# Comparative Analysis of Fatty Acid Desaturases in  Cyanobacterial Genomes

Authors: Xiaoyuan Chi; Qingli Yang; Fangqing Zhao; Song Qin; Yu Yang; Junjun Shen; Hanzhi Lin
Journal: Comparative and Functional Genomics
 (2008)

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## 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 orN2-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.


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## Body

## 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 [5–7]. 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 thesn 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 cyanobacteriumSynechocystis
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
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
Section2 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 generaSynechocystis, 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.
SpeciesLocus tagAccessionDNA coordinatesLengthProposed functionAnabaena sp. PCC 7120all4991NP_4890315963080⋯5963937857d9all1599NP_4856391879629⋯1880447818d9all1598NP_4856381878346⋯18793981052d12all1597NP_4856371876897⋯18779761079d15alr3189NP_4872293858986⋯3859762776crtWalr4009NP_4880494829483⋯4830322839crtRAnabaena variabilis ATCC 29413Ava_2277YP_3227902832413⋯2833270857d9Ava_4212YP_3247065282348⋯5283166818d9Ava_4211YP_3247055281066⋯52821181052d12Ava_4210YP_3247045279614⋯52806931079d15Ava_2048YP_3225652535646⋯2536410764crtWAva_3888YP_3243884842189⋯4842965776crtWAva_1693YP_3222102121129⋯2122049920crtRCrocosphaera
watsonii WH 8501CwatDRAFT_1377ZP_005181703068⋯3892824d9CwatDRAFT_3226ZP_0051684322017⋯230661049d12CwatDRAFT_5150ZP_00515010150888⋯1519821049d12CwatDRAFT_3625ZP_0051618110760⋯118091049d15CwatDRAFT_1857ZP_005177001398⋯2231834hypothetical
proteinCwatDRAFT_5424ZP_00514501315629⋯316522893crtRGloeobacter
violaceus strain PCC 7421gvip390NP_9258123057506⋯3058357851d9gvip170NP_9241811312274⋯1313095822d9gll1946NP_9248922071551⋯2072504953d9gll1947NP_9248932072509⋯2073507998d9gll1938NP_9248842060880⋯2061839959d9gll1940NP_9248862063884⋯2064876992d9gvip364NP_9255692779580⋯27806381058d12gvip506NP_9266813944843⋯39459101058d12gll0171NP_923117161268⋯1624401173hypothetical
proteingll2501NP_9254472660474⋯26614751001mocDgvip239NP_9246741833712⋯1834485773crtWNostoc
punctiforme ATCC 29133(PCC 73102)Npun02000467ZP_00345918175651⋯176532881d9Npun02005010ZP_0010858241108⋯41929821d9Npun02005011ZP_0010858342265⋯433261061d12Npun02005012ZP_0010858443524⋯446031080d15Npun02001904ZP_0034576563255⋯643101056hypothetical
proteinNpun02001905ZP_0011089064537⋯655741038hypothetical
proteinNpun02002344ZP_0011054977763⋯788631101hypothetical
proteinNpun02003462ZP_0010937176020⋯76964945mocDNpun02000865ZP_00345866139810⋯140571762crtWNpun02001326ZP_0011125855604⋯56392788crtWNpun02006805ZP_0010683223657⋯24556899crtRProchlorococcus
marinus str.
NATL1ANATL1_21421YP_0010159621799954⋯1800733780d9NATL1_10821YP_001014905992775⋯9939921218d12NATL1_03151YP_001014144291853⋯2928841032crtRProchlorococcus
marinus strain NATL2APMN2A_1271YP_2924641227545⋯1228474929d9PMN2A_0393YP_291588388657⋯3898741217d12PMN2A_1603YP_2927941566557⋯15675881031crtRProchlorococcus marinus MIT 9211P9211_09157ZP_010063631417821⋯1418765944d9P9211_05577ZP_01005647779723⋯780334611d12P9211_05582ZP_01005648780304⋯780729425d12P9211_07547ZP_010060411108444⋯11094691015crtRProchlorococcus
marinus str.
