Search

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

Bacterial mechanosensitive channels gate in response to membrane tension, driven by shifts in environmental osmolarity. The mechanosensitive channels of small conductance (MscS) and large conductance (MscL) from Escherichia coli (Ec) gate in response to mechanical force applied to the membrane. Ec-MscS is the foundational member of the MscS superfamily of ion channels, a diverse family with at least fifteen subfamilies identified by homology to the pore lining helix of Ec-MscS, as well as significant diversity on the N- and C-termini. The MscL family of channels are homologous to Ec-MscL. In a rhizosphere associated bacterium, Paraburkholderia graminis C4D1M, mechanosensitive channels are essential for cell survival during changing osmotic environments such as a rainstorm. Utilizing bioinformatics, we predicted six MscS superfamily members and a single MscL homologue. The MscS superfamily members fall into at least three subfamilies: bacterial cyclic nucleotide gated, multi-TM, and extended N-terminus. Osmotic downshock experiments show that wildtype P. graminis cells contain a survival mechanism that prevents cell lysis in response to hypoosmotic shock. To determine if this rescue is due to mechanosensitive channels, we developed a method to create giant spheroplasts of P. graminis to explore the single channel response to applied mechanical tension. Patch clamp electrophysiology on these spheroplasts shows two unique conductances: MscL-like and MscS-like. These conductances are due to likely three unique proteins. This indicates that channels that gate in response to mechanical tension are present in the membrane. Here, we report the first single channel evidence of mechanosensitive ion channels from P. graminis membranes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The treatment of hospital- and community-associated infections by methicillin-resistant Staphylococcus aureus (MRSA) is a perpetual challenge. This Gram-positive bacterium is resistant specifically to β-lactam antibiotics, and generally to many other antibacterial agents. Its resistance mechanisms to β-lactam antibiotics are activated only when the bacterium encounters a β-lactam. This activation is regulated by the transmembrane sensor/signal transducer proteins BlaR1 and MecR1. Neither the transmembrane/metalloprotease domain, nor the complete MecR1 and BlaR1 proteins, are isolatable for mechanistic study. Here we propose a model for full-length MecR1 based on homology modeling, residue coevolution data, a new extensive experimental mapping of transmembrane topology, partial structures, molecular simulations, and available NMR data. Our model defines the metalloprotease domain as a hydrophilic transmembrane chamber effectively sealed by the apo-sensor domain. It proposes that the amphipathic helices inserted into the gluzincin domain constitute the route for transmission of the β-lactam-binding event in the extracellular sensor domain, to the intracellular and membrane-embedded zinc-containing active site. From here, we discuss possible routes for subsequent activation of proteolytic action. This study provides the first coherent model of the structure of MecR1, opening routes for future functional investigations on how β-lactam binding culminates in the proteolytic degradation of MecI.

We compared the gene expression levels of the blue-light-responsive genes, appA (encoding photosynthesis promoting protein AppA), ppsR (encoding photosynthesis suppressing protein PpsR), and EL368 (encoding a blue-light-activated histidine kinase with a light, oxygen, or voltage domain) between aerobic and anaerobic conditions in spheroplasts of the aerobic photosynthetic bacterium Erythrobacter litoralis. The spheroplasts conducted photosynthesis under red light but not under blue light. All three blue-light-responsive genes showed higher expression under aerobic conditions than under anaerobic conditions under blue light. In contrast, under red light, although the expression level of appA was higher in the presence of oxygen than in the absence of oxygen, the expression levels of ppsR and EL368 were similar in the presence and absence of oxygen. Our findings demonstrate that the expression of blue-light-responsive genes is strongly affected by oxygen in E. litoralis spheroplasts.

