Petra Hildebrandt

Universitätsmedizin Greifswald

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Uwe Völker

Universitätsmedizin Greifswald

Kristin Surmann

Universitätsmedizin Greifswald

Frank Schmidt

Weill Cornell Medical College in Qatar

Stephan Michalik

Universitätsmedizin Greifswald

Maren Depke

Universitätsmedizin Greifswald

Vishnu M. Dhople

Universitätsmedizin Greifswald

Manuela Gesell Salazar

Universitätsmedizin Greifswald

Henrike Pförtner

Universitätsmedizin Greifswald

Elke Hammer

Universitätsmedizin Greifswald

Jörg Bernhardt

Universität Greifswald

Cooperative Institutions

Universität Greifswald

Universitätsmedizin Greifswald

University of Rostock

University of Groningen

Institute of Marine Biotechnology

University of Graz

German Centre for Cardiovascular Research

University Medical Center

Genomic (Brazil)

Institute for Systems Biology

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Efficient resolution of prostereogenic arylaliphatic ketones using a recombinant alcohol dehydrogenase from Pseudomonas fluorescens

Tetrahedron Asymmetry (2001)

Petra Hildebrandt Thomas H. Riermeier Josef Altenbuchner Uwe T. Bornscheuer

Pseudomonas fluorescens

Acetophenone

10.1016/s0957-4166(01)00201-4

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Citations (27)

Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis

eLife (2021)

Tjorven Hinzke Manuel Kleiner Mareike Meister Rabea Schlüter Christian Hentschker

The hydrothermal vent tubeworm Riftia pachyptila hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.

Holdfast

10.7554/elife.58371

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Citations (28)

Proteomic analysis of doxorubicin‐induced changes in the proteome of HepG2cells combining 2‐D DIGE and LC‐MS/MS approaches

PROTEOMICS (2009)

Elke Hammer Sandra Bien Manuela Gesell Salazar Leif Steil Christian Scharf

HepG-2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2-D gel-based and gel-free methods. The analysis of crude HepG2 cell extracts by 2-D DIGE provided data on 1835 protein spots which was then complemented by MS-centered analysis of stable isotope labeling by amino acids in cell culture-labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin-induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin-associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.

Proteome

10.1002/pmic.200800626

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Citations (43)

A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria

Methods (2013)

Henrike Pförtner Juliane Wagner Kristin Surmann Petra Hildebrandt S M Ernst

Proteome

Internalization

Cell Sorting

10.1016/j.ymeth.2013.04.009

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Citations (26)

Alternative fluorescent labeling strategies for characterizing gram-positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis ofStaphylococcus aureusretrieved from infected host cells

Cytometry Part A (2016)

Petra Hildebrandt Kristin Surmann Manuela Gesell Salazar Nicole Normann Uwe Völker

Abstract Staphylococcus aureus is a Gram‐positive opportunistic pathogen that is able to cause a broad range of infectious diseases in humans. Furthermore, S. aureus is able to survive inside nonprofessional phagocytic host cell which serve as a niche for the pathogen to hide from the immune system and antibiotics therapies. Modern OMICs technologies provide valuable tools to investigate host‐pathogen interactions upon internalization. However, these experiments are often hampered by limited capabilities to retrieve bacteria from such an experimental setting. Thus, the aim of this study was to develop a labeling strategy allowing fast detection and quantitation of S. aureus in cell lysates or infected cell lines by flow cytometry for subsequent proteome analyses. Therefore, S. aureus cells were labeled with the DNA stain SYTO ® 9, or Vancomycin BODIPY ® FL (VMB), a glycopeptide antibiotic binding to most Gram‐positive bacteria which was conjugated to a fluorescent dye. Staining of S. aureus HG001 with SYTO 9 allowed counting of bacteria from pure cultures but not in cell lysates from infection experiments. In contrast, with VMB it was feasible to stain bacteria from pure cultures as well as from samples of infection experiments. VMB can also be applied for histocytochemistry analysis of formaldehyde fixed cell layers grown on coverslips. Proteome analyses of S. aureus labeled with VMB revealed that the labeling procedure provoked only minor changes on proteome level and allowed cell sorting and analysis of S. aureus from infection settings with sensitivity similar to continuous gfp expression. Furthermore, VMB labeling allowed precise counting of internalized bacteria and can be employed for downstream analyses, e.g., proteomics, of strains not easily amendable to genetic manipulation such as clinical isolates. © 2016 International Society for Advancement of Cytometry

