Cellulose production in bacteria is of potential economic interest because of its impact in medical settings and in the paper and food industry
. In addition, cellulose biosynthesis in a wide variety of bacteria has increased the possibility for the elucidation of regulation and molecular mechanisms of cellulose biosynthesis, which still remains largely unknown. The role of cellulose in attachment and biofilm formation during the interaction of the bacteria with the environment and its hosts confers further importance to this polymer as a potential target to design methods against harmful biofilm proliferation. In this work, we report how the alterations in the levels of endogenous cellulase contribute to biofilm architecture.
Although Congo red can also bind outer membrane proteins in some animal pathogens
, several authors have noted that among Rhizobium spp., staining with this dye correlated with the cellulose content in the bacterial cultures
[16, 40]. The significant reduction in Congo red binding to engineered strains of Rhizobium impaired in cellulose production (Figure
1D) suggests that these bacteria do not produce other substances that strongly bind Congo red and contribute to their red colony pigmentation. Both the binding to Congo red in diverse species of the family Rhizobiaceae and the presence of genes encoding cellulose synthases in their sequenced genomes strongly suggest that the ability to synthesize cellulose is fairly common in this taxonomic group of plant-associated bacteria. Several lines of evidence further support that cellulase activity is commonly involved in the cellulose production pathway used by these species: i) the existence of at least a cellulase-encoding gene associated to a glycosil transferase-encoding gene in all Rhizobiaceae species with accessible data in GeneBank, ii) the presence of the celABC operon within Rhizobiaceae, and iii) the high degree of conservation of CelC cellulase-encoding genes between Rhizobium species
The ability of most legume root-nodulating microorganisms to synthesize cellulose implicates the importance of this polymer in their eco-physiology. Furthermore, although the inactivation of cellulose biosynthesis in R. leguminosarum bv. trifolii does not affect the ability to nodulate clover in controlled laboratory conditions
, the ability of rhizobia to adhere firmly to a substrate using cellulose micro fibrils facilitates host root colonization
[1, 8]. Moreover, it is very possible that cellulose micro fibril-mediated firm adhesion during legume host root colonization is important under natural conditions in the rhizosphere (or rhizospheric soil), where bacteria have to compete for survival and colonization to successfully gain access to plant carbon sources.
During the course of these studies on the CelC2 protein, we detected by Congo Red and Calcofluor staining microscopy and enzymatic treatment that the celC over-expression derivative strain lost the ability to make extracellular cellulose micro fibrils, and that the extracellular micro fibrils of the celC knockout mutant were significantly longer than those seen in the wild type parent (Figure
2) suggesting that R. leguminosarum bv. trifolii cellulase CelC2 is involved in determining the longitude of cellulose extracellular micro fibrils.
Ausmees et al. found that cloned genes involved in cellulose biosynthesis have similar homology and the same organization in R. leguminosarum bv. trifolii strain R200 and A. tumefaciens. These authors identified these genes by Tn5 mutagenesis followed by screening with Calcofluor staining for mutants showing less ability to synthesize cellulose. They obtained celA, celB, and celE mutants but not celC, consistent with our finding that a celC knock-out mutant overproduces external cellulose micro fibrils. Therefore, we conclude that the celC gene is involved in the formation and elongation of cellulose micro fibrils. It is likely that cellulose oligomers synthesized by CelA elongate indefinitely in the absence of the CelC endoglucanase, producing very long micro fibrils that entangle ANU843ΔC2 mutant bacterial cells into very large aggregates causing them to flocculate and settle in liquid medium cultures (Figures
3A). By contrast, an excess of endoglucanase activity leads to an uncontrolled degradation of CelA-synthesized cellulose oligomers, preventing their transport and subsequent maturation into microcrystalline micro fibrils that extend outside the cell (Figure
2I). The fluorescence intensity of these CelC2-degraded oligomer products suggest that they may concentrate at both cell poles (Figure