Gene replacement is one of the most essential approaches in construction of the genetically modified yeast strains. However, the fidelity of gene targeting and the effort needed for construction of a particular strain can vary significantly. We investigated the influence of two important factors-the choice of the transformation method and the design of the transforming DNA fragment, which can vary in overall length (including flanking regions and selectable marker) compared to the length of the targeted region in the genome. Gene replacement fidelity was determined in several assays using electroporation and spheroplast transformation, and compared with our previous results obtained by lithium acetate. We have demonstrated clearly that gene targeting fidelity depends on the transformation protocol, being highest for lithium acetate method. In contrast, lower fidelity was observed with electroporation and spheroplast transformation. Additionally, the fidelity also depends on a design of the transformation assay, since a higher overall length ratio of the transforming DNA and targeted region results in higher fidelity. Moreover, the karyotype analysis of the aberrant transformants by qPCR demonstrates that gene targeting can result in diploidisation of haploid strains, most likely via targeted chromosome duplication followed by subsequent duplication of other chromosomes., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A Gram-stain-negative, vibrio-shaped, spheroplast-forming, motile, aerobic bacterium was isolated from the soil of a salt desert in Kutch, Gujarat, India. The strain, designated JC232(T), was oxidase- and catalase-positive. 16S rRNA gene sequence analysis indicated that strain JC232(T) was a member of the genus Caenispirillum and was related most closely to Caenispirillum salinarum AK4(T) (98.9% similarity) and Caenispirillum bisanense K92(T) (96.8%). Genome relatedness based on DNA-DNA hybridization of strain JC232(T) with the type strains of closely related species was less than 40%. The DNA G+C content of strain JC232(T) was 70 mol%. Phosphatidylglycerol, phosphatidylethanolamine, phosphotidylcholine, diphosphatidylglycerol, two unidentified amino lipids (AL1 and 2) and four unidentified lipids (UL1-4) were the polar lipids of strain JC232(T). C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and C16 : 1ω7c/C16 : 1ω6c were the major (>15%) fatty acids of strain JC232(T), with minor amounts of C12 : 0, C14 : 0 3-OH/iso-C16 : 0 I, C18 : 1 2-OH, C18 : 0, C16 : 0 3-OH and C19 : 0cycloω8c. Although strain JC232(T) shared the predominant ubiquinone system (Q10) with the type strains of C. salinarum and C. bisanense, it differed from the latter in polar lipid profile, NaCl growth range and other phenotypic/physiological properties. On the basis of morphological, physiological, genotypic, phylogenetic and chemotaxonomic analyses, strain JC232(T) is considered to represent a novel species of the genus Caenispirillum, for which the name Caenispirillum deserti sp. nov. is proposed. The type strain is JC232(T) ( = KCTC 42064(T) = NBRC 110150(T)).

Phenotypical analysis of the lipid interacting residues in the closed state of the mechanosensitive channel of small conductance (MscS) from Escherichia coli (E. coli) has previously shown that these residues are critical for channel function. In the closed state, mutation of individual hydrophobic lipid lining residues to alanine, thus reducing the hydrophobicity, resulted in phenotypic changes that were observable using in vivo assays. Here, in an analogous set of experiments, we identify eleven residues in the first transmembrane domain of the open state of MscS that interact with the lipid bilayer. Each of these residues was mutated to alanine and leucine to modulate their hydrophobic interaction with the lipid tail-groups in the open state. The effects of these changes on channel function were analyzed using in vivo bacterial assays and patch clamp electrophysiology. Mutant channels were found to be functionally indistinguishable from wildtype MscS. Thus, mutation of open-state lipid interacting residues does not differentially stabilize or destabilize the open, closed, intermediate, or transition states of MscS. Based on these results and other data from the literature, we propose a new gating paradigm for MscS where MscS acts as a "Jack-In-The-Box" with the intrinsic bilayer lateral pressure holding the channel in the closed state. In this model, upon application of extrinsic tension the channel springs into the open state due to relief of the intrinsic lipid bilayer pressure., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We generated spheroplasts from Escherichia coli carrying a broad-host-range plasmid. In the presence of penicillin, the spheroplasts did not divide but grew and enlarged in marine broth, whereas, in the absence of penicillin, they elongated. We quantified cellular DNA at different time points by using real-time quantitative PCR. Both chromosomal and plasmid DNA had replicated during spheroplast growth not only in the absence but also in the presence of penicillin. Thus, plasmid DNA and chromosomal DNA replication might be regulated synchronously during the growth of spheroplasts.

Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed.

Introducing human genes into mice offers the opportunity to analyze their in vivo function or to obtain therapeutic molecules. For proper gene regulation, or in case of multigene families, megabase (Mb)-sized DNA fragments often have to be used. Yeast artificial chromosome (YAC)-mediated transgenesis is irreplaceable for this purpose, because alternative methods such as the use of bacterial artificial chromosomes (BACs) cannot introduce DNA fragments larger than 500 kb into the mouse germ line. However, YAC libraries often contain only partial gene loci. Time-consuming reconstruction of YACs, genetic instability and the difficulty in obtaining intact YAC DNA above a certain size impede the generation of humanized mice. Here we describe how to reconstruct YACs containing Mb-sized human DNA, such as the T cell receptor-α (TRA) gene locus, thus facilitating the introduction of large DNA fragments into the mouse germ line. Fusion of YAC-containing yeast and embryonic stem (ES) cells avoids the need for YAC DNA purification. These ES cells are then used to stably introduce the functional TRA gene locus into the mouse germ line. The protocol takes ∼1 year to complete, from reconstruction of the entire TRA gene locus from YACs containing partial but overlapping TRA regions to germline transmission of the YAC.

Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future.

Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown. To address this question, we treated Escherichia coli with lysozyme to remove the peptidoglycan wall while leaving intact the inner and outer membranes and periplasm. The resulting lysozyme-induced (LI) spheroplasts recovered a rod shape after four to six generations. Recovery proceeded via a series of cell divisions that produced misshapen and branched intermediates before later progeny assumed a normal rod shape. Importantly, mutants defective in mounting the Rcs stress response and those lacking penicillin binding protein 1B (PBP1B) or LpoB could not divide or recover their cell shape but instead enlarged until they lysed. LI spheroplasts from mutants lacking the Lpp lipoprotein or PBP6 produced spherical daughter cells that did not recover a normal rod shape or that did so only after a significant delay. Thus, to regenerate normal morphology de novo, E. coli must supplement the classic FtsZ- and MreBCD-directed cell wall systems with activities that are otherwise dispensable for growth under normal laboratory conditions. The existence of these auxiliary mechanisms implies that they may be required for survival in natural environments, where bacterial walls can be damaged extensively or removed altogether.

Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.

Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50-5 μg/ml), were shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), the dimeric cyanine dye TOTO-1, or fluorescent DNA-binding protein appeared as cloud-like structures, in the absence of phase contrast. Because UV-irradiation disrupted the DAPI-stained nucleoids within 5-10 s, they were imaged by time-lapse microscopy with exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spheroplasts and minimally exposed to UV (<2 s) was on average ∼42 μm(3). Lysozyme at concentrations above 1 μg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20-200 μg/ml) and sodium dodecyl sulfate (SDS; 0.001-0.01%) caused a twofold volume increase and showed a granular nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 μM ethidium bromide, but not with chloroquine. While DNase (1 μg/ml) caused a rapid disruption of the nucleoids, RNase (0.1-400 μg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after prolonged (>4 s) UV irradiation. We interpret the measured volume in terms of a physical model of the nucleoid viewed as a branched DNA supercoil crosslinked by adhering proteins into a homogeneous network., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Chromatin is dynamically regulated, and proteomic analysis of its composition can provide important information about chromatin functional components. Many DNA replication proteins for example bind chromatin at specific times during the cell cycle. Proteomic investigation can also be used to characterize changes in chromatin composition in response to perturbations such as DNA damage, while useful information is obtained by testing the effects on chromatin composition of mutations in chromosome stability pathways. We have successfully used the method of stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomic analysis of normal and pathological changes to yeast chromatin. Here we describe this proteomic method for analyzing changes to Saccharomyces cerevisiae chromatin, illustrating the procedure with an analysis of the changes that occur in chromatin composition as cells progress from a G1 phase block (induced by alpha factor) into S phase (in the presence of DNA replication inhibitor hydroxyurea)., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The type VI secretion system (T6SS) with diversified functions is widely distributed in pathogenic Proteobacteria. The IcmF (intracellular multiplication protein F) family protein TssM is a conserved T6SS inner membrane protein. Despite the conservation of its Walker A nucleotide-binding motif, the NTPase activity of TssM and its role in T6SS remain obscure. In this study, we characterized TssM in the plant pathogen Agrobacterium tumefaciens and provided the first biochemical evidence for TssM exhibiting ATPase activity to power the secretion of the T6SS hallmark protein, hemolysin-coregulated protein (Hcp). Amino acid substitutions in the Walker A motif of TssM caused reduced ATP binding and hydrolysis activity. Importantly, we discovered the Walker B motif of TssM and demonstrated that it is critical for ATP hydrolysis activity. Protein-protein interaction studies and protease susceptibility assays indicated that TssM undergoes an ATP binding-induced conformational change and that subsequent ATP hydrolysis is crucial for recruiting Hcp to interact with the periplasmic domain of the TssM-interacting protein TssL (an IcmH/DotU family protein) into a ternary complex and mediating Hcp secretion. Our findings strongly argue that TssM functions as a T6SS energizer to recruit Hcp into the TssM-TssL inner membrane complex prior to Hcp secretion across the outer membrane.

Many bacterial and archaeal genomes are of a similar size to molecules that have been cloned in the yeast Saccharomyces cerevisiae and thus might be clonable as single, circular episomes in this host. Yeast offers a variety of efficient tools for the manipulation and study of cloned DNA. One strategy to clone a genome in yeast is to cotransform yeast spheroplasts with the genome of interest and a linear yeast vector whose termini are homologous to a spot in the genome. Clones are selected on auxotrophic medium and then screened for completeness and size; they may also be sequenced.

Only a small fraction of the bacterial diversity present in natural microbial communities is regularly cultured in the laboratory. Those bacteria that remain recalcitrant to culturing cannot be examined for the production of bioactive secondary metabolites using standard pure-culture approaches. The screening of genomic DNA libraries containing DNA isolated directly from environmental samples (environmental DNA (eDNA)) provides an alternative approach for studying the biosynthetic capacities of these organisms. One drawback of this approach has been that most eDNA isolation procedures do not permit the cloning of DNA fragments of sufficient length to capture large natural product biosynthetic gene clusters in their entirety. Although the construction of eDNA libraries with inserts big enough to capture biosynthetic gene clusters larger than ∼40kb remains challenging, it is possible to access large gene clusters by reassembling them from sets of smaller overlapping fragments using transformation-associated recombination in Saccharomyces cerevisiae. Here, we outline a method for the reassembly of large biosynthetic gene clusters from captured sets of overlapping soil eDNA cosmid clones. Natural product biosynthetic gene clusters reassembled using this approach can then be used directly for functional heterologous expression studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mouse G258 mutant stopped both cell growth and the synthesis of lipid-linked oligosaccharide at the Man(3)GlcNAc(2)-P-P-Dolichol at a restricted temperature with a single gene mutation. To clarify the lesion in the G258 mutant, we isolated human genomic DNA transformants of the G258 mutant, which recovered from both defects by way of cell hybridization with X-ray irradiated HeLa cells. We detected a common 1.3-kb product by inter-human specific sequence in the L1 (L1Hs) PCR in the transformants (Kataoka et al., Somat. Cell Mol. Genet., 24, 235-243 (1998)). In the present study, we screened a human mega yeast artificial chromosome (YAC) library by PCR with primers designed according to the 1.3-kb DNA, and selected YAC clone 923f5. Moreover, we found by spheroplast fusion that YAC clone 923f5 complemented both defects of the G258 mutant. Since the human counterpart of the yeast ALG11 gene is localized in the region, the G258 mutant might have a defect in the mouse ALG11 gene.

The β(2) subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe(2)(III)-Tyr(•)) that is essential for nucleotide reduction. The β(2) subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β'). Although only β is capable of iron binding and Tyr(•) formation, cells lacking β' are either dead or exhibit extremely low Tyr(•) levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β' is required for iron loading and Tyr(•) formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr(•)-reducing agent hydroxyurea. Transient induction of β' in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr(•) levels in β. Tyr(•) can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β' and is inhibited by adding an iron chelator prior to, but not after, β' supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr(•) levels and ββ' activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr(•) cofactor in RNR.

Immense diversity of prion strains is observed, but its underlying mechanism is less clear. Three [PSI] prion strains--named VH, VK, and VL--were previously isolated in the wild-type yeast genetic background. Here we report the generation and characterization of eight new [PSI] isolates, obtained by propagating the wild-type strains with Sup35 proteins containing single amino-acid alterations. The VH strain splits into two distinct strains when propagated in each of the three genetic backgrounds, harboring respectively single mutations of N21L, R28P, and Gi47 (i.e. insertion of a glycine residue at position 47) on the Sup35 N-terminal prion-forming segment. The six new strains exhibit complex inter-conversion patterns, and one of them continuously mutates into another. However, when they are introduced back into the wild-type background, all 6 strains revert to the VH strain. We obtain two more [PSI] isolates by propagating VK and VL with the Gi47 and N21L backgrounds, respectively. The two isolates do not transmit to other mutant backgrounds but revert to their parental strains in the wild-type background. Our data indicate that a large number of [PSI] strains can be built on three basic Sup35 amyloid structures. It is proposed that the three basic structures differ by chain folding topologies, and sub-strains with the same topology differ in distinct ways by local structural adjustments. This "large number of variations on a small number of basic themes" may also be operative in generating strain diversities in other prion elements. It thus suggests a possible general scheme to classify a multitude of prion strains., Competing Interests: The authors have declared that no competing interests exist.

Two strains (JA317(T) and JA559) of spiral shaped, spheroplast-forming, anaerobic, gram-negative, motile purple non-sulfur bacteria were isolated from rhizosphere soils of paddy and were characterized by a polyphasic taxonomic approach. Bacteriochlorophyll a and carotenoids, rhodopin, lycopene and rhodopin glucoside, were present as photosynthetic pigments. Intracellular photosynthetic membranes were of stacked type. The major fatty acids were C(18 : 1)ω7c, C(16 : 0) and C(16 : 1)ω6c/C(16 : 1)ω7c in both strains. The genomic DNA G+C content was 63.3±0.8 mol%. The two strains were closely related (mean DNA-DNA hybridization >85 %). Phylogenetic analysis showed that the strains clustered with the species of the genus Phaeospirillum, which belongs to the family Rhodospirillaceae within the class Alphaproteobacteria. Based on 16S rRNA gene sequence analysis, strains JA317(T) and JA559 showed highest sequence similarity with the type strains of Phaeospirillum chandramohanii (98.2 %), Phaeospirillum molischianum (97.4 %) and Phaeospirillum fulvum (97.1 %) of the family Rhodospirillaceae. Strain JA317(T) can be clearly distinguished from P. chandramohanii with respect to spheroplast formation and several other morphological and physiological properties. DNA-DNA relatedness of strain JA317(T) with its closest relatives of the genus Phaeospirillum was less than 55 %. It is evident from the phenotypic, chemotaxonomic and molecular genetic evidence that strain JA317(T) represents a novel species of the genus Phaeospirillum, for which the name Phaeospirillum oryzae sp. nov., is proposed. The type strain of the species is JA317(T) ( = NBRC 104938(T)  = KCTC 5704(T)).

We have described a protocol for performing high-throughput immunofluorescence microscopy on microarrays of yeast cells. This approach employs immunostaining of spheroplasted yeast cells printed as high-density cell microarrays, followed by imaging using automated microscopy. A yeast spheroplast microarray can contain more than 5,000 printed spots, each containing cells from a given yeast strain, and is thus suitable for genome-wide screens focusing on single cell phenotypes, such as systematic localization or co-localization studies or genetic assays for genes affecting probed targets. We demonstrate the use of yeast spheroplast microarrays to probe microtubule and spindle defects across a collection of yeast strains harboring tetracycline-down-regulatable alleles of essential genes.

Transformation (i.e., genetic modification of a cell by the incorporation of exogenous DNA) is indispensable for manipulating fungi. Here, we review the transformation methods for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris and Aspergillus species and discuss some common modifications to improve transformation efficiency. We also present a model of the mechanism underlying S. cerevisiae transformation, based on recent reports and the mechanism of transfection in mammalian systems. This model predicts that DNA attaches to the cell wall and enters the cell via endocytotic membrane invagination, although how DNA reaches the nucleus is unknown. Polyethylene glycol is indispensable for successful transformation of intact cells and the attachment of DNA and also possibly acts on the membrane to increase the transformation efficiency. Both lithium acetate and heat shock, which enhance the transformation efficiency of intact cells but not that of spheroplasts, probably help DNA to pass through the cell wall., (© 2010 Landes Bioscience)

The Trypanosoma brucei genome has four highly similar genes encoding sphingolipid synthases (TbSLS1-4). TbSLSs are polytopic membrane proteins that are essential for viability of the pathogenic bloodstream stage of this human protozoan parasite and, consequently, can be considered as potential drug targets. TbSLS4 was shown previously to be a bifunctional sphingomyelin/ethanolamine phosphorylceramide synthase, whereas functions of the others were not characterized. Using a recently described liposome-supplemented cell-free synthesis system, which eliminates complications from background cellular activities, we now unambiguously define the enzymatic specificity of the entire gene family. TbSLS1 produces inositol phosphorylceramide, TbSLS2 produces ethanolamine phosphorylceramide, and TbSLS3 is bifunctional, like TbSLS4. These findings indicate that TbSLS1 is uniquely responsible for synthesis of inositol phosphorylceramide in insect stage parasites, in agreement with published expression array data (17). This approach also revealed that the Trypanosoma cruzi ortholog (TcSLS1) is a dedicated inositol phosphorylceramide synthase. The cell-free synthesis system allowed rapid optimization of the reaction conditions for these enzymes and site-specific mutagenesis to alter end product specificity. A single residue at position 252 (TbSLS1, Ser(252); TbSLS3, Phe(252)) strongly influences enzymatic specificity. We also have used this system to demonstrate that aureobasidin A, a potent inhibitor of fungal inositol phosphorylceramide synthases, does not significantly affect any of the TbSLS activities, consistent with the phylogenetic distance of these two clades of sphingolipid synthases. These results represent the first application of cell-free synthesis for the rapid preparation and functional annotation of integral membrane proteins and thus illustrate its utility in studying otherwise intractable enzyme systems.

Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.

We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.

The complexes of repair DNA polymerase beta with 3'-exonuclease and some other proteins were isolated from the chromatin of hepatocytes of normal rats for the first time. Biopolymers were extracted from the chromatin by the solution of NaCl and Triton X-100. The extract was fractionated by gel-filtration on Sephacryl S-300 columns successively in low and high ionic strength solutions, on hydroxyapatite, and on Sephadex G-100 columns. The complexes have molecular weights of 100 and 300 kDa. They dissociate to DNA polymerase and exonuclease in the course of chromatography on a DNA-cellulose column or after gel-filtration in the presence of 1 M NaCl. The co-purification of the polymerase and exonuclease is reconstituted in 0.1 M NaCl. The fidelity of monomeric and composite DNA polymerase beta was measured using phage phiX174 amber 3 as a primer/template. The products of the synthesis were transfected into Escherichia coli spheroplasts, and the frequency of reverse mutations was determined. The complex of DNA polymerase beta with 3'-exonuclease was shown to be 30 times more accurate than the monomeric polymerase, which can decrease the probability of repair mutagenesis and carcinogenesis.

Introduction of sequences of interest into an intercistronic spacer (ICS) of dual reporter plasmids is the main experimental set-up used to identify and study internal ribosome entry sites (IRESs). We studied internal initiation of translation in yeast using the dicistronic approach. Three viral sequences and a polylinker-derived reference sequence were inserted into the ICS of a dual reporter plasmid upstream of the firefly luciferase gene, luc. LUC expression was taken as a putative indication of internal translation initiation from the studied sequences. Interestingly, all sequences mediated 3' LUC expression. However, northern blot analysis revealed that in addition to the dicistronic mRNAs, transcripts containing only the LUC-encoding sequences were produced from the plasmids. Electroporation studies with in vitro synthesized mRNAs showed that expression from the 3' cistron of the dicistronic mRNAs was below the level of detection. This suggested that the observed LUC expression from yeast transformed with the dicistronic expression plasmids did not originate from dicistronic messages. Deletion of the promoter increased 3' LUC expression. Similarly, repression of transcription prevented 5' cistron expression whereas 3' LUC expression was stimulated. These results suggested that the observed LUC expression did not result from partially degraded or spliced dicistronic RNAs but rather from transcripts synthesized from cryptic promoters. Despite high LUC activity, northern blot analysis detected few transcripts from yeast transformed with the promoterless constructs. Therefore, our data indicate that the functional assay is more revealing than RNA analysis in the case of very sensitive reporter genes generally used in IRES studies. Furthermore, our studies show that there is a clear need for detailed analysis prior to concluding the mechanism of 3' cistron expression.

The mechanosensitive channel of small conductance (MscS) is a bacterial mechanosensitive channel that opens in response to rapid hypoosmotic stress. Since MscS can be opened solely by membrane stretch without help from any accessory protein, the lipid-protein interface must play a crucial role in sensing membrane tension. In this study, the hydrophobic residues in the lipid-protein interface were substituted one by one with a hydrophilic amino acid, asparagine, to modify the interaction between the protein and the lipid. Function of the mutant MscSs was examined by patch-clamp and hypoosmotic shock experiments. An increase in the gating threshold and a decrease in the viability on hypoosmotic shock were observed when the hydrophobic residues near either end of the first or the second transmembrane helix (TM1 or TM2) were replaced with asparagine. This observation indicates that the lipid-protein interaction at the ends of both helices (TM1 and TM2) is essential to MscS function.

In cells, the expression of Gag protein, one of the major structural proteins of retroviruses, is sufficient for budding virus-like particles (VLPs) from the cell surface. We have previously reported that spheroplasts of Saccharomyces cerevisiae expressing HIV-1 Gag proteins from an episomal plasmid constitutively released a large amount of VLPs into culture media; however, commercially available ELISA kits which detect mature capsid of HIV-1 could not detect uncleaved 55-kDa Gag proteins released from budding yeast. We therefore developed a method to quantitate VLP levels released from budding yeast by using fusion protein from HIV-1 Gag and Firefly Luciferase. This system is useful for screening cellular factor(s) involved in retrovirus budding from S. cerevisiae.

We present methods to prepare infectious Sup35 protein aggregates and use them for genetic transformation of yeast. The protein aggregates are prepared from bacterially expressed recombinant protein, which is converted to amyloid fibers by extended incubation or by nucleated growth using yeast prion particles as seeds. The aggregates are introduced into yeast by a modified spheroplast transformation protocol. The phenotype of the yeast transformants is further characterized by robust prion strain typing methods. The methodology can be used to introduce different [PSI(+)] particles to many laboratory yeast genetic backgrounds. It can be adapted for applications in other yeast prion systems as well.

Study of Saccharomyces cerevisiae killer toxin-sensitive strains with the deltakre2 phenotype (resistant to toxin K1, sensitive to toxin K2) showed that the phenotype is complemented by the KRE2 gene not only in intact cells but also in spheroplasts, and resistance to K1 thus resides very probably in the plasma membrane. deltakre1 deletant displays a faulty interaction with both K1 and K2 toxin. Hence, Kre1p probably serves as plasma membrane receptor for both toxins. Deletants in seven other genes (GDA1, SAC1, LUV1, KRE23, SAC2, KRE21, ERG4) exhibit different degrees of the deltakre2-like resistance pattern, but the phenotype in deltagda1 and deltasac1 is not connected with a defect in K1 toxin interaction with the plasma membrane, similarly as in deltakre6 and deltakre11 strains with a higher resistance to K2 toxin. Differences between the K1 and K2 killer toxin thus occur on the level of both the plasma membrane and the cell wall.

Background: Transformation-associated recombination (TAR) cloning in yeast is a unique method for selective isolation of large chromosomal fragments or entire genes from complex genomes. The technique involves homologous recombination, during yeast spheroplast transformation, between genomic DNA and a TAR vector that has short (approximately 60 bp) 5' and 3' gene targeting sequences (hooks)., Result: TAR cloning requires that the cloned DNA fragment carry at least one autonomously replicating sequence (ARS) that can function as the origin of replication in yeast, which prevents wide application of the method. In this paper, we describe a novel TAR cloning system that allows isolation of genomic regions lacking yeast ARS-like sequences. ARS is inserted into the TAR vector along with URA3 as a counter-selectable marker. The hooks are placed between the TATA box and the transcription initiation site of URA3. Insertion of any sequence between hooks results in inactivation of URA3 expression. That inactivation confers resistance to 5-fluoroorotic acid, allowing selection of TAR cloning events against background vector recircularization events., Conclusion: The new system greatly expands the area of application of TAR cloning by allowing isolation of any chromosomal region from eukaryotic and prokaryotic genomes regardless of the presence of autonomously replicating sequences.

A transformation protocol mediated by Agrobacterium tumefaciens is described for the sapstaining fungus Ophiostoma piceae. We compared transformants obtained from Agrobacterium with those obtained from yeast-like cells made into spheroplasts and treated with CaCl2. For all putative transformants analyzed, Southern hybridization confirmed that the hygromycin resistance gene had been integrated into the genomic DNA. While all transformants obtained from the treated spheroplasts had multiple copy vector insertion, 85% of the Agrobacterium-mediated transformants had single copy vector insertion.

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (10(2) to 10(3) transformants per micro g of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.

Killer toxin K1 of Saccharomyces cerevisiae kills sensitive cells of the same species by disturbing the ion gradient across the plasma membrane after binding to the receptor at cell wall beta-1,6-glucan. Killer protein K2 is assumed to act by a similar mechanism. To identify the putative plasma membrane receptors for both toxins we mutagenized three sensitive S. cerevisiae strains and searched for clones with killer-resistant spheroplasts. The well diffusion assay identified three phenotypically different groups of clones: clones resistant simultaneously to both toxins, clones with lowered sensitivity to only K1 toxin and those with strongly lowered sensitivity to K2 and partially lowered sensitivity to K1 toxin. These phenotypes are controlled by recessive mutations that belong to at least four different complementation groups. This indicates certain differences at the level of interaction of K1 and K2 toxin with sensitive cells.

The yeast artificial chromosome (YAC) cloning system makes it possible to clone large pieces of genomic DNA into yeast. Libraries have been made containing clones with inserts in the megabase-pair range. The basic protocol in this unit describes preparation of YAC vectors and transformation of ligated DNA into yeast spheroplasts. A support protocol describes titration of Lyticase to make spheroplasts. Additional support protocols detail two methods for partial digestion of genomic DNA: EcoRI restriction endonuclease-EcoRI methylase competition and the partial digestion of genomic DNA by use of limiting amounts of Mg2+, respectively.

Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of approximately 0.5%; the positive clones are identified by PCR or colony hybridization. This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection. This system utilizes a TAR vector with two targeting hooks, HIS3 as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence. The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector. When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is lost at high frequency by direct repeat recombination involving the loop sequence. Positive clones are identified by selecting against URA3. This method produces positive YAC recombinants at a frequency of approximately 40%. This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.

This unit provides a set of protocols for introducing large insert DNA into cultured mammalian cells and embryos. Two different methods, spheroplast fusion and lipofection, are described for effecting transfer of YACs or gel-purified YAC DNA into cells. Additional protocols discuss preparing and transferring BACs into cells by lipofection and into embryos by microinjection.

The most commonly used yeast transformation protocol is the lithium acetate procedure (described here). It is reasonably fast and provides a transformation efficiency of 10(5) to 10(6) transformants/microg. This efficiency rivals that achieved for most, but not all, strains with the more difficult and time-consuming spheroplast procedure presented here. However, the fastest and easiest of the transformation methods is electroporation, as described in this unit. For a number of strains, electroporation offers the highest transformation efficiency, and may prove especially useful with limiting quantities of transforming DNA. Unlike the lithium acetate procedure, however, electroporation saturates at low DNA levels, restricting its general utility.

Cell wall deficient forms of mycobacteria may be important in the pathogenesis of Crohn's disease and sarcoidosis. However, no method has been available to localize this type of organisms in tissue sections. We developed an in situ hybridization method for the demonstration of Mycobacterium paratuberculosis spheroplasts (cell wall deficient forms) in paraffin embedded tissue sections.M. paratuberculosis spheroplasts were prepared by treatment with glycine and lysozyme. Pieces of beef were injected with the prepared spheroplasts. The samples were fixed in buffered formalin and paraffin embedded. A M. paratuberculosis-specific probe derived from the IS900 gene was used. Specificity was controlled by using an irrelevant probe and by hybridizing sections with spheroplasts from other bacteria. Beef samples injected with M. paratuberculosis spheroplasts were the only samples that hybridized with the probe. Beef samples containing acid-fast or spheroplast forms of M. smegmatis and M. tuberculosis as well as the acid-fast forms of M. paratuberculosis did not hybridize with the probe. Unrelated bacterial controls, i.e. Helicobacter pylori and Escherichia coli were also negative in the assay. In situ hybridization with the IS900 probe provides a specific way to localize M. paratuberculosis spheroplasts in tissue sections and may be useful for studies of the connection between M. paratuberculosis and Crohn's disease and sarcoidosis. The assay may also be valuable for studies on Johne's diseased animals.

To study Bax-induced release of cytochrome c in vivo, we have expressed a cytochrome c-GFP (green fluorescent protein) fusion in Saccharomyces cerevisiae cells null for the expression of the endogenous cytochrome. We show here that cytochrome c-GFP is efficiently localised to mitochondria and able to function as an electron carrier between complexes III and IV of the respiratory chain. Strikingly, while natural cytochrome c is released into the cytoplasm upon expression of Bax, the cytochrome c-GFP fusion is not. Nevertheless, cells co-expressing Bax and the cytochrome c-GFP fusion die, indicating that mitochondrial release of cytochrome c is not essential for cell death to occur in yeast. The failure to release cytochrome c-GFP is presumed to arise from increased bulk due to the GFP moiety. We propose that in intact yeast cells, Bax-induced release of cytochrome c into the cytoplasm occurs through a selective pore and not as a consequence of the non-specific breakage of the mitochondrial outer membrane.

In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency. Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50-100 transformants/microg DNA), while spheroplast transformation, although more efficient ( approximately 300 transformants/microg integrative DNA and 10(3)-10(4) transformants/microg replicative DNA), is complicated and time-consuming. In this study we applied various yeast transformation techniques to C. albicans and selected an electroporation procedure for further optimization. Transformation efficiencies of up to 300 transformants/microg were obtained for an integrative plasmid and up to 4500 transformants/microg for a CARS-carrying plasmid. This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C. albicans., (Copyright 1999 John Wiley & Sons, Ltd.)

A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purified and its sequence directly determined using thermal cycle sequencing. Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days. Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed.

The development of a system for shuttling DNA cloned as yeast artificial chromosomes (YACs) between yeast and mammalian cells requires that the DNA is maintained as extrachromosomal elements in both cell types. We have recently shown that circular YACs carrying the Epstein-Barr virus origin of plasmid replication (oriP) are maintained as stable, episomal elements in a human kidney cell line constitutively expressing the viral transactivator protein EBNA-1. Here, we demonstrate that a 90-kb episomal YAC can be isolated intact from human cells by a simple alkaline lysis procedure and shuttled back into Saccharomyces cerevisiae by spheroplast transformation. In addition, we demonstrate that the 90-kb YAC can be isolated intact from yeast cells. The ability to shuttle large, intact fragments of DNA between yeast and human cells should provide a powerful tool in the manipulation and analysis of functional regions of mammalian DNA.

The Saccharomyces cerevisiae killer toxin K1 is a secreted alpha/beta-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to beta-1,6-D-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of the KRE genes whose products are involved in synthesis and/or assembly of cell wall beta-D-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strains indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), rendering kre12 mutants incapable of binding significant amounts of toxin to the membrane. Since kre12 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from a kre12 mutant and from an isogenic Kre12(+) strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in the kre12 mutant. A model for K1 killer toxin action is presented in which the gene product of KRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.