Deletion Mutants Research Articles

The NEDD4-binding protein N4BP1 degrades mRNA substrates through the coding sequence independent of nonsense-mediated decay

3'-Untranslated regions (3'UTRs) are recognized for their role in regulating mRNA turnover while the turnover of a specific group of mRNAs mediated by coding sequences (CDS) remains poorly understood. N4BP1 is a critical inflammatory regulator in vivo with a molecular mechanism that is not yet clearly defined. Our study reveals that N4BP1 efficiently degrades its mRNA targets via CDS rather than the 3'-UTR. This CDS-dependent mRNA turnover mechanism appears to be a general feature of N4BP1, as evidenced by testing multiple mRNA substrates, such as Fos-C, Fos-B, Jun-B and CXCL1. Detailed mapping of the motif identified a crucial 33nt (289-322) sequence near the 5'-end of Fos-C-CDS, where the presence of polyC is necessary for N4BP1-mediated degradation. Functional studies involving domain deletion and point mutations showed that both the KH and NYN domains are essential for N4BP1 to restrict mRNA substrates. The function of N4BP1 in mRNA turnover is not dependent on nonsense-mediated decay as it efficiently restricts mRNA substrates even in cells deficient in UPF1, UPF3A, and UPF3B. Additionally, the function of N4BP1 is not reliant on LUC7L3 despite its known association with this protein. Our findings suggest that N4BP1 acts as an endoribonuclease to degrade mRNA substrates primarily through coding sequences containing a C-rich motif.

Putative mechanisms of primary resistance to EGFR-targeted therapies: A retrospective study

BackgroundsAdvanced lung adenocarcinoma (LUAD) patient with EGFR mutations often experience resistance to first-line epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs) therapy. Nonetheless, the mechanism and biomarkers of primary resistance remain unclear. Further exploration of independent prognostic factors will help clinicians identify patients who may not respond to EGFR-TKIs and select appropriate treatment strategies. MethodsA retrospective study involving 124 patients with stage IV LUAD harboring a common sensitizing EGFR mutation (exon 19 deletion or L858R mutation) who received EGFR-TKIs as first-line therapy was performed. All participants were tested by DNA-targeted sequencing in baseline samples, and there were 19 patients with progression-free survival (PFS) ≤ 3 months (cohort 1, C1, primary resistance), 22 patients with 3 < PFS < 8 months (cohort 2, C2, poor response) without known mutations associated with resistance, and 83 patients with PFS ≥ 8 months (cohort 3, C3, normal). ResultsThe most commonly mutated genes at baseline in patients prior to treatment within the entire study population. were TP53 (65 %), MYC (19 %), CDKN2A (12 %), MUC16 (12 %) and RBM10 (12 %). The baseline characteristics, except for the proportions of patients with EGFR L858R mutation and exon 19 deletion in C1 plus C2 compared to C3 (p = 0.036), were not significantly different among the cohorts. The frequencies of PIK3C2G, STK11, EPAS1, RARA and BTG2 variation were significantly higher in C1, the primary resistance group. Multivariate Cox analysis revealed that PIK3C2G (HR 15.70 95 % CI 3.24–76.05, p < 0.001), STK11 (HR 17.04, 95 % CI 3.68–78.92, p < 0.001), EPAS1 (HR 11.99, 95 % CI 2.57–56.03, p = 0.002), and BTG2 amplification (HR 9.53, 95 % CI 1.67–54.28, p = 0.011) were significantly associated with shorter PFS. ConclusionsThe genomic landscape varies significantly among patients with LUAD, which should be considered when making personalized treatment decisions. This information could provide insights into molecular changes and their effects on clinical treatment in diverse patients with LUAD harboring sensitizing EGFR mutations.

Osteoprotegerin secretion and its inhibition by RANKL in osteoblastic cells visualized using bioluminescence imaging

Bone remodeling is regulated by the interaction between receptor activator of nuclear factor kappa-B ligand (RANKL) and its receptor RANK on osteoblasts and osteoclasts, respectively. Osteoprotegerin (OPG) is secreted from osteoblasts and inhibits osteoclast differentiation by acting as a decoy receptor for RANKL. Despite its importance, the mechanism underlying the secretion of OPG remains poorly understood. Here, we applied a method of video-rate bioluminescence imaging using a fusion protein with Gaussia luciferase (GLase) and visualized the secretion of OPG from living mouse osteoblastic MC3T3-E1 cells. The bioluminescence imaging revealed that the secretion of OPG fused to GLase (OPG-GLase) occurred frequently and widely across the cell surface. Notably, co-expression of RANKL significantly reduced the secretion of OPG-GLase, indicating an inhibitory role of RANKL on OPG secretion within cells. Further imaging and biochemical analyses using deletion mutants of OPG and RANKL, as well as RANKL mutants that cause autosomal recessive osteopetrosis, demonstrated the essential role of protein-protein interaction between OPG and RANKL in the inhibition of OPG secretion. Treatment with proteasome inhibitors resulted in increased levels of OPG in both culture medium and cell lysates. However, the fold-increase of OPG was similar regardless of the presence or absence of RANKL, suggesting that the regulation of OPG secretion by RANKL is independent of proteasome activity. This report visualized the secretion of OPG from living cells and provided evidence for a novel intracellular inhibitory effect of RANKL on OPG secretion.

Optimized psilocybin production in tryptophan catabolism-repressed fungi.

The high therapeutic potential of psilocybin, a prodrug of the psychotropic psilocin, holds great promise for the treatment of mental disorders such as therapy-refractory depression, alcohol use disorder and anorexia nervosa. Psilocybin has been designated a 'Breakthrough Therapy' by the US Food and Drug Administration, and therefore a sustainable production process must be established to meet future market demands. Here, we present the development of an invivo psilocybin production chassis based on repression of l-tryptophan catabolism. We demonstrate the proof of principle in Saccharomyces cerevisiae expressing the psilocybin biosynthetic genes. Deletion of the two aminotransferase genes ARO8/9 and the indoleamine 2,3-dioxygenase gene BNA2 yielded a fivefold increase of psilocybin titre. We transferred this knowledge to the filamentous fungus Aspergillus nidulans and identified functional ARO8/9 orthologs involved in fungal l-tryptophan catabolism by genome mining and cross-complementