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. 2007 Aug 6;2007:25751. doi: 10.1155/2007/25751

Comparison of Envelope-Related Genes in Unicellular and Filamentous Cyanobacteria

Yu Yang 1, 2,2, Song Qin 1,*, Fangqing Zhao 1, 2,2, Xiaoyuan Chi 1, 2,2, Xiaowen Zhang 1, 2,2
PMCID: PMC2211374  PMID: 18253473

Abstract

To elucidate the evolution of cyanobacterial envelopes and the relation between gene content and environmental adaptation, cell envelope structures and components of unicellular and filamentous cyanobacteria were analyzed in comparative genomics. Hundreds of envelope biogenesis genes were divided into 5 major groups and annotated according to their conserved domains and phylogenetic profiles. Compared to unicellular species, the gene numbers of filamentous cyanobacteria expanded due to genome enlargement effect, but only few gene families amplified disproportionately, such as those encoding waaG and glycosyl transferase 2. Comparison of envelope genes among various species suggested that the significant variance of certain cyanobacterial envelope biogenesis genes should be the response to their environmental adaptation, which might be also related to the emergence of filamentous shapes with some new functions.

1. INTRODUCTION

As the oldest oxygenic phototrophs on the earth, cyanobacteria originated 2.8∼3.5 billion years ago [1]. Cyanobacteria are usually considered gram negative in traditional classification of prokaryotic envelopes [2], for the existence of outer membrane and lack of teichoic acid in cell walls. However, many unusual features exist in their envelopes. Cyanobacteria have a thick (15∼35 nm or more) and high cross-linking peptidoglycan layer, similar to gram-positive bacteria [3]. Some rare composition of gram-negative walls, such as carotenoid [4] and β-hydroxypalmitic acid [5], has been found from in lipopolysaccharide (LPS) of cyanobacteria. The archaic organisms contain cellulose indicative of vascular plants [6].

Phylum cyanobacteria has been well diverged in evolution. Some cyanobacteria have been evolved in a multicellular filamentous form, while others remained unicellular. Filamentous cyanobacteria are the oldest known multicellular organisms [7], and the divergence of cyanobacteria is a landmark in biological evolution. Transition from unicellular to filamentous cyanobacteria was a significant evolutionary event, as the organisms were equipped with an advantageous interior nutrition system able to interact with ambient factors [8].

The rise of genomics greatly promoted biological research, of which comparative genomics became an effective tool to explore different species. So far, 25 cyanobacterial genomes, both unicellular and filamentous, have been sequenced, ranging from 1.6 to 9.1 Mb [9]. However, a large difference exists in cell envelope between unicellular and filamentous species. At present, few comparative analyses have been made concerning the structure and function of cell envelopes of both. Therefore, to understand the diversity in cyanobacterial envelope, comparative genomic analysis is conducted in this paper by comparing envelope biogenetic genes between unicellular and filamentous species. As each of them occupies own ecological niche, cyanobacterial genome, the envelope structure, and environment adaptability were associated for inferring multicellular selection of cyanobacteria.

2. MATERIALS AND METHODS

2.1. The information management system

At the time of this study, 25 sequenced cyanobacterial genomes, including 21 unicellular and 4 filamentous were available for public online access into the Integrated Microbial Genomes (IMG) system provided by Joint Genome Institute (JGI) (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi) [10]. Unicellular Prochlorococcus marinus MED4 and Synechocystis sp. PCC 6803, and filamentous Trichodesmium erythraeum IMS101 and Anabaena sp. PCC 7120 (also called Nostoc sp. PCC 7120) were chosen for this research. In each species, over 60% of genes have been already included into the database of Clusters of Orthologous Groups (COGs) [11] based on orthology concept [12]. In a COG under the directory of “Cell wall/membrane/envelope biogenesis,” gene sequences in FASTA amino acid format were selected, exported, and downloaded in November, 2006 (as IMG version often updates, the data may change).

