Penetrating the air-liquid interface is the key to colonization and wrinkly spreader fitness

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

Extending an eco-evolutionary understanding of biofilm-formation at the air-liquid interface to community biofilms

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

Three biofilm types produced by a model pseudomonad are differentiated by structural characteristics and fitness advantage

Model bacterial biofilm systems suggest that bacteria produce one type of biofilm which is then modified by environmental and physiological factors, though 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 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. Wrinkly Spreader, 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 advantage 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 air-liquid interface, most probably O2 and nutrients, though VM and CBFS cells located lower down in the liquid column might provide an additional fitness advantage through the colonisation 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

Predicted structure and homology models for key orphan proteins

This dataset contains Protein Database (PDB) files of predicted structure and homology models for key Orphan proteins. The models were generated as part of a research project for which a manuscript has been prepared and is about to be submitted to PLoS ONE (Andrew J. Spiers, Helge C. Dorfmueller, Robyn Jerdan, Jessica McGregor, Abbie Nicoll, Kenzie Steel, and Scott Cameron). The models were produced by AlphaFold Colab Notebook, InterFold6, Phyre2, RoseTTAFold, TrRosetta and SWISSMODEL following the suggested approaches and default options and using the full-length protein sequences (some of these models might not be able to be recreated as the servers will update their template databases etc.)

Community biofilm-formation, stratification and productivity in serially-transferred microcosms

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

Bioinformatics characterization of BcsA-like orphan proteins suggest they form a novel family of pseudomonad cyclic-β-glucan synthases

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.<br/

Azithromycin possesses biofilm–inhibitory activity and potentiates non-bactericidal colistin methanesulfonate (CMS) and polymyxin B against <i>Klebsiella pneumonia</i>

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

Three biofilm types produced by a model pseudomonad are differentiated by structural characteristics and fitness advantage supplementary information

This is the Supplementary Information for our paper 'Three biofilm types produced by a model pseudomonad are differentiated by structural characteristics and fitness advantage', Anna Koza, Robyn Jerdan, Scott Cameron, and Andrew J. Spiers; Microbiology 2020; DOI 10.1099/mic.0.000938. This contains the Supplementary Sections S1 - S6 and Figures S1 - S4