Integrative transformation of the zygomycete Rhizomucor pusillus by homologous recombination

 For development of a homologous transformation system for the zygomycete fungus, Rhizomucor pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R. pusillus IFO 4578 by the DNA-probing method with the leuA sequence of Mucor circinelloides as probe. The nucleotide sequence revealed that leuA of R. pusillus encoded a 755-amino-acid protein of 82.5 kDa with no intron. The leuA gene on pUC19 (plasmid pRPLeu10) was introduced by polyethyleneglycol-assisted transformation into protoplasts of a leuA ^- mutant of R. pusillus that was obtained by UV mutagenesis. Transformation under optimal conditions yielded 20 Leu⁺ transformants (μg pRPLeu10 DNA)^-1 (1×10⁶ viable protoplasts)^-1. Blot analysis of DNA from the transformants showed that the pRPLeu10 sequence was integrated into the genome by homologous recombination at the leuA locus. Received: 2 October 1995/Received last revision: 5 December 1995/Accepted: 11 December 1995     

Optimization of electroporation conditions for Arthrobacter with plasmid PART2

A prerequisite for genetic studies of Arthrobacter is a high efficiency transformation system that allows for DNA transfer, transposon mutagenesis, and expression of specific genes. In this study, we develop a detailed electroporation method through a systematic examination of the factors involved in the entire electroporation process. Key features of this procedure, including the addition of penicillin to cells during the early log phase of growth and the presence of 0.5 M sorbitol in the electroporation and recovery media, produced the greatest increases in transformation efficiency and consistency of results. The transformation rate also varied depending on the electrical parameters, DNA concentration, and recovery time period. Using optimum conditions, we generally achieved an efficiency of 6.8 × 10⁷ transformants per microgram of PART2 for Arthrobacter sp. A3. This protocol was also successfully applied to other Arthrobacter species. Therefore, we conclude that the proposed method is rapid, simple and convenient, which allows a transformation trial to be accomplished in minutes.     

Canthaxanthin production with modified Mucor circinelloides strains

Canthaxanthin is a natural diketo derivative of β-carotene primarily used by the food and feed industries. Mucor circinelloides is a β-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. In this study, the β-carotene ketolase gene (crtW) of the marine bacterium Paracoccus sp. N81106 fused with fungal promoter and terminator regions was integrated into the M. circinelloides genome to construct stable canthaxanthin-producing strains. Different transformation methods including polyethylene glycol-mediated transformation with linear DNA fragments, restriction enzyme-mediated integration and Agrobacterium tumefaciens-mediated transformation were tested to integrate the crtW gene into the Mucor genome. Mitotic stability, site of integration and copy number of the transferred genes were analysed in the transformants, and several stable strains containing the crtW gene in high copy number were isolated. Carotenoid composition of selected transformants and effect of culturing conditions, such as temperature, carbon sources and application of certain additives in the culturing media, on their carotenoid content were analysed. Canthaxanthin-producing transformants were able to survive at higher growth temperature than the untransformed strain, maybe due to the effect of canthaxanthin on the membrane fluidity and integrity. With the application of glucose, trehalose, dihydroxyacetone and l-aspartic acid as sole carbon sources in minimal medium, the crtW-expressing M. circinelloides strain, MS12+pCA8lf/1, produced more than 200 μg/g (dry mass) of canthaxanthin.     

Transformation of a methionine auxotrophic mutant of Mucor circinelloides by direct cloning of the corresponding wild type gene

Summary A transformation system has been developed for Mucor circinelloides, by direct cloning of a wild-type methionine gene that complements the auxotrophic mutation. The marker gene isolated was associated with an autonomous replication sequence (ARS) functional in this zygomycete. Southern hybridisation analyses of transformants showed sequence homology both with vector DNA and with Mucor wild-type DNA. The transformation frequency (up to 6000 per g DNA) and the mitotic instability of the transformed cells were studied. The hybridisation pattern of undigested DNA from the transformants suggests that the inserts contain a novel autonomous replication element for this filamentous fungus.     

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per μg of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per μg of vector DNA. However, a 380-fold improvement (3,417 transformants per μg of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.     

