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Comparative and Functional Genomics logoLink to Comparative and Functional Genomics
. 2008 Dec 2;2008:284508. doi: 10.1155/2008/284508

Comparative Analysis of Fatty Acid Desaturases in Cyanobacterial Genomes

Xiaoyuan Chi 1, 2, 3,2,3, Qingli Yang 1, 2, 3,2,3, Fangqing Zhao 1, 2,2, Song Qin 1,*, Yu Yang 1, 2,2, Junjun Shen 1, 2,2, Hanzhi Lin 1, 2,2
PMCID: PMC2593844  PMID: 19096516

Abstract

Fatty acid desaturases are enzymes that introduce double bonds into the hydrocarbon chains of fatty acids. The fatty acid desaturases from 37 cyanobacterial genomes were identified and classified based upon their conserved histidine-rich motifs and phylogenetic analysis, which help to determine the amounts and distributions of desaturases in cyanobacterial species. The filamentous or N2-fixing cyanobacteria usually possess more types of fatty acid desaturases than that of unicellular species. The pathway of acyl-lipid desaturation for unicellular marine cyanobacteria Synechococcus and Prochlorococcus differs from that of other cyanobacteria, indicating different phylogenetic histories of the two genera from other cyanobacteria isolated from freshwater, soil, or symbiont. Strain Gloeobacter violaceus PCC 7421 was isolated from calcareous rock and lacks thylakoid membranes. The types and amounts of desaturases of this strain are distinct to those of other cyanobacteria, reflecting the earliest divergence of it from the cyanobacterial line. Three thermophilic unicellular strains, Thermosynechococcus elongatus BP-1 and two Synechococcus Yellowstone species, lack highly unsaturated fatty acids in lipids and contain only one Δ9 desaturase in contrast with mesophilic strains, which is probably due to their thermic habitats. Thus, the amounts and types of fatty acid desaturases are various among different cyanobacterial species, which may result from the adaption to environments in evolution.

1. Introduction

In living organisms, the regulation of membrane fluidity is necessary for the proper function of biological membranes, which is important in the tolerance and acclimatization to environmental stresses such as heat, cold, desiccation, salinity, nitrogen starvation, photooxidation, anaerobiosis, and osmosis, and so forth. Unsaturated fatty acids are essential constituents of polar glycerolipids in biological membranes and the unsaturation level of membrane lipids is important in controlling the fluidity of membranes [1]. Fatty acid desaturases are enzymes that introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated and polyunsaturated fatty acids [2], thus these enzymes play an important role during the process of environmental adaptation.

Cyanobacteria, prokaryotes capable of carrying out a plant-like oxygenic photosynthesis, represent one of the oldest known bacterial lineages, with fossil evidence suggesting an appearance around 3–3.5 billion years ago [3]. Cyanobacteria comprise over 1600 species with various morphologies and species-specific characteristics such as cell movement, cell differentiation, and nitrogen fixation [4]. Extant cyanobacteria can be found in virtually all ecosystem habitats on Earth, ranging from the freshwater lakes and rivers through to the oceans, and also in hot springs and deserts, ranging from the hottest to the cold dry valleys of Antarctica [3].

Polyunsaturated membrane lipids play important roles in the growth, respiration, and photosynthesis of cyanobacteria. It is well documented that the content of polyunsaturated fatty acids in membrane lipids of cyanobacteria can be altered by changing the temperature [57]. The mechanism that regulates the fatty acid desaturation of membrane lipids in response to temperature has been demonstrated to be the result of the up- or downregulation of the expression of the desaturase genes [8]. Furthermore, it has been demonstrated that the position of double bonds in fatty acids is more influential on the fluidity of membrane lipids than the number of double bonds in fatty acids [9]. It is also found that the temperature of the phase transition dramatically decreased when the first and second double bonds are introduced into fatty acids, whereas the introduction of the third and fourth double bonds do not further lower the temperature of phase transition of membrane lipids [10].

Exposure of cyanobacteria to high PAR (photosynthetically active radiation) or UV radiation leads to photoinhibition of photosynthesis, thereby limiting the efficient fixation of light energy [11, 12]. In Synechocystis sp. PCC 6803, the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed the growth of the cells at low temperature, and it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri- and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures [13]. Transformation of the cyanobacterium Synechococcus sp. PCC 7942 with the desA gene for a Δ12 desaturase has been reported to increase the unsaturation of membrane lipids and thereby enhance the tolerance of cyanobacterium to intense light. These findings demonstrate that the ability of membrane lipids to desaturate fatty acids is important for the photosynthetic organisms to be able to tolerate high-light stress by accelerating the synthesis of the D1 protein de novo [14].

Cyanobacteria have been classified into four groups in terms of the composition of fatty acids, the distribution of fatty acids at the sn position of the glycerol moiety, and the position of double bonds in the fatty acids [15]. Strains in Group 1 (e.g., Prochlorothrix hollandica, Synechococcus sp. PCC 6301, Synechococcus sp. PCC 7942, Synechococcus elongatus, Thermosynechococcus elongates, and Thermosynechococcus vulcanus) introduce a double bond only at the Δ9 position of fatty acids at the sn-1 or sn-2 position of glycerolipids. Strains in Group 2 (e.g., Anabaena variabilis, Anabaena sp. PCC 7120, Synechococcus sp. PCC 7002, Nostoc punctiforme, and Nostoc sp. SO-36) introduce double bonds at the Δ9, Δ12, and Δ15 (ω3) positions of C18 acids at the sn-1 position, and at the Δ9 position of C16 acids at the sn-2 position. Strains in Group 3 (e.g., Synechocystis sp. PCC 6714 and Spirulina platensis) can also introduce three double bonds, but these are at the Δ6, Δ9, and Δ12 positions of C18 acids at the sn-1 position. Strains in Group 4 (e.g., Synechocystis sp. PCC 6803 and Tolypothrix tenuis) introduce double bonds at the Δ6, Δ9, Δ12, and Δ15 (ω3) positions of C18 acids at the sn-1 position. The C16 acids at the sn-2 position are not desaturated in Groups 3 and 4.

The entire genome sequence of a unicellular cyanobacterium Synechocystis sp. strain PCC 6803 was first described in 1996 [16]. To date, 37 cyanobacterial genomes have been sequenced (Figure 1). These genomes are those of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, the thermophilic strain Thermosynechococcus elongatus BP-1, the thylakoid-free strain Gloeobacter violaceus PCC 7421, the marine cyanobacterium Synechococcus sp. strain WH8102, the Prochlorococcus marinus strains SS120, MED4, MIT 9313, Synechococcus sp. CC9311, and others. These genome-sequencing projects undoubtedly bring a great convenience to obtain a comprehensive dataset of genes involved in unsaturated fatty acid biosynthesis in cyanobacteria. In this work, we identified all the putative fatty acid desaturases using bioinformatic tools and presented a genomic comparison of the fatty acid desaturases from 37 cyanobacterial genomes. The identification of novel desaturases and the reconstruction of the pathways for unsaturated fatty acid biosynthesis in cyanobacteria will guide the experimental analysis and provide clues in study of the relationship between the unsaturation level of membrane lipids and environmental adaptation in higher plants.

Figure 1.

Figure 1

Phylogenetic tree of the sequenced cyanobacterial strains. A Neighbor-joining tree for 33 sequenced cyanobacteria constructed based on 16 S rRNA as was described in Section 2 and about 1300 positions were employed. To maximize the number of sites available for analysis, three partial sequences from Synechococcus sp. RS9917 (170 bp), Synechococcus sp. RS9916 (865 bp), and Synechococcus sp. BL107 (296 bp) were excluded. Moreover, no 16 S rRNA sequence was found in Cyanothece sp. CCY0110.

2. Materials and Methods

2.1. Computational Search for Novel Fatty Acid Desaturase Genes

The genomes of 37 cyanobacteria including genera Synechocystis, Synechococcus, Prochlorococcus, Anabaena, Nostoc, Trichodesmium, Gloeobacter, Crocosphaera, Cyanothece, and Lyngbya were downloaded from IMG database (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi). The dataset comprised of well-characterized fatty acid desaturases from Synechocystis PCC 6803 (NP_442430, NP_441489, NP_441622, NP_441824), Nostoc sp. SO-36 (CAF18426), Synechococcus sp. PCC 7002 (AAB61353, AAF21445, AAB61352), Arthrospira platensis (CAA05166, Q54794, CAA60573), Synechococcus vulcanus (AAD00699), Synechococcus s elongatus sp. PCC 6301 (YP_172259), Synechococcus elongatus sp. PCC 7942 (YP_401578), Phaeodactylum tricorutum (AAW70158, AY082393, AAO23565, AY165023), Chlamydomonas reinhardtii (AB007640, ABL09485, EDP04777), and Chlorella vulgaris (AB075526, AB075527) was used to construct a query protein set. Each protein in this query dataset was used to search the potential novel sequences in 37 cyanobacterial species with whole genome sequences available, by using the BLASTP and TBLASTN programs, with E-value < 1e − 10. The searches were repeated until no novel sequences were detected at the e value threshold used. The putative desaturase genes across 37 genomes were summarized in Table 1. The other amino acid sequences beyond the 37 cyanobacterial species were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/). The accession number of these sequences and the names of corresponding cyanobacteria, eukaryotic algae, higher plants, fungi, and animals were indicated in Table 2.

Table 1.

Lists of putative desaturase genes from thirty seven cyanobacterial genomes.

