DNA-mediated transformation system in a bipolar basidiomycete, <Emphasis Type="Italic">Pholiota microspora</Emphasis> (<Emphasis Type="Italic">P. nameko</Emphasis>)

We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was about 88.8 transformants/μg pMBsip2 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the efficiency of hygromycin B resistance transformation was about 122.4 transformants/μg pMBhph1 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more.     

Analysis of β-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

The anti-cancer drug taxol binds to β-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC₅₀ greater than 11.7?μM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC₅₀ 0.1?μM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding β-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of β-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of β-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [³H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [³H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various β-tubulin sequences showed that β-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but β-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and β-tubulin.     

A versatile PCR marker for pungency in Capsicum spp.

Pungency in Capsicum spp. is an important quality trait for pepper breeding. The perception of pungency in pepper is due to the presence of a group of compounds named capsaicinoids, only found within the Capsicum genus. How pungency is controlled at genetic and molecular levels has not been completely elucidated. The use of molecular markers to assess pungency trait is required for molecular breeding, despite the difficulty of development of universal markers for this trait. In this work, a DNA sequence possibly related to pungency with a high similarity to Pun1 locus was studied, and sequence analysis of this homolog revealed a 15?bp deletion in non-pungent pepper accessions. An allele-specific pair of primers was designed and specific fragments of 479?bp from non-pungent and 494?bp from pungent accessions were obtained. Polymorphism of this marker, named MAP1, was tested in a wide range of accessions, belonging to several Capsicum species, including pungent and non-pungent accessions of C. annuum L., and pungent accessions of C. chinense, C. baccatum, C. frutescens, C. pubescens, C. galapagoense, C. eximium, C. tovarii, C. cardenasii, and C. chacoense. All these Capsicum accessions were correctly discriminated. The marker suitability to assess pungency in domesticated and wild Capsicum species was demonstrated, and therefore it will be very useful in marker assisted selection (MAS). Moreover, MAP1 was located in a saturated pepper linkage map and its possible relationship with the Pun1 locus has been discussed. Among the available markers for this complex quality trait, the marker developed in this study is the most universal so far.     

Veluticeps microspora sp. nov. and V. ambigua new to Asia with a preliminary phylogenetic study on the genus

Veluticeps microspora sp. nov. and V. ambigua new to Asia are described and illustrated based on morphological and molecular characters. V. microspora was collected from Yunnan Province, southwestern China, and is characterized by its special anatomical structure (without tomentum, cutis, and subiculum), simple septate hyphae, and small basidiospores. V. ambigua was collected from Jilin Province, northeastern China, and is reported in Asia for the first time. The molecular phylogeny of Veluticeps was preliminarily studied based on the analysis of ITS sequences from 18 species of Gloeophyllales. Two distinct clades of Veluticeps were formed in the phylogenetic tree. While V. microspora and V. ambigua clustered together to form a strongly supported clade, V. abietina, V. fimbriata, and V. berkeleyi grouped with Chaetodermella luna to form another clade.     

New PCR-based sequence-tagged site marker for bacterial blight resistance gene Xa38 of rice

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae, is a major disease of rice managed largely through the deployment of resistance genes. Xa38, a BB resistance gene identified from Oryza nivara acc. IRGC 81825, was mapped on chromosome 4L in a 38.4-kb region. The closely linked markers for this gene, identified earlier, were simple sequence repeat marker RM17499 and sequence-tagged site markers developed from loci Os04g53060 and Os04g53120. Marker Os04g53060 is dominant while the other two markers show smaller size differences difficult to resolve accurately on agarose gel. Based on gene annotation, three nucleotide binding site?Cleucine-rich repeat genes present in the target region were cloned from O. nivara and sequenced. One of the loci, LOC_Os04g53050, had a 48-base-pair deletion in O. nivara acc. IRGC 81825 compared to the cultivated rice. Primers were designed around the deletion and the resulting marker is codominant and easy to score in agarose gel. The newly designed marker co-segregated with Xa38, amplifying products of 269?bp in O. nivara and 317?bp in cultivated rice. This marker could be more useful for marker-assisted selection than ones reported earlier.     

