Biofilms最新文献

Biofilms are typically characterized as a consortium of microorganisms, which adhere to each other and often to surfaces. This adhesion is realized by extracellular polymeric substances (EPS), which are secreted by the microorganisms and mainly consist of water, polysaccharides, proteins and lipids as well as nucleic acids and lysis products [1]. Although cultured plant cells are not typically considered biofilms, parallels can be found in the properties of plant calli. These callus cells tend to form cohesive aggregates, owing to their extracellular matrix, and often strongly adhere to the agar plates they are kept on. The extracellular matrix of plant cells is mainly composed of structural polysaccharides, such as xyloglucans, arabinogalactans [2], homogalacturonan and extensins [3] among others. Cultured plant cells were found to adhere to surfaces before [4]. Surface-associated plant cell culture may have potential in a (semi‑)continuous cultivation including product secretion, as was shown in principle for alginate-embedded plant cells [5]. For cyanobacterial biofilms, an efficient strategy for EPS extraction was recently developed [6]. The transferability of these protocols to biofilm-like growing plant calli of Ocimum basilicum is currently being investigated. Subsequently, the composition of the extracellular matrix extracted from cultured O. basilicum cells is of interest. Furthermore, the adhesive properties of O. basilicum suspension cultures to microstructured surfaces and the potential role of the extracellular matrix are under investigation. An investigation of culture properties in an aerosol photobioreactor [7] is planned as well.

This project is financially supported by the German research foundation (DFG, project number SFB 926-C03).

References:

[1]      H. C. Flemming, T. R. Neu, and D. J. Wozniak, “The EPS matrix: The ‘House of Biofilm Cells,’” J. Bacteriol., vol. 189, no. 22, pp. 7945–7947, 2007.

[2]      I. M. Sims, K. Middleton, A. G. Lane, A. J. Cairns, and A. Bacic, “Characterisation of extracellular polysaccharides from suspension cultures of members of the Poaceae,” Planta, vol. 210, no. 2, pp. 261–268, Jan. 2000.

[3]      M. Popielarska-Konieczna, K. Sala, M. Abdullah, M. Tuleja, and E. Kurczyńska, “Extracellular matrix and wall composition are diverse in the organogenic and non-organogenic calli of Actinidia arguta,” Plant Cell Rep., no. 0123456789, 2020.

[4]      R. J. Robins, D. O. Hall, D. ‐J Shi, R. J. Turner, and M. J. C. Rhodes, “Mucilage acts to adhere cyanobacteria and cultured plant cells to biological and inert surfaces,” FEMS Microbiol. Lett., vol. 34, no. 2, pp. 155–160, 1986.

[5]      Y. Kobayashi, H. Fukui, and M.

Planktonic bacterial cells are by definition not aggregated. However, our previous work, where we have demonstrated the invisible mechanical connections between bacterial cells in dilute planktonic suspensions, challenged this assumption. Here we provide an experimental evidence using autocorrelation analysis of micrographs that in planktonic suspensions of B. subtilis a size continuum of aggregated structures is formed. In the microbial aggregates viable cells were embedded in the nucleic acid network. The eDNA was released during regular cell lysis events. To determine the size distribution of planktonic bacterial aggregates a pair-wise spatial correlations of bacterial cells in microscopic images were calculated. The monotonously decreasing shape of the autocorrelation function indicated a continuous distribution of bacterial aggregate sizes from monomer to multimers. Soft bacterial aggregates in dilute suspensions provide a missing link in a continuum of organic matter in aqueous environments and can significantly improve our understanding how non-attached biofilms form during planktonic growth.

Shewanella oneidensis MR1 is the best understood model organism with regards to dissimilatory metal reduction and extracellular electron transfer onto carbon electrodes in bioelectrochemical systems (BES)¹. However, under anoxic conditions S. oneidensis is known to form very thin biofilms resulting in low current density output. In contrast, another exoelectrogenic model organism Geobacter surfurreduscens can form electroactive biofilms up to 100 µm in thickness. This organism is known for its ability to transport electrons over a long range (> 10 µm) along a network of protein filaments, called microbial nanowires. Although still controversial, it was recently reported that OmcS has a special importance for the conductivity of these nanowires². One of the key differences between G. surfurreduscens and S. oneidensis lies in how cell-to-cell electronic communication occurs, which dictate the range of electronic communication between distant cells. S. oneidensis relies on direct cell-to-cell communication via electron transfer between outer membrane cytochromes or via soluble redox active flavins that are secreted by the cells³. Our research is based on the question, what if the S. oneidensis biofilm formation could be improved by introducing an artificial electronic network, similar to the native microbial nanowires for G. sulfurreducens?

