Electrotransformation of Foodborne Pathogen Cronobacter sakazakii by a Simple Method.

Cronobacter sakazakii is an opportunistic foodborne pathogen that mainly infects infants and immunocompromised people, with a high mortality rate. However, the efficient transformation method of this bacterium has not been systematically reported. In this study, we developed a fast and efficient transformation method for C. sakazakii by cold sucrose treatment. Compared with CaCl2 or glycerol treatment, the transformation efficiency of this method is significantly high when bacteria were cultured overnight at 42°C before cold sucrose treatment. Furthermore, applying this method, we successfully knocked out the pppA gene by direct electroporation. Collectively, our study provides a simple, time-saving, and efficient method for competent cell preparation of C. sakazakii, which is conducive to the further research of C. sakazakii.

CRISPR/Cas12a-Mediated Genome Editing in Thioalkalivibrio versutus.

Haloalkaliphilic Thioalkalivibrio versutus, a dominant species for sulfide removal, has attracted increasing attention. However, research on T. versutus is limited by the lack of genetic manipulation tools. In this work, we developed a CRISPR/AsCas12a-mediated system in T. versutus for an efficient and implementable genome editing workflow. Compared to the CRISPR/Cas9-mediated system, the CRISPR/AsCas12a system exhibited enhanced editing efficiency. Additionally, as Cas12a is capable of processing the crRNA maturation independently, the CRISPR/AsCas12a system allowed multiplex gene editing and large-fragment DNA knockout by expressing more than one crRNA under the control of one promoter. Using the CRISPR/AsCas12a system, five key genes of the elemental sulfur oxidation pathway were knocked out. Simultaneous deletion of the rhd and tusA genes disrupted the ability of T. versutus to metabolize elemental sulfur, resulting in a 24.7% increase in elemental sulfur generation and a 15.2% reduction in sulfate production. This genome engineering strategy significantly improved our understanding of sulfur metabolism in Thioalkalivibrio spp.

Genome engineering of the Corynebacterium glutamicum chromosome by the Extended Dual-In/Out strategy.

A novel genome editing method for repeated introduction of foreign DNA, including insertion of rather large DNA fragments, into predesigned points in the Corynebacterium glutamicum chromosome was developed. The method is based on the implementation of the Dual-In/Out strategy, which was previously provided in Escherichia coli according to recombineering-based methods (Minaeva et al., 2008) and allowed step-by-step construction of marker-less plasmid free recombinant strains. The strategy, suggested in the current study, is based on (i) E. coli Rac prophage RecE564/RecT-dependent recombineering; (ii) corynephage ϕ16 (Int/Xis)- and E. coli phage P1 Cre-mediated site-specific recombination systems; and (iii) the development of a C. glutamicum electrotransformation protocol with donor chromosomal DNA for combining of obtained modifications. It was found, that for each tested C. glutamicums strain, the efficiency of the different modifications for electrotransformation fluctuated significantly (up to two orders of magnitude), likely due to the recombinogenic accessibility of the corresponding locus of the bacterial chromosome. To avoid this difficulty, we proposed the phage Mu-driven transposition as a powerful approach for pre-selection of chromosomal regions convenient for single insertions and their further combination in a one strain. Additionally, it was found that the expression of RecE564/RecT coding genes in the recipient strain facilitated the inheritance of the penetrated DNA. It is proposed that the developed strategy in general and its separate elements should be helpful for broadening the genetic toolbox needed for genome editing of targeted C. glutamicum strains.

Stable transformation of fluorescent proteins into Nosema bombycis by electroporation.

BACKGROUND: Microsporidia are a group of intracellular parasitic eukaryotes, serious pathogens that cause widespread infection in humans, vertebrates, and invertebrates. Because microsporidia have a thick spore wall structure, the in vitro transformation, cell culture, and genetic operation technology of microsporidia are far behind that of other parasites. METHODS: In this study, according to an analysis of the life-cycle of microsporidia, Nosema bombycis, and different electro-transformation conditions, the transduction efficiency of introducing foreign genes into N. bombycis was systematically determined. RESULTS: We analyzed the direct electro-transformation of foreign genes into germinating N. bombycis using reporters under the regulation of different characteristic promoters. Furthermore, we systematically determined the efficiency of electro-transformation into N. bombycis under different electro-transformation conditions and different developmental stages through an analysis of the whole life-cycle of N. bombycis. These results revealed that foreign genes could be effectively introduced through a perforation voltage of 100 V pulsed for 15 ms during the period of N. bombycis sporeplasm proliferation. CONCLUSIONS: We present an effective method for electro-transformation of a plasmid encoding a fluorescent protein into N. bombycis, which provides new insight for establishing genetic modifications and potential applications in these intracellular parasites.

