Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins.

Organelle acidification plays a demonstrable role in intracellular protein processing, transport, and sorting in animal cells. We investigated the relationship between acidification and protein sorting in yeast by treating yeast cells with ammonium chloride and found that this lysosomotropic agent caused the mislocalization of a substantial fraction of the newly synthesized vacuolar (lysosomal) enzyme proteinase A (PrA) to the cell surface. We have also determined that a subset of the vpl mutants, which are deficient in sorting of vacuolar proteins (Rothman, J. H., and T. H. Stevens. 1986. Cell. 47:1041-1051; Rothman, J. H., I. Howald, and T. H. Stevens. EMBO [Eur. Mol. Biol. Organ.] J. In press), failed to accumulate the lysosomotropic fluorescent dye quinacrine within their vacuoles, mimicking the phenotype of wild-type cells treated with ammonium. The acidification defect of vpl3 and vpl6 mutants correlated with a marked deficiency in vacuolar ATPase activity, diminished levels of two immunoreactive subunits of the protontranslocating ATPase (H+-ATPase) in purified vacuolar membranes, and accumulation of the intracellular portion of PrA as the precursor species. Therefore, some of the VPL genes are required for the normal function of the yeast vacuolar H+-ATPase complex and may encode either subunits of the enzyme or components required for its assembly and targeting. Collectively, these findings implicate a critical role for acidification in vacuolar protein sorting and zymogen activation in yeast, and suggest that components of the yeast vacuolar acidification system may be identified by examining mutants defective in sorting of vacuolar proteins.

Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase.

The TFP1 gene of the yeast Saccharomyces cerevisiae encodes two proteins: the 69-kilodalton (kD) catalytic subunit of the vacuolar proton-translocating adenosine triphosphatase (H(+)-ATPase) and a 50-kD protein. The 69-kD subunit is encoded by the 5' and 3' thirds of the TFP1 coding region, whereas the 50-kD protein is encoded by the central third. Evidence is presented that both the 69-kD and 50-kD proteins are obtained from a single translation product that is cleaved to release the 50-kD protein and spliced to form the 69-kD subunit.

Protein targeting to the yeast vacuole.

Mutational and gene fusion studies have identified localization signals that target proteins to the yeast lysosome-like vacuole. Genetic analyses have also identified groups of genes (VPS and PEP) whose products are required for recognition of these signals, and sorting and transport of proteins to the vacuole. One of the components involved in protein sorting has been shown to be the vacuolar H+-ATPase, presumably via its role in vacuolar acidification.

Characterization of rco-1 of Neurospora crassa, a pleiotropic gene affecting growth and development that encodes a homolog of Tup1 of Saccharomyces cerevisiae.

The filamentous fungus Neurospora crassa undergoes a well-defined developmental program, conidiation, that culminates in the production of numerous asexual spores, conidia. Several cloned genes, including con-10, are expressed during conidiation but not during mycelial growth. Using a previously described selection strategy, we isolated mutants that express con-10 during mycelial growth. Selection was based on expression of an integrated DNA fragment containing the con-10 promoter-regulatory region followed by the initial segment of the con-10 open reading frame fused in frame with the bacterial hygromycin B phosphotransferase structural gene (con10'-'hph). Resistance to hygromycin results from mutational alterations that allow mycelial expression of the con-10'-'hph gene fusion. A set of drug-resistant mutants were isolated; several of these had abnormal conidiation phenotypes and were trans-acting, i.e., they allowed mycelial expression of the endogenous con-10 gene. Four of these had alterations at a single locus, designated rco-1 (regulation of conidiation). Strains with the rco-1 mutant alleles were aconidial, female sterile, had reduced growth rates, and formed hyphae that coiled in a counterclockwise direction, opposite that of the wild type. The four rco-1 mutants had distinct conidiation morphologies, suggesting that conidiation was blocked at different stages. Wild-type rco-1 was cloned by a novel procedure employing heterokaryon-assisted transformation and ligation-mediated PCR. The predicted RCO1 polypeptide is a homolog of Tup1 of Saccharomyces cerevisiae, a multidomain protein that mediates transcriptional repression of genes concerned with a variety of processes. Like tup1 mutants, null mutants of rco-1 are viable and pleiotropic. A promoter element was identified that could be responsible for RCO1-mediated vegetative repression of con-10 and other conidiation genes.

Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase.

Vacuolar acidification has been proposed to play a key role in a number of cellular processes, including protein sorting, zymogen activation, and maintenance of intracellular pH. We investigated the significance of vacuolar acidification by cloning and mutagenizing the gene for the yeast vacuolar proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying a vat2 null allele were viable; however, these cells were severely defective for growth in medium buffered at neutral pH. Vacuoles isolated from cells bearing the vat2 null allele were completely devoid of vacuolar ATPase activity. The pH of the vacuolar lumen of cells bearing the vat2 mutation was 7.1, compared with the wild-type pH of 6.1, as determined by a flow cytometric pH assay. These results indicate that the vacuolar proton-translocating ATPase complex is essential for vacuolar acidification and that the low-pH state of the vacuole is crucial for normal growth. The vacuolar acidification-defective vat2 mutant exhibited normal zymogen activation but displayed a minor defect in vacuolar protein sorting.

The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities.

The TaqMan LS-50B PCR Detection System facilitates the automated and direct detection of polymerase chain reaction (PCR) products. The system employs the 5' nuclease activity of Taq DNA polymerase to hydrolyse a Salmonella specific internal fluorogenic probe for monitoring the amplification of a 287-bp region of the Salmonella invA gene. Using the fluorogenic 5' nuclease assay, 164 Salmonella strains representing all the subspecies of Salmonella enterica were detected while over 50 non-Salmonella strains were not detected. The detection limit of the assay was two colony forming units (cfu) per PCR reaction when a pure culture of S. typhimurium was used. Six protocols for the isolation of PCR-amplifiable DNA were evaluated using chicken carcass rinses, ground beef, ground pork and raw milk contaminated with Salmonella. Of the six DNA isolation protocols, a modified sample preparation protocol using the EnviroAmp kit was chosen for subsequent studies because it was reliable, easy to use and efficient for the isolation of PCR-amplifiable DNA from foods. A detection limit of 3-7 cfu per PCR reaction was obtained using food samples that were pre-enriched overnight and then inoculated with Salmonella. The detection limit was below 3 cfu/25 g or 25 ml when foods inoculated with Salmonella were pre-enriched overnight. Naturally contaminated foods (50 chicken carcass rinses and 60 raw milk samples) were examined using both the fluorogenic 5' nuclease assay and a modified semi-solid rappaport vassiliadis (MSRV) culture method. Thirty four of the 110 samples tested were Salmonella-positive and 74 were Salmonella-negative by both the 5' nuclease assay and the MSRV method. Two samples were Salmonella-positive by the 5' nuclease assay, but negative by the MSRV method. The correlation between the 5' nuclease assay and the MSRV method was over 98%.

Effects of the tol mutation on allelic interactions at het loci in Neurospora crassa.

A mutant at the tol locus of Neurospora crassa can suppress heterokaryon (vegetative) incompatibility associated with differences at the mating-type locus. We tested the ability of this allele to suppress the vegetative incompatibility reactions that can occur when strains differ at one of nine het loci (het-C, -D, -E, -5, -6, -7, -8, -9, and -10). We found no cases in which the tol mutant suppresses a heteroallelic het locus interaction. Thus, the interaction(s) that leads to vegetative incompatibility because of differences at the mating-type locus is distinct from the interaction(s) that leads to vegetative incompatibility because of differences at any of these nine het loci.

A dominant selectable marker that is meiotically stable in Neurospora crassa: the amdS gene of Aspergillus nidulans.