MIT 9301P9301_18621YP_0010920861588713⋯1589651939d9P9301_15761YP_0010918001328773⋯13299391167d12P9301_15721YP_0010917961326076⋯13271821107d12P9301_02581YP_001090482239249⋯239974726crtRProchlorococcus
marinus str. MIT 9303P9303_28951YP_0010188902560285⋯2561250966d9P9303_28931YP_0010188882558615⋯2559535921d9P9303_14121YP_0010174241208715⋯12098001086d12P9303_21081YP_0010181081869188⋯18703301143d12P9303_24321YP_0010184282137288⋯21383281041crtRProchlorococcus
marinus str. MIT 9312PMT9312_1764YP_3982611656076⋯1657014938d9PMT9312_1476YP_3979721385670⋯13868451175d12PMT9312_1473YP_3979691382796⋯13839021106d12PMT9312_0238YP_396735229042⋯229842800crtRProchlorococcus
marinus str. MIT 9313PMT2172NP_8959962299082⋯2300002920d9PMT2174NP_8959982300938⋯2301717779d9PMT0249NP_894082278544⋯2796831139d12PMT0797NP_894629872385⋯8734701085d12PMT1816NP_8956431920323⋯19213631040crtRProchlorococcus
marinus str. AS9601A9601_18811YP_0010102711616719⋯1617657939d9A9601_15921YP_0010099821355480⋯13565141035d12A9601_15871YP_0010099771352826⋯13539321107d12A9601_02571YP_001008652238284⋯239117834crtRProchlorococcus
marinus str. MIT 9515P9515_18621YP_0010121761650943⋯1651929987d9P9515_15601YP_0010118741376566⋯13776931128d12P9515_15521YP_0010118661371646⋯13727521107d12P9515_02681YP_001010584247534⋯248433900crtRProchlorococcus marinus subsp. marinus str. CCMP1375 (SS120)Pro1833NP_8762241690865⋯1691797932d9Pro1208NP_8756001116904⋯11180161112d12Pro1214NP_8756061121144⋯11222501106d12Pro0266NP_874660261189⋯2622231034crtRProchlorococcus marinus subsp. marinus str. CCMP1986 (MED4)PMM1672NP_8937891604745⋯1605731986d9PMM1382NP_8934991331162⋯13323401178d12PMM1378NP_8934951325388⋯13264941106d12PMM0236/228281⋯229270989crtRSynechococcus
elongatus strain PCC 7942Synpcc7942_2561YP_4015782639146⋯2639982836d9Synpcc7942_1713YP_4007301781317⋯1782219902mocDSynpcc7942_2439YP_4014562514276⋯2515271995crtRSynechococcus
elongatus strain PCC 6301syc1549_dYP_1722591676804⋯1677640837d9Syc2378_cYP_1730882534831⋯2535691861mocDsyc1667_cYP_1723771801757⋯1802752996crtRSynechococcus sp. BL107BL107_07284ZP_01469203490784⋯491566782d9BL107_07289ZP_01469204491936⋯492721785d9BL107_06084ZP_01468963247334⋯2483561022d12BL107_14110ZP_01468055331111⋯331884773crtWBL107_08054ZP_01469357636707⋯6377381031crtRSynechococcus sp. CC9311sync_2793YP_7319812458778⋯2459710932d9sync_2791YP_7319792457075⋯2457986911d9sync_0336YP_729569344430⋯3454491019crtRsync_0396YP_729627408306⋯4095051199d12sync_1804YP_7310081621108⋯1621869761crtWSynechococcus sp. CC9605Syncc9605_2541YP_3828242358792⋯2359703911d9Syncc9605_1972YP_3822681793076⋯17942211145d12Syncc9605_0286YP_380617292821⋯2938701049crtRSynechococcus sp. CC9902Syncc9902_2191YP_3781922099771⋯2100673902d9Syncc9902_2192YP_3781932100902⋯2101825923d9Syncc9902_0141YP_376159149723⋯1507241001d12Syncc9902_0972YP_376982954015⋯954788773crtWSyncc9902_2058YP_3780591964618⋯19657301112crtRSynechococcus sp. JA-2-3B′a(2-13)CYB_0861YP_477105894187⋯895071884d9CYB_2914YP_4790963011594⋯3012520926mocDCYB_0102YP_476366118335⋯119306971crtRSynechococcus sp. JA-3-3AbCYA_2349YP_4757392357019⋯2357912893d9CYA_1931YP_4753401944066⋯1945040974crtRSynechococcus sp.
RCC307SynRCC307_2395YP_0012286512091372⋯2092274903d9SynRCC307_2393YP_0012286492089667⋯2090581915d9SynRCC307_1757YP_0012280131538507⋯15395621056d12SynRCC307_1993YP_0012282491729342⋯1730103762crtWSynRCC307_2209YP_0012284651915148⋯19161671020crtRSynechococcus sp. RS9916RS9916_36767ZP_014713841050409⋯1051341932d9RS9916_36757ZP_014713821048603⋯1049568965d9RS9916_39311ZP_01472905116650⋯1176751025crtRSynechococcus sp. RS9917RS9917_06370ZP_01079314447782⋯448705923d9RS9917_06360ZP_01079312446060⋯446992932d9RS9917_03333ZP_0108084999968⋯1010471079d12RS9917_00687ZP_0108054164826⋯65563737crtWRS9917_03663ZP_01080915166940⋯167902962crtRSynechococcus sp. WH 5701WH5701_02025ZP_01084898299319⋯300257787d9WH5701_02015ZP_01084896297579⋯298532953d9WH5701_14646ZP_01083974104382⋯1055391157d12WH5701_16535ZP_01086617164⋯11861022d12WH5701_06521ZP_0108593565353⋯66231878hypothetical proteinWH5701_02369ZP_0108432242300⋯43271971mocDWH5701_04005ZP_0108342143734⋯44519785crtWWH5701_01215ZP_01084736138584⋯1396151031crtRSynechococcus