Proteome

Cell Sorting

10.1002/cyto.a.22981

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Citations (23)

Activation of the alternative sigma factor SigB of Staphylococcus aureus following internalization by epithelial cells – An in vivo proteomics perspective

International Journal of Medical Microbiology (2013)

Henrike Pförtner Marc Burian Stephan Michalik Maren Depke Petra Hildebrandt

Proteome

Sigma factor

Internalization

Virulence factor

10.1016/j.ijmm.2013.11.014

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Citations (28)

Lysophosphatidic acid depletes intracellular calcium stores different from those mediating capacitative calcium entry in C6 rat glioma cells

Glia (1997)

Jan‐Peter Hildebrandt Petra Hildebrandt

Lysophosphatidic acid (LPA) functions as an extracellular lipid mediator stimulating phospholipase C and affecting the structure of the cytoskeleton in several cell types. In rat glioma C6 cells, LPA mobilizes calcium from intracellular calcium stores and reverts morphological changes induced by elevated cytosolic cAMP-concentrations. Here we show that LPA-stimulation of C6 cells loaded with the calcium-sensitive fluorescent dye indo-1 results in calcium release from a subset of intracellular calcium stores that are not sensitive to the tumor promoter thapsigargin and do not overlap with calcium stores depleted during purinergic receptor stimulation with ATP. Furthermore, depletion of LPA-sensitive calcium stores does not induce capacitative calcium entry from the extracellular space into the cytosol to the same extent as ATP. These results indicate that inositol phosphate signaling induced by LPA or ATP may differ in kinetics or in spatial organisation within the cell. This may represent a possible explanation for the previous observation that only LPA, but not other calcium-mobilizing agonists, reverts cAMP-induced changes in the cytoskeletal organization in C6 cells. GLIA 19:67–73, 1997. © 1997 Wiley-Liss, Inc.

Calcium in biology

Autotaxin

Calcium Signaling

Thapsigargin

10.1002/(sici)1098-1136(199701)19:1<67::aid-glia7>3.0.co;2-7

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Citations (14)

Emulation of Racemase Activity by Employing a Pair of Stereocomplementary Biocatalysts

Chemistry - A European Journal (2007)

Christian Gruber Bettina M. Nestl Johannes Gross Petra Hildebrandt Uwe T. Bornscheuer

Abstract Racemization is the key step to turn a kinetic resolution process into dynamic resolution. A general strategy for racemization under mild reaction conditions by employing stereoselective biocatalysts is presented, in which racemization is achieved by employing a pair of stereocomplementary biocatalysts that reversibly interconvert an sp 3 to a sp 2 center. The formal interconversion of the enantiomers proceeds via a prochiral sp 2 intermediate the formation of which is catalyzed either by two stereocomplementary enzymes or by a single enzyme with low stereoselectivity. By choosing appropriate reaction conditions, the amount of the prochiral intermediate is kept to a minimum. This general strategy, which is applicable to redox enzymes (e.g., by acting on R 2 CHOH and R 2 CHNHR groups) and lyase‐catalyzed addition–elimination reactions, was proven for the racemization of secondary alcohols by employing alcohol dehydrogenases. Thus, enantiopure chiral alcohols were used as model substrates and were racemized either with highly stereoselective biocatalysts or by using (rarely found) non‐selective enzymes.

Racemization

Enantiopure drug

Kinetic resolution

Biocatalysis

Enantiomeric excess

Enzyme Catalysis

10.1002/chem.200700528

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Citations (39)

Effects of Staphylococcus aureus-hemolysin A on calcium signalling in immortalized human airway epithelial cells

Cell Calcium (2008)

Stefanie Eichstaedt Karoline Gäbler Sabine Below Christian Müller Christian Köhler

Calcium in biology

Calcium Signaling

10.1016/j.ceca.2008.09.001

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Citations (53)

Analyses of competent and non‐competent subpopulations of Bacillus subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence

Environmental Microbiology (2020)

Mirjam Boonstra Marc Schaffer Joana Sousa Luiza P. Morawska Siger Holsappel

Upon competence-inducing nutrient-limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence-assisted cell sorting (FACS). Using RNA-seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post-transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK-binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ΔyhfW mutant. RNA-seq analysis of ΔyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC.

Regulon

10.1111/1462-2920.15005

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Citations (15)