2.2. Gene retrieval and annotation

Quite a number of genes directly available online have only accession or gene ID, but complete description. So it was hard to know their roles in cyanobacterial envelope biogenesis. What we tried to solve the problem was to online-use software InterProScan from the EMBL of European Bioinformatics Institute (EBI) (http://www.ebi.ac.uk/InterProScan) [13]. Unfortunately, this action alone could not provide enough information, such as the family to which the gene belongs and the impact by envelope biogenesis. Therefore, two online tools in NCBI, protein-protein BLAST (blastp), (http://www.ncbi.nlm.nih.gov/BLAST) [14] and reverse position specific BLAST (RPS-BLAST) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [15], were also used as assistants.

Putative conserved domains of the genes (without detailed description) were detected; and the genes were aligned up with other known genes, commonly with score > 80 bits and expect < 1e-10 at least. Finally, present references to the roles of particular domains or gene families involved in bacterial envelope biogenesis were combined; the unclear genes would be retrieved and annotated.

2.3. Sequence alignment and phylogenetic analysis

The sequences with similar domains were input and completely aligned using ClustalX 1.8. The produced files with “*.aln” extension were opened by BioEdit at the option of “Graphic View.” The same or similar residues were highlighted in black or dark shade. In this paper, only the most conserved area of gene sequences is shown in figures.

In addition to Trichodesmium erythraeum IMS101 and Anabaena sp. PCC 7120, 15 FAS1-containing genes from other cyanobacteria, archaebacteria, eubacteria, yeast, filamentous fungi, and high plants were gained from NCBI. Sequence alignments of genes predicted for the same families were used as an input file for MEGA3 program [16]. Phylogenetic tree was built via the Neighbor-Joining (NJ) method in evaluation with 1000 rounds of bootstrapping test [17, 18].

3. RESULTS

One hundred envelope biogenesis genes were obtained from Prochlorococcus marinus MED4, 186 from Synechocystis sp. PCC 6803, 266 from Trichodesmium erythraeum IMS101, and 294 from Anabaena sp. PCC 7120, which are shown in the “total” column in Table 1. Known constituents of cyanobacterial cell walls, including peptidoglycan, lipopolysaccharide (LPS), exopolysaccharide (EPS), outer membrane protein, and so on, were respectively synthesized under the control of different genes. Thus we might as well divide above 846 envelope biogenesis genes into 5 major types: peptidoglycan biosynthesis-related (PBR) genes, lipopolysaccharide biosynthesis-related (LBR) genes, exopolysaccharide biosynthesis-related (EBR) genes, outer membrane proteins (OMP) coding genes, and other unknown (OU) genes. The OU ones were loaded from the COG “Cell wall/membrane/envelope biogenesis;” but not enough information was available to annotate them using the methods mentioned in the section “Gene retrieval and annotation.”

Table 1.

Absolute and relative numbers of envelope related genes in four cyanobacteria. PBR, LBR, EBR, OMP, and OU represent peptidoglycan biosynthesis-related, lipopolysaccharide biosynthesis-related, exopolysaccharide biosynthesis-related, outer membrane proteins coding, and other unknown genes, respectively. The data in the brackets were the percentage of each group within the total envelope-related genes.

Species Total PBR LBR EBR OMP OU
Prochlorococcus marinus MED4 100 29 (29.0%) 40 (40.0%) 14 (15.0%) 16 (15.0%) 2 (2.0%)
Synechocystis sp. PCC 6803 186 37 (19.9%) 73 (39.2%) 28 (15.1%) 40 (21.5%) 8 (4.3%)
Trichodesmium erythraeum IMS101 266 47 (17.7%) 90 (33.1%) 48 (18. 0%) 63 (23.7%) 18 (6.8%)
Anabaena sp. PCC 7120 294 48 (16.3%) 113 (38.4%) 61 (20.7%) 60 (20.4%) 12 (4.1%)
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Table 1 shows the absolute and relative amounts of classified genes from unicellular and filamentous species. The appearance of filament naturally resulted in the enlargement of genome sizes and the addition of gene numbers; however, the percentage of each type of “total” varied, too. Therefore, the percentage of EBR increased in filamentous species (EBR percentage of Trichodesmium erythraeum IMS101 and Anabaena sp. PCC 7120 was 18.0% and 21.2% respectively, compared with 15.0% of Prochlorococcus marinus MED4 and 15.1% of Synechocystis sp. PCC 6803). The percentage of other types changed simultaneously, which were discussed in detail in Section 4.

