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Bacterial cellulose (BC) is an extracellular polysaccharide with myriad unique properties, such as high purity, water-holding capacity and biocompatibility, making it attractive in materials science. However, genetic engineering techniques for BC-producing microorganisms are rare. Herein, the electroporation-based gene transformation and the λ Red-mediated gene knockout method with a nearly 100 % recombination efficiency were established in the fast-growing and BC hyperproducer Enterobacter sp. FY-07. This genetic manipulation toolkit was validated by inactivating the protein subunit BcsA in the cellulose synthase complex. Subsequently, the inducible BC-producing strains from glycerol were constructed through inducible expression of the key gene fbp in the gluconeogenesis pathway, which recovered >80 % of the BC production. Finally, the BC properties analysis results indicated that the induced-synthesized BC pellicles were looser, more porous and reduced crystallinity, which could further broaden the application prospects of BC. To our best knowledge, this is the first attempt to construct the completely inducible BC-producing strains. Our work paves the way for increasing BC productivity by metabolic engineering and broadens the available fabrication methods for BC-based advanced functional materials.

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Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

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Manipulating viral genomes is an essential technique in reverse genetics and recombinant vaccine development. A strategy for manipulating large viral genomes involves introducing their entire genome into bacterial artificial chromosomes and employing Escherichia coli genetic tools. For sequence manipulation on bacterial artificial chromosomes (bacterial artificial chromosomes recombineering), a well-established method that relies on the Escherichia coli strain GS1783, and the template plasmid, pEPKan-S, is often used. This method, known as markerless DNA manipulation, allows for the generation of a recombinant bacterial artificial chromosome that does not retain the selection markers used during recombination. Although this method is highly innovative, there remains room for improvement as the plasmid is currently only available for positive selection. Additionally, differentiating true recombinants from false negatives often proves time-consuming. Consequently, an improved method for bacterial artificial chromosomes recombineering, which utilizes fluorescent proteins, has been developed. This method's core comprises three plasmids containing the I-SceI recognition site, antibiotic resistance genes (ampicillin, kanamycin, and zeocin), and fluorescent genes (YPet, mOrange, and mScarlet). The success or failure of Red recombination can be confirmed via fluorescent signals. To validate this method, the Lassa virus genes were introduced into the bacterial artificial chromosomes, containing the entire genome of the vaccinia virus strain LC16m8. Consequently, the expression of fluorescent protein genes contributed to positive selection, such as blue-white screening and counter-selection during the first and second Red recombination.

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Porcine epidemic diarrhea virus (PEDV) is a member of the genera Alphacoronavirus that has been associated with acute watery diarrhea and vomiting in swine. Unfortunately, no effective vaccines and antiviral drugs for PEDV are currently available. Reverse genetics systems are crucial tools for these researches. Here, a PEDV full-length cDNA clone was constructed. Furtherly, three PEDV reporter virus plasmids containing red fluorescent protein (RFP), Nano luciferase (Nluc), or green fluorescence protein (GFP) were generated using Red recombination with the GS1783 E. coli strain. These reporter-expressing recombinant (r) PEDVs showed similar growth properties to the rPEDV, and the foreign genes were stable to culture up to P9 in Vero cells. Using the Nluc-expressing rPEDV, the replication of PEDV was easily quantified, and a platform for rapid anti-PEDV drug screening was constructed. Among the three drugs, Bergenin, Umifenovir hydrochloride (Arbidol), and Ganoderma lucidum triterpenoids (GLTs), we found that GLTs inhibited PEDV replication mainly after the stage of virus "Entry". Overall, this study will broaden insight into the method for manipulating the PEDV genome and provide a powerful tool for screening anti-PEDV agents.

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Horizontal gene transfer is an important mechanism in bacterial evolution and can occur at striking frequencies when mediated by mobile genetic elements. Conjugative plasmids are mobile genetic elements that are main drivers of horizontal transfer and a major facilitator in the spread of antibiotic resistance genes. However, conjugative plasmid models that readily can be genetically modified with the aim to study horizontal transfer are not currently available. The aim of this study was to develop a conjugative plasmid model where the insertion of gene cassettes such as reporter genes (e.g., fluorescent proteins) or antibiotic resistance genes would be efficient and convenient. Here, we introduced a single attTn7 site into the conjugative broad-host-range IncP-1 plasmid pKJK5 in a non-disruptive manner. Furthermore, a version with lower transfer rate and a non-conjugative version of pKJK5-attTn7 were also constructed. The advantage of having the attTn7 sites is that genes of interest can be introduced in a single step with very high success rate using the Tn7 transposition system. In addition, larger genetic fragments can be inserted. To illustrate the efficacy of the constructed pKJK5 plasmids, they were complemented with sfGFP (a gene encoding superfolder green fluorescent protein) in addition to seven different ß-lactamase genes representing the four known classes of ß-lactamases.