When Neurospora crassa is transformed using a Neurospora gene as the selectable marker, the vegetatively stable transformants obtained cannot be used successfully in a cross because the selectable marker will be inactivated by the process of RIP (repeat-induced point mutation). Introduction of the acetamidase-encoding gene amdS of Aspergillus nidulans into N. crassa by transformation yielded transformants that would grow in minimal medium containing acetamide as a sole nitrogen source. In mitotically stable transformants containing a single copy of the amdS gene, the capacity to utilize acetamide as a sole nitrogen source was maintained in the progeny of a sexual cross. Therefore, the A. nidulans amdS gene is an appropriate dominant selectable marker for use in transformation analyses with N. crassa in which sexual crosses will be subsequently performed.

Biochemical characterization of the yeast vacuolar H(+)-ATPase.

The yeast vacuolar proton-translocating ATPase was isolated by two different methods. A previously reported purification of the enzyme (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095) was repeated. This procedure consisted of isolation of vacuoles, solubilization with the zwitterionic detergent ZW3-14, and glycerol gradient centrifugation of the solubilized vacuoles. The fraction with the highest specific activity (11 mumol of ATP hydrolyzed mg-1 min-1) included eight polypeptides of apparent molecular masses of 100, 69, 60, 42, 36, 32, 27, and 17 kDa, suggesting that the enzyme may be more complex than the three-subunit composition proposed from the original purification. The 69-kDa polypeptide was recognized by antisera against the catalytic subunits of two other vacuolar ATPases and labeled with the ATP analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, indicating that it contains all or part of the catalytic site. A monoclonal antibody was prepared against this subunit. Under nondenaturing conditions, the antibody immunoprecipitated eight polypeptides, of the same molecular masses as those seen in the glycerol gradient fraction, from solubilized vacuolar vesicles. All eight of these polypeptides are therefore good candidates for being genuine subunits of the enzyme. The structure and function of the yeast vacuolar H+-ATPase were further characterized by examining the inhibition of ATPase activity by KNO3. In the presence of 5 mM MgATP, 100 mM KNO3 inhibited 71% of the ATPase activity of vacuolar vesicles, and the 69- and 60-kDa subunits (and possibly the 42-kDa subunit) were removed from the vacuolar membrane to a similar extent. At concentrations of less than 200 mM KNO3, the stripping of the ATPase subunits and the inhibition of ATPase activity were dependent on the presence of MgATP, suggesting that this is a conformation-specific disassembly of the enzyme. The yeast vacuolar H+-ATPase is a multisubunit enzyme, consisting of a combination of peripheral and integral membrane subunits. Its structure and subunit composition are very similar to other vacuolar ATPase, and it shares some characteristics with the F1F0-ATPases.

Protein sorting in yeast: the role of the vacuolar proton-translocating ATPase.

We are investigating the physiological roles of organelle acidification in yeast by two different approaches. First, we have identified two mutants which are defective in acidification of the yeast lysosome-like vacuole from among a collection of mutants which mis-sort soluble vacuolar proteins to the cell surface. These mutants have been helpful in identifying other cellular functions linked to acidification, such as the activation of vacuolar zymogens. We have complemented this classical genetic approach to acidification with direct biochemical and reverse genetic studies on the yeast vacuolar proton-translocating ATPase (H+-ATPase), the enzyme responsible for vacuolar network acidification. Our biochemical characterization of this enzyme indicates that it is a multisubunit complex with many structural similarities to other vacuolar H+-ATPases. Like the other vacuolar H+-ATPases characterized, it also shares some structural features with the F1F0-type ATPases of mitochondria, chloroplasts, and Escherichia coli. We are currently cloning the genes for the subunits of the yeast vacuolar H+-ATPase. Mutagenesis of the cloned genes will allow us to determine the phenotype of yeast cells expressing a vacuolar H+-ATPase altered in well controlled ways. We are also beginning to investigate how the subunits of the vacuolar H+-ATPase are assembled into the enzyme complex and targeted to their proper cellular location.

A ras homologue of Neurospora crassa regulates morphology.

