Counterselection System Research Articles

Exploiting phenotypic heterogeneity to improve production of glutathione by yeast

BackgroundGene expression noise (variation in gene expression among individual cells of a genetically uniform cell population) can result in heterogenous metabolite production by industrial microorganisms, with cultures containing both low- and high-producing cells. The presence of low-producing individuals may be a factor limiting the potential for high yields. This study tested the hypothesis that low-producing variants in yeast cell populations can be continuously counter-selected, to increase net production of glutathione (GSH) as an exemplar product.ResultsA counter-selection system was engineered in Saccharomyces cerevisiae based on the known feedback inhibition of gamma-glutamylcysteine synthetase (GSH1) gene expression, which is rate limiting for GSH synthesis: the GSH1 ORF and the counter-selectable marker GAP1 were expressed under control of the TEF1 and GSH-regulated GSH1 promoters, respectively. An 18% increase in the mean cellular GSH level was achieved in cultures of the engineered strain supplemented with D-histidine to counter-select cells with high GAP1 expression (i.e. low GSH-producing cells). The phenotype was non-heritable and did not arise from a generic response to D-histidine, unlike that with certain other test-constructs prepared with alternative markers.ConclusionsThe results corroborate that the system developed here improves GSH production by targeting low-producing cells. This supports the potential for exploiting end-product/promoter interactions to enrich high-producing cells in phenotypically heterogeneous populations, in order to improve metabolite production by yeast.

A convenient broad-host counterselectable system endowing rapid genetic manipulations of Kluyveromyces lactis and other yeast species

Being generally regarded as safe, Kluyveromyces lactis has been widely taken for food, feed, and pharmaceutical applications, owing to its ability to achieve high levels of protein secretion and hence being suitable for industrial production of heterologous proteins. Production platform strains can be created through genetic engineering; while prototrophic cells without chromosomally accumulated antibiotics resistance genes have been generally preferred, arising the need for dominant counterselection. We report here the establishment of a convenient counterselection system based on a Frs2 variant, Frs2v, which is a mutant of the alpha-subunit of phenylalanyl-tRNA synthase capable of preferentially incorporating a toxic analog of phenylalanine, r-chloro-phenylalanine (4-CP), into proteins to bring about cell growth inhibition. We demonstrated that expression of Frs2v from an episomal plasmid in K. lactis could make the host cells sensitive to 2 mM 4-CP, and a Frs2v-expressing plasmid could be efficiently removed from the cells immediately after a single round of cell culturing in a 4-CP-contianing YPD medium. This Frs2v-based counterselection helped us attain scarless gene replacement in K. lactis without any prior engineering of the host cells. More importantly, counterselection with this system was proven to be functionally efficient also in Saccharomyces cerevisiae and Komagataella phaffii, suggestive of a broader application scope of the system in various yeast hosts. Collectively, this work has developed a strategy to enable rapid, convenient, and high-efficiency construction of prototrophic strains of K. lactis and possibly many other yeast species, and provided an important reference for establishing similar methods in other industrially important eukaryotic microbes.

Development of a Recombineering System for the Acetogen Eubacterium limosum with Cas9 Counterselection for Markerless Genome Engineering.

Eubacterium limosum is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO2-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in E. limosum have been hindered by the inability to rapidly make precise genomic modifications. Here, we screened a diverse library of recombinase proteins to develop a highly efficient oligonucleotide-based recombineering system based on the viral recombinase RecT. Following optimization, the system is capable of catalyzing ssDNA recombination at an efficiency of up to 2%. Addition of a Cas9 counterselection system eliminated unrecombined cells, with up to 100% of viable cells encoding the desired mutation, enabling creation of genomic point mutations in a scarless and markerless manner. We deployed this system to create a clean knockout of the extracellular polymeric substance (EPS) gene cluster, generating a strain incapable of biofilm formation. This approach is rapid and simple, not requiring laborious homology arm cloning, and can readily be retargeted to almost any genomic locus. This work overcomes a major bottleneck in E. limosum genetic engineering by enabling precise genomic modifications, and provides both a roadmap and associated recombinase plasmid library for developing similar systems in other Clostridia of interest.