Asymmetric Effects of Deletions and Substitutions on High Negative Interference in Coliphage Lambda

Experiments have been performed to help clarify the role of nonhomologies in phage λ recombination. Three-factor crosses were carried out, and the frequencies of single and double recombinants in the two adjoining intervals were compared when the central marker was either a double point mutation (v1v3) or deletion (rex-cI deletion) or nonhomologous substitution (imm434). In all cases the lefthand marker was a bio substitution (Fec^- phenotype, which does not permit plating on recA^-), and the righthand marker was an amber mutation in gene O. Experiments were performed in all four possible arrangements of the central and rightward markers, while selecting for the Fec⁺ phenotype on the recA^- host. As anticipated, high negative interference (HNI) was observed with point mutations, but when the central marker was a substitution nonhomology, HNI was reduced about tenfold. Surprisingly, when the central marker was a simple deletion, a dramatic asymmetry in results was observed, with HNI being exhibited only when the central deletion marker was acquired by the double recombinant. These results indicate that under normal conditions (red⁺, gam⁺, rec⁺) and with noninhibited DNA replication, recombination in coliphage λ entails a highly asymmetric step that could be at the level of strand transfer or mismatch repair.     

Polypeptone Induces Dramatic Cell Lysis in ura4 Deletion Mutants of Fission Yeast

Polypeptone is widely excluded from Schizosaccharomyces pombe growth medium. However, the reasons why polypeptone should be avoided have not been documented. Polypeptone dramatically induced cell lysis in the ura4 deletion mutant when cells approached the stationary growth phase, and this phenotype was suppressed by supplementation of uracil. To determine the specificity of this cell lysis phenotype, we created deletion mutants of other genes involved in de novo biosynthesis of uridine monophosphate (ura1, ura2, ura3, and ura5). Cell lysis was not observed in these gene deletion mutants. In addition, concomitant disruption of ura1, ura2, ura3, or ura5 in the ura4 deletion mutant suppressed cell lysis, indicating that cell lysis induced by polypeptone is specific to the ura4 deletion mutant. Furthermore, cell lysis was also suppressed when the gene involved in coenzyme Q biosynthesis was deleted. This is likely because Ura3 requires coenzyme Q for its activity. The ura4 deletion mutant was sensitive to zymolyase, which mainly degrades (1,3)-beta-D glucan, when grown in the presence of polypeptone, and cell lysis was suppressed by the osmotic stabiliser, sorbitol. Finally, the induction of cell lysis in the ura4 deletion mutant was due to the accumulation of orotidine-5-monophosphate. Cell wall integrity was dramatically impaired in the ura4 deletion mutant when grown in the presence of polypeptone. Because ura4 is widely used as a selection marker in S. pombe, caution needs to be taken when evaluating phenotypes of ura4 mutants.     

Adaptation of the Yeast URA3 Selection System to Gram-Negative Bacteria and Generation of a ΔbetCDE Pseudomonas putida Strain

A general procedure for efficient generation of gene knockouts in gram-negative bacteria by the adaptation of the Saccharomyces cerevisiae URA3 selection system is described. A Pseudomonas putida strain lacking the URA3 homolog pyrF (encoding orotidine-5′-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the pyrF gene and promoter, a conditional origin of replication (oriR6K), an origin of transfer (mobRK2), and an antibiotic selection marker flanked by multiple sites for cloning appropriate DNA segments. The versatility of pyrF as a selection system, allowing both positive and negative selection of the marker, and the robustness of the selection, where pyrF is associated with uracil prototrophy and fluoroorotic acid sensitivity, make this setup a powerful tool for efficient homologous gene replacement in gram-negative bacteria. The system has been instrumental for complete deletion of the P. putida choline-O-sulfate utilization operon betCDE, a mutant which could not be produced by any of the other genetic strategies available.     

Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration

Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens. This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167, sigF, and sigK) or disruptions (ca_p0157 and sigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.     

Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii.     

Chalmicrin,a mannitol ether of methylated monocyclofarnesol,from Chalara microspora

The structure of chalmicrin, isolated from Chalara microspora, has been determined. It consists of 10-methyl-trans-monocyclofarnesol linked to d-mannitol as an allyl ether.     

Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker

With increasing application of Hansenula polymorpha in fundamental research and biotechnology, many more genetic manipulations are required. However, these have been restricted for the finiteness of selectable markers. Here, MazF, a toxin protein from Escherichia coli, was investigated as a counter-selectable marker in H. polymorpha. The lethal effect of MazF on yeast cells suggested that it is a candidate for counter-selection in H. polymorpha. Markerless or scarless gene deletion in H. polymorpha was conducted based on selectable markers cassette mazF-zeoR, in which the zeocin resistance cassette and mazF expression cassette were used as positive and counter-selectable markers, respectively. For markerless deletion, the target region can be replaced by CYC1TT via two-step homologous recombination. For scarless deletion, the innate upstream region (5′UP) of target genes rather than CYC1TT mediates homologous recombination to excise both selectable markers and 5′ sequence of target genes. Moreover, scarless deletion can be accomplished by using short homologous arms for the effectiveness of mazF as a counter-selectable marker. The applicability of the strategies in markerless or scarless deletion of PEP4, LEU2, and TRP1 indicates that this study provides easy, time-efficient, and host-independent protocols for single or multiple genetic manipulations in H. polymorpha.     

An efficient gene disruption method using a positive–negative split-selection marker and <Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-mediated transformation for <Emphasis Type="Italic">Nomuraea rileyi</Emphasis>

Targeted gene disruption via Agrobacterium tumefaciens-mediated transformation (ATMT) and homologous recombination is the most common method used to identify and investigate the functions of genes in fungi. However, the gene disruption efficiency of this method is low due to ectopic integration. In this study, a high-efficiency gene disruption strategy based on ATMT and the split-marker method was developed for use in Nomuraea rileyi. The β-glucuronidase (gus) gene was used as a negative selection marker to facilitate the screening of putative transformants. We assessed the efficacy of this gene disruption method using the NrCat1, NrCat4, and NrPex16 genes and found that the targeting efficiency was between 36.2 and 60.7%, whereas the targeting efficiency using linear cassettes was only 1.0–4.2%. The efficiency of negative selection assays was between 64.1 and 82.3%. Randomly selected deletion mutants exhibited a single copy of the hph cassette. Therefore, high-throughput gene disruption could be possible using the split-marker method and the majority of ectopic integration transformants can be eliminated using negative selection markers. This study provides a platform to study the function of genes in N. rileyi.     

Functional molecular markers and high-resolution melting curve analysis of low phytic acid mutations for marker-assisted selection in rice

Phytic acid (PA, myo-inositol-1,2,3,4,5,6-hexakis-phosphate) and its salt form (phytate) are the principal storage forms of phosphorus in cereal grains. Since PA and phytates cannot be efficiently digested by monogastric animals, the abundance of PA in cereal and legume grains causes nutritional and environmental problems. The present study aimed at developing breeder-friendly functional molecular markers of five low phytic acid (LPA) mutant alleles of three rice (Oryza sativa L.) genes: viz., XQZ-lpa (a 1,475-bp deletion) and KBNT-lpa (a C→T single nucleotide polymorphism [SNP]) of LOC_Os02g57400, Z9B-lpa (a 6-bp deletion) and MH-lpa (a 1-bp deletion) of LOC_Os04g55800, and XS-lpa (a C→T SNP) of LOC_Os03g04920. First, markers for gel-based length polymorphism analysis were developed: viz., two insertion–deletion markers for XQZ-lpa and Z9B-lpa, two cleaved amplified polymorphic sequence (CAPS) markers for KBNT-lpa and XS-lpa, and one derived CAPS marker for MH-lpa. Second, the high-resolution melting (HRM) curve analysis method was explored for distinguishing plants with wild-type (WT) and LPA alleles (except XQZ-lpa). Plants of genotypes with homozygous mutant allele and WT, and with heterozygous alleles, could be directly differentiated by HRM for KBNT-lpa, XS-lpa and MH-lpa; only heterozygous individuals could be directly distinguished from homozygous WT and mutant plants for Z9B-lpa. However, by adding 15 % WT DNA templates to test samples before PCR, amplicons of three genotypes of the Z9B-lpa allele could also be differentiated by HRM analysis. Third, it was demonstrated that these markers could be effectively used for marker-assisted selection of LPA rice, and breeding lines with two non-allelic LPA mutations were developed with PA contents significantly lower than their respective parental LPA lines. Taken together, the present study developed functional molecular markers for efficient selection of LPA plants and demonstrated that double mutant LPA lines with significantly lower PA levels than primary LPA mutants (with single mutations) could be developed by pyramiding two non-allelic LPA mutations.     

TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes^1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons^1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene⁴. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)⁵ (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism⁶ and other representative of the genus Vibrio such as V. fischeri⁷. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region⁸. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences⁵. Several optimization steps concerning the initial protocol of chitin-induced natural transformation⁶ are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes⁸, the donation of PCR-derived DNA as transforming material⁹, and the addition of FLP-recombination target sites (FRT)⁵. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase¹⁰.     

Application of counter-selectable marker PIGA in engineering designer deletion cell lines and characterization of CRISPR deletion efficiency

The CRISPR/Cas9 system is a technology for genome engineering, which has been applied to indel mutations in genes as well as targeted gene deletion and replacement. Here, we describe paired gRNA deletions along the PIGA locus on the human X chromosome ranging from 17 kb to 2 Mb. We found no compelling linear correlation between deletion size and the deletion efficiency, and there is no substantial impact of topologically associating domains on deletion frequency. Using this precise deletion technique, we have engineered a series of designer deletion cell lines, including one with deletions of two X-chromosomal counterselectable (negative selection) markers, PIGA and HPRT1, and additional cell lines bearing each individual deletion. PIGA encodes a component of the glycosylphosphatidylinositol (GPI) anchor biosynthetic apparatus. The PIGA gene counterselectable marker has unique features, including existing single cell level assays for both function and loss of function of PIGA and the existence of a potent counterselectable agent, proaerolysin, which we use routinely for selection against cells expressing PIGA. These designer cell lines may serve as a general platform with multiple selection markers and may be particularly useful for large scale genome engineering projects such as Genome Project-Write (GP-write).     

Schizosaccharomyces pombe KAT5 contributes to resection and repair of a DNA double-strand break

Chromatin remodeling is essential for effective repair of a DNA double-strand break (DSB). KAT5 (Schizosaccharomyces pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that coordinates various DNA damage response activities at a DNA DSB, including histone remodeling and activation of the DNA damage checkpoint. In S. pombe, mutations in mst1⁺ causes sensitivity to DNA damaging drugs. Here we show that Mst1 is recruited to DSBs. Mutation of mst1⁺ disrupts recruitment of repair proteins and delays resection. These defects are partially rescued by deletion of pku70, which has been previously shown to antagonize repair by homologous recombination (HR). These phenotypes of mst1 are similar to pht1-4KR, a nonacetylatable form of histone variant H2A.Z, which has been proposed to affect resection. Our data suggest that Mst1 functions to direct repair of DSBs toward HR pathways by modulating resection at the DSB.     

pyrF as a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola

The pathophysiology of Treponema denticola, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate that pyrF, a gene encoding orotidine-5′-monophosphate decarboxylase, can be used as a counterselectable marker in T. denticola to construct marker-free mutants. T. denticola is susceptible to 5-fluoroorotic acid (5-FOA). To establish a pyrF-based counterselectable knockout system in T. denticola, the pyrF gene was deleted. The deletion conferred resistance to 5-FOA in T. denticola. Next, a single-crossover mutant was constructed by reintroducing pyrF along with a gentamicin resistance gene (aacC1) back into the chromosome of the pyrF mutant at the locus of choice. In this study, we chose flgE, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. The obtained single-crossover mutant (named FlgEⁱⁿ) regained the susceptibility to 5-FOA. Finally, FlgEⁱⁿ was plated on solid agar containing 5-FOA. Numerous colonies of the 5-FOA-resistant mutant (named FlgE^out) were obtained and characterized by PCR and Southern blotting analyses. The results showed that the flgE gene was deleted and FlgE^out was free of selection markers (i.e., pyrF and aacC1). Compared to previously constructed flgE mutants that contain an antibiotic selection marker, the deletion of flgE in FlgE^out has no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity of T. denticola.