3.1. Percentage variation of peptidoglycan biosynthesis-related (PBR) genes

Being an important component of cyanobacterial envelope, peptidoglycan forms a covalently closed and net-like layer, for protecting cells against detrimental environmental influences, maintaining a high internal osmotic pressure, and serving as a barrier to transenvelope transport sometimes [19]. As the amount of envelope biogenesis gene from Prochlorococcus marinus MED4 to Anabaena sp. PCC 7120 increased, this increase was exclusively reflected on one gene family, which encodes class A high-molecular-weight penicillin binding proteins [20]. However, the percentage of PBR decreased instead. In filamentous cyanobacteria, envelope components (besides peptidoglycan) and structures could also protect the cells, such as exopolysaccharide and filamentous sheaths; so relatively fewer peptidoglycan genes were expressed.

3.2. Uneven increase of LBR genes in filamentous cyanobacteria

LPS also has a function of the protection, so the percentage of LBR genes of “total” decreased from unicellular to filamentous cyanobacteria, which is like PBR genes. This course is clearly expressed among Prochlorococcus marinus MED4, Synechocystis sp. PCC 6803, and Trichodesmium erythraeum IMS101. However, Anabaena sp. PCC 7120 did not obey the “trend.” It expressed relatively more LBR genes than that of Trichodesmium erythraeum IMS101, which is probable due to differentiation of some cells into heterocysts, forming special N2-fixing cells within O2-producing filamentous cyanobacteria [20, 21]. For nitrogen fixing, the heterocysts need extracellular LPS layers to protect oxygen invasion [22].

In terms of absolute amounts, Anabaena sp. PCC 7120 had most of the LBR genes. Interesting is that most increased genes had the common conserved domain waaG (formerly RfaG). There were 43 waaG-containing genes found in Anabaena sp. PCC 7120 (while only 5 in Prochlorococcus marinus MED4, 17 in Synechocystis sp. PCC 6803, and 24 in Trichodesmium erythraeum IMS101). The 43 genes and their multiple alignments in similar domain were shown in Table 2 and Figure 1, about 20 residues out of the 43 sequences were in common (black shading areas). These residues may have formed typical spatial structures that could be defined as active sites of waaG domain.

Table 2.

waaG homologous genes in Anabaena sp. PCC 7120. Information of the 43 genes was provided.

