A family of multiple autonomously replicating sequences (ARSs) which are located at several chromosomal ends of Hansenula polymorpha DL-1 has been identified and characterized. Genomic Southern blotting with an ARS, HARS36, originating from the end of a chromosome, as a probe showed several homologues in the genome of H. polymorpha. Nucleotide sequences of the three fragments obtained by a selective cloning for chromosomal ends were nearly identical to that of HARS36. All three fragments harbored an ARS motif and ended with 18 to 23 identical repetitions of 5′-GGGTGGCG-3′ which resemble the telomeric repeat sequence in other eukaryotes. Transformation of H. polymorpha with nonlinearized plasmids containing the newly obtained telomeric ARSs almost exclusively resulted in the targeted integration of a single copy or multiple tandem copies of the plasmid into the chromosomes. The sensitivity to exonuclease Bal31 digestion of the common DNA fragment in all integrants confirmed the telomeric origin of HARS36 homologues, suggesting that several chromosomal ends, if not all of them, consisted of the same ARS motif and highly conserved sequences observed in HARS36. Even though the frequencies of targeted recombination were varied among the ends of the chromosomes, the overall frequency was over 96%. The results suggested that the integration of the plasmids containing telemeric ARSs occurred largely through homologous recombination at the telomeric repeats, which serve as high-frequency recombination targets.
Several autonomously replicating sequences of Hansenula polymorpha DL-1 (HARSs) with the characteristics of tandem integration were cloned by an enrichment procedure and analyzed for their functional elements to elucidate the mechanism of multiple integration in tandem repeats. All plasmids harboring newly cloned HARSs showed a high frequency of transformation and were maintained episomally before stabilization. After stabilization, the transforming DNA was stably integrated into the chromosome. HARS36 was selected for its high efficiency of transformation and tendency for integration. Several tandemly repeated copies of the transforming plasmid containing HARS36 (pCE36) integrated into the vicinity of the chromosomal end. Bal 31 digestion of the total DNA from the integrants followed by Southern blotting generated progressive shortening of the hybridization signal, indicating the telomeric localization of the transforming plasmids on the chromosome. The minimum region of HARS36 required for its HARS activity was analyzed by deletion analyses. Three important regions, A, B, and C, for episomal replication and integration were detected. Analysis of the DNA sequences of regions A and B required for the episomal replication revealed that region A contained several AT-rich sequences that showed sequence homology with the ARS core consensus sequence of Saccharomyces cerevisiae. Region B contained two directly repeated sequences which were predicted to form a bent DNA structure. Deletion of the AT-rich core in region A resulted in a complete loss of ARS activity, and deletion of the repeated sequences in region B greatly reduced the stability of the transforming plasmid and resulted in retarded cell growth. Region C was required for the facilitated chromosomal integration of transforming plasmids.
The methylotrophic yeast, Hansenula polymorpha is an industrially important microorganism, and belongs to the best studied yeast species with well-developed tools for molecular research. The complete genome sequence of the strain NCYC495 of H. polymorpha is publicly available. Some of the well-studied strains of H. polymorpha are known to ferment glucose, cellobiose and xylose to ethanol at elevated temperature (45 – 50°C) with ethanol yield from xylose significantly lower than that from glucose and cellobiose. Increased yield of ethanol from xylose was demonstrated following directed metabolic changes but, still the final ethanol concentration achieved is well below what is considered feasible for economic recovery by distillation.
In this work, we describe the construction of strains of H. polymorpha with increased ethanol production from xylose using an ethanol-non-utilizing strain (2EthOH−) as the host. The transformants derived from 2EthOH− overexpressing modified xylose reductase (XYL1m) and native xylitol dehydrogenase (XYL2) were isolated. These transformants produced 1.5-fold more ethanol from xylose than the original host strain. The additional overexpression of XYL3 gene coding for xylulokinase, resulted in further 2.3-fold improvement in ethanol production with no measurable xylitol formed during xylose fermentation. The best ethanol producing strain obtained by metabolic engineering approaches was subjected to selection for resistance to the known inhibitor of glycolysis, the anticancer drug 3-bromopyruvate. The best mutant selected had an ethanol yield of 0.3 g/g xylose and produced up to 9.8 g of ethanol/l during xylose alcoholic fermentation at 45°C without correction for ethanol evaporation.
Our results indicate that xylose conversion to ethanol at elevated temperature can be significantly improved in H. polymorpha by combining methods of metabolic engineering and classical selection.
