Mutant Phenotype Research Articles

Analysis of randomly mutated AlSRKb genes reveals that most loss-of-function mutations cause defects in plasma membrane localization.

Only very limited information is available on why some nonsynonymous variants severely alter gene function while others have no effect. To identify the characteristic features of mutations that strongly influence gene function, this study focused on SRK which encodes a highly polymorphic receptor kinase expressed in stigma papillary cells that underlies a female determinant of self-incompatibility in Brassicaceae. A set of 300 Arabidopsis thaliana transformants expressing mutated SRKb from A. lyrata was constructed using error-prone PCR and the genotype and self-incompatibility phenotype of each transformant were determined. Almost all the transformants showing the self-incompatibility defect contained mutations in AlSRKb that altered localization to the plasma membrane. The observed mutations occurred in amino acid residues that were highly conserved across S haplotypes and whose predicted locations were in the interior of the protein. Our findings suggested that mutations causing the self-incompatibility defect were more likely to result from changes to AlSRKb biosynthesis than from loss of AlSRKb function. In addition, we examined whether the RandomForest and Extreme Gradient Boosting methods could predict the self-incompatibility phenotypes of SRK mutants.

Anti-Müllerian hormone deficiency leads to two distinct ovarian phenotypes with different alterations of sex hormones in gynogenetic carp

Anti-Müllerian hormone (Amh) plays conserved roles in gonadal development and function in vertebrates. Previous studies have demonstrated that amh deficiency results in severe gonadal hypertrophy in teleost. Here, we report a novel phenotype of gonadal atrophy caused by amh knockout in fish. Firstly, it was found that the amh gene had three alleles and was expressed in the cytoplasm of follicle cells in gynogenetic amphiploid gibel carp (Carassius gibelio). Subsequently, we constructed a mutant line of amh and identified two distinct ovarian abnormalities in gibel carp. Most mutants exhibited hypertrophic ovaries with increased proliferation and decreased differentiation of early germ cells, which is consistent with previously reported findings. Intriguingly, a small number of mutants displayed atrophic ovaries with decreased proliferation and premature differentiation of germ cells, which represents a novel phenotype in fish species. Transcriptome analysis revealed that these two distinct ovaries of amh mutants showed different alterations in the sex hormone biosynthesis pathway, when compared with wild-type ovaries. Furthermore, diverse gonadal sex hormone levels were also detected in these two abnormal ovaries, which were consistent with the transcriptional expressions of corresponding genes in the sex hormone synthesis pathway. These findings identify a novel atrophic ovarian phenotype of amh mutants and confirm the amh function in controlling the balance between proliferation and differentiation of germ cells in fish.

Unveiling the genetic basis and metabolic rewiring behind the galactose-positive phenotype in a Streptococcus thermophilus mutant

Streptococcus thermophilus (S. thermophilus) is a widely used starter culture in dairy fermentation, but most strains are galactose-negative and only metabolize glucose from lactose hydrolysis. In this study, we aimed to uncover the mechanisms underlying the acquisition of a stable galactose-positive (Gal+) phenotype in a mutant strain of S. thermophilus IMAU10636. By treating the wild-type strain with the mutagenic agent N-methyl-N-nitro-N-nitrosoguanidine, we successfully isolated a Gal+ mutant, S. thermophilus IMAU10636Y. Comparative enzyme activity assays revealed that the mutant exhibited higher β-galactosidase and galactokinase activities, but lower glucokinase and pyruvate kinase activities compared to the wild-type. High-performance liquid chromatography analysis confirmed the mutant’s enhanced ability to utilize lactose and galactose, leading to increased glucose secretion. Integrated genome and transcriptomics analyses provided deeper insights into the underlying genetic and metabolic mechanisms. We found that the metabolism regulatory network of the glycolysis / Leloir pathway was altered in the mutant, possibly due to the upregulation of the gene expression in the galR-galK intergenic region. This likely led to increased RNA polymerase binding and transcription of the gal operon, ultimately promoting the Gal+ phenotype. Additionally, we identified a mutation in the scrR gene, encoding a LacI family transcriptional repressor, which also contributed to the Gal+ phenotype. These findings offer new perspectives on the metabolic rewiring and regulatory mechanisms that enable S. thermophilus to acquire the ability to metabolize galactose. This knowledge can inform strategies for engineering and selecting Gal+ strains with desirable fermentation characteristics for dairy applications.