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Shigellosis remains a major cause of severe diarrheal disease and death throughout the world. Vaccine development against shigellosis has been hampered by an incomplete understanding of the molecular mechanisms by which Shigella spp. causes disease and difficulties in manipulating Shigella spp. genomes. While homologous recombination protocols for the construction of precise gene deletions exist, construction of mutants in S. flexneri has not become commonplace. We describe the steps for construction of gene deletions using λ-red recombination using tools that we have developed in our laboratory.

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Escherichia coli K-12, being one of the best understood and thoroughly analyzed organisms, is the preferred platform for genetic and biochemical research. Among all genetic engineering approaches applied on E. coli, the homologous recombination approach is versatile and precise, which allows engineering genes or large segments of the chromosome directly by using polymerase chain reaction (PCR) products or synthetic oligonucleotides. The previously explained approaches for random insertion and deletions were reported as technically not easy and laborious. This study, first, finds the minimum length of homology extension that is efficient and accurate for homologous recombination, as 30 nt. Second, proposes an approach utilizing PCR products flanking ambiguous NNN-sequence (30-nt) extensions, which facilitate the homologous recombination to recombine them at multiple regions on the genome and generate insertion-deletion mutations. Further analysis found that these mutations were varying in number, that is, multiple genomic regions were deleted. Moreover, evaluation of the phenotype of all the multiple random insertion-deletion mutants demonstrated no significant changes in the normal metabolism of bacteria. This study not only presents the efficiency of ambiguous sequences in making random deletion mutations, but also demonstrates their further applicability in genomics.

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The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA-binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the genetic engineering and fluorescent labeling of strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types.

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BACKGROUND: Seamless modification of bacterial chromosomes is widely performed in both theoretical and practical research. For this purpose, excellent counter-selection marker genes with high stringency are needed. MAIN METHODS AND MAJOR RESULTS: The lysis gene E was first constructed under the control of the PL promoter and the cI857 repressor. At 42°C, it could effectively kill Escherichia coli and seamless modification in this bacterium using E as a counter-selection marker was successfully conducted. It also works in another gram-negative strain, Serratia marcescens, under the control of the Arac/PBAD regulatory system. By combining lysis gene E and kil, the counter-selection frequencies of the PL -kil-sd-E cassette in E. coli reached 4.9 × 10-8 and 3.2 × 10-8 at two test loci, which are very close to frequencies observed with the best counter-selection systems reported, the inducible toxin systems. Under the control of the Arac/PBAD , the counter-selection frequency of PBAD -kil-sd-E in S. marcescens reached the level of 10-7 at four test loci. By expressing the araC gene from plasmid pKDsg-ack, 5- to 17-fold improvements in counter-selection stringency were observed at these loci. A surprisingly low counter-selection frequency of 4.9 × 10-9 was obtained at the marR-1 locus, which reflects the highest stringency for a counter-selection cassette reported thus far. Similarly, at the araB locus of E. coli, the counter-selection frequency of PBAD -kil-sd-E was 3 × 10-9 after introducing plasmid pKDsg-ack. CONCLUSIONS AND IMPLICATIONS: We have developed and optimized a new universal counter-selection marker based on lysis gene E. The best counter-selection stringency of this new marker exceeds the inducible toxin system several fold. Our work can also provide inspiration for improving counter-selection stringency based on existing markers.

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The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.

