.48 434.12 507.39 485.8 504.113 477.40 468.78 502.192 504.12 466.40 466.40 468.70 357.8 501.187 441.3 501.122 472.34 – 507.12 454.43 429.13 344.2 465.53 623 439.16 493.50 493.51 493.52 623 507.250 – 423.49 505.36 498.7 501.53 – 464.53 431.22
765 1200 1406 341 1139 1594 1670 1671 – 1877 331 600 – 1390 412 646 1078 858 715 542 160 1167 1196 1418 1961 398 1631 1975 2126 1885 1530 281 2047 2048 2049 88 318 910 514 1241 1467 1036 1958 977 925
2886 1210 1052 1921 384 1515 2786 3564 2774 2347 3845 474 1079 980 220 – 1305 2830 2525 2695 2219 1395 1198 2919 2117 194 2988 2131 2553 2361 3330 2413 2045 2046 2047 475 – 2622 289 914 3805 3462 2103 2718 777
82 28 3501 2774 2939 2420 2040 2043 446 1095 2764 1588 121 241 1456 1752 570 1656 821 3096 2543 879 3180 3527 908 1545 3388 927 1582 1110 1814 1160 2296 2297 2298 2221 1224 2612 1511 155 2716 629 897 3536 1720
55.6548 12.1088 10.339 32.4372 10.349 70.7692 100.511 100.512 94.8798 46.5725 16.1883 14.1557 13.1355 1.151 69.7448 51.6329 20.2700 – 10.455 50.6270 2.2628 8.2226 40.5311 17.2115 – 50.6231 2.2572 38.4925 29.3820 1.53 7.759 15.1731 8.8173 8.8174 8.8175 1.138 57.6669 6.6850 3.4146 2.2455 – 22.3033 13.1241 4.5055 11.782
tlr1594 tlr0325 tlr0493 tlr1240 tll2191 tll2101 tll1109 tll1108 tlr2434 tll2053 tll1724 tll1388 tll1572 tsr2273 tlr0429 tll1499 tsr0524 tlr1421 tll0399 tll0396 tlr2269 tll2409 tll1435 tlr1208 tsr0804 tsr1916 tll0748 tll0146 tll1300 tll1717 tlr0806 tlr1249 tll1695 tsr1541 tsr1542 tll1699 tlr1140 tlr2324 tll2044 tlr2348 tll2113 tlr0289 tll1632 tll2476 tlr1507
213 Table 1. Continued Synechosystis sp. PCC6803
Anabaena sp. PCC7120
N. punctiforme
P. marinus MED4
P. marinus MIT9313
Synechococcus sp. WH8102
Trichodesmium erythraeum
T. elongatus
slr1702 sll1578 sll1577 sll1926 sll1071 ssl3451 sll1797 slr1896 slr1900 slr1195 slr1206 sll1968 ssl3712 sll1737 slr1834 slr1841 slr1535 ssr2595 sll1632 slr1220 sll1271 sll0860 sll1317 ssl2598 smr0009 ssl3379 ssl3364 sll2013 sll2002 slr2144 sll1702 sll0854 sll0851 sll1162 slr1263 slr1990 sll1902 sll1586 ssl0461 sll0247 sll1979 ssl2781 slr1506 sll1418 sll1414 slr0816 slr1342 sll1898
all4574 alr0529 alr0528 all2971 all2545 asl2557 all0938 alr4351 alr4979 alr2014 all2750 alr3655 all7022 all4804 alr5154 all7614 all0967 asl0514 alr3857 alr1129 alr2231 alr2431 all2452 asl0846 all4665 all0404 asl2850 all4333 alr4888 all0476 alr2762 all3378 alr4291 alr1535 alr1370 alr1215 all4902 all0462 asl3112 all4001 all4118 asr2932 all0969 all3076 all0646 alr3417 alr3454 all0949
508.79 313.2 313.1 474.14 498.172 494.69 501.62 463.41 399.18 458.41 506.114 430.13 509.351 493.57 456.7 373.16 463.50 397.9 502.59 479.57 – 489.37 506.87 501.108 501.138 505.20 372.10 423.22 507.62 494.32 492.31 352.13 496.2 405.5 453.53 441.1 362.22 463.65 489.111 363.3 506.216 409.17 445.50 493.23 378.2 507.98 504.121 493.119
