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Rhodococcus spp. have broad potential applications related to the degradation of organic contaminants and the transformation or synthesis of useful compounds. However, some Gram-positive bacteria are difficult to manipulate genetically due to low transformation efficiency. In this study, we investigated the effects of chemicals including glycine, isonicotinic acid hydrazide (INH), Tween 80 and penicillin G, as well as cell growth status, competent cell concentration, electroporation field strength, electroporation time and heat shock time, on the electrotransformation efficiency of the tetrahydrofuran-degrading bacterium Rhodococcus ruber YYL with low transformation efficiency. The highest electrotransformation efficiency was 1.60 × 106 CFU/µg DNA after parameter optimization. GmhD (D-glycero-D-manno-heptose 1-phosphate guanosyltransferase) gene, which is important in the biosynthesis of lipopolysaccharide, was deleted via the optimized electrotransformation method. Compared with wild-type strain, YYL ΔgmhD showed extremely high electrotransformation efficiency because the surface of it had no mushroom-like extracellular polymeric substances (EPS). In addition, the results showed that cell wall-weakening reagents might cause some translucent substances like EPS, to detach from the cells, increasing the electrotransformation efficiency of strain YYL. We propose that these results could provide a new strategy for unique bacteria that are rich in EPS, for which genetic manipulation systems are difficult to establish.

It is well established that efflux pumps play important roles in bacterial pathogenicity and efflux inhibitors (EIs) have been proved to be effective in suppressing bacterial virulence properties. However, little is known regarding the EI of Streptococcus mutans, a well-known caries-inducing bacterium. In this study, we identified the EI of S. mutans through ethidium bromide efflux assay and investigated how EI affected S. mutans virulence regarding the cariogenicity and stress response. Results indicated that reserpine, the identified EI, suppressed acid tolerance, mutacin production and transformation efficiency of S. mutans, and modified biofilm architecture and extracellular polysaccharide distribution. Suppressed glycosyltransferase activity was also noted after reserpine exposure. The data from quantitative real-time-PCR demonstrated that reserpine significantly altered the expression profile of quorum-sensing and virulence-associated genes. These findings suggest that reserpine represents a promising adjunct anticariogenic agent in that it suppresses virulence properties of S. mutans.

Schizophyllum commune is the only mushroom-forming fungus in which targeted gene deletions by homologous recombination have been reported. However, these deletions occur with a low frequency. To overcome this, the ku80 gene of S. commune was deleted. This gene is involved in the nonhomologous end-joining system for DNA repair. The Δku80 strain was not affected in growth and development. However, the transformation efficiency was reduced up to 100-fold. This was accompanied by a strong increase in the relative number of transformants with a homologous integration of a knockout construct. Genes sc15, jmj3 and pri2 were deleted in the Δku80 strain. In total, seven out of 10 transformants showed a gene deletion. This frequency will facilitate a systematic analysis of gene function in S. commune.

The plant hormone ethylene has been reported to inhibit the Agrobacterium tumefaciens-mediated transformation efficiency of many plants. In this study, an acdS gene that encodes 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, an enzyme that breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, was introduced into A. tumefaciens GV3101∷pMP90. It was found that the presence of active ACC deaminase in A. tumefaciens reduced ethylene levels produced by plant tissues during the process of infection and cocultivation, and significantly increased the transformation efficiency of three commercial canola cultivars: Brassica napus cv. Westar, B. napus cv. Hyola 401 and B. napus cv. 4414RR.

Genetic transformation is an indispensable tool for basic fungal research, as well as a useful technique for directed improvement of industrial strains. Here we describe a simple and reproducible transformation system for the filamentous fungus Hypocrea jecorina . The system is based on hxk1 (encoding hexokinase) as selectable marker, a hexokinase-negative strain and d-mannitol, which is used as selective carbon source and osmotic stabilizer. Following transformation with the hxk1 gene, the obtained transformants were able to grow on d-mannitol as sole carbon source. Transformation efficiency achieved using d-mannitol as carbon source and osmotic stabilizer was roughly five times higher than that using d-sorbitol. The utility of this system was further demonstrated by transformation of H. jecorina with the egfp (encoding the enhanced green fluorescent protein) gene. Fluorescence microscopy revealed EGFP fluorescence in positive transformants. Our results demonstrated the feasibility of exploiting a carbon source metabolic pathway for the development of promising fungal transformation systems, which provides a new molecular toolbox for genetic modifications of the cell factory H. jecorina .