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Komagataeibacter xylinus has received increasing attention as an important microorganism for the conversion of several carbon sources to bacterial cellulose (BC). However, BC productivity has been impeded by the lack of efficient genetic engineering techniques. In this study, a lambda Red and FLP/FRT-mediated site-specific recombination system was successfully established in Komagataeibacter xylinus. Using this system, the membrane bound gene gcd, a gene that encodes glucose dehydrogenase, was knocked out to reduce the modification of glucose to gluconic acid. The engineered strain could not produce any gluconic acid and presented a decreased bacterial cellulose (BC) production due to its restricted glucose utilization. To address this problem, the gene of glucose facilitator protein (glf; ZMO0366) was introduced into the knockout strain coupled with the overexpression of the endogenous glucokinase gene (glk). The BC yield of the resultant strain increased by 63.63-173.68%, thus reducing the production cost.

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In this study, we established the Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV) bacmid system for the construction of a Bac-to-Bac expression system and the generation of virus mutants. The CopyRight pSMART BAC cloning vector harboring the chloramphenicol resistance gene was introduced into the AnpeNPV genome to produce the AnpeNPV bacmid that could be propagated in Escherichia coli with stable replication. The enhanced green fluorescent protein (EGFP) was successfully expressed in both Tn-Hi5 cells and A. pernyi pupae using the AnpeNPV Bac-to-Bac expression system. To generate the AnpeNPV mutants, we developed the AnpeNPV bacmid/λ Red recombination system that facilitated the deletion of viral genes from the AnpeNPV genome. The genes cathepsin and chitinase were deleted and a derivative AnpeNPV Bac-to-Bac expression system was constructed. Furthermore, we demonstrated that the novel expression system could be used to express human epidermal growth factor in A. pernyi pupae. Taken together, the AnpeNPV bacmid system provides a powerful tool to create the AnpeNPV Bac-to-Bac expression system for protein expression in A. pernyi pupae. Further, it helps to knock-out genes from the AnpeNPV genome with λ Red recombination system for identification of the role of viral genes involved in regulating gene expression, DNA replication, virion structure, and infectivity during the AnpeNPV infection process.

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Over the past two decades, several coronavirus (CoV) infectious clones have been engineered, allowing for the manipulation of their large viral genomes (~30 kb) using unique reverse genetic systems. These reverse genetic systems include targeted recombination, in vitro ligation, vaccinia virus vectors, and bacterial artificial chromosomes (BACs). Quickly after the identification of Middle East respiratory syndrome-CoV (MERS-CoV), both in vitro ligation and BAC-based reverse genetic technologies were engineered for MERS-CoV to study its basic biological properties, develop live-attenuated vaccines, and test antiviral drugs. Here, I will describe how lambda red recombination can be used with the MERS-CoV BAC to quickly and efficiently introduce virtually any type of genetic modification (point mutations, insertions, deletions) into the MERS-CoV genome and recover recombinant virus.

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Seamless modification is a popular genomic manipulation technique in genetic engineering. Selection stringency of the counter-selection system determines the efficiency of the seamless modification. Recently, a novel counter-selection system, kil, was constructed. It is reported that the selection selectivity of kil is higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, indicating that recombinants could be selected out more efficiently by combining kil counter-selection system and plasmid pSim6. In order to confirm this speculation, four different loci (lacI, dbpa, ack, glk) in Escherichia coli strains W3110, MG1655 and DH10B were selected for testing: dsDNA fragments of different sizes (500 bp, 1 000 bp, and 2 000 bp) were used to substitute tet/kil. As expected, recombination efficiency was higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, and the results were more obvious with the length of dsDNA increasing. Specifically, recombination efficiency was 1.2 to 2 fold higher in pSim6 harboring bacteria than in pKD46 harboring bacteria when dsDNA fragments were 1 000 bp in length. With the length of dsDNA increasing up to 2 000 bp, the gap increased to 2.2-5 fold. In conclusion, it is easier to perform seamless modification by combining kil counter-selection system and plasmid pSim6 than combining kil and pKD46. An alternative tool in genomic engineering is provided in this study.

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This chapter provides a detailed protocol for construction of DNA barcode-tagged isogenic strains of Salmonella. The protocol is illustrated with S. Enteritidis in this chapter. However, this protocol should be widely applicable to other Salmonella serotypes. A series of the DNA barcode-tagged strains thus constructed can be used in combination with next generation sequencing or quantitative PCR to study the population dynamics of the bacterial pathogen during infection within the host or transmission within a population of the host in a quantitative manner.

