[Abstract unavailable

During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

Tub4p is a novel tubulin found in Saccharomyces cerevisiae. It most resembles gamma‐tubulin and, like it, is localized to the yeast microtubule organizing centre, the spindle pole body (SPB). In this paper we report the identification of SPC98 as a dosage‐dependent suppressor of the conditional lethal tub4–1 allele. SPC98 encodes an SPB component of 98 kDa which is identical to the previously described 90 kDa SPB protein. Strong overexpression of SPC98 is toxic, causing cells to arrest with a large bud, defective microtubule structures, undivided nucleus and replicated DNA. The toxicity of SPC98 overexpression was relieved by co‐overexpression of TUB4. Further evidence for an interaction between Tub4p and Spc98p came from the synthetic toxicity of tub4–1 and spc98–1 alleles, the dosage‐dependent suppression of spc98–4 by TUB4, the binding of Tub4p to Spc98p in the two‐hybrid system and the co‐immunoprecipitation of Tub4p and Spc98p. In addition, Spc98–1p is defective in its interaction with Tub4p in the two‐hybrid system. We suggest a model in which Tub4p and Spc98p form a complex involved in microtubule organization by the SPB.

Do cargo proteins influence this process? Recent work * Department of Molecular and Cell Biology and on the exchange of proteins between the endoplasmic † Howard Hughes Medical Institute reticulum (ER) and the Golgi apparatus in Saccharo-University of California myces cerevisiae and higher eukaryotes suggests that Berkeley, California 94720 all three requirements for functional vesicle budding could be regulated in a single mechanistic step, the formation of a "priming complex" of a small GTPase, a Transport vesicles carry lumenal and membrane cargo membrane protein, and a coat subunit (Figure 1). proteins between secretory organelles in eukaryotic To Begin: Recruit a GTPase cells. To make such a vesicle, cytosolic coat proteins to the Donor Membrane assemble on a donor membrane and deform it into a COPII (coat protein complex II)-coated vesicles transbud. Cargo proteins are sorted into the originating vesiport proteins from the ER to the Golgi. The COPII coat cle, which then separates from the donor membrane, consists of the small GTPase Sar1p and the heteroditravels a certain distance, and finally fuses with the acmeric protein complexes Sec23/24p and Sec13/31p ceptor organelle. (Barlowe, 1998). These five proteins are necessary and At the time of vesicle budding, three specific requiresufficient to produce COPII vesicles from ER microments have to be fulfilled in order to produce a functional somes (Barlowe et al., 1994) and from chemically defined vesicle. First, different vesicular transport processes are liposomes (Matsuoka et al., 1998). mediated by different coat protein complexes, and Proteins can also be recycled to the ER from the cistherefore a donor membrane must attract the correct Golgi via a backward, or retrograde, route. Such proteins species of cytosolic coat proteins. Second, a transport include ER residents that have escaped ER retention, vesicle needs to be equipped with certain membrane and functional components of COPII ("anterograde") proteins that have essential tasks at a later stage. vesicles that return to participate in another round of v-SNAREs, for example, are required for fusion with the COPII vesicle formation. Retrograde vesicles are coated acceptor membrane (reviewed by Nichols and Pelham, with the COPI coat (Cosson and Letourneur, 1997; 1998). These proteins must be included into the vesicles Gaynor et al., 1998), which consists of the small GTPase with high fidelity. And third, cargo proteins have to be recognized and included into the originating vesicles.

We examined the kinetics and the nature of the association of two herpes simplex virus proteins, the major DNA-binding protein (ICP8) and the major capsid protein (ICP5), with the nuclei of infected cells. We defined a series of stages in the association of the ICP8 protein with the cell nucleus. (i) Immediately after synthesis, the protein was found in the cytoplasmic fraction but associated rapidly with the crude nuclear fraction. (ii) The initial association of ICP8 with the crude nuclear fraction was detergent sensitive but DNase resistant, and, thus, the protein was either bound to structures attached to the outside of the nucleus and had not penetrated the nuclear envelope or was loosely bound in the nucleus. (iii) At intermediate times, a low level of an intermediate form was observed in which the association of ICP8 with the nuclear fraction was resistant to both detergent and DNase treatment. The protein may be bound to the nuclear matrix at this stage. Inhibition of viral DNA synthesis caused the DNA-binding protein to accumulate in this form. (iv) At late times during the chase period, the association of ICP8 with the cell nucleus was resistant to detergent treatment but sensitive to DNase treatment. Our results argue that at this stage ICP8 was bound to viral DNA. Thus, nuclear association of the DNA-binding protein did not require viral DNA replication. More important is the observation that there is a series of stages in the nuclear association of this protein, and, thus, there may be a succession of binding sites for this protein in the cell during its movement to its final site of action in the nucleus. The major capsid protein showed some similar stages of association with the cell nucleus but the initial association with the nucleus followed a lag period. Its early association with the crude nuclear fraction was also detergent sensitive but was resistant to detergent treatment at later times. Its association with the cell nucleus was almost completely resistant to DNase treatment at all times. Inhibition of viral DNA replication blocked the nuclear transport of this protein. Thus, these two viral proteins share some stages in nuclear transport, although their requirements for nuclear association are different.