3-Bromopyruvate; High-temperature fermentation; Xylose; Fuel ethanol; Hansenula polymorpha
Genetic transformation of the dimorphic pathogenic fungus Histoplasma capsulatum can result in chromosomal integration of the transforming DNA or the generation of multicopy linear plasmids carrying the transforming DNA. We showed previously that Escherichia coli plasmids do not replicate autonomously in H. capsulatum without significant modifications, one of which is the in vivo addition of Histoplasma telomeres at the termini of linear DNA. To address the requirements for autonomous replication in H. capsulatum, we constructed a circular E. coli plasmid containing adjacent inverted stretches of Histoplasma telomeric repeats separated by a unique restriction site. The linearized plasmid bearing telomeric termini was maintained in H. capsulatum without modification other than the addition of more telomeric sequence. We recovered the original plasmid in E. coli after removal of the telomeric termini by using engineered restriction sites. Thus, no special Histoplasma modification or sequence other than the telomeres was needed for autonomous replication in H. capsulatum. Additionally, this plasmid provides a shuttle vector that replicates autonomously in E. coli (as a circular plasmid) and in H. capsulatum (as a linear plasmid).
RecQ helicases, including Saccharomyces cerevisiae Sgs1p and the human Werner syndrome protein, are important for telomere maintenance in cells lacking telomerase activity. How maintenance is accomplished is only partly understood, although there is evidence that RecQ helicases function in telomere replication and recombination. Here we use two-dimensional gel electrophoresis (2DGE) and telomere sequence analysis to explore why cells lacking telomerase and Sgs1p (tlc1 sgs1 mutants) senesce more rapidly than tlc1 mutants with functional Sgs1p. We find that apparent X-shaped structures accumulate at telomeres in senescing tlc1 sgs1 mutants in a RAD52- and RAD53-dependent fashion. The X-structures are neither Holliday junctions nor convergent replication forks, but instead may be recombination intermediates related to hemicatenanes. Direct sequencing of examples of telomere I-L in senescing cells reveals a reduced recombination frequency in tlc1 sgs1 compared with tlc1 mutants, indicating that Sgs1p is needed for tlc1 mutants to complete telomere recombination. The reduction in recombinants is most prominent at longer telomeres, consistent with a requirement for Sgs1p to generate viable progeny following telomere recombination. We therefore suggest that Sgs1p may be required for efficient resolution of telomere recombination intermediates, and that resolution failure contributes to the premature senescence of tlc1 sgs1 mutants.
Because telomeres are situated at the ends of chromosomes, they are both essential for chromosome integrity and particularly susceptible to processes that lead to loss of their own DNA sequences. The enzyme telomerase can counter these losses, but there are also other means of telomere maintenance, some of which depend on DNA recombination. The RecQ family of DNA helicases process DNA recombination intermediates and also help ensure telomere integrity, but the relationship between these activities is poorly understood. Family members include yeast Sgs1p and human WRN and BLM, which are deficient in the Werner premature aging syndrome and the Bloom cancer predisposition syndrome, respectively. We have found that the telomeres of yeast cells lacking both telomerase and Sgs1p accumulate structures that resemble recombination intermediates. Further, we provide evidence that the inability of cells lacking Sgs1p to process these telomere recombination intermediates leads to the premature arrest of cell division. We predict that similar defects in the processing of recombination intermediates may contribute to telomere defects in human Werner and Bloom syndrome cells.
Yeast cells lacking the RecQ helicase Sgs1p show an accumulation of telomere recombination intermediates associated with premature senescence.
Many repair and recombination proteins play essential roles in telomere function and chromosome stability, notwithstanding the role of telomeres in “hiding” chromosome ends from DNA repair and recombination. Among these are XPF and ERCC1, which form a structure-specific endonuclease known for its essential role in nucleotide excision repair and is the subject of considerable interest in studies of recombination. In contrast to observations in mammalian cells, we observe no enhancement of chromosomal instability in Arabidopsis plants mutated for either XPF (AtRAD1) or ERCC1 (AtERCC1) orthologs, which develop normally and show wild-type telomere length. However, in the absence of telomerase, mutation of either of these two genes induces a significantly earlier onset of chromosomal instability. This early appearance of telomere instability is not due to a general acceleration of telomeric repeat loss, but is associated with the presence of dicentric chromosome bridges and cytologically visible extrachromosomal DNA fragments in mitotic anaphase. Such extrachromosomal fragments are not observed in later-generation single-telomerase mutant plants presenting similar frequencies of anaphase bridges. Extensive FISH analyses show that these DNAs are broken chromosomes and correspond to two specific chromosome arms. Analysis of the Arabidopsis genome sequence identified two extensive blocks of degenerate telomeric repeats, which lie at the bases of these two arms. Our data thus indicate a protective role of ERCC1/XPF against 3′ G-strand overhang invasion of interstitial telomeric repeats. The fact that the Atercc1 (and Atrad1) mutants dramatically potentiate levels of chromosome instability in Attert mutants, and the absence of such events in the presence of telomerase, have important implications for models of the roles of recombination at telomeres and is a striking illustration of the impact of genome structure on the outcomes of equivalent recombination processes in different organisms.
