IntroductionPeriodontal diseases are known to be associated with polymicrobial biofilms and inflammasome activation. A deeper understanding of the subgingival cytological (micro) landscape, the role of extracellular DNA (eDNA) during periodontitis, and contribution of the host immune eDNA to inflammasome persistence, may improve our understanding of the mechanisms underlaying severe forms of periodontitis.MethodsIn this work, subgingival biolfilms developing on biologically neutral polyethylene terephthalate films placed in gingival cavities of patients with chronic periodontitis were investigated by confocal laser scanning microscopy (CLSM). This allowed examination of realistic cytological landscapes and visualization of extracellular polymeric substances (EPS) including amyloids, total proteins, carbohydrates and eDNA, as well as comparison with several single-strain in vitro model biofilms produced by oral pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus gordonii, S. sanguinis and S. mitis. Fluorescence in situ hybridization (FISH) analysis was also used to identify eDNA derived from eubacteria, streptococci and members of the Bacteroides–Porphyromonas–Prevotella (BPP) group associated with periodontitis.ResultsAnalysis of subgingival biofilm EPS revealed low levels of amyloids and high levels of eDNA which appears to be the main matrix component. However, bacterial eDNA contributed less than a third of the total eDNA observed, suggesting that host-derived eDNA released in neutrophil extracellular traps may be of more importance in the development of biofilms causing periodontitis.DiscussioneDNA derived from host immunocompetent cells activated at the onset of periodontitis may therefore be a major driver of bacterial persistence and pathogenesis.

Bacteria produce a variety of polysaccharides with functional roles in cell surface coating, surface and host interactions, and biofilms. We have identified an 'Orphan' bacterial cellulose synthase catalytic subunit (BcsA)-like protein found in four model pseudomonads, P. aeruginosa PA01, P. fluorescens SBW25, P. putida KT2440 and P. syringae pv. tomato DC3000. Pairwise alignments indicated that the Orphan and BcsA proteins shared less than 41% sequence identity suggesting they may not have the same structural folds or function. We identified 112 Orphans among soil and plant-associated pseudomonads as well as in phytopathogenic and human opportunistic pathogenic strains. The wide distribution of these highly conserved proteins suggest they form a novel family of synthases producing a different polysaccharide. In silico analysis, including sequence comparisons, secondary structure and topology predictions, and protein structural modelling, revealed a two-domain transmembrane ovoid-like structure for the Orphan protein with a periplasmic glycosyl hydrolase family GH17 domain linked via a transmembrane region to a cytoplasmic glycosyltransferase family GT2 domain. We suggest the GT2 domain synthesises β-(1,3)-glucan that is transferred to the GH17 domain where it is cleaved and cyclised to produce cyclic-β-(1,3)-glucan (CβG). Our structural models are consistent with enzymatic characterisation and recent molecular simulations of the PaPA01 and PpKT2440 GH17 domains. It also provides a functional explanation linking PaPAK and PaPA14 Orphan (also known as NdvB) transposon mutants with CβG production and biofilm-associated antibiotic resistance. Importantly, cyclic glucans are also involved in osmoregulation, plant infection and induced systemic suppression, and our findings suggest this novel family of CβG synthases may provide similar range of adaptive responses for pseudomonads.

Early Mortality Syndrome (EMS) has been a major problem for shrimp aquaculture in Southeast Asia due to its epizootic prevalence within the region since the first reported case in 2009. This study explores the application of halophilic marine bacilli isolated from coral mucus and their quorum-quenching abilities as potential biocontrol agents in aquaculture systems to combat the causative agent of EMS, Vibrio parahaemolyticus. N-acylhomoserine lactone (AHL)-degrading (AiiA) activity was first screened by PCR then confirmed by bio-reporter assay, and a combination of 16S rDNA sequence analysis and quantitative phenotype assays including biofilm-formation and temperature-growth responses were used to demonstrate diversity amongst these quorum-quenching isolates. Three phenotypically distinct strains showing notable potential were chosen to undergo co-cultivation as a method for strain improvement via long term exposure to the pathogenic V. parahaemolyticus. The novel approach taken led to significant improvements in antagonism and quorum quenching activities as compared to the ancestral wild-type strains and offers a potential solution as well as pathway to improve existing beneficial microbes for one of the most pressing issues in shrimp aquacultures worldwide.