NCBI accession IMG accession Locus Tag Product Position in Genome
NP_484203 4210510 Alr0159 Alr0159 protein 163382–164575
NP_484204 4210520 All0160 All0160 protein 164558–165712
NP_484626 4214800 Alr0582 Alr0582 protein 676349–677545
NP_484628 4214820 Alr0584 Alr0584 protein 679928–681130
NP_484962 4218190 All0919 All0919 protein 1063224–1064513
NP_485043 4219010 Alr1000 Alr1000 protein 1171949–1173031
NP_485160 4220180 Alr1117 Alr1117 protein 1308038–1309267
NP_485388 4222480 All1345 All1345 protein 1596626–1597858
NP_485708 4225730 Alr1668 Alr1668 protein 1990621–1991904
NP_486077 4229490 All2037 All2037 protein 2435914–2437014
NP_486305 4231820 SqdX Sulfolipid sulfoquinovosyldiacylglycerol biosynthesis protein 2725143–2726279
NP_486331 4232080 All2291 Glycosyltransferase 2760187–2761173
NP_486332 4232090 All2292 All2292 protein 2761170–2762348
NP_486547 4234260 All2507 All2507 protein 3008236–3009423
NP_486589 4234680 All2549 All2549 protein 3051362–3052363
NP_486760 4236410 All2720 All2720 protein 3315625–3316713
NP_486872 4237530 Alr2832 Alr2832 protein 3448705–3449793
NP_486879 4237600 Alr2839 Glycosyltransferase 3459432–3460577
NP_486904 4237850 Alr2864 Alr2864 protein 3488401–3489579
NP_486907 4237880 Alr2867 Alr2867 protein 3491419–3492636
NP_487097 4239800 Alr3057 Alr3057 protein 3703378–3704592
NP_487098 4239810 Alr3058 Alr3058 protein 3704628–3705854
NP_487104 4239870 Alr3064 Alr3064 protein 3712759–3714171
NP_487465 4243510 Alr3425 Alr3425 protein 4133859–4135025
NP_487738 4246270 HepB Heterocyst envelope polysaccharide synthesis protein 4465828–4466997
NP_487739 4246280 Alr3699 Alr3699 protein 4467059–4468207
NP_488208 4251030 Alr4168 Alr4168 protein 5015231–5016502
NP_488218 4251140 Alr4178 Alr4178 protein 5025948–5027096
NP_488463 4253590 All4423 All4423 protein 5300887–5302026
NP_488466 4253620 All4426 All4426 protein 5304172–5305425
NP_488476 4253720 All4436 All4436 protein 5320348–5321526
NP_488534 4254300 Alr4494 Mannosyltransferase 5380744–5381811
NP_489234 4261400 All5194 Glycosyltransferase 6192395–6193555
NP_489235 4261410 All5195 Glycosyltransferase 6193736–6194992
NP_489241 4261470 Alr5201 Glycosyltransferase 6201983–6203275
NP_489242 4261480 Alr5202 Glycosyltransferase 6203285–6204574
NP_489263 4261690 Alr5223 Glycosyltransferase 6236642–6237991
NP_489275 4261810 Alr5235 Alr5235 protein 6247505–6248551
NP_489277 4261830 Alr5237 Alr5237 protein 6249905–6251158
NP_489278 4261840 Alr5238 Glycosyltransferase 6251167–6252315
NP_489279 4261850 Alr5239 Alr5239 protein 6252417–6253586
NP_489347 4262550 Alr5307 Alr5307 protein 6328387–6329490
NP_489381 4262900 All5341 All5341 protein 6373814–6375079
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Figure 1.

Figure 1

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Multiple sequence alignments of the 43 waaG homologous genes in Anabaena sp. PCC 7120. Only most conserved areas were shown. The number following the genus name was the gene accession in IMG database. NCBI accessions and other information of genes were provided in Table 2.

The waa family includes many members, such as waaP, waaY, waaA, waaT, waaO, waaQ, waaA, and waaC, and helps synthesize the LPS core oligosaccharide. At present, we only knew that the waaG product is a glucosyltransferase, and its mutation can truncated LPS at the phosphorylation sites and destabilized the outer membrane [23]. Probably, waaG can provide a selective advantage to Anabaena sp. PCC 7120.

3.3. Analysis of EBR

During the progress from unicellular to filamentous cyanobacteria, the percentage of EBR genes increased clearly but unevenly in some particular genes. Most extra genes of filamentous species belonged to the family encoding glycosyl transferase 2 that involved in many metabolic processes, mainly in the cellulose biosynthesis [24]. The common conserved domain Glycos_transf_2 was detected for 36 times in Anabaena sp. PCC 7120, and 27 in Trichodesmium erythraeum IMS101, as shown in Tables 3 and 4 and Figure 2, whereas it was only 8 times in Prochlorococcus marinus MED4 and 14 times in Synechocystis sp. PCC 6803. It is believed that certain member in the family glycosyl transferase 2 was a key enzyme synthesizing cellulose in filamentous cyanobacteria.

Table 3.

Genes encoding GT2 domain in Anabaena sp. PCC 7120. Information of the 36 genes was provided.