Telomeres are the specialised nucleoprotein structures evolved to avoid progressive replicative shortening and recombinational instability of the ends of linear chromosomes. Notwithstanding this role of telomeres in “hiding” chromosome ends from DNA repair and recombination, many repair and recombination proteins play essential roles in telomere function and chromosome stability. Among these are XPF and ERCC1, which form a structure-specific endonuclease known for its essential role in nucleotide excision repair and that is the subject of considerable interest in studies of recombination. In this study, we analyse the roles of the XPF/ERCC1 in telomere function and chromosome stability in the plant Arabidopsis thaliana, which, with its remarkable tolerance to genomic instability and sequenced genome, is an excellent higher eukaryotic model for these studies. Surprisingly, and in striking contrast to observations in mammalian cells, we observe no enhancement of chromosomal instability in Arabidopsis plants lacking either of these two proteins, which develop normally and show wild-type telomere length. However, Atercc1 (and Atrad1) mutants profoundly affect the recombination of de-protected telomeres, dramatically potentiating chromosome instability. These results provide a striking illustration of the different outcomes and genomic impacts of the same recombination processes in different organisms.
It has been previously shown that linear plasmids bearing Tetrahymena telomeric sequences are able to replicate autonomously in the filamentous fungus Podospora anserina (1). However, autonomous replication occurs in only 50-70% of the transformants, suggesting a defect in the recognition of the Tetrahymena telomeric template by the putative P. anserina telomerase so that only a fraction of entering DNA is stabilized into linear extrachromosomal molecules. We have cloned DNA sequences added to the Tetrahymena (T2G4)n ends of the linear plasmid. Nucleotide sequencing showed that these sequences are exclusively composed of T2AG3 repeat units. Hybridization experiments of Bal31 treated DNA showed that T2AG3 repeats are confined within 200 bp in chromosomal P. anserina telomeres. A new plasmid has been constructed so that after linearization, the terminal sequences contain T2AG3 repeats. This linear molecule transforms P. anserina with a high frequency (up to 1.75 x 10(4) transformants/micrograms), autonomous replication occurs in 100% of the transformants and the plasmid copy number is about 2-3 per nucleus. These results underscore the importance of the telomeric repeat nucleotide sequence for efficient recognition as functional telomeric DNA in vivo and provide the first step toward the development of an artificial chromosome cloning system for filamentous fungi.
Hansenula polymorpha DL1 is a methylotrophic yeast, widely used in fundamental studies of methanol metabolism, peroxisome biogenesis and function, and also as a microbial cell factory for production of recombinant proteins and metabolic engineering towards the goal of high temperature ethanol production.
We have sequenced the 9 Mbp H. polymorpha DL1 genome and performed whole-genome analysis for the H. polymorpha transcriptome obtained from both methanol- and glucose-grown cells. RNA-seq analysis revealed the complex and dynamic character of the H. polymorpha transcriptome under the two studied conditions, identified abundant and highly unregulated expression of 40% of the genome in methanol grown cells, and revealed alternative splicing events. We have identified subtelomerically biased protein families in H. polymorpha, clusters of LTR elements at G + C-poor chromosomal loci in the middle of each of the seven H. polymorpha chromosomes, and established the evolutionary position of H. polymorpha DL1 within a separate yeast clade together with the methylotrophic yeast Pichia pastoris and the non-methylotrophic yeast Dekkera bruxellensis. Intergenome comparisons uncovered extensive gene order reshuffling between the three yeast genomes. Phylogenetic analyses enabled us to reveal patterns of evolution of methylotrophy in yeasts and filamentous fungi.
Our results open new opportunities for in-depth understanding of many aspects of H. polymorpha life cycle, physiology and metabolism as well as genome evolution in methylotrophic yeasts and may lead to novel improvements toward the application of H. polymorpha DL-1 as a microbial cell factory.