Pseudomonas fluorescens SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air–liquid interface biofilms in experimental microcosms and on plant surfaces. Previous investigations have shown that cellulose is the primary structural matrix component in the robust and well-attached Wrinkly Spreader biofilm, as well as in the fragile Viscous Mass biofilm. Here, we demonstrate that both biofilms include extracellular DNA (eDNA) which can be visualized using confocal laser scanning microscopy (CLSM), quantified by absorbance measurements, and degraded by DNase I treatment. This eDNA plays an important role in cell attachment and biofilm development. However, exogenous high-molecular-weight DNA appears to decrease the strength and attachment levels of mature Wrinkly Spreader biofilms, whereas low-molecular-weight DNA appears to have little effect. Further investigation with CLSM using an amyloid-specific fluorophore suggests that the Wrinkly Spreader biofilm might also include Fap fibers, which might be involved in attachment and contribute to biofilm strength. The robust nature of the Wrinkly Spreader biofilm also allowed us, using MALDI-TOF mass spectrometry, to identify matrix-associated proteins unable to diffuse out of the structure, as well as membrane vesicles which had a different protein profile compared to the matrix-associated proteins. CLSM and DNase I treatment suggest that some vesicles were also associated with eDNA. These findings add to our understanding of the matrix components in this model pseudomonad, and, as found in other biofilms, biofilm-specific products and material from lysed cells contribute to these structures through a range of complex interactions.

Novel antibiotic combinations may act synergistically to inhibit the growth of multidrug-resistant bacterial pathogens but predicting which combination will be successful is difficult, and standard antimicrobial susceptibility testing may not identify important physiological differences between planktonic free-swimming and biofilm-protected surface-attached sessile cells. Using a nominally macrolide-resistant model Klebsiella pneumoniae strain (ATCC 10031) we demonstrate the effectiveness of several macrolides in inhibiting biofilm growth in multi-well plates, and the ability of azithromycin (AZM) to improve the effectiveness of the antibacterial last-agent-of-choice for K. pneumoniae infections, colistin methanesulfonate (CMS), against biofilms. This synergistic action was also seen in biofilm tests of several K. pneumoniae hospital isolates and could also be identified in polymyxin B disc-diffusion assays on azithromycin plates. Our work highlights the complexity of antimicrobial-resistance in bacterial pathogens and the need to test antibiotics with biofilm models where potential synergies might provide new therapeutic opportunities not seen in liquid culture or colony-based assays.

The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA–PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.

The establishment of O2 gradients in liquid columns by bacterial metabolic activity produces a spatially-structured environment. This produces a high-O2 region at the top that represents an un-occupied niche which could be colonised by biofilm-competent strains. We have used this to develop an experimental model system using soil-wash inocula and a serial-transfer approach to investigate changes in community-based biofilm-formation and productivity. This involved 10 transfers of mixed-community or biofilm-only samples over a total of 10-60 days incubation. In all final-transfer communities the ability to form biofilms was retained, though in longer incubations the build-up of toxic metabolites limited productivity. Measurements of microcosm productivity, biofilm-strength and attachment levels were used to assess community-aggregated traits which showed changes at both the community and individual-strain levels. Final-transfer communities were stratified with strains demonstrating a plastic phenotype when migrating between the high and low-O2 regions. The majority of community productivity came from the O2-depleted region rather than the top of the liquid column. This model system illustrates the complexity we expect to see in natural biofilm-forming communities. The connection between biofilms and the liquid column seen here has important implications for how these structures form and respond to selective pressure.

