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101.
Yeast secretory mutants that block the formation of active cell surface enzymes 总被引:35,自引:11,他引:24 下载免费PDF全文
Yeast cells secrete a variety of glycosylated proteins. At least two of these proteins, invertase and acid phosphatase, fail to be secreted in a new class of mutants that are temperature-sensitive for growth. Unlike the yeast secretory mutants previously described (class A sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell., 21:205-420), class B sec mutants (sec 53, sec 59) fail to produce active secretory enzymes at the restrictive temperature (37 degrees C). sec 53 and sec 59 appear to be defective in reactions associated with the endoplasmic reticulum. Although protein synthesis continues at a nearly normal rate for 2 h at 37 degrees C, incorporation of [3H]mannose into glycoprotein is reduced. Immunoreactive polypeptide forms of invertase accumulate within the cell which have mobilities on SDS PAGE consistent with incomplete glycosylation: sec 53 produces little or no glycosylated invertase, and sec 59 accumulates forms containing 0-3 of the 9-10 N-linked oligosaccharide chains that are normally added to the protein. In addition to secreted enzymes, maturation of the vacuolar glycoprotein carboxypeptidase Y, incorporation of the plasma membrane sulfate permease activity, and secretion of the major cell wall proteins are blocked at 37 degrees C. 相似文献
102.
Steve Novick David Christopher Monisha Dey Svetlana Lyapustina Michael Golden Stefan Leiner Bruce Wyka Hans-Joachim Delzeit Chris Novak Gregory Larner 《AAPS PharmSciTech》2009,10(3):841-849
This article examines the effects of changing parameters in the test which was proposed by the FDA at the October 2005 Advisory
Committee meeting for confirming delivered dose uniformity in orally inhaled and nasal drug products. This article is an extension
of the characterization study presented in an accompanying article (Part 1). The goal of this study is to understand how parameters
of the test affect the test performance. The effects of changing test parameters such as target interval, maximum allowable
proportion in the tail area, and sample size are examined. The results show that changing the maximum allowable tail area
and/or the target interval have the largest impact on the test outcomes, i.e., probability of acceptance for a given batch
mean and standard deviation. The presented information may provide potential users of the test with a set of tools for optimizing
the test characteristics for a particular product. 相似文献
103.
We have investigated the specificity of replication origin recognition by the initiator proteins of a set of six closely related Staphylococcus aureus plasmids, the pT181 family. These plasmids replicate by an asymmetric rolling-circle mechanism using plasmid-coded initiators that nick the replication origins and form a phosphotyrosine bond at the 5' nick terminus. Five of the plasmids are in different incompatibility groups and their initiator proteins do not cross-complement the cloned origins of any but their own plasmid. One pair is weakly incompatible and their initiator proteins and origins do cross-complement for replication in vivo. This pattern of cross-reactivity led to the prediction that the determinant of specificity would correspond to a homologously positioned set of six residues in the C-terminal domain of the protein, some 80 residues away from the active site tyrosine, that are divergent for all of the compatible plasmids and identical for the incompatible pair. Site-directed mutagenesis was used to exchange these six residues among three pairs of plasmids and these exchanges brought about the predicted switching of origin recognition specificity. Single substitution within this six residue set reduced or eliminated the activity of the protein but did not alter the origin recognition specificity. These six and flanking residues cannot form an amphipathic alpha-helix nor do they conform to the classical helix-turn-helix or other known DNA binding motifs. A novel type of interaction is suggested in which the protein binds to its recognition site, bends and melts the DNA, and causes or enhances the extrusion of an adjacent cruciform containing the nick site. This configuration would juxtapose the nicking target and the active site tyrosine residue and would unwind the highly G + C-rich replication origin. 相似文献
104.
105.
R P Novick 《Microbiological reviews》1969,33(2):210-263
106.
