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Session Overview
Session
SESSION 10: Functional Heterogeneity in Microbial Processes
Time:
Friday, 17/Oct/2014:
10:50am - 12:20pm

Chairs: Susann Müller, Leipzig, Germany; Christian Dusny, Dortmund, Germany


Session Abstract

New methods for single cell analysis show improved possibilities not only for the in depth analysis of individual cells but also to investigate what is happening in microbial populations and communities. Today, there are several powerful technologies available to investigate and describe the dynamics of the cells in populations or communities. Completed by other techniques it extends the understanding of structure and function relationships by investigating phylogenetic, genomic, transcriptomic and proteomic levels at the scale of the single microorganism or that of sub-communities. The new information collected helps to better understand the functioning of the cells and their intra- or inter-relationships. The session will contribute to reveal principles of microbial ecosystems or to interpret even simpler systems like pure cultures as are used in microbial (white) biotechnology. Potential applications will also be addressed.


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Presentations

Quantitative Physiology of Single Cells for Linking Phenotype and Environment

Christian Dusny1, Alexander Grünberg2, Katrin Rosenthal1, Wolfgang Wiechert2, Andreas Schmid1

1TU Dortmund, Germany; 2Forschungszentrum Jülich GmbH, Jülich, Germany

The single cell represents the basal self‐sustaining unit of life. Each single cell constitutes an independent and complex system that shows remarkable individuality in a multitude of physiological traits. Consequently, a microbial population exhibits physiological diversity despite of clonality, which can be exclusively accessed by single cell analysis. This especially concerns the cellular repertory of adaptation mechanisms reacting to physicochemical changes in the extracellular environment. Such mechanisms include stochastic alterations of regulatory circuits as well as specific adaptations to external stimuli, which are highly individual and strongly depend on frequency and extent of fluctuations in the cells surrounding environment. Therefore, the control of the extracellular (micro‐) environment is of fundamental importance for the unambiguous identification of the origins of intrapopulation heterogeneity of an actually clonal population. Only with this, the occurrence of cell‐to‐cell differences can be assigned to either environmental factors or intrinsic stochasticity. To date, only microfluidic single cell cultivation systems allow the necessary degree of control over the extracellular environment. In this talk, we present a systematic comparison of principally different cultivation technologies in order to quantify cellular responses to the environment at a single cell level. We compared contactless cell retention based on negative dielectrophoresis (nDEP), allowing constant environmental conditions by continuous cell perfusion, with flow‐through contact‐based hydrodynamic single‐cell cultivation systems (MGC) and static solid agarose‐based

cultivation pads. To enable a quantitative comparison of these three systems on the basis of physiological and morphological parameters, universally applicable analytical approaches for the precise quantification of specific growth rates, cell morphology and division characteristics of single microorganisms were developed. Corynebacterium glutamicum ATCC13032 was harnessed as a model organism and cultivated under otherwise identical cultivation conditions with all three systems. Interestingly, all cultivated cells exhibited virtually identical growth capacity and showed exceptionally robust and high specific growth rates of 0.6 h‐1 for micropopulations of up to 8 cells, independent of the applied cultivation technology. The observed growth rates of single cells exceeded population growth rates in shake flasks by up to

120%. These results lead to the conclusion that optimal nutrient supply was present with all employed systems and the rate of growth of a cell is tightly regulated and conserved. It could therefore be argued that the metabolic capacity of the cells is rate‐limiting rather than environmental factors. Unlike growth rates, division rates, snapping division angle and cell length distributions showed considerable differences in the static environment of agarose pads, whereas cells cultivated with nDEP and MGC had similar length and division angle distributions. To the best of our knowledge, this is the first investigation of physiological responses to steady and static extracellular environments at the single cell level. Moreover, this study underlines the importance of exploring cellular physiology at a single cell level, eliminating bias by uncontrollable fluctuations in the extracellular environment and population activity.