The effect of temperature conditions during growth on the transformation frequency of Coccomyxa subellipsoidea C-169 obtained by electroporation.

In this study, we have shown that transformation efficiency of Coccomyxa subellipsoidea C-169 obtained by electroporation can be significantly increased by either supra- or sub-optimal growth temperatures.

Electrotransformation of thermophilic bacterium Caldimonas manganoxidans.

Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 108 colony-forming units/µg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species.

Monitoring Tritrophic Biocontrol Interactions Between Bacillus spp., Fusarium oxysporum f. sp. cubense, Tropical Race 4, and Banana Plants in vivo Based on Fluorescent Transformation System.

Bacillus spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one B. subtilis strain, three B. velezensis strains, and three B. amyloliquefaciens strains that are proven to significantly inhibit TR4 in vitro, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD600 of bacteria concentration=0.7, plasmid concentration=50ng/µl, plasmid volume=2µl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory Bacillus spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. In vivo colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that Bacillus spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that B. subtilis, B. velezensis, and B. amyloliquefaciens can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.

Molecular Biology and Genetic Tools to Investigate Functional Redundancy Among Fe-S Cluster Carriers in E. coli.

Iron-sulfur (Fe-S) clusters are among the oldest protein cofactors, and Fe-S cluster-based chemistry has shaped the cellular metabolism of all living organisms. Over the last 30 years, thanks to molecular biology and genetic approaches, numerous actors for Fe-S cluster assembly and delivery to apotargets have been uncovered. In prokaryotes, Escherichia coli is the best-studied for its convenience of growth and its genetic amenability. During evolution, redundant ways to secure the supply of Fe-S clusters to the client proteins have emerged in E. coli. Disrupting gene expression is essential for gene function exploration, but redundancy can blur the interpretations as it can mask the role of important biogenesis components. This chapter describes molecular biology and genetic strategies that have permitted to reveal the E. coli Fe-S cluster conveying component network, composition, organization, and plasticity. In this chapter, we will describe the following genetic methods to investigate the importance of E. coli Fe-S cluster carriers: one-step inactivation of chromosomal genes in E. coli using polymerase chain reaction (PCR) products, P1 transduction, arabinose-inducible expression system, mevalonate (MVA) genetic by-pass, sensitivity tests to oxidative stress and iron starvation, ß-galactosidase assay, gentamicin survival test, and Hot Fusion cloning method.

Efficient electrotransformation of Rhodococcus ruber YYL with abundant extracellular polymeric substances via a cell wall-weakening strategy.

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.

Optimization of Electrotransformation Parameters and Engineered Promoters for Lactobacillus plantarum from Wine.

Robust and versatile promoters for Lactobacillus plantarum found in wine are necessary gene expression tools for genetic research involving wine stress. We optimized the electrotransformation parameters for L. plantarum XJ25 isolated from wine and engineered five promoters based on the promoter P23; these promoters showed significantly different transcriptional activities under nonstress conditions. The activities of these promoters in vivo and the resulting growth burden to the host strain under different wine stresses were also evaluated. A range of colors (from white to dark pink) of the developing colonies with the plasmid pNZ8148 carrying an X-mCherry expression cassette, namely, P23-mCherry, trcP23-mCherry, POL1-mCherry, POL2-mCherry, POL3-mCherry, or POL4-mCherry, were analyzed. The applicability of the optimized electrotransformation parameters and synthetic promoters with different activities were also verified in several L. plantarum strains. Therefore, the optimized electrotransformation and these characterized promoters were determined to be suitable for applications in wine research in the future.

The optimization system for preparation of TG1 competent cells and electrotransformation.

An efficient electrotransformation system that includes electrocompetent cells is a critical component for the success of large-scale gene transduction and replication. The conditions of TG1 competent cell preparation and optimal electrotransformation were evaluated by investigating different parameters. Certain parameters for preparation of TG1 competent cells (≥8 × 1010 colony forming units (cfu)/µg DNA) include optimum culture time of monoclonal bacteria (8-10 hr), amplification growth concentration (approximately OD600  = 0.45), and culture volume (400 ml in 2 L conical flask). With increased storage of competent cells at -80°C, electrotransformation efficiency gradually decreased, but it remains greater than ≥ 1010  cfu/µg DNA 3 months later. Moreover, the recovery time of electrotransformation also influenced electrotransformation efficiency (1.5-2 hr for optimization). The optimized transformation efficiency of TG1 (≥8 × 1010  cfu/µg DNA) was observed under suitable electric voltage (2.5 kV), electric intensity (15 kV/cm), and electric time (3.5 ms) of electricity for plasmid transformation. Optimized DNA amount (0.01-100 ng) dissolved in water led to the high efficiency of plasmid transformation (≥8 × 1010  cfu/µg DNA), but had low efficiency when dissolved in T4 ligation buffer (≤3 × 1010  cfu/µg DNA). These results indicated that an optimized TG1 transformation system is useful for high electrotransformation efficiency under general laboratory conditions. The optimized TG1 transformation system might facilitate large-scale gene transduction for phage display library construction.

Optimization of electrotransformation (ETF) conditions in lactic acid bacteria (LAB).

Lactic acid bacteria (LAB) play important roles in the food industry, animal husbandry and medicine which are closely related to human life. Modern gene engineering technology is the major means to reveal gene functions, study metabolic pathways and metabolomes of strains and improve the properties of strains. However, up to now, the molecular technologies that can be applied to LAB are still very scarce. One of the main reasons for this phenomenon is the low efficiency of transformation, furthermore, the transformation protocols developed are strain specific. At present, the most common method in the transformation of LAB is electrotransformation (ETF), which has relatively high transformation efficiency, convenient operation and good reproducibility. In the process of ETF, many factors may be involved in the regulation of the ETF efficiency of the strains, including the characteristics of the strains, the properties of exogenous plasmids, the parameters of electric pulse, the application of cell wall weakening agents, the composition of washing and electroporation buffers, the resuspending culture media and heat treatment. In recent years, other methods have also been utilized to boost the transformation efficiency of strains, such as combined chemical-physical methods and bacterial conjugation. Various experimental parameters and alternative technologies to ETF which may impact the transformation efficiency are summarized in this paper. This review describes some meaningful factors for the development of transformation systems, and provides direction and foundations for the optimization and establishment of novel transformation strategies of LAB.

Stable transformation of unicellular green alga Coccomyxa subellipsoidea C-169 via electroporation.

In this study, we have shown the applicability of electroporation and hygromycin B as a convenient selectable marker for stable nuclear transformation of Coccomyxa subellipsoidea C-169. Since it is the first sequenced eukaryotic microorganism from polar environment, this offers unique opportunities to study adaptation mechanisms to cold.

Electroporation Procedures for Genetic Modification of Green Algae (Chlorella spp.).

The green algae of Chlorella spp. are usually very small (about 3-6 µm), typically have solid and thick cell wall tissue; thus, neither the gene-gun method based on particle carrier nor the glass-bead transformation method is suitable enough. Selecting the proper, effective strategy has always attracted researcher's attention. Electroporation is currently the most widely used method for the transformation of algal species. The principle of electroporation is that the cell membrane produces tiny holes by high-voltage pulses, which lead to the introduction of exogenous DNA into cells. The method was proved by simple in principle and effective in introducing foreign genes in several Chlorella species.

Electrotransformation of Saccharomyces cerevisiae.

Intact yeast cell transformation is easily achieved by gene electrotransfer (GET). The procedure is fast and efficient in terms of transformants/µg DNA. Yeast cells in exponential growth phase are washed, treated for a short period with dithiothreitol (DTT) and then mixed with the plasmid DNA in a buffer with a low conductivity. A single well defined electric pulsed is delivered. After a 1 h incubation in the growth medium without selection, transformants are obtained on a selective plate medium. After a short description of the present knowledge on the events affecting the yeast cell as a consequence of the pulsed electric field, a step-by-step protocol is reported for Saccharomyces cerevisiae.

[Construction of Tn5 transposon insertion mutants of Ralstonia solanacearum isolated from Pogostemon cablin].

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.

Introduction of Phage Genome into Escherichia coli by Electroporation.