In the model proposed for the synthesis of cellulose in plants
, CesA protein is a polymerase that catalyzes ß-1,4-glucan chain elongation by transferring UDP-glucose moieties to the sitosterol-ß-sitosterol-glucoside intermediate, forming cellodextrins. The other protein that has been proposed to participate in this process is Korrigan (Kor) cellulase, which may act by releasing the sitosterol-ß-glucoside from the newly synthesized cellulose polymer chain. How the final cellulose chain is formed, what other molecule(s) intervene in this step, the mechanism of its export and components of its anchoring apparatus on the cell surface all remain unknown. However, in the model for cellulose synthesis in bacteria
, CelA activity adds glucose monomers from the UDP-glucose substrate to the intermediate lipid-glucose to form lipid-glucosex (x = 2–4 glucose). Bacterial CelA and plant CesA are both glycosyl transferases, whereas CelC and Korrigan present a glycosyl hydrolase domain. According to Matthysse et al., bacterial CelC cellulase may act in this case as a translocase, by incorporating the lipid-linked oligosaccharide into the cellulose polymer chain being formed. According to our findings, the role of CelC cellulase in cellulose biosynthesis is similar to the role that has been proposed for Korrigan cellulase. This CelC cellulase may catalyze the hydrolysis reaction, in which cellotriose are released from the lipid-glucose4 intermediary, thus providing the substrate for the translocase to transfer it to the internal growing point of another lipid-intermediate, thereby elongating the cellulose microfibril product by 3 glucose units at one time.
This function for the celC gene is distinctly different from its established role as an endoglucanase involved in the infection process
. The fact that core celC has both colonization and infection functions, i.e., in cellulose biosynthesis and independently as a hydrolytic enzyme that creates the portal of Rhizobium entry into the host root hair and their liberation from infection threads into the symbiosomes within nodule cells, implies the likely existence of two different sets of control mechanisms, functional designs and target cellular locations. Furthermore, the possible role in cellulose biosynthesis of cellulase CelC1, that it is not involved in plant root hair tip erosion
, has not been yet characterized. We propose that cellulose production may reflect an earlier evolutionary development because this property is encoded by an operon common to all nodulating rhizobia, including Burkholderia and Cupriavidus strains, the so-called β-rhizobia
, whereas the cellulase celC gene function for the infection may develope later.
Previous results have shown that Tn5 mutation in nodC diminishes extracellular microfibril production by ANU843 on white clover roots
 and that Rhizobium common nod genes are required for biofilm formation
. Nevertheless, the role of cellulose in Rhizobium biofilm establishment has not been fully recognized. To our knowledge, this is the first time that Rhizobium mutants either lacking or over-expressing cellulase have been analysed with respect to cellulose production and biofilm formation on both abiotic surfaces and the root epidermis.
Our results show that biofilm formation ability was markedly reduced in ANU843ΔC2 and ANU843C2+ strains not only on abiotic surfaces (PVC, microtiter plate assays, and sand Figures
4), but also on plant roots (Figure
5). Since ANU843C2+ is impaired to produce external micro fibrils, it cannot tightly bind to the substrate preventing subsequent mature biofilm formation. On the other hand, it seems that ANU843ΔC2 cells, which produced more cellulose micro fibrils, tended to associate themselves more than to the plastic surfaces. However, no such caps are observed on sand grains. Since the PVC surface is hydrophobic while sand surface is hydrophilic, it might be possible that adhesion to each of these surfaces has different physicochemical constraints, and cellulose might play different roles in these scenarios.
Microtiter well, sand attachment and microscopy assays of GFP-tagged bacterial cultures confirmed the role of cellulose in affecting biofilm structure. We propose that cellulase CelC is associated with cellulose cleavage and processing, and that extracellular cellulose micro fibrils, but not in excess, are needed to build the three-dimensional structure of a mature biofilm of R. leguminosarum bv. trifolii on plastic, sand and white clover host root surfaces.