779 2053 2053 – 939 1295 1857 1555 592 1242 – 766 1673 1231 374 910 – 462 1657 1923 636 773 1061 118 1133 1255 138 387 1023 154 993 378 1563 222 1632 1946 1009 1192 – – 2130 – 1506 1528 881 596 317 1113
2959 22 21 1141 999 1060 2289 3392 878 915 2023 2887 3568 2351 1878 1722 3235 101 1267 3718 654 2948 3500 1823 374 992 3665 877 3439 2216 2752 1885 3402 2536 3366 1394 2781 1179 1855 2809 926 365 1500 3322 989 3536 1749 343
3357 2746 2745 3531 259 90 1011 1960 1870 156 2266 83 2054 1098 734 2609 373 1880 548 2206 2609 3345 671 2948 2930 254 2669 2855 608 2541 424 742 1973 583 1943 878 460 3173 1303 1973 896 2922 2412 1806 251 703 1221 2904
13.1258 4.5118 4.5134 43.5529 28.3708 1.258 70.7710 3.4123 2.2487 16.1844 56.6664 13.1249 10.338 1.110 12.1150 6.6851 10.402 – 16.1948 23.3115 6.6850 3.4005 29.3867 1.235 62.7131 16.1906 2.2613 14.1574 12.1104 34.4556 9.8694 3.4011 14.1466 13.1245 – 18.2224 3.4058 3.4041 36.4704 57.6686 77.7948 1.74 48.5830 15.1653 26.3452 12.1036 20.2820 87.8443
tlr2221 tlr1958 tlr1957 tlr2451 tlr0249 tsl2428 tll1562 tlr1934 tll0916 tlr0208 tll1929 tlr1461 tlr1849 tlr1073 tlr0731 tll1706 tll1668 tsl2208 tlr0136 tll0853 tlr2324 tll0315 tlr0960 tsl1386 tsr1387 tsr1087 tsr1820 tll1711 tlr1986 tlr2015 tll1092 tll2274 tlr1631 tlr2008 tlr2402 tll0200 tlr0207 tll2375 tsl2468 tlr1050 tsl0866 tsr0968 tll1717 tlr2075 tlr1134 tll0077 tll2430 tll1894
214 Table 1. Continued Synechosystis sp. PCC6803
Anabaena sp. PCC7120
N. punctiforme
P. marinus MED4
P. marinus MIT9313
Synechococcus sp. WH8102
Trichodesmium erythraeum
T. elongatus
slr1978 sll1390 slr1470 sll1382 sll1376 sll1372 slr1273 slr1287 slr1530 slr1160 slr1677 ssr2831 slr0941 slr0954 slr1623 slr1636 slr1638 slr1645 slr1649 sll0427 slr0443 sll0272 slr0280 sll0258 sll0359 ssr0657 slr1926 slr1946 slr1949 ssl0563 sll0295 slr0169 sll0169 sll0157 sll0350 slr0376 ssl1417 slr0013 sll0208 sll0199 slr0438 sll0071 ssr0109 sll0456 slr0630 sll0609 sll0608 slr0418
alr0484 alr4100 alr3414 all2919 alr1909 alr0296 alr2060 alr0786 alr3399 all4869 alr4005 asr4319 all2381 alr1121 all1732 alr4066 all5165 all1258 all5339 all3854 alr3855 alr3297 all4343 all0259 alr0946 asl3656 all4664 all4180 all4162 asr3463 alr3863 all3257 all2707 all5037 alr1278 all1871 asr0062 alr4373 alr5283 all0258 alr3231 all2549 asl0272 all3908 alr3419 asl4507 all4508 alr4674
468.81 486.28 507.95 477.92 476.85 484.94 454.61 478.98 506.25 443.26 504.190 452.35 506.63 477.5 434.47 477.39 498.72 429.9 378.29 502.159 502.58 428.21 423.12 497.55 501.182 430.31 464.2 366.10 501.118 507.20 372.20 464.26 493.84 457.38 492.17 506.229 472.16 498.167 468.71 497.56 502.3 454.38 – 508.91 473.96 455.49 455.48 382.16