A catenated intermediate in plasmid replication 总被引:8,自引:0,他引:8
R P Novick K Smith R J Sheehy E Murphy 《Biochemical and biophysical research communications》1973,54(4):1460-1469
An intermediate in the replication of three different staphylococcal plasmids is a catenated mixed dimer consisting of an open circular and a closed circular monomer. The mixed dimer, having a buoyant density intermediate between that of circular duplex and relaxed DNA's in dye-buoyant density gradients is rapidly and transiently labeled and appears to be a precursor of mature closed circular monomers. Whether it is an obligatory intermediate or a step on an alternate replication pathway remains uncertain. 相似文献
107.
Background
Amino acid repeat-containing proteins have a broad range of functions and their identification is of relevance to many experimental biologists. In human-infective protozoan parasites (such as the Kinetoplastid and Plasmodium species), they are implicated in immune evasion and have been shown to influence virulence and pathogenicity. RepSeq is a new database of amino acid repeat-containing proteins found in lower eukaryotic pathogens. The RepSeq database is accessed via a web-based application which also provides links to related online tools and databases for further analyses. 相似文献108.
John Chen Nuria Carpena Nuria Quiles-Puchalt Geeta Ram Richard P Novick José R Penadés 《The ISME journal》2015,9(5):1260-1263
Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.Classically, transducing phages use the pac site-headful system for DNA packaging. Packaging is initiated on concatemeric post-replicative DNA by terminase cleavage at the sequence-specific pac site, a genome slightly longer than unit length is packaged, and packaging is completed by non-sequence-specific cleavage (reviewed in Rao and Feiss, 2008). Generalized transduction results from the initiation of packaging at pac site homologs in host chromosomal or plasmid DNA, and typically represents ∼1% of the total number of phage particles. In the alternative cos site mechanism packaging is also initiated on concatemeric post-replicative DNA by terminase cleavage at a sequence-specific (cos) site. Here, however, packaging is completed by terminase cleavage at the next cos site, generating a precise monomer with the cohesive termini used for subsequent circularization (Rao and Feiss, 2008). Although cos site homologs may exist in host DNA, it is exceedingly rare that two such sites would be appropriately spaced. Consequently, cos phages, of which lambda is the prototype, do not engage in generalized transduction. For this reason, cos-site phages have been preferred for possible phage therapy, since they would not introduce adventitious host DNA into target organisms.The Staphylococcus aureus pathogenicity islands (SaPIs) are the best-characterized members of the phage-inducible chromosomal island family of mobile genetic elements (MGEs; Novick et al., 2010). SaPIs are ∼15 kb mobile elements that encode virulence factors and are parasitic on specific temperate (helper) phages. Helper phage proteins are required to lift their repression (Tormo-Más et al., 2010, 2013), thereby initiating their excision, circularization and replication. Phage-induced lysis releases vast numbers of infectious SaPI particles, resulting in high frequencies of transfer. Most SaPI helper phages identified to date are pac phages, and many well-studied SaPIs are packaged by the headful mechanism (Ruzin et al., 2001; Ubeda et al., 2007). Recently, we have reported that some SaPIs, of which the prototype is SaPIbov5 (Viana et al., 2010), carry phage cos sequences in their genomes, and can be efficiently packaged and transferred by cos phages to S. aureus strains at high frequencies (Quiles-Puchalt et al., 2014). Here we show that this transfer extends to non-aureus staphylococci and to Listeria monocytogenes.Since the pac phages transfer SaPIs to non-aureus staphylococci and to the Gram-positive pathogen Listeria monocytogenes (Maiques et al., 2007; Chen and Novick, 2009), we reasoned that cos phages might also be capable of intra- and intergeneric transfer. We tested this with SaPIbov5, into which we had previously inserted a tetracycline