Efficient markerless genetic manipulation of Pasteurella multocida using lacZ and pheSm as selection markers.

Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida. Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB, opa, and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheSm screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p-chloro-phenylalanine, demonstrating the feasibility of pheSm as a counter-selective marker. The pheSm system was used for markerless deletions of glgB, opa, and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheSm counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida.IMPORTANCEPasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheSm screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheSm as counterselection markers to improve counterselection efficiency in P. multocida. This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida.

Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses.

Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

Versatile allelic replacement and self-excising integrative vectors for plasmid genome mutation and complementation.

In spite of the dissemination of multidrug-resistant plasmids among Gram-negative pathogens, including those carrying virulence genes, vector tools for studying plasmid-born genes are lacking. The allelic replacement vectors can be used to generate plasmid or chromosomal mutations including markless point mutations. This is the first report describing a self-excising integrative vector that can be used as a stable single-copy complementing tool to study medically important pathogens including in vivo studies without the need for antibiotic selection. Overall, our newly developed vectors can be applied for the assessment of the function of plasmid-encoded genes by specifically creating mutations, moving large operons between plasmids and to/from the chromosome, and complementing phenotypes associated with gene mutation. Furthermore, the vectors express chromophores for the detection of target gene modification or colony isolation, avoiding time-consuming screening procedures.

Development of a counterselectable system for rapid and efficient CRISPR-based genome engineering in Zymomonas mobilis

BackgroundZymomonas mobilis is an important industrial bacterium ideal for biorefinery and synthetic biology studies. High-throughput CRISPR-based genome editing technologies have been developed to enable targeted engineering of genes and hence metabolic pathways in the model ZM4 strain, expediting the exploitation of this biofuel-producing strain as a cell factory for sustainable chemicals, proteins and biofuels production. As these technologies mainly take plasmid-based strategies, their applications would be impeded due to the fact that curing of the extremely stable plasmids is laborious and inefficient. Whilst counterselection markers have been proven to be efficient for plasmid curing, hitherto only very few counterselection markers have been available for Z. mobilis.ResultsWe constructed a conditional lethal mutant of the pheS gene of Z. mobilis ZM4, clmPheS, containing T263A and A318G substitutions and coding for a mutated alpha-subunit of phenylalanyl-tRNA synthetase to allow for the incorporation of a toxic analog of phenylalanine, p-chloro-phenylalanine (4-CP), into proteins, and hence leading to inhibition of cell growth. We demonstrated that expression of clmPheS driven by a strong Pgap promoter from a plasmid could render the Z. mobilis ZM4 cells sufficient sensitivity to 4-CP. The clmPheS-expressing cells were assayed to be extremely sensitive to 0.2 mM 4-CP. Subsequently, the clmPheS-assisted counterselection endowed fast curing of genome engineering plasmids immediately after obtaining the desired mutants, shortening the time of every two rounds of multiplex chromosome editing by at least 9 days, and enabled the development of a strategy for scarless modification of the native Z. mobilis ZM4 plasmids.ConclusionsThis study developed a strategy, coupling an endogenous CRISPR-based genome editing toolkit with a counterselection marker created here, for rapid and efficient multi-round multiplex editing of the chromosome, as well as scarless modification of the native plasmids, providing an improved genome engineering toolkit for Z. mobilis and an important reference to develope similar genetic manipulation systems in other non-model organisms.

Exploitation of a Type 1 Toxin-Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen Eubacterium limosum.

Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in Eubacterium limosum B2. The genes targeted in this study were those encoding the histidine biosynthesis gene hisI, the methanol methyltransferase and corrinoid protein mtaA and mtaC, and mtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within hisI brought about the expected histidine auxotrophy, and deletions of mtaA and mtaC both abolished autotrophic growth on methanol. Deletion of mtcB was shown to abolish the growth of E. limosum on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of E. limosum.

Adaptation of Prokaryotic Toxins for Negative Selection and Cloning-Independent Markerless Mutagenesis in Streptococcus Species.