2119 2118 1584 1382 1050 1109 1016 422 1978 122 225 68 195 753 1973 1310 982 1033 – 133 816 812 1599 1510 1072 1404 1134 – 823 309 1808 1171 533 1459 2026 402 1952 – 1162 1000 761 1609 1992 999 1834 1389 1388 1248
2545 2987 3534 2301 3489 337 2940 232 2134 1828 104 1384 1731 1007 2129 1078 2730 345 3810 1857 1985 3005 1970 3579 2777 1047 375 1173 3037 1737 1899 449 2677 3551 2014 200 2094 2962 428 2761 1035 1755 2170 2760 294 979 977 2927
1573 3387 701 226 658 289 3335 1467 932 1815 1327 2692 2622 50 925 120 3546 626 2747 1306 2233 3402 2845 2069 686 3498 2931 3165 1694 1209 770 2997 3082 1020 2258 3349 888 3358 2986 433 – 1226 959 432 1520 240 239 3320
15.1804 21.2881 12.1033 5.6163 20.2798 23.3109 69.7432 1.31 26.3534 5.6050 8.8130 56.6628 68.7417 88.8459 1.67 11.756 9.8638 20.2791 21.2953 1.186 1.149 29.3858 – 32.4411 14.1452 13.1378 62.7132 37.4809 39.4981 3.4159 7.7572 2.2608 21.2839 15.1659 22.2992 24.3302 1.137 13.1261 13.1351 7.7632 10.405 – 44.5634 20.2779 1.206 20.2811 20.2810 35.4689
tlr0284 tll0404 tll0601 tlr1656 tll1891 tlr1402 tlr0757 tlr0672 tll1658 tll0135 tll1550 tsl1567 tll0336 tlr0428 tll0447 tll1133 tll2024 tll2464 tlr2156 tll0444 tll1059 tlr0472 tll1850 tll1285 tll1150 tsl0253 tlr0863 tll2292 tll1052 tsl1013 tlr1691 tlr0311 tlr0758 tlr2433 tlr1075 tlr1877 tsr1483 tlr0729 tll1313 – tsr1840 tll0418 tsr1584 tlr0742 tlr0872 tsr2284 tlr1437 tll0771
215 Table 1. Continued Synechosystis sp. PCC6803
Anabaena sp. PCC7120
N. punctiforme
P. marinus MED4
P. marinus MIT9313
Synechococcus sp. WH8102
Trichodesmium erythraeum
T. elongatus
sll0372 ssl0353 ssl0352 slr0204 slr0208 slr0906 sll0544 slr0575 sll0832 sll0827 ssr1425 sll0822 slr0503 ssr1041 sll0584 slr0116 ssl0546 sll0288 slr0304 sll0286 sll0662 sll0661 ssl1263 slr0022 sll0031 slr0042 sll0509 sll1340 slr1459 slr0651 sll0621 slr1557 slr1579 slr1655 slr1660 slr1177 sll1109 slr0589 slr0590 slr0598
all2849 asl0940 asr0654 alr2465 all1363 all0138 alr3444 alr3596 alr4394 alr3827 asr3137 all2080 alr0942 asr2378 alr4170 alr3707 asr3457 alr3455 alr3097 alr0113 alr0045 alr0044 asr0043 alr5129 alr2308 alr2231 alr4466 asl4395 all2327 all4101 alr3122 all1338 all4042 all0107 all4118 alr3362 alr3101 alr3980 alr3874 alr2454
472.78 501.60 356.8 382.21 379.13 494.8 505.39 479.98 493.20 479.30 475.30 388.32 501.179 506.212 353.4 493.86 504.124 504.122 478.108 320.6 506.101 506.100 506.99 435.38 481.68 362.17 472.37 493.128 481.91 486.93 469.12 448.41 479.48 423.1 374.6 507.131 509.283 357.7 432.18 506.182
126 224 311 1989 1546 80 1840 1037 2023 – 184 1007 1926 1565 825 1296 79 77 967 467 403 402 401 807 1807 910 788 120 2053 201 1771 586 – 377 1974 – 862 327 1844 1125
1837 103 1739 2165 1449 3796 1452 3463 2012 3454 1710 3518 3722 3407 – 1062 3784 3782 832 1183 201 200 199 2999 1897 899 2973 1826 22 2512 3615 320 1692 1959 2130 1550 2834 3840 3313 382