Outer membrane vesicles (OMVs) and extracellular DNA (eDNA) are important for biofilm formation for many bacteria. OMVs are a perfect transport system to deliver biofilm-related components including eDNA beyond the boundaries of cells, and eDNA itself is an important structural component of biofilms as well as enabling horizontal gene transfer and local adaptation. Both OMVs and eDNA are found in the biofilms produced by the opportunistic human pathogen P. aeruginosa and the plant pathogen P. syringae, but as yet, they have not been reported in the cellulose matrix-based biofilms produced by the related model rhizosphere bacterium Pseudomonas fluorescens SBW25. In this work we have gone back to re-assess the complexity of SBW25 biofilms by looking for evidence of OMVs and eDNA associated with biofilm–formation. OMVs were first imaged by SEM and LC-MC analysis used to identify 51 biofilm matrix-associated proteins of which 12 were also identified in biofilm OMVs. Interestingly, only 5 proteins were identified in both biofilm matrix and OMV samples, but not in planktonic OMVs, suggesting that these may be biofilm-specific components. We also observed eDNA by CLSM in both the weak and poorly-attached Viscous Mass (VM) and robust and well-attached Wrinkly Spreader (WS) air-liquid (A-L) interface biofilms produced by wild-type SBW25 and the Wrinkly Spreader mutant. The eDNA fraction could be precipitated from biofilm cell-free supernatant samples which demonstrated that WS biofilms had two-fold–higher levels than VM biofilms. DNAse treatment effected the development of both types of biofilm and reduced the strength and attachment levels when added to mature VM and WS biofilms. Testing with exogenous DNA suggests that high molecular weight (HMW) DNA is involved in both strength and attachment, perhaps by surface conditioning and interactions with the primary cellulose matrix common to both biofilms. HMW eDNA could be isolated directly from biofilm supernatants whereas two different HMW size fractions could be isolated from OMVs, presumably, from the outer OMV surface because DNAse treatment led to a substantially reduced DNA signal. This suggest that eDNA persistence and degradation in SBW25 biofilms is complex and eDNA fractions may play different roles in biofilm development, protection and adaptation.

Growing bacterial populations diversify to produce a number of competing lineages. In the Pseudomonas fluorescens SBW25 model system, Wrinkly Spreader mutant lineages, capable of colonising the air-liquid interface of static microcosms by biofilm-formation, rapidly appear in diversifying populations with a fitness advantage over the ancestral wild-type strain. Similarly, a biofilm is rapidly produced by a community containing many biofilm-competent members, and selection by serial transfer of biofilm samples across microcosms results in a gradually changing community structure. Both the adaptive radiation producing Wrinkly Spreaders and the succession of biofilm communities in these static microcosms can be understood through evolutionary ecology in which ecological interactions and evolutionary processes are combined. Such eco-evolutionary dynamics are especially important for bacteria, as rapid growth, high population densities and strong selection in the context of infections can lead to fast changes in disease progression and resistance phenotypes, while similar changes in community function may also affect many microbially mediated biotechnological and industrial processes. Evolutionary ecology provides an understanding of why bacterial biofilms are so prevalent and why they are such a successful colonisation strategy, and it can be directly linked to molecular analyses to understand the importance of pathways and responses involved in biofilm-formation.

Static incubation of liquid microcosms results in a physically heterogeneous environment, where depletion of O2 in the lower region creates a relatively high-O2 niche directly below the air-liquid (A-L) interface. This has been investigated using the model bacterium Pseudomonas fluorescens SBW25 and the biofilm-forming adaptive mutant known as the Wrinkly Spreader. In this system, colonisation of the A-L interface by the Wrinkly Spreader provides a fitness advantage over non-biofilm-forming competitors, including the ancestral SBW25, due to better access to O2 in an otherwise O2-growth limiting environment. Our current research seeks to understand how the ecological interactions of this simple system applies in more complex communities, where biofilms can be produced by multiple competing or co-operative strains and the low-O2 region colonised by a range of strains capable of micro-aerobic growth. Here we report the effect of selection on the productivity of A-L interface biofilm-forming communities initiated by soil-wash (SW) inocula, which were serially transferred across ten microcosms and sixty days with mixed-community or biofilm-only samples. Initial analysis of the serial transfer experiments shows a decrease in community productivity which is explained by the accumulation of toxic metabolites, though small increases in community biofilm strength and attachment were also observed. Isolate-level analysis revealed a decrease in community diversity and a biofilm-associated phenotypic shift between the SW inocula and final-transfer communities, and these changes provide evidence of selection within our system. Cell-localisation experiments confirm enrichment at the top of the liquid column in the high-O2 region, but also show high cell densities in the low-O2 region, even within the biofilm-only final-transfer communities. Samples taken from the biofilm and lower region of these communities were able to re-colonise both in fresh microcosms, indicating that community members were capable of migration within the liquid column. Despite the over-all decrease seen in community productivity in the serial transfer experiments, we suggest that communities maximised productivity by colonising both regions of the liquid column, with a resource trade-off between fast growth in the highly competitive high-O2 region and slower growth in the less-competitive low-O2 region. Many isolates from the final-transfer communities could occupy both regions and were capable of migration, with almost all isolates capable of flagella-mediated motility, and we interpret this ability to move between regions as a fitness advantage in A-L interface biofilm-forming communities. Although we have not been able to test this directly using the final-transfer communities or isolates, we have been able to demonstrate a fitness advantage in the less complex P. fluorescens SBW25 system, where biofilm-forming mutants capable of colonising both regions had a greater competitive fitness advantage over those with a poor ability to colonise the liquid column.

