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Abstract: Enrichments with [Fe(II)EDTA]2− as electron donor and nitrate or nitrite as electron acceptor were established using an inoculum from a bioreactor performing denitrification. A nitrate-reducing, [Fe(II)EDTA]2− oxidizing strain was isolated and named strain BDN-1. The G+C content of strain BDN-1 was 67%, and the organism was closely affiliated to Paracoccus denitrificans, P. pantotrophus and P. versutus by 16S rRNA sequence comparison. Results from DNA–DNA hybridization, rep-PCR, and whole cell protein analysis gave congruent results confirming the genotypic and phenotypic differences between strain BDN-1 and the other species of Paracoccus. From these results, we considered strain BDN-1 as a novel species for which we propose the name Paracoccus ferrooxidans. Apart from [Fe(II)EDTA]2−, BDN-1 could also use thiosulfate and thiocyanate as inorganic electron donors. Nitrate, nitrite, N2O, [Fe(II)EDTA·NO]2− and oxygen could be used by strain BDN-1 as electron acceptors. Repeated transfer on a culture medium with bicarbonate as the sole carbon source confirmed that strain BDN-1 was a facultative autotroph. [Fe(II)EDTA]2− oxidation dependent denitrification was also performed by other Paracoccus species, that were closely affiliated to P. ferrooxidans. [Copyright &y& Elsevier]

We subjected methanol-grown cells of wild type Hansenula polymorpha simultaneously to nitrogen depletion and excess glucose conditions. Both treatments induce the degradation of peroxisomes, either selective (via excess glucose) or non-selective (via nitrogen limitation). Our combined data strongly suggest that both processes occur simultaneously under these conditions. The implications of these findings on studies of autophagy and related transport pathways to the vacuole in yeast are discussed. [Copyright &y& Elsevier]

Saccharomyces cerevisiae contains three dynamin-related-proteins, Vps1p, Dnm1p and Mgm1p. Previous data from glucose-grown VPS1 and DNM1 null mutants suggested that Vps1p, but not Dnm1p, plays a role in regulating peroxisome abundance. Here we show that deletion of DNM1 also results in reduction of peroxisome numbers. This was not observed in glucose-grown dnm1 cells, but was evident in cells grown in the presence of oleate. Similar observations were made in cells lacking Fis1p, a protein involved in Dnm1p function. Fluorescence microscopy of cells producing Dnm1-GFP or GFP-Fis1p demonstrated that both proteins had a dual localization on mitochondria and peroxisomes. Quantitative analysis revealed a greater reduction in peroxisome number in oleate-induced vps1 cells relative to dnm1 or fis1 cells. A significant fraction of oleate-induced vps1 cells still contained two or more peroxisomes. Conversely, almost all cells of a dnm1 vps1 double-deletion strain contained only one, enlarged peroxisome. This suggests that deletion of DNM1 reinforces the vps1 peroxisome phenotype. Time-lapse imaging indicated that during budding of dnm1 vps1 cells, the single peroxisome present in the mother cell formed long protrusions into the developing bud. This organelle divided at a very late stage of the budding process, possibly during cytokinesis. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE C103S gene in P. chrysogenum gave rise to an unprocessed inactive variant of IAT (IATC103S) located inside peroxisomes, which indicates that transport of the proIAT inside these organelles is not dependent on the processing state of the protein. Co-expression of the penDE C103S and wild-type penDE genes in P. chrysogenum (Wis54-DE C103S strain) led to a decrease in benzylpenicillin levels. Changes in the wild-type IAT processing profile (β subunit formation) were observed in the Wis54-DE C103S strain, suggesting a regulatory role of the unprocessed IATC103S in the processing of the wild-type IAT. This was confirmed in Escherichia coli, where a delay in the processing of IAT in presence of the unprocessable IAT C103S was observed. Our results indicate that IAT is post-translationally regulated by its preprotein, which interferes with the self-processing. [Copyright &y& Elsevier]

Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids l-α-aminoadipate, l-cysteine, and l-valine into the tripeptide ACV. ACV synthetase has previously been localized to the vacuole where it is thought to utilize amino acids from the vacuolar pools. We localized ACV synthetase by subcellular fractionation and immuno-electron microscopy under conditions that prevented proteolysis and found it to co-localize with isopenicillin N synthetase in the cytosol, while acyltransferase localizes in microbodies. These data imply that the key enzymatic steps in penicillin biosynthesis are confined to only two compartments, i.e., the cytosol and microbody. [Copyright &y& Elsevier]

Lipoplexes and polyplexes, that is, assemblies of cationic lipids and polymers with nucleic acids, respectively, are popular nanocarriers for delivery of genes or siRNA into cells for therapeutic or cell biological purposes. Although endocytosis represents a major mechanism for their cellular entry, very little is known about parameters that govern early events in the initial interaction of such delivery devices with the cell surface. Here, we demonstrate that prior to entry, poly- and lipoplexes are captured by thin, actin-rich filopodial extensions, protruding from the cell surface. Subsequent additional recruitment and local clustering of filopodia-localized syndecans, presumably driven by multivalent interactions with the polycationic nanocarriers, appear instrumental in their processing to the cell body. Detailed microscopic analyses reveal that the latter relies on either directional surfing along or retraction of the filopodia. By interfering with actin polymerization or inhibiting the motor protein myosin II, localized at the base of filopodia, our data reveal that the binding of the nanocarriers to and subsequent clustering of syndecans initiates actin retrograde flow, which moves the syndecan-bound nanocarriers to the cell body. At the present experimental conditions, inhibition of this process inhibits nanocarrier-mediated transfection by 50-90%. The present findings add novel insight to our understanding of the mechanism of nanocarrier-cell surface interaction, which may be instrumental in further improving delivery efficiency. In addition, the current experimental approach may also be of relevance to improving our understanding of cellular infection by viruses and pathogenic bacteria, given a striking parallel in filopodia-mediated processing of these infectious particles and nanocarriers.

Understanding where, when, and how biomolecules (inter)act is crucial to uncover fundamental mechanisms in cell biology. Recent developments in fluorescence light microscopy (FLM) allow protein imaging in living cells and at the near molecular level. However, fluorescence microscopy only reveals selected biomolecules or organelles but not the (ultra)structural context, as can be examined by electron microscopy (EM). LM and EM of the same cells, so-called correlative (or correlated) light and electron microscopy (CLEM), allow examining rare or dynamic events first by LM, and subsequently by EM. Here, we review progress in CLEM, with focus on matching the areas between different microscopic modalities. Moreover, we introduce a method that includes a virtual overlay and automated large-scale imaging, allowing to switch between most microscopes. Ongoing developments will revolutionize and standardize CLEM in the near future, which thus holds great promise to become a routine technique in cell biology., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Strain ALEN 2(T) was isolated from a mixed culture capable of complete autotrophic denitrification with thiosulfate as electron donor at pH 10; the mixed culture was enriched from sediment from Lake Fazda (Wadi Natrun, Egypt), a hypersaline alkaline lake. The isolate had large, non-motile, coccoid or barrel-shaped cells with intracellular sulfur globules. The bacterium was obligately chemolithoautotrophic. It grew with reduced sulfur compounds aerobically and anaerobically with nitrate as electron acceptor, nitrate being reduced to nitrite. It was moderately halophilic and obligately alkaliphilic. On the basis of genetic analysis and its unique phenotype, strain ALEN 2(T) (=DSM 14787(T)=UNIQEM 213(T)) is proposed as the type strain of a novel species of the genus Thialkalivibrio, Thialkalivibrio nitratireducens.