1295 1326 1213 951 1243 2708 2366 631 2255 623 2596 2815 2210 589 1696 91 2705 2703 1716 2768 1554 1553 1552 3396 767 2837 3374 1289 85 2631 2117 2876 2579 2830 926 3563 1660 2758 2372 2914
2.2614 66.7305 26.3516 9.8547 84.8351 12.1153 34.4609 8.8215 54.6491 49.5949 24.3272 37.4783 66.7316 68.7392 15.1780 35.4695 2.2477 2.2475 38.4877 47.5821 7.7540 7.7539 7.7538 13.1337 52.6407 6.6850 80.8228 1.240 40.5258 21.2884 19.2279 16.1838 3.4044 118.973 77.7948 16.1901 11.816 1.189 53.6441 29.3790
tlr1952 tlr1577 tlr0636 tll0488 tlr0320 tlr1530 tlr1573 tll0792 tlr0651 tlr1856 tsl2457 tll2172 tlr2014 tsl1557 tll1063 tll2308 tlr2018 tlr2016 tll1363 tlr1682 tlr2140 tlr2139 tsr2138 tlr0653 tlr2308 tlr2324 tll0991 tsl2214 tlr2034 tlr0351 tlr0052 tlr0610 tll1913 tlr2404 tlr1444 tlr0353 tll1867 tll0625 tlr2271 tll0958
were Synechocystis sp. PCC 6803 (3.6 MB) (Kaneko et al. 2001), Anabaena PCC 7120 (7.2 MB), and Thermosynechococcus elongates BP-1 (2.6 MB) available at http://www.kazusa.or.jp/cyano/cyano.html, and Synechococcus WH8102 (2.72 MB), Prochlorococcus
marinus MED4 (1.6 MB), Prochlorococcus marinus MIT9313 (2.4 Mb), Nostoc punctiforme (9.2 MB), and Trichodesmium erythraeum IMS101 (6.5 MB) available at http://jgi.doe.gov/JGI_microbial/html/index. html. Some of these sequences are currently in draft
216
Figure 1. A representative phylogenetic tree of cyanobacteria showing the position of strains whose complete genome has been sequenced and used in this study. The cyanobacterial 16S rRNA tree was constructed from 1063 unambiguously aligned nucleotides under the Kimura 2-parameter using the Neighbor Joining tree making algorithm in Bioedit (Hall 1999). The branches are designated with order-level nomenclature (Turner et al. 1999 with modifications). Bold names indicate the position of the strains whose genomic sequences were publicly available in October of 2002.
form. At the time this work was undertaken, the Synechocystis sp. PCC 6803 was by far the best annotated genome among these and, as such, was used as a reference sequence for much of the work reported here. Except for Anabaena PCC 7120, each genome had previously been inter-compared with the other seven
genomes and the results have been posted on the respective genome sites. The genes found to be common to at least seven cyanobacterial genomes were extracted and assembled into individual sequence files using the Bioedit platform (Hall 1999). Multigene sequence alignment was performed using CLUSTALW
217 in Bioedit (Higgins et al. 1994) and the results examined to verify that the genes extracted from the various genomes were likely homologs or orthologs. Each of the conserved genes was next compared against the NCBI protein database by use of BLASTP. BLASTP tables were individually examined for score and corresponding organism. Proteins with E-values
