Dörte Gade

Complete genome sequence of the marine planctomycete Pirellula sp strain 1

Pirellula sp. strain 1 (“Rhodopirellula baltica”) is a marine representative of the globally distributed and environmentally important bacterial order Planctomycetales. Here we report the complete genome sequence of a member of this independent phylum. With 7.145 megabases, Pirellula sp. strain 1 has the largest circular bacterial genome sequenced so far. The presence of all genes required for heterolactic acid fermentation, key genes for the interconversion of C1 compounds, and 110 sulfatases were unexpected for this aerobic heterotrophic isolate. Although Pirellula sp. strain 1 has a proteinaceous cell wall, remnants of genes for peptidoglycan synthesis were found. Genes for lipid A biosynthesis and homologues to the flagellar L- and P-ring protein indicate a former Gram-negative type of cell wall. Phylogenetic analysis of all relevant markers clearly affiliates the Planctomycetales to the domain Bacteria as a distinct phylum, but a deepest branching is not supported by our analyses.

Evaluation of Two-Dimensional Difference Gel Electrophoresis for Protein Profiling

Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes™ and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder™ software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10–9) were identified. For comparison, another experiment was carried out with four SYPRO®-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster™ 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 µg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.

Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines.

Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2‐D DIGE and nanoHPLC‐nanoESI‐MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2‐D gels of the proteomes of uninfected and influenza‐infected host cells, 16 quantitatively altered protein spots (at least ±1.7‐fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon‐induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome‐wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.

Identification of Planctomycetes with Order-, Genus-, and Strain-Specific 16S rRNA-Targeted Probes

A specific 16S rRNA-targeted oligonucleotide probe (PIR1223) for the genus Pirellula and a species-specific probe (RB454) for Pirellula sp. strain SH1 have been designed and optimized. Together with the already existing order-specific probe PLA886, the two newly designed probes were used to detect and identify planctomycetes, pirellulae, and close relatives of Pirellula sp. strain SH1 in different habitats. With the help of these probes for detection and identification, bacteria of the genus Pirellula were detected and cultivated from tissue of the Mediterranean sponge Aplysinaaerophoba and from the water column of the Kiel Fjord. An unexpected result was the close phylogenetic relationship of the isolate from the sponge and the brackish water habitat Kiel Fjord as revealed by DNA/DNA hybridization.

Analysis of N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1 by a proteomic approach

Pirellula sp. strain 1 is a marine bacterium that can grow with the chitin monomer N‐acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes. As a basis for the proteomic studies we quantified growth of strain 1 with N‐acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively. Studies with dense cell suspensions indicated that the capacity to degrade N‐acetylglucosamine and glucose may not be tightly regulated. Proteins from soluble extracts prepared from exponential cultures grown either with N‐acetylglucosamine or glucose were separated by two‐dimensional gel electrophoresis and visualized by fluorescence staining (Sypro® Ruby). Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N‐acetylglucosamine grown cells. Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp. strain 1. This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine‐6‐phosphate isomerase from B. subtilis. Genes of two other proteins expressed during growth on N‐acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde‐3‐phosphate‐dehydrogenase and a NADH‐dehydrogenase, respectively. Thus the coding genes of three proteins expressed during growth of Pirellula sp. strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism.

Proteomic analysis of influenza A virus infected mammalian cells by 2D-DIGE

Host cell protein expression changes in canine MDCK and human A549 cells after infection with human influenza A/PR/8/34 (H1N1) virus were charac- terized by two-dimensional differential gel electrophoresis (2D-DIGE). A dynamic host cell response specific for both host cell lines but also differences of the same virus strain obtained from different suppliers (RKI, Germany; NIBSC, UK) were observed.