Species Locus tag Accession DNA coordinates Length Proposed function
Anabaena sp. PCC 7120 all4991 NP_489031 5963080⋯5963937 857 d9
all1599 NP_485639 1879629⋯1880447 818 d9
all1598 NP_485638 1878346⋯1879398 1052 d12
all1597 NP_485637 1876897⋯1877976 1079 d15
alr3189 NP_487229 3858986⋯3859762 776 crtW
alr4009 NP_488049 4829483⋯4830322 839 crtR

Anabaena variabilis ATCC 29413 Ava_2277 YP_322790 2832413⋯2833270 857 d9
Ava_4212 YP_324706 5282348⋯5283166 818 d9
Ava_4211 YP_324705 5281066⋯5282118 1052 d12
Ava_4210 YP_324704 5279614⋯5280693 1079 d15
Ava_2048 YP_322565 2535646⋯2536410 764 crtW
Ava_3888 YP_324388 4842189⋯4842965 776 crtW
Ava_1693 YP_322210 2121129⋯2122049 920 crtR

Crocosphaera watsonii WH 8501 CwatDRAFT_1377 ZP_00518170 3068⋯3892 824 d9
CwatDRAFT_3226 ZP_00516843 22017⋯23066 1049 d12
CwatDRAFT_5150 ZP_00515010 150888⋯151982 1049 d12
CwatDRAFT_3625 ZP_00516181 10760⋯11809 1049 d15
CwatDRAFT_1857 ZP_00517700 1398⋯2231 834 hypothetical protein
CwatDRAFT_5424 ZP_00514501 315629⋯316522 893 crtR

Gloeobacter violaceus strain PCC 7421 gvip390 NP_925812 3057506⋯3058357 851 d9
gvip170 NP_924181 1312274⋯1313095 822 d9
gll1946 NP_924892 2071551⋯2072504 953 d9
gll1947 NP_924893 2072509⋯2073507 998 d9
gll1938 NP_924884 2060880⋯2061839 959 d9
gll1940 NP_924886 2063884⋯2064876 992 d9
gvip364 NP_925569 2779580⋯2780638 1058 d12
gvip506 NP_926681 3944843⋯3945910 1058 d12
gll0171 NP_923117 161268⋯162440 1173 hypothetical protein
gll2501 NP_925447 2660474⋯2661475 1001 mocD
gvip239 NP_924674 1833712⋯1834485 773 crtW

Nostoc punctiforme ATCC 29133(PCC 73102) Npun02000467 ZP_00345918 175651⋯176532 881 d9
Npun02005010 ZP_00108582 41108⋯41929 821 d9
Npun02005011 ZP_00108583 42265⋯43326 1061 d12
Npun02005012 ZP_00108584 43524⋯44603 1080 d15
Npun02001904 ZP_00345765 63255⋯64310 1056 hypothetical protein
Npun02001905 ZP_00110890 64537⋯65574 1038 hypothetical protein
Npun02002344 ZP_00110549 77763⋯78863 1101 hypothetical protein
Npun02003462 ZP_00109371 76020⋯76964 945 mocD
Npun02000865 ZP_00345866 139810⋯140571 762 crtW
Npun02001326 ZP_00111258 55604⋯56392 788 crtW
Npun02006805 ZP_00106832 23657⋯24556 899 crtR

Prochlorococcus marinus str. NATL1A NATL1_21421 YP_001015962 1799954⋯1800733 780 d9
NATL1_10821 YP_001014905 992775⋯993992 1218 d12
NATL1_03151 YP_001014144 291853⋯292884 1032 crtR

Prochlorococcus marinus strain NATL2A PMN2A_1271 YP_292464 1227545⋯1228474 929 d9
PMN2A_0393 YP_291588 388657⋯389874 1217 d12
PMN2A_1603 YP_292794 1566557⋯1567588 1031 crtR

Prochlorococcus marinus MIT 9211 P9211_09157 ZP_01006363 1417821⋯1418765 944 d9
P9211_05577 ZP_01005647 779723⋯780334 611 d12
P9211_05582 ZP_01005648 780304⋯780729 425 d12
P9211_07547 ZP_01006041 1108444⋯1109469 1015 crtR

Prochlorococcus marinus str. MIT 9301 P9301_18621 YP_001092086 1588713⋯1589651 939 d9
P9301_15761 YP_001091800 1328773⋯1329939 1167 d12
P9301_15721 YP_001091796 1326076⋯1327182 1107 d12
P9301_02581 YP_001090482 239249⋯239974 726 crtR

Prochlorococcus marinus str. MIT 9303 P9303_28951 YP_001018890 2560285⋯2561250 966 d9
P9303_28931 YP_001018888 2558615⋯2559535 921 d9
P9303_14121 YP_001017424 1208715⋯1209800 1086 d12
P9303_21081 YP_001018108 1869188⋯1870330 1143 d12
P9303_24321 YP_001018428 2137288⋯2138328 1041 crtR

Prochlorococcus marinus str. MIT 9312 PMT9312_1764 YP_398261 1656076⋯1657014 938 d9
PMT9312_1476 YP_397972 1385670⋯1386845 1175 d12
PMT9312_1473 YP_397969 1382796⋯1383902 1106 d12
PMT9312_0238 YP_396735 229042⋯229842 800 crtR

Prochlorococcus marinus str. MIT 9313 PMT2172 NP_895996 2299082⋯2300002 920 d9
PMT2174 NP_895998 2300938⋯2301717 779 d9
PMT0249 NP_894082 278544⋯279683 1139 d12
PMT0797 NP_894629 872385⋯873470 1085 d12
PMT1816 NP_895643 1920323⋯1921363 1040 crtR

Prochlorococcus marinus str. AS9601 A9601_18811 YP_001010271 1616719⋯1617657 939 d9
A9601_15921 YP_001009982 1355480⋯1356514 1035 d12
A9601_15871 YP_001009977 1352826⋯1353932 1107 d12
A9601_02571 YP_001008652 238284⋯239117 834 crtR

Prochlorococcus marinus str. MIT 9515 P9515_18621 YP_001012176 1650943⋯1651929 987 d9
P9515_15601 YP_001011874 1376566⋯1377693 1128 d12
P9515_15521 YP_001011866 1371646⋯1372752 1107 d12
P9515_02681 YP_001010584 247534⋯248433 900 crtR

Prochlorococcus marinus subsp. marinus str. CCMP1375 (SS120) Pro1833 NP_876224 1690865⋯1691797 932 d9
Pro1208 NP_875600 1116904⋯1118016 1112 d12
Pro1214 NP_875606 1121144⋯1122250 1106 d12
Pro0266 NP_874660 261189⋯262223 1034 crtR

Prochlorococcus marinus subsp. marinus str. CCMP1986 (MED4) PMM1672 NP_893789 1604745⋯1605731 986 d9
PMM1382 NP_893499 1331162⋯1332340 1178 d12
PMM1378 NP_893495 1325388⋯1326494 1106 d12
PMM0236 / 228281⋯229270 989 crtR

Synechococcus elongatus strain PCC 7942 Synpcc7942_2561 YP_401578 2639146⋯2639982 836 d9
Synpcc7942_1713 YP_400730 1781317⋯1782219 902 mocD
Synpcc7942_2439 YP_401456 2514276⋯2515271 995 crtR

Synechococcus elongatus strain PCC 6301 syc1549_d YP_172259 1676804⋯1677640 837 d9
Syc2378_c YP_173088 2534831⋯2535691 861 mocD
syc1667_c YP_172377 1801757⋯1802752 996 crtR

Synechococcus sp. BL107 BL107_07284 ZP_01469203 490784⋯491566 782 d9
BL107_07289 ZP_01469204 491936⋯492721 785 d9
BL107_06084 ZP_01468963 247334⋯248356 1022 d12
BL107_14110 ZP_01468055 331111⋯331884 773 crtW
BL107_08054 ZP_01469357 636707⋯637738 1031 crtR

Synechococcus sp. CC9311 sync_2793 YP_731981 2458778⋯2459710 932 d9
sync_2791 YP_731979 2457075⋯2457986 911 d9
sync_0336 YP_729569 344430⋯345449 1019 crtR
sync_0396 YP_729627 408306⋯409505 1199 d12
sync_1804 YP_731008 1621108⋯1621869 761 crtW

Synechococcus sp. CC9605 Syncc9605_2541 YP_382824 2358792⋯2359703 911 d9
Syncc9605_1972 YP_382268 1793076⋯1794221 1145 d12
Syncc9605_0286 YP_380617 292821⋯293870 1049 crtR

Synechococcus sp. CC9902 Syncc9902_2191 YP_378192 2099771⋯2100673 902 d9
Syncc9902_2192 YP_378193 2100902⋯2101825 923 d9
Syncc9902_0141 YP_376159 149723⋯150724 1001 d12
Syncc9902_0972 YP_376982 954015⋯954788 773 crtW
Syncc9902_2058 YP_378059 1964618⋯1965730 1112 crtR

Synechococcus sp. JA-2-3B′a(2-13) CYB_0861 YP_477105 894187⋯895071 884 d9
CYB_2914 YP_479096 3011594⋯3012520 926 mocD
CYB_0102 YP_476366 118335⋯119306 971 crtR

Synechococcus sp. JA-3-3Ab CYA_2349 YP_475739 2357019⋯2357912 893 d9
CYA_1931 YP_475340 1944066⋯1945040 974 crtR

Synechococcus sp. RCC307 SynRCC307_2395 YP_001228651 2091372⋯2092274 903 d9
SynRCC307_2393 YP_001228649 2089667⋯2090581 915 d9
SynRCC307_1757 YP_001228013 1538507⋯1539562 1056 d12
SynRCC307_1993 YP_001228249 1729342⋯1730103 762 crtW
SynRCC307_2209 YP_001228465 1915148⋯1916167 1020 crtR

Synechococcus sp. RS9916 RS9916_36767 ZP_01471384 1050409⋯1051341 932 d9
RS9916_36757 ZP_01471382 1048603⋯1049568 965 d9
RS9916_39311 ZP_01472905 116650⋯117675 1025 crtR

Synechococcus sp. RS9917 RS9917_06370 ZP_01079314 447782⋯448705 923 d9
RS9917_06360 ZP_01079312 446060⋯446992 932 d9
RS9917_03333 ZP_01080849 99968⋯101047 1079 d12
RS9917_00687 ZP_01080541 64826⋯65563 737 crtW
RS9917_03663 ZP_01080915 166940⋯167902 962 crtR

Synechococcus sp. WH 5701 WH5701_02025 ZP_01084898 299319⋯300257 787 d9
WH5701_02015 ZP_01084896 297579⋯298532 953 d9
WH5701_14646 ZP_01083974 104382⋯105539 1157 d12
WH5701_16535 ZP_01086617 164⋯1186 1022 d12
WH5701_06521 ZP_01085935 65353⋯66231 878 hypothetical protein
WH5701_02369 ZP_01084322 42300⋯43271 971 mocD
WH5701_04005 ZP_01083421 43734⋯44519 785 crtW
WH5701_01215 ZP_01084736 138584⋯139615 1031 crtR