The Streptococcus mutans genetic system offers a variety of strategies to rapidly engineer targeted chromosomal mutations. Previously, we reported the first S. mutans negative selection system that functions in a wild-type background. This system utilizes induced sensitivity to the toxic amino acid analog p-chlorophenylalanine (4-CP) as a negative selection mechanism and was developed for counterselection-based cloning-independent markerless mutagenesis (CIMM). While we have employed this system extensively for our ongoing genetic studies, we have encountered a couple limitations with the system, mainly its narrow host range and the requirement for selection on a toxic substrate. Here, we report the development of a new negative selection system that addresses both limitations, while still retaining the utility of the previous 4-CP-based markerless mutagenesis system. We placed a variety of toxin-encoding genes under the control of the xylose-inducible gene expression cassette (Xyl-S) and found the Fst-sm and ParE toxins to be suitable candidates for inducible negative selection. We combined the inducible toxins with an antibiotic resistance gene to create several different counterselection cassettes. The most broadly useful of these contained a wild-type fst-sm open reading frame transcriptionally fused to a point mutant form of the Xyl-S expression system, which we subsequently named IFDC4. IFDC4 was shown to exhibit exceptionally low background resistance, with 3- to 4-log reductions in cell number observed when plating on xylose-supplemented medium. IFDC4 also functioned similarly in multiple strains of S. mutans as well as with Streptococcus gordonii and Streptococcus sanguinis. We performed CIMM with IFDC4 and successfully engineered a variety of different types of markerless mutations in all three species. The counterselection strategy described here provides a template approach that should be adaptable for the creation of similar counterselection systems in many other bacteria. IMPORTANCE Multiple medically significant Streptococcus species, such as S. mutans, have highly sophisticated genetic systems available, largely as a consequence of their amenability to genetic manipulation via natural competence. Despite this, few options are available for the creation of markerless mutations in streptococci, especially within wild-type strains. Markerless mutagenesis is a critical tool for genetic studies, as it allows the user to explore many fundamental questions that are not easily addressable using marked mutagenesis. Here, we describe a new approach for streptococcal markerless mutagenesis that offers a variety of advantages over the current approach, which employs induced sensitivity to the toxic substrate 4-CP. The approach employed here should be readily adaptable for the creation of similar markerless mutagenesis systems in other organisms.

HicA Toxin-Based Counterselection Marker for Allelic Exchange Mutations in Fusobacterium nucleatum.

The study of fusobacterial virulence factors has dramatically benefited from the creation of various genetic tools for DNA manipulation, including galK-based counterselection for in-frame deletion mutagenesis in Fusobacterium nucleatum, which was recently developed. However, this method requires a host lacking the galK gene, which is an inherent limitation. To circumvent this limitation, we explored the possibility of using the hicA gene that encodes a toxin consisting of a HicAB toxin-antitoxin module in Fusobacterium periodonticum as a new counterselective marker. Interestingly, the full-length hicA gene is not toxic in F. nucleatum, but a truncated hicA gene version lacking the first six amino acids is functional as a toxin. The toxin expression is driven by an rpsJ promoter and is controlled at its translational level by using a theophylline-responsive riboswitch unit. As a proof of concept, we created markerless in-frame deletions in the fusobacterial adhesin radD gene within the F. nucleatum rad operon and the tnaA gene that encodes the tryptophanase for indole production. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of colonies grown on theophylline-added plates and resulted in in-frame deletions in 50% of the screened isolates. This hicA-based counterselection system provides a robust and reliable counterselection in wild-type background F. nucleatum and should also be adapted for use in other bacteria. IMPORTANCE Fusobacterium nucleatum is an indole-producing human oral anaerobe associated with periodontal diseases, preterm birth, and several cancers. Little is known about the mechanisms of fusobacterial pathogenesis and associated factors, mainly due to the lack of robust genetic tools for this organism. Here, we showed that a mutated hicA gene from Fusobacterium periodonticum expresses an active toxin and was used as a counterselection marker. This hicA-based in-frame deletion system efficiently creates in-frame deletion mutations in the wild-type background of F. nucleatum. This is the first report to use the hicA gene as a counterselection marker in a bacterial genetic study.

Development of an efficient ClosTron system for gene disruption in Ruminiclostridium papyrosolvens.

Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic clostridia, promising consolidated bioprocessing (CBP) candidate for producing renewable green chemicals from cellulose, but its metabolic engineering is limited by lack of genetic tools. Here, we firstly employed the endogenous xylan-inducible promoter to control ClosTron system for gene disruption of R. papyrosolvens. The modified ClosTron can be easily transformed into R. papyrosolvens and specifically disrupt targeting genes. Furthermore, a counter selectable system based on uracil phosphoribosyl-transferase (Upp) was successfully established and introduced into the ClosTron system, which resulted in plasmid curing rapidly. Thus, the combination of xylan-inducible ClosTron and upp-based counter selectable system makes the gene disruption more efficient and convenient for successive gene disruption in R. papyrosolvens. KEY POINTS: • Limiting expression of LtrA enhanced the transformation of ClosTron plasmids in R. papyrosolvens. • DNA targeting specificity can be improved by precise management of the expression of LtrA. • Curing of ClosTron plasmids was achieved by introducing the upp-based counter selectable system.

129. LiaX, a Member of the LiaFSR System, is Essential for Cell Envelope Adaptation System in Enterococcus faecium

Abstract Background Daptomycin (DAP) is an important antibiotic for enterococci. Our previous studies identified LiaX as a surface-exposed protein that is a mediator of cell envelope homeostasis in Enterococcus faecalis (Efs) upon exposure to DAP via activation of the LiaFSR system. LiaX encodes for a soluble protein with an N-terminal of α-helices, and a C-terminus which is β-pleated sheets. Its role in E. faecium, a species of clinical relevance, remains unknown. In this work, we aim to elucidate the function of LiaX of E. faecium (Efm). Methods We used the commensal strain of Efm TX1330RF (DAP minimum inhibitory concentration [MIC] 3 μg/ml), its ΔliaR derivative (TX1330RFΔliaR [DAP MIC 0.125 μg/ml]) and targeted the liaX gene for mutagenesis. Using the PheS* counterselection system, we aimed to create a truncation or in-frame deletion of liaX. Mutants were characterized by determination of DAP MIC and visualization of anionic phospholipid domains by 10-N-nonyl-acridine orange (NAO). The nisin-controlled expression vector, pMSP3535, was used to express LiaX from Efm (LiaXTX1330RF) in Efs host OG1RFΔliaX, which lacks liaX. Expression of LiaX of Efs (LiaXOG1RF) was used as a control. Results In the presence of liaR, we were unable to create truncation or in-frame deletion of liaX after many attempts. In Efm TX1330RFΔliaR, we successfully created an in-frame deletion of liaX, TX1330RFΔliaRliaX (DAP MIC 0.125 μg/ml). NAO staining of all strains of TX1330RF, TX1330RFΔliaR and its ΔliaX mutant showed localization of anionic phospholipid domains at septa of the cells. Interestingly, multiple attempts to complement liaR in TX1330RFΔliaRliaX were also futile. NAO staining of Efs OG1RFΔliaX demonstrated that anionic phospholipid microdomains redistributed away from septa. Expression of LiaXOG1RF in Efs OG1RFΔliaX successfully restored microdomains to septal and polar regions of Efs while expression of LiaXTX1330RF did not show any significant difference in the fluorescence staining compared to its parental host (Efs OG1RFΔliaX). Conclusion Our findings suggest that LiaX has divergent functions as a mediator of cell envelope homeostasis in enterococci and may be essential in the Efm redponse to DAP. Efforts to elucidate its role in DAP resistance in Efm are ongoing. Disclosures Cesar A. Arias, MD, PhD, Entasis Phramceuticals: Grant/Research Support|MeMed Diagnostics: Grant/Research Support|Merck: Grant/Research Support.

Recombineering in Non-Model Bacteria.

