Identification and characterization of coenzyme B12-dependent glycerol dehydratase- and diol dehydratase-encoding genes from metagenomic DNA libraries derived from enrichment cultures

To isolate genes encoding coenzyme B(12)-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

Specific targeting of a DNA-binding protein to the SPP1 procapsid by interaction with the portal oligomer

The icosahedral procapsid of tailed bacteriophages is composed of a large number of identical subunits and of minor proteins found in a few copies. Proteins present in a very low copy number are targeted to the viral procapsid by an unknown mechanism. Bacteriophage SPP1 procapsids and mature virions contain two copies of gp7 on average. Gp7 forms stable complexes with the SPP1 portal protein gp6. Deletion of the gp6 carboxyl-terminus and the mutation Y467-->C localized in the same region prevent gp6-gp7 complex formation. Gp7 binds double-stranded and single-stranded DNA. Gp6 competes for this interaction, and purified gp6-gp7 complexes do not bind DNA. Procapsid structures assembled in the absence of gp6 or carrying the mutant gp6 Y467-->C lack gp7. The gp6-gp7 interaction thus targets gp7 to the procapsid where the portal protein is localized asymmetrically at a single vertex of the icosahedral structure. The interaction between the two proteins is disrupted during viral assembly. Proteins homologous to gp6 and gp7 are coded by contiguous genes in a variety of phage genomes from Gram-positive bacteria, suggesting that the gp6-gp7 complex is widespread in this group of phages. Transient association with the portal protein, an essential component of tailed bacteriophages and herpes viruses, provides a novel strategy to target minor proteins to the virion structure that might be operative in a large number of viruses.     

Uteroglobin promoter-targeted c-MYC expression in transgenic mice cause hyperplasia of Clara cells and malignant transformation of T-lymphoblasts and tubular epithelial cells

To investigate the influence of the proto-oncogene c-MYC on tumor development in different epithelial tissues which secrete Clara Cell Secretory Protein (uteroglobin, UG), transgenic mouse lines were established expressing the human c-MYC proto-oncogene under the control of the rabbit UG-promoter. These mice expressed the c-MYC transgene in Clara cells and other UG expressing tissues like uterus and prostate. In the bronchioalveolar epithelium of the lung hyperplasias developed originating from Clara cells. Surprisingly, transgenics most frequently developed T-lymphoblastic lymphomas, a polycystic kidney phenotype and renal cell carcinoma derived from tubular epithelial cells, which are both tissues that had so far not been known to express UG. Immunohistological studies in UG/MYC transgenics and in a transgenic line (UG/eGFP) expressing Green Fluorescent Protein confirmed that the uteroglobin promoter is not only active in Clara cells, but also in tubular epithelial cells of the kidney and in lymphatic tissue. The UG/MYC transgenics will be useful to investigate the biochemical mechanisms underlying the development of carcinomas and the oncogenic properties of c-MYC in epithelial cells of various tissues.     

Induction of Raf kinase inhibitor protein contributes to macrophage differentiation

Differential gene expression analysis of human blood monocytes has identified the Raf kinase inhibitor protein (RKIP) as a continuously upregulated gene in macrophage and dendritic cell maturation. Using realtime RT-PCR and Western blot analysis we were able to confirm the initial DNA-microarray findings of RKIP induction on mRNA and protein levels. RKIP upregulation in primary cells and overexpression in THP-1 cells did not alter ERK activity but strongly reduced the amount of the NFkappaB subunit p65 in the nucleus. mRNA levels and cell surface expression of maturation markers including the integrin CD11c and the scavenger receptor CD36 were significantly increased in RKIP transfected THP-1 cells. Our data show for the first time that RKIP is upregulated during macrophage and dendritic cell differentiation on mRNA and protein levels and we conclude that RKIP contributes to the monocytic differentiation process via inhibition of the NFkappaB signaling cascade independent from the canonical Ras/Raf/MEK/ERK pathway.     

The DNA translocase RAD5A acts independently of the other main DNA repair pathways,and requires both its ATPase and RING domain for activity in Arabidopsis thaliana

Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error‐free branch of post‐replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication‐associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (AtRAD1), single‐strand break repair (AtPARP1), as well as microhomology‐mediated double‐strand break repair (AtTEB). Moreover, RAD5A can partially complement for a deficient AtATM‐mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects.     

A molecular biological protocol to distinguish potentially human pathogenic Stenotrophomonas maltophilia from plant-associated Stenotrophomonas rhizophila

In recent years, the importance of the Gram-negative bacterium Stenotrophomonas as an opportunistic pathogen as well as in biotechnology has increased. The aim of the present study was to develop new methods for distinguishing between strains closely related to the potentially human pathogenic Stenotrophomonas maltophilia and those closely related to the plant-associated Stenotrophomonas rhizophila. To accomplish this, 58 strains were characterized by 16S rDNA sequencing and amplified ribosomal DNA restriction analysis (ARDRA), and the occurrence of specific functional genes. Based on 16S rDNA sequences, an ARDRA protocol was developed which allowed differentiation between strains of the S. maltophilia and the S. rhizophila group. As it was known that only salt-treated cells of S. rhizophila were able to synthesize the compatible solute glucosylglycerol (GG), the ggpS gene responsible for GG synthesis was used for differentiation between both species and it was confirmed that it only occurred in S. rhizophila strains. As a further genetic marker the smeD gene, which is part of the genes coding for the multidrug efflux pump SmeDEF from S. maltophilia, was used. Based on the results we propose a combination of fingerprinting techniques using the 16S rDNA and the functional genes ggpS and smeD to distinguish both Stenotrophomonas species.     

Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.     

TNF but not Fas ligand provides protective anti-L. major immunity in C57BL/6 mice

The pro-inflammatory cytokine TNF is essential for a protective immune response to some but not all strains of Leishmania major. TNF-deficient mice of a resistant genetic background succumbed rapidly to an infection with L. major BNI. Another member of the TNF superfamily, Fas ligand (FasL), has also been reported to be critical for the immune response to L. major. To test the relative importance of TNF versus FasL for the control of L. major BNI, we infected wildtype C57BL/6 (B6.WT), B6.TNF(-/-), B6.gld and C57BL/6.gld x TNF(-/-) (B6.gld.TNF(-/-)) double-negative mice. Visceral, fatal disease was only observed in B6.TNF(-/-) mice, but not in B6 gld mice. The course of infection and the immune response of B6.gld.TNF(-/-) mice were similar to those of B6.TNF(-/-) mice. B6.gld.TNF(-/-) mice had a high tissue parasite burden and expressed prominent amounts of inducible nitric oxide synthase (iNOS) in the skin, the lymph nodes (LN) and the spleen as previously reported for B6.TNF(-/-) mice, whereas the tissue parasite load and the iNOS expression of B6.gld mice resembled that of B6.WT controls. Neither the TNF- nor the FasL-deficiency exerted a detectable intrinsic effect on the proliferation of T cells. Thus, TNF, but not FasL is essential for the control of L. major BNI. The discrepancy between these and other published data are most likely due to the use of different strains of the pathogen.