Synechococcus sp. WH 7803 SynWH7803_2417 YP_001226140 2249293⋯2250087 795 d9
SynWH7803_2415 YP_001226138 2247475⋯2248386 912 d9
SynWH7803_0589 YP_001224312 594539⋯595603 1065 d12
SynWH7803_1625 YP_001225348 1496144⋯1497139 996 d15
SynWH7803_0928 YP_001224651 871421⋯872167 747 crtW
SynWH7803_0337 YP_001224060 361336⋯362337 1002 crtR

Synechococcus sp. WH 7805 WH7805_10184 ZP_01125021 209067⋯209999 932 d9
WH7805_10194 ZP_01125023 210769⋯211680 911 d9
WH7805_06186 ZP_01124768 405535⋯406059 524 d12
WH7805_04931 ZP_01124517 184338⋯185516 1178 d12
WH7805_01197 ZP_01123773 3991⋯4734 743 crtW
WH7805_07481 ZP_01123496 193165⋯194193 1028 crtR

Synechococcus sp. WH 8102 SYNW2377 NP_898466 2286168⋯ 2287028 860 d9
SYNW0696 NP_896789 679330⋯680478 1148 d12
SYNW1696 NP_897787 1631011⋯1632147 1136 d12
SYNW1368 NP_897461 1354793⋯1355527 734 crtW
SYNW0291 NP_896386 291323⋯292354 1031 crtR

Synechocystis sp. PCC 6803 sll0541 NP_442430 2822579⋯2823535 956 d9
slr1350 NP_441489 1746308⋯1747363 1055 d12
sll1441 NP_441622 1895520⋯1896599 1079 d15
sll0262 NP_441824 2120067⋯2121146 1079 d6
Sll1611 NP_441220 1462136⋯1463245 1110 hypothetical protein
sll1468 NP_440788 981691⋯982629 938 crtR

Thermosynechococcus elongatus strain BP-1 tll1719 NP_682509 1800682⋯1801521 839 d9
tlr2380 NP_683170 2490209⋯2491048 839 d9
tlr1653 NP_682443 1733919⋯1734767 848 d9
tlr1254 NP_682044 1300388⋯1301308 920 mocD
tlr1900 NP_682690 1986642⋯1987529 887 crtR

Trichodesmium erythraeum IMS101 Tery_1437 YP_721205 2173203⋯2174015 812 d9
Tery_0142 YP_720110 207806⋯208861 1055 d12
Tery_4492 YP_723951 6931402⋯6932475 1073 d15
Tery_3898 YP_723406 6024293⋯6025342 1050 hypothetical protein
Tery_2925 YP_722564 4543239⋯4544114 875 crtR

Lyngbya sp. PCC 8106 L8106_03152 ZP_01624678 2253⋯3071 818 d9
L8106_27002 ZP_01621185 94912⋯95955 1043 d12
L8106_10697 ZP_01624560 6961⋯8043 1082 d15
L8106_14825 ZP_01619238 100018⋯101133 1115 d6
L8106_06180 ZP_01620148 172993⋯173604 611 hypothetical protein
L8106_18641 ZP_01624278 13290⋯14111 821 hypothetical protein
L8106_30215 ZP_01622578 23391⋯24185 794 crtR

Nodularia spumigena CCY9414 N9414_19077 ZP_01631817 16235⋯17026 791 d9
N9414_07494 ZP_01632615 317⋯1135 818 d9
N9414_07499 ZP_01632616 1303⋯2427 1124 d12
N9414_07504 ZP_01632617 2618⋯3688 1070 d15
N9414_07509 ZP_01632618 4087⋯5178 1091 d6
N9414_18293 ZP_01629726 29633⋯30223 590 hypothetical protein
N9414_07726 ZP_01632305 4851⋯5633 782 crtW
N9414_01572 ZP_01632726 697⋯1587 890 crtR

Cyanothece sp. CCY0110 CY0110_10577 ZP_01726409 185891⋯186724 834 d9
CY0110_05582 ZP_01729213 74180⋯75004 825 d9
CY0110_10917 ZP_01732458 7951⋯9000 1050 d12
CY0110_00445 ZP_01728541 90142⋯91191 1050 d15
CY0110_24056 ZP_01727982 158769⋯159887 1119 d6
CY0110_13441 ZP_01729024 60390⋯61220 831 hypothetical protein
CY0110_27283 ZP_01731934 15787⋯16914 1128 hypothetical protein
CY0110_11357 ZP_01729279 9512⋯10513 1002 mocD
CY0110_08481 ZP_01731007 25752⋯26747 996 crtR

Table 2.

List of organisms (except the above thirty seven cyanobacteria) and protein sequences analyzed in this study. Note: micro represents Microsomal, chl represents Chloroplastic, “uncertain” means that the function of the gene is uncertain.

Species Accession no/locus tag Label Accession no/locus tag Label
Arabidopsis thaliana BAA25180 d9 AAB60302 chld15
Q949X0 d7 BAA05514 microd15
AAA92800 chld12 CAA11858 d8
NP_187819 microd12

Thalassiosira pseudonana Tp22511 d9 AY817152 d5
Tp23798 d12 AY817155 d6
Tp3143 d12 AY817154 d8
AY817156 d4

Phaeodactylum tricorutum AAW70158 d9 AY082393 d6
AAO23565 chld12 AY082392 d5
AY165023 microd12 Pt22459 d5

Chlamydomonas reinhardtii Cr117883 uncertain ABL09485 d15
AB007640 chld12 AY860820 crtW
EDP04777 microd12

Synechococcus sp. PCC 7002 AAB61353 d9 AAF21445 d12
AAF21447 uncertain AAB61352 d15

Nostoc sp. SO-36 CAF18426 d9 CAF18425 d15
CAF18423 d9 CAF18424 d12

Mortierella alpina CAB38177 d9 AAF08684 d12
AAF08685 d6 AAC39508 d5

Cyanidioschyzon merolae BAA28834 d9 CMK291C d12
CMJ201C d9 BAC76126 crtR

Arthrospira platensis CAA05166 d9 Q54794 d12
ABN11122 d6

Ostreococcus lucimarinus Ol51664 uncertain Ol24150 d12
Ol18582 d12

Caenorhabditis elegans AAF97550 d9 AAC15586 d6
AAC95143 d5

Rattus norvegicus NP_114029 d9 BAA75496 d6
AAG35068 d5

Homo sapiens XP_005719 d9 AAD20018 d6
AAF29378 d5

Brassica napus AAA50157 chl d12 AAF78778 microd12
CAA11857 d8

Chlorella vulgaris AB075526 microd12 AB075527 microd15
Chlamydomonas sp. W80 AB031546 chld12
Synechocystis sp. PCC 6714 BAA02921 d12
Mucor circinelloides AAD55982 d12 BAB69055 d6
Emericella nidulans AAG36933 d12
Glycine max BAD89862 microd12
Calendula officinalis AAK26633 microd12
Gossypium hirsutum AAL37484 microd12
Nicotiana tabacum BAC01274 chld15 BAC01273 microd15
Brassica juncea CAB85467 chld15
Picea abies CAC18722 chld15
Ricinus communis AAA73511 chld15 AAC49010 12-hydroxylase
Triticum aestivum BAA28358 microd15
Oryza sativa BAA11397 microd15
Vernicia fordii AAN87573 microd12 AAN87574 12-conjugase
Punica granatum CAD24671 microd12 AAO37753 12-conjugase
Lesquerella fendleri AAC32755 12-hydroxylase/desaturase
Physaria lindheimeri ABQ01458 12-hydroxylase
Crepis palaestina CAA76156 12-epoxygenase
Stokesia laevis AAR23815 12-epoxygenase
Daucus carota AAO38033 12-acetylenase
Foeniculum vulgare AAO38034 12-acetylenase
Hedera helix AAO38031 12-acetylenase
Helianthus annuus AAO38032 12-acetylenase CAA60621 d8
Helichrysum bracteatum AAO38037 12-acetylenase
Rudbeckia hirta AAO38035 12-acetylenase
Crepis alpina CAA76158 12-acetylenase
Calendula officinalis AAK26632 12-conjugase
Trichosanthes kirilowii AAO37751 12-conjugase
Acheta domesticus AAK25797 d9
Cyprinus carpio CAB57858 d9
Drosophila simulans CAB52475 d9
Gallus gallus CAA42997 d9
Helicoverpa zea AAF81790 d9
Rosa hybrid cultivar BAA23136 d9
Saccharomyces cerevisiae AAA34826 d9
Limnanthes douglasii AAG28599 d9
Prochlorothrix hollandica AAG16761 d9
Lyngbya majuscula AAS98775 d9
Synechococcus vulcanus AAD00699 d9
Thraustochytrium sp. ATCC21685 AAM09688 d4 AAM09687 d5
Euglena gracilis AAQ19605 d4 AF139720 d8
Pavlova lutheri AY332747 d4
Isochrysis galbana strain CCMP1323 AY630574 d4
Marchantia polymorpha AAT85663 d5 AAT85661 d6
Nitzschia closterium f. minutissima AY603475 d5
Dictyostelium discoideum BAA37090 d5
Bacillus subtilis AAC38355 d5
Danio rerio Q9DEX7 d5/d6
Borago officinalis AAD01410 d6 AAG43277 d8
Oncorhynchus mykiss AAK26745 d6
Mus musculus NP_062673 d6
Glossomastix chrysoplasta AAU11444 d6
Ostreococcus tauri AY746357 d6
Physcomitrella patens CAA11033 d6
Echium pitardii AAL23581 d6
Chlorella zofingiensis AY772713 crtW
Cyanidium caldarium AAB82698 crtR
Haematococcus pluvialis CAA60478 crtW
Myxococcus xanthus DK 1622 YP_634431 uncertain
Stigmatella aurantiaca DW4/3-1 ZP_01463016 uncertain
Bradyrhizobium japonicum USDA 110 NP_771234 uncertain

2.2. Multiple Sequence Alignment and Phylogenetic Analysis

Sequence alignments were generated using Clustal W program [17]. The SMART (http://smart.embl-heidelberg.de/) and PFAM (http://pfam.sanger.ac.uk/) databases were used to search the conserved domains of the putative desaturase enzymes. The conserved amino acid residues of different conserved domains were manually identified using the BioEdit sequence editor. The final alignment was further refined after excluding the poorly conserved regions at the protein ends, and consisted of sequences spanning the conserved domains. The neighbor-joining (NJ) and minimum-evolution (ME) methods in MEGA4 [18] were used to construct the phylogenetic tree. To maximize the number of sites available for analysis, two partial sequences from Synechococcus sp. WH 7805 (ZP_01124768, 174 aa) and Nodularia spumigena CCY9414 (ZP_01629726, 196 aa) were excluded. Bootstrap with 1000 replicates was used to establish the confidence limit of the tree branches.