NCBI Accession IMG Accession Locus Tag Product Position in Genome
NP_484086 4209330 all0042 All0042 protein 44511–45458
NP_484118 4209650 alr0074 Alr0074 protein 78171–79187
NP_484187 4210350 all0143 All0143 protein 148503–149681
NP_484819 4216740 alr0776 Alr0776 protein 899704–900894
NP_484957 4218140 all0914 All0914 protein 1057871–1058884
NP_484958 4218150 all0915 All0915 protein 1058947–1059852
NP_485777 4226430 all1737 All1737 protein 2088106–2089074
NP_485802 4226680 all1762 All1762 protein 2117622–2118518
NP_485806 4226720 all1766 All1766 protein 2121006–2122007
NP_485807 4226730 all1767 All1767 protein 2122000–2123007
NP_485926 4227930 all1886 All1886 protein 2252568–2253362
NP_486328 4232050 all2288 Glucosyltransferase 2756810–2757841
NP_486329 4232060 all2289 Glucosyltransferase 2757927–2758916
NP_486448 4233260 alr2408 Alr2408 protein 2888194–2888949
NP_486868 4237490 alr2828 Alr2828 protein 3444428–3445441
NP_486876 4237570 alr2836 Putative glycosyl transferase 3456248–3457216
NP_486877 4237580 alr2837 Glycosyltransferase 3457336–3458310
NP_486880 4237610 alr2840 Glycosyltransferase 3460577–3461524
NP_486906 4237870 alr2866 Glycosyltransferase 3490561–3491400
NP_487103 4239860 alr3063 Alr3063 protein 3711770–3712762
NP_487109 4239920 alr3069 Alr3069 protein 3718782–3719963
NP_487110 4239930 alr3070 Alr3070 protein 3719986–3720942
NP_487111 4239940 alr3071 Alr3071 protein 3720982–3721938
NP_487113 4239960 alr3073 Alr3073 protein 3723391–3724365
NP_487216 4241000 alr3176 Alr3176 protein 3844812–3845753
NP_487217 4241010 alr3177 Alr3177 protein 3845774–3846715
NP_487420 4243050 alr3380 Dolichol-phosphate mannosyltransferase 4091498–4092511
NP_488471 4253670 all4431 Glycosyl transferase 5310064–5311017
NP_488532 4254280 alr4492 Alr4492 protein 5378788–5379816
NP_488897 4257980 all4857 All4857 protein 5785088–5786275
NP_488973 4258750 all4933 All4933 protein 5886142–5887548
NP_489142 4260480 all5102 All5102 protein 6079688–6080410
NP_489158 4260640 all5118 All5118 protein 6114366–6115355
NP_489280 4261860 alr5240 Glycosyltransferase 6253630–6254397
NP_489382 4262910 all5342 All5342 protein 6375223–6376452
NP_489383 4262920 all5343 All5343 protein 6376587–6377849
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Table 4.

Genes encoding GT2 domain in Trichodesmium erythraeum IMS101. Information of the 27 genes was provided.