A simple bacterial transformation method using magnesium- and calcium-aminoclays

An efficient and user-friendly bacterial transformation method by simple spreading cells with aminoclays was demonstrated. Compared to the reported transformation approaches using DNA adsorption or wrapping onto (in)organic fibers, the spontaneously generated clay-coated DNA suprastructures by mixing DNA with aminoclay resulted in transformants in both Gram-negative (Escherichia coli) and Gram-positive cells (Streptococcus mutans). Notably, the wild type S. mutans showed comparable transformation efficiency to that of the E. coli host for recombinant DNA cloning. This is a potentially promising result because other trials such as heat-shock, electroporation, and treatment with sepiolite for introducing DNA into the wild type S. mutans failed. Under defined conditions, the transformation efficiency of E. coli XL1-Blue and S. mutans exhibited ~ 2 × 10⁵ and ~ 6 × 10³ CFU/μg of plasmid DNA using magnesium-aminoclay. In contrast, transformation efficiency was higher in S. mutans than that in E. coli XL1-Blue for calcium-aminoclay. It was also confirmed that each plasmid transformed into E. coli and S. mutans was stably maintained and that they expressed the inserted gene encoding the green fluorescent protein during prolonged growth of up to 80 generations.     

Transformation of Heat-Treated Clostridium acetobutylicum Protoplasts with pUB110 Plasmid DNA

Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55°C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44°C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55°C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55°C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44°C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 55°C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heatsensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycin-resistant (Km^r) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Km^r transformants.     

Natural transformation as a mechanism of horizontal gene transfer among environmental Aeromonas species

Aeromonas species are common inhabitants of aquatic environments and relevant as human pathogens. Their potential as pathogens may be related in part to lateral transfer of genes associated with toxin production, biofilm formation, antibiotic resistance, and other virulence determinants. Natural transformation has not been characterized in aeromonads. DNA from wild-type, prototrophic strains that had been isolated from environmental sources was used as donor DNA in transformation assays with auxotrophs as the recipients. Competence was induced in 20% nutrient broth during the stationary phase of growth. Optimal transformation assay conditions for one chosen isolate were in Tris buffer with magnesium or calcium, pH 5–8, and a saturating concentration of 0.5 μg of DNA per assay (3.3 ng of DNA μl⁻¹) at 30 °C. Sodium was also required and could not be replaced with ammonium, potassium, or lithium. The maximal transformation frequency observed was 1.95 × 10⁻³ transformants (recipient cell)⁻¹. A survey of environmental Aeromonas auxotrophic recipients (n = 37), assayed with donor DNA from other wild-type environmental aeromonads under optimal assay conditions, demonstrated that 73% were able to act as recipients, and 100% were able to act as donors to at least some other aeromonads. Three different transformation groups were identified based on each isolates’ ability to transform other strains with its DNA. The transformation groups roughly corresponded to phylogenetic groups. These results demonstrate that natural transformation is a general property of Aeromonas environmental isolates with implications for the genetic structures of coincident Aeromonas populations.     

Simultaneous enrichment of cereals with polyunsaturated fatty acids and pigments by fungal solid state fermentations

Four Mucor strains were tested for their ability to grow on four cereal substrates and enriched them with gamma-linolenic acid (GLA) and β-carotene. M. circinelloides CCF-2617 as the best producer accumulated of both GLA and β-carotene in high amounts during utilization of rye bran/spent malt grains (3:1). The first growth phase was characterized by rapid GLA biosynthesis, while distinct β-carotene formation was found in the stationary fungal growth. Therefore various cultivation conditions were tested in order to optimize the yield of either GLA or β-carotene. The fungus grown on cereal substrate supplemented with glucose produced maximal 8.5 mg β-carotene and 12.1 g GLA in 1 kg fermented substrate, respectively. On the other hand, the highest amount of GLA in the fermented substrate (24.2 g/kg) was achieved when 30% of sunflower oil was employed to the substrate. Interestingly, β-carotene biosynthesis was completely inhibited when either whey or linseed oil were added to the substrate.     

Cloning of the <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene and its heterologous expression in <Emphasis Type="Italic">Mucor circinelloides</Emphasis>

In this study, the gene hmgR encoding the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) was cloned and characterized in the zygomycete fungus Rhizomucor miehei. The hmgR gene comprises a total of 3,585 bp including the coding sequence of a 1,058 amino acids length putative protein and five introns (137, 83, 59, 60 and 69 bp in length) dispersed in the whole coding region. Southern hybridization analysis revealed that the gene is present only in one copy in the R. miehei genome. The isolated Rhizomucor gene was expressed in the related fungus, Mucor circinelloides. Transformants harbouring the Rhizomucor hmgR gene in an autoreplicative plasmid proved to be more tolerant to statins (e.g. lovastatin, simvastatin, and fluvastatin), the competitive inhibitors of the HMG-CoA reductase, than the original M. circinelloides strain. At the same time, heterologous expression of the Rhizomucor hmgR did not affect the carotenoid production of M. circinelloides.     