Plasmid Copy Number Variation in Pseudomonas putida Analysed by Cell Sorting and Digital Droplet PCR

Michael Jahn1, Carsten Vorpahl1, Dominique Türkowsky1, Martin Lindmeyer2, Bruno Bühler2, Hauke Harms1, Susann Müller1

1Helmholtz-Centre for Environmental Research, Leipzig, Germany; 2Laboratory of Chemical Biotechnology, TU Dortmund University, Dortmund, Germany

Whole microbial cells are cheap, efficient and self-reproducing catalysts for the enzymatic conversion of chemicals or production of proteins. For expression of the gene of interest plasmids are widely used in academia and industry. However, the overall yield and efficiency of production processes is easily disturbed by fluctutations in plasmid copy number (PCN), leading to population heterogeneity. Here, we investigated the effect of PCN on heterologous expression of a model enzyme in Pseudomonas putida. Conventionally, plasmid retention is estimated by plate counting on selective media or roughly determined by gel electrophoresis. In contrast to that, we used an EGFP reporter fused to the target protein, allowing real time fluorescence screening of individual cells by flow cytometry. Furthermore, we developed a work flow for direct PCN analysis using cell sorting and cutting edge Droplet Digital PCR (ddPCR). The work flow using a duplex TaqMan® ddPCR setup was tested and optimized for as little as 1,000 sorted cells, and gave insights into population heterogeneity on the DNA level not seen before.

We found, that a large proportion of cells (30-60%) was not able to produce the desired protein even under high induction regimes, dividing the population into high- and low-fluorescent cells. The ddPCR analysis revealed that plasmid distribution was remarkably unequal, directly correlating the strength of EGFP expression to low and high PCN. We further exploited this technique to probe the influence of antibiotics on PCN, as well as the combination of different expression plasmids and microbial strains to select an optimal production system.


Flow cytometry for energy balances of phytoplankton organisms

Susanne Dunker, Torsten Jakob, Christian Wilhelm

University of Leipzig, Germany

Phytoplankton organisms (cyanobacteria and algae) are microscopic small organisms having an enormous impact on the water quality and ecology in waters. Their ability to convert light energy to biomass via photosynthesis determines the extent of primary production. The efficiency of this conversion highly varies within the taxonomic groups (e.g. cyanobacteria, green algae, dinophytes, cryptophytes and diatoms). This means that the same quantity of light not necessarily leads to the same amount of biomass for different taxa. In contrast to green algae, cyanobacteria can use extremely low light intensities for biomass production but due to their toxicity they are not an appropriate food source for primary consumers and decrease water quality. Until now a resolution of energy use efficiency in a complex community for different taxa is not possible, but quite important for water-quality-management. The idea behind the project was to use cellular characteristics to resolve the energy use efficiency for different species in a mixed culture. On a basic level we cultured the cyanobacterium ( Microcystis aeruginosa ) with one of two green algae ( Scenedesmus obliquus or Oocystis marsonii ). By the means of flow cytometry (FC) both species in a mixed culture could be distinguished due to their different pigmentation. FC delivered the following cell parameters: cell volume, dry weight and chl a -content per cell. These data allowed the quantification of absorbed light energy in relation to created biomass separately for the species in the mixed culture. It could be shown that energy use efficiency (numbers of photons needed to assimilate one molecule of carbon) of the green algae Oocystis marsonii in a mixture with Microcystis aeruginosa was strongly reduced in comparison to uni-algal control culture, while energy use efficiency of Scenedesmus obliquus was unaffected in growth by the mixture with the cyanobacterium. In consequence it could be shown how energy is distributed and converted into biomass in a phytoplankton community. The estimation of these taxonomical resolved energy balances was only possible by the means of flow cytometry. In future this single-cell-approach could be a valuable tool for water-quality-management.


Enhanced viability of microalgal populations by photoperiodic cycles

Felix Krujatz, Thomas Bley, Jost Weber

TU Dresden, Institute of Food Technology and Bioprocess Engineering, Germany

Microalgae represent a promising raw material for various industries due to their wide range of valuable ingredients. By photosynthetic processes carbon dioxid is fixed under the influence of light and converted into products like proteins, pigments, biopolymers, lipids or drugs. Despite the great potential of algae research only 150 of the estimated 400,000 algae strains are used for industrial applications.

