Efficient DNA transformation of Bradyrhizobium japonicum by electroporation

Intact cells of Bradyrhizobium japonicum USDA 110 were transformed with a 30-kilobase plasmid to efficiencies of 10(6) to 10(7) transformants per microgram by high-voltage electroporation. The technique was reliable and simple, with single colonies arising from transformed cells within 5 days of antibiotic selection. Plasmid DNA from B. japonicum transformed the Bradyrhizobium (Arachis) sp. with high efficiency, while the same plasmid extracted from Escherichia coli transformed B. japonicum at very low efficiency. The electrical conditions that resulted in the highest efficiencies were high voltage (10.5 to 12.5 kV/cm) and short pulse length (6 to 7 ms). A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude; saturation appeared to begin between 120 ng/ml and 1.2 micrograms/ml. This novel method of transformation should enhance B. japonicum genetic research by providing a valuable alternative to conjugal mating, which is currently the only efficient, widely used means of introducing DNA into this organism.     

Efficient DNA transformation of Bradyrhizobium japonicum by electroporation.

Intact cells of Bradyrhizobium japonicum USDA 110 were transformed with a 30-kilobase plasmid to efficiencies of 10(6) to 10(7) transformants per microgram by high-voltage electroporation. The technique was reliable and simple, with single colonies arising from transformed cells within 5 days of antibiotic selection. Plasmid DNA from B. japonicum transformed the Bradyrhizobium (Arachis) sp. with high efficiency, while the same plasmid extracted from Escherichia coli transformed B. japonicum at very low efficiency. The electrical conditions that resulted in the highest efficiencies were high voltage (10.5 to 12.5 kV/cm) and short pulse length (6 to 7 ms). A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude; saturation appeared to begin between 120 ng/ml and 1.2 micrograms/ml. This novel method of transformation should enhance B. japonicum genetic research by providing a valuable alternative to conjugal mating, which is currently the only efficient, widely used means of introducing DNA into this organism.     

Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

ABSTRACT: BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.     

High efficiency electroporation of intact Corynebacterium glutamicum cells

High-frequency electroporation of whole Corynebacterium glutamicum cells without enzymatic pretreatment was achieved. Under optimized conditions concerning growth stage, washing of cells, cell concentration and pulse parameter transformation efficiencies of far more than 10(7) transformants per microgram pWST4B plasmid DNA were reached. Using electroporation, linearised and subsequently religated plasmid as well as chimeric ligase reaction products were directly introduced into C. glutamicum with reasonable efficiencies. Electrotransformation efficiency was reduced about 10(5)-fold for plasmid DNA cycled through E. coli JM83. Restriction deficient mutants of C. glutamicum were isolated which could be efficiently transformed with foreign DNA.     

Efficient electrotransformation of Enterococcus hirae with a new Enterococcus-Escherichia coli shuttle vector

In the present study, an Enterococcus-Escherichia coli shuttle vector, pC3, was constructed that allows efficient transformation by electroporation of Enterococcus hirae ATCC9790. 5 x 10(6) transformants per microgram of plasmid DNA were obtained, using a commercial capacitor discharge device with an improved circuitry and a home-made electrode assembly, delivering pulses of 24 kV/cm across the cell suspension. The transformants were stable without selective pressure and plasmid DNA reisolated from transformed cells displayed no alterations in restriction enzyme analysis. Chromosomal DNA from E coli or E hirae, carried by pC3, was stably maintained in E hirae, making cloning and genetic manipulation in this organism feasible.     

Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.     

An improved Eschehchia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation

The genetic studies of metabolically diverse Rhodococcus spp. have been hampered by the lack of a system of introducing exogenous DNA. The authors improved an existing Escherichia coli-Rhodococcus shuttle vector (pMVS301) by removing much of the DNA not needed for replication and adding a multicloning site. This improved vector (pBS305) is 7·9 kb in length. Its ability to transform Rhodococcus was tested using electroporation parameters optimized for introduction of pMVS301 into Rhodococcus. Transformation efficiencies as high as 10⁵ cfu μg^-1 DNA were obtained although efficiencies varied depending on the Rhodococcus strain tested. The improved vector pBS305 offers great utility for genetic studies of Rhodococcus because its small size enables movement of large inserts of DNA into Rhodococcus , it has multicloning sites, contains a highly selective thiostrepton marker, and can be replicated in both E. coli and Rhodococcus.     

Methylated Bases of Bacillus megaterium KM Nucleic Acids: Comparison with Escherichia coli

Parallel studies were performed with methionineless derivatives of Escherichia coli 15 T(-) and Bacillus megaterium KM: T(-). Methylated bases are present in the total cell ribonucleic acid (RNA) of B. megaterium. The level of RNA methylation in E. coli is about 60% greater than that in B. megaterium. Although E. coli deoxyribonucleic acid (DNA) was found to contain 0.12% 5-methylcytosine (5-MC) and 0.24% 6-methylaminopurine (6-MA), methylated bases were not detected in the DNA of B. megaterium. Assuming a molecular weight of 7 x 10(9) daltons for B. megaterium DNA, it was calculated that this organism could not contain more than one molecule of 5-MC or 6-MA per genome, and that possibly no methylated bases were present. Methylated bases were also not detected in the DNA of thymine-starved B. megaterium. Crude extracts of this organism possess RNA methylase activity but no detectable DNA methylase activity.     

Efficient cloning in Bacillus megaterium: comparison to Bacillus subtilis and Escherichia coli cloning hosts

Quantitative cloning efficiencies for B. megaterium, B. subtilis , and E. coli were compared. Transformation of B. megaterium is less efficient than transformation of B. subtilis or E. coli . The frequency of recombinant clones was equal in E. coli and B. megaterium ; both somewhat higher than in B. subtilis . Equivalent average insert sizes were found in B. megaterium and E. coli clones, but significantly smaller inserts were obtained in B. subtilis clones. Clones obtained and propagated in B. megaterium were structurally stable when grown under plasmid selection.     

Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region.

Recombinant plasmids containing all or part of the genetic region of Escherichia coli coding for the two subunits of ribonucleoside diphosphate reductase (proteins B1 and B2) were constructed with the aid of the multicopy plasmid pBR322. Two of these plasmids (pPS1 and pPS2) appeared to carry both a regulator and the complete structural information for the enzyme and, after transformation of E. coli, directed a 10- to 20-fold overproduction of both proteins B1 and B2. The other plasmids (pPS101 and pPS201) carried structural information for only protein B2. Cells carrying pPS1 and pPS2 showed a 5- to 500-fold increased resistance against the drug hydroxyurea. This establishes that in E. coli the inhibition of deoxyribonucleic acid synthesis by hydroxyurea is fully explained by its action on ribonucleotide reductase.     

一种含双歧杆菌种属特异性复制子的新型大肠杆菌-双歧杆菌穿梭质粒的构建

目前,双歧杆菌的转化是一个技术难题,与大肠杆菌等宿主菌的高转化效率不同,采用普通的原核质粒无法转化双歧杆菌.为此,本文提出双歧杆菌转化对质粒复制子具有"种属特异性"要求,并通过构建含有双歧杆菌特异复制子的新型穿梭质粒,以求解决这一难题.首先从GenBank获取长双歧杆菌隐性质粒pMB1的序列信息,采用Overlap-PCR方法获得其全长DNA,作为拟构建质粒的复制子;继而采用重组技术,将其与pMK4质粒片段(含大肠杆菌复制子pUC和抗氯霉素基因Cat)重组,构建大肠杆菌-双歧杆菌穿梭质粒;用电穿孔法将重组质粒转化双歧杆菌,通过观察不同电转参数下的转化效率,选择双歧杆菌转化的最佳条件.结果,成功获得全长1899bp的pMB1复制子并构建成功含有pMB1和pUC双复制子的原核重组质粒,经酶切和测序鉴定正确,命名为pCMB1.以重组质粒成功转化了长双歧杆菌NCC2705和NQ1501,而其它3种野生型双歧杆菌(包括1株长双歧杆菌)未能转化成功.结论:质粒中含有双歧杆菌种属特异的复制子是实现双歧杆菌转化的必要条件;即使是含有特异复制子的质粒也只能转化有限数量种型甚至有限数量种株的双歧杆菌;选择最佳电转化条件能显著提高转化效率.     

Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli.

The effects of competence-inducing treatments on the composition and organization of membrane lipids in Escherichia coli K-12, DH1, DH5, HB101, and RR1 were investigated for two widely used protocols in which transformability is developed at low temperatures in Ca2+ buffers. At stages during each procedure, the lipid compositions of the cells were determined, and the thermotropic lipid phase transitions were observed in whole cell culture by fluorescence assay with the hydrophobic probe N-phenyl-1-naphthylamine. Competence was evaluated by determining transformation efficiencies with plasmid pBR322 DNA. The competence-inducing procedures effected only slight changes in phospholipid compositions which did not correlate with transformability. However, the induction of competence was coincident with de novo synthesis and incorporation of poly-beta-hydroxybutyrate into the cytoplasmic membranes and with the appearance of a sharp lipid phase transition above physiological temperatures. Transformation efficiencies correlated with poly-beta-hydroxybutyrate concentrations and with the intensity of the new phase transition. Transformability, poly-beta-hydroxybutyrate synthesis and the new phase transition were not significantly affected by inhibition of protein synthesis with chloramphenicol or inhibition of respiration or ATP synthesis with azide, cyanide, arsenate, or 2,4-dinitrophenol; however, when poly-beta-hydroxybutyrate synthesis was inhibited with acetaldehyde, the new phase transition was not observed, and competence failed to develop. These studies suggest that genetic transformability in E. coli may be physiologically regulated.     

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis. II. Transfer of sequences propagated in Escherichia coli to B. subtilis

Recombinant plasmid DNA cloned in E. coli via the bifunctional vector pDH5060 suffered deletions when returned to B. subtilis. However, DNA preparations of identical chimeras containing homologous or heterologous sequences stably transformed B. subtilis at high efficiency when isolated from B. subtilis. The vector pDH5060, however, was not affected and could be stably shuttled between E. coli and B. subtilis at high frequency. These problems affected the transfer of clone pools and individual chimeras, irrespective of the restriction or recombination phenotype of B. subtilis recipients. Deleted chimeras lost at least one end of cloned inserts, and in most cases, flanking plasmid sequences. Single plasmid forms (intact or deleted) were isolated from several hundred individual Cmr-transformants this suggests that events leading to deletion of chimeric plasmid DNA occur during transformation by restriction of unmodified insert sequences propagated in the intermediate host, E. coli. This conclusion is discussed with regard to the mechanism of plasmid transformation in B. subtilis.     

Plasmid-directed synthesis of genuine adenovirus 2 early-region 1A and 1B proteins in Escherichia coli.

The transforming region of human adenovirus 2 is located in the left 11.2% of the viral genome and is comprised of two distinct genetic units termed E1A and E1B. cDNAs containing the entire nucleotide sequence of the mature E1A 13S and E1B 22S mRNAs that are complementary to these genetic units have been introduced into bacterial plasmids a short distance downstream from the Escherichia coli lac promoter. Upon transformation into appropriate E. coli hosts, one of these plasmids, pKHAO, directed the synthesis of a 45-kilodalton (kd) protein, and the other, pKHBO, synthesized a protein of 54.9 kd. Both of these plasmid-encoded proteins constituted 0.1 to 0.3% of the total cellular protein and were virtually identical to the authentic adenovirus 2 E1A 42- to 50-kd and E1B 53- to 58-kd tumor antigens (T antigen) as determined by gel electrophoresis, immunoprecipitation, and tryptic fingerprint analysis. With the use of our pKHBO expression plasmid we were also able to demonstrate that the second AUG sequence appearing in the E1B 22S mRNA corresponded to the start of the gene encoding the large adenovirus 2 T antigen. This confirms theoretical deductions based on DNA sequencing analysis that translation of the large T antigen initiates translation at an internal ATG rather than at the 5'-proximal AUG.     

Construction of a marker rescue system in Bacillus subtilis for detection of horizontal gene transfer in food

A marker rescue system based on the repair of the kanamycin resistance gene nptII was constructed for use in Gram-positive bacteria and established in Bacillus subtilis 168. Marker rescue was detected in vitro using different types of donor DNA containing intact nptII. The efficiency of marker rescue using chromosomal DNA of E. coli Sure as well as plasmids pMR2 or pSR8-30 ranged from 3.8 x 10(-8) to 1.5 x 10(-9) transformants per nptII gene. Low efficiencies of ca. 10(-12) were obtained with PCR fragments of 792 bp obtained from chromosomal DNA of E. coli Sure or DNA from a transgenic potato. B. subtilis developed competence during growth in milk and chocolate milk, and marker rescue transformation was detected with frequencies of ca. 10(-6) and 10(-8), respectively, using chromosomal DNA of E. coli Sure as donor DNA. Although the copy number of nptII genes of the plant DNA exceeded that of chromosomal E. coli DNA in the marker rescue experiments, a transfer of DNA from the transgenic plant to B. subtilis was detectable neither in vitro nor in situ.     

Micronuclei and sister chromatid exchanges induced by capsaicin in human lymphocytes

Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA. Changes in triplet repeat length during growth following transformation in E. coli have been used as a measure of repeat instability. However, very little is known about the molecular and biological changes that may occur on transformation. Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells. To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E. coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E. coli. Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic. The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2 x 10(5) following transformation. The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent. Thus, the process of transformation was required to observe the instability. Instabilities of (CAG).(CTG) repeats are also dramatically elevated upon transformation. The magnitude of the instability is dependent on the nature and length of the repeat. Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements. Moreover, different E. coli genetic backgrounds show different levels of instability during transformation.     

Incorporation of plasmid DNA into liposomes from the total lipid of E. coli and determination of their stability

Large monolamellar liposomes were constructed from the total E. coli lipid by ultrasonication and consecutive treatment with Ca2+ and EDTA. Serum albumin and plasmid DNA were incorporated into the liposomes with the efficiency of 6.3 and 4.7%, respectively. The plasmid DNA remained intact after incorporation, as was demonstrated by gel electrophoresis and transformation of E. coli with the DNA extracted from the liposomes, About one half of DNA-containing liposomes remained undamaged after 10 hr incubation at 4 degrees C. Possible implications of E. coli lipid liposomes in genetic transformation are discussed.     

