Up-dating the Cholodny method using PET films to sample microbial communities in soil

The aim of this work was to investigate the use of PET (polyethylene terephtalate) films as a modern development of Cholodny’s glass slides, to enable microscopy and molecular-based analysis of soil communities where spatial detail at the scale of microbial habitats is essential to understand microbial associations and interactions in this complex environment. Methods. Classical microbiological methods; attachment assay; surface tension measurements; molecular techniques: DNA extraction, PCR; confocal laser scanning microscopy (CLSM); micro- focus X-ray computed tomography (μCT). Results. We first show, using the model soil and rhizosphere bacteria Pseudomonas fluorescens SBW25 and P. putida KT2440, that bacteria are able to attach and detach from PET films, and that pre-conditioning with a filtered soil suspension improved the levels of attachment. Bacteria attached to the films were viable and could develop substantial biofilms. PET films buried in soil were rapidly colonised by microorganisms which could be investigated by CLSM and recovered onto agar plates. Secondly, we demonstrate that μCT can be used to non-destructively visualise soil aggregate contact points and pore spaces across the surface of PET films buried in soil. Conclusions. PET films are a successful development of Cholodny’s glass slides and can be used to sample soil communities in which bacterial adherence, growth, biofilm and community development can be investigated. The use of these films with μCT imaging in soil will enable a better understanding of soil micro-habitats and the spatially-explicit nature of microbial interactions in this complex environment

Adaptive radiation of <i>Pseudomonas fluorescens</i> SBW25 in experimental microcosms provides an understanding of the evolutionary ecology and molecular biology of A-L interface biofilm formation

Combined experimental evolutionary and molecular biology approaches have been used to investigate the adaptive radiation of Pseudomonas fluorescens SBW25 in static microcosms leading to the colonisation of the air-liquid interface by biofilm–forming mutants such as the Wrinkly Spreader. In these microcosms, the ecosystem engineering of the early wild-type colonists establish the niche space for subsequent WS evolution and colonisation. Random WS mutations occurring in the developing population that de-regulate diguanylate cyclases and c-di-GMP homeostasis result in cellulose-based biofilms at the air-liquid interface. These structures allow Wrinkly Spreaders to intercept O2 diffusing into the liquid column and limit the growth of competitors lower down. As the biofilm matures, competition increasingly occurs between WS lineages, and niche divergence within the biofilm may support further diversification before system failure when the structure finally sinks. A combination of pleiotropic and epistasis effects, as well as secondary mutations, may explain variations in WS phenotype and fitness. Understanding how mutations subvert regulatory networks to express intrinsic genome potential and key innovations providing a selective advantage in novel environments is key to understanding the versatility of bacteria, and how selection and ecological opportunity can rapidly lead to substantive changes in phenotype and in community structure and function

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

Adaptation of the autotrophic acetogen <i>Sporomusa ovata</i> to methanol accelerates the conversion of CO₂ to organic products

Acetogens are efficient microbial catalysts for bioprocesses converting C1 compounds into organic products. Here, an adaptive laboratory evolution approach was implemented to adapt Sporomusa ovata for faster autotrophic metabolism and CO(2) conversion to organic chemicals. S. ovata was first adapted to grow quicker autotrophically with methanol, a toxic C1 compound, as the sole substrate. Better growth on different concentrations of methanol and with H(2)-CO(2) indicated the adapted strain had a more efficient autotrophic metabolism and a higher tolerance to solvent. The growth rate on methanol was increased 5-fold. Furthermore, acetate production rate from CO(2) with an electrode serving as the electron donor was increased 6.5-fold confirming that the acceleration of the autotrophic metabolism of the adapted strain is independent of the electron donor provided. Whole-genome sequencing, transcriptomic, and biochemical studies revealed that the molecular mechanisms responsible for the novel characteristics of the adapted strain were associated with the methanol oxidation pathway and the Wood-Ljungdahl pathway of acetogens along with biosynthetic pathways, cell wall components, and protein chaperones. The results demonstrate that an efficient strategy to increase rates of CO(2) conversion in bioprocesses like microbial electrosynthesis is to evolve the microbial catalyst by adaptive laboratory evolution to optimize its autotrophic metabolism

Is genotyping of single isolates sufficient for population structure analysis of <i>Pseudomonas aeruginosa</i> in cystic fibrosis airways?

The primary cause of morbidity and mortality in cystic fibrosis (CF) patients is lung infection by Pseudomonas aeruginosa. Therefore much work has been done to understand the adaptation and evolution of P. aeruginosa in the CF lung. However, many of these studies have focused on longitudinally collected single isolates, and only few have included cross-sectional analyses of entire P. aeruginosa populations in sputum samples. To date only few studies have used the approach of metagenomic analysis for the purpose of investigating P. aeruginosa populations in CF airways. We analysed five metagenomes together with longitudinally collected single isolates from four recently chronically infected CF patients. With this approach we were able to link the clone type and the majority of SNP profiles of the single isolates to that of the metagenome(s) for each individual patient. Based on our analysis we find that when having access to comprehensive collections of longitudinal single isolates it is possible to rediscover the genotypes of the single isolates in the metagenomic samples. This suggests that information gained from genome sequencing of comprehensive collections of single isolates is satisfactory for many investigations of adaptation and evolution of P. aeruginosa to the CF airways