MYB Transcription Factor CDC5 Activates CBF3 Expression to Positively Regulates Freezing Tolerance via Cooperating With ICE1 and Histone Modification in Arabidopsis.

The freezing temperature greatly limits the growth, development and productivity of plants. The C-repeat/DRE binding factor (CBF) plays a major role in cold acclimation, enabling plants to increase their freezing tolerance. Notably, the INDUCER OF CBF EXPRESSION1 (ICE1) protein has garnered attention for its pivotal role in bolstering plants' resilience against freezing through transcriptional upregulation of DREB1A/CBF3. However, the research on the interaction between ICE1 and other transcription factors and its function in regulating cold stress tolerance is largely inadequate. In this study, we found that a R2R3 MYB transcription factor CDC5 interacts with ICE1 and regulates the expression of CBF3 by recruiting RNA polymerase II, overexpression of ICE1 can complements the freezing deficient phenotype of cdc5 mutant. CDC5 increases the expression of CBF3 in response to freezing. Furthermore, CDC5 influences the expression of CBF3 by altering the chromatin status through H3K4me3 and H3K27me3 modifications. Our work identified a novel component that regulates CBF3 transcription in both ICE1-dependent and ICE1-independent manner, improving the understanding of the freezing signal transduction in plants.

Identification of Transposon Insertion Sites in MaizeMu-Tagged Mutants Using Mu-Seq

Mutator(Mu) transposons facilitate untargeted insertional mutagenesis in maize by moving within the genome and disrupting genes. Such an approach has been used to generate collections such as theBonnMuresource, aMu-tagged maize population for functional genomics studies. Mutant-Seq (Mu-Seq) is a sequencing-based method for the high-throughput identification and mapping ofMuinsertion sites. The approach involves the construction of multiplexed sequencing libraries (known as Mu-Seq libraries) fromMu-tagged populations, followed by high-throughput sequencing and data processing using the Mu-Seq Workflow Utility (MuWU) tool, to determine the location ofMuinsertions. Here, we provide a detailed protocol for Mu-Seq, from the generation of the maizeMu-tagged mutant population to data analysis. Researchers can use this approach to develop mutant collections customized to specific genetic backgrounds of interest, which can aid in characterizing genotype-specific mutations and identifying candidate genes linked to visible mutant phenotypes.

Use of Maize (Zea maysL.) Mutator Transposon-Induced Mutants of theBonnMuResource for Forward and Reverse Genetics Studies

TheBonnMuresource represents a tagged collection of maize (Zea maysL.)Mutator(Mu) transposon-induced mutants, designed for functional genomics studies. Here, we describe the use of theBonnMucollection for identifying and characterizing mutations. Specifically, we describe workflows for use in both reverse and forward genetics strategies in maize. For reverse genetics, users first acquire aBonnMuF2stock of interest based on data accessible at the Maize Genetics and Genomics Database (MaizeGDB). We provide details here for their subsequent propagation and for the confirmation ofMuinsertions by genotyping via PCR, with the ultimate goal of establishing genotype–phenotype relationships of interest. For forward genetics studies, we describe a workflow that involves a combined approach of Mutant-Seq (Mu-Seq) and bulked segregant RNA-seq (BSR-Seq), to identify the causal gene underlying a mutant phenotype of interest.

You Don't Always Get What You Want!

Mutations in several genes of Caenorhabditis elegans confer altered sensitivities to volatile anesthetics. A mutation in one gene, gas-1(fc21), causes animals to be immobilized at lower concentrations of all volatile anesthetics than in the wild type, and it does not depend on mutations in other genes to control anesthetic sensitivity. gas-1 confers different sensitivities to stereoisomers of isoflurane, and thus may be a direct target for volatile anesthetics. The authors have cloned and characterized the gas gene and the mutant allele fc21. Genetic techniques for nematodes were as previously described. Polymerase chain reaction, sequencing, and other molecular biology techniques were performed by standard methods. Mutant rescue was done by injecting DNA fragments into the gonad of mutant animals and scoring the offspring for loss of the mutant phenotype. The gas-1 gene was cloned and identified. The protein GAS-1 is a homologue of the 49-kd (IP) subunit of the mitochondrial NADH-ubiquinone-oxidoreductase (complex I of the respiratory chain). gas-1(fc21) is a missense mutation replacing a strictly conserved arginine with lysine. The function of the 49-kd (IP) subunit of complex I is unknown. The finding that mutations in complex I increase sensitivity of C. elegans to volatile anesthetics may implicate this physiologic process in the determination of anesthetic sensitivity. The hypersensitivity of animals with a mutation in the gas-1 gene may be caused by a direct anesthetic effect on a mitochondrial protein or secondary effects at other sites caused by mitochondrial dysfunction.