Hansenula polymorpha; Genome; Methylotrophic yeasts; RNA-seq; Yeast evolution
Yeasts provide attractive expression platforms in combining ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. However, early restriction to a single yeast platform can result in costly and time-consuming failures. It is therefore advisable to assess several selected systems in parallel for the capability to produce a particular protein in desired amounts and quality. A suitable vector must contain a targeting sequence, a promoter element and a selection marker that function in all selected organisms. These criteria are fulfilled by a wide-range integrative yeast expression vector (CoMed™) system based on A. adeninivorans- and H. polymorpha-derived elements that can be introduced in a modular way.
The vector system and a selection of modular elements for vector design are presented. Individual single vector constructs were used to transform a range of yeast species. Various successful examples are described. A vector with a combination of an rDNA sequence for genomic targeting, the E. coli-derived hph gene for selection and the A. adeninivorans-derived TEF1 promoter for expression control of a GFP (green fluorescent protein) gene was employed in a first example to transform eight different species including Hansenula polymorpha, Arxula adeninivorans and others. In a second example, a vector for the secretion of IL-6 was constructed, now using an A. adeninivorans-derived LEU2 gene for selection of recombinants in a range of auxotrophic hosts. In this example, differences in precursor processing were observed: only in A. adeninivorans processing of a MFα1/IL-6 fusion was performed in a faithful way.
rDNA targeting provides a tool to co-integrate up to 3 different expression plasmids by a single transformation step. Thus, a versatile system is at hand that allows a comparative assessment of newly introduced metabolic pathways in several organisms or a comparative co-expression of bottleneck genes in cases where production or secretion of a certain product is impaired.
Recombination cloning encompasses a set of technologies that transfer gene sequences between vectors through site-specific recombination. Due in part to the instability of linear DNA in bacteria, both the initial capture and subsequent transfer of gene sequences is often performed using purified recombination enzymes. However, we find linear DNAs flanked by loxP sites recombine efficiently in bacteria expressing Cre recombinase and the lambda Gam protein, suggesting Cre/lox recombination of linear substrates can be performed in vivo. As one approach towards exploiting this capability, we describe a method for constructing large (>1 × 106 recombinants) libraries of gene mutations in a format compatible with recombination cloning. In this method, gene sequences are cloned into recombination entry plasmids and whole-plasmid PCR is used to produce mutagenized plasmid amplicons flanked by loxP. The PCR products are converted back into circular plasmids by transforming Cre/Gam-expressing bacteria, after which the mutant libraries are transferred to expression vectors and screened for phenotypes of interest. We further show that linear DNA fragments flanked by loxP repeats can be efficiently recombined into loxP-containing vectors through this same one-step transformation procedure. Thus, the approach reported here could be adapted as general cloning method.
Telomeres are nucleoprotein structures located at the linear ends of eukaryotic chromosomes. Telomere integrity is required for cell proliferation and survival. Although the vast majority of eukaryotic species use telomerase as a primary means for telomere maintenance, a few species can use recombination or retrotransposon-mediated maintenance pathways. Since Saccharomyces cerevisiae can use both telomerase and recombination to replicate telomeres, budding yeast provides a useful system with which to examine the evolutionary advantages of telomerase and recombination in preserving an organism or cell under natural selection. In this study, we examined the life span in telomerase-null, post-senescent type II survivors that have employed homologous recombination to replicate their telomeres. Type II recombination survivors stably maintained chromosomal integrity but exhibited a significantly reduced replicative life span. Normal patterns of cell morphology at the end of a replicative life span and aging-dependent sterility were observed in telomerase-null type II survivors, suggesting the type II survivors aged prematurely in a manner that is phenotypically consistent with that of wild-type senescent cells. The shortened life span of type II survivors was extended by calorie restriction or TOR1 deletion, but not by Fob1p inactivation or Sir2p over-expression. Intriguingly, rDNA recombination was decreased in type II survivors, indicating that the premature aging of type II survivors was not caused by an increase in extra-chromosomal rDNA circle accumulation. Reintroduction of telomerase activity immediately restored the replicative life span of type II survivors despite their heterogeneous telomeres. These results suggest that telomere recombination accelerates cellular aging in telomerase-null type II survivors and that telomerase is likely a superior telomere maintenance pathway in sustaining yeast replicative life span.
