Pili-Induced Clustering of N. gonorrhoeae Bacteria

Type IV pili (Tfp) are prokaryotic retractable appendages known to mediate surface attachment, motility, and subsequent clustering of cells. Tfp are the main means of motility for Neisseria gonorrhoeae, the causative agent of gonorrhea. Tfp are also involved in formation of the microcolonies, which play a crucial role in the progression of the disease. While motility of individual cells is relatively well understood, little is known about the dynamics of N. gonorrhoeae aggregation. We investigate how individual N. gonorrhoeae cells, initially uniformly dispersed on flat plastic or glass surfaces, agglomerate into spherical microcolonies within hours. We quantify the clustering process by measuring the area fraction covered by the cells, number of cell aggregates, and their average size as a function of time. We observe that the microcolonies are also able to move but their mobility rapidly vanishes as the size of the colony increases. After a certain critical size they become immobile. We propose a simple theoretical model which assumes a pili-pili interaction of cells as the main clustering mechanism. Numerical simulations of the model quantitatively reproduce the experimental data on clustering and thus suggest that the agglomeration process can be entirely explained by the Tfp-mediated interactions. In agreement with this hypothesis mutants lacking pili are not able to form colonies. Moreover, cells with deficient quorum sensing mechanism show similar aggregation as the wild-type bacteria. Therefore, our results demonstrate that pili provide an essential mechanism for colony formation, while additional chemical cues, for example quorum sensing, might be of secondary importance.     

The role of absC,a novel regulatory gene for secondary metabolism,in zinc‐dependent antibiotic production in Streptomyces coelicolor A3(2)

The availability of zinc was shown to have a marked influence on the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2). Production of actinorhodin and undecylprodigiosin was abolished when a novel pleiotropic regulatory gene, absC, was deleted, but only when zinc concentrations were low. AbsC was shown to control expression of the gene cluster encoding production of coelibactin, an uncharacterized non‐ribosomally synthesized peptide with predicted siderophore‐like activity, and the observed defect in antibiotic production was found to result from elevated expression of this gene cluster. Promoter regions in the coelibactin cluster contain predicted binding motifs for the zinc‐responsive regulator Zur, and dual regulation of coelibactin expression by zur and absC was demonstrated using strains engineered to contain deletions in either or both of these genes. An AbsC binding site was identified in a divergent promoter region within the coelibactin biosynthetic gene cluster, adjacent to a putative Zur binding site. Repression of the coelibactin gene cluster by both AbsC and Zur appears to be required to maintain appropriate intracellular levels of zinc in S. coelicolor.     

Dimeric H-ATP synthase in the chloroplast of Chlamydomonas reinhardtii

H⁺-ATP synthase is the dominant ATP production site in mitochondria and chloroplasts. So far, dimerization of ATP synthase has been observed only in mitochondria by biochemical and electron microscopic investigations. Although the physiological relevance remains still enigmatic, dimerization was proposed to be a unique feature of the mitochondrion [Biochim. Biophys. Acta 1555 (2002) 154]. It is hard to imagine, however, that closely related protein complexes of mitochondria and chloroplast should show such severe differences in structural organization. We present the first evidences for dimerization of chloroplast ATP synthases within the thylakoid membrane.By investigation of the thylakoid membrane of Chlamydomonas reinhardtii by blue-native polyacrylamide gel electrophoresis, dimerization of the chloroplast ATP synthase was detected. Chloroplast ATP synthase dimer dissociates into monomers upon incubation with vanadate or phosphate but not by incubation with molybdate, while the mitochondrial dimer is not affected by the incubation. This suggests a distinct dimerization mechanism for mitochondrial and chloroplast ATP synthase. Since vanadate and phosphate bind to the active sites, contact sites located on the hydrophilic CF₁ part are suggested for the chloroplast ATP synthase dimer. As the degree of dimerization varies with phosphate concentration, dimerization might be a response to low phosphate concentrations.     

Comparing minimally invasive funnel chest repair versus the conventional technique: an outcome analysis in children

