**7. Biofilm formation and cellulose expression amongst other pseudomonads**

Having discovered that *P. fluorescens* SBW25 could express cellulose in the WS biofilm (and subsequently in the VM biofilm), we were interested to see if related environmental pseudomonads could produce similar cellulose-matrix–based A-L interface biofilms. We therefore undertook a survey of environmental pseudomonads, including water, soil, plantassociated and plant pathogenic isolates (we did not include human or other animal pathogens in this survey) [15]. The ability of each to produce an A-L interface biofilm was assessed in static King's B liquid media microcosms. Importantly, this assay did not differentiate between isolates that constitutively produced biofilms, with those that might utilise quorum sensing-like signalling to initiate biofilm-formation, or those that had mutated into a biofilm-forming genotype. Of the 185 environmental pseudomonads tested, 76% were found to produce observable biofilms within 15 days incubation. The phenotypes of these were variable, with biofilm strengths ranging 1500*x*, but could be categorised into the physically-cohesive (PC), floccular mass (FM), waxy aggregate (WA) and viscous mass (VM)-class biofilms described in Table 2 (see also Figure 2) [15, 46].

Calcofluor-fluorescent microscopy identified cellulose as the matrix component of 20% of the biofilm-forming isolates, indicating that at least seven *Pseudomonas* species were capable of expressing cellulose under the conditions tested. These included *P. corrugata* (tomato pathogens), *P. fluorescens* (plant-associated isolates), *P. marginalis* (alfalfa and parsnip pathogens), *P. putida* (rhizosphere and soil isolates), *P. savastanoi* (olive pathogens), *P. stutzeri* (represented by a single clinical isolate), and *P. syringae* (celery, cucumber, tobacco, and tomato isolates or pathogens) (isolates from another eleven *Pseudomonas* spp. were tested, including *P. aeruginosa* PA01 and PA14, and were not found to produce cellulose). For two of the celluloseexpressing isolates, *P. putida* KT2440 and *P. syringae* DC3000, the whole genome sequences were available and SBW25 *wss*-like cellulose synthase operons had been annotated [74-75], though no experimental reports of either expressing cellulose had been made.

14 Cellulose – Medical, Pharmaceutical and Electronic Applications

attachment factor that is also required for the WS phenotype.

**pseudomonads** 

**7. Biofilm formation and cellulose expression amongst other** 

(VM)-class biofilms described in Table 2 (see also Figure 2) [15, 46].

**Figure 7. Activation of the WS phenotype.** The Wrinkly Spreader (WS) phenotype is controlled by the membrane-associated Wsp complex and associated DGC response regulator WspR. **(A)** In wild-type *Pseudomonas fluorescens* SBW25, when an appropriate environmental signal is received the Wsp complex phosphorylates WspR which then results in the production of c-*di*-GMP. However, in the absence of signal, the Wsp complex is silent and c-*di*-GMP levels remain low. **(B)** In the Wrinkly Spreader a mutation in a Wsp subunit (WspF) alters the sensitivity of the Wsp complex such that it activates WspR in the absence of the environmental signal. The resulting increase in c-*di*-GMP activates the membraneassociated cellulose synthase complex to produce cellulose, and also activates the unidentified

Having discovered that *P. fluorescens* SBW25 could express cellulose in the WS biofilm (and subsequently in the VM biofilm), we were interested to see if related environmental pseudomonads could produce similar cellulose-matrix–based A-L interface biofilms. We therefore undertook a survey of environmental pseudomonads, including water, soil, plantassociated and plant pathogenic isolates (we did not include human or other animal pathogens in this survey) [15]. The ability of each to produce an A-L interface biofilm was assessed in static King's B liquid media microcosms. Importantly, this assay did not differentiate between isolates that constitutively produced biofilms, with those that might utilise quorum sensing-like signalling to initiate biofilm-formation, or those that had mutated into a biofilm-forming genotype. Of the 185 environmental pseudomonads tested, 76% were found to produce observable biofilms within 15 days incubation. The phenotypes of these were variable, with biofilm strengths ranging 1500*x*, but could be categorised into the physically-cohesive (PC), floccular mass (FM), waxy aggregate (WA) and viscous mass Many environmental pseudomonads can also be induced to form A-L interface biofilms and to express cellulose using WspR19. When expressed *in trans* in wild-type SBW25 it produces the WS phenotype [60, 70], though in other pseudomonads the impact was found to be more variable. In a test of 16 pseudomonads known to form biofilms and express cellulose, WspR19 was found to significantly increase biofilm attachment, strength, and cellulose expression in *P. fluorescens* 54/96, *P. syringae* DC3000, *P. syringae* T1615 and *P. syringae* 6034 [15] (WspR19 induction of cellulose production by SBW25 and DC3000 is shown in Figure 5). WspR19 also induced a WS-like phenotype in *P. putida* KT2440, despite the fact that biofilm-formation or cellulose expression in this pseudomonad had not been observed in the initial survey (cellulose expression was subsequently reported for both wild-type and WspR19-carrying strains under different experimental conditions by [76]). Similarly, nine of ten non-biofilm– forming and non-cellulose expressing *P. syringae* isolates were found to produce biofilms when induced with WspR19, and two of these also expressed detectable levels of cellulose [15]. These findings suggest that biofilm-formation and cellulose expression in pseudomonads closely related to *P. fluorescens* SBW25 are probably controlled by the same c-*di*-GMP– mediated regulatory system or are sensitive to non-specific increases in c-*di*-GMP levels.


\* Estimated from a sub-sample of 25 isolates. Data compiled from [15] and unpublished research from **a,** R. Ahmed, AK & AJS; **b,** B. Varun, AK & AJS; **c,** D.S. Kumar, AK & AJS; **d,** M. Robertson & AJS; and **e,** AK & AJS.

**Table 4.** Prevalence of A-L interface biofilm formation and cellulose expression amongst environmental pseudomonads

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 matrix components [77-80].

Cellulose Expression in *Pseudomonas fluorescens* SBW25 and Other Environmental Pseudomonads 17

cellulose expression under some environmental conditions, or that such gene duplications

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

**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].

phenotype no longer of functional value or under positive selection.

may persist for some time before deletion.