sp. WH 7803SynWH7803_2417YP_0012261402249293⋯2250087795d9SynWH7803_2415YP_0012261382247475⋯2248386912d9SynWH7803_0589YP_001224312594539⋯5956031065d12SynWH7803_1625YP_0012253481496144⋯1497139996d15SynWH7803_0928YP_001224651871421⋯872167747crtWSynWH7803_0337YP_001224060361336⋯3623371002crtRSynechococcus sp. WH 7805WH7805_10184ZP_01125021209067⋯209999932d9WH7805_10194ZP_01125023210769⋯211680911d9WH7805_06186ZP_01124768405535⋯406059524d12WH7805_04931ZP_01124517184338⋯1855161178d12WH7805_01197ZP_011237733991⋯4734743crtWWH7805_07481ZP_01123496193165⋯1941931028crtRSynechococcus sp. WH 8102SYNW2377NP_8984662286168⋯
2287028860d9SYNW0696NP_896789679330⋯6804781148d12SYNW1696NP_8977871631011⋯16321471136d12SYNW1368NP_8974611354793⋯1355527734crtWSYNW0291NP_896386291323⋯2923541031crtRSynechocystis sp. PCC 6803sll0541NP_4424302822579⋯2823535956d9slr1350NP_4414891746308⋯17473631055d12sll1441NP_4416221895520⋯18965991079d15sll0262NP_4418242120067⋯21211461079d6Sll1611NP_4412201462136⋯14632451110hypothetical proteinsll1468NP_440788981691⋯982629938crtRThermosynechococcus
elongatus strain BP-1tll1719NP_6825091800682⋯1801521839d9tlr2380NP_6831702490209⋯2491048839d9tlr1653NP_6824431733919⋯1734767848d9tlr1254NP_6820441300388⋯1301308920mocDtlr1900NP_6826901986642⋯1987529887crtRTrichodesmium
erythraeum IMS101Tery_1437YP_7212052173203⋯2174015812d9Tery_0142YP_720110207806⋯2088611055d12Tery_4492YP_7239516931402⋯69324751073d15Tery_3898YP_7234066024293⋯60253421050hypothetical proteinTery_2925YP_7225644543239⋯4544114875crtRLyngbya sp. PCC 8106L8106_03152ZP_016246782253⋯3071818d9L8106_27002ZP_0162118594912⋯959551043d12L8106_10697ZP_016245606961⋯80431082d15L8106_14825ZP_01619238100018⋯1011331115d6L8106_06180ZP_01620148172993⋯173604611hypothetical proteinL8106_18641ZP_0162427813290⋯14111821hypothetical
proteinL8106_30215ZP_0162257823391⋯24185794crtRNodularia
spumigena CCY9414N9414_19077ZP_0163181716235⋯17026791d9N9414_07494ZP_01632615317⋯1135818d9N9414_07499ZP_016326161303⋯24271124d12N9414_07504ZP_016326172618⋯36881070d15N9414_07509ZP_016326184087⋯51781091d6N9414_18293ZP_0162972629633⋯30223590hypothetical
proteinN9414_07726ZP_016323054851⋯5633782crtWN9414_01572ZP_01632726697⋯1587890crtRCyanothece sp. CCY0110CY0110_10577ZP_01726409185891⋯186724834d9CY0110_05582ZP_0172921374180⋯75004825d9CY0110_10917ZP_017324587951⋯90001050d12CY0110_00445ZP_0172854190142⋯911911050d15CY0110_24056ZP_01727982158769⋯1598871119d6CY0110_13441ZP_0172902460390⋯61220831hypothetical proteinCY0110_27283ZP_0173193415787⋯169141128hypothetical proteinCY0110_11357ZP_017292799512⋯105131002mocDCY0110_08481ZP_0173100725752⋯26747996crtRTable 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.
SpeciesAccession no/locus tagLabelAccession no/locus tagLabelArabidopsis
thalianaBAA25180d9AAB60302chld15Q949X0d7BAA05514microd15AAA92800chld12CAA11858d8NP_187819microd12Thalassiosira
pseudonanaTp22511d9AY817152d5Tp23798d12AY817155d6Tp3143d12AY817154d8AY817156d4Phaeodactylum
tricorutumAAW70158d9AY082393d6AAO23565chld12AY082392d5AY165023microd12Pt22459d5Chlamydomonas
reinhardtiiCr117883uncertainABL09485d15AB007640chld12AY860820crtWEDP04777microd12Synechococcus sp. PCC 7002AAB61353d9AAF21445d12AAF21447uncertainAAB61352d15Nostoc sp. SO-36CAF18426d9CAF18425d15CAF18423d9CAF18424d12Mortierella
alpinaCAB38177d9AAF08684d12AAF08685d6AAC39508d5Cyanidioschyzon
merolaeBAA28834d9CMK291Cd12CMJ201Cd9BAC76126crtRArthrospira
platensisCAA05166d9Q54794d12ABN11122d6OstreococcuslucimarinusOl51664uncertainOl24150d12Ol18582d12Caenorhabditis
elegansAAF97550d9AAC15586d6AAC95143d5Rattus
norvegicusNP_114029d9BAA75496d6AAG35068d5Homo
sapiensXP_005719d9AAD20018d6AAF29378d5Brassica
napusAAA50157chl d12AAF78778microd12CAA11857d8Chlorella
vulgarisAB075526microd12AB075527microd15Chlamydomonas sp. W80AB031546chld12Synechocystis sp. PCC
6714BAA02921d12Mucor
circinelloidesAAD55982d12BAB69055d6Emericella
nidulansAAG36933d12Glycine
maxBAD89862microd12Calendula
officinalisAAK26633microd12Gossypium
hirsutumAAL37484microd12Nicotiana
tabacumBAC01274chld15BAC01273microd15Brassica