Development and Characterization of a New TILLING Population for Forward and Reverse Genetics in Barley (Hordeum vulgare L.).

Mutagenesis is an important tool in crop improvement and free of the regulatory restrictions imposed on genetically modified organisms. Barley (Hordeum vulgare L.) is a diploid species with a genome smaller than those of other members of the Triticeae crops, making it an attractive model for genetic studies in Triticeae crops. In this study, we report an ethyl methane sulfonate (EMS)-mutagenized population in the Chinese barley landrace TX9425, which is tolerant to both abiotic and biotic stress. A TILLING (Targeting Induced Locus Lesion in Genomes) population consisting of 2000 M2 lines was also constructed based on the CEL I enzyme with subsequent polyacrylamide electrophoresis, which decreased the cost and labor investment. The mutant phenotypes of the M2 and M3 generations were scored and revealed the presence of a wide spectrum of morphological diversity. The population was evaluated by screening for induced mutations in five genes of interest. A detailed analysis was performed for the HvGLR3.5 gene and three mutations were identified by screening in 2000 M2 lines. Two of three mutations displayed tuft and yellow striped leaves compared to the wild type. Altogether, our study shows the efficiency of screening and the great potential of the new TILLING population for genetic studies in the barley crop model system.

YjbH contributes to Staphylococcus aureus skin pathology and immune response through Agr-mediated α-toxin regulation

ABSTRACT Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.

Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase.

Here, we introduce 'TICIT', targeted integration by CRISPR-Cas9 and integrase technologies, which utilizes the site-specific DNA recombinase - phiC31 integrase - to insert large DNA fragments into CRISPR-Cas9 target loci. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to repeatedly perform site-specific transgenesis at the exact genomic location with high precision and efficiency. We applied this approach to devise a method for the instantaneous determination of a zebrafish's genotype simply by examining its color. When a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, employing this method allows facile identification of a population of homozygous mutant embryos even before the mutant phenotypes manifest. Thus, it should facilitate various downstream applications, such as large-scale chemical screens. We demonstrated that TICIT could also create reporter fish driven by an endogenous promoter. Further, we identified a landing site in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types. In sum, TICIT enables site-specific DNA integration without requiring complex donor DNA construction. It can yield consistent transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms.

Proteomic Insights into Trichome Responses to Elevated Elemental Stress in Cation Exchanger (CAX) Mutants.

Research on elemental distribution in plants is crucial for understanding nutrient uptake, environmental adaptation, and optimizing agricultural practices for sustainable food production. Plant trichomes, with their self-contained structures and easy accessibility, offer a robust model system for investigating elemental repartitioning. Transport proteins, such as the four functional cation exchangers (CAXs) in Arabidopsis, are low-affinity, high-capacity transporters primarily located on the vacuole. Mutants in these transporters have been partially characterized, with one of the phenotypes of the CAX1 mutant being altered tolerance to low-oxygen conditions. A simple visual screen demonstrated trichome density and morphology in cax1 and quadruple CAX (cax1-4: qKO) mutants remained unaltered. Here we used SXRF (Synchrotron X-Ray Fluorescence) to show that trichomes in CAX-deficient lines accumulated high levels of chlorine, potassium, calcium, and manganese. Proteomic analysis on isolated Arabidopsis trichomes. showed changes in protein abundance in response to changes in element accumulation. The CAX mutants showed an increased abundance of plasma membrane ATPase and vacuolar H-pumping proteins, and proteins associated with water movement and endocytosis, while also showing changes in proteins associated with the regulation of plasmodesmata. These findings advance our understanding of the integration of CAX transport with elemental homeostasis within trichomes and shed light on how plants modulate protein abundance under conditions of altered elemental levels.

