Transfer of plasmid DNA to Brevibacterium lactofermentum by electrotransformation

The Escherichia coli-Brevibacterium lactofermentum shuttle vector pBLA was introduced into intact cells of B. lactofermentum by electrotransformation. Several parameters of this procedure such as voltage and cell concentration were analysed. Optimal conditions gave an efficiency of 10(6) transformants per microgram of DNA. Two recalcitrant strains could be electrotransformed when an ampicillin pretreatment step was used. Electrotransformation experiments using DNAase or different structural forms of plasmid DNA showed that the electrotransformation process is quite different from natural transformation involving competence development. Restriction-modification-proficient B. lactofermentum could be efficiently electrotransformed with pBLA DNA isolated from E. coli. This restriction-modification system therefore seems to be overcome by electrotransformation. Thus electrotransformation may efficiently replace the protoplast bacterial transformation method.     

Development of a shuttle vector for Moraxella catarrhalis

Efforts to perform genetic analysis in Moraxella catarrhalis have been hampered by the lack of a cloning vector. M. catarrhalis strain E22 was previously shown to contain plasmid pLQ510 which lacked a selectable antibiotic resistance marker. Several methods were used to eliminate unnecessary DNA from pLQ510. Then, a 1.2 kb spectinomycin resistance cartridge, a multiple cloning site, and the origin of replication from pACYC184 were cloned into this plasmid backbone to obtain the 7.2 kb plasmid pWW102B. This new plasmid could replicate in M. catarrhalis as well as in both Escherichia coli and Haemophilus influenzae. This shuttle vector was used to clone and express two different M. catarrhalis genes, respectively, encoding an adhesin and a protein involved in serum resistance. When these two plasmids were introduced into appropriate M. catarrhalis mutants, they complemented the phenotypic deficiency of each mutant. This is the first report of functional complementation in trans in this pathogen.     

Plasmid transformation of Ruminococcus albus by means of high-voltage electroporation

To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal bacteria, a plasmid vector transformation system must be available. The objective of this work was to develop a system for plasmid transformation of Ruminococcus albus. Using high voltage electrotransformation, pSC22 and pCK17 plasmid vectors, derived from lactic acid bacteria plasmids and replicating via single-stranded DNA intermediate, were successfully introduced into three freshly isolated R. albus strains and into R. albus type strain ATCC 27210. The optimization of the electrotransformation condition raised the electroporation efficiency up to 3 x 10(5) transformants per microgram of pSC22 plasmid.     

Electrotransformation of the stable L-form of Proteus mirabilis

Abstract To improve the transformability of stable protoplast type L-forms of Proteus mirabilis for recombinant plasmid DNA, conditions for efficient electrotransformation were explored. Exposing cells from the exponential phase of growth at a density of 6−8 × 10⁹/ml in electrotransformation buffer having a conductivity of 1.4 mS/cm to a field strength of 6.5 kV/cm for a mean pulse duration time of 1.2 ms reproducibly yielded transformation efficiencies in the order of 5 × 10⁴ transformants per μg of DNA. Compared to the polyethylene glycol method for transformation, electrotransformation appeared to be the method of choice for introduction of plasmid DNA into L-form cells.     

Release of extracellular transformable plasmid DNA from Escherichia coli cocultivated with algae

We studied the effects of cocultivation with either Euglena gracilis (Euglenophyta), Microcystis aeruginosa (Cyanophyta), Chlamydomonas neglecta (Chlorophyta), or Carteria inversa (Chlorophyta) on the production of extracellular plasmid DNA by Escherichia coli LE392(pKZ105). Dot blot hybridization analysis showed a significant release of plasmid DNA by cocultivation with all the algae tested. Further analysis by electrotransformation confirmed the release of transformable plasmid DNA by cocultivation with either E. gracilis, M. aeruginosa, or C. inversa. These results suggest algal involvement in bacterial horizontal gene transfer by stimulating the release of transformable DNA into aquatic environments.     

Plasmid pWW115, a cloning vector for use with Moraxella catarrhalis

The plasmid shuttle vector pWW102B is able to replicate in only a modest number of Moraxella catarrhalis strains. Plasmid pWW115, a spontaneous deletion mutant of pWW102B, was shown to lack both the pACYC184-derived origin of replication and the associated chloramphenicol-resistance gene but was able to replicate in every M. catarrhalis strain tested in this study, including one strain that had been previously refractory to all types of genetic manipulations. To test the utility of this plasmid, a M. catarrhalis gene encoding the UspA2 serum-resistance factor was cloned into pWW115 and the resultant recombinant plasmid was shown to confer serum-resistance on a serum-sensitive M. catarrhalis uspA2 mutant.     

Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA

Electrotransformation was used to introduce both plasmid and linear DNA into Streptococcus pyogenes. The method was optimized using strain NZ131, for which transformation frequencies up to 10(7) per micrograms of plasmid DNA were obtained. A linear fragment of DNA, containing the streptokinase gene (ska) in which an internal fragment had been replaced with an erythromycin resistance gene (erm), was transformed into strain NZ131 with a frequency of 10(3) per micrograms DNA. The introduction of linear DNA into S. pyogenes by electrotransformation should be useful for future genetic analyses as well as targeted gene replacement.     

Efficient electrotransformation system and gene targeting in pyogenic streptococci

Hemolytic streptococci are lacking in natural competence for uptake of DNA, and existing electrotransformation methods are still ineffective for most strains. By optimizing biological and electric parameters of electroporation, we established a simple, efficient, and reproducible transformation method for streptococcal cells. The major factor was an increase in the electric field strength. All tested streptococci (6 group A strains and one group C strain) were successfully transformed, and the maximal efficiency was higher than 1 x 10(7) transformants per mug of plasmid DNA. Targeted inactivation of the chromosomal genes of group A and C streptococci was achieved, using the electrotransformation method. The slo- or sagB- mutants constructed by the gene-targeting showed elevated competence for electrotransformation. Availability of the electrotransfer system for cloning and analysis of streptococcal genes is discussed.     

A novel plasmid (pEMCJH03) isolated from moraxella catarrhalis possibly useful as a cloning and expression vector within this species

A preliminary screening study of six Moraxella catarrhalis isolates from primary school children in the Netherlands identified a small 3.5 kb plasmid (pEMCJH03), containing four open reading frames, which encoded three mobilizing and one replicase protein. Insertion of a kanamycin containing transposon (yielding pEMCJH04) allowed selection and isolation of the plasmid in Escherichia coli. Natural transformation of pEMCJH04 into M. catarrhalis was successful for 25% (3/12) of non-isogenic isolates, with no link between (lack of) transformability and genetic lineage or (lack of) transformability and complement phenotype, though the transformation efficiency was found to be rather low at approximately 615CFU/microg (range=60-1040CFU/microg ). This is only the second publication detailing a plasmid isolated from this important respiratory pathogen, and the ability to clone and express foreign proteins in M. catarrhalis using pEMCJH04 could help in the development of a vaccine expression vector, as well as providing a useful tool for studying promoter activity and in complementation studies of gene knockout mutants.     

嗜热厌氧乙醇菌JW200转化条件的研究

摘要 嗜热厌氧乙醇菌遗传转化系统的缺少,制约了对该菌理论基础和应用领域的进一步研究。利用聚乙二醇（PEG6000）转化和电转化技术国际首次实现了嗜热厌氧乙醇菌JW200外源基因的导入。PEG转化效率很低,因此选择对电转化条件进行优化,转化效率从4±3.2个转化子/μg质粒DNA提高到50±7.4个转化子/μg质粒DNA。实验表明获得较高的转化效率的必要条件是在细胞密度为OD660 0.2时添加甘氨酸与蔗糖后继续培养2h以及细胞在电击前的收集与洗涤保持低温。本研究为利用基因工程手段改造嗜热厌氧乙醇菌和从分子水平研究胞内乙醇代谢途径奠定了基础。     

Efficient Electrotransformation System and Gene Targeting in Pyogenic Streptococci

Hemolytic streptococci are lacking in natural competence for uptake of DNA, and existing electrotransformation methods are still ineffective for most strains. By optimizing biological and electric parameters of electroporation, we established a simple, efficient, and reproducible transformation method for streptococcal cells. The major factor was an increase in the electric field strength. All tested streptococci (6 group A strains and one group C strain) were successfully transformed, and the maximal efficiency was higher than 1×10⁷ transformants per μg of plasmid DNA. Targeted inactivation of the chromosomal genes of group A and C streptococci was achieved, using the electrotransformation method. The slo ^- or sagB ^- mutants constructed by the gene-targeting showed elevated competence for electrotransformation. Availability of the electrotransfer system for cloning and analysis of streptococcal genes is discussed.     

Plasmid transformation of Clostridium perfringens by electroporation methods

Two electroporation methods were compared and modified to improve the frequencies of transfer of plasmid DNA into Clostridium perfringens. A plasmid shuttle vector, pSB92A2, containing chloramphenicol and ampicillin resistance genes and a clostridial origin of replication isolated from a cryptic C. perfringens plasmid, was constructed and successfully introduced into C. perfringens by both electrotransformation methods. Modifications which improved frequencies by 15-28 fold are described and may improve frequencies sufficiently for some vector/host combinations to consider the future use of more direct cloning strategies for the clostridia.     