resistance (tetM) marker to enable selection, and with lysogens of two helper cos phages, φ12 and φSLT, carrying SaPIbov5 (strains JP11010 and JP11194, respectively; Supplementary Table 1). The prophages in these strains were induced with mitomycin C, and the resulting lysates were adjusted to 1 μg ml−1 DNase I and RNase A, filter sterilized (0.2 μm pore), and tested for SaPI transfer with tetracycline selection, as previously described (Ubeda et al., 2008). To test for trans-specific or trans-generic transduction, coagulase-negative staphylococci species and L. monocytogenes strains were used as recipients for SaPIbov5 transfer, respectively, as previously described (Maiques et al., 2007; Chen and Novick, 2009). As shown in Figure 1 and Supplementary Table 2). In contrast, deletion of the SaPIbov5 cos site (strains JP11229 and JP11230) did not affect SaPI replication (Supplementary Figure 1), but completely eliminated SaPIbov5 transfer (Supplementary Table 2). The TerS protein is essential for φ12 and SaPIbov5 DNA packaging, but not for phage-mediated lysis (Quiles-Puchalt et al., 2014). As expected, this mutation abolished SaPIbov5 transfer (Open in a separate windowFigure 1(a) Map of SaPIbov5. Arrows represent the localization and orientation of ORFs greater than 50 amino acids in length. Rectangles represent the position of the ori (in purple) or cos (in red) sites. Positions of different primers described in the text are shown. (b) Amplimers generated for detection of SaPIbov5 in the different recipient strains. Supplementary Table 2 lists the sequence of the different primers used. The element was detected in S. epidermidis JP829 (Se-1), S. epidermidis JP830 (Se-2), L. monocytogenes SK1351 (Lm-1), L. monocytogenes EGDe (Lm-2), S. xylosus C2a (Sx) and S. aureus JP4226 (Sa).
Open in a separate windowAbbreviation: SAPI, Staphylococcus aureus pathogenicity island.aThe means of results from three independent experiments are shown. Variation was within ±5% in all cases.bNo. of transductants per ml induced culture.Because plaque formation is commonly used to determine phage host range, we next determined the ability of phages φ12 and φSLT to parasitize and form plaques on S. xylosus, S. epidermidis and L. monocytogenes strains. As shown in Supplementary Figure 2, phages φ12 and φSLT can parasitize and form plaques on their normal S. aureus hosts, but are completely unable to lyse the non-aureus strains. Therefore, as previously observed with pac phages (Chen and Novick, 2009), these results indicate that the overall host range of a cos phage may also be much wider if it includes infection without plaque formation.Previous studies have demonstrated pac phage-mediated transfer of MGEs between S. aureus and other bacterial species (Maiques et al., 2007; Chen and Novick, 2009; Uchiyama et al., 2014); however, no previous studies have described the natural intra- or intergeneric transfer of pathogenicity islands by cos phages. As bacterial pathogens become increasingly antibiotic resistant, lytic and poorly transducing phages, such as cos phages, have been proposed for phage therapy, on the grounds that they would not introduce adventitious host DNA into target organisms and that the phages are so restricted in host range that the resulting progeny are harmless and will not result in dysbiosis of human bacterial flora. Because plaque formation was once thought to determine the host range of a phage, the evolutionary impact of phages on bacterial strains they can transduce, but are unable to parasitize, has remained an unrecognized aspect of phage biology and pathogen evolution. Our results add to the recently recognized concept of ‘silent transfer'' of pathogenicity factors carried by MGEs (Maiques et al., 2007; Chen and Novick, 2009) by phages that cannot grow on the target organism. They extend this capability to cos phages, which have hitherto been unrecognized as mediators of natural genetic transfer.The potential for gene transfer of MGEs by this mechanism is limited by the ability of cos phages to adsorb and inject DNA into recipient strains, and also by the presence of suitable attachment sites in recipient genomes. However, since different bacterial genera