Nonfunctional coq10 mutants maintain the ERMES complex and reveal true phenotypes associated with the loss of the coenzyme Q chaperone protein Coq10

Coenzyme Q (CoQ) is a redox-active lipid molecule that acts as an electron carrier in the mitochondrial electron transport chain. In Saccharomyces cerevisiae, CoQ is synthesized in the mitochondrial matrix by a multi-subunit protein-lipid complex termed the CoQ synthome, the spatial positioning of which is coordinated by the Endoplasmic Reticulum-Mitochondria Encounter Structure (ERMES). The MDM12 gene encoding the cytosolic subunit of ERMES, is co-expressed with COQ10, which encodes the putative CoQ chaperone Coq10, via a shared bidirectional promoter. Deletion of COQ10 results in respiratory deficiency, impaired CoQ biosynthesis, and reduced spatial coordination between ERMES and the CoQ Synthome. While Coq10 protein content is maintained upon deletion of MDM12, we show that deletion of COQ10 by replacement with a HIS3 marker results in diminished Mdm12 protein content. Since deletion of individual ERMES subunits prevents ERMES formation, we asked whether some or all of the phenotypes associated with COQ10 deletion result from ERMES dysfunction. To identify the phenotypes resulting solely due to the loss of Coq10, we constructed strains expressing a functionally impaired (coq10-L96S) or truncated (coq10-R147*) Coq10 isoform using CRISPR-Cas9. We show that both coq10 mutants preserve Mdm12 protein content and exhibit impaired respiratory capacity like the coq10Δ mutant, indicating that Coq10's function is vital for respiration regardless of ERMES integrity. Moreover, the maintenance of CoQ synthome stability and efficient CoQ biosynthesis observed for the coq10-R147* mutant suggests these deleterious phenotypes in the coq10Δ mutant result from ERMES disruption. Overall, this study clarifies the role of Coq10 in modulating CoQ biosynthesis.

A Plant biostimulant prepared from Ascophyllum nodosum Induces Flowering by Regulating the MIR156-mediated Age Pathway in Arabidopsis.

Flowering, the change from vegetative development to the reproductive phase, represents a crucial and intricate stage in the life cycle of plants, which is tightly controlled by both internal and external factors. In this study, we investigated the effect of Ascophyllum nodosum extract (ANE) on the flowering time of Arabidopsis. We found that a 0.1% concentration of ANE induced flowering in Arabidopsis, accompanied by the upregulation of key flowering time genes: FT (FLOWERING LOCUS T), SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1), and LFY (LEAFY). Further investigation showed that ANE specifically promotes flowering through the MIR156-mediated age pathway. ANE treatment resulted in the repression of negative regulator genes, MIR156, while simultaneously enhancing the expression of positive regulator genes, including SPLs and MIR172. This, in turn, led to the downregulation of AP2-like genes, which are known as floral repressors. It is worth noting that ANE did not alleviate the late flowering phenotype of MIR156-overexpressing plants and spl mutants. Furthermore, ANE-derived fucoidan mimics the function of sugars in regulating MIR156, closely mirroring the effects induced by ANE treatments. It suppresses the transcript levels of MIR156 and AP2-like genes while inducing those of SPLs and MIR172, thereby reinforcing the involvement of fucoidan in the control of flowering by ANE. In summary, our results demonstrate that ANE induces flowering by modulating the MIR156-SPL module within the age pathway, and this effect is mediated by fucoidan.

Rhizobial secretion of truncated exopolysaccharides severely impairs the Mesorhizobium-Lotus symbiosis.