3. Results and Discussions

3.1. The Conserved Motifs

Using BlastP and TBlastN programs with the query sequences to search the 37 genomes of cyanobacteria, 193 protein sequences were identified including fatty acid desaturase, fatty acid dehydrogenase, hypothetical protein, β-carotene ketolase, β-carotene hydroxylase, and hydrocarbon oxygenase. PFAM and SMART domain analyses could not distinguish fatty acid desaturase from fatty acid dehydrogenase, β-carotene ketolase, β-carotene hydroxylase, or hydrocarbon oxygenase. Moreover, most of the protein sequences which were originally annotated as fatty acid desaturase were not classified into Δ9, Δ12, Δ15, or Δ6 desaturase categories. To facilitate the classification of different types of desaturases, the conserved motifs of different enzymes were identified by multiple sequence alignments with Clustal W.

There were three typical histidine-rich motifs existed in all the proteins similar to proven cyanobacterial fatty acid desaturases (Table 3). Moreover, there were different conserved residues in the same histidine-boxes of different kinds of proteins, suggesting that these proteins might have acquired different functions from a common ancestor during the evolution. According to the different conserved residues of three histidine-motifs and phylogenetic profile, 16 β-carotene ketolases, 36 β-carotene hydroxylases, and 8 hydrocarbon oxygenases (MocD, a rhizopine oxygenase for the conversion of 3-O-MSI to SI)) were identified from the 37 cyanobacterial genomes (Figures 2, 4, and 5).

Table 3.

Conserved motifs of membrane desaturases in cyanobacteria. Note: X represents an unspecified amino acid. Δ9-1: clade 1 of Δ9 homologous genes, Δ9-2: clade 2 of Δ9 homologous genes, Δ9-3: clade 3 of Δ9 homologous genes, Δ9-4: clade 4 of Δ9 homologous genes, Δ9-5: clade 5 of Δ9 homologous genes, Δ12a : clade 3 of Δ12 homologous genes, Δ12b: clade 1 of Δ12 homologous genes, Δ12c : clade 4 of Δ12 homologous genes, Δ15: Δ15 desaturase, Δ6: Δ6 desaturase.

Name H-box1 H-box2 H-box3
β-carotene ketolase TGLFIX2HDXMH K(N)HX2HH CY(F)H(N)FGYHXEHH
β-carotene hydroxylase GTVIHDAS(C)HX2AH RVHL(M)Q(E)HHXHVN GQNYHLI(V)HHLWPSI(V)PW
hydrocarbon oxygenase HECXHRTAFA FY(F)RRYHXWHHRXT MWNMPF(Y)HXEHHL(F)
Δ9-1 GICLGYHRLLXHKSF WX3HRXHHAX3D YGEGWHNNHHX2PX5GX2WWE
Δ9-2 GXTLGXHRX3HRSF WXGXHRXHHX2SD GEGWHNNHHX4SARHGXXWWE
Δ9-3 TVLGVTLGLHRLXAHRS WX2LHRHHHX2SDQ WVAXLSFGEGWHNNHHAXPXSARHGL
Δ9-4 CLGVTXGYHRLLXHRX2 WXGLHRHHHXFSDT WVAALTFGEGWHNNHHAXPXSA
Δ9-5 GX4GXHRXFXHX2F WX3HRXHHX3D GESWHNNHHXFX3AX2G
Δ12a FVXGHDCGHRSF WRX2HX2HHX2TN HXPHHX4IPXYNLR
Δ12b WVXAHECGHXAFH WX2SHX2HHX3N HX2HHX4PHYXA
Δ12c FSLMHDCGHXSLF WSX2HAXHHX2NG HX2HHLXERIPNYXL
Δ15 FWXLFVVGHDCGHXSFS HGWRISHRTHHXNTGN IHHXIGTHVAHHIF
Δ6 HDX2HX3S WX3HX2LHHXYTNI GGLNXQ(H)X2HHLFPXICH

Figure 2.

Figure 2

Comparison of the three conserved histidine-rich motifs of proteins from cyanobacteria, eukaryotic algae, and higher plants, including Δ12 fatty acid desaturase, Δ15 fatty acid desaturase, β-carotene ketolase, β-carotene hydroxylase, hydrocarbon oxygenase, Δ12 fatty acid epoxygenase, Δ12 fatty acid acetylenase, Δ12 fatty acid conjugase, and Δ12 fatty acid hydroxylase. The conserved amino acid residues are in black. “Microsomal” represents the microsome-type desaturases, “Chloroplast” represents the chloroplast-type desaturases.

Figure 4.

Figure 4

Neighbor-joining tree of β-carotene ketolase, β-carotene hydroxylase, and hydrocarbon oxygenase homologs of cyanobacteria and eukaryotic algae. About 220 positions spanning the three histidine-boxes were employed. Colored branches indicate different groups of proteins. Red: β-carotene hydroxylase, green: β-carotene ketolase, magenta: hydrocarbon oxygenase. Sequences from 37 sequenced cyanobacterial genomes are shown by their acronyms and accession numbers (locus tags). Other sequences are shown by their accession numbers, labels, and strain names. Desaturase genes that have been functionally characterized are indicated on the tree by their labels. Bootstrap values from neighbor-joining analyses are listed to the left of each node, with values more than 50 are shown.

Figure 5.

Figure 5

Minimum-evolution tree of β-carotene ketolase, β-carotene hydroxylase, and hydrocarbon oxygenase homologs of cyanobacteria and eukaryotic algae. About 220 positions spanning the three histidine-boxes were employed. Colored branches indicate different groups of proteins. Red: β-carotene hydroxylase, green: β-carotene ketolase, magenta: hydrocarbon oxygenase. Sequences from 37 sequenced cyanobacterial genomes are shown by their acronyms and accession numbers (locus tags). Other sequences are shown by their accession numbers, labels, and strain names. Desaturase genes that have been functionally characterized are indicated on the tree by their labels. Bootstrap values from minimum-evolution analyses are listed to the left of each node, with values more than 50 are shown.

3.2. Discovery of Candidate Genes for Δ9 Desaturases

To elucidate the phylogenetic relationships among different membrane desaturases, genes from cyanobacteria, eukaryotic algae, higher plants, fungi, invertebrates, and vertebrates were analyzed using neighbor-joining (NJ) and minimum-evolution (ME) methods. Observation of the tree revealed that all the desaturases fell into three distinct subfamilies (Figures 12 and 13): Δ9 desaturase subfamily, Δ12/ω3 desaturases subfamily, and the front-end desaturases subfamily.

Figure 12.

Figure 12

Neighbor-joining tree of membrane desaturases. About 330 positions spanning the three histidine-boxes were employed. Sequences from 37 sequenced cyanobacterial genomes are shown by their acronyms and accession numbers (locus tags). Other sequences are shown by their accession numbers, labels, and strain names. Desaturase genes that have been functionally characterized are indicated on the tree by their labels. Bootstrap values from neighbor-joining analyses are listed to the left of each node, with values more than 50 are shown.

Figure 13.

Figure 13

Minimum-evolution tree of membrane desaturases. About 330 positions spanning the three histidine-boxes were employed. Sequences from 37 sequenced cyanobacterial genomes are shown by their acronyms and accession numbers (locus tags). Other sequences are shown by their accession numbers, labels, and strain names. Desaturase genes that have been functionally characterized are indicated on the tree by their labels. Bootstrap values from minimum-evolution analyses are listed to the left of each node, with values more than 50 are shown.

As shown in Figures 12 and 13, Δ9 desaturases clustered into a single-monophyletic group, thus were analyzed separately from other types of desaturases. Six clades could be identified within the Δ9 desaturase homologs from cyanobacteria based on high-bootstrap support values and a large degree of within-clade sequence identity (Figures 3, 6, and 7). Except for the genes from Clade 6 (ZP_01620148, ZP_01085935, and AAF21447) whose second residue of the second histidine-box was not arginine, the genes from other clades all matched the standard for Δ9 desaturase, that is, HR-X3-H, HR-X-HH, and HN-X-HH. Thus, genes from Clade 6 are assigned as hypothetical proteins with functions unknown.

Figure 3.

Figure 3

Alignment of the complete deduced amino acid sequences of Δ9-homologous genes. Amino acid residues that are conserved are highlighted in black boxes. The conserved His clusters and their associated conserved domains are underlined. The limits of the domains are indicated by the residue positions, on top of the sequence. The sequences are denoted by their strain names and the clades they belong to.

Figure 6.

Figure 6

Neighbor-joining tree of Δ9-homologous genes of cyanobacteria and eukaryotic algae. About 250 positions spanning the three histidine-boxes were employed. Colored branches indicate different groups of proteins. Dark blue: Clade 1, magenta: Clade 2, green: Clade 3, red: Clade 4, light blue: Clade 5, orange: Clade 6. Sequences from 37 sequenced cyanobacterial genomes are shown by their acronyms and accession numbers (locus tags). Other sequences are shown by their accession numbers, labels, and strain names. Desaturase genes that have been functionally characterized are indicated on the tree by their labels. Bootstrap values from neighbor-joining analyses are listed to the left of each node, with values more than 50 are shown.

Figure 7.

Figure 7

Minimum-evolution tree of Δ9-homologous genes of cyanobacteria and eukaryotic algae. About 250 positions spanning the three histidine-boxes were employed. Colored branches indicate different groups of proteins. Dark blue: Clade 1, magenta: Clade 2, green: Clade 3, red: Clade 4, light blue: Clade 5, orange: Clade 6. Sequences from 37 sequenced cyanobacterial genomes are shown by their acronyms and accession numbers (locus tags). Other sequences are shown by their accession numbers, labels, and strain names. Desaturase genes that have been functionally characterized are indicated on the tree by their labels. Bootstrap values from minimum-evolution analyses are listed to the left of each node, with values more than 50 are shown.