Static microcosms are a well-established system used to study the adaptive radiation of Pseudomonas fluorescens SBW25 and the adaptive biofilm-forming mutants known as the Wrinkly Spreaders (WS). We have developed this system to investigate selection within multi-species communities using a soil-wash inoculum dominated by biofilm-competent pseudomonads. Here we present community and isolate-level analyses of one serial-transfer experiment in which replicate populations were selected for over ten transfers and 60 days. Although no significant trends in improving community biofilm characteristics or total microcosm productivity were observed, a significant shift in biofilm-formation and microcosm growth by individual isolates recovered from the initial soil-wash inoculum and final transfers indicated that these communities were subject to selection for growth in these microcosms. Surprisingly, the fitness of the archetypal WS was poor when competing against community samples, and having compared the cell densities in the low-O2 region of liquid column below the biofilm, we suggest that part of the community’s fitness advantage comes from the ability to colonise this under-utilised niche as well as to compete at the A-L interface. Samples from the community biofilms and the low-O2 region were able to re-colonize both niches and many final transfer isolates grew throughout the liquid column as well as forming A-L interface biofilms. This suggests that there is a trade-off between fast growth under highly competitive conditions at the A-L interface and slower growth with less competition in the low-O2 region, with some isolates taking a bet-hedging approach a colonizing both niches in our microcosm system.

Model bacterial biofilm systems suggest that bacteria produce one type of biofilm, which is then modified by environmental and physiological factors, although the diversification of developing populations might result in the appearance of adaptive mutants producing altered structures with improved fitness advantage. Here we compare the air-liquid (A-L) interface viscous mass (VM) biofilm produced by Pseudomonas fluorescens SBW25 and the wrinkly spreader (WS) and complementary biofilm-forming strain (CBFS) biofilm types produced by adaptive SBW25 mutants in order to better understand the link between these physical structures and the fitness advantage they provide in experimental microcosms. WS, CBFS and VM biofilms can be differentiated by strength, attachment levels and rheology, as well as by strain characteristics associated with biofilm formation. Competitive fitness assays demonstrate that they provide similar advantages under static growth conditions but respond differently to increasing levels of physical disturbance. Pairwise competitions between biofilms suggest that these strains must be competing for at least two growth-limiting resources at the A-L interface, most probably O2 and nutrients, although VM and CBFS cells located lower down in the liquid column might provide an additional fitness advantage through the colonization of a less competitive zone below the biofilm. Our comparison of different SBW25 biofilm types illustrates more generally how varied biofilm characteristics and fitness advantage could become among adaptive mutants arising from an ancestral biofilm-forming strain and raises the question of how significant these changes might be in a range of medical, biotechnological and industrial contexts where diversification and change may be problematic.

Background: The use of different chemicals for agriculture, industry and mining has caused pollution of agrarian soils which provokes changes in rhizosphere microflora. We have studied 10 bacterial strains isolated from a winter wheat Cd-polluted field in Ukraine by their taxonomic position, biochemical properties and resistance to 3 classes of toxicants: heavy metals (Cu2+, Cd2+), non-metals (perchlorate-ion), organic xenobiotic (1-chloro-4-nitrobenzene, CNB). Objectives: To study the effect of toxicants on the biofilm–formation ability of individual strains and a mixed community. Methods: Biofilm characteristics (total microcosm growth, biofilm strength and attachment to the microcosm walls) were studied by combined biofilm assay (n = 4) with four treatments including 100 mg/L Cu2+, 25 mg/L Cd2+, 300 mg/L ClO4-, and 100 mg/L CNB, with correlations and Principal component analysis (PCA) used to investigate data. Results: We found that microbial community had a greater resistance to the toxicants compare to individual strains. The presence of heavy metals increased the strength of biofilms, and in most cases growth and biofilm strength were positively correlated. However, in the presence of Cd2+ this correlation was lost. Perchlorate affected bacteria, increasing mucus production and biofilm strength though it also reduced attachment levels. Finally, we also found that CNB could be used as a source of Carbon and energy during biofilm–formation.