The symbiosis between Mesorhizobium japonicum R7A and Lotus japonicus Gifu is an important model system for investigating the role of bacterial exopolysaccharides (EPS) in plant-microbe interactions. Previously we showed that R7A exoB mutants that are affected at an early stage of EPS synthesis and in lipopolysaccharide (LPS) synthesis induce effective nodules on L. japonicus Gifu after a delay, whereas exoU mutants affected in the biosynthesis of the EPS side chain induce small uninfected nodule primordia and are impaired in infection. The presence of a halo around the exoU mutant when grown on Calcofluor-containing media suggested the mutant secreted a truncated version of R7A EPS. A non-polar ΔexoA mutant defective in the addition of the first glucose residue to the EPS backbone was also severely impaired symbiotically. Here we used a suppressor screen to show that the severe symbiotic phenotype of the exoU mutant was due to secretion of an acetylated pentasaccharide, as both monomers and oligomers, by the same Wzx/Wzy system that transports wild-type exopolysaccharide. We also present evidence that the ΔexoA mutant secretes an oligosaccharide by the same transport system, contributing to its symbiotic phenotype. In contrast, ΔexoYF, and polar exoA and exoL mutants have a similar phenotype to exoB mutants, forming effective nodules after a delay. These studies provide substantial evidence that secreted incompatible EPS is perceived by the plant leading to abrogation of the infection process.

Roles of microbiota in autoimmunity in Arabidopsis leaves

Over the past three decades, researchers have isolated plant mutants that show constitutively activated defence responses in the absence of pathogen infection. These mutants are called autoimmune mutants and are typically dwarf and/or bearing chlorotic/necrotic lesions. Here, from a genetic screen for Arabidopsis genes involved in maintaining a normal leaf microbiota, we identified TIP GROWTH DEFECTIVE 1 (TIP1), which encodes an S-acyltransferase, as a key player in guarding leaves against abnormal microbiota level and composition under high-humidity conditions. The tip1 mutant has several characteristic phenotypes of classical autoimmune mutants, including a dwarf stature, showing lesions, and having a high basal level of defence gene expression. Gnotobiotic experiments revealed that the autoimmune phenotypes of the tip1 mutant are largely dependent on the presence of microbiota as axenic tip1 plants have markedly reduced autoimmune phenotypes. We found that the microbiota dependency of autoimmune phenotypes is shared by several ‘lesion mimic’-type autoimmune mutants in Arabidopsis. It is worth noting that autoimmune phenotypes caused by mutations in two Nucleotide-Binding, Leucine-Rich Repeat (NLR) genes do not require the presence of microbiota and can even be partially alleviated by microbiota. Our results therefore suggest the existence of at least two classes of autoimmunity (microbiota-dependent versus microbiota-independent) in plants. The observed interplay between autoimmunity and microbiota in the lesion mimic class of autoimmunity is reminiscent of the interactions between autoimmunity and dysbiosis in the animal kingdom. These parallels highlight the intricate relationship between host immunity and microbial communities across various biological systems.

Mutation of KAP, which encodes a keratin-associated protein, affects grain size and yield production in rice.

Grain size and shape are critical agronomic traits that directly impact rice grain yield. Identifying genes that control these traits can provide new strategies for yield improvement. In this study, we characterized a rice mutant, reduced grain length (rgl), which exhibited decreased grain length due to reduced cell proliferation. Map-based cloning identified a base deletion in OsRGL2, a gene encoding a keratin-associated protein (KAP), as the cause of the mutant phenotype. CRISPR-Cas9-generated OsRGL2 knockout mutants also displayed reduced grain length, confirming its role. OsRGL2 transcripts were detected in various tissues, with relative higher gene expression in young panicles, and OsRGL2 was localized to the plasma membrane. Overexpression of OsRGL2 increased grain size by promoting cell proliferation in the spikelet hull and significantly enhanced grain yield per plant. Importantly, OsRGL2 was found to interact with RGB1, indicating that OsRGL2 positively regulates grain size and yield through its interaction with RGB1. Additionally, OsRGL2 regulated the expression of cell cycle-related genes, further elucidating its role in grain development. These findings demonstrate that OsRGL2 is a positive regulator of grain size in rice, and manipulating its expression may offer a novel strategy for enhancing rice grain yield.

A chloroplast sulphate transporter modulates glutathione-mediated redox cycling to regulate cell division.