NCBI accession IMG accession Locus Tag Product Position in Genome
YP_720085 636810880 Tery_0115 Glycosyl transferase, family 2 155085–157763
YP_720116 636811045 Tery_0148 Glycosyl transferase, family 2 217777–218829
YP_720694 636814360 Tery_0804 Glycosyl transferase, family 2 1279953–1280891
YP_720758 636814755 Tery_0883 Glycosyl transferase, family 2 1403156–1404088
YP_720935 636815825 Tery_1097 Glycosyl transferase, family 2 1725763–1726743
YP_721031 636816345 Tery_1201 Glycosyl transferase, family 2 1875929–1876612
YP_721128 636817045 Tery_1340 Glycosyl transferase, family 2 2040749–2041705
YP_721156 636817205 Tery_1372 Glycosyl transferase, family 2 2104828–2106021
YP_721969 636821740 Tery_2268 Glycosyl transferase, family 2 3529656–3534458
YP_722405 636824155 Tery_2749 Glycosyl transferase, family 2 4257314–4258822
YP_722496 636824655 Tery_2849 Glycosyl transferase, family 2 4430305–4432779
YP_722503 636824690 Tery_2856 Glycosyl transferase, family 2 4447185–4448186
YP_722586 636825160 Tery_2950 Glycosyl transferase, family 2 4584744–4585874
YP_722664 636825630 Tery_3040 Glycosyl transferase, family 2 4692416–4693294
YP_722816 636826565 Tery_3225 Glycosyl transferase, family 2 4937986–4938924
YP_722946 636827300 Tery_3371 Glycosyl transferase, family 2 5168831–5170021
YP_722999 636827610 Tery_3433 Glycosyl transferase, family 2 5251339–5252268
YP_723000 636827615 Tery_3434 Glycosyl transferase, family 2 5252486–5253415
YP_723155 636828495 Tery_3609 Glycosyl transferase, family 2 5550523–5551482
YP_723304 636829395 Tery_3784 Dolichyl-phosphate beta-D-mannosyltransferase 5816905–5817705
YP_723576 636830965 Tery_4095 Glycosyl transferase, family 2 6315001–6316008
YP_723603 636831105 Tery_4122 Glycosyl transferase, family 2 6360766–6361701
YP_723606 636831120 Tery_4125 Glycosyl transferase, family 2 6363768–6364736
YP_723897 636832695 Tery_4437 Glycosyl transferase, family 2 6839236–6842421
YP_724037 636833455 Tery_4588 Glycosyl transferase, family 2 7057924–7058847
YP_724197 636834370 Tery_4771 Glycosyl transferase, family 2 7329873–7332980
YP_724341 636835285 Tery_4954 Glycosyl transferase, family 2 7547130–7548305
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Figure 2.

Figure 2

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Multiple sequence alignments of homologous genes encoding glycosyl transferase 2 (GT2) domains in Trichodesmium erythraeum IMS101 (27 genes) and Anabaena sp. PCC 7120 (36 genes). Only most conserved areas were shown. The number following the genus name was the gene accession in IMG database. NCBI accession and other information of genes were provided in Tables 3 and 4.

Fasciclin-like (FAS1) family has been identified as hemicellulose synthase in fungi and high plants [25], and it was involved in the secondary wall biosynthesis [26]. Homologues of this conserved domain, closely relative to the formation of filaments and extracellular polysaccharides, has been found in archaebacteria, eubacteria, actinomycetes, yeast, filamentous fungi, and vascular plants. It was found that 2 genes in Trichodesmium erythraeum IMS-101 and 6 in Anabaena sp. PCC 7120 contained the domain. Representative FAS1-containing genes were found from NCBI, including Synechococcus, Crocosphaera, Nostoc, Methanosarcina, Dehalococcoides, Aspergillus, Cryptococcus, Flavobacteria, Physcomitrella, Streptomyces, Chaetomium, Magnaapothe, Arabidopsis, Gossypium, and Zea, as shown in Table 5. Phylogenetic tree of all 23 FAS1-containing genes in many species was constructed (See Figure 3). It is clear that genes in Trichodesmium erythraeum IMS101 and Anabaena sp. PCC 7120 were distant from other cyanobacteria (Synechocystis, Synechococcus, Crocosphaera, and Nostoc); and all the cyanobacterial genes were separated from fungi and plants. The FAS1-containing genes were paralogous in the Phylum Cyanobacteria.

Table 5.

FAS1-containing genes from Trichodesmium erythraeum IMS101, Anabaena sp. PCC 7120, and other 15 species.