Electroporation has been an established tool for DNA delivery into prokaryotic and eukaryotic cells, thus facilitating basic research studies and improving medical treatments. Here we describe its use for introduction of phage genomic DNA into Escherichia coli cells, including preparation of electrocompetent cells, electric pulse optimization and recovery of electrotransformed cells. The technique can also be adapted for other bacterial species.

Construction of Tn5 transposon insertion mutants of Ralstonia solanacearum isolated from Pogostemon cablin / 中国中药杂志

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.

A practical random mutagenesis system for Ralstonia solanacearum strains causing bacterial wilt of Pogostemon cablin using Tn5 transposon.

A practical random mutagenesis system of Ralstonia solanacearum by electroporation with Tn5 transposon was established, which may be utilized to provide genetic approach to study virulence genes of R. solanacearum strains and create nonpathogenic mutants for biological control of bacterial wilt in Pogostemon cablin. R. solanacearum strain PRS-84 used in this study was isolated from P. cablin plants infected with bacterial wilt. The bacterial suspension of R. solanacearum strain PRS-84 was mixed with Tn5 transposome complex and the mixture was transformed by electroporation. The electroporated cells were then spread on the 2, 3, 5-triphenyltetrazolium chloride agar plates containing kanamycin to select the kanamycin-resistant colonies. Several factors which determined the bacterial transformation efficiency were optimized. The transformation process was shown to be optimal at the electric field strength of 12.5 kV cm-1. Bacterial cells harvested at mid-exponential phase gave the highest transformation efficiency. 10 µg mL-1 kanamycin was found to be the optimal concentration for transformant selection. Tn5 insertion mutants of R. solanacearum strain PRS-84 were identified by PCR amplification and Southern blot analysis. Mutants subcultured for 100 passages were also detected by PCR amplification and Southern blot analysis. Furthermore, pathogenicity screening test of mutants was performed by inoculating in vitro regenerated patchouli plants. Results revealed that mutants with a single Tn5 insertion in their genomes were obtained from R. solanacearum strain PRS-84, and the Tn5 insertion could be stably inherited in the mutants. Then, mutants with reduced pathogenicity were selected.

Mutations in Peptidoglycan Synthesis Gene ponA Improve Electrotransformation Efficiency of Corynebacterium glutamicum ATCC 13869.

Corynebacterium glutamicum is frequently engineered to serve as a versatile platform and model microorganism. However, due to its complex cell wall structure, transformation of C. glutamicum with exogenous DNA is inefficient. Although efforts have been devoted to improve the transformation efficiency by using cell wall-weakening agents, direct genetic engineering of cell wall synthesis for enhancing cell competency has not been explored thus far. Herein, we reported that engineering of peptidoglycan synthesis could significantly increase the transformation efficiency of C. glutamicum Comparative analysis of C. glutamicum wild-type strain ATCC 13869 and a mutant with high electrotransformation efficiency revealed nine mutations in eight cell wall synthesis-related genes. Among them, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA dramatically increased the electrotransformation of strain ATCC 13869 by 19.25-fold in the absence of cell wall-weakening agents, with no inhibition on growth. The Y489C mutation had no effect on the membrane localization of PonA but affected the peptidoglycan structure. Deletion of the ponA gene led to more dramatic changes to the peptidoglycan structure but only increased the electrotransformation by 4.89-fold, suggesting that appropriate inhibition of cell wall synthesis benefited electrotransformation more. Finally, we demonstrated that the PonAY489C mutation did not cause constitutive or enhanced glutamate excretion, making its permanent existence in C. glutamicum ATCC 13869 acceptable. This study demonstrates that genetic engineering of genes involved in cell wall synthesis, especially peptidoglycan synthesis, is a promising strategy to improve the electrotransformation efficiency of C. glutamicumIMPORTANCE Metabolic engineering and synthetic biology are now the key enabling technologies for manipulating microorganisms to suit the practical outcomes desired by humankind. The introduction of exogenous DNA into cells is an indispensable step for this purpose. However, some microorganisms, including the important industrial workhorse Corynebacterium glutamicum, possess a complex cell wall structure to shield cells against exogenous DNA. Although genes responsible for cell wall synthesis in C. glutamicum are known, engineering of related genes to improve cell competency has not been explored yet. In this study, we demonstrate that mutations in cell wall synthesis genes can significantly improve the electrotransformation efficiency of C. glutamicum Notably, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA increased electrotransformation efficiency by 19.25-fold by affecting peptidoglycan synthesis.