The first clade was composed by one Δ9-homologous gene from eight N2-fixing cyanobacterial species (such as Nostoc sp. strain SO-36 and Anabaena sp. PCC 7120), Thermosynechococcus elongatus BP-1, Synechococcus vulcanus, and two genes from Gloeobacter violaceus. The amino acid identity of these genes ranged from 50% to 98% among various cyanobacterial species. It has been proven by previous research that the Δ9 desaturase gene from Nostoc sp. strain SO-36 in this clade introduced double bonds into fatty acids that are bound to the sn-2 position of the glycerol moiety of membrane glycerolipids [19]. Moreover, the three histidine-boxes of the gene from Nostoc sp. SO-36 were consistent with those of genes in Clade 1. Therefore, the genes of Clade 1 are presumed to act on fatty acids esterified to the sn-2 position of glycerolipids.

In Clade 2, one Δ9-homologous gene from Prochlorothrix hollandica, Synechococcus sp. PCC 7942, and Synechococcus sp. PCC 6301 clustered together with two genes from Thermosynechococcus elongatus, apart from the subgroup comprised of genes from nine N2-fixing cyanobacterial species (such as Anabaena variabilis and Trichodesmium erythraeum), Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7002, and Arthrospira platensis. It has been demonstrated that Thermosynechococcus elongatus has three Δ9-homologous genes that consist of one c-type and two unspecified types. By contrast, Synechococcus sp. PCC 7942, Synechococcus sp. PCC 6301, and Prochlorothrix hollandica have only one Δ9-homologous gene, which is nonspecific with respect to sn positions, acting on fatty acids at both the sn-1 and sn-2 positions [19]. Δ9 homologs from another subgroup showed high similarity with amino acid identity from 53% to 98% among various cyanobacterial species. They are strongly homologous to the genes of Synechocystis sp. PCC 6803 (NP_442430), Synechococcus sp. PCC 7002 (AAB61353), and Arthrospira platensis (CAA05166) that encode Δ9 desaturases acting on C18 fatty acids at the sn-1 position. Moreover, the three histidine-boxes of these Δ9-homologous genes (HRX3HRSF, WXGXHRXHH, GEGWHNNHH) accorded with those inferred by Chintalapati et al. (2006) [19].

The Δ9-homologous genes from two unicellular marine cyanobacteria Synechococcus and Prochlorococcus constituted the third and fourth clades. Amino acid identity of genes from these two clades ranged from 54% to 98% and 65% to 99%, respectively. In addition, the two groups are closely related to Clade 2. Therefore, it is possible that these genes are homologous to the gene that encodes a Δ9 desaturase acting on C18 fatty acids at the sn-1 position or sn-1 and sn-2 positions of glycerolipids. In these two clades, 11 strains (nine Synechococcus and two low light-adapted Prochlorococcus strains) contained two Δ9-homologous genes, which clustered separately into two subgroups. It is possible that there are two paralogous genes of a common ancestor in some evolutionary lineages, such as Synechococcus sp. CC9605; however, one of them has been lost. Alternatively, acquirement of one gene from other organisms could have occurred in the evolutionary lineage, in which horizontal gene transfer (HGT) might have taken place.

Four genes of Gloeobacter violaceus PCC 7421 as well as JamB gene of Lyngbya majuscula integrated the fifth clade. JamB is a gene of jamaicamide biosynthetic gene cluster, and similar to a large family of membrane-associated desaturases that utilize a diiron active site to execute Δ5- or Δ9-fatty acid desaturation [20]. These genes fell into the group of proteobacterial stearoyl-CoA desaturases, far away from the other desaturase genes of cyanobacteria as analyzed by BLASTP program of NCBI (data not shown). It is probable that horizontal gene transfer (HGT) from other organisms like proteobacteria might have occurred.

Phylogenetic analyses from Figures 12 and 13 showed that Δ9 desaturases from cyanobacteria were grouped to those from green algae and higher plants, apart from red algae, diatoms, fungi, and animals. Among cyanobacterial Δ9 desaturases, the desaturase genes acting on fatty acids esterified to the sn-1 or sn-1 and sn-2 positions of glycerolipids (b-type or a-type) were placed in a basal position, while desaturase genes acting on fatty acids esterified to the sn-2 position of glycerolipids (c-type) were in the exoteric position, which indicates that a-type or b-type Δ9 desaturases may be ancestral to c-type desaturase.

3.3. Discovery of Candidate Genes for Δ12/ω3 Desaturases

Observation on the phylogenetic tree of different membrane desaturases showed that Δ12 desaturases and Δ15 desaturases fell into the same clade (Figures 12 and 13), thus were analyzed together. As could be seen in Figures 8 and 9, the Δ12/ω3 desaturase homologs from cyanobacteria were classified into five different clades.

Figure 8.

Figure 8

Neighbor-joining tree of Δ12 and Δ15 homologous genes of cyanobacteria and eukaryotic algae. About 300 positions spanning the three histidine-boxes were employed. Colored branches indicate different groups of proteins. Red: Clade 1, green: Clade 2, magenta: Clade 3, blue: Clade 4, orange: Clade 5. Sequences from 37 sequenced cyanobacterial genomes are shown by their acronyms and accession numbers (locus tags). Other sequences are shown by their accession numbers, labels, and strain names. Desaturase genes that have been functionally characterized are indicated on the tree by their labels. Bootstrap values from neighbor-joining analyses are listed to the left of each node, with values more than 50 are shown.

Figure 9.

Figure 9

Minimum-evolution tree of Δ12 and Δ15 homologous genes of cyanobacteria and eukaryotic algae. About 300 positions spanning the three histidine-boxes were employed. Colored branches indicate different groups of proteins. Red: Clade 1, green: Clade 2, magenta: Clade 3, blue: Clade 4, orange: Clade 5. Sequences from 37 sequenced cyanobacterial genomes are shown by their acronyms and accession numbers (locus tags). Other sequences are shown by their accession numbers, labels, and strain names. Desaturase genes that have been functionally characterized are indicated on the tree by their labels. Bootstrap values from minimum-evolution analyses are listed to the left of each node, with values more than 50 are shown.

It was surprising that the first clade was constituted by the Δ12 homologs of marine cyanobacteria Synechococcus, Prochlorococcus, and the microsomal Δ12 desaturases of eukaryotic algae. Moreover, three histidine-boxes of the genes from cyanobacteria were represented as AHECGH, WX2SHX2HHX3N, and HX2HH (Figure 2 and Table 3), which were similar to those of microsome-type desaturases. Two partial amino acid sequences homologous to microsome-type Δ12 desaturases were revealed in Prochlorococcus marinus MIT 9211 (ZP_01005647 and ZP_01005648). One encoded an N-terminus region and the other encoded a C-terminus region. They may represent a single gene inferred from their close chromosome location of the graft genome, thus were designated as a unique gene with the accession number ZP_01005647.

The microsomal Δ12 desaturases are members of a large class of membrane-bound enzymes that contain a tripartite histidine sequence motif and two putative membrane-spanning domains. This group of membrane-bound enzymes includes desaturases, hydroxylases, epoxygenases, acetylenases, methyl oxidases and ketolases found in animals, fungi, plants, and bacteria [2123]. The diverse reactions that these enzymes catalyze probably use a common reactive center [24]. Histidine-rich motifs are thought to form a part of the diiron center, where oxygen activation and substrate oxidation occur [25].

To further clarify the role of genes in Clade 1, anotherphylogenetic tree was constructed by neighbor-joining (NJ) and minimum-evolution (ME) methods (Figures 10 and 11). It could be seen evidently from Figures 10 and 11 that the microsomal Δ12 desaturases from higher plants and some eukaryotic algae (such as green algae, chlorella, and chlamydomonas) fell into one group with Δ12 fatty acid hydroxylase, epoxygenase, acetylenase, and conjugase, while the genes of marine cyanobacteria clustered only with diatom plastidial and microsomal Δ12 desaturases [26]. Therefore, the microsomal Δ12 desaturases of some eukaryotic algae (such as diatom) might originate from cyanobacterial orthologs in Clade 1, and possibly horizontal gene transfer might have occurred from eukaryotic algae to Synechococcus and Prochlorococcus strains.

Figure 10.

Figure 10

Neighbor-joining tree of Δ12 homologous genes of cyanobacteria, eukaryotic algae, and higher plants. About 300 positions spanning the three histidine-boxes were employed. Sequences from 37 sequenced cyanobacterial genomes are shown by their acronyms and accession numbers (locus tags). Other sequences are shown by their accession numbers, labels, and strain names. Desaturase genes that have been functionally characterized are indicated on the tree by their labels. Bootstrap values from neighbor-joining analyses are listed to the left of each node, with values more than 50 are shown.

Figure 11.

Figure 11

Minimum-evolution tree of Δ12 homologous genes of cyanobacteria, eukaryotic algae, and higher plants. About 300 positions spanning the three histidine-boxes were employed. Sequences from 37 sequenced cyanobacterial genomes are shown by their acronyms and accession numbers (locus tags). Other sequences are shown by their accession numbers, labels, and strain names. Desaturase genes that have been functionally characterized are indicated on the tree by their labels. Bootstrap values from minimum-evolution analyses are listed to the left of each node, with values more than 50 are shown.

The ω3-homologous genes of cyanobacteria and eukaryotic algae constituted the second clade. Moreover, three histidine-boxes of the genes from cyanobacteria (FVVGHDCGHXSFS, HGWRISHRTHHXNTGN, and IHHXIGTHVAHHIF) established the standard for prokaryotic Δ15 desaturase (Figure 2 and Table 3). The third clade was integrated by the Δ12 homologs of cyanobacteria and the chloroplastic Δ12 desaturases of eukaryotic algae. Moreover, three histidine-boxes of these genes were consistent with those of plastidial Δ12 desaturase that were represented as HDCGH, HX2HH, and HXPHH.

The homologous genes from Clade 4 also had three histidine-motifs (FSLMHDCGHXSLF, WSX2HAXHHX2NG, and HX2HHLXERIPNYXL) (Figure 2 and Table 3) that were similar to those of the Δ12 desaturase. As shown in Figures 12 and 13, the genes of this clade clustered with Bacillus subtilis Δ5 desaturase. Aguilar et al. (1998) demonstrated that Bacillus subtilis possessed a single desaturase. Expression of the gene in Escherichia coli resulted in desaturation of palmitic acid moieties of the membrane phospholipids to give the novel mono-UFA cis-5-hexadecenoic acid, indicating that the gene product was a Δ5 acyl-lipid desaturase [27]. However, it is well known from freshwater cyanobacteria that only four distinct desaturases, Δ9, Δ12, Δ15, and Δ6, exist in cyanobacterial cells. Therefore, the relatively close phylogenetic relationship between genes of Clade 4 and Δ5 desaturase gene of Bacillus subtilis may be due to horizontal gene transfer and the function of these genes would require further work to fully characterize.