We are investigating characteristics associated with oil degradation amongst bacteria isolated from clean and hydrocarbon contaminated soils from Nigeria and the UK.Our focus has been to identify bacteria expressing surfactants following isolation on Pseudomonas selective (PSA-CFC) and non-selective nutrient media and investigate the nature of surfactants, heavy metal resistance and hydrocarbon-degrading enzymes expressed by the bacteria. Of five sites sampled, a total of 1460 colonies were tested using the drop collapse assay, and 110 were found to express surfactants reducing liquid surface tensions as assessed by quantitative tensiometry to between 24.7 and 26.7 mN.m-1 (Tukey-Kramer HSD, α=0.05). We undertook a range of growth and behaviour-based assays on 60 selected strain which, when investigated by Hierarchical cluster analysis (HCA) demonstrated that this collection showed considerable phenotypic diversity. Eight out of the 60 strains could grow at a high temperature (50 °C), 35 of the 60 strains utilized diesel as a sole carbon source, and most of the strains could tolerate high concentrations (up to 20 mM) of heavy metals. Identification by 16S rDNA sequencing revealed that some of the strains belong to Pseudomonas, Bacillus, and Stenotrophomonas genera. We found using bioinformatics analysis of eight-selected draft genome sequences (AntiSMASH and RAST) NRPS-like (probable surfactants), cytochrome P450, catechol-1,2/2,3-dioxygenase, lipase, and heavy metal resistance gene sequences. We intend to use the information provided in this research to select strains for potential applications in in-situor ex-situ bioremediation of hydrocarbon-contaminated soils.

Several model plants are known to respond to bacterial quorum sensing molecules with altered root growth and gene expression patterns and induced resistance to plant pathogens. These compounds may represent novel elicitors that could be applied as seed primers to enhance cereal crop resistance to pathogens and abiotic stress and to improve yields. We investigated whether the acyl-homoserine lactone N-hexanoyl-L-homoserine lactone (C6-HSL) impacted winter wheat (Triticum aestivum L.) seed germination, plant development and productivity, using two Ukrainian varieties, Volodarka and Yatran 60, in both in vitro experiments and field trials. In vitro germination experiments indicated that C6-HSL seed priming had a small but significant positive impact on germination levels (1.2x increase, p < 0.0001), coleoptile and radicle development (1.4x increase, p < 0.0001). Field trials over two growing seasons (2015-16 and 2016-17) also demonstrated significant improvements in biomass at the tillering stage (1.4x increase, p < 0.0001), and crop structure and productivity at maturity including grain yield (1.4-1.5x increase, p < 0.0007) and quality (1.3x increase in good grain, p < 0.0001). In some cases variety effects were observed (p ≤ 0.05) suggesting that the effect of C6-HSL seed priming might depend on plant genetics, and some benefits of priming were also evident in F1 plants grown from seeds collected the previous season (p ≤ 0.05). These field-scale findings suggest that bacterial acyl-homoserine lactones such as C6-HSL could be used to improve cereal crop growth and yield and reduce reliance on fungicides and fertilisers to combat pathogens and stress.

The adaptive radiation of Pseudomonas fluorescens SBW25 populations in static liquid microcosms results in the appearance of the biofilm–forming Wrinkly Spreader. This adaptive mutant is able to colonise the high O2-rich region at the top of the liquid column established by the metabolic activity of earlier wild-type colonists, and by doing so, enjoys a significant fitness advantage over non-biofilm–forming competitors including the ancestral Pf. SBW25. Although the underlying molecular biology and evolutionary ecology of the Wrinkly Spreader is well understood, we have recently questioned the need for expensive biofilm–formation to colonise the air-liquid (A-L) interface, as O2-directed flagella-mediated swimming (aerotaxis) should be sufficient to maintain cells in this region. Our investigations show that swimming can overcome displacement by Brownian diffusion and microcurrents within the liquid column. However, it is not sufficient to explain the high levels of enrichment at the A-Linterface shown by Wrinkly Spreader cells. A comparison of the liquid surface tension of wild-type and Wrinkly Spreader cultures, supernatants, and washed cells, suggests that the Wrinkly Spreader produces a surface-active compound weakly associated with the cell which helps penetration of the A-L interface and allows cells to remain in the high-O2 region without further expenditure of energy. Our results suggest that this penetration is key to the following biofilm–formation which supports higher populations at the A-L interface and that this explains the adaptive advantage of the Wrinkly Spreader.

