**Cellulose Expression in**  *Pseudomonas fluorescens* **SBW25 and Other Environmental Pseudomonads**

Andrew J. Spiers, Yusuf Y. Deeni, Ayorinde O. Folorunso, Anna Koza, Olena Moshynets and Kamil Zawadzki

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53736

## **1. Introduction**

Bacterial cellulose was first isolated from the air-liquid (A-L) interface biofilm produced by *Bacterium xylinum* in 1886 [1], an acetic acid bacterium strain which would probably now be recognised as *Gluconacetobacter xylinus* (formerly *Acetobacter xylinum*) or a related species. Over the following century, more acetic acid bacteria and additional *Proteobacter* were found to produce cellulose (reviewed in [2-3]). Cellulose-producing bacteria include a mixture of gut commensals, plant and animal pathogens (these are listed in Table 1), and all share soil as a common secondary habitat. It is likely that cellulose provides protection against physical disturbance, predation or other environmental stresses common to these diverse environments. The biochemistry of bacterial cellulose expression has been studied extensively for *Gluconacetobacter,* and this understanding has been used as a model for enteric bacteria and pseudomonads [4-5] (for a range of bacterial cellulose reviews, see [2-3, 6-9]). Experimental reports of bacteria expressing cellulose are increasing, as well as the annotation of putative cellulose synthase-like operons in bacterial whole-genome sequences, suggesting that an increasingly wider range of bacteria may be capable of producing cellulose.

Our interest in bacterial cellulose began with the experimental evolution of the soil and plant-associated pseudomonad, *Pseudomonas fluorescens* SBW25 [10-12]. This resulted in a novel biofilm–forming adaptive mutant known as the Wrinkly Spreader (WS) and shown in Figure 1. Subsequent investigation of the WS phenotype identified partially-acetylated cellulose as the main matrix component of the biofilm. The pseudomonads are a highly diverse genus (see recent reviews by [13-14]), and biofilm-formation and celluloseexpression are now known to be common amongst the water, soil, plant-associated and

© 2013 Spiers et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Spiers et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

plant-pathogenic environmental pseudomonads [15]. However, the ecological role of cellulose and the fitness advantage it confers to these bacteria is poorly understood.

Cellulose Expression in *Pseudomonas fluorescens* SBW25 and Other Environmental Pseudomonads 3

partially and fully-saturated aggregations growing on solid surfaces, as well as those that are poorly-attached or 'free-floating', after the early and broad definition of Costerton *et al.* [16].

**Figure 1. The Wrinkly Spreader mutant of** *Pseudomonas fluorescens* **SBW25.** The Wrinkly Spreader (WS) mutant was isolated from evolving populations of wild-type SBW25 in static King's B microcosms. **(A)** Wild-type SBW25 (*left*) grows throughout the liquid column; in comparison, the WS (*right*) occupies the air-liquid (A-L) interface by producing a robust biofilm 1-2mm thick. **(B)** Wild-type SBW25 (smooth and rounded) and WS (wrinkled) colonies are readily differentiated on agar plates. Images from A. Spiers.

The importance of biofilms (aggregations) in nature is reflected by their prevalence in aquatic, soil, fungal, plant and animal ecosystems, and their role in many chronic human diseases and antibiotic resistance. Many natural biofilms are multi-species structures with complex interactions, and in earlier literature they were often referred to as zoogleal mats. Bacteria found within biofilms are profoundly different from those growing in suspension, differing in both gene expression and physiology and more resistant to desiccation, physical

Biofilm research has largely focussed on submerged, solid-liquid (S-L) interface biofilms to provide archetypal models of biofilm structure, function and allow genetic investigation (e.g. *Pseudomonas aeruginosa* PA01 flow-cell biofilms). In these, a surface-attached exopolysaccharide (EPS) polymer matrix-based structure develops away from the solid surface, into the flow of a nutrient and O2-rich growth medium, and where fluid flow and mass transfer affects biofilm

Biofilm formation begins when planktonic bacterial cells initiate attachment to a solid surface. Attached bacteria start to move across the surface, grow and form micro-colonies, which then develop slowly into the mature biofilm structure in which bacterial cells are embedded in an exopolysaccharide polymer matrix. When conditions become unfavourable within the biofilm, single bacteria or large lumps of biofilm material detach

disturbance and predation. A range of biofilm reviews are provided by [16-31].

development, structure and rheology (for reviews, see [19, 28-29]).

**3. Archetypal 'flow-cell' biofilms** 


Adapted from [2-3]. **\*,** Also known as the acetic acid bacteria; **†,** Referred to here as the enteric bacteria.

**Table 1.** Cellulose-expressing bacterial genera

Here we provide a review of our work focussing on biofilm-formation and cellulose expression by SBW25 and other environmental pseudomonads. We do not provide an extensive list of primary literature or current reviews, but hope that the citations we have made will allow others to access the growing wealth of publications relevant to the subjects raised in this review.

## **2. Bacterial assemblages and biofilms**

The formation of biofilms by bacteria is a key strategy in the colonisation of many environments, though biofilms are only one of a range of bacterial assemblages involved in this process. Bacterial assemblages range from isolated surface-attached bacteria, monolayers of associated bacteria forming micro-colonies, larger and more complex structures including differentiated biofilms, as well as poorly-attached or free-floating flocs and slime. At times the differences between assemblage types may be minor and will depend on local environmental conditions. These differences are frequently ignored by many who prefer the simple dichotomy of individual, free-swimming planktonic bacteria verses the structurally complex and genetically-determined biofilms. Here we use the term 'biofilm' to include partially and fully-saturated aggregations growing on solid surfaces, as well as those that are poorly-attached or 'free-floating', after the early and broad definition of Costerton *et al.* [16].

**Figure 1. The Wrinkly Spreader mutant of** *Pseudomonas fluorescens* **SBW25.** The Wrinkly Spreader (WS) mutant was isolated from evolving populations of wild-type SBW25 in static King's B microcosms. **(A)** Wild-type SBW25 (*left*) grows throughout the liquid column; in comparison, the WS (*right*) occupies the air-liquid (A-L) interface by producing a robust biofilm 1-2mm thick. **(B)** Wild-type SBW25 (smooth and rounded) and WS (wrinkled) colonies are readily differentiated on agar plates. Images from A. Spiers.

The importance of biofilms (aggregations) in nature is reflected by their prevalence in aquatic, soil, fungal, plant and animal ecosystems, and their role in many chronic human diseases and antibiotic resistance. Many natural biofilms are multi-species structures with complex interactions, and in earlier literature they were often referred to as zoogleal mats. Bacteria found within biofilms are profoundly different from those growing in suspension, differing in both gene expression and physiology and more resistant to desiccation, physical disturbance and predation. A range of biofilm reviews are provided by [16-31].