Three genes from Nostoc punctiforme ATCC 29133, two genes from Cyanothece sp. CCY0110, and one gene from Synechocystis sp. PCC 6803, Crocosphaera watsonii WH 8501, Lyngbya sp. PCC 8106 constituted the fifth clade. It has been proven by experiments that there is only one Δ12 desaturase in Synechocystis sp. PCC 6803 [13]. Additionally, the three histidine-motifs of these genes were HXXXH, HXXXHH, HXXHH, among which the amounts of residues between histidines from the second histidine-box were three, while that of known cyanobacterial Δ12 desaturase were two (HXXXH, HXXHH, HXXHH). Therefore, in our analysis they are assigned as hypothetical proteins and their functions need to be further investigated.

As indicated by Figures 12 and 13, the Δ12/ω3 desaturase subfamily was integrated by two main groups. Group 1 included the Δ12 desaturases from Synechococcus, Prochlorococcus and Δ5 desaturase from Bacillus subtilis. In Group 2, the Δ12 desaturases of cyanobacteria and the chloroplastic Δ12 desaturases of green algae, higher plants were in the basal position, leading to Cluster 1. In Cluster 2, the microsomal Δ12 desaturases of fungi, green algae, and higher plants set apart from Δ12 desaturases of Synechococcus, Prochlorococcus, Cyanidioschyzon merolae, Ostreococcus, Thalassiosira pseudonana, and Phaeodactylum tricorutum. Cluster 3 included the ω3 desaturases of cyanobacteria at the basal position, ω3 desaturases of green algae and both microsomal and chloroplastic ω3 desaturases of higher plants. Thus, the plastidial Δ12 desaturases are ancestral to the ω3 and microsomal Δ12 desaturases, and the ω3 desaturase of higher plants and green algae arose by independent gene duplication events from prokaryotic ω3 desaturase [28].

3.4. Discovery of Candidate Genes for Δ6 Desaturases

The “front-end” desaturases (Δ4, Δ5, Δ6, and Δ8 desaturases) formed a separate clade, and their phylogeny is complicated (Figures 12 and 13). It has been speculated that front-end desaturases may have the same origin, but their precise lineages are still unclear. There were just four prokaryotic Δ6 desaturases found from cyanobacterial genomes in our analysis: Synechocystis sp. PCC 6803 (NP_441824), Cyanothece sp. CCY0110 (ZP_01727982), Lyngbya sp. PCC 8106 (ZP_01619238), Nodularia spumigena CCY9414 (ZP_01632618), among which the function and molecular characteristics of Δ6 acyl-lipid desaturases from Synechocystis sp. PCC 6803 had been fully analyzed [13].

3.5. Occurrence and Phyletic Distribution of Fatty Acid Desaturases in Thirty Seven Cyanobacteria

In this study, thirty one unicellular and six filamentous cyanobacterial genomes were searched by bioinformatic approach for the putative fatty acid desaturases involved in polyunsaturated fatty acid synthesis. 193 protein sequences were obtained from the 37 cyanobacterial genomes, 120 of which were annotated as fatty acid desaturase. The pathway of acyl-lipid desaturation and the distribution of desaturases among different cyanobacterial species were speculated and summarized in Figures 14 and 15. Among these cyanobacteria, the Δ9 desaturase existed in 37 species of cyanobacteria. The Δ12, Δ15 and Δ6 desaturases existed in 31, 9, and 4 species of cyanobacteria, respectively. Based on functional criteria and the position of the clade integrated by Δ9 desaturases, Δ9 desaturase is assumed to be the ancestor of the remaining desaturases [28]. The functions performed by the latter three desaturases could have been obtained in some organisms along the evolutionary lineages.

Figure 14.

Figure 14

Diversity of different enzymes in thirty seven cyanobacteria. Distributions and amounts of different enzymes are marked by colors. One: red, two: green, three: magenta, four: orange. Names of nitrogen-fixing strains are marked in red. “HypoPr” represents hypothetical protein.

Figure 15.

Figure 15

The acyl-lipid desaturation of fatty acids in cyanobacteria. Numbers around arrowhead indicate the positions at which a double bond is introduced. Δ9a : desaturation occurring on both the sn-1 and the sn-2 positions of glycerolipids, Δ9b: desaturation occurring on the sn-1 position of glycerolipids, Δ9c : desaturation occurring on the sn-2 position of glycerolipids, Δ9d: genes with desaturation sn-position of glycerolipids unspecified. Δ12a : Clade 3 of Δ12 homologous genes, Δ12b: Clade 1 of Δ12 homologous genes, Δ12c : Clade 4 of Δ12 homologous genes.

Twenty seven of the investigated cyanobacteria come from the marine environment. These are 11 unicellular Prochlorococcus strains, 11 unicellular marine Synechococcus strains, Cyanothece sp. CCY0110, Crocosphaera watsonii WH 8501, Trichodesmium erythraeum IMS101, Lyngbya sp. PCC 8106, and Nodularia spumigena CCY9414. The other strains are from freshwater, soil, rock, hot spring, or symbiont.

In the 16S rRNA tree, marine Synechococcus and Prochlorococcus make a monophyletic group supported by a comparatively high-statistical confidence value, 100% (Figure 1). The two genera are proposed to diverge from a common phycobilisome-containing ancestor. While marine Synechococcus still uses phycobilisomes as light-harvesting antennae, members of the Prochlorococcus genus lack phycobilisomes and use a different antenna complex that possesses derivatives of chlorophyll a and b. They are the dominant picophytoplankton in the world’s open oceans. Carbon fixation is dominated by them and together they have been shown to contribute between 32 and 80% of the primary production in oligotrophic oceans [2932]. Synechococcus are distributed ubiquitously throughout oceanic regions, ranging from polar through temperate to tropical waters and are generally more abundant in nutrient-rich surface waters than oligotrophic areas, whilst Prochlorococcus are largely confined to a 40°N∼40°S latitudinal band, being generally absent from brackish or well-mixed waters. Prochlorococcus also generally extend deeper in the water column than Synechococcus [33, 34].

Prochlorococcus have been divided into two genetically and physiologically distinct groups: high- and low-B/A ecotypes, which were originally named for their difference in optimal growth irradiance (low- and high-light adapted, resp.) [35, 36]. High-B/A isolates, with larger ratios of chl b/a 2, are able to grow at extremely low irradiances (less than 10 umol of quanta [Q] m−2  s−1) and preferentially thrive at the bottom of the euphotic zone (80–200 m) at dimmer light but in a nutrient-rich environment [37, 38]. Low-B/A isolates, have lower chl b/a 2 ratios, are able to grow maximally at higher light intensities, and occupy the upper, well illuminated but nutrient-poor 100-m layer of the water column [37, 38]. In the 16S rRNA tree, high-light-adapted Prochlorococcus sp. arises from a low-light-adapted clade (Figure 1). Prochlorococcus marinus strains AS9601, MIT 9312, MIT 9301, MIT 9515, and CCMP1986 belong to low-B/A ecotype. Their genome sizes vary from 1.6 Mb to 1.7 Mb, smaller than that of the low light-adapted strains (1.7 Mb to 2.6 Mb). They all contain two types of desaturases, one Δ9 desaturases and two Δ12 desaturases (b-type and c-type). Strains NATL1A, NATL2A, MIT 9211, CCMP1375, MIT 9303, and MIT 9313 belong to high-B/A ecotype. Only b-type Δ12 desaturase exists in strain NATL1A, NATL2A, and MIT 9211; while two Δ9 desaturases exist in strain MIT 9303 and MIT 9313, which have larger genome size (2.6 Mb and 2.4 Mb) compared to other high-B/A ecotypes.

The marine Synechococcus isolates have themselves been classified into three groups, designated marine cluster -A, -B, and -C (MC-A, MC-B, MC-C), based on the composition of the major light harvesting pigments, an ability to perform a novel swimming motility, whether they have an elevated salt requirement for growth, and G+C content [39]. The marine cluster A group (mol% G+C = 55–62), phycoerythrin-containing strains, has an elevated salt (Na+, Cl, Mg2+ and Ca2+) requirement for growth and occur abundantly within the euphotic zone of both open-ocean and coastal waters [4044]. This cluster is additionally diverse in that ratios of phycourobilin to phycoerythrobilin chromophores differ among phycoerythrins of different strains [45, 46]. The marine cluster B (mol% G+C = 63–69.5) includes halotolerant strains that possess phycocyanin but lack phycoerythrin and appear confined to coastal waters. A further cluster, marine cluster C (MC-C) has been distinguished by its low % G+C (47.5–49.5) containing strains from brackish or coastal marine waters [39]. These latter environments have been relatively poorly studied so far and are likely underrepresented in cultured Synechococcus isolates [33]. The b-type Δ12 desaturase only exists in strains WH 7803, WH 7805, WH 8102, and CC9605. Except for strains RS9916 and CC9605, other strains all contain c-type Δ12 desaturase, two copies of which exist in strain WH 5701 (MC-B) whose genome (30 Mb) is larger than other Synechococcus strains (22 Mb–26 Mb). The unique characteristics can be observed in strain RS9916 that contains only Δ9 fatty acid desaturase.

The pathway of acyl-lipid desaturation for marine cyanobacteria Prochlorococcus and Synechococcus differs obviously from that of other cyanobacteria, indicating the different phylogenetic histories of the two genera from other cyanobacteria. At present, few fatty acid composition of these unicellular cyanobacteria has been determined yet, as functionally characterized genes. Therefore, the analysis on fatty acids in these cyanobacteria should provide more meaningful information for further research.

The two closely related freshwater Synechococcus elongatus strains PCC 6301 and PCC 7942 branch outside the marine picophytoplankton group (Figure 1), which suggests that marine cyanobacteria may diverge from the freshwater cyanobacterial ancestor. The gene arrangement and nucleotide sequence of Synechococcus elongatus PCC 6301 are nearly identical to those of Synechococcus elongatus PCC 7942, except for the presence of a 188.6 kb inversion. Genome-wide screening only recognizes one a-type Δ9 desaturase in these two strains.