Telomeres are the specialized structures at the ends of eukaryotic linear chromosomes. The simple guanine-rich DNA repeats at telomeres and their associated proteins are important for chromosome stability. Most eukaryotic species have evolved an enzyme named telomerase to replicate their telomeric DNA. Telomerase usually contains a protein catalytic subunit and a RNA template subunit. A few eukaryotic species can use either telomere recombination or retrotransposon-mediated transposition to accomplish telomere elongation. Interestingly, the baker's yeast Saccharomyces cerevisiae can use both telomerase and recombination to replicate telomeres. In this study, we utilize this unique eukaryotic model system to compare the efficiency of these two mechanisms in the maintenance of cellular function and life span. Telomerase-null cells that used recombination to elongate telomeres were able to maintain relatively stable chromosomes; however, they exhibited a shortened replicative life span which may represent a novel aging pathway. Reintroduction of telomerase inhibited telomere recombination and restored the replicative life span of these cells, implying that telomerase is superior to telomere recombination in the regulation of yeast replicative life span.
The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved.
Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Δxyl1 background. This resulted in significant increase in the KM for NADPH in the mutated xylose reductase (K341 → R N343 → D), while KM for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Δxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg × (L × h)-1 as compared to 4.2 mg × (L × h)-1 for the wild type strain.
Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation.
Telomeres are protein–DNA structures found at the ends of linear chromosomes and are crucial for genome integrity. Telomeric DNA length is primarily maintained by the enzyme telomerase. Cells lacking telomerase will undergo senescence when telomeres become critically short. In Saccharomyces cerevisiae, a very small percentage of cells lacking telomerase can remain viable by lengthening telomeres via two distinct homologous recombination pathways. These “survivor” cells are classified as either Type I or Type II, with each class of survivor possessing distinct telomeric DNA structures and genetic requirements. To elucidate the regulatory pathways contributing to survivor generation, we knocked out the telomerase RNA gene TLC1 in 280 telomere-length-maintenance (TLM) gene mutants and examined telomere structures in post-senescent survivors. We uncovered new functional roles for 10 genes that affect the emerging ratio of Type I versus Type II survivors and 22 genes that are required for Type II survivor generation. We further verified that Pif1 helicase was required for Type I recombination and that the INO80 chromatin remodeling complex greatly affected the emerging frequency of Type I survivors. Finally, we found the Rad6-mediated ubiquitination pathway and the KEOPS complex were required for Type II recombination. Our data provide an independent line of evidence supporting the idea that these genes play important roles in telomere dynamics.
Homologous recombination is a means for an organism or a cell to repair damaged DNA in its genome. Eukaryotic chromosomes have a linear configuration with two ends that are special DNA–protein structures called telomeres. Telomeres can be recognized by the cell as DNA double-strand breaks and subjected to repair by homologous recombination. In the baker's yeast Saccharomyces cerevisiae, cells that lack the enzyme telomerase, which is the primary factor responsible for telomeric DNA elongation, are able to escape senescence and cell death when telomeres undergo repair via homologous recombination. In this study, we have performed genetic screens to identify genes that affect telomeric DNA recombination. By examining the telomere structures in 280 mutants, each of which lacks both a telomere-length-maintenance gene and telomerase RNA gene, we identified 32 genes that were not previously known to be involved in telomere recombination. These genes have functions in a variety of cellular processes, and our work provides new insights into the regulation of telomere recombination in the absence of telomerase.
Telomerase is a ribonucleoprotein, the main function of which is to synthesize
telomeres, i.e. repetitive sequences which are localized at the ends of
eukaryotic chromosomes. Telomerase maintains the stability of the genome in
eukaryotic cells by replicating chromosomal ends. The structural and functional
investigation of the telomerase complex is significantly restricted due to
difficulties connected with the isolation of its main catalytic subunit in
recombinant form. Herein, we describe a method developed for the isolation of
the recombinant telomerase reverse transcriptase from thermotolerant
yeastHansenula polymorpha. A functional test performed for
the isolated protein and the RNA/DNA duplex, simulating the interaction of
telomerase RNA and telomere, reveals that the isolated catalytic subunit of
telomerase possesses limited reverse transcriptase activity.
telomerase reverse transcriptase; recombinant proteins; thermotolerant yeastHansenula polymorpha.
We have investigated two reactions that occur on telomeric sequences introduced into Saccharomyces cerevisiae cells by transformation. The elongation reaction added repeats of the yeast telomeric sequence C1-3A to telomeric sequences at the end of linear DNA molecules. The reaction worked on the Tetrahymena telomeric sequence C4A2 and also on the simple repeat CA. The reaction was orientation specific: it occurred only when the GT-rich strand ran 5' to 3' towards the end of the molecule. Telomere elongation occurred by non-template-directed DNA synthesis rather than any type of recombination with chromosomal telomeres, because C1-3A repeats could be added to unrelated DNA sequences between the CA-rich repeats and the terminus of the transforming DNA. The elongation reaction was very efficient, and we believe that it was responsible for maintaining an average telomere length despite incomplete replication by template-directed DNA polymerase. The resolution reaction processed a head-to-head inverted repeat of telomeric sequences into two new telomeres at a frequency of 10(-2) per cell division.
Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of the reverse transcriptase telomerase. In both tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Genetic studies have led to the identification of DNA polymerases, cell cycle checkpoint proteins, and telomere binding proteins involved in the telomerase pathway. However, how these proteins affect telomere-telomere recombination has not been identified to date. Using an assay to trace the in vivo recombinational products throughout the course of survivor development, we show here that three major replicative polymerases, α, δ, and ɛ, play roles in telomere-telomere recombination and that each causes different effects and phenotypes when they as well as the telomerase are defective. Polymerase δ appears to be the main activity for telomere extension, since neither type I nor type II survivors arising via telomere-telomere recombination were seen in its absence. The frequency of type I versus type II is altered in the polymerase α and ɛ mutants relative to the wild type. Each prefers to develop a particular type of survivor. Moreover, type II recombination is mediated by the cell cycle checkpoint proteins Tel1 and Mec1, and telomere-telomere recombination is regulated by telomere binding protein Cdc13 and the Ku complex. Together, our results suggest that coordination between DNA replication machinery, DNA damage signaling, DNA recombination machinery, and the telomere protein-DNA complex allows telomere recombination to repair telomeric ends in the absence of telomerase.
Mammalian DNAs cloned as artificial chromosomes in yeast (YACs) frequently are chimeras formed between noncontiguous DNAs. Using pairs of human and mouse YACs we examined the contribution of recombination during transformation or subsequent mitotic growth to chimeric YAC formation. The DNA from pairs of yeast strains containing homologous or heterologous YACs was transformed into a third strain under conditions typical for the development of YAC libraries. One YAC was selected and the presence of the second was then determined. Co-penetration of large molecules, as deduced from co-transformation of markers identifying the different YACs, was > 50%. In approximately half the cells receiving two homologous YACs, the YACs had undergone recombination. Co-transformation depends on recombination since it was reduced nearly 10-fold when the YACs were heterologous. While mitotic recombination between homologous YACs is nearly 100-fold higher than for yeast chromosomes, the level is still much lower than observed during transformation. To investigate the role of commonly occurring Alu repeats in chimera formation, spheroplasts were transformed with various human YACs and an unselected DNA fragment containing an Alu at one end and a telomere at the other. When unbroken YACs were used, between 1 and 6% of the selected YACs could incorporate the fragment as compared to 49% when the YACs were broken. We propose that Alu's or other commonly occurring repeats could be an important source of chimeric YACs. Since the frequency of chimeras formed between YACs or a YAC and an Alu-containing fragment was reduced when a rad52 mutant was the recipient and since intra-YAC deletions are reduced, rad52 and possibly other recombination-deficient mutants are expected to be useful for YAC library development.
We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional β-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus.
Yeast mating type is determined by the genotype at the mating type locus (MAT). In homothallic (self-fertile) Saccharomycotina such as Saccharomyces cerevisiae and Kluveromyces lactis, high-efficiency switching between a and α mating types enables mating. Two silent mating type cassettes, in addition to an active MAT locus, are essential components of the mating type switching mechanism. In this study, we investigated the structure and functions of mating type genes in H. polymorpha (also designated as Ogataea polymorpha). The H. polymorpha genome was found to harbor two MAT loci, MAT1 and MAT2, that are ∼18 kb apart on the same chromosome. MAT1-encoded α1 specifies α cell identity, whereas none of the mating type genes were required for a identity and mating. MAT1-encoded α2 and MAT2-encoded a1 were, however, essential for meiosis. When present in the location next to SLA2 and SUI1 genes, MAT1 or MAT2 was transcriptionally active, while the other was repressed. An inversion of the MAT intervening region was induced by nutrient limitation, resulting in the swapping of the chromosomal locations of two MAT loci, and hence switching of mating type identity. Inversion-deficient mutants exhibited severe defects only in mating with each other, suggesting that this inversion is the mechanism of mating type switching and homothallism. This chromosomal inversion-based mechanism represents a novel form of mating type switching that requires only two MAT loci.