In radiating populations of Pseudomonas fluorescens SBW25, adaptive wrinkly spreader (WS) mutants are able to gain access to the air-liquid (A-L) interface of static liquid microcosms and achieve a significant competitive fitness advantage over other non-biofilm-forming competitors. Aerotaxis and flagella-based swimming allows SBW25 cells to move into the high-O2 region located at the top of the liquid column and maintain their position by countering the effects of random cell diffusion, convection and disturbance (i.e. physical displacement). However, wild-type cells showed significantly lower levels of enrichment in this region compared to the archetypal WS, indicating that WS cells employ an additional mechanism to transfer to the A-L interface where displacement is no longer an issue and a biofilm can develop at the top of the liquid column. Preliminary experiments suggest that this might be achieved through the expression of an as yet unidentified surface active agent that is weakly associated with WS cells and alters liquid surface tension, as determined by quantitative tensiometry. The effect of physical displacement on the colonization of the high-O2 region and A-L interface was reduced through the addition of agar or polyethylene glycol to increase liquid viscosity, and under these conditions the competitive fitness of the WS was significantly reduced. These observations suggest that the ability to transfer to the A-L interface from the high-O2 region and remain there without further expenditure of energy (through, for example, the deployment of flagella) is a key evolutionary innovation of the WS, as it allows subsequent biofilm development and significant population increase, thereby affording these adaptive mutants a competitive fitness advantage over non-biofilm-forming competitors located within the liquid column.

Bacterial success in colonizing complex environments requires individual response to micro-scale conditions as well as community-level cooperation to produce large-scale structures such as biofilms. Connecting individual and community responses could be achieved by linking the intracellular sensory and regulatory systems mediated by bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) and other compounds of individuals with intercellular quorum sensing (QS) regulation controlling populations.There is growing evidence to suggest that biofilm formation by many pseudomonads is regulated by both intra and intercellular systems, though in the case of the model Pseudomonas fluorescens SBW25 Wrinkly Spreader in which mutations increasing c-di-GMP levels result in the production of a robust cellulose-based air-liquid interface biofilm, no evidence for the involvement of QS regulation has been reported.However, our recent review of the P. fluorescens SBW25 genome has identified a potential QS regulatory pathway and other QS-associated genes linked to c-di-GMP homeostasis, and QS signal molecules have also been identified in culture supernatants.These findings suggest a possible link between c-di-GMP and QS regulation in P. fluorescens SBW25 which might allow a more sophisticated and responsive control of cellulose production and biofilm formation when colonising the soil and plant-associated environments P. fluorescens SBW25 normally inhabits.

Bacterial biosurfactants have a wide range of biological functions and biotechnological applications. Previous analyses had suggested a limit to their reduction of aqueous liquid surface tensions (gamma(Min)), and here we confirm this in an analysis of 25 Pseudomonas spp. strains isolated from soil which produce high-strength surfactants that reduce surface tensions to 25.2 +/- 0.1-26.5 +/- 0.2 mN m(-1) (the surface tension of sterile growth medium and pure water was 52.9 +/- 0.4 mN m(-1) and 72.1 +/- 1.2 mN m(-1), respectively). Comparisons of culture supernatants produced using different growth media and semi-purified samples indicate that the limit of 24.2-24.7 mN m(-1) is not greatly influenced by culture conditions, pH or NaCl concentrations. We have used foam, emulsion and oil-displacement behavioural assays as a simple and cost-effective proxy for in-depth biochemical characterisation, and these suggest that there is significant structural diversity amongst these surfactants that may reflect different biological functions and offer new biotechnological opportunities. Finally, we obtained a draft genome for the strain producing the highest strength surfactant, and identified a cluster of non-ribosomal protein synthase genes that may produce a cyclic lipopeptide (CLP)-like surfactant. Further investigation of this group of related bacteria recovered from the same site will allow a better understanding of the significance of the great variety of surfactants produced by bacterial communities found in soil and elsewhere.