Three thermophilic unicellular strains, Thermosynechococcus elongatus BP-1 and two Synechococcus Yellowstone species, are most closely related to Gloeobacter violaceus sp. PCC 7421, and phylogenetically distinct from other cyanobacterial lineages (Figure 1). They were all isolated from the hot spring. Additionally, the latter two thermophilic strains are capable of N2 fixation with a diurnal rhythm. Genes for three types of fatty acid desaturases (desA, desB, and desD) are missing in contrast with mesophilic Synechocystis, although the fourth type (desC) is found in Synechococcus and Thermosynechococcus elongtus. This agrees with the absence of highly unsaturated fatty acids in lipids, which are popular in many thermophiles [47]. Synechococcus sp. JA-2-3B′a(2-13) as well as JA-3-3Ab contains one c-type Δ9 desaturase, whereas Thermosynechococcus elongtus contains three copies, one c-type and two unspecified types. At lower temperatures, cyanobacteria desaturate the fatty acids of membrane lipids to compensate for the decrease in membrane fluidity [48]. While at higher temperatures, the membrane fluidity increased, it is unnecessary to desaturate the fatty acids of membrane lipids to produce more unsaturated fatty acids. So the thermophilic strains lack highly unsaturated fatty acids in lipids and contain only one Δ9 desaturase in contrast with mesophilic strains, which probably due to their thermic habitats.

Gloeobacter violaceus sp. PCC 7421 was originally isolated from calcareous rock in Switzerland [49, 50]. It is an unusual unicellular cyanobacterium for the absence of thylakoid membranes, and its phycobilisomes and photosystem reaction centers are localized in the plasma membrane [51, 52]. It is also remarkable that Sulfoquinovosyl diacylglycerol (SQDG), which is thought to have an important role in photosystem stabilization, is absent in Gloeobacter while the content of polyunsaturated fatty acids (PUFA) is high [53]. The data of the fatty acid composition of Gloeobacter violaceus are few in number and contradictory. In one case, linoleic and α-linolenic acids were found [53]. In other work, linoleic and γ-linolenic acids were identified [54]. The occurrence of α-linolenic or γ-linolenic acid confirms that there must be a gene in the strain that is functionally similar to the ω3 desaturase or Δ6 desaturase. Two types of desaturases, six Δ9 desaturases (two c-types and four unspecified types) and two Δ12 desaturases (a-type), were recognized from this strain. One hypothetical protein (NP_923117) was also found, but the three histidine-motifs of it (HDAGH, HNQLHH, HTAHH) did not agree with the standards for a front-end or ω3 desaturase. It is this protein or another protein that performs the same function as the front-end or ω3 desaturase, which need further investigation. The types and amounts of desaturases in Gloeobacter violaceus sp. PCC 7421 are distinct to those of other cyanobacteria (Figure 14). This result may accord with the conclusion that this organism is one of the earliest ones that diverged from the cyanobacterial line [55].

Nine of the 37 cyanobacteria studied here are known to fix nitrogen (Figure 1). Four Nostocales, Nostoc punctiforme ATCC 29133, Anabaena sp. PCC 7120, Anabaena variabilis ATCC 29413, and Nodularia spumigena CCY9414, are heterocyst-forming filamentous diazotroph; the other five are nonheterocystous nitrogen fixers, which are filamentous strains Trichodesmium erythraeum IMS101, Lyngbya sp. PCC 8106, unicellular strains Crocosphaera watsonii WH 8501, Cyanothece sp. CCY0110 along with thermophic Synechococcus strains JA-2-3B′a(2-13) and JA-3-3Ab.

The diazotrophic filamentous cyanobacteria, which can form terminally differentiated, nondividing heterocysts in response to nitrogen deprivation and the ensuing intracellular accumulation of 2-oxoglutarate [56], have almost the largest genome sizes (53 Mb–90 Mb) and are isolated from soil (Anabaena PCC7120), from fresh water (Anabaena variabilis ATCC 29413), from a plant-cyanobacterial symbionsis (Nostoc punctiforme PCC73102), or from the surface of Baltic sea (Nodularia spumigena CCY9414). Three types of desaturases (Δ9, Δ12, and Δ15) exist in Anabaena sp. PCC 7120, Anabaena variabilis ATCC 29413, and Nostoc punctiforme ATCC 29133, with the exception that Nodularia spumigena CCY9414 contains four types of desaturases (Δ9, Δ12, Δ15, and Δ6). Moreover, phylogenetic analysis shows that the desaturase genes of the same type all cluster together for these four strains, indicating a recent common ancestor for Anabaena and Nostoc [57].

Trichodesmium erythraeum IMS101 and Lyngbya sp. PCC 8106, which belong to the Oscillatoriales, both fix N2 and do not form heterocysts (Figure 1). Trichodesmium, but not Lyngbya, is known to fix nitrogen in differentiated cells called diazocytes. Like heterocysts, diazocytes are the exclusive carriers of nitrogenase and fix nitrogen aerobically in the light, and show morphological and physiological changes [58].

Unicellular strains Crocosphaera watsonii WH 8501, Cyanothece sp. CCY0110, and Synechocystis sp. PCC 6803 belong to the Chroococcaces (Figure 1), among which the former two strains fix nitrogen presumably at night while growing photosynthetically during the day. Three types of desaturases (Δ9, Δ12, and Δ15) exist in Crocosphaera watsonii WH 8501 and Trichodesmium erythraeum, while four types of desaturases (Δ9, Δ12, Δ15, and Δ6) exist in Lyngbya sp. PCC 8106, Cyanothece sp. CCY0110 and Synechocystis sp. PCC 6803. It is worth noting that the c-type Δ12 desaturase is identified exclusively in Crocosphaera watsonii WH 8501, which may be due to horizontal gene transfer (HGT) from marine cyanobacteria Prochlorococcus and Synechococcus.

In conclusion, the filamentous or N2-fixing cyanobacteria usually possess more types of fatty acid desaturases than unicellular species. The main role of fatty acid desaturase of cyanobacteria is to modulate the fluidity of membranes, which helps to improve tolerance to physiological stressors such as low temperature, high light-induced photoinhibition, salt-induced damage, or desiccation. Thus, the amounts and types of fatty acid desaturases are various among different cyanobacterial species. This evolution scheme might have formed under the force adapting to distinct environments.

Acknowledgments

This work was supported by the Key Innovative Project of Chinese Academy of Science (KZCX2-YW-209, KZCX2-YW-216), Hi-Tech Research and Development Program (2006AA090303) of China, and the CAS/SAFEA International Partnership Program for Creative Research Teams (Research and Applications of Marine Functional Genomics). Xiaoyuan Chi and Qingli Yang contributed equally to this paper.