Surgical correction of pectus excavatum in children has gained new momentum since the introduction of the new minimally invasive repair by Nuss. To date, no studies directly evaluate the outcome of the new technique versus that of the conventional technique. From 2000 to 2002, 28 patients underwent pectus excavatum correction in the authors' hospital. Twenty-one were treated by minimally invasive repair of pectus excavatum and seven patients had open correction. Intraoperative and postoperative complications, clinical outcome, and patient satisfaction were evaluated. In the minimally invasive repair of pectus excavatum group, the children were younger (14.4 +/- 2.9 versus 17.8 +/- 3.2 years), had shorter operation times (53 +/- 18 versus 125 +/- 6 minutes), and had less blood loss (minimal versus 380 +/- 175 ml). No intraoperative complications were recorded. In the conventional group, two pleural lacerations occurred. Early postoperative complications in the minimally invasive repair group included two pneumothoraces and one case of pleural effusion. In the conventional group, one pneumothorax and one case of pleural effusion occurred. Late postoperative complications in the Nuss group included one costal erosion, two bar dislocations, one severe wound infection requiring bar removal, one hematothorax, and one case of postpericardiotomy syndrome; in the conventional group, there was one severe wound infection. In both groups, the patients rated their cosmetic results as good to very good. Minimally invasive repair of pectus excavatum is a novel method with clear advantages, such as limited surgical trauma and small scars. The high rate of postoperative complications may decrease with growing experience in the future. In well-selected patients (age, symmetric deformity), the Nuss procedure may become the method of choice. However, there is still a lack of long-term follow-up.     

The Cfd1-Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol

Biogenesis of iron-sulfur ([Fe-S]) proteins in eukaryotes requires the function of complex proteinaceous machineries in both mitochondria and cytosol. In contrast to the mitochondrial pathway, little is known about [Fe-S] protein assembly in the cytosol. So far, four highly conserved proteins (Cfd1, Nbp35, Nar1 and Cia1) have been identified as members of the cytosolic [Fe-S] protein assembly machinery, but their molecular function is unresolved. Using in vivo and in vitro approaches, we found that the soluble P-loop NTPases Cfd1 and Nbp35 form a complex and bind up to three [4Fe-4S] clusters, one at the N terminus of Nbp35 and one each at a new C-terminal cysteine-rich motif present in both proteins. These labile [Fe-S] clusters can be rapidly transferred and incorporated into target [Fe-S] apoproteins in a Nar1- and Cia1-dependent fashion. Our data suggest that the Cfd1-Nbp35 complex functions as a novel scaffold for [Fe-S] cluster assembly in the eukaryotic cytosol.     

Identification of novel dendritic cell subset markers in human blood

Human dendritic cells (DC) are key regulators of innate and adaptive immunity that can be divided in at least three major subpopulations: plasmacytoid DC (pDC), myeloid type 1 DC (mDC1) and myeloid type 2 DC (mDC2) exhibiting different functions. However, research, diagnostic and cell therapeutic studies on human DC subsets are limited because only few DC subset markers have been identified so far. Especially mDC2 representing the rarest blood DC subset are difficult to be separated from mDC1 and pDC due to a paucity of mDC2 markers. We have combined multiparameter flow cytometry analysis of human blood DC subsets with systematic expression analysis of 332 surface antigens in magnetic bead-enriched blood DC samples. The initial analysis revealed eight novel putative DC subset markers CD26, CD85a, CD109, CD172a, CD200, CD200R, CD275 and CD301 that were subsequently tested in bulk peripheral blood mononuclear cell (PBMC) samples from healthy blood donors. Secondary analysis of PBMC samples confirmed three novel DC subset markers CD26 (dipeptidyl peptidase IV), CD85a (Leukocyte immunoglobulin-like receptor B3) and CD275 (inducible costimulator ligand). CD85a is specifically expressed in mDC1 and CD26 and CD275 represent novel mDC2 markers. These markers will facilitate human DC subset discrimination and additionally provide insight into potentially novel DC subset-specific functions.     

Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.     

Proteomic approach: Identification of <Emphasis Type="Italic">Medicago truncatula</Emphasis> proteins induced in roots after infection with the pathogenic oomycete <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>

The legume root rot disease caused by the oomycete pathogen Aphanomyces euteiches is one major yield reducing factor in legume crop production. A comparative proteomic approach was carried out in order to identify proteins of the model legume Medicago truncatula which are regulated after an infection with A. euteiches. Several proteins were identified by two dimensional gel electrophoresis to be differentially expressed after pathogen challenge. Densitometric evaluation of expression values showed different regulation during the time-course analysed. Proteins regulated during the infection were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Among the differentially expressed proteins, two encoded putative cell wall proteins and two were designated as small heat shock proteins. Furthermore, an isoform of the chalcone-O-methyltransferase was found to be increased in infected roots. The majority of induced proteins belonged to the family of class 10 of pathogenesis related proteins (PR10). Previously, various PR10-like proteins have been shown to be regulated by general stress or abscisic acid (ABA). Therefore, these proteins were further investigated concerning their regulation in response to drought stress and exogenous ABA-application. Complex regulation patterns were identified: three of the A. euteiches-induced PR10-like proteins were also induced by exogenous ABA- but none of them is induced after drought stress. In contrast, three of these proteins are down-regulated by drought stress. Hence, the strong expression of different PR10-family members and their regulation profiles indicates that this set of proteins plays a major role during root adaptations to various stress conditions.