**10. Concluding statement**

18 Cellulose – Medical, Pharmaceutical and Electronic Applications

cellulose expression via these repressors.

instead of natural soil.

**9. Ecological role and fitness advantage of cellulose** 

Attempts to understand the importance of cellulose expression by *P. fluorescens* SBW25 in soil and the phytosphere have involved investigation of the regulators controlling *wss* operon transcription [5, 84], and measurement of fitness advantage using plant microcosms [85]. Cellulose expression is clearly regulated at two levels in SBW25. First, the activity of the cellulose synthase complex is regulated by c-*di*-GMP levels, but it is not known what environmental signals control WspR, other DGCs or their antagonists, though c-*di*-GMP is known to be involved in a range of surface colonisation and pathogenicity systems in a variety of bacteria (reviewed in [86-87]). Second, cellulose expression is regulated at the level of *wss* operon transcription, with mini-transposon analysis identifying AlgR, AwsR and

AwsXR was a previously unrecognised regulatory system, first identified in SBW25 where the mutational activation of the DGC AwsR results in the WS phenotype [47, 72], though the normal means of regulating AwsR activity remains unknown. FleQ is a c-*di*-GMP– responsive transcriptional regulator, and in *P. aeruginosa* PA01 it controls the hierarchical regulatory cascade for flagella biosynthesis and the repression of the PEL biosynthesis genes [88-89]. AlgR and AmrZ (also referred to as AlgZ) are transcription factors involved in the regulation of a number of systems including alginate biosynthesis and twitching motility [79, 90]. It is possible that AlgR, AmrZ, and AwsR directly repress SBW25 *wss* transcription, whilst AwsX, FleQ, and WspR act indirectly to regulate transcription and therefore cellulose expression [84]. However, no environmental signals have been identified that induce

Sugar beet (*Beta vulgaris*) seedlings have been used to determine the competitive fitness advantage cellulose-expression may provide wild-type SBW25 compared to a cellulosedeficient mutant [85]. In these experiments, seeds were first inoculated with a mixture of wild-type SBW25 and SM-13, a mutant containing a mini-Tn*5* insertion cassette derived from WS-13 [5]. These were then germinated and grown for four weeks in an artificial soil substrate. Bacteria were then recovered from the stems and leaves (the phyllosphere), from roots and adherent vermiculite (the rhizosphere), and from un-planted containers ('bulk soil') to allow the calculation of competitive fitness (W) [91] (we report W for SM-13 *cf* wildtype SBW25 here for clarity). In the phyllosphere and rhizosphere, SBW25 was found to have a significant fitness advantage over SM-13, with W ≈ 1.8 and W ≈ 1.11, respectively, but not in bulk soil where W ≈ 1.05. These findings suggest that the appropriately-controlled expression of cellulose by wild-type SBW25 provides some benefit on plant surfaces. It is possible that the mechanistic nature of this benefit may be an improved tolerance to waterlimiting conditions rather than resistance to physical disturbance and predation, as the seedlings were watered using a tray rather than from a sprinkler, and vermiculite was used

Comparisons of the survival of wild-type SBW25 and a cellulose-deficient mutant similar to SM-13 under water-limiting conditions have shown that the loss of cell viability is faster for the mutant than for wild-type SBW25 (A Koza & A Spiers, unpublished observations). A

WspR as positive regulators, and AmrZ and FleQ as negative regulators [5, 84].

Bacterial cellulose production and air-liquid (A-L) interface biofilm-formation was first described 1886 for *Bacterium xylinum*, and subsequently observed by ourselves and colleagues in the evolution of the *Pseudomonas fluorescens* SBW25 Wrinkly Spreader (WS) some 116 years later. It is clear that this type of biofilm-formation is common-place amongst the environmental pseudomonads, many of which also utilise cellulose as the main matrix component of the biofilm. The fitness advantage of cellulose matrix-based biofilm-formation by SBW25 in static microcosms is well-proven, but the fitness in natural environments, and the true function of cellulose, is poorly researched and not yet understood. It may be that bacterial cellulose is used to form small biofilms in water bodies, acting to retain bacteria at the A-L interface or to maintain them on solid surfaces against water flow. Appositely, cellulose fibres may resist desiccation stress in water-limited environments, or even provide protection from protist and nematode predation. It is of course possible that cellulose performs a number of functions, which might explain the wide distribution of cellulose synthase operons amongst the *Proteobacter* inhabiting a diverse array of environments.
