Andre Schmidt

Interaction of HapX with the CCAAT‐binding complex—a novel mechanism of gene regulation by iron

Iron homeostasis requires subtle control systems, as iron is both essential and toxic. In Aspergillus nidulans, iron represses iron acquisition via the GATA factor SreA, and induces iron‐dependent pathways at the transcriptional level, by a so far unknown mechanism. Here, we demonstrate that iron‐dependent pathways (e.g., heme biosynthesis) are repressed during iron‐depleted conditions by physical interaction of HapX with the CCAAT‐binding core complex (CBC). Proteome analysis identified putative HapX targets. Mutual transcriptional control between hapX and sreA and synthetic lethality resulting from deletion of both regulatory genes indicate a tight interplay of these control systems. Expression of genes encoding CBC subunits was not influenced by iron availability, and their deletion was deleterious during iron‐depleted and iron‐replete conditions. Expression of hapX was repressed by iron and its deletion was deleterious during iron‐depleted conditions only. These data indicate that the CBC has a general role and that HapX function is confined to iron‐depleted conditions. Remarkably, CBC‐mediated regulation has an inverse impact on the expression of the same gene set in A. nidulans, compared with Saccharomyces cerevisae.

Involvement of siderophores in the reduction of metal-induced inhibition of auxin synthesis in Streptomyces spp

Unlike synthetic metal chelators, microbe-assisted phytoremediation provides plants with natural metal-solubilizing chelators which do not constitute a potential source of environmental pollution. Concurrently with microbial chelators, plant growth promotion can be enhanced through bacterially-produced phytohormones. In this work, the simultaneous production of siderophores and auxins by Streptomyces was studied to gain insight for future application in plant growth and phytoremediation in a metal-contaminated soil. Standard auxin and siderophore detection assays indicated that all of the investigated Streptomyces strains can produce these metabolites simultaneously. However, Al(3+), Cd(2+), Cu(2+), Fe(3+) and Ni(2+), or a combination of Fe(3+) and Cd(2+), and Fe(3+) and Ni(2+) affected auxin production negatively, as revealed by spectrophotometry and gas chromatography-mass spectrometry. This effect was more dramatic in a siderophore-deficient mutant. In contrast, except for Fe, all the metals stimulated siderophore production. Mass spectrometry showed that siderophore and auxin-containing supernatants from a representative Streptomyces species contain three different hydroxamate siderophores, revealing the individual binding responses of these siderophores to Cd(2+) and Ni(2+), and thus, showing their auxin-stimulating effects. We conclude that siderophores promote auxin synthesis in the presence of Al(3+), Cd(2+), Cu(2+) and Ni(2+) by chelating these metals. Chelation makes the metals less able to inhibit the synthesis of auxins, and potentially increases the plant growth-promoting effects of auxins, which in turn enhances the phytoremediation potential of plants.

Two-dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life‐threatening infections in immunosuppressed patients. We established a 2‐D reference map for A. fumigatus. Using MALDI‐TOF‐MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.

Identification of virulence determinants of the human pathogenic fungi Aspergillus fumigatus and Candida albicans by proteomics.

Both fungi Candida albicans and Aspergillus fumigatus can cause a number of life-threatening systemic infections in humans. The commensal yeast C. albicans is one of the main causes of nosocomial fungal infectious diseases, whereas the filamentous fungus A. fumigatus has become one of the most prevalent airborne fungal pathogens. Early diagnosis of these fungal infections is challenging, only a limited number of antifungals for treatment are available, and the molecular details of pathogenicity are hardly understood. The completion of both the A. fumigatus and C. albicans genome sequence provides the opportunity to improve diagnosis, to define new drug targets, to understand the functions of many uncharacterised proteins, and to study protein regulation on a global scale. With the application of proteomic tools, particularly two-dimensional gel electrophoresis and LC/MS-based methods, a comprehensive overview about the proteins of A. fumigatus and C. albicans present or induced during environmental changes and stress conditions has been obtained in the past 5 years. However, for the discovery of further putative virulence determinants, more sensitive and targeted proteomic methods have to be applied. Here, we review the recent proteome data generated for A. fumigatus and C. albicans that are related to factors required for pathogenicity.

In silico analysis of nickel containing superoxide dismutase evolution and regulation

Superoxide dismutases are essential enzymes involved in detoxification of reactive oxygen by dismutation of the superoxide radical anion. A class of nickel containing superoxide dismutases has been described for streptomycetes and cyanobacteria. In silico analysis was used to study the distribution of genes coding for NiSOD in other taxa and to elucidate signals linked to nickel incorporation and maturation of NiSOD. Data mining revealed homologous proteins from actinobacteria, proteobacteria, chlamydiae, and eukarya (green algae) thus allowing a comparison of protein structural elements. Nickel ligands and maturation signals for N‐terminal proteolysis were highly conserved. Genomic sequences surrounding genes encoding NiSOD homologs were compared in order to detect putative accessory enzymes involved in maturation. An endopeptidase gene linked to sodN coding for NiSOD was found in actinobacteria and cyanobacteria, but not in other taxa. The distribution of NiSOD encoding sequences showed four clusters which are not consistent with the phylogeny of the species. In addition, the different genomic context argues for heterologous gene transfer, most likely from actinobacteria to other taxa. In order to address regulation by nickel availability and incorporation into the mature protein, we present first evidence for putative regulatory nucleotide sequences which will be useful in future studies on nickel uptake and incorporation. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

In silico prediction of potential metallothioneins and metallohistins in actinobacteria.

