Genome Sequence of Staphylococcus epidermidis Strain AU12-03, Isolated from an Intravascular Catheter

In recent years, Staphylococcus epidermidis has become a major nosocomial pathogen and the most common cause of intravascular catheter-related bacteremia, which can increase morbidity and mortality and significantly affect patient recovery. We report a draft genome sequence of Staphylococcus epidermidis AU12-03, isolated from an intravascular catheter tip.     

Defective Expression of β1-Integrins in Cells with Constitutively Active αLβ2-Integrins

We have investigated a potential relationship between expression of β1-integrins and adhesiveness of the β2-integrin LFA-1 (αLβ2, CD11a/CD18). By an approach of random mutagenesis and selection we established clones from the human acute lymphatic leukemia cell line HPB-ALL with (i) constitutively active LFA-1 and (ii) with no apparent integrin-β1 cell surface expression. Thirty-seven of 42 clones selected for activated LFA-1 were found to have lost apparent integrin-β1 expression. Conversely, 7 of 21 clones selected for lack of β1 expression were found to have activated LFA-1. Since this pointed toward a possible coupling between β1 expression and LFA-1 activity, we further analyzed at which level β1 expression was blocked. We focused on one clone, HAP4, with activated LFA-1 and no detectable β1 cell surface expression and found, surprisingly, that it expressed wild-type levels of β1 mRNA and, in Western blots of whole cell lysates, apparently normal levels of β1 protein. However, in addition to β1 of the expected molecular weight, HAP4 expressed a unique 48-kDa band recognized by the polyclonal anti-β1 antiserum. Immunoprecipitation experiments revealed that the epitope recognized by the anti-β1 antibody 4B4 was hidden or lost. The α4-chain was found in its precursor form but it did not associate with any β-chain, and it was not processed to its mature form. Instead α4-chains were eventually degraded. Taken together this showed that β1-chains were produced but not properly processed in HAP4. From this we propose that HAP4 is deficient in a gene product required both for proper β1 folding and for repression of LFA-1 adhesiveness.     

Maximization of Candida utilis cytochromes by pulse additions of ethanol to continuous cultures

Candida utilis was grown with glucose as growth-limiting carbon source in batch culture, steady-state continuous culture, and non-steady-state continuous culture. High cytochrome concentrations were routinely recorded in cells harvested in the latter stages of batch culture. They were occasionally recorded in cells from imprecisely controlled steady-state cultures, but precise control of the steady-state dissolved oxygen tension stabilized the cytochromes at relatively low levels. Controlled non-steady-state continuous cultures, imposed by pulse additions of ethanol, routinely produced cells with high cytochrome concentrations. A mechanism is proposed whereby maintenance of cytochrome derepression in continuous culture is dependent upon indefinitely prolonging an “overshoot” response in gene expression.     

Pre-lysosomal divergence of alpha 2-macroglobulin and transferrin: a kinetic study using a monoclonal antibody against a lysosomal membrane glycoprotein (LAMP-1)

We have used electron microscopic immunocytochemistry to compare the distribution of LAMP-1, a marker for lysosomal membranes, with the intracellular localization of alpha 2-macroglobulin (alpha 2-M) and transferrin at various time points after their endocytosis into cultured NIH 3T3 cells. The purposes of this study were (a) to determine how soon endocytic ligands reach lysosomal organelles, (b) to examine whether the intermediate endocytic vesicles gained lysosomal markers gradually or in a precipitous, discrete event, and (c) to examine the relationship, if any, between the pathway of recycling ligands and lysosomes. At early time points (0-5 min) after initiation of endocytosis, most structures containing alpha 2-M labeled with colloidal gold (receptosomes) were not labeled by anti-LAMP-1 detected using ferritin bridge or peroxidase immunocytochemistry. At late time points (greater than or equal to 15 min), the structures containing alpha 2-M (lysosomes) were strongly labeled by anti-LAMP-1. In contrast, transferrin that was directly labeled with ferritin was mostly located in LAMP-1-negative structures at all time points studied. The proportion of alpha 2-M-gold containing vesicles strongly labeled for LAMP-1 roughly paralleled the proportion of alpha 2-M-gold-containing structures positive for cytochemically detectable acid phosphatase. Our data indicate that ligands such as transferrin that are internalized through coated pits and receptosomes, but not delivered to lysosomes, do not traverse a lysosomal organelle compartment as marked by LAMP-1 content. Ligands such as alpha 2-M that are destined for lysosomal delivery reach a LAMP-1-positive organelle compartment only after they traverse LAMP-1-negative, non-lysosomal vesicles previously described as receptosomes.     