The technology of recombineering, in vivo genetic engineering, was initially developed in Escherichia coli and uses bacteriophage-encoded homologous recombination proteins to efficiently recombine DNA at short homologies (35 to 50 nt). Because the technology is homology driven, genomic DNA can be modified precisely and independently of restriction site location. Recombineering uses linear DNA substrates that are introduced into the cell by electroporation; these can be PCR products, synthetic double-strand DNA (dsDNA), or single-strand DNA (ssDNA). Here we describe the applications, challenges, and factors affecting ssDNA and dsDNA recombineering in a variety of non-model bacteria, both Gram-negative and -positive, and recent breakthroughs in the field. We list different microbes in which the widely used phage λ Red and Rac RecET recombination systems have been used for in vivo genetic engineering. New homologous ssDNA and dsDNA recombineering systems isolated from non-model bacteria are also described. The Basic Protocol outlines a method for ssDNA recombineering in the non-model species of Shewanella. The Alternate Protocol describes the use of CRISPR/Cas as a counter-selection system in conjunction with recombineering to enhance recovery of recombinants. We provide additional background information, pertinent considerations for experimental design, and parameters critical for success. The design of ssDNA oligonucleotides (oligos) and various internet-based tools for oligo selection from genome sequences are also described, as is the use of oligo-mediated recombination. This simple form of genome editing uses only ssDNA oligo(s) and does not require an exogenous recombination system. The information presented here should help researchers identify a recombineering system suitable for their microbe(s) of interest. If no system has been characterized for a specific microbe, researchers can find guidance in developing a recombineering system from scratch. We provide a flowchart of decision-making paths for strategically applying annealase-dependent or oligo-mediated recombination in non-model and undomesticated bacteria. © 2022 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: ssDNA recombineering in Shewanella species Alternate Protocol: ssDNA recombineering coupled to CRISPR/Cas9 in Shewanella species.

A pyrF-Based Efficient Genetic Manipulation Platform in Acinetobacter baumannii To Explore the Vital DNA Components of Adaptive Immunity for I-F CRISPR-Cas.

ABSTRACTAcinetobacter baumannii is an important pathogenic bacterium with multidrug resistance which causes infections with high mortality rates. In-depth genetic analysis of A. baumannii virulence and drug-resistant genes is highly desirable. In this study, we utilized the conserved pyrF-flanking fragment to rapidly generate uracil auxotrophy hosts with pyrF deleted in model and clinical A. baumannii strains and then introduced the pyrF gene as the selectable and counterselectable marker to establish a series of gene manipulation vectors. For gene deletion with the suicide pyrF-based plasmid, the second-crossover colonies screened with the pyrF/5-fluoroorotic acid (5-FOA) system were obtained more quickly and efficiently than those screened with the sacB/sucrose system. By using the replicative plasmid, the recognized protospacer-adjacent motif (PAM) bias for type I-F CRISPR was experimentally revealed in A. baumannii AYE. Interestingly, interference recognized only the PAM-CC sequence, whereas adaptation priming tolerates 4 PAM sequences. Furthermore, we also performed a rapid and extensive modification of the I-F CRISPR-Cas elements and revealed that the role of double-nucleotide sequence mutants at the end of the repeat could be critical during both CRISPR interference and priming; we also found strong biases for A and demonstrated that adaptation could tolerate certain sequence and size variations of the leader in A. baumannii. In conclusion, this pyrF-based genetic manipulation system was readily applicable and efficient for exploring the genetic characteristics of A. baumannii.IMPORTANCE In this study, we developed the widely applicable and efficient pyrF-based selection and counterselection system in A. baumannii for gene manipulation. In most cases, this pyrF/5-FOA genetic manipulation system was very effective and enabled us to obtain marker-free mutants in a very short period of time. Utilizing this system and the separate mechanism of interference and/or primed adaptation, our experiments revealed some recognition mechanism differences for the key DNA elements of PAM, leader, and repeat in the priming adaptation process of the I-F CRISPR-Cas systems of A. baumannii, which provided some new and original insights for the study of the molecular mechanisms of these processes and laid a foundation for further studies.

Development and optimization of Lysis gene E as a counter-selection marker with high stringency.

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. 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. 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.

Generation of Markerless Gene Deletion Mutants in Listeria monocytogenes Using a Mutated pheS for Counterselection.

Gene alteration/deletion by allelic exchange is the preferred strategy for gene manipulation in bacteria. Here we present the fundamentals for an efficient allelic exchange gene deletion method in the bacterial pathogen Listeria monocytogenes. Combining vector generation by Gibson assembly with a counterselection system based on the mutated phenylalanine synthetase (pheS*) makes the generation of gene deletion mutants straightforward and time efficient.

Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria.

Here we describe a universal approach for plasmid-free genome engineering in cyanobacteria that exploits the polyploidy of their chromosomes as a natural counterselection system. Rather than being delivered via replicating plasmids, genes encoding for DNA modifying enzymes are instead integrated into essential genes on the chromosome by allelic exchange, as facilitated by antibiotic selection, a process that occurs readily and with only minor fitness defects. By virtue of the essentiality of these integration sites, full segregation is never achieved, with the strain instead remaining as a merodiploid so long as antibiotic selection is maintained. As a result, once the desired genome modification is complete, removal of antibiotic selection results in the gene encoding for the DNA modifying enzyme to then be promptly eliminated from the population. Proof of concept of this new and generalizable strategy is provided using two different site-specific recombination systems, CRE-lox and DRE-rox, in the fast-growing cyanobacterium Synechococcus sp. PCC 7002, as well as CRE-lox in the model cyanobacterium Synechocystis sp. PCC 6803. Reusability of the method, meanwhile, is demonstrated by constructing a high-CO2 requiring and markerless Δndh3 Δndh4 ΔbicA ΔsbtA mutant of Synechococcus sp. PCC 7002. Overall, this method enables the simple and efficient construction of stable and unmarked mutants in cyanobacteria without the need to develop additional shuttle vectors nor counterselection systems.

Development of a Positive Selection High Throughput Genetic Screen in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum is a powerful model organism that can be utilized to investigate human health and disease. One particular strength of Dictyostelium is that it can be utilized for high throughput genetic screens. For many phenotypes, one limitation of utilizing Dictyostelium is that screening can be an arduous and time-consuming process, limiting the genomic depth one can cover. Previously, we utilized a restriction enzyme-mediated integration screen to identify suppressors of polyglutamine aggregation in Dictyostelium. However, due to the time required to perform the screen, we only obtained ∼4% genome coverage. Here we have developed an efficient screening pipeline that couples chemical mutagenesis with the 5-fluoroorotic acid counterselection system to enrich for mutations in genes of interest. Here we describe this new screening methodology and highlight how it can be utilized for other biological systems.

Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus.

Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications. The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host (ΔpyrF) resistant to the highly toxic uracil analog of 5-fluoroorotic acid (5-FOA) converted by PyrF, and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase. The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications. Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutant ΔpyrF at the targeted locus. Double-crossover recombinants were generated, from which the pyrF gene, plasmid backbone, and targeted gene were excised through homologous recombination exchange. These recombinants were rapidly screened by the counterselection agent, 5-FOA. We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018. This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.

Development and application of a highly efficient CRISPR-Cas9 system for genome engineering in Bacillus megaterium

Bacillus megaterium has become increasingly important for the biotechnological production of valuable compounds of industrial and pharmaceutical importance. Despite recent advances in rational strain design of B. megaterium, these studies have been largely impaired by the lack of molecular tools that are not state-of-the-art for comprehensive genome engineering approaches. In the current work, we describe the adaptation of the CRISPR-Cas9 vector pJOE8999 to enable efficient genome editing in B. megaterium. Crucial modifications comprise the exchange of promoter elements and associated ribosomal binding sites as well as the implementation of a 5-fluorouracil based counterselection system to facilitate proper plasmid curing. In addition, the functionality and performance of the new CRISPR-Cas9 vector pMOE was successfully evaluated by chromosomal disruption studies of the endogenous β-galactosidase gene (BMD_2126) and demonstrated an outstanding efficiency of 100 % based on combinatorial pheno- and genotype analyses. Furthermore, pMOE was applied for the genomic deletion of a steroid esterase gene (BMD_2256) that was identified among several other candidates as the gene encoding the esterase, which prevented accumulation of pharmaceutically important glucocorticoid esters. Recombinant expression of the bacterial chloramphenicol acetyltransferase 1 gene (cat1) in the resulting esterase deficient B. megaterium strain ultimately yielded C21-acetylated as well as novel C21-esterified derivates of cortisone.