Metallothioneins and metallohistins are short peptides with a high cysteine and/or histidine content able to coordinate metals intracellularly, thereby increasing the tolerance against elevated concentrations of metals. Because of their features, they can be detected by in silico prediction from proteomes annotated from sequenced genomes. Here, we analyzed 73 sequenced actinobacterial genomes for peptides (≤100 amino acids) with a high content of cysteine and histidine (≥15%) and identified 103 putative metallothioneins and metallohistins. For 45 of these peptides, we found similarities to metal binding protein domains, including zinc fingers, heavy metal transporters or eukaryotic metallothioneins, which can serve as proof‐of‐principle in underscoring a potential function as metal binding peptides. An evolutionary origin from metal containing domains of enzymes is discussed and metallohistins not containing cysteine are described for the first time for bacteria. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

GC content-independent amino acid patterns in bacteria and archaea.

Every organism can be characterized by the amino acid composition of its proteome. So far it was assumed that these compositions are determined by the GC content of the DNA or, in some cases, by extreme lifestyles, like thermophily or halophily. Here, we focussed our analysis on eight amino acids, each of which is encoded by both, GC and AT rich codons, to identify finer amino acid patterns beyond the GC dominance. We investigated the conceptually translated proteomes of 1029 bacterial and archaeal strains with sequenced genomes for amino acid composition. Using correspondence analysis, we found that phylogenetic groups within bacteria and archaea generally can be discriminated from other groups due to their amino acid composition. In some cases, single organisms, e.g. Treponema pallidum strains or Mycoplasma penetrans, are characterized by extreme amino acid compositions. We assume that our data could provide a basis for a new approach to analyze evolution of bacterial and archaeal groups. Furthermore, for single organisms, the detailed knowledge of the amino acid composition of the entire proteome encoded in the genome could lead to a better understanding, important for pharmaceutical or biotechnological applications. We recommend that information about amino acid compositions should be provided in databases, comparable to the GC content of genomes. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Human serum alters cell culture behavior and improves spheroid formation in comparison to fetal bovine serum

Background The use of fetal bovine serum (FBS) as growth supplement for human cell and tissue culture is widely spread in basic research as well as in clinical approaches, although several limitations must be considered, such as unstable composition and availability, biosafety and ethical aspects. Regarding interspecies differences, xenogeneic growth factors may evoke incompatibilities and non‐desired interactions with human cells resulting in imprecise outcome of human‐relevant data. Methods In this study the functionality of human serum (HS) has been investigated in comparison to FBS by assessing proliferation, migration and invasion of the human cervical cancer cell lines SiHa and HeLa. The effects of both sera on spheroid formation were analyzed microscopically. Results Both, FBS and HS, stimulate cell proliferation and migration similarly, whereas HS significantly enhanced cell invasion. The spheroid formation assay revealed remarkable differences between both sera, especially for SiHa cells. While in FBS supplemented medium cells only formed loose aggregates, HS induced regularly shaped spheroids under all tested conditions. Conclusion We were able to demonstrate that HS and FBS differently influence behavior of cells in culture which may have an impact on experimental results, especially in 3D cultures.

Comparison of sample preparation techniques and data analysis for the LC-MS/MS-based identification of proteins in human follicular fluid

The proteomic analysis of complex body fluids by liquid chromatography tandem mass spectrometry (LC‐MS/MS) analysis requires the selection of suitable sample preparation techniques and optimal parameter settings in data analysis software packages to obtain reliable results. Proteomic analysis of follicular fluid, as a representative of a complex body fluid similar to serum or plasma, is difficult as it contains a vast amount of high abundant proteins and a variety of proteins with different concentrations. However, the accessibility of this complex body fluid for LC‐MS/MS analysis is an opportunity to gain insights into the status, the composition of fertility‐relevant proteins including immunological factors or for the discovery of new diagnostic and prognostic markers for, for example, the treatment of infertility. In this study, we compared different sample preparation methods (FASP, eFASP and in‐solution digestion) and three different data analysis software packages (Proteome Discoverer with SEQUEST, Mascot and MaxQuant with Andromeda) combined with semi‐ and full‐tryptic databank search options to obtain a maximum coverage of the follicular fluid proteome. We found that the most comprehensive proteome coverage is achieved by the eFASP sample preparation method using SDS in the initial denaturing step and the SEQUEST‐based semi‐tryptic data analysis. In conclusion, we have developed a fractionation‐free methodical workflow for in depth LC‐MS/MS‐based analysis for the standardized investigation of human follicle fluid as an important representative of a complex body fluid. Taken together, we were able to identify a total of 1392 proteins in follicular fluid.