NCBI Accession IMG Accession Gene Species
NP_485363 4222220 Alr1320 Alr1320 protein Anabaena sp. PCC 7120
NP_485859 4227250 Alr1819 Alr1819 protein Anabaena sp. PCC 7120
NP_487837 4247260 All3797 All3797 protein Anabaena sp. PCC 7120
NP_488687 4255850 All4647 All4647 protein Anabaena sp. PCC 7120
NP_488934 4258350 All4894 All4894 protein Anabaena sp. PCC 7120
NP_489304 4262100 All5264 All5264 protein Anabaena sp. PCC 7120
YP_722947 636827305 Tery_3372 beta-Ig-H3/fasciclin Trichodesmium erythraeum IMS101
YP_722948 636827310 Tery_3373 beta-Ig-H3/fasciclin Trichodesmium erythraeum IMS101
AAF02137 — Unknown protein Arabidopsis thaliana
CAF32145 — Fasciclin I family protein, putative Aspergillus fumigatus
EAQ86204 — Hypothetical protein CHGG_07457 Chaetomium globosum CBS 148.51
EAM48409 — Beta-Ig-H3/fasciclin Crocosphaera watsonii WH 8501
AAW46332 — Hypothetical protein CNK01730 Cryptococcus neoformans var. neoformans JEC21
CAI83309.1 — Fasciclin domain protein Dehalococcoides sp. CBDB1
EAS19928 — Putative cell adhesion protein, fasciclin domain Flavobacteria bacterium BBFL7
AAO92753 — Arabinogalactan protein Gossypium hirsutum
BAC65875 — Putative membrane-associated or secreted protein Magnaporthe grisea
AAM05399 — Hypothetical protein MA_1996 Methanosarcina acetivorans C2A
ZP_00108174 — COG2335 Nostoc punctiforme PCC 73102
CAH58718 — Fasciclin-like protein precursor Physcomitrella patens
CAA20163 — Putative secreted protein Streptomyces coelicolor A3(2)
AAB62187 — Putative secreted protein MPB70 Synechococcus sp. PCC 7002
AAC49869 — Endosperm specific protein Zea mays
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Figure 3.

Figure 3

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The phylogenetic tree of genes containing FAS1 domain in 17 species. Besides Trichodesmium erythraeum IMS101 and Anabaena sp. PCC 7120, other 15 species were from cyanobacteria, archaebacteria, eubacteria, actinomycetes, yeast, filamentous fungi, and vascular plants. To keep the figure clear and direct, the species were written in their genus name for short. The detailed information was described in Section 3 and Table 5.

4. DISCUSSION

4.1. General descriptions of 5 types of genes

In Table 1, remarkable changes could be seen from top to bottom lines, especially in columns of PBR, EBR, and OMP, which should be easily understood: to adopt better external environment and improve own nutrition status, cyanobacterial envelopes have to be modified. Adding outer membrane proteins could be a choice, as happened in Synechocystis sp. PCC 6803. From unicellular to filamentous cyanobacteria, the number of envelope biogenesis gene has increased. However, the increase was uneven, and gene duplication focused on in very few families. It is shown that in the evolution, only few families of genes expressed excessively, and they could be involved in generating novel structures and functions.

4.2. Role of waaG in filamentous cyanobacterial regulation

LPS is a characteristic component of gram-negative bacteria, which shows architecture of 3 covalently linked domains, namely hydrophobic lipid A, core oligosaccharide, and hydrophilic O-antigen [27]. In structural feature, the region of phosphorylated core oligosaccharide can be subdivided into inner and outer cores [28]. During LPS biosynthesis, waaG produces transferases, a glucosyl group from D-glucose I (Glc I) I to L-glycero-D-manno-heptose II (Hep II). The mutation of waaG destabilizes the LPS layer by interfering with core phosphorylation [23]; and the stability of LPS layer is necessary to the stabilization of heterocysts' external layers [22]. Unlike marine filamentous Trichodesmium erythraeum IMS101, Anabaena sp. PCC 7120 usually lives in freshwater or wetland, which is considered as a less stable environment than marine ecosystem, with drastic changes of temperature and light, abundant but inconstant nutrient resources and more potential hazards. Anabaena sp. PCC 7120 is also able to produce heterocysts to fix N2 and actively adapt environment, making itself more mutable than in the ocean environment. Over-expression of waaG homologous genes would help stabilize the heterocysts, and improve the N2-fixing in Anabaena sp. PCC 7120.

