UV hypersensitivity of yeast linear plasmids

The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. Killer yeasts secrete toxins lethal to sensitive yeasts but are immune to their own toxins. Since first discovered in Saccharomyces cerevisiae (2), killer strains have been isolated from several yeast genera, including Candida (46), Cryptococcus (10), Hanseniaspora (33), Kluyveromyces (14), Pichia (27), Torulopsis (7), Ustilago (30), Williopsis (45), and Zygosaccharomyces (32). Based on killing and immunity interactions among killer yeasts, killer phenotypes are classified into at least 10 groups (48) and the responsible genes may be carried on a chromosome (S. cerevisiae KHS, KHR, Williopsis mrakii) (11,12,21), on a double-stranded RNA (dsRNA) (S. cerevisiae K1, K2, K28, Ustilage maydis, Hanseniaspora uvarum) (5,15,22,35,49), or on a linear double-stranded DNA (dsDNA) (Kluyveromyces lactis, Pichia inositovora, Pichia acaciae) (14,16,44).Schwanniomyces occidentalis produces amylolytic enzymes, including ␣-amylase and glucoamylase (8). It is one of the few yeasts capable of completely hydrolyzing soluble starch. Moreover, it can grow to high cell mass by utilizing cheap starch from plants such as cassava, corn, potato, sorghum, and wheat as the carbon source (40). S. occidentalis has been used to produce ethanol and single cell protein from starch fermentation (19,42). S. occidentalis has no detectable extracellular proteases and can secrete large proteins (40). It also has an established transformation system and available inducible promoters, which could make it a commercially important system for the production of heterologous proteins (40). For example, endoglucanase D recently has been successfully expressed and secreted in this system (31).Wild killer yeasts sometimes contaminate cultures of industrial yeasts, resulting in lagging or stopped fermentation and poor product quality (39). To avoid such complications, the use of industrial killer strains as starters has been suggested (17). Commercially interesting killer strains for sake brewing, wine making, alcohol fermentation, and lager, beer, and ale production have been constructed (29,36,39,47). Furthermore, the W. mrakii mycocin expressed by Aspergillus niger can reduce silage and yogurt spoilage caused by yeasts (25).The killer phenotype of S. occidentalis has not been described previously. Thus, our objectives in this study were as follows: (i) to screen killer strains from S. occidentalis for a killer phenotype; (ii...

Section: Methodsmentioning

confidence: 99%

Isolation, Purification, and Characterization of a Killer Protein fromSchwanniomyces occidentalis

Chen¹,

Han²,

Jong

et al. 2000

“…73, 2007 P. ACACIAE pPac1-2 ORF4 ENCODES IMMUNITY 4377 (15) killer systems and probably also for the pPac1-2 homologous killer plasmid pWR1A from Debaryomyces robertsiae, in which a pPac1-2 ORF4 homologue is present (22). Thus, the immunity factor constitutes an autoselection system for the nonautonomous element, which can otherwise be accidentally lost (7,40).…”

mentioning

confidence: 99%

Pichia acaciae Killer System: Genetic Analysis of Toxin Immunity

Paluszynski

Klassen

Meinhardt

2007

Self Cite

The gene responsible for self-protection in the Pichia acaciae killer plasmid system was identified by heterologous expression in Saccharomyces cerevisiae. Resistance profiling and conditional toxin/immunity coexpression analysis revealed dose-independent protection by pPac1-2 ORF4 and intracellular interference with toxin function, suggesting toxin reinternalization in immune killer cells.Killer toxin production is a frequently realized intra-and interspecies strategy among yeasts to restrict the growth of competitors. While target cell killing is the common purpose, the structures of toxins, their mechanisms of action, and the organizations of encoding genes are rather diverse (25,32). The Kluyveromyces lactis and Pichia acaciae toxin systems depend on double-stranded DNA elements (8,16,40). The two species each harbor a pair of extranuclear linear plasmids, i.e., pGKL1 (8.9 kb) and pGKL2 (13.5 kb) (K. lactis) and pPac1-1 (13.6 kb) and pPac1-2 (6.8 kb) (P. acaciae) (1,8,37). The larger plasmids are autonomous elements displaying almost identical gene contents that include loci essential for cytoplasmic replication, transcription, and transcript modification (14,15). In contrast, the smaller plasmids carry structurally distinct toxin genes (32); these elements are nonautonomous and rely on the respective larger autonomous plasmid for extranuclear replication and transcription (29).The K. lactis toxin, termed zymocin, consists of three subunits encoded by the pGKL1-borne ORF2 (the ␣ and ␤ subunits) and ORF4 (the ␥ subunit) (36). Docking to the primary cell wall receptor chitin is facilitated by the ␣ subunit (12), and the remarkably hydrophobic ␤ subunit presumably assists in the uptake of the ␥ subunit, which is a tRNase (24,27,37).Like the K. lactis toxin zymocin, the P. acaciae toxin (PaT) comprises a heteromeric complex (26). The polypeptide encoded by pPac1-2 ORF1, possessing both chitin-binding and hydrophobic domains, is akin to the K. lactis counterparts; however, the intracellularly acting toxic subunit (encoded by pPac1-2 ORF2) is obviously unrelated to the K. lactis ␥ subunit (22).Zymocin action depends on the protein complex Elongator (4). Recently published data indicate that Elongator is instrumental in tRNA modification, i.e., in placing 5-methoxycarbonylmethyl (mcm 5 ) and 5-carbamoylmethyl (ncm 5 ) moieties on uridines at the wobble position (11, 24). Loss of Elongatordependent wobble nucleoside modifications in tRNA Glu , tRNA Lys , and tRNA Gln prevents recognition and cleavage by the zymocin ␥ subunit and confers exotoxin resistance (13, 24).For PaT function, in contrast, Elongator is not required, indicating the functional diversity of the toxins. Moreover, terminal toxin responses to PaT and zymocin differ: while the latter toxin arrests target cells in G 1 , PaT has been shown to induce S-phase arrest and DNA damage checkpoint induction followed by apoptotic cell death (20,21). It has been shown that self-protection from zymocin is mediated by pGKL1 ORF3; however, in the pPac killer system...

“…Since yeast cells carrying linear cytoplasmic plasmids can efficiently be cured by UV irradiation (21,22), approximately 2 ϫ 10 3 cells of Saccharomyces cerevisiae 301 grown overnight in YPD at 30°C were plated on YPD agar and exposed to UV light essentially as described previously (23,24). Following incubation for 24 h at 30°C, arising colonies of S. cerevisiae 301 ⌬pGKL were analyzed for the presence of linear elements by gel electrophoresis and Southern analysis.…”

Section: Methodsmentioning

confidence: 99%

Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

Wemhoff

Klassen

Meinhardt

2014

Self Cite

Zymocin is a Kluyveromyces lactis protein toxin composed of ␣␤␥ subunits encoded by the cytoplasmic virus-like element k1 and functions by ␣␤-assisted delivery of the anticodon nuclease (ACNase) ␥ into target cells. The toxin binds to cells' chitin and exhibits chitinase activity in vitro that might be important during ␥ import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either ␣␤ (k1ORF2) or ␥ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of ␣'s chitinase domain and the sole cysteine residue of ␤ (Cys250). Since ␤␥ are reportedly disulfide linked, the requirement for the conserved ␥ C231 was probed. Toxicity of intracellularly expressed ␥ C231A indicated no major defect in ACNase activity, while complementation of k1⌬ORF4 by ␥ C231A was lost, consistent with a role of ␤ C250 and ␥ C231 in zymocin assembly. To test the capability of ␣␤ to carry alternative cargos, the heterologous ACNase from Pichia acaciae (P. acaciae Orf2 [PaOrf2]) was expressed, along with its immunity gene, in k1⌬ORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1⌬ORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying ␣␤'s capability to deliver other cargo proteins into target cells.T he protein toxin zymocin, produced by the yeast Kluyveromyces lactis, was identified as the first known anticodon nuclease toxin from a eukaryote, selectively cleaving tRNA within the anticodon loop due to highly specific anticodon nuclease (ACNase) activity (1). Zymocin production is correlated with the presence of a pair of linear cytoplasmic genetic double-stranded DNA (ds-DNA) elements, termed pGKL1 (k1 in short) and pGKL2 (k2 in short) (2). The larger k2 is required for cytoplasmic maintenance of k1, which encodes the toxin as well as an immunity determinant (2, 3). k2 provides essential functions for cytoplasmic replication, transcription, and transcript processing, and several of these components show phylogenetic proximity to viruses (4, 5). Since the proposed mode of replication via protein priming is typically found in viruses, the cytoplasmic linear dsDNA elements were termed virus-like elements (VLE) (6). The zymocin toxin is a heterotrimeric ␣␤␥ complex, the smallest subunit of which (␥) exhibits the cytotoxic ACNase activity (1, 7-9). The ␣ and ␤ subunits are generated from the 128-kDa k1ORF2 gene product, which is first translocated to the endoplasmic reticulum (ER), where it becomes glycosylated and cleaved by signal peptidase and then travels to the Golgi apparatus, where it is processed by the Kex1/2 endopeptidase internally at the N-terminal end to produce mature ␣ (99-kDa) and ␤ (30-kDa) subunits (7, ...