Cellulose in cyanobacteria. Origin of vascular plant cellulose synthase?

Abstract

Although cellulose biosynthesis among the cyanobacteria has been suggested previously, we present the first conclusive evidence, to our knowledge, of the presence of cellulose in these organisms. Based on the results of x-ray diffraction, electron microscopy of microfibrils, and cellobiohydrolase I-gold labeling, we report the occurrence of cellulose biosynthesis in nine species representing three of the five sections of cyanobacteria. Sequence analysis of the genomes of four cyanobacteria revealed the presence of multiple amino acid sequences bearing the DDD35QXXRW motif conserved in all cellulose synthases. Pairwise alignments demonstrated that CesAs from plants were more similar to putative cellulose synthases from Anabaena sp. Pasteur Culture Collection 7120 and Nostoc punctiforme American Type Culture Collection 29133 than any other cellulose synthases in the database. Multiple alignments of putative cellulose synthases from Anabaena sp. Pasteur Culture Collection 7120 and N. punctiforme American Type Culture Collection 29133 with the cellulose synthases of other prokaryotes, Arabidopsis, Gossypium hirsutum, Populus alba x Populus tremula, corn (Zea mays), and Dictyostelium discoideum showed that cyanobacteria share an insertion between conserved regions U1 and U2 found previously only in eukaryotic sequences. Furthermore, phylogenetic analysis indicates that the cyanobacterial cellulose synthases share a common branch with CesAs of vascular plants in a manner similar to the relationship observed with cyanobacterial and chloroplast 16s rRNAs, implying endosymbiotic transfer of CesA from cyanobacteria to plants and an ancient origin for cellulose synthase in eukaryotes.

Figure 1

A through F, Various cellulose microfibrils isolated from cyanobacteria (all negatively stained with 1% [v/v] aqueous uranyl acetate and labeled with CBHI-gold; the gold complex is 10 nm in diameter). A, Oriented bundles of microfibrils from Gloeocapsa sp. L795, many of which are stained with the CBHI-gold complex, which specifically binds to the surface of crystalline cellulose (Okuda et al., 1993; Tomme et al., 1995). B, Cellulose microfibrils from N. muscorum UTEX 2209. These microfibrils frequently aggregate into twisted ribbon-like structures reminiscent of the cellulose ribbons of A. xylinum. C, Cellulose microfibrils from C. epipsammum ATCC 49662. These microfibrils appear to be very thin in both dimensions in comparison with the other cyanobacteria tested. D, Microfibrils from P. autumnale, which appear more discontinuous and irregular. This could be caused by amorphous regions or regions of low crystallinity rendering the microfibrils more altered from acid treatments. E, Microfibrils from Nostoc punctiforme ATCC 29133. These microfibrils are shorter and many have tapered ends, suggesting the possibility of cellulose II. F, Large bundles of elongated microfibrils from Anabaena UTEX 2576. A through E, Bar = 20 nm; F, bar = 30 nm.

Figure 2

Negatively stained thin section of S. hofmanni UTEX 2349 with Epon removed and labeled with CBHI-gold. Note the layering of the sheath and the position of the cellulose region near the outer boundary of the sheath. Sheath thickness is designated by the white line. Bar = 60 nm.

Figure 3

A, Untreated slime tube material collected from the liquid culture O. princeps. The microfibrils are somewhat masked in the EPS matrix. Bar = 20 nm. B, Trifluoroacetic-digested slime tube material collected from liquid media. Microfibrils are more apparent here as is a decrease in matrix material. Bar = 40 nm.

Figure 4

X-ray analysis of cellulose from four different cyanobacterial strains. A, Gloeocapsa; B, Scytonema; C, Phormidium; D, Anabaena. All genera exhibit a typical cellulose I pattern specified by the overlapping peaks. Peak 1 is the 101 d-spacing; peak 2 is the −101 d-spacing; and peak 3 is the 002 d-spacing. For the 002 spacings, all genera with the exception of S. hofmanni (4.0 ) have reflections of 3.9 . For the −101 spacings, all genera with the exception of Gloeocapsa (5.4 ) have 5.3- reflections. For the 101 spacings, all genera with the exception of P. autumnale (5.9 ) have 6.0- reflections. The presence of contaminating crystalline materials is evidenced by the existence of peaks not related to cellulose. Note that these peaks do not always produce uniform overlaps with all four genera.

Figure 5

Multiple alignment of amino acid sequences from 17 prokaryotic cellulose synthase homologs with CesA sequences from Arabidopsis, Gossypium hirsutum, corn, Populus tremulus × Populus alba, and D. discoideum. The alignment demonstrates the presence of a CR-P region between the U1 and U2 domains present only in eukaryotic and cyanobacterial sequences.

Figure 6

Comparison of NJ and MP trees. The tree shown is an NJ tree subjected to 5,000 bootstrap trials. Bootstrap values are shown as percentages with MP bootstrap values shown in parentheses. Differences in the MP tree are denoted by bold lines (multifurcations) and dashed arrow (variable position), and an asterisk indicates rooting at the base of the tree. Note the distribution of cyanobacterial sequences in the tree: two sequences from Anabaena sp. PCC 7120 and N. punctiforme branch with vascular plants; two sequences from N. punctiforme and Synechococcus branch distantly with Thermotogales and Proteobacteria; and three sequences from Synechocystis, N. punctiforme, and Anabaena, which are most likely CSL proteins, group with Bacillus subtilis. The high bootstrap values support the validity of the tree.

Figure 7

The maximum likelihood phylogeny for the cellulose synthase sequences showing confidence values and the log likelihood. With the exception of D. discoideum (which groups with the Euryarchea in this tree), the relationships in this tree are nearly identical to those shown in Figure 6.

Figure 8

Four distinct phylogenies alternative to the maximum likelihood phylogeny for N. punctiforme 1 and Anabaena 1 sequences (underlined). The log likelihood and the se are shown for each tree.