junceaCAB85467chld15Picea
abiesCAC18722chld15Ricinus
communisAAA73511chld15AAC4901012-hydroxylaseTriticum
aestivumBAA28358microd15Oryza
sativaBAA11397microd15Vernicia
fordiiAAN87573microd12AAN8757412-conjugasePunica
granatumCAD24671microd12AAO3775312-conjugaseLesquerella
fendleriAAC3275512-hydroxylase/desaturasePhysaria
lindheimeriABQ0145812-hydroxylaseCrepis
palaestinaCAA7615612-epoxygenaseStokesia
laevisAAR2381512-epoxygenaseDaucus
carotaAAO3803312-acetylenaseFoeniculum
vulgareAAO3803412-acetylenaseHedera
helixAAO3803112-acetylenaseHelianthus
annuusAAO3803212-acetylenaseCAA60621d8Helichrysum
bracteatumAAO3803712-acetylenaseRudbeckia
hirtaAAO3803512-acetylenaseCrepis
alpinaCAA7615812-acetylenaseCalendula
officinalisAAK2663212-conjugaseTrichosanthes
kirilowiiAAO3775112-conjugaseAcheta
domesticusAAK25797d9Cyprinus
carpioCAB57858d9Drosophila
simulansCAB52475d9Gallus
gallusCAA42997d9Helicoverpa
zeaAAF81790d9Rosa
hybrid cultivarBAA23136d9Saccharomyces
cerevisiaeAAA34826d9Limnanthes
douglasiiAAG28599d9Prochlorothrix
hollandicaAAG16761d9Lyngbya
majusculaAAS98775d9Synechococcus
vulcanusAAD00699d9Thraustochytrium sp. ATCC21685AAM09688d4AAM09687d5Euglena
gracilisAAQ19605d4AF139720d8Pavlova
lutheriAY332747d4Isochrysis
galbana strain
CCMP1323AY630574d4Marchantia
polymorphaAAT85663d5AAT85661d6Nitzschia
closterium f. minutissimaAY603475d5Dictyostelium
discoideumBAA37090d5Bacillus
subtilisAAC38355d5Danio
rerioQ9DEX7d5/d6Borago
officinalisAAD01410d6AAG43277d8Oncorhynchus
mykissAAK26745d6Mus
musculusNP_062673d6Glossomastix
chrysoplastaAAU11444d6Ostreococcus
tauriAY746357d6Physcomitrella
patensCAA11033d6Echium
pitardiiAAL23581d6Chlorella
zofingiensisAY772713crtWCyanidium
caldariumAAB82698crtRHaematococcus
pluvialisCAA60478crtWMyxococcus
xanthus DK 1622YP_634431uncertainStigmatella
aurantiaca DW4/3-1ZP_01463016uncertainBradyrhizobium
japonicum USDA 110NP_771234uncertain


### 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.


## 2.1. Computational Search for Novel Fatty Acid Desaturase Genes

The
genomes of 37 cyanobacteria including generaSynechocystis, 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.
SpeciesLocus tagAccessionDNA coordinatesLengthProposed functionAnabaena sp. PCC 7120all4991NP_4890315963080⋯5963937857d9all1599NP_4856391879629⋯1880447818d9all1598NP_4856381878346⋯18793981052d12all1597NP_4856371876897⋯18779761079d15alr3189NP_4872293858986⋯3859762776crtWalr4009NP_4880494829483⋯4830322839crtRAnabaena variabilis ATCC 29413Ava_2277YP_3227902832413⋯2833270857d9Ava_4212YP_3247065282348⋯5283166818d9Ava_4211YP_3247055281066⋯52821181052d12Ava_4210YP_3247045279614⋯52806931079d15Ava_2048YP_3225652535646⋯2536410764crtWAva_3888YP_3243884842189⋯4842965776crtWAva_1693YP_3222102121129⋯2122049920crtRCrocosphaera
watsonii WH 8501CwatDRAFT_1377ZP_005181703068⋯3892824d9CwatDRAFT_3226ZP_0051684322017⋯230661049d12CwatDRAFT_5150ZP_00515010150888⋯1519821049d12CwatDRAFT_3625ZP_0051618110760⋯118091049d15CwatDRAFT_1857ZP_005177001398⋯2231834hypothetical
proteinCwatDRAFT_5424ZP_00514501315629⋯316522893crtRGloeobacter
violaceus strain PCC 7421gvip390NP_9258123057506⋯3058357851d9gvip170NP_9241811312274⋯1313095822d9gll1946NP_9248922071551⋯2072504953d9gll1947NP_9248932072509⋯2073507998d9gll1938NP_9248842060880⋯2061839959d9gll1940NP_9248862063884⋯2064876992d9gvip364NP_9255692779580⋯27806381058d12gvip506NP_9266813944843⋯39459101058d12gll0171NP_923117161268⋯1624401173hypothetical
proteingll2501NP_9254472660474⋯26614751001mocDgvip239NP_9246741833712⋯1834485773crtWNostoc
punctiforme ATCC 29133(PCC 73102)Npun02000467ZP_00345918175651⋯176532881d9Npun02005010ZP_0010858241108⋯41929821d9Npun02005011ZP_0010858342265⋯433261061d12Npun02005012ZP_0010858443524⋯446031080d15Npun02001904ZP_0034576563255⋯643101056hypothetical
proteinNpun02001905ZP_0011089064537⋯655741038hypothetical
proteinNpun02002344ZP_0011054977763⋯788631101hypothetical
proteinNpun02003462ZP_0010937176020⋯76964945mocDNpun02000865ZP_00345866139810⋯140571762crtWNpun02001326ZP_0011125855604⋯56392788crtWNpun02006805ZP_0010683223657⋯24556899crtRProchlorococcus
marinus str.
