CzcD from Ralstonia metallidurans and ZitB from Escherichia coli are prototypes of bacterial members of the cation diffusion facilitator (CDF) protein family. Expression of the czcD gene in an E. coli mutant strain devoid of zitB and the gene for the zinc-transporting P-type ATPase zntA rendered this strain more zinc resistant and caused decreased accumulation of zinc. CzcD, purified as an amino-terminal streptavidin-tagged protein, . Conserved amino acyl residues that might be involved in binding and transport of zinc were mutated in CzcD and/or ZitB, and the influence on Zn 2؉ resistance was studied. Charged or polar amino acyl residues that were located within or adjacent to membrane-spanning regions of the proteins were essential for the full function of the proteins. Probably, these amino acyl residues constituted a pathway required for export of the heavy metal cations or for import of counter-flowing protons.The cation diffusion facilitators (CDF) (T.C.2.A.4.1.1) (26) are a family of metal transport proteins found in a variety of organisms (16,17,19,23). In contrast to other protein families, such as P-type ATPases or ABC transporters (26), all CDF proteins characterized to date transport only metals, and the majority are involved in Zn 2ϩ transport (3,16,23). One of the first two CDF identified was the CzcD protein (15, 18) from the gram-negative bacterium Ralstonia metallidurans strain CH34 (previously Alcaligenes eutrophus [5]). This transporter is part of a cobalt-zinc-cadmium resistance system (Czc) and decreases the intracellular zinc concentration (1). The CzcD protein is composed of a hydrophobic, membrane-bound domain consisting of about 200 amino acid residues with probably six transmembrane ␣-helices and a 115-amino-acid hydrophilic domain located in the cytoplasm (1).Escherichia coli detoxifies excess Zn 2ϩ by using ZntA, a P-type ATPase, and the CDF protein ZitB (6), which is closely related to CzcD. Previously, site-directed mutagenesis was used to identify amino acyl residues H53, H159, D163, and D186 of ZitB as essential residues (12). These residues were located within predicted transmembrane domains (TMs) of this protein. In this study we compared a variety of additional mutations in ZitB and CzcD to better understand functional aspects of zinc binding and efflux. ZitB-dependent 65 Zn 2ϩ transport into everted membrane vesicles was driven by the proton motive force (PMF). These kinetics suggest that this protein is mainly responsible for zinc homeostasis under most physiological conditions. MATERIALS AND METHODSBacterial strains, growth conditions, and plasmid construction. The media used to cultivate E. coli strains W3110 (wild type) (7), GG48 (⌬zitB::Cm zntA::Km) (6), and GR362 (⌬zntA::Km ⌬zitB ⌬zupT ⌬znuABC ⌬zntB::Cm) (8) were Tris-buffered mineral salts medium (13) containing 2 g of glucose liter Ϫ1 plus 1 g of yeast extract liter Ϫ1 (TGY), the same medium with 2 g of glycerol liter Ϫ1 plus 3 g of Casamino Acids liter Ϫ1 (TGC), and Luria-Bertani broth (LB) (27). Solid Tris-buffered m...

Intracellular Staphylococcus aureus has been implicated in the establishment of chronic infections. It is therefore imperative to understand by what means S. aureus is able to survive within cells. Here we use two expression systems with a fluorescent readout to assay alpha-toxin expression and function within phagolysosomes of infected upper-airway epithelial cells: avirulent Staphylococcus carnosus TM300 and phenotypically alpha-toxin-negative S. aureus laboratory strains. Data from CFU recovery assays suggest that the presence of alpha-toxin is not beneficial for the intracellular survival of recombinant Staphylococcus strains. This finding was corroborated by immunofluorescence studies: whereas S. carnosus and S. aureus are able to deliver S. aureus alpha-toxin to lumina of host cell phagolysosomes, the membrane integrity of these organelles was not affected. Alpha-toxin-expressing strains were detected exclusively within lysosome-associated membrane protein 1 (LAMP1)-yellow fluorescent protein (YFP)-positive vesicles. Measurements of intraphagosomal pH illustrated that all infected phagolysosomes acidified regardless of alpha-toxin expression. In contrast, S. aureus expressing Listeria monocytogenes listeriolysin O leads to the breakdown of the phagolysosomal membrane, as indicated by staphylococci that are not associated with LAMP1-YFP-decorated vesicles and that do not reside within an acidic cellular environment. Thus, our results suggest that staphylococcal alpha-toxin is not sufficient to mediate phagolysosomal escape in upper-airway epithelial cells.

Novel chitosan (CHS) and cellulose sulfates (CSs) are studied regarding their mitogenic activity and their protective effect against proteolytic digestion of FGF-2. An intermediate degree of sulfation (DS(S) ) and lower concentration of CHS have superior effect on 3T3 cell growth while the mitogenic activity of CS increases with DS(S) and concentration. Experiments with trypsin as model proteinase show that protection of FGF-2 from proteolytic digestion depends on DS(S) and the concentration of derivatives in the same manner as cell growth. Studies on stability of FGF-2 added to cultures of 3T3 cells show that the FGF-2 concentration remains higher in the presence of derivatives. Results indicate that the mitogenic activity of CHS and CS is due to protection of FGF-2 from proteolytic cleavage.

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