We hypothesize that synthetic biofilms containing conductive nanostructure additives would allow S. oneidensis to build multilayer thick biofilms under anoxic conditions on solid electron acceptors. To answer this question of how conductive materials affect the formation of anoxic S. oneidensis biofilms, we integrated both biological and synthetic conductive nanostructures into these biofilms. As biological additive, the c-type cytochrome OmcS purified from G. sulfurreducens was utilized. As synthetic additives, both commercially available biotinylated gold nanorods and in-house electrochemically synthesized metal nanostructures were added to anoxic S. oneidensis biofilms.

Cultivation and characterization of the biofilms was performed using our newly developed microfluidic bioelectrochemical platform. Microbial cultivation with the aid of microfluidic flow chambers has a great potential to form biofilms on an easy to handle laboratory scale with simultaneously ongoing multianalytical analysis⁴. In our bioelectrochemical microfluidic, system S. oneidensis biofilms can be grown under anoxic conditions using an anode as sole electron acceptor. The growth behavior and bioelectrochemical performance was evaluated by a combination of electrochemical techniques (chronoamperometry, electrochemical impedance spectroscopy, cyclic voltammetry) and optical analyses (confocal laser scanning microscopy and optical coherence tomo

A shift from petrochemical processes to a bio-based economy is inevitable to establish a sustainable industry. Bioelectrochemical systems (BESs) are a future technology for the environment-friendly production of platform chemicals. In BESs exoelectrogenic bacteria such as Shewanella oneidensis can directly transfer respiratory electrons to the anode, which serves as a non-depletable electron acceptor. So far, the main limiting factor in BESs is the achievable current density which correlates to some extend with the density, thickness and metabolic activity of anode biofilms composed of exoelectrogenic microorganisms. This is especially true for S. oneidensis as the organism forms rather thin biofilms under anoxic conditions on anode surfaces.

In order to enhance the organisms’ biofilm formation capabilities Bursac et al. deleted the λ-prophage from the genome. The deletion of the λ-prophage led to a 2.3-fold increased cell number on the anode ongoing with a 1.34-fold increased mean current density (Bursac et al., 2017). Furthermore, we just recently discovered that exogenous riboflavin enhances biofilm formation by the upregulation of the Ornithine-decarboxylase speC. This is probably based on a quorum sensing effect of riboflavin. Taken together the upregulation of speC ongoing with the deletion of the λ-prophage leads to a 4-fold increase in current density ongoing with a 6.1-fold increased biofilm formation on the anode.

However, to ensure an optimal performance of the biofilm in BESs, biofilm thickness itself is not sufficient. The biofilm also needs to be conductive. Our aim is to establish the Spytag-/Spycatcher-tool to synthetically steer biofilm conductivity. Spytag and Spycatcher are two protein residues from the fibronectin binding protein of Streptococcus pyogenes (Spy). These two protein residues form a spontaneous isopeptide bond under a variety of temperatures, pH values and buffers (Zakeri et al., 2012). By coupling Spytag and Spyctacher to different outer membrane c-type cytochromes of S. oneidensis the cells are covalently bound to each other while the biofilm remains conductive. In a first application the production of acetoin as one of the top 30 platform chemicals world-wide is desired (US Department of Energy, 2004).

In order to render S. oneidensis producing acetoin instead of the native end product acetate, Bursac et al. deleted the key genes for acetate production and introduced the acteoin production pathway (Bursac et al., 2017). To broaden the substrate spectrum of S. oneidensis further genes for glucose metabolism were introduced. Through a long term adaption, the glucose degradation, the biofilm formation abilities and the bioelectrochemical performance were significantly enhanced.

Merging all genetic optimizations

Staphylococcus aureus biofilms play important roles during infection. The main components of these biofilms are well studied; however, we lack the full understanding of factors and genes involved in regulation of biofilm formation. To screen for essential and non-essential biofilm regulatory genes in S. aureus, we have created a pooled inducible CRISPR interference library. The pooled library is designed to allow knockdown of every transcriptional unit in the S. aureus genome, thus targeting both essential and non-essential genes. We used our library in S. aureus Newman, a strain which forms structured macrocolonies on agar plates. We performed an unbiased screen of 1500 macrocolonies and found 10 macrocolonies with stably altered structures. The genotypes of these macrocolonies were determined by sequencing the single guide RNAs of the CRISPR interference system. As a proof of the validity of the approach, we identified several genes previously reported to be implicated in biofilm and macrocolony formation, including ica-genes, and metabolic genes of the TCA-cycle and gluconeogenesis. In addition, three new genes (two encoding putative enzymes and one hypothetical genes) whose depletion resulted in completely altered macrocolonies were also identified. The molecular mechanisms explaining the roles of these proteins in biofilm formation are currently under investigation.