The mating system of Saccharomycotina has evolved from the ancestral heterothallic system as seen in Yarrowia lipolytica to homothallism as seen in Saccharomyces cerevisiae. The acquisition of silent cassettes was an important step towards homothallism. However, some Saccharomycotina species that diverged from the common ancestor before the acquisition of silent cassettes are also homothallic, including Hansenula polymorpha. We investigated the structure and functions of the mating type locus (MAT) in H. polymorpha, and found two MAT loci, MAT1 and MAT2. Although MAT1 contains both a and α information, the results suggest that it functions as MATα. MATa is represented by MAT2, which is located at a distance of 18 kb from MAT1. The functional repression of MAT1 or MAT2 was required to establish a or α mating type identity in individual cells. The chromosomal location of MAT1 and MAT2 was found to influence their transcriptional status, with only one locus maintained in an active state. An inversion of the MAT intervening region resulted in the switching of the two MAT loci and hence of mating type identity, which was required for homothallism. This chromosomal inversion-based mechanism represents a novel form of mating type switching that requires two MAT loci, of which only one is expressed.
Homologous recombination (HR) is required for both genome maintenance and generation of diversity in eukaryotes and prokaryotes. This process initiates from single-stranded (ss) DNA and is driven by a universal recombinase, which promotes strand exchange between homologous sequences. The bacterial recombinase, RecA, is loaded onto ssDNA by recombinase loaders, RecBCD and RecFOR for genome maintenance. DprA was recently proposed as a third loader dedicated to genetic transformation. Here we assessed the role of RecFOR in transformation of the human pathogen Streptococcus pneumoniae. We firstly established that RecFOR proteins are not required for plasmid transformation, strongly suggesting that DprA ensures annealing of plasmid single-strands internalized in the process. We then observed no reduction in chromosomal transformation using a PCR fragment as donor, contrasting with the 10,000-fold drop in dprA- cells and demonstrating that RecFOR play no role in transformation. However, a ∼1.45-fold drop in transformation was observed with total chromosomal DNA in recFOR mutants. To account for this limited deficit, we hypothesized that transformation with chromosomal DNA stimulated unexpectedly high frequency (>30% of cells) formation of chromosome dimers as an intermediate in the generation of tandem duplications, and that RecFOR were crucial for dimer resolution. We validated this hypothesis, showing that the site-specific recombinase XerS was also crucial for dimer resolution. An even higher frequency of dimer formation (>80% of cells) was promoted by interspecies transformation with Streptococcus mitis chromosomal DNA, which contains numerous inversions compared to pneumococcal chromosome, each potentially promoting dimerization. In the absence of RecFOR and XerS, dimers persist, as confirmed by DAPI staining, and can limit the efficiency of transformation, since resulting in loss of transformant chromosome. These findings strengthen the view that different HR machineries exist for genome maintenance and transformation in pneumococci. These observations presumably apply to most naturally transformable species.
Homologous recombination (HR) is a widespread process which maintains genome integrity and promotes diversity. In bacteria, HR mends damaged DNA to ensure genome integrity and is also involved in transformation, a mechanism of horizontal gene transfer allowing acquisition of new genetic traits. HR is driven by recombinases, which are loaded onto single-stranded DNA by the recombinase loaders RecBCD and RecFOR for genome maintenance. DprA was recently proposed as another loader dedicated to transformation. During transformation, foreign DNA is taken up as single strands and integrated into the chromosome by HR. In this study, we show that RecFOR is not involved in transformation in Streptococcus pneumoniae. These results provide further support to the existence of different HR machineries dedicated to genetic transformation and genome maintenance in this pathogen. In addition, we show that transformation with chromosomal DNA generates chromosome dimers with unexpectedly high frequency, and that their resolution requires RecFOR and the site-specific recombinase XerS. In cells lacking these proteins, dimers persist and have a detrimental effect on the efficiency of transformation. Since the HR mechanisms leading to dimer formation are most likely conserved, this effect is presumably general to naturally transformable species.
The Ku heterodimer associates with the Saccharomyces cerevisiae telomere, where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel, its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions, we constructed DNA end binding–defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners, such as Sir4, a factor required for telomeric silencing, and TLC1, the RNA component of telomerase. Despite these interactions, we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection, length regulation, and silencing functions. In contrast to Ku null strains, the DNA end binding defective Ku mutants resulted in increased, rather than markedly decreased, imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap, where it promotes a specific architecture that is central to telomere function and maintenance.