express wall teichoic acid with similar structures, which can act as bacteriophage receptors governing the routes of horizontal gene transfer between major bacterial pathogens, horizontal gene transfer even across long phylogenetic distances is possible (Winstel et al., 2013). In addition, our previous results also demonstrated that the SaPI integrases have much lower sequence specificity than other typical integrases, and SaPIs readily integrate into alternative sites in the absence of the cognate attC site, such that any bacterium that can adsorb SaPI helper phage is a potential recipient (Chen and Novick, 2009). Thus, we anticipate that cos phages can have an important role in spreading MGEs carrying virulence and resistance genes. We also predict that cos sites will be found on many other MGEs, enabling cos phage-mediated transfer of any such element that can generate post-replicative concatemeric DNA. 相似文献
Table 1
Intra- and intergeneric SaPIbov5 transferaDonor strain | |||
---|---|---|---|
Phage | SaPI | Recipient strain | SaPI titreb |
φ12 | SaPIbov5 | S. aureus JP4226 | 8.3 × 104 |
S. epidermidis JP829 | 2.4 × 104 | ||
S. epidermidis JP830 | 4.7 × 104 | ||
L. monocytogenes SK1351 | 6.6 × 103 | ||
L. monocytogenes EGDe | 2.1 × 104 | ||
S. xylosus C2a | 7.1 × 104 | ||
φ12 | SaPIbov5 Δcos | S. aureus JP4226 | <10 |
S. epidermidis JP829 | <10 | ||
S. epidermidis JP830 | <10 | ||
L. monocytogenes SK1351 | <10 | ||
L. monocytogenes EGDe | <10 | ||
S. xylosus C2a | <10 | ||
φ12 ΔterS | SaPIbov5 | S. aureus JP4226 | <10 |
S. epidermidis JP829 | <10 | ||
S. epidermidis JP830 | <10 | ||
L. monocytogenes SK1351 | <10 | ||
L. monocytogenes EGDe | <10 | ||
S. xylosus C2a | <10 | ||
φSLT | SaPIbov5 | S. aureus JP4226 | 4.1 × 103 |
S. epidermidis JP829 | 1.1 × 103 | ||
S. epidermidis JP830 | 2.1 × 103 | ||
L. monocytogenes SK1351 | 3.6 × 102 | ||
L. monocytogenes EGDe | 3.1 × 103 | ||
S. xylosus C2a | 4.0 × 103 | ||
φSLT | SaPIbov5 Δcos | S. aureus JP4226 | <10 |
S. epidermidis JP829 | <10 | ||
S. epidermidis JP830 | <10 | ||
L. monocytogenes SK1351 | <10 | ||
L. monocytogenes EGDe | <10 | ||
S. xylosus C2a | <10 |
109.
Bloodstream infection with Staphylococcus aureus is common and can be fatal. However, virulence factors that contribute to lethality in S. aureus bloodstream infection are poorly defined. We discovered that LukED, a commonly overlooked leucotoxin, is critical for S. aureus bloodstream infection in mice. We also determined that LukED promotes S. aureus replication in vivo by directly killing phagocytes recruited to sites of haematogenously seeded tissue. Furthermore, we established that murine neutrophils are the primary target of LukED, as the greater virulence of wild-type S. aureus compared with a lukED mutant was abrogated by depleting neutrophils. The in vivo toxicity of LukED towards murine phagocytes is unique among S. aureus leucotoxins, implying its crucial role in pathogenesis. Moreover, the tropism of LukED for murine phagocytes highlights the utility of murine models to study LukED pathobiology, including development and testing of strategies to inhibit toxin activity and control bacterial infection. 相似文献
110.
Studies in the yeast Saccharomyces cerevisiae have shown that the inheritance of endoplasmic reticulum (ER), mitochondria, and vacuoles involves the capture of a tubular structure at the bud tip. Ptc1p, a serine/threonine phosphatase, has previously been shown to regulate mitochondrial inheritance by an unknown mechanism. Ptc1p regulates the high osmolarity glycerol mitogen-activated protein kinase (MAPK) pathway and has also been implicated in the cell wall integrity (CWI) MAPK pathway. Here we show that the loss of Ptc1p or the Ptc1p binding protein, Nbp2p, causes a prominent delay in the delivery of ER tubules to the periphery of daughter cells and results in a dramatic increase in the level of phosphorylated Slt2p, the MAPK in the CWI pathway. Either loss of Slt2p or inhibition of the CWI pathway by addition of sorbitol, suppresses the ER inheritance defect in the ptc1Delta and nbp2Delta mutants. Our findings indicate that Ptc1p and Nbp2p regulate ER inheritance through the CWI MAPK pathway by modulating the MAPK, Slt2p. 相似文献