Anna Koza

Characterization of a novel air-liquid interface biofilm of Pseudomonas fluorescens SBW25

Pseudomonads are able to form a variety of biofilms that colonize the air-liquid (A-L) interface of static liquid microcosms, and differ in matrix composition, strength, resilience and degrees of attachment to the microcosm walls. From Pseudomonas fluorescens SBW25, mutants have evolved during prolonged adaptation-evolution experiments which produce robust biofilms of the physically cohesive class at the A-L interface, and which have been well characterized. In this study we describe a novel A-L interface biofilm produced by SBW25 that is categorized as a viscous mass (VM)-class biofilm. Several metals were found to induce this biofilm in static Kings B microcosms, including copper, iron, lead and manganese, and we have used iron to allow further examination of this structure. Iron was demonstrated to induce SBW25 to express cellulose, which provided the matrix of the biofilm, a weak structure that was readily destroyed by physical disturbance. This was confirmed in situ by a low (0.023-0.047 g) maximum deformation mass and relatively poor attachment as measured by crystal violet staining. Biofilm strength increased with increasing iron concentration, in contrast to attachment levels, which decreased with increasing iron. Furthermore, iron added to mature biofilms significantly increased strength, suggesting that iron also promotes interactions between cellulose fibres that increase matrix interconnectivity. Whilst weak attachment is important in maintaining the biofilm at the A-L interface, surface-interaction effects involving cellulose, which reduced surface tension by approximately 3.8 mN m(-1), may also contribute towards this localization. The fragility and viscoelastic nature of the biofilm were confirmed by controlled-stress amplitude sweep tests to characterize critical rheological parameters, which included a shear modulus of 0.75 Pa, a zero shear viscosity of 0.24 Pa s(-1) and a flow point of 0.028 Pa. Growth and morphological data thus far support a non-specific metal-associated physiological, rather than mutational, origin for production of the SBW25 VM biofilm, which is an example of the versatility of bacteria to inhabit optimal niches within their environment.

Environmental modification and niche construction: developing O2 gradients drive the evolution of the Wrinkly Spreader.

The evolutionary success of the novel Wrinkly Spreader (WS) genotypes in diversifying Pseudomonas fluorescens SBW25 populations in static liquid microcosms has been attributed to the greater availability of O2 at the air–liquid (A–L) interface where the WS produces a physically cohesive-class biofilm. However, the importance of O2 gradients in SBW25 adaptation has never been examined. We have explicitly tested the role of O2 in evolving populations using microsensor profiling and experiments conducted under high and low O2 conditions. Initial colonists of static microcosms were found to establish O2 gradients before significant population growth had occurred, converting a previously homogenous environment into one containing a resource continuum with high and low O2 regions. These gradients were found to persist for long periods by which time significant numbers of WS had appeared colonising the high O2 niches. Growth was O2 limited in static microcosms, but high O2 conditions like those found near the A–L interface supported greater growth and favoured the emergence of WS-like genotypes. A fitness advantage to biofilm formation was seen under high but not low O2 conditions, suggesting that the cost of biofilm production could only be offset when O2 levels above the A–L interface were high. Profiling of mature WS biofilms showed that they also contained high and low O2 regions. Niches within these may support further diversification and succession of the developing biofilm population. O2 availability has been found to be a major factor underlying the evolutionary success of the WS genotype in static microcosms and illustrates the importance of this resource continuum in microbial diversification and adaptation.

Adaptation of the autotrophic acetogen Sporomusa ovata 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 CO2 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 H2-CO2 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 CO2 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 CO2 conversion in bioprocesses like microbial electrosynthesis is to evolve the microbial catalyst by adaptive laboratory evolution to optimize its autotrophic metabolism.

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

L-serine is a promising building block biochemical with a high theoretical production yield from glucose. Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasing L-serine concentration from 3 to 100g/L using adaptive laboratory evolution (ALE). Genome sequencing of isolated clones revealed multiplication of genetic regions, as well as mutations in thrA, thereby showing a potential mechanism of serine inhibition. Additional mutations were evaluated by MAGE combined with amplicon sequencing, revealing role of rho, lrp, pykF, eno, and rpoB on tolerance and fitness in minimal medium. Production using the tolerant strains resulted in 37g/L of L-serine with a 24% mass yield. The resulting titer is similar to the highest production reported for any organism thereby highlighting the potential of ALE for industrial biotechnology.