The telomeric cap modulates telomere replication and prevents natural chromosome ends from being processed as DNA double-strand breaks (DSBs). In multiple species, including budding yeast, a detailed picture exists of the factors that comprise the telomeric cap and how they associate with telomeric DNA. It is less clear where to place Ku, a conserved heterodimer involved in multiple aspects of telomere biology and DSB repair. Although Ku avidly binds DNA ends, its access to telomeric ends might be restricted by telomere binding proteins and/or higher-order telomere structure. Ku might also be recruited to telomeres via its telomere-associated binding partners. Here, we address whether Ku loads directly onto telomeric ends and whether direct DNA binding is crucial for its telomeric functions. Using structure-guided mutagenesis, we generated end binding–defective yeast Ku heterodimers that retained the ability to associate with Ku's known telomeric binding partners. These end binding–defective heterodimers showed a dramatic reduction in telomere association and were defective for all of Ku's telomeric functions. Our findings indicate that Ku is indeed a component of the telomere cap and that its loading onto telomeric ends is crucial for its telomeric functions and, perhaps, a specific telomere architecture.
Lactic acid bacteria (LAB) play an important role in agricultural as well as industrial biotechnology. Development of improved LAB strains using e.g. library approaches is often limited by low transformation efficiencies wherefore one reason could be differences in the DNA methylation patterns between the Escherichia coli intermediate host for plasmid amplification and the final LAB host. In the present study, we examined the influence of DNA methylation on transformation efficiency in LAB and developed a direct cloning approach for Lactobacillus plantarum CD033. Therefore, we propagated plasmid pCD256 in E. coli strains with different dam/dcm-methylation properties. The obtained plasmid DNA was purified and transformed into three different L. plantarum strains and a selection of other LAB species.
Best transformation efficiencies were obtained using the strain L. plantarum CD033 and non-methylated plasmid DNA. Thereby we achieved transformation efficiencies of ~ 109 colony forming units/μg DNA in L. plantarum CD033 which is in the range of transformation efficiencies reached with E. coli. Based on these results, we directly transformed recombinant expression vectors received from PCR/ligation reactions into L. plantarum CD033, omitting plasmid amplification in E. coli. Also this approach was successful and yielded a sufficient number of recombinant clones.
Transformation efficiency of L. plantarum CD033 was drastically increased when non-methylated plasmid DNA was used, providing the possibility to generate expression libraries in this organism. A direct cloning approach, whereby ligated PCR-products where successfully transformed directly into L. plantarum CD033, obviates the construction of shuttle vectors containing E. coli-specific sequences, as e.g. a ColEI origin of replication, and makes amplification of these vectors in E. coli obsolete. Thus, plasmid constructs become much smaller and occasional structural instability or mutagenesis during E. coli propagation is excluded. The results of our study provide new genetic tools for L. plantarum which will allow fast, forward and systems based genetic engineering of this species.
Lactobacillus plantarum; DNA methylation; mrr; Direct cloning; Library efficiency; Reduced plasmid size
We marked a large number of yeast telomeres within their Y' regions by transforming strains with a fragment of Y' DNA into which the URA3 gene had been inserted. A few of the Ura+ transformants obtained were very unstable and were found to contain autonomously replicating URA3-marked circular Y' elements in high copy number. These marked extrachromosomal circles were capable of reintegrating into the chromosome at other telomeric locations. In contrast, most of the Ura+ transformants obtained were quite stable mitotically and were marked at bona fide chromosomal ends. These stable transformants gave rise to mitotically unstable URA3-marked circular Y' elements at a low frequency (up to 2.5%). The likelihood that such excisions and integrations represent a natural process in Saccharomyces cerevisiae is supported by our identification of putative Y' circles in untransformed strains. The transfer of Y' information among telomeres via a circular intermediate may be important for homogenizing the sequences at the ends of yeast chromosomes and for generating the frequent telomeric rearrangements that have been observed in S. cerevisiae.
Yeast mutants lacking telomerase are capable of maintaining telomeres by an alternate mechanism that depends on homologous recombination. We show here, by using Kluyveromyces lactis cells containing two types of telomeric repeats, that recombinational telomere elongation generates a repeating pattern common in most or all telomeres in survivors that retain both repeat types. We propose that these patterns arise from small circles of telomeric DNA being used as templates for rolling-circle gene conversion and that the sequence from the lengthened telomere is spread to other telomeres by additional, more typical gene conversion events. Consistent with this, artificially constructed circles of DNA containing telomeric repeats form long tandem arrays at telomeres when transformed into K. lactis cells. Mixing experiments done with two species of telomeric circles indicated that all of the integrated copies of the transforming sequence arise from a single original circular molecule.
In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing.
It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion.
These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The previously unrecognized prevalence and persistence of multiply transformed yeast cells have important implications for yeast library screens. The quantitative information described herein should increase awareness of this issue, and the rapid sequencing approach developed for these studies should be widely useful for identifying multiple vector transformants and avoiding complications associated with cells that have acquired more than one unique plasmid.