NATL1ANATL1_21421YP_0010159621799954⋯1800733780d9NATL1_10821YP_001014905992775⋯9939921218d12NATL1_03151YP_001014144291853⋯2928841032crtRProchlorococcus
marinus strain NATL2APMN2A_1271YP_2924641227545⋯1228474929d9PMN2A_0393YP_291588388657⋯3898741217d12PMN2A_1603YP_2927941566557⋯15675881031crtRProchlorococcus marinus MIT 9211P9211_09157ZP_010063631417821⋯1418765944d9P9211_05577ZP_01005647779723⋯780334611d12P9211_05582ZP_01005648780304⋯780729425d12P9211_07547ZP_010060411108444⋯11094691015crtRProchlorococcus
marinus str.
MIT 9301P9301_18621YP_0010920861588713⋯1589651939d9P9301_15761YP_0010918001328773⋯13299391167d12P9301_15721YP_0010917961326076⋯13271821107d12P9301_02581YP_001090482239249⋯239974726crtRProchlorococcus
marinus str. MIT 9303P9303_28951YP_0010188902560285⋯2561250966d9P9303_28931YP_0010188882558615⋯2559535921d9P9303_14121YP_0010174241208715⋯12098001086d12P9303_21081YP_0010181081869188⋯18703301143d12P9303_24321YP_0010184282137288⋯21383281041crtRProchlorococcus
marinus str. MIT 9312PMT9312_1764YP_3982611656076⋯1657014938d9PMT9312_1476YP_3979721385670⋯13868451175d12PMT9312_1473YP_3979691382796⋯13839021106d12PMT9312_0238YP_396735229042⋯229842800crtRProchlorococcus
marinus str. MIT 9313PMT2172NP_8959962299082⋯2300002920d9PMT2174NP_8959982300938⋯2301717779d9PMT0249NP_894082278544⋯2796831139d12PMT0797NP_894629872385⋯8734701085d12PMT1816NP_8956431920323⋯19213631040crtRProchlorococcus
marinus str. AS9601A9601_18811YP_0010102711616719⋯1617657939d9A9601_15921YP_0010099821355480⋯13565141035d12A9601_15871YP_0010099771352826⋯13539321107d12A9601_02571YP_001008652238284⋯239117834crtRProchlorococcus
marinus str. MIT 9515P9515_18621YP_0010121761650943⋯1651929987d9P9515_15601YP_0010118741376566⋯13776931128d12P9515_15521YP_0010118661371646⋯13727521107d12P9515_02681YP_001010584247534⋯248433900crtRProchlorococcus marinus subsp. marinus str. CCMP1375 (SS120)Pro1833NP_8762241690865⋯1691797932d9Pro1208NP_8756001116904⋯11180161112d12Pro1214NP_8756061121144⋯11222501106d12Pro0266NP_874660261189⋯2622231034crtRProchlorococcus marinus subsp. marinus str. CCMP1986 (MED4)PMM1672NP_8937891604745⋯1605731986d9PMM1382NP_8934991331162⋯13323401178d12PMM1378NP_8934951325388⋯13264941106d12PMM0236/228281⋯229270989crtRSynechococcus
elongatus strain PCC 7942Synpcc7942_2561YP_4015782639146⋯2639982836d9Synpcc7942_1713YP_4007301781317⋯1782219902mocDSynpcc7942_2439YP_4014562514276⋯2515271995crtRSynechococcus
elongatus strain PCC 6301syc1549_dYP_1722591676804⋯1677640837d9Syc2378_cYP_1730882534831⋯2535691861mocDsyc1667_cYP_1723771801757⋯1802752996crtRSynechococcus sp. BL107BL107_07284ZP_01469203490784⋯491566782d9BL107_07289ZP_01469204491936⋯492721785d9BL107_06084ZP_01468963247334⋯2483561022d12BL107_14110ZP_01468055331111⋯331884773crtWBL107_08054ZP_01469357636707⋯6377381031crtRSynechococcus sp. CC9311sync_2793YP_7319812458778⋯2459710932d9sync_2791YP_7319792457075⋯2457986911d9sync_0336YP_729569344430⋯3454491019crtRsync_0396YP_729627408306⋯4095051199d12sync_1804YP_7310081621108⋯1621869761crtWSynechococcus sp. CC9605Syncc9605_2541YP_3828242358792⋯2359703911d9Syncc9605_1972YP_3822681793076⋯17942211145d12Syncc9605_0286YP_380617292821⋯2938701049crtRSynechococcus sp. CC9902Syncc9902_2191YP_3781922099771⋯2100673902d9Syncc9902_2192YP_3781932100902⋯2101825923d9Syncc9902_0141YP_376159149723⋯1507241001d12Syncc9902_0972YP_376982954015⋯954788773crtWSyncc9902_2058YP_3780591964618⋯19657301112crtRSynechococcus sp. JA-2-3B′a(2-13)CYB_0861YP_477105894187⋯895071884d9CYB_2914YP_4790963011594⋯3012520926mocDCYB_0102YP_476366118335⋯119306971crtRSynechococcus sp. JA-3-3AbCYA_2349YP_4757392357019⋯2357912893d9CYA_1931YP_4753401944066⋯1945040974crtRSynechococcus sp.