Basically, cultivation of microalgae is performed in open or closed photobioreactor systems, varying in size and geometry. Light energy for photosynthetic processes is commonly provided by external or internal illumination devices. Self-shading and inhomogeneous illumination of photobioreactors are responsible for light gradients within microalgae suspensions which consequently causes the formation of population heterogeneity. It has been proven that population heterogeneity of microbial cultures can significantly influence the productivity of biotechnological processes. An important cell-specific parameter for the overall productivity of microbial cultures is given by the viability which can be defined as the differentiation of metabolic active and inactive cells.

_words in italics_Chlamydomonas reinhardtti_words in italics_ 11.32b and _words in italics_Chlorella sorokiniana_words in italics_ UTEX1230 are widely used and robust model organisms for photosynthesis and algae research due to their high growth rates and temperature resistance. In this study, a flow cytometry viability staining procedure was established using bis-(1,3-dibarbituric acid)-trimethine oxonol (DiBAC4(3)), a slow response membrane potential sensitive probe. Above mentioned microalgal strains were cultivated in shake flasks under varying temperature (26°C, 30°C and 37°C) and photoperiodic light cycles (150 µmol m-2 s-1, ligt/dark: 24/0 or 14/10). We found that for all culture conditions _words in italics_C. sorokiniana_words in italics_ UTEX1230 obtained higher cell densities compared to _words in italics_C.reinhardtti_words in italics_ 11.32b even at 30°C and 37°C. In comparison to full light conditions (light/dark cycles of 24/0) photoperiodic cycles of 14/10 hours resulted in lower cell densities of _words in italics_C.reinhardtti 11.32b_words in italics_ and _words in italics_C. sorokiniana_words in italics_ UTEX1230 at all considered cultivation temperatures.

After a period of stable population viability (ca. 50 hours) the amount of DiBAC4(3) positive _words in italics_C.reinhardtti_words in italics_ 11.32b and _words in italics_C. sorokiniana_words in italics_ UTEX1230 cells increased at 30°C and 37°C. For both strains it could be observed, that the viability of microalgal populations remained at constant high values by adjusting photoperiodic cycles of 14/10 hours.

Thus, the amount of metabolic active cells can directly be influenced by photoperiodic cycles.


Staphylococcus aureus infection induces human T cell apoptosis and increased mortality in humanized mice

Janin Knop1, Frank Hanses2, Nancie Archin3, Joachim Gläsner4, Andre Gessner4, Anja Kathrin Wege1

1University Medical Hospital Regensburg, Department of Gynecology and Obstetrics, Germany; 2Medical Hospital Regensburg, Department of Internal Medicine I, Germany; 3University of North Carolina at Chapel Hill, Department of Medicine, USA; 4University of Regensburg, Institute for Medical Micobiology and Hygiene, Germany

Background: S. aureus is a common pathogen causing infections in humans worldwide with an increasing prevalence of multidrug resistance. Unfortunately, mouse and human immune systems exhibit some substantial differences and the immune responses against pathogens and thereby the outcome of infectious diseases differs dramatically between species. As many as 150 clinical studies failed in clinical settings although they showed promising results in mice studies. In addition, the rate of methicillin-resistant S. aureus (MRSA) strains is increasing and therefore, new treatment strategies and an appropriate animal model to test these approaches under human-like conditions are urgently needed.

Methods: In this study we established an S. aureus infection model in mice engrafted with a human immune system and investigated the role of apoptosis induction for pathogenesis.

Results: S. aureus infection was aggravated in these humanized mice (HM) compared to wild type or non- engrafted mice. The HM displayed significantly reduced survival, increased weight loss and accelerated bacterial burden. S. aureus infection in HM elucidated the negative impact of a human immune response on disease progression which was characterized by increased human cytokine production, T cell apoptosis and FAS receptor expression.

Conclusion. Our findings clearly demonstrate the different effects obtained in wild type and humanized mice and discuss the possible benefit of including humanized mice in future studies involving S. aureus as a prior step to human clinical trials.



 
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