In order to study the role of signal transduction pathways in the regulation of morphology in Neurospora crassa, we cloned and characterized a ras homologue, termed NC-ras2. The predicted protein product of this gene is composed of 229 amino acid residues and contains all the consensus sequences shared by the ras protein family. The gene is located in linkage group V. An NC-ras2 disruptant showed morphological characteristics very similar to those of the smco7 mutant, which also maps to linkage group V. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in the NC-ras2 gene, which is predicted to result in the truncation of the protein product. Introduction into the smco7 mutant of an NC-ras2 clone yielded stable transformants with a wild-type phenotype. The smco7 mutant exhibited very slow hyphal growth and the rate of conidial formation was approximately one two-hundredth of wild type. The smco7 mutation causes both the changes in the pattern of hyphal growth and the defects in cell wall synthesis. Both the diameter and the length of the apical compartment were shorter in the hyphae of the smco7 mutant. These results suggest that NC-ras2 is identical to smco7, and that the signal transduction pathway mediated by the NC-ras2 protein regulates the apical growth of hyphae, cell wall synthesis, and conidial formation in N. crassa.

A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef.

A detection system based on the PCR has been developed for Escherichia coli strains which harbor the Shiga-like toxin genes. This quantitative detection system involves the 5'-->3' nuclease activity of Thermus aquaticus DNA polymerase, which cleaves an internal oligonucleotide probe that has been labeled with both a fluorescent reporter dye (6-carboxy-fluorescein [FAM]) and a quencher dye (6-carboxytetramethyl-rhodamine [TAMRA]). Parameters which affected the performance of the assay included primer probe distance, probe concentration, and probe target sequence homology. The optimized assay format includes two PCR primers that generate a 497-bp amplicon specific for the sltI gene with the fluorogenic probe located 19 bp from the upstream PCR primer. When the distance between the upstream PCR primer and the probe was reduced from 190 to 19 bp, delta RQ values increased from approximately 1.5 to 3.0. The delta RQ for Shiga-like toxin I probe 102 reached a maximum of 4.15 at concentrations between 25 and 50 nM. The assay is sensitive and can detect approximately 10 +/- 5 CFU per PCR. As few as 0.5 CFU of Shiga-like toxin I-producing E. coli per g could be detected in ground beef with only 12 h of enrichment in modified E. coli broth.

Photoregulation of cot-1, a kinase-encoding gene involved in hyphal growth in Neurospora crassa.

Blue light plays a key role as an environmental signal in the regulation of growth and development of fungi and plants. Here we demonstrate that in Neurospora crassa hyphae branch more frequently in cultures grown in light. Previous studies had identified cot-1 as a gene that controls apical hyphal cell elongation. In the cot-1 mutant, cessation of elongation is accompanied by hyperbranching. Here we demonstrate that the cot-1 gene encodes two transcript species of about 2100 nt (cot-1 (s)) and about 2400 nt (cot-1 (l)) in length and that the ratio of both transcript species abundance is photoregulated. The origin of the difference between cot-1 (l) and cot-1 (s) was localized to the 5' end of the cot-1 transcripts, suggesting that two COT1 isoforms with different activities may be formed. Both light effects, on branching and on cot-1 expression, were dependent on functional wc-1 and wc-2 gene products. In addition to light, L-sorbose comprises another environmental cue that controls hyphal branching in N. crassa. In the presence of L-sorbose, photoregulation of cot-1 was blocked, suggesting the involvement of alternative and potentially interdependent signaling pathways for the regulation of hyphal elongation/branching.

PCR-based DNA amplification and presumptive detection of Escherichia coli O157:H7 with an internal fluorogenic probe and the 5' nuclease (TaqMan) assay.

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC) eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5' nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5' nuclease assay system was sufficient to correctly identify all E. coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed, eaeA-targeted 5' nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coli O157:H7 when >/=10(3) CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when >/=10(4) CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to >/=10(2) CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when >/=10(4) CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed, eaeA-targeted 5' nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.