RCC307SynRCC307_2395YP_0012286512091372⋯2092274903d9SynRCC307_2393YP_0012286492089667⋯2090581915d9SynRCC307_1757YP_0012280131538507⋯15395621056d12SynRCC307_1993YP_0012282491729342⋯1730103762crtWSynRCC307_2209YP_0012284651915148⋯19161671020crtRSynechococcus sp. RS9916RS9916_36767ZP_014713841050409⋯1051341932d9RS9916_36757ZP_014713821048603⋯1049568965d9RS9916_39311ZP_01472905116650⋯1176751025crtRSynechococcus sp. RS9917RS9917_06370ZP_01079314447782⋯448705923d9RS9917_06360ZP_01079312446060⋯446992932d9RS9917_03333ZP_0108084999968⋯1010471079d12RS9917_00687ZP_0108054164826⋯65563737crtWRS9917_03663ZP_01080915166940⋯167902962crtRSynechococcus sp. WH 5701WH5701_02025ZP_01084898299319⋯300257787d9WH5701_02015ZP_01084896297579⋯298532953d9WH5701_14646ZP_01083974104382⋯1055391157d12WH5701_16535ZP_01086617164⋯11861022d12WH5701_06521ZP_0108593565353⋯66231878hypothetical proteinWH5701_02369ZP_0108432242300⋯43271971mocDWH5701_04005ZP_0108342143734⋯44519785crtWWH5701_01215ZP_01084736138584⋯1396151031crtRSynechococcus sp. WH 7803SynWH7803_2417YP_0012261402249293⋯2250087795d9SynWH7803_2415YP_0012261382247475⋯2248386912d9SynWH7803_0589YP_001224312594539⋯5956031065d12SynWH7803_1625YP_0012253481496144⋯1497139996d15SynWH7803_0928YP_001224651871421⋯872167747crtWSynWH7803_0337YP_001224060361336⋯3623371002crtRSynechococcus sp. WH 7805WH7805_10184ZP_01125021209067⋯209999932d9WH7805_10194ZP_01125023210769⋯211680911d9WH7805_06186ZP_01124768405535⋯406059524d12WH7805_04931ZP_01124517184338⋯1855161178d12WH7805_01197ZP_011237733991⋯4734743crtWWH7805_07481ZP_01123496193165⋯1941931028crtRSynechococcus sp. WH 8102SYNW2377NP_8984662286168⋯
2287028860d9SYNW0696NP_896789679330⋯6804781148d12SYNW1696NP_8977871631011⋯16321471136d12SYNW1368NP_8974611354793⋯1355527734crtWSYNW0291NP_896386291323⋯2923541031crtRSynechocystis sp. PCC 6803sll0541NP_4424302822579⋯2823535956d9slr1350NP_4414891746308⋯17473631055d12sll1441NP_4416221895520⋯18965991079d15sll0262NP_4418242120067⋯21211461079d6Sll1611NP_4412201462136⋯14632451110hypothetical proteinsll1468NP_440788981691⋯982629938crtRThermosynechococcus
elongatus strain BP-1tll1719NP_6825091800682⋯1801521839d9tlr2380NP_6831702490209⋯2491048839d9tlr1653NP_6824431733919⋯1734767848d9tlr1254NP_6820441300388⋯1301308920mocDtlr1900NP_6826901986642⋯1987529887crtRTrichodesmium
erythraeum IMS101Tery_1437YP_7212052173203⋯2174015812d9Tery_0142YP_720110207806⋯2088611055d12Tery_4492YP_7239516931402⋯69324751073d15Tery_3898YP_7234066024293⋯60253421050hypothetical proteinTery_2925YP_7225644543239⋯4544114875crtRLyngbya sp. PCC 8106L8106_03152ZP_016246782253⋯3071818d9L8106_27002ZP_0162118594912⋯959551043d12L8106_10697ZP_016245606961⋯80431082d15L8106_14825ZP_01619238100018⋯1011331115d6L8106_06180ZP_01620148172993⋯173604611hypothetical proteinL8106_18641ZP_0162427813290⋯14111821hypothetical
proteinL8106_30215ZP_0162257823391⋯24185794crtRNodularia
spumigena CCY9414N9414_19077ZP_0163181716235⋯17026791d9N9414_07494ZP_01632615317⋯1135818d9N9414_07499ZP_016326161303⋯24271124d12N9414_07504ZP_016326172618⋯36881070d15N9414_07509ZP_016326184087⋯51781091d6N9414_18293ZP_0162972629633⋯30223590hypothetical
proteinN9414_07726ZP_016323054851⋯5633782crtWN9414_01572ZP_01632726697⋯1587890crtRCyanothece sp. CCY0110CY0110_10577ZP_01726409185891⋯186724834d9CY0110_05582ZP_0172921374180⋯75004825d9CY0110_10917ZP_017324587951⋯90001050d12CY0110_00445ZP_0172854190142⋯911911050d15CY0110_24056ZP_01727982158769⋯1598871119d6CY0110_13441ZP_0172902460390⋯61220831hypothetical proteinCY0110_27283ZP_0173193415787⋯169141128hypothetical proteinCY0110_11357ZP_017292799512⋯105131002mocDCY0110_08481ZP_0173100725752⋯26747996crtRTable 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.