Evolution in a test tube: rise of the Wrinkly Spreaders

Understanding evolutionary mechanisms is fundamental to a balanced biological education, yet practical demonstrations are rarely considered. In this paper we describe a bacterial liquid microcosm which can be used to demonstrate aspects of evolution, namely adaptive radiation, niche colonisation and competitive fitness. In microcosms inoculated with Pseudomonas fluorescens SBW25, evolved mutants such as the Wrinkly Spreader (WS) rapidly arise to form biofilms covering the air–liquid (A–L) interface. WS are readily isolated due to a distinctive colony morphology and reach ∼30% of the population within five days. When re‐inoculated into static microcosms, WS preferentially colonises the A–L interface by producing a biofilm, demonstrating a niche preference distinct from the ancestral SBW25 which grows throughout the liquid column. This ability provides the WS with a ∼2.5× competitive fitness advantage over the non‐biofilm forming ancestral SBW25. However, WS and SBW25 have similar fitness in shaken microcosms where biofilms cannot form. These practical demonstrations of WS evolution, suitable for secondary or tertiary‐level classes, can be linked with a literature‐based review of the underlying molecular biology of the WS phenotype to provide a true exemplar of the modern evolutionary synthesis, the current paradigm in evolutionary biology.

Rapid resistome mapping using nanopore sequencing

Abstract The emergence of antibiotic resistance in human pathogens has become a major threat to modern medicine. The outcome of antibiotic treatment can be affected by the composition of the gut. Accordingly, knowledge of the gut resistome composition could enable more effective and individualized treatment of bacterial infections. Yet, rapid workflows for resistome characterization are lacking. To address this challenge we developed the poreFUME workflow that deploys functional metagenomic selections and nanopore sequencing to resistome mapping. We demonstrate the approach by functionally characterizing the gut resistome of an ICU (intensive care unit) patient. The accuracy of the poreFUME pipeline is with >97% sufficient for the annotation of antibiotic resistance genes. The poreFUME pipeline provides a promising approach for efficient resistome profiling that could inform antibiotic treatment decisions in the future.

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae

Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the β-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L-1 of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.

Cellulose expression in Pseudomonas fluorescens SBW25 and other environmental Pseudomonads

© 2013 Spiers et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads

Surfactants expressed by soil pseudomonads alter local soil-water distribution, suggesting a hydrological role for these compounds.

Several biological roles have been demonstrated for surfactants expressed by soil and rhizosphere Pseudomonas spp., but the impact of these powerful surface-active agents on the local soil-water distribution within the partially saturated soil pore network has not been examined. To investigate this potential hydrological role, the liquid surface tension (γ)-reducing activities (LSTRA) of 72 pseudomonads isolated from a sandy loam soil by tensiometry of culture supernatants were characterized. Of these, 67% exhibited LSTRA, reducing γ to a minimum (γ(Min)) of 24 mN m(-1) established by individual distribution identification analysis. Soil microcosms were then used to examine the impact of surfactant expression on the local soil-water distribution. The volumetric water content (θ) of soil microcosms was significantly lowered (0.78 ×) by Pseudomonas fluorescens SBW25 expressing the surfactant viscosin compared with a surfactant-deficient mutant (P<0.002). Six of 15 soil pseudomonad isolates examined were found to have a similar impact on θ when compared with sterile microcosms (P<0.05). These findings indicate that surfactant-expressing pseudomonads could modify the local soil-water distributions and that surfactants may therefore play a significant hydrological role in soils, in addition to their recognized biological activities.

Genome-wide identification of tolerance mechanisms towards p-coumaric acid in Pseudomonas putida

The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p‐coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome‐wide approach, Tn‐seq. Libraries containing a large number of miniTn5‐Km transposon insertion mutants were grown in the presence and absence of p‐coumaric acid, and the enrichment or depletion of mutants was quantified by high‐throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p‐coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p‐coumaric acid is related to transport and membrane integrity.