德氏乳杆菌保加利亚亚种电转化平台的构建和优化

德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisub sp.bulgaricus)是最具经济价值的乳酸菌之一,在世界上广泛应用于酸奶和其它发酵乳生产。当前对该菌的代谢机制研究甚少。外源基因的转化效率是制约其分子代谢机制研究的重要因素。本研究以pMG36c为材料,对L.delbrueckiisub sp.bulgaricus CH3进行电转化条件研究。结果表明,在电转化过程中,电场强度、质粒的浓度、细胞生长状态均对转化效率有明显影响,得到了该菌株的最适电转化条件为:对数初期的细胞,质粒浓度为100 ng/50μl菌液,在10 kV/cm电场强度下电转化,转化后细胞在复壮培养液中培养3 h后涂布选择性培养基,转化率可达2.6×103CFU/μg DNA。以甘氨酸、醋酸锂、二硫苏糖醇处理细胞壁,发现醋酸锂和二硫苏糖醇共同处理对转化效率有明显改善,可提高转化效率。     

Improvement of transformation and electroduction in avermectin high-producer,<Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

Factors affecting the PEG-mediated transformation and electrotransformation of Streptomyces avermitilis protoplasts, an industrial avermectin high-producer, were evaluated. The maximum protoplast transformation efficiency under optimum conditions with PEG was 3 x 106 transformants per microg plasmid pIJ702 DNA. The efficiency of electrotransformation with the same plasmid the intact cells grown in medium with 0.5 mmol/L CaCl2, suspended in buffer with 0.5 mol/L sucrose +1 mmol/L MgCl2, and pulsed at an electric field strength of 10 kV/cm, 800 ohms, 25 microF, was of 2 x 10(3) transformants per microg DNA. When the cells were electroporated after mild lysozyme-treatment, the efficiency was up to 10(4) transformants per microg DNA. Electroporation of protoplasts and germlings had a lower efficiency (10(2) transformants per microg DNA). We report that electroporation under optimum conditions can be used for direct transfer of nonconjugative plasmid pIJ699 between two different Streptomyces species, S. avermitilis and S. lividans.     

A Simple and Rapid Method of Transformation of Streptomyces rimosus R6 and Other Streptomycetes by Electroporation

Usually plasmid DNA is introduced into Streptomyces strains by polyethylene glycol-mediated transformation of protoplasts. However, many Streptomyces strains are only poorly or not at all transformable via protoplasts. Therefore, we have optimized the parameters critical for the application of electrotransformation of plasmid DNA into Streptomyces species. The most critical parameters evaluated for electrotransformation of the model strain Streptomyces rimosus R6 were the pretreatment of mycelia, buffer composition, and electric field strength. The electrocompetent mycelia were prepared from 24-h-old cultures, treated mildly with lysozyme, resuspended in sucrose-glycerol-polyethylene glycol buffer, and stored in aliquots at -70 deg C. The electric field strength of 10 kV/cm at 400 (Omega) and a capacitance of 25 (mu)F was applied. The method is simple and rapid, yielding transformant colonies in 48 to 72 h. Efficiencies of 10(sup5) to 10(sup6) transformants per (mu)g of plasmid DNA were reproducibly achieved for S. rimosus R6 and its mutants, and these numbers were 10(sup2) to 10(sup3) higher than those attained by polyethylene glycol-assisted transformation of protoplasts. In addition, we show that electroporation can be applied to other Streptomyces species, such as S. lividans 66, S. coelicolor A3(2), and an S. venezuelae strain. This last one could not be transformed by the standard protoplast procedure. Our data suggest that, because of the diversity of streptomycetes, the conditions have to be optimized for each strain.     

Exploration of horizontal gene transfer between transplastomic tobacco and plant-associated bacteria

The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of chloroplast genomes in a plant cell as well as the prokaryotic origin of the transgene may thus significantly increase the likelihood of gene transfer from transplastomic plants to bacteria. In order to assess the probability of such a transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity to the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to take up extracellular DNA (broad host-range pBBR1MCS-3-derived plasmid, transplastomic plant DNA and PCR products containing the genes accD-aadA-rbcL) by natural or electrotransformation. The results showed that among the 16 bacterial isolates tested, six were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid, but none of them managed to integrate transgenic DNA in their chromosome. Our results provide no indication that the theoretical gene transfer-enhancing properties of transplastomic plants cause horizontal gene transfer at rates above those found in other studies with nuclear transgenes.     

Possible relationship of PFGE patterns of Moraxella catarrhalis between hospital- and community-acquired respiratory infections in a community hospital

We describe a prospective study of molecular analysis of Moraxella catarrhalis isolated from a community hospital. Our study was designed to investigate the possible relationship of pulsed-field gel electrophoresis (PFGE) patterns of M. catarrhalis between hospital- and community-acquired respiratory infections. A nosocomial outbreak of M. catarrhalis was observed between September 2000 and September 2001. During the study period, 40 strains of M. catarrhalis were isolated from a total of 32 patients with respiratory infections (26 strains from 18 inpatients, and 14 strains from 14 outpatients). We compared the PFGE patterns in 40 strains of M. catarrhalis isolated from the respiratory tract of the study patients. The genomic types of M. catarrhalis were classified into three PFGE patterns (A, B, and C). Interestingly, the nosocomial outbreak of M. catarrhalis included two patterns (A and B). Of the three patterns, two patterns (A and B) were found in both inpatients and outpatients. More interestingly, two subtypes of pattern B (B1 and B4) were simultaneously found in both inpatients and outpatients. Our results indicated that PFGE with SmaI chromosomal digestion is a suitable technique to establish the inter-strain genetic relatedness of M. catarrhalis, and suggested that the outbreak of M. catarrhalis occasionally included miscellaneous PFGE patterns. The results also showed that PFGE patterns of M. catarrhalis isolates were similar between hospital- and community-acquired respiratory infections. Analysis of the subtypes suggested that there might be some association between hospital- and community-acquired respiratory infections caused by M. catarrhalis.     

Laboratory-scale evidence for lightning-mediated gene transfer in soil

Electrical fields and current can permeabilize bacterial membranes, allowing for the penetration of naked DNA. Given that the environment is subjected to regular thunderstorms and lightning discharges that induce enormous electrical perturbations, the possibility of natural electrotransformation of bacteria was investigated. We demonstrated with soil microcosm experiments that the transformation of added bacteria could be increased locally via lightning-mediated current injection. The incorporation of three genes coding for antibiotic resistance (plasmid pBR328) into the Escherichia coli strain DH10B recipient previously added to soil was observed only after the soil had been subjected to laboratory-scale lightning. Laboratory-scale lightning had an electrical field gradient (700 versus 600 kV m(-1)) and current density (2.5 versus 12.6 kA m(-2)) similar to those of full-scale lightning. Controls handled identically except for not being subjected to lightning produced no detectable antibiotic-resistant clones. In addition, simulated storm cloud electrical fields (in the absence of current) did not produce detectable clones (transformation detection limit, 10(-9)). Natural electrotransformation might be a mechanism involved in bacterial evolution.     

Mobilization and location of the genetic determinant of chloramphenicol resistance from Lactobacillus plantarum caTC2R.

The mobilization of a nonconjugative plasmid (pCaT) that mediates chloramphenicol resistance in Lactobacillus plantarum caTC2R was achieved by comobilization with the conjugative plasmid pAM beta 1. The conjugation studies confirmed that the 8.5-kb pCaT in L. plantarum caTC2R contains the gene responsible for chloramphenicol resistance and that the plasmid has several unique restriction sites which make it useful for genetic studies in Carnobacterium spp. Cloning studies showed that the gene responsible for chloramphenicol resistance is located in the 2.6-kb EcoRV-SalI region of pCaT. This was confirmed by probing the 3.0-kb BglII fragment of pCaT with a biotin-labeled 1.6-kb BstEII-HpaII fragment from the streptococcal-derived plasmid pVA797(Cmr). Expression of chloramphenicol resistance in Carnobacterium as well as in other Lactobacillus species was achieved by electrotransformation using donor DNA from pCaT.     

Replacement recombination in Lactococcus lactis.

In the pUC18-derived integration plasmid pML336 there is a 5.3-kb chromosomal DNA fragment that carries the X-prolyl dipeptidyl aminopeptidase gene (pepXP). The gene was inactivated by the insertion of an erythromycin resistance determinant into its coding sequence. Covalently closed circular DNA of pML336 was used for the electrotransformation of Lactococcus lactis. In 2% of the erythromycin-resistant transformants the pepXP gene was inactivated by a double-crossover event (replacement recombination) between pML336 and the L. lactis chromosome. The other transformants in which the pepXP gene had not been inactivated carried a Campbell-type integrated copy of the plasmid. Loss of part of the Campbell-type integrated plasmid via recombination between 1.6-kb nontandem repeats occurred with low frequencies that varied between less than 2.8 x 10(-6) and 8.5 x 10(-6), producing cells with a chromosomal structure like that of cells in which replacement recombination had taken place.