SpeciesAccession no/locus tagLabelAccession no/locus tagLabelArabidopsis
thalianaBAA25180d9AAB60302chld15Q949X0d7BAA05514microd15AAA92800chld12CAA11858d8NP_187819microd12Thalassiosira
pseudonanaTp22511d9AY817152d5Tp23798d12AY817155d6Tp3143d12AY817154d8AY817156d4Phaeodactylum
tricorutumAAW70158d9AY082393d6AAO23565chld12AY082392d5AY165023microd12Pt22459d5Chlamydomonas
reinhardtiiCr117883uncertainABL09485d15AB007640chld12AY860820crtWEDP04777microd12Synechococcus sp. PCC 7002AAB61353d9AAF21445d12AAF21447uncertainAAB61352d15Nostoc sp. SO-36CAF18426d9CAF18425d15CAF18423d9CAF18424d12Mortierella
alpinaCAB38177d9AAF08684d12AAF08685d6AAC39508d5Cyanidioschyzon
merolaeBAA28834d9CMK291Cd12CMJ201Cd9BAC76126crtRArthrospira
platensisCAA05166d9Q54794d12ABN11122d6OstreococcuslucimarinusOl51664uncertainOl24150d12Ol18582d12Caenorhabditis
elegansAAF97550d9AAC15586d6AAC95143d5Rattus
norvegicusNP_114029d9BAA75496d6AAG35068d5Homo
sapiensXP_005719d9AAD20018d6AAF29378d5Brassica
napusAAA50157chl d12AAF78778microd12CAA11857d8Chlorella
vulgarisAB075526microd12AB075527microd15Chlamydomonas sp. W80AB031546chld12Synechocystis sp. PCC
6714BAA02921d12Mucor
circinelloidesAAD55982d12BAB69055d6Emericella
nidulansAAG36933d12Glycine
maxBAD89862microd12Calendula
officinalisAAK26633microd12Gossypium
hirsutumAAL37484microd12Nicotiana
tabacumBAC01274chld15BAC01273microd15Brassica
junceaCAB85467chld15Picea
abiesCAC18722chld15Ricinus
communisAAA73511chld15AAC4901012-hydroxylaseTriticum
aestivumBAA28358microd15Oryza
sativaBAA11397microd15Vernicia
fordiiAAN87573microd12AAN8757412-conjugasePunica
granatumCAD24671microd12AAO3775312-conjugaseLesquerella
fendleriAAC3275512-hydroxylase/desaturasePhysaria
lindheimeriABQ0145812-hydroxylaseCrepis
palaestinaCAA7615612-epoxygenaseStokesia
laevisAAR2381512-epoxygenaseDaucus
carotaAAO3803312-acetylenaseFoeniculum
vulgareAAO3803412-acetylenaseHedera
helixAAO3803112-acetylenaseHelianthus
annuusAAO3803212-acetylenaseCAA60621d8Helichrysum
bracteatumAAO3803712-acetylenaseRudbeckia
hirtaAAO3803512-acetylenaseCrepis
alpinaCAA7615812-acetylenaseCalendula
officinalisAAK2663212-conjugaseTrichosanthes
kirilowiiAAO3775112-conjugaseAcheta
domesticusAAK25797d9Cyprinus
carpioCAB57858d9Drosophila
simulansCAB52475d9Gallus
gallusCAA42997d9Helicoverpa
zeaAAF81790d9Rosa
hybrid cultivarBAA23136d9Saccharomyces
cerevisiaeAAA34826d9Limnanthes
douglasiiAAG28599d9Prochlorothrix
hollandicaAAG16761d9Lyngbya
majusculaAAS98775d9Synechococcus
vulcanusAAD00699d9Thraustochytrium sp. ATCC21685AAM09688d4AAM09687d5Euglena
gracilisAAQ19605d4AF139720d8Pavlova
lutheriAY332747d4Isochrysis
galbana strain
CCMP1323AY630574d4Marchantia
polymorphaAAT85663d5AAT85661d6Nitzschia
closterium f. minutissimaAY603475d5Dictyostelium
discoideumBAA37090d5Bacillus
subtilisAAC38355d5Danio
rerioQ9DEX7d5/d6Borago
officinalisAAD01410d6AAG43277d8Oncorhynchus
mykissAAK26745d6Mus
musculusNP_062673d6Glossomastix
chrysoplastaAAU11444d6Ostreococcus
tauriAY746357d6Physcomitrella
patensCAA11033d6Echium
pitardiiAAL23581d6Chlorella
zofingiensisAY772713crtWCyanidium
caldariumAAB82698crtRHaematococcus
pluvialisCAA60478crtWMyxococcus
xanthus DK 1622YP_634431uncertainStigmatella
aurantiaca DW4/3-1ZP_01463016uncertainBradyrhizobium
japonicum USDA 110NP_771234uncertain


## 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
(Table3). 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.