Glutathione redox cycling is important for cell cycle regulation, but its mechanisms are not well understood. We previously identified a small-sized mutant, suppressor of mat3 15-1 (smt15-1) that has elevated cellular glutathione. Here, we demonstrated that SMT15 is a chloroplast sulphate transporter. Reducing expression of γ-GLUTAMYLCYSTEINE SYNTHETASE, encoding the rate-limiting enzyme required for glutathione biosynthesis, corrected the size defect of smt15-1 cells. Overexpressing GLUTATHIONE SYNTHETASE (GSH2) recapitulated the small-size phenotype of smt15-1 mutant, confirming the role of glutathione in cell division. Hence, SMT15 may regulate chloroplast sulphate concentration to modulate cellular glutathione levels. In wild-type cells, glutathione and/or thiol-containing molecules (GSH/thiol) accumulated in the cytosol at the G1 phase and decreased as cells entered the S/M phase. While the cytosolic GSH/thiol levels in the small-sized mutants, smt15-1 and GSH2 overexpressors, mirrored those of wild-type cells (accumulating during G1 and declining at early S/M phase), GSH/thiol was specifically accumulated in the basal bodies at early S/M phase in the small-sized mutants. Therefore, we propose that GSH/thiol-mediated redox signalling in the basal bodies may regulate mitotic division number in Chlamydomonas reinhardtii. Our findings suggest a new mechanism by which glutathione regulates the multiple fission cell cycle in C. reinhardtii.

Strigolactone signalling inhibits trehalose 6-phosphate signalling independently of BRC1 to suppress shoot branching.

The phytohormone strigolactone (SL) inhibits shoot branching, whereas the signalling metabolite trehalose 6-phosphate (Tre6P) promotes branching. How Tre6P and SL signalling may interact and which molecular mechanisms might be involved remains largely unknown. Transcript profiling of Arabidopsis SL mutants revealed a cluster of differentially expressed genes highly enriched in the Tre6P pathway compared with wild-type (WT) plants or brc1 mutants. Tre6P-related genes were also differentially expressed in axillary buds of garden pea (Pisum sativum) SL mutants. Tre6P levels were elevated in the SL signalling mutant more axillary (max) growth 2 compared with other SL mutants or WT plants indicating a role of MAX2-dependent SL signalling in regulating Tre6P levels. A transgenic approach to increase Tre6P levels demonstrated that all SL mutant lines and brc1 flowered earlier, showing all of these mutants were responsive to Tre6P. Elevated Tre6P led to increased branching in WT plants but not in max2 and max4 mutants, indicating some dependency between the SL pathway and Tre6P regulation of shoot branching. By contrast, elevated Tre6P led to an enhanced branching phenotype in brc1 mutants indicating independence between BRC1 and Tre6P. A model is proposed whereby SL signalling represses branching via Tre6P and independently of the BRC1 pathway.

Cell-free synthesis of infective phages from in vitro assembled phage genomes for efficient phage engineering and production of large phage libraries.

Bacteriophages are promising alternatives to traditional antimicrobial treatment of bacterial infections. To further increase the potential of phages, efficient engineering methods are needed. This study investigates an approach to phage engineering based on phage rebooting and compares selected methods of assembly and rebooting of phage genomes. GG assembly of phage genomes and subsequent rebooting by cell-free transcription-translation reactions yielded the most efficient phage engineering and allowed production of a proof-of-concept T7 phage library of 1.8 × 107 phages. We obtained 7.5 × 106 different phages, demonstrating generation of large and diverse libraries suitable for high-throughput screening of mutant phenotypes. Implementing versatile and high-throughput phage engineering methods allows vastly accelerated and improved phage engineering, bringing us closer to applying effective phages to treat infections in the clinic.

Mycobacterium smegmatis MfpC is a GEF that regulates mfpA translationally to alter the fluoroquinolone efficacy

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a serious threat to global public health. Fluoroquinolones (FQs) are effective against M. tuberculosis; however, resistant strains have limited their efficacy. Mycobacteriumfluoroquinolone resistance protein A (MfpA) confers intrinsic resistance to FQs; however, its regulatory mechanisms remain largely unknown. Using M. smegmatis as a model, we investigated whether MfpC is necessary for FQ susceptibility. MfpC mutants were sensitive to moxifloxacin, indicating that MfpC is involved in FQ susceptibility. By testing the mfpC inactivation phenotype in different mutants and using mycobacterial protein fragment complementation, we demonstrated that the function of MfpC depends on its interactions with MfpB. Guanine nucleotide exchange assays and site-directed mutagenesis confirmed that MfpC acts as a guanine nucleotide exchange factor to regulate MfpB. We propose that MfpB influences MfpA at the translational level. In summary, we reveal the role of MfpC in regulating the function of MfpA in FQ resistance.