References

  • 1.Chapman D. Phase transitions and fluidity characteristics of lipids and cell membranes. Quarterly Reviews of Biophysics. 1975;8(2):185–235. doi: 10.1017/s0033583500001797. [DOI] [PubMed] [Google Scholar]
  • 2.Singh SC, Sinha RP, Häder DP. Role of lipids and fatty acids in stress tolerance in cyanobacteria. Acta Protozoologica. 2002;41(4):297–308. [Google Scholar]
  • 3.Scanlan D. Cyanobacteria: ecology, niche adaptation and genomics. Microbiology Today. 2001;28:128–130. [Google Scholar]
  • 4.Bryant DA, editor. The Molecular Biology of Cyanobacteria. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1994. [Google Scholar]
  • 5.Sato N, Murata N, Miura Y, Ueta N. Effect of growth temperature on lipid and fatty acid compositions in the blue-green algae, Anabaena variabilis and Anacystis nidulans . Biochimica et Biophysica Acta. 1979;572(1):19–28. [PubMed] [Google Scholar]
  • 6.Sato N, Murata N. Studies on the temperature shift induced desaturation of fatty acids in monogalactosyl diacylglycerol in the blue-green alga (cyanobacterium), Anabaena variabilis . Plant and Cell Physiology. 1981;22:1043–1050. [Google Scholar]
  • 7.Wada H, Murata N. Temperature-induced changes in the fatty acid composition of the cyanobacterium, Synechocystis PCC6803. Plant Physiology. 1990;92(4):1062–1069. doi: 10.1104/pp.92.4.1062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Murata N, Wada H. Acyl-lipid desaturases and their importance in the tolerance and acclimatization to cold of cyanobacteria. Biochemical Journal. 1995;308(1):1–8. doi: 10.1042/bj3080001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Stubbs CD, Smith AD. The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochimica et Biophysica Acta. 1984;779(1):89–137. doi: 10.1016/0304-4157(84)90005-4. [DOI] [PubMed] [Google Scholar]
  • 10.Coolbear KP, Berde CB, Keough KMW. Gel to liquid-crystalline phase transitions of aqueous dispersions of polyunsaturated mixed-acid phosphatidylcholines. Biochemistry. 1983;22(6):1466–1473. doi: 10.1021/bi00275a022. [DOI] [PubMed] [Google Scholar]
  • 11.Han T, Sinha RP, Häder DP. UV-A/blue light-induced reactivation of photosynthesis in UV-B irradiated cyanobacterium, Anabaena sp. Journal of Plant Physiology. 2001;158(11):1403–1413. [Google Scholar]
  • 12.Nishiyama Y, Yamamoto H, Allakhverdiev SI, Inaba M, Yokota A, Murata N. Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery. EMBO Journal. 2001;20(20):5587–5594. doi: 10.1093/emboj/20.20.5587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Tasaka Y, Gombos Z, Nishiyama Y, et al. Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis. EMBO Journal. 1996;15(23):6416–6425. [PMC free article] [PubMed] [Google Scholar]
  • 14.Gombos Z, Kanervo E, Tsvetkova N, Sakamoto T, Aro E-M, Murata N. Genetic enhancement of the ability to tolerate photoinhibition by introduction of unsaturated bonds into membrane glycerolipids. Plant Physiology. 1997;115(2):551–559. doi: 10.1104/pp.115.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Murata N, Wada H, Gombos Z. Modes of fatty-acid desaturation in cyanobacteria. Plant and Cell Physiology. 1992;33:933–941. [Google Scholar]
  • 16.Kaneko T, Sato S, Kotani H, et al. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Research. 1996;3(3):109–136. doi: 10.1093/dnares/3.3.109. [DOI] [PubMed] [Google Scholar]
  • 17.Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research. 1994;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution. 2007;24(8):1596–1599. doi: 10.1093/molbev/msm092. [DOI] [PubMed] [Google Scholar]
  • 19.Chintalapati S, Prakash JSS, Gupta P, et al. A novel Δ9 acyl-lipid desaturase, DesC2, from cyanobacteria acts on fatty acids esterified to the sn-2 position of glycerolipids. Biochemical Journal. 2006;398(2):207–214. doi: 10.1042/BJ20060039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Edwards DJ, Marquez BL, Nogle LM, et al. Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya majuscula . Chemistry & Biology. 2004;11(6):817–833. doi: 10.1016/j.chembiol.2004.03.030. [DOI] [PubMed] [Google Scholar]
  • 21.Shanklin J, Cahoon EB. Desaturation and related modifications of fatty acids. Annual Review of Plant Biology. 1998;49:611–641. doi: 10.1146/annurev.arplant.49.1.611. [DOI] [PubMed] [Google Scholar]
  • 22.Broadwater JA, Whittle E, Shanklin J. Desaturation and hydroxylation. Residues 148 and 324 of Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of catalytic specificity. Journal of Biological Chemistry. 2002;277(18):15613–15620. doi: 10.1074/jbc.M200231200. [DOI] [PubMed] [Google Scholar]
  • 23.Lee M, Lenman M, Banaś A, et al. Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation. Science. 1998;280(5365):915–918. doi: 10.1126/science.280.5365.915. [DOI] [PubMed] [Google Scholar]
  • 24.van de Loo FJ, Fox BG, Somerville C. Unusual fatty acids. In: Moore TS, editor. Lipid Metabolism in Plants. BocaRaton, Fla, USA: CRC Press; 1993. pp. 91–126. [Google Scholar]
  • 25.Shanklin J, Achim C, Schmidt H, Fox BG, Münck E. Mössbauer studies of alkane ω-hydroxylase: evidence for a diiron cluster in an integral-membrane enzyme. Proceedings of the National Academy of Sciences of the United States of America. 1997;94(7):2981–2986. doi: 10.1073/pnas.94.7.2981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Domergue F, Spiekermann P, Lerchl J, et al. New insight into Phaeodactylum tricornutum fatty acid metabolism. Cloning and functional characterization of plastidial and microsomal Δ12-fatty acid desaturases. Plant Physiology. 2003;131(4):1648–1660. doi: 10.1104/pp.102.018317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Aguilar PS, Cronan JE, Jr., De Mendoza D. A Bacillus subtilis gene induced by cold shock encodes a membrane phospholipid desaturase. Journal of Bacteriology. 1998;180(8):2194–2200. doi: 10.1128/jb.180.8.2194-2200.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.López Alonso D, García-Maroto F, Rodríguez-Ruiz J, Garrido JA, Vilches MA. Evolution of the membrane-bound fatty acid desaturases. Biochemical Systematics and Ecology. 2003;31(10):1111–1124. [Google Scholar]
  • 29.Goericke R, Welschmeyer NA. The marine prochlorophyte Prochlorococcus contributes significantly to phytoplankton biomass and primary production in the Sargasso Sea. Deep Sea Research Part I. 1993;40(11-12):2283–2294. [Google Scholar]
  • 30.Li WK. Composition of ultraphytoplankton in the central north Atlantic. Marine Ecology Progress Series. 1995;122(1–3):1–8. [Google Scholar]
  • 31.Liu H, Nolla HA, Campbell L. Prochlorococcus growth rate and contribution to primary production in the equatorial and subtropical North Pacific Ocean. Aquatic Microbial Ecology. 1997;12(1):39–47. [Google Scholar]
  • 32.Veldhuis MJW, Kraay GW, Van Bleijswijk JDL, Baars MA. Seasonal and spatial variability in phytoplankton biomass, productivity and growth in the northwestern Indian ocean: the southwest and northeast monsoon, 1992-1993. Deep Sea Research Part I. 1997;44(3):425–449. [Google Scholar]
  • 33.Scanlan DJ, West NJ. Molecular ecology of the marine cyanobacterial genera Prochlorococcus and Synechococcus . FEMS Microbiology Ecology. 2002;40(1):1–12. doi: 10.1111/j.1574-6941.2002.tb00930.x. [DOI] [PubMed] [Google Scholar]
  • 34.Partensky F, Hess WR, Vaulot D. Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiology and Molecular Biology Reviews. 1999;63(1):106–127. doi: 10.1128/mmbr.63.1.106-127.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Rocap G, Moore LR, Chisholm SW. Molecular phylogeny of Prochlorococcus ecotypes. In: Charpy L, Larkum AWD, editors. Marine Cyanobacteria. Monaco, France: Bulletin de l'Institut Océanographique; 1999. pp. 107–116. [Google Scholar]
  • 36.Moore LR, Rocap G, Chisholm SW. Physiology and molecular phytogeny of coexisting Prochlorococcus ecotypes. Nature. 1998;393(6684):464–467. doi: 10.1038/30965. [DOI] [PubMed] [Google Scholar]
  • 37.Moore LR, Chisholm SW. Photophysiology of the marine cyanobacterium Prochlorococcus: ecotypic differences among cultured isolates. Limnology and Oceanography. 1999;44(3):628–638. [Google Scholar]
  • 38.Dufresne A, Salanoubat M, Partensky F, et al. Genome sequence of the cyanobacterium Prochlorococcus marinus SS120, a nearly minimal oxyphototrophic genome. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(17):10020–10025. doi: 10.1073/pnas.1733211100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Waterbury JB, Rippka R. Subsection 1. Order Croococcales Wettsten 1924, emend. Rippka et al., 1979. In: Staley JT, Bryant MP, Pfenning N, Holt JG, editors. Bergey's Manual of Systematic Bacteriology. Vol. 3. Baltimore, Md, USA: Williams and Wilkins; 1989. pp. 1728–1746. [Google Scholar]
  • 40.Ferris MJ, Palenik B. Niche adaptation in ocean cyanobacteria. Nature. 1998;396(6708):226–228. [Google Scholar]
  • 41.Partensky F, Blanchot J, Vaulot D. Differential distribution and ecology of Prochlorococcus and Synechococcus in oceanic waters: a review. Bulletin de l'Institut Oceanographique. 1999;19:457–475. [Google Scholar]
  • 42.Uysal Z. Pigments, size and distribution of Synechococcus spp. in the Black Sea. Journal of Marine Systems. 2000;24(3-4):313–326. [Google Scholar]
  • 43.Wilmotte A, Demonceau C, Goffart A, Hecq J-H, Demoulin V, Crossley AC. Molecular and pigment studies of the picophytoplankton in a region of the Southern Ocean (42–54∘S, 141–144∘E) in March 1998. Deep Sea Research Part II. 2002;49(16):3351–3363. [Google Scholar]
  • 44.Wood AM, Phinney DA, Yentsch CS. Water column transparency and the distribution of spectrally distinct forms of phycoerythrin-containing organisms. Marine Ecology Progress Series. 1998;162:25–31. [Google Scholar]
  • 45.Wood AM, Horan PK, Muirhead K, Phinney DA, Yentsch CM, Waterbury JB. Discrimination between types of pigments in marine Synechococcus sp. by scanning spectroscopy, epifluorescence microscopy, and flow cytometry. Limnology and Oceanography. 1985;30:1303–1315. [Google Scholar]
  • 46.Waterbury JB, Watson SW, Valois FW, Franks DG. Biological and ecological characterisation of the marine unicellular cyanobacterium Synechococcus . Canadian Bulletin of Fisheries and Aquatic Sciences. 1986;214:71–120. [Google Scholar]
  • 47.Nakamura Y, Kaneko T, Sato S, et al. Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. DNA Research. 2002;9(4):123–130. doi: 10.1093/dnares/9.4.123. [DOI] [PubMed] [Google Scholar]
  • 48.Murata N, Nishida I. Lipids of blue-green algae (cyanobacteria) In: Stumpf PK, editor. The Biochemistry of Plants. San Diego, Calif, USA: Academic Press; 1987. pp. 315–347. [Google Scholar]
  • 49.Rippka R, Waterbury J, Cohen-Bazire G. A cyanobacterium which lacks thylakoids. Archives of Microbiology. 1974;100(4):419–436. [Google Scholar]
  • 50.Rippka R, Herdman M. Pasteur Culture Collection (PCC) of Cyanobacterial Strains in Axenic Culture. Vol. 1. Paris, France: Institute Pasteur; 1992. (Catalogue of Strains). [Google Scholar]
  • 51.Nakamura Y, Kaneko T, Sato S, et al. Complete genome structure of Gloeobacter violaceus PCC 7421, a cyanobacterium that lacks thylakoids. DNA Research. 2003;10(4):137–145. doi: 10.1093/dnares/10.4.137. [DOI] [PubMed] [Google Scholar]
  • 52.Guglielmi G, Cohen-Bazire G, Bryant DA. The structure of Gloeobacter violaceus and its phycobilisomes. Archives of Microbiology. 1981;129(3):181–189. [Google Scholar]
  • 53.Selstam E, Campbell D. Membrane lipid composition of the unusual cyanobacterium Gloeobacter violaceus sp. PCC 7421, which lacks sulfoquinovosyl diacylglycerol. Archives of Microbiology. 1996;166(2):132–135. [Google Scholar]
  • 54.Rippka R, Waterbury J, Cohen-Bazire G. A cyanobacterium which lacks thylakoids. Archives of Microbiology. 1974;100(4):419–436. [Google Scholar]
  • 55.Honda D, Yokota A, Sugiyama J. Detection of seven major evolutionary lineages in cyanobacteria based on the 16S rRNA gene sequence analysis with new sequences of five marine Synechococcus strains. Journal of Molecular Evolution. 1999;48(6):723–739. doi: 10.1007/pl00006517. [DOI] [PubMed] [Google Scholar]
  • 56.Zhang C-C, Laurent S, Sakr S, Peng L, Bédu S. Heterocyst differentiation and pattern formation in cyanobacteria: a chorus of signals. Molecular Microbiology. 2006;59(2):367–375. doi: 10.1111/j.1365-2958.2005.04979.x. [DOI] [PubMed] [Google Scholar]
  • 57.McGuire RF. A numerical taxonomic study of Nostoc and Anabaena . Journal of Phycology. 1984;20:454–460. [Google Scholar]
  • 58.El-Shehawy R, Lugomela C, Ernst A, Bergman B. Diurnal expression of hetR and diazocyte development in the filamentous non-heterocystous cyanobacterium Trichodesmium erythraeum . Microbiology. 2003;149(5):1139–1146. doi: 10.1099/mic.0.26170-0. [DOI] [PubMed] [Google Scholar]

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