4.3. Relation between EBR and cyanobacterial evolution

Cyanobacterial filaments were made up of mainly diverse polysaccharide molecules, containing cellulose and matrix polysaccharide. Most of the genes are from the glycosyl transferase 2 (GT2) family. In model plant Arabidopsis thaliana, over 10 members of the family catalyze glucan-chain elongation in cellulose, and they belong to the group of genes encoding catalytic subunit of cellulose synthase (CESA) [29]. Since cellulose and other EPS were also the main components of cyanobacterial filamentous sheath, the GT2 family may play a vital role in the formation of filaments. In the meanwhile, these results could further prove that the cellulose produced by cyanobacteria is, at least one of, the earliest origins of the most abundant biopolymer on the earth today [30].

At present, a little is known about the matrix polysaccharide (hemicellulose, pectin, and so on) in cyanobacteria. Surprisingly, several matrix polysaccharide biogenesis genes or their homologues were discovered in this study. The phylogenetic tree (see Figure 3) shows that the genes of fasciclin-like (FAS1) family are duplicated in evolution among different cyanobacteria, suggesting that the FAS1 family occurred after the branch point where cyanobacteria separated from other archaic species but before the divergence of different cyanobacteria. The family is very rare in oceanic unicellular cyanobacteria, but in filamentous Anabaena, Nostoc, and Trichodesmium, it cannot be neglected. Large difference in content of the family between unicellular and filamentous cyanobacteria implied the family's contribution to filament formation, which provides us a clue to understanding the evolution of cyanobacteria.

4.4. Species selection and gene classification

Contrast to Prochlorococcus marinus MED4, Trichodesmium erythraeum IMS101, and Anabaena sp. PCC 7120, the selection of Synechocystis sp. PCC 6803 is more or less special. Although Synechocystis sp. PCC 6803 is usually unicellular in entire lifetime, it can hardly be recognized as a “pure” unicellular cyanobacterium. Synechocystis sp. PCC 6803 can frequently congregate in dimer or in a loose group. The group members must communicate with each other in special mechanism with similar actions to multicellular filaments. Strong light or other external factors can stimulate numerous single cells to arrange in filamentous shape, showing semi-filamentous feature. That was why Synechocystis sp. PCC 6803 was chosen for this study as it has clear transitional character. Selection of and comparison among the 4 cyanobacteria with own particular shape and status should be more persuasive on the issue of this paper.

In addition to major components of typical gram-negative bacteria, the existence of EPS (mainly refers to cellulose and hemicellulose) in cyanobacteria is significant. Therefore, peptidoglycan, LPS, EPS, and outer membrane proteins become 4 major components of cyanobacterial envelopes. Over 93% of biogenesis genes of each cyanobacterial envelope were placed in correct place, leaving only <7% of other unknown genes, showing that the classification is scientifically acceptable and also practical. However, problem still remains as it is difficult to eliminate error or misplacement until all cyanobacterial genes are correctly annotated. For instance, some LBR coding proteins were localized in the outer membrane; so these LBR genes can also be considered as OMP genes. Therefore, the genes of the OMP defined in this paper represented mostly those genes whose expressing products are located in outer membrane and carry out functions other than the biosynthesis by peptidoglycan, LPS, and EPS.

Moreover, previous reports believed that cyanobacterial cell wall did not contain teichoic acid [3], but the gene alr4011 in Anabaena sp. PCC 7120 put the issue in argument. The amino acid sequence of alr4011 has a conserved domain DltE that is a short-chain dehydrogenase involved in the teichoic acid synthesis [31]; and alr4011 showed great similarity to the gene dltE in gram-positive Bacillus subtilis (146 bits [Expect = 7e-34]). No DltE-containing gene was found in Prochlorococcus marinus MED4, Synechocystis sp. PCC 6803, or Trichodesmium erythraeum IMS101. A possible explanation is that alr4011 was transferred horizontally from gram-positive bacteria, or that the gene was regulated via a special pathway to produce another envelope constituent but teichoic acid. Whether teichoic acid exists in cyanobacterial envelopes is currently an open question that needs further research and experiment.

ACKNOWLEDGMENTS

This work was supported by the Key Innovative Project of Chinese Academy of Sciences (KZCX2-YW-209) and the Open-End Funds of Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology (2005HS002).

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