NameH-box1H-box2H-box3β-carotene
ketolaseTGLFIX2HDXMHK(N)HX2HHCY(F)H(N)FGYHXEHHβ-carotene
hydroxylaseGTVIHDAS(C)HX2AHRVHL(M)Q(E)HHXHVNGQNYHLI(V)HHLWPSI(V)PWhydrocarbon
oxygenaseHECXHRTAFAFY(F)RRYHXWHHRXTMWNMPF(Y)HXEHHL(F)Δ9-1GICLGYHRLLXHKSFWX3HRXHHAX3DYGEGWHNNHHX2PX5GX2WWEΔ9-2GXTLGXHRX3HRSFWXGXHRXHHX2SDGEGWHNNHHX4SARHGXXWWEΔ9-3TVLGVTLGLHRLXAHRSWX2LHRHHHX2SDQWVAXLSFGEGWHNNHHAXPXSARHGLΔ9-4CLGVTXGYHRLLXHRX2WXGLHRHHHXFSDTWVAALTFGEGWHNNHHAXPXSAΔ9-5GX4GXHRXFXHX2FWX3HRXHHX3DGESWHNNHHXFX3AX2GΔ12aFVXGHDCGHRSFWRX2HX2HHX2TNHXPHHX4IPXYNLRΔ12bWVXAHECGHXAFHWX2SHX2HHX3NHX2HHX4PHYXAΔ12cFSLMHDCGHXSLFWSX2HAXHHX2NGHX2HHLXERIPNYXLΔ15FWXLFVVGHDCGHXSFSHGWRISHRTHHXNTGNIHHXIGTHVAHHIFΔ6HDX2HX3SWX3HX2LHHXYTNIGGLNXQ(H)X2HHLFPXICHFigure 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.


### 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 (Figures12 and 13): Δ9 desaturase subfamily,
Δ12/ω3 desaturases subfamily, and the front-end desaturases subfamily.As shown in Figures12
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.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.
(a)(b)(c)(d)Figure 4
Neighbor-joining tree ofβ-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
Minimum-evolution tree ofβ-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.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
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 eightN2-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 fromProchlorothrix 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 cyanobacteriaSynechococcus 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 ofGloeobacter 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 Figures12 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 (Figures12 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
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
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 cyanobacteriaSynechococcus, 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 Prochlorococcusmarinus 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 [21–23]. 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 (Figures10 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
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
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.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
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.Theω3-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 fromNostoc
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
Figures12 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 (Figures12 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 Figures14 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
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
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 thesn-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 unicellularProchlorococcus 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, marineSynechococcus 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 [29–32]. 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/a2, are able to grow at
extremely low irradiances (less than 10 umol of quanta [Q] m−2s−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/a2 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 marineSynechococcus 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 [40–44]. 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 cyanobacteriaProchlorococcus 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 freshwaterSynechococcus 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
(Figure1). 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 strainsCrocosphaera 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 orN2-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.


## 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
(Table3). 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.
NameH-box1H-box2H-box3β-carotene
ketolaseTGLFIX2HDXMHK(N)HX2HHCY(F)H(N)FGYHXEHHβ-carotene
hydroxylaseGTVIHDAS(C)HX2AHRVHL(M)Q(E)HHXHVNGQNYHLI(V)HHLWPSI(V)PWhydrocarbon
oxygenaseHECXHRTAFAFY(F)RRYHXWHHRXTMWNMPF(Y)HXEHHL(F)Δ9-1GICLGYHRLLXHKSFWX3HRXHHAX3DYGEGWHNNHHX2PX5GX2WWEΔ9-2GXTLGXHRX3HRSFWXGXHRXHHX2SDGEGWHNNHHX4SARHGXXWWEΔ9-3TVLGVTLGLHRLXAHRSWX2LHRHHHX2SDQWVAXLSFGEGWHNNHHAXPXSARHGLΔ9-4CLGVTXGYHRLLXHRX2WXGLHRHHHXFSDTWVAALTFGEGWHNNHHAXPXSAΔ9-5GX4GXHRXFXHX2FWX3HRXHHX3DGESWHNNHHXFX3AX2GΔ12aFVXGHDCGHRSFWRX2HX2HHX2TNHXPHHX4IPXYNLRΔ12bWVXAHECGHXAFHWX2SHX2HHX3NHX2HHX4PHYXAΔ12cFSLMHDCGHXSLFWSX2HAXHHX2NGHX2HHLXERIPNYXLΔ15FWXLFVVGHDCGHXSFSHGWRISHRTHHXNTGNIHHXIGTHVAHHIFΔ6HDX2HX3SWX3HX2LHHXYTNIGGLNXQ(H)X2HHLFPXICHFigure 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.


## 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 (Figures12 and 13): Δ9 desaturase subfamily,
Δ12/ω3 desaturases subfamily, and the front-end desaturases subfamily.As shown in Figures12
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.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.
(a)(b)(c)(d)Figure 4
Neighbor-joining tree ofβ-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
Minimum-evolution tree ofβ-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.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
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 eightN2-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 fromProchlorothrix 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 cyanobacteriaSynechococcus 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 ofGloeobacter 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 Figures12 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 (Figures12 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
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
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 cyanobacteriaSynechococcus, 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 Prochlorococcusmarinus 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 [21–23]. 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 (Figures10 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
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
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.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
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.Theω3-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 fromNostoc
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
Figures12 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 (Figures12 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 Figures14 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
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
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 thesn-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 unicellularProchlorococcus 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, marineSynechococcus 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 [29–32]. 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/a2, are able to grow at
extremely low irradiances (less than 10 umol of quanta [Q] m−2s−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/a2 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 marineSynechococcus 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 [40–44]. 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 cyanobacteriaProchlorococcus 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 freshwaterSynechococcus 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
(Figure1). 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 strainsCrocosphaera 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 orN2-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.



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*Source: 284508-2008-12-02.xml*