The mechanism of mutation induction by a hydrogen bond ambivalent, bicyclic N4-oxy-2'-deoxycytidine in Escherichia coli.

The triphosphate of the nucleoside deoxyribosyl dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is known to be incorporated into DNA efficiently by Taq polymerase and is a useful tool for polymerase-mediated in vitro mutagenesis. It is shown here that dP is a potent mutagen in Escherichia coli and Salmonella typhimurium . In E.coli , this deoxycytidine analog induces both GC-->AT and AT-->GC transitions. No induced transversions are observed. It is highly mutagenic in wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase. Mutagenesis induced by dP is efficiently inhibited by the addition of thymidine. Partially purified thymidine kinase from E.coli catalyzes phosphorylation of dP to its 5'-monophosphate. When E.coli was grown in the presence of dP, the nucleoside analog was incorporated into its DNA. The content of dP in DNA was dependent on the concentration of dP added to the medium. The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment. The results confirm that this triphosphate can be incorporated opposite A and G in the template with similar efficiencies. This indicates that dP is metabolized as a thymidine analog and that the resulting triphosphate induces a high rate of mutagenesis through replicational errors.     

Determination of free and liposomal Amphotericin B in human plasma by liquid chromatography–mass spectroscopy with solid phase extraction and protein precipitation techniques

Amphotericin B is available in various drug delivery systems such as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The separation and measurement of free drug (drug which is not bound with liposomal lipids) and liposomal drug (drug which is entrapped in liposomes) in the human plasma after injection of liposomal Amphotericin B is of prime importance due to toxicity concerns. A robust, specific and sensitive method has been developed to effectively separate and then quantify the free drug and liposomal drug, present in human plasma. This method utilizes solid phase extraction Oasis HLB cartridges, which retains the free drug and the liposomal Amphotericin B was eluted from the cartridge in first step. The eluted liposomal Amphotericin B was then extracted from lipids by protein precipitation method using 2% dimethylsulfoxide (DMSO) in acetonitrile. After separation and extraction, the quantification of free and liposomal fractions of Amphotericin B was performed by HPLC–MS–MS technique. The chromatographic separation was performed using Chromolith Performance RP 18e column. The mobile phase composed of 5 mM ammonium acetate, methanol and acetonitrile and a gradient elution program was used. The calibration curves were found to be linear for free Amphotericin B (0.25–15.0 μg/ml) and liposomal Amphotericin B (1.0–100.0 μg/ml). The recovery was about 96% for free Amphotericin B and about 92% for liposomal Amphotericin B. Recoveries were consistent over the linearity ranges defined. The intra-batch and inter-batch accuracy and precision fulfilled the international requirements. The stability of free and liposomal Amphotericin B was assessed under different storage conditions.     

Utilization of nucleoside monophosphates per Se for intraperiplasmic growth of Bdellovibrio bacteriovorus.

During growth of Bdellovibrio bacteriovorus on Escherichia coli, there was a marked preferential use of E. coli phosphorus over exogenous orthophosphate even though the latter permeated into the intraperiplasmic space where the bdellovibrio was growing. This preferential use occurred to an equal extent for lipid phosphorus and nucleic acid phosphorus. Exogenous thymidine-5'-monophosphate competed effectively with [3H]thymine residues of E. coli as a precursor for bdellovibrio deoxyribonucleic acid; exogenous thymidine competed less effectively and thymine and uridine not at all. A mixture of exogenous nucleoside-5'-monophosphates equilibrated effectively with E. coli phosphorus as a phosphorus source for B. bacteriovorus; the nucleotide phosphorus entered preferentially into bdellovibrio nucleic acids. A comparable mixture of exogenous nucleosides plus orthophosphate had only a small effect on utilization of E. coli phosphorus by B. bacteriovorus, as did orthophosphate alone. A mixture of exogenous deoxyriboside monophosphates equilibrium effectively with E. coli phosphorus as a phosphorus source for bdellovibrio growth; the phosphorus from this source entered preferentially into deoxyribonucleic acid. These data show that nucleoside monophosphates derived from the substrate organism are utilized directly for n-cleic acid biosynthesis by B. bacteriovorus growing intraperiplasmically. As a consequence, the phosphate ester bonds preexisting in the nucleic acids of the substrate organism are conserved by the bdellovibrio, presumably lessening its energy requirement for intraperiplasmic growth. The data also suggest, but do not prove, that the phosphate ester bonds of phospholipids are also conserved.