Kinetic properties and contribution to cellulose saccharification of a cloned Pseudomonas beta-glucosidase

The plasmid pND71, which encodes beta-glucosidase (cellobiase) activity, cloned from the cellulolytic Pseudomonad, PS2-2, was mobilized by conjugation into 10 Pseudomonas strains. The highest specific activity was produced by 17498 (pND71) and the properties of the enzyme produced from this transconjugant were studied. The enzyme was shown to be cell associated, to have a temperature optimum of 37 degrees C, a pH optimum of 7.0 and Km values of 1.33 and 2.94 mM for pNPG and cellobiose respectively. It was competitively inhibited by glucose, with a Ki of 30 mM. Evidence was obtained which suggested that the enzyme was produced constitutively and that synthesis was not repressed by glucose. When culture preparations were used in combination with Trichoderma reesei QM9414 and C30 enzyme preparations to saccharify cellulose, 17498 (pND71) was more effective than preparations of PS2-2 in acting synergistically with T. reesei to solubilize more carbohydrate and produce more glucose.     

Cyanide and methylisocyanide binding to the isolated iron-molybdenum cofactor of nitrogenase

19F NMR and x-ray absorption experiments have been performed with both the isolated FeMo cofactor and the MoFe protein of nitrogenase in search of direct evidence for substrate or inhibitor binding. Using 19F NMR as a probe and p-CF3C6H4S- as the receptor ligand, the data show that the nitrogenase inhibitors CN- and CH3NC bind to the isolated FeMo cofactor-RFS- complex in N-methylformamide with a finite formation constant. Their binding increases the electronic relaxation time of the complex and increases the life-time of the FeMo cofactor-p-CF3C6H4S- bond, Parallel molybdenum K edge and extended x-ray absorption fine structure experiments show that CH3NC does not bind to molybdenum. Although CO and N3- both relieve CN- and CH3NC inhibition of electron flow through nitrogenase, unlike the latter, they do not appear to bind to isolated FeMo cofactor. In experiments with the dithionite-reduced MoFe protein, we did not detect any changes in the molybdenum K edge or extended x-ray absorption fine structure spectra upon addition of CO, N2, C2H2, NaCN, CH3NC, or azide demonstrating that either these substrates and inhibitors do not bind to molybdenum or that the FeMo cofactor site of nitrogenase is inaccessible to substrate binding except under turnover conditions.     

CLIP-170 links endocytic vesicles to microtubules.

Binding of endocytic carrier vesicles to microtubules depends on the microtubule-binding protein CLIP-170 in vitro. In vivo, CLIP-170 colocalizes with a subset of transferrin receptor-positive endocytic structures and, more extensively, with endosomal tubules induced by brefeldin A. The structure of CLIP-170 has been analyzed by cloning its cDNA. The predicted non-helical C- and N-terminal domains of the homodimeric protein are connected by a long coiled-coil domain. We have identified a novel motif present in a tandem repeat in the N-terminal domain of CLIP-170 that is involved in binding to microtubules. This motif is also found in the Drosophila Glued and yeast BIK1 proteins. These features, together with its very elongated structure, suggest that CLIP-170 belongs to a novel class of proteins, cytoplasmic linker proteins (CLIPs), mediating interactions of organelles with microtubules.