The REMI method was used to introduce the plasmid pV2 harboring the hygromycin B phosphotransferase (hph) gene controlled by the Aspergillus nidulans trpC promoter and the trpC terminator into a taxol-producing endophytic fungus BT2. REMI transformation yielded stable transformants capable of continuing to grow on PDA medium containing 125 mug mL(-1) hygromycin B. The transformation efficiency was about 5-6 transformants mug(-1) plasmid DNA. The presence of hph gene in transformants was confirmed by PCR and Southern blot analyses. To the authors' knowledge, this is the first report on the transformation of taxol-producing endophytic fungi by the REMI technique. This study provides an effective approach for improving taxol production of endophytic fungi by the genetic engineering of taxol biosynthetic pathway genes in the future.

A preincubation of fission yeast cells with hyperosmotic solution improved the electro-transformation efficiency. The efficiency increased approximately five-fold when the cells were preincubated with 2.0 M sorbitol and 1.5 M NaCl at 30 degrees C for 60 min before an applied high electric pulse. Losses in the efficiency of the cells after hyperosmotic stress above 2.5 M sorbitol and 2.0 M NaCl were directly related to the marked reduction of viability. The efficiency at 2.0 M sorbitol gradually increased until 60 min of the preincubation period, but longer exposure resulted in a gradual decrease. On the other hand, when the cells of the osmotic-sensitive mutant were preincubated with isosmotic solution of 0.5 M sorbitol, the efficiency was also dramatically increased by approximately 15-fold. These improvements in efficiency were observed in sublethal conditions of osmotic stress regardless of osmoticums and strains.

Streptococcus pneumoniae strain JNR.7/87 is a highly virulent, type 4 encapsulated Gram-positive bacterium whose transformability has not been tested previously, and whose genome is currently being sequenced. The strain was transformed at very low efficiency by addition of exogenous competence-stimulating peptide: However, the efficiency was too low and irreproducible to be useful in many genetic studies. Therefore, the effects on transformation efficiency of changing different components of competence-stimulating peptide-induced transformation have been examined. Screening of growth media was followed by optimization of pre-induction culture acidification, glycine concentration, and induction time. An optimized protocol was developed whereby S. pneumoniae strain JNR.7/87 was transformed reproducibly with a streptomycin resistance (SmR) marker at an efficiency of approximately 10(5) colony forming units per 10(8) cells.

Oxygen is a limiting factor in the production of γ-PGA by the glutamic acid-independent strain Bacillus amyloliquefaciens LL3 because of the high viscosity of the culture broth. The vgb gene encoding Vitreoscilla hemoglobin (VHb) was introduced into LL3 to overcome the low concentration of dissolved oxygen (DO). First, recombinant plasmid pWHV was constructed by cloning vgb into the Bacillus expression vector pWH1520 and transformed into LL3. Carbon monoxide difference spectral analysis confirmed the expression of VHb. The γ-PGA yield of LL3 (pWHV) under the optimized fermentation conditions increased by 9.56%. To overcome the instability of pWH1520 and to establish stable expression of VHb, the engineered strain LL3-PVK was constructed by homologous recombination between the integration vector pKSVPVK and the 16S rRNA gene of LL3. The temperature-sensitive plasmid was used to perform the integration, which successfully circumvented the obstacle of the low transformation efficiency of B. amyloliquefaciens LL3. Bacillus amyloliquefaciens LL3-PVK showed an increase of 30% in γ-PGA production, while the biomass was increased by 7.9%. To our knowledge, this is the first report describing enhancement of γ-PGA production in a glutamic acid-independent strain as a result of vgb expression.

A procedure for isolation of genomic DNA from the zygomycete Cunninghamella elegans and other filamentous fungi and yeasts is reported. This procedure involves disruption of cells by grinding using dry ice, removal of polysaccharides using cetyltrimethylammonium bromide and by phenol extractions, and precipitation of DNA with isopropanol at room temperature. The isolation method produced large scale (approximate 1 mg DNA/5 g wet cells) and highly purified high molecular mass DNA. Sau3AI partially digested DNA showed high transformation efficiency ( 10 6 100 ng DNA ) when ligated to ZAP-express λ vector.

Pseudomonas putida KT2440 is a saprophytic and generally recognized as safe microorganism that plays important roles in the biodegradation and production of value-added chemicals. Chromosomal gene deletion of P. putida KT2440 usually involves time-consuming gene cloning, conjugal transfer and counterselection. Recently, we developed a P. putida KT2440 markerless gene deletion method based on recombineering and Cre/loxP site-specific recombination. PCR-based λ Red recombineering circumvents the tedious cloning steps and is more amenable to high-throughput manipulation. Here we report an improved scarless gene deletion strategy based on recombineering and intron-encoded homing endonuclease I-SceI-mediated double-strand break repair. Sixteen drug exporter gene(s) were deleted and the minimal inhibition concentrations of the mutants to a variety of antibiotics were determined. The robustness of the procedure was also demonstrated by sequential deletion of five large genomic regions. Up to 59% recombination efficiency was achieved for a 54.8 kb deletion, and the efficiency of RecA-mediated double-strand break repair, which was boosted by λ Red recombinase, was nearly 100%. The strain with a 3.76% genome reduction showed an improved growth rate and transformation efficiency. The straightforward, time-saving and highly efficient scarless deletion approach has the potential to facilitate the genetic study, and biotechnological and environmental applications of P. putida KT2440.

A prepulse incubation with 2% glucose (mannose, fructose) strongly inhibited electrotransformation of intact yeast cells. This inhibitory effect was not due to alterations of cell viability or to cell membrane electropermeabilization, and was not affected by the solution buffer properties of preincubation and pulsing media. The electrotransformation efficiency was not modified by non-metabolized substances, such as sorbitol or alose. The glucose inhibition of electrotransformation was fast, occurring after only a 3-5-min preincubation. The postpulse incubation with substrate also decreased transformation efficiency, but was pH-dependent. We observed a similar, strong inhibitory effect of glucose when cells were transformed chemically. A pH dependence of yeast electrotransformation was established.

The transformation efficiency of competent Bacillus subtilis degU32(Hy) strains was found to depend on the marker that was selected. Protrophic transformants were obtained at frequencies similar to those in the wild type control, but Spo− transformants oere rare also when a spoOA::erm insertion that produces a selectable marker (ErmR) was used. The ErmR transformants obtained within the degU32(Hy) background were Spo+ and had lost the characteristics of the DegU(Hy) parental recipient strain i.e., secretion of exo-enzymes and sporulation resistance to catabolites. The spoOA::erm insertion was mapped to a location near degU. The similarities between the spoOA and degU sequences and the metabolic interferences between the mutated products which result in this unexpected recombination, are discussed.

Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl(2)) and 'one-step' (polyethylene glycol, dimethyl sulfoxide and MgSO(4)) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5+/-0.2 x 10(4) transformants ng(-1) plasmid DNA) than with the CaCl(2) (8.1+/-6.1 x 10(-1) transformants ng(-1) plasmid) and the 'one-step transformation' protocol (2.5+/-2.7 transformants ng(-1) plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl(2) and one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.

A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 108 transformants per μg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm−1), pulse length (2.4 ms), plasmid DNA concentration (0.25 μg ml−1 and cell density (1010 cells ml−1). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites down-stream of the SP6 RNA polymerase promoter.

Plasmid pPG1 from Staphylococcus aureus coding for ampicillin (Apr), gentamicin (Gmr) and amikacin (Akr) resistance was transformed into Escherichia coli. Transformation efficiency was about 2 x 10(3) transformants/micrograms of plasmid DNA. The plasmids present in the E. coli transformants were identical to pPG1 according to their restriction patterns. The copy number of pPG1 was estimated to be at least 20-times less in E. coli than in S. aureus. The minimal inhibitory concentrations (MICs) for Ap and Gm were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus. pPG1 was maintained in the E. coli transformants for at least 80 generations at 37 degrees C without antibiotic selection pressure.

The following factors were found to improve the efficiency of transformation of Clostridium perfringens 3624A Rifr Strr: (1) a reduction in cuvette sample volume (DNA and cell suspension) to 0.8 ml, (2) use of a 1 microgram/ml concentration of transforming DNA, (3) use of late-logarithmic phase cells, (4) 3-fold concentration of cell density (3.0 x 10(8) CFU/ml), and (5) a reduction in the pH of the expression and selective plating medium to 6.4. Application of the improved conditions resulted in transformation efficiencies for C. perfringens 3624A Rifr Strr ranging from 7.1 transformants/microgram DNA for plasmic pIP401 to 9.2 x 10(4) transformants per microgram DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 x 10(6) transformants/micrograms DNA for C. perfringens strain 13. Using the improved protocol, pAM beta 1 was transformed at a 42-fold greater level when compared with the values reported earlier [1]. In addition to C. perfringens 3624A Rifr Strr, strains 13, 10543A, 3628C, NTG-4, and 3624A were successfully transformed. Nuclease does not appear to be a factor in the C. perfringens strain-specific electro-transformation protocol.

A transformation procedure for Myobacterium aurum using electroporation was developed and optimized. Effects of glycine and lysozyme treatments were studied, and isonicotinic acid hydrazide was shown to increase transformation efficiency ten-fold.

Candida parapsilosis is a human pathogenic fungus with increasing importance, particularly in nosocomial infections. For detailed molecular genetic explorations of prototrophic clinical isolates of C. parapsilosis, we developed an efficient transformation system based on a dominant selectable marker. The gene encoding resistance to mycophenolic acid (MPA) was used for selection in yeast transformation. C. parapsilosis cells were transformed with a plasmid vector containing the Candida albicans inosine monophosphate dehydrogenase gene (IMH3) responsible for mycophenolic acid resistance. Transformation was carried out both by electroporation and by the lithium acetate (LiAc) method. The LiAc method resulted in very poor transformation efficiency, while the modified electroporation method yielded a high number of mitotically stable transformants exhibiting unambiguous MPA resistance. Two hundred transformants were analysed for the presence of the C. albicans IMH3(r) gene by polymerase chain reaction. Integration of single or multiple plasmid copies into the genomic DNA of C. parapsilosis was determined by Southern hybridization. To our knowledge, the present study is the first report about a method based on a dominant selectable marker for the transformation of a prototrophic, clinical isolate of C. parapsilosis. The described technique may prove to be an efficient tool for the examination of the biology and virulence of this pathogenic yeast.

The broad host range vector pBBR1MCS-2 has been evaluated as an expression vector for Zymomonas mobilis. The transformation efficiency of this vector was 2 · 10 3 CFU per lg of DNA in a recombinant strain of Z. mobilis ZM4/Ac R containing the plasmid pZB5. Stable replication for this expression vector was demonstrated for 50 generations. This vector was used to study xylose metabolism in acetate resistant Z. mobilis ZM4/Ac R (pZB5) by over-expression of xylulokinase (XK), as previous studies had suggested that XK could be the rate-limiting enzyme for such strains. Based on the above vector, a recombinant plasmid pJX1 harboring xylB (expressing XK) under control of a native Z. mobilis promotor Ppdc was constructed. When this plasmid was introduced into ZM4/ Ac R (pZB5) a 3-fold higher XK expression was found compared to the control strain. However, fermentation studies with ZM4/Ac R (pZB5, pJX1) on xylose medium did not result in any increase in rate of growth or xylose metabolism, suggesting that XK expression was not rate-limiting for ZM4/Ac R (pZB5) and related strains.

Streptomyces griseus does not readily take up foreign DNA isolated from other Streptomyces species or Escherichia coli, presumably due to its unique restriction-modification systems that function as a barrier for interspecific DNA transfer. To efficiently transform S. griseus by avoiding the restriction barriers, we methylated incoming DNA in vivo and in vitro and treated protoplasts with heat prior to transformation. Whereas heat treatment of protoplasts or methylation of the E. coli-Streptomyces shuttle vectors (pXE4 and pKK1443) did not prominently improve the transformation efficiency, HpaII methylation of the vectors from any E. coli strains tested in this study highly increased the transformation efficiency. The highest transformation efficiency was observed when the shuttle vectors were isolated from the dam, hsd strain of E. coli (GM161) and methylated by AluI and HpaII methyltransferases, and the efficiency was approximately the same as that of the vectors from S. griseus. We identified several restriction-modification systems that decrease the transformation efficiency. This research also led us to understand methylation profiles and restriction-modification systems in S. griseus.

The cyanobacterium Synechocystis sp. PCC 6803 is transformable at high efficiency and integrates DNA by homologous double recombination. However, several genetic mapping procedures depend on the ability to generate transformants even with very small amounts of added DNA. This study is aimed at optimizing the transformation efficiency at limiting concentrations of exogenous DNA. The transformation efficiency showed little sensitivity to experimental conditions. Transformation with circular plasmid DNA was found to be no more than 30% more efficient than with linearized plasmid DNA. The efficiency of transformation remained essentially the same in the presence of competing DNA, indicating that the capacity of DNA uptake by the cells is not limiting. The incubation time of cells with DNA before plating (0–8 h) affected the transformation efficiency by up to 3-fold. Only minor changes in the efficiency were observed as a function of the presence of a membrane filter on the plate or the presence of TAE or TBE gel buffer residues in the transformation mixture. However, transformability of the host strain of Synechocystis sp. PCC 6803 was increased by two orders of magnitude if the sll1354 gene encoding the exonuclease RecJ was deleted. Therefore, the transformation efficiency of Synechocystis sp. PCC 6803 with exogenous DNA appears to be determined primarily by intracellular processes such as the efficiency of DNA processing and homologous recombination.

To assess the likelihood that the bla gene present in a transgenic maize line may transfer from plant material to the microflora associated with animal feeds, we have examined the survival of free DNA in maize silage effluent, ovine rumen fluid and ovine saliva. Plasmid DNA that had previously been exposed to freshly sampled ovine saliva was capable of transforming competent Escherichia coli cells to ampicillin resistance even after 24 h, implying that DNA released from the diet could provide a source of transforming DNA in the oral cavity of sheep. Although target DNA sequences could be amplified by polymerase chain reaction from plasmid DNA after a 30-min incubation in silage effluent and rumen contents, only short term biological activity, lasting less than 1 min, was observed in these environments, as shown by transformation to antibiotic resistance. These experiments were performed under in vitro conditions; therefore further studies are needed to elucidate the biological significance of free DNA in the rumen and oral cavities of sheep and in silage effluent.

Uracil auxotrophs of Pleurotus ostreatus were isolated using the selectable marker, resistance to 5'-fluoro-orotic acid (5'-FOA). Two of the nine uracil auxotrophs obtained were transformed to prototrophy using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from Trichoderma reesei. Southern blot analyses of the transformants showed that the transforming DNA had integrated into the genome of the protoplasts. Using 2 x 10(7) protoplasts, this system gave a transformation efficiency of about 30 transformants per microg of DNA. Normal fruiting bodies were induced in the transformants by crossing them with wild-type monokaryons, and the basidiospores collected from these fruiting bodies showed a biased segregation rate to prototrophy. These results indicate the integrated DNA was stably inherited.

The effects of electroporation temperature, biochemical pretreatment of cells and stage of culture on electroporation efficiency for slow-growing mycobacteria were investigated. The efficiency of transformation into Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium intracellulare increased markedly with temperature. In contrast, the efficiency of transformation into Mycobacterium smegmatis, a fast-growing species, was higher at 0 degree C and decreased with temperature. While stage of culture had little effect, a further increase in efficiency of 2-4-fold was obtained following glycine or ethionamide pretreatment. Electroporation at 37 degrees C has been chosen as a standard condition for slow-growing species as it usually resulted in a transformation efficiency several orders of magnitude higher than that obtained at 0 degree C.

Attempts to transform wilt type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were note successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type of DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.

A method for transformation of whole Bacillus amyloliquefaciens cells by electroporation was developed. The procedure is as efficient as the protoplast transformation method, resulting in up to 105 transformants/μg plasmid DNA, but requires less effort and time. Cells for electroporation were grown to late exponential phase in a rich medium supplemented with 0.25 M sucrose, washed with and resuspended in 0.25 M sucrose, 1 mM HEPES, 1 mM MgCl2, 10% (v/v) glycerol, pH 7.0, at 3–5 × 1010 cells/ml for storage at -80%°C. The highest transformation frequency was obtained at 7.5 kV/cm with a 25 μF capacitor. The transformation efficiency increased linearly with DNA concentration at least over the range 10 ng-12.5 μg/ml. Transformations with ligated DNA and of industrial strains were also successful. In addition, B. subtilis cells treated as above could be transformed by electroporation, resulting in 104 transformants/μg DNA at 12.5 kV/cm.

The transformability of batch cultures of Escherichia coli with pBR322 plasmid DNA was found to be dependent on the inoculum size used as starter. Highest rates of competence were achieved in cultures from the early exponential phase (OD660= 0.15) following > 250-fold dilution of an overnight (37°C) culture into prewarmed broth. Modal cell volume and transformation frequency changed with similar periodicity throughout growth and the highest transformability occurred when recipient cells had the greatest volumes. The range over which transformability fluctuated (ca. 100-fold) vastly exceeded the range (ca. 2-fold) in cell volume observed. This suggests that simple increase in surface area available for DNA uptake alone cannot explain these findings. It is more likely that stresses caused by cell division make the cell envelope expose more binding sites for DNA uptake or otherwise make the surface more permeable to DNA during transformation.

Transformation of Ca²⁺-heat shocked Escherichia coli cells with plasmid DNA has become an indispensable technique in the study of plasmids and in genetic engineering. The process of induction of competence by Ca²⁺-heat shock is still poorly understood, but it has been argued that only a small proportion of the shocked cells is effectively rendered competent in the process. This has been deduced from the fact that in transformation with two different plasmids as donor DNA, the observed frequency of double transformants is higher than would be expected from the observed transformation frequencies of each of the plasmids separately.