**8. Distribution of** *wss***-like cellulose synthase operons amongst the proteobacteria**

We are undertaking a bioinformatics analysis of all publicly-available fully-sequenced bacterial genomes in order to determine the phylogenetic distribution and structural variation of *P. fluorescens* SBW25 *wss*-like cellulose biosynthetic operons amongst the proteobacteria. Protein (TBLASTN) homology searches were run against the GenBank complete genome database [81] using the SBW25 Wss proteins as the query sequences in October of 2011. From this, we have identified over 50 bacteria with gene clusters that showed significant protein sequence homology (≥ 25% ID) to three or more Wss proteins, including a minimum of two key cellulose synthase subunits, WssB, WssC, or WssE. Putative SBW25 *wss*-like operons were then manually curated for accuracy to provide Wss homologue protein sequences and operon structures. Phylogentic trees were constructed using the multiple sequence alignment program ClustalW 2.0 [82], and neighbour-joining and minimal evolution methods implemented by Geneious 5.5.5 (Biomatters Ltd, NZ).

Although this bioinformatics analysis is on-going and the final results expected to be published elsewhere, we make the following preliminary observations. First, whilst *wssB*  tends to be followed by *wssC* in the *wss*-like cellulose synthase operons as found previously [5, 8, 83], there are examples within the *Burkholderia* and in *Cupriavidus metallidurans* CH34 where *wssB* is separated from the rest of the operon. Second, we note that only the *P. fluorescens* SBW25 and *P. syringae* DC3000 *wss* operons include the *wssG-I* alginate acetylation-like genes, but not the closely-related pseudomonad *P. putida* KT2440. This suggests that DC3000 may also be able to express partially-acetylated cellulose, and that *wssF-I* genes may be narrowly restricted to the *P. fluorescens–syringae* complex. Third, there is considerable variation in cellulose synthesis operon structure amongst the *Enterobacteriacea*, with many having two clusters of genes (e.g. *Erwinia billingiae* Eb66, *Klebsiella pneumoniae* MGH78578, *Pantoea* sp. At-9b). Many enteric bacteria also include the additional genes *bcsEFG* which have no *wss* homologues (e.g. *Escherichia coli* K-12 MG1655, *Salmonella enterica* Typhimurium LT2, *Vibrio fisheri* MJ11). These have been reported to be associated with cellulose production in *Salmonella enteritidis* 3934 [4]. However, *Escherichia coli* K-12 DH10B contains only *bcsFG*, raising the possibility that *bcsE-G* are not essential for cellulose production in these bacteria. Although the *Gluconacetobacter* are not closely related to the enteric bacteria, we note that *G. xylinus* NBRC 3288 has a small *wssBCE* operon plus a larger *wssDBCE* operon. It is possible that such duplications might enable higher levels of cellulose expression under some environmental conditions, or that such gene duplications may persist for some time before deletion.

16 Cellulose – Medical, Pharmaceutical and Electronic Applications

matrix components [77-80].

**proteobacteria** 

We have conducted additional surveys of pseudomonads isolated from other habitats, including pond water, pitcher plant (*Sarracenia* spp.) deadfall trap-water, spoilt cold-stored meat and mushrooms (Table 4). These confirm the wide-spread ability of environmental pseudomonads to form A-L interface biofilms and to express cellulose under the experimental conditions used previously [15]. It is also evident that pseudomonads are capable of producing a wide range of EPS in addition to cellulose, including alginate, levan, marginalan, PEL, PSL, and a number of other polymers, which may be utilised as biofilm

**8. Distribution of** *wss***-like cellulose synthase operons amongst the** 

We are undertaking a bioinformatics analysis of all publicly-available fully-sequenced bacterial genomes in order to determine the phylogenetic distribution and structural variation of *P. fluorescens* SBW25 *wss*-like cellulose biosynthetic operons amongst the proteobacteria. Protein (TBLASTN) homology searches were run against the GenBank complete genome database [81] using the SBW25 Wss proteins as the query sequences in October of 2011. From this, we have identified over 50 bacteria with gene clusters that showed significant protein sequence homology (≥ 25% ID) to three or more Wss proteins, including a minimum of two key cellulose synthase subunits, WssB, WssC, or WssE. Putative SBW25 *wss*-like operons were then manually curated for accuracy to provide Wss homologue protein sequences and operon structures. Phylogentic trees were constructed using the multiple sequence alignment program ClustalW 2.0 [82], and neighbour-joining and minimal evolution methods implemented by Geneious 5.5.5 (Biomatters Ltd, NZ).

Although this bioinformatics analysis is on-going and the final results expected to be published elsewhere, we make the following preliminary observations. First, whilst *wssB*  tends to be followed by *wssC* in the *wss*-like cellulose synthase operons as found previously [5, 8, 83], there are examples within the *Burkholderia* and in *Cupriavidus metallidurans* CH34 where *wssB* is separated from the rest of the operon. Second, we note that only the *P. fluorescens* SBW25 and *P. syringae* DC3000 *wss* operons include the *wssG-I* alginate acetylation-like genes, but not the closely-related pseudomonad *P. putida* KT2440. This suggests that DC3000 may also be able to express partially-acetylated cellulose, and that *wssF-I* genes may be narrowly restricted to the *P. fluorescens–syringae* complex. Third, there is considerable variation in cellulose synthesis operon structure amongst the *Enterobacteriacea*, with many having two clusters of genes (e.g. *Erwinia billingiae* Eb66, *Klebsiella pneumoniae* MGH78578, *Pantoea* sp. At-9b). Many enteric bacteria also include the additional genes *bcsEFG* which have no *wss* homologues (e.g. *Escherichia coli* K-12 MG1655, *Salmonella enterica* Typhimurium LT2, *Vibrio fisheri* MJ11). These have been reported to be associated with cellulose production in *Salmonella enteritidis* 3934 [4]. However, *Escherichia coli* K-12 DH10B contains only *bcsFG*, raising the possibility that *bcsE-G* are not essential for cellulose production in these bacteria. Although the *Gluconacetobacter* are not closely related to the enteric bacteria, we note that *G. xylinus* NBRC 3288 has a small *wssBCE* operon plus a larger *wssDBCE* operon. It is possible that such duplications might enable higher levels of Finally, the clustering of WssB homologue sequences (Figure 8) generally follows the 16S phylogenetic relationships between bacteria. However, we are surprised to find that *P. fluorescens* SBW25 and *P. syringae* DC300 cluster with many of the *Burkholderia* and *Xanthomonas*, whilst *P. putida* and *P. stutzeri* strains cluster with the enteric bacteria. We have yet to compare the clustering patterns of the WssC, WssD and WssE homologues, where conserved patterns may reflect different functional roles for cellulose and host lifestyles, whilst aberrant placements of single proteins might reflect the random mutation of a phenotype no longer of functional value or under positive selection.

**Figure 8. Cladogram of WssB homologues.** The structure of the WssB cladogram is similar to that constructed using 16s rRNA sequences, with the enteric bacteria and pseudomonads forming two distinct clusters. Within the pseudomonads, the *P. fluorescens-syringae* complex has diverged earlier than the *P. putida-stutzeri* group. The cladogram was constructed using Geneious 5.5.5 (Biomatters Ltd, NZ) default parameters after multiple sequence alignment of 58 WssB proteins by ClustalW 2.0 [82].

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

Cellulose Expression in *Pseudomonas fluorescens* SBW25 and Other Environmental Pseudomonads 19

similar observation has been made for *P. putida* mt-2 (the progenitor of KT2440) where water stress was also found to increase cellulose expression [76]. Many bacteria respond to desiccation by producing exopolysaccharides, many of which are hygroscopic and retain water entropically [92], and amorphous cellulose is more hygroscopic and retains more liquid than crystalline cellulose [62]. Support for an anti-predation role for cellulose comes from the finding that the competitive fitness of WS genotypes in static microcosms increases

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.

, Ayorinde O. Folorunso and Kamil Zawadzki

*Laboratory of Microbial Ecology, Institute of Molecular Biology and Genetics of the National* 

AJS is a member of the Scottish Alliance for Geoscience Environment and Society (SAGES) and AK was a SAGES-associated PhD student with AJS. KZ undertook his MSc research

*The SIMBIOS Centre, University of Abertay Dundee, Dundee, UK* 

*School of Contemporary Sciences, University of Abertay Dundee, Dundee, UK* 

*Novo Nordisk Foundation Center for Biosustainability, Hørsholm, Denmark* 

in the presence of the grazing protist *Tetrahymena thermophile* [93].

**10. Concluding statement** 

**Author details** 

Andrew J. Spiers\*

Yusuf Y. Deeni

Olena Moshynets

**Acknowledgement** 

Corresponding Author

*Academy of Sciences of Ukraine, Kiev, Ukraine* 

Anna Koza

 \*

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 cellulose expression via these repressors.

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 instead of natural soil.

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 similar observation has been made for *P. putida* mt-2 (the progenitor of KT2440) where water stress was also found to increase cellulose expression [76]. Many bacteria respond to desiccation by producing exopolysaccharides, many of which are hygroscopic and retain water entropically [92], and amorphous cellulose is more hygroscopic and retains more liquid than crystalline cellulose [62]. Support for an anti-predation role for cellulose comes from the finding that the competitive fitness of WS genotypes in static microcosms increases in the presence of the grazing protist *Tetrahymena thermophile* [93].