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The lambda red recombination system makes it suitable for screening virulence gene utility in avian pathogenic Escherichia coli (APEC) on account of its wide applicability, simplicity and high efficiency. In APEC E058 (O2 serogroup), there are two copies of the outer membrane protease (ompT) gene, compT encoding cOmpT that is located on the chromosome and pompT encoding pOmpT that is located on a ColV plasmid. However, the relationship between pathogenesis and pompT expression in APEC E058 has yet to be elucidated. Here, we successfully constructed two pompT gene mutants: E058ΔpompT containing a chloramphenicol (cat) resistance gene and E058ΔpompT' without the cat gene. By RT-PCR and sequencing analysis, an unexpected transcriptome pompT' was detected in mutant strain E058ΔpompT' after deletion of the cat gene induced by the lambda red recombination system. Surprisingly, the pathogenicity of mutant E058ΔpompT was significantly attenuated compared to its parental strain in the chicken infection model and HD11 cell model then the pompT gene was knocked out, while the pathogenicity of the other mutant strain E058ΔpompT' had no difference. Furthermore, the presence of unexpected transcriptome pompT' influenced the bactericidal activity of SPF chicken serum and decreased the transcription level of TLR2 in the heart tissue of chickens. Our study identifies the pompT gene plays an important role in the virulence of APEC E058, and the unexpected transcriptome pompT' contributes to the increased pathogenicity of APEC E058 mutants following deletion of the cat gene induced by the lambda red recombination system, which suggests that this system still has some limitations for construction of mutant strains particularly where these are used in development of live vaccine.

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Seamless modification is a popular genomic manipulation technique in genetic engineering. Selection stringency of the counter-selection system determines the efficiency of the seamless modification. Recently, a novel counter-selection system, kil, was constructed. It is reported that the selection selectivity of kil is higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, indicating that recombinants could be selected out more efficiently by combining kil counter-selection system and plasmid pSim6. In order to confirm this speculation, four different loci (lacI, dbpa, ack, glk) in Escherichia coli strains W3110, MG1655 and DH10B were selected for testing: dsDNA fragments of different sizes (500 bp, 1 000 bp, and 2 000 bp) were used to substitute tet/kil. As expected, recombination efficiency was higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, and the results were more obvious with the length of dsDNA increasing. Specifically, recombination efficiency was 1.2 to 2 fold higher in pSim6 harboring bacteria than in pKD46 harboring bacteria when dsDNA fragments were 1 000 bp in length. With the length of dsDNA increasing up to 2 000 bp, the gap increased to 2.2-5 fold. In conclusion, it is easier to perform seamless modification by combining kil counter-selection system and plasmid pSim6 than combining kil and pKD46. An alternative tool in genomic engineering is provided in this study.

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In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis (S. Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). L-Arabinose, a common inducer for gene expression, modulated S. Choleraesuis efflux pump activity in a dose-dependent manner. At low L-arabinose concentrations, increasing L-arabinose led to a corresponding increase in fluorophore efflux, while at higher L-arabinose concentrations, increasing L-arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (ΔtolC), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude L-arabinose. Further, due to the rapid export of L-arabinose, an efficient method for recombination-mediated gene knockout, the L-arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of L-arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that L-arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of L-arabinose should facilitate the optimization of L-arabinose induction in strains with high efflux activity.

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Objective To construct a highly efficient approach to the introduction of the single-base mutation in a plasmid containing the adenovirus whole genome larger than 40 kb.Methods The target DNA with a mutation site was achieved by over-lapping PCR.The large plasmid with adenovirus genome and target DNA were co-transformed into Escherichia coli strain DY330 carrying a high rate Red recombination system.The positive clone was selected via colony PCR in combination with enzyme identification.The site-mutation large plasmid was transformed into E.coli strain DH10B in which the backbone of the large plasmid remained was stable.Results Two mutations were continuously introduced into the adenovirus genome,the location of which was pos.9171 and pos.24410 respectively.The integrality and stability of the plasmid backbone were verified by enzyme cutting identification.The two mutations on the plasmid were verified by DNA sequencing.Conclusion An efficient approach to the introduction of the single-base mutation in positions 9171 and 24410 from the adenovirus genome which was integrated into a plasmid is successfully established.The positive selection efficiency ranges from 5%to 15%.The construction of the approach will facilitate the study of adenovirus infection mechanism.

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The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA-binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types.

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