**3.2. Cellulose**

378 The Complex World of Polysaccharides

**3.1. Bacterial alginates** 

**Figure 3.** Structure of alginate

uronic acid sequence.

**matrix** 

biofilms.

**3. Structure and function of different polysaccharides from the biofilm** 

The most famous exopolysaccharides present inside biofilms are alginate, cellulose and poly-N-acetyl glucosamine. This section focuses on their structures and their function inside

Alginate, a polysaccharide which occurs in brown algae and in different bacteria like *Azotobacter vinelandii* [30] and *P. aeruginosa* [44] has been extensively studied. Alginate is an exopolysaccharide with a relatively high molecular mass (104-106 g/ml). It consists of the uronic acid residues β-D-mannuronate (M) and its C-5 epimer, α-L-guluronate (G) [45] (Figure 3).

Generally, the monomers form a block copolymer with homopolymeric regions of poly-β-Dmannuronate (M-blocks) and poly-α-L-guluronate (G-blocks) as well as heteropolymeric regions (MG-blocks). The absence of G-blocks differentiates alginates produced by *P. aeruginosa* from alginates expressed by algae or by *A. vinelandii* [46]. The functional properties of the alginates strongly correlate with the composition (M/G ratio) and with the

There are 24 genes located on the bacterial chromosome, involved in the production and secretion of alginate in *P. aeruginosa* [45]. Eight genes are implicated in the exportation of alginic acids (Figure 4), and twelve in the biosynthesis of the polysaccharide [47]. The four

Alginates can form a gel in the presence of chelating divalent cations. This structure formed is called a Grant "egg-box" [47]. The alginate gel is formed by ionic bonds between the Grich blocks and divalent cations. The mechanical properties of alginate gels can vary depending on the amounts of guluronic acid present in the polymer. Moreover, alginate gels

remaining genes are involved in the regulation of the synthesis.

can be formed in vitro in the presence of proteins such as gelatin [48].

Cellulose is the most abundant sugar polymer found on the surface of the planet. It is found throughout the living world: in plants, animals, fungi and in bacteria such as *Salmonella, E. coli, Acetobacter, Agrobacterium* and *Rhizobium* [49].

*Salmonella* and *E. coli* produce cellulose as a crucial component of the extracellular matrix [50]. Cellulose consists of a β-1-4 linked linear glucose (Figure 5). The formation of cellulose fibers is provided by hydrogen bonds between the chains of glucose. These formed sheets are very stable and their number varies depending on the nature of the environment.

Cellulose has a crystalline structure. Each crystal of cellulose contains numerous glycan chains in parallel orientation. The reducing ends are at one terminus while the non-reducing ends are at the opposite terminus. The structure is not uniform and amorphous regions cohabit with highly crystalline regions.

**Figure 5.** Structure of cellulose

Genes involved in the production of cellulose in *E. coli* and *S. typhimurium* are called *bcs* for bacterial cellulose synthesis (Figure 6). The four *bcs* genes called *bcsA*, *bcsB*, *bcsZ* and *bcsC* are organized as an operon. The *bcs* operon is partially regulated by AgfD, a thin aggregative fimbriae which increases the production of cellulose and curli [49].

**Figure 6.** Cellulose biosynthesis

Cellulose can form a gel at adequate temperatures. Cellulose solutions are liquid at room temperature. Gels can form in a cellulose solution at either high temperature (above 50 °C) or low temperature (less than 10 °C). After gelification, cellulose solutions remain more or less stable in the gel state at room temperature [51, 52]. The gel structure of cellulose may explain the mechanical properties of biofilms formed by bacterial species producing this polymer.

## **3.3. Poly-N-acetyl glucosamine**

380 The Complex World of Polysaccharides

**Figure 5.** Structure of cellulose

**Figure 6.** Cellulose biosynthesis

Genes involved in the production of cellulose in *E. coli* and *S. typhimurium* are called *bcs* for bacterial cellulose synthesis (Figure 6). The four *bcs* genes called *bcsA*, *bcsB*, *bcsZ* and *bcsC* are organized as an operon. The *bcs* operon is partially regulated by AgfD, a thin

aggregative fimbriae which increases the production of cellulose and curli [49].

The polysaccharide intercellular adhesin (PIA) or the related poly-N-acetyl glucosamine (PNAG) polymer is required for bacterial adherence and biofilm formation of some bacterial species. This polysaccharide family was first described in *Saphylococcus* species [53], and further in *E. coli* [54]. PNAG is a positively charged linear homoglycan composed of β-1,6-Nacetylglucosamine residues with approximately 20% deacetylated residues [55] (figure 7).

**Figure 7.** Structure of PNAG

The genes involved in the biosynthesis of PIA are named *ica* for intercellular adhesion. This operon is composed of a regulation gene *icaR* and four biosynthetic genes: *icaADBC* [56, 57] (Figure 8).

PNAG forms a protective matrix around bacterial cells that is also involved in cell-to-cell interactions [53, 54]. PNAG can also interact with eDNA, reinforcing the biofilm matrix structure [58].

## **3.4. Other polysaccharides involved in biofilm formation**

Individual strains or one strain put in different environmental conditions, are able to produce several different extracellular polysaccharides. In mucoid strains of *P. aeruginosa* isolated from patients with cystic fibrosis, mucoidy is due to the overproduction of alginate that is the major constituent of the biofilm matrix. Nevertheless, biofilms formed by non

mucoid strains do not contain alginate. A glucose-rich polymer named PEL (pellicle), and a mannose-rich polymer named PSL (polysaccharide synthesis locus), makes significant contribution to *P. aeruginosa* biofilm structure [59]. The *pel* genes are present in several strains but some commonly used reference strains, such as PAK and PAO1 do not express strongly these genes under common laboratory conditions. The *psl* genes are only present in some strains, but not in the well known PA14 laboratory strain. In some instance, PEL, and PSL can be present together in the biofilm matrix of *P. aeruginosa*.

Other polymers are present in the matrix of the biofilm of *S. epidermidis* for example: teichoic acid [60]. There are two types of teichoic acid in *S. epidermidis*: teichoic acid associated with the bacterial membrane (CW TA) and extracellular teichoic acid (EC TA). The EC TA is responsible for the increased viscosity of the colony. The EC TA is a (1-3)-linked poly(glycerol phosphate), substituted at the 2-position with α-glucose, α-N-acetylglucose, D-alanine and α-glucose-6-alanine (Figure 9).

**Figure 8.** PNAG biosynthesis

**Figure 9.** Structure of teichoic acid

In *E. coli*, colanic acid, a sugar polymer composed of galactose, fructose and glucose, is regularly found in the biofilm matrix [61] (Figure 10).

**Figure 10.** Structure of colanic acid

mucoid strains do not contain alginate. A glucose-rich polymer named PEL (pellicle), and a mannose-rich polymer named PSL (polysaccharide synthesis locus), makes significant contribution to *P. aeruginosa* biofilm structure [59]. The *pel* genes are present in several strains but some commonly used reference strains, such as PAK and PAO1 do not express strongly these genes under common laboratory conditions. The *psl* genes are only present in some strains, but not in the well known PA14 laboratory strain. In some instance, PEL, and

Other polymers are present in the matrix of the biofilm of *S. epidermidis* for example: teichoic acid [60]. There are two types of teichoic acid in *S. epidermidis*: teichoic acid associated with the bacterial membrane (CW TA) and extracellular teichoic acid (EC TA). The EC TA is responsible for the increased viscosity of the colony. The EC TA is a (1-3)-linked poly(glycerol phosphate), substituted at the 2-position with α-glucose, α-N-acetylglucose,

In *E. coli*, colanic acid, a sugar polymer composed of galactose, fructose and glucose, is

PSL can be present together in the biofilm matrix of *P. aeruginosa*.

D-alanine and α-glucose-6-alanine (Figure 9).

**Figure 8.** PNAG biosynthesis

**Figure 9.** Structure of teichoic acid

regularly found in the biofilm matrix [61] (Figure 10).

It must be remembered that although different strains can apparently synthesize the same EPS, there can be differences in physical properties especially with respect to viscosity and gel formation. Several biofilm studies have used colanic acid-producing *E. coli* [61]. Prigent-Combaret, C. et al. [62], yet this polymer can vary greatly in mass and viscosity, as can bacterial alginates.