Ultrasensitive DNA sensor based on gold nanoparticles/reduced graphene oxide/glassy carbon electrode

We have designed a simple and novel electrochemical biosensor based on glassy carbon electrode (GCE) for DNA detection. GCE was modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs) by the electrochemical method, which is helpful for immobilization of thiolated bioreceptors. The electrode modification processes were characterized by scanning electron microscopy (SEM) and electrochemical methods. Then a single-stranded DNA (ssDNA) probe for BRCA1 5382 insC mutation detection was immobilized on the modified electrode for a specific time. The experimental conditions, such as probe immobilization time and target DNA (complementary DNA) hybridization time and temperature with probe DNA, were optimized using electrochemical methods. The electrochemical response for DNA hybridization and synthesis was measured using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) methods. The calibration graph contains two linear ranges; the first part is in the range of 3.0 × 10⁻²⁰ to 1.0 × 10⁻¹² M, and the second segment part is in the range of 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ M. The biosensor showed excellent selectivity for the detection of the complementary sequences from noncomplementary sequences, so it can be used for detection of breast cancer.     

High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei

The cellobiase gene from Aspergillus niger was cloned and connected with the strong promoter Pcbh1 from Trichoderma reesei to construct a recombinant plasmid pHB9 with the hygromycin B resistance marker. The plasmid was transformed into conidia of T. reesei using the modified PEG-CaCl₂ method. Main factors effecting the transformation were discussed and about 99-113 transformants/μg DNA could be obtained under optimal conditions. It was found that the molecular mass of the recombinant cellobiase was about 120 kDa by SDS-PAGE analysis. The activity of cellobiase could reach 5.3 IU/ml after 48 h fermentation, which was as high as 106 times compared with that of the host strain. Meanwhile, the filter paper activity of recombinant T. reesei was 1.44-fold of the host strain. Saccharification of corncob residue with the crude enzyme showed that the hydrolysis yield (84.2%) of recombinant T. reesei was 21% higher than that (69.5%) of the host strain.     

Remarkable photocytotoxicity in hypoxic HeLa cells by a dipyridophenazine copper(II) Schiff base thiolate

Copper(II) complexes [Cu(satp)(L)] (1-3) of a Schiff base thiolate (salicylidene-2-aminothiophenol, H₂satp) and phenanthroline bases (L), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq in 2) and dipyrido[3,2-a:2′,3′-c]phenazine (dppz in 3), were prepared, characterized and their anaerobic DNA photocleavage activity and hypoxic photocytotoxicity studied. The redox active complexes show the Cu(II)-Cu(I) couple near − 0.5 V for 1 and near 0.0 V vs. SCE (saturated calomel electrode) for 2 and 3. The one-electron paramagnetic complexes (~ 1.85 μ_B) are avid DNA binders giving K_b values within 1.0 × 10⁵ − 8.0 × 10⁵ M^− 1. Thermal melting and viscosity data along with molecular docking calculations suggest DNA groove and/or partial intercalative binding of the complexes. The complexes show anaerobic DNA cleavage activity in red light under argon via type-I pathway, while DNA photocleavage in air proceeds via hydroxyl radical pathway. The DFT (density functional theory) calculations reveal a thyil radical pathway for the anaerobic DNA photocleavage activity and suggest the possibility of generation of a transient copper(I) species due to bond breakage between the copper and sulfur to generate the thyil radical. An oxidation of the copper(I) species is likely by oxygen in an aerobic medium or by the buffer medium in an anaerobic condition. Complex 3 exhibits significant photocytotoxicity in HeLa cells (IC₅₀ = 8.3(± 1.0) μM) in visible light, while showing lower dark toxicity (IC₅₀ = 17.2(± 1.0) μM). A significant reduction in the dark toxicity is observed under hypoxic cellular conditions (IC₅₀ = 30.0(± 1.0) μM in dark), while retaining its photocytotoxicity (IC₅₀ = 8.0(± 1.0) μM).     

Single-strand DNA translation initiation step analyzed by Isothermal Titration Calorimetry

Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K_d = 3.62 ± 2.1 × 10⁻⁸ M) or the RNA corresponding sequence (K_d = 2.7 ± 0.82 × 10⁻⁸ M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.     

Effect of the central metal ion on the cleavage of DNA by [M(TPA)Cl]ClO4 complexes (M = Co, Cu and Zn, TPA = tris(2-pyridylmethyl)amine): An efficient artificial nuclease for DNA cleavage

[M(TPA)Cl]ClO₄·nH₂O complexes (1: M = Co^II, n = 0; 2: M = Cu^II, n = ½; 3: M = Zn^II, n = 0) where TPA = tris(2-pyridylmethyl)amine, were synthesized and structurally characterized. The molecular structure of [Cu(TPA)Cl]ClO₄·½H₂O was determined by single crystal X-ray crystallography. In aqueous solution, the complex ions [M(TPA)Cl]⁺ (M = Co^II or Cu^II) are hydrolyzed to the corresponding aqua species [M(TPA)(H₂O)]²⁺. In contrast to the TBP [Cu(TPA)(H₂O)]²⁺, the corresponding TBP cobalt(II) species showed severe distortion towards tetrahedral geometry. The interactions of the three complexes with DNA have been investigated at pH 7.0 (1.0 mM Tris-Cl buffer) and 37 °C. Significant DNA cleavages were obtained for complexes 1 and 2, whereas complex 3 did not show any detectable cleavage for DNA. Under pseudo Michaelis-Menten kinetic conditions, the kinetic parameters k_cat and K_M were determined as k_cat = 6.59 h⁻¹ and K_M = 2.20 × 10⁻⁴ M for 1 and the corresponding parameters for 2 are k_cat = 5.7 × 10⁻² h⁻¹ and K_M = 6.9 × 10⁻⁵ M, and the reactivity of the complexes in promoting the cleavage of DNA decreases in the order 1 > 2 ? 3. The rate enhancements for the DNA cleavage by 1 and 2 correspond to 1.8 × 10⁸ and 1.6 × 10⁶, respectively, over the non-catalyzed DNA. The reactivity of the two complexes was discussed in relation to other related artificial nucleases.     

Quantitative PCR method for detection of mycoplasma spp. DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii)

Mycoplasma agassizii and M. testudineum have been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Because microbiological culture methods have proven difficult to employ in wild desert tortoises, our goal was to develop a sensitive and specific qPCR method for detecting and quantifying mycoplasma DNA in nasal lavage fluid collected in the field. Primers for 16S ribosomal RNA gene sequences specific for M. agassizii and M. testudineum were designed, together with primers that recognize conserved sequences of both microorganisms. Standard curves generated with DNA extracted from known numbers of mycoplasma cells revealed a lower detection limit of approximately 5 fg. The qPCR method did not recognize normal flora DNA, and nasal lavage fluid contained no interfering substances. Nasal lavage samples collected from 20 captive desert tortoises housed at the Desert Tortoise Conservation Center (Clark County, Nevada, USA) revealed the presence of M. agassizii DNA in 100% of the tortoises. Concentrations ranged from a low of 6 pg ml^− 1 to a high of 72,962 pg ml^− 1. Only one of the tortoises was positive for M. testudineum. Interestingly, not all of the qPCR positive tortoises showed evidence of seroconversion, suggesting that they were colonized but not infected. This new quantitative method will provide a critical tool for managing threatened populations of the desert tortoise.     

Carbon nanotube-enhanced DNA biosensor for DNA hybridization detection using manganese(II)-Schiff base complex as hybridization indicator

A Mn(II) complex, MnL (L = sodium (E)-3-((1-carboxyethylimino)methyl)-4-hydroxybenzenesulfonate), was synthesized and characterized using elemental analysis and IR spectroscopy. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction between MnL and salmon sperm DNA. It was revealed that MnL presented high electrochemical activity on glassy carbon electrode (GCE), and it could be intercalated into the double helices of double-stranded DNA (dsDNA). Using MnL as the hybridization indicator, a novel and sensitive electrochemical DNA biosensor based on multiwall carbon nanotubes functionalized with carboxyl groups (MWCNTs-COOH, on which DNA probes were covalently immobilized) was prepared. The target single-stranded DNA (ssDNA) could be quantified ranging from 6.7 × 10⁻¹⁰ M to 8.4 × 10⁻⁹ M with good linearity (r = 0.9922). A detection limit of 1.4 × 10⁻¹⁰ M (3σ, n = 9) was achieved.     

Efficient ligation and cloning of DNA fragments with 2-bp overhangs

Various methods of ligation are currently available and routinely used by molecular biologists, such as blunt end ligation, cohesive end (two and four overhangs), and ligation of Taq polymerase-derived products. However, there is no efficient method for the cloning of DNA fragments with 2-bp overhangs. We present a simple method for the efficient ligation of DNA fragments with 2-bp overhanging ends, ranging in size from 0.7 to 2.5 kbp. Our method involves the initial heating and flash freezing of the vector-insert DNA mix, and a subsequent unique ligation reaction. This method provides a new molecular biology tool for researchers.     

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.