David Nobles

Cellulose Biosynthesis in the Cyanobacteria

David Nobles, grad student in Dr. Malcolm Brown's lab

Cyanobacteria were the first organisms, and the only prokaryotes, to evolve oxygen-evolving photosynthesis. Fossil records and geological evidence of an oxidizing atmosphere show their ancestry to date back 2.8 to 3.5 billion years. Cyanobacteria represent one of the most diverse groups of Eubacteria, having both unicellular and filamentous species demonstrating differentiated cell types (eg. heterocysts, hormogonia, and akinetes) for specialized functions such as nitrogen fixation, gliding motility, and desiccation tolerance.

Our work centers on the synthesis of cellulose by cyanobacteria. Recently, using x-ray diffraction and CBHI-gold staining (CBHI stands for cellobiohydrolase, an enzyme which specifically binds to crystalline cellulose), we have shown that cellulose biosynthesis exists in nine strains from five genera of cyanobacteria. Three of the five sections of cyanobacteria are represented among the cellulose producing strains. Thus, cellulose biosynthesis is likely widespread among this group of organisms.

Recent genome sequencing projects allowed us to mine databases of cyanobacteria and other prokaryotes for protein sequences with similarity to cellulose synthases. In all, 17 prokaryotic (five of which were cyanobacterial) and eight eukaryotic cellulose synthase homologues were aligned and compared. The results show that vascular plant and cyanobacterial cellulose synthases share a common branch in phylogenetic trees. This evidence indicates the likelihood of a synologous acquisition of cellulose synthase by plants from cyanobacteria.

David Nobles
Figure 1

Current research involves gene disruption of cellulose synthases in Nostoc punctiforme. N. punctiforme is a nitrogen-fixing, symbiotically competent, facultatively heterotrophic cyanobacterium capable of differentiating multiple cell types. We hope to discover if cellulose has a role in specific functions of differentiated cells eg. gliding motility or symbiotic competence in hormogonium, or nitrogen fixation in heterocysts. Ultimately our goal is to elucidate the entire biosynthetic and secretory machinery involved in the biosynthesis of cellulose in cyanobacteria.

Figure 1A-F shows various cellulose microfibrils isolated from cyanobacteria (all negatively stained with 1% aqueous uranyl acetate and labeled with CBHI-gold, the gold complex is 10 nm in diameter). Figure 1A shows 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). Figure 1B shows cellulose microfibrils from Nostoc muscorum UTEX 2209. These microfibrils frequently aggregate into twisted ribbon-like structures reminiscent of the cellulose ribbons of A. xylinum. Figure 1C shows cellulose microfibrils from Crinalium epipsammum ATCC 49662. These microfibrils appear to be very thin in both dimensions in comparison with the other cyanobacteria tested. Figure 1D shows 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. Figure 1E represents microfibrils from N. punctiforme ATCC 29133. These microfibrils are shorter and many have tapered ends, suggesting the possibility of cellulose II. (A-E Bar = 20nm). Figure 1F represents large bundles of elongated microfibrils from Anabaena UTEX 2576. (Bar = 30nm)

Figure 2

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 spacing. For the 002 spacings, all genera with the exception of S. hofmanni (4.0 Ǻ) have reflections of 3.9 Ǻ. For the 101 spacings, all genera with the exception of Gloeocapsa (5.4 Ǻ) have 5.3 Ǻ reflections. For the 101 spacings all genera with the exception of P. autumnale (5.9 Ǻ) have 6.0 Ǻ 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 3

The maximum likelihood phylogeny for the cellulose synthase sequences showing confidence values and the log likelihood. The contig listings and amino acid accession numbers of the sequence legend in the phylognetic trees are as follows: E coli (AAC76558.1), B cepacia (contig 411), A xylinus (2019362A), R leguminosarum (AAD28574.1), R sphaeroides (contig 168), A aeolicus (D70422), Anabaena1 (contig 326), Anabaena2 (contig 294), Synechocystis (D90912), N punctiforme1 (contig 499), N punctiforme2 (contig 640), N punctiforme3 (contig 583), Ath1 (BAB09693.1), Zma (AAF89961.1), Ghi1 (T10797), Pt/Pa (AAC78476.1), Ath2 (AAC29067.1), Ath3 (T51579), Ghi2 (AAD39534.2), D discoideum  (AAF00200.1),     B subtilus (D69769), T acidophilum (CAC11626.1), A tumefaciens (I39714), and F acidarmanus (contig 137).

Plant Biology images. (Photo credit: Dr. Z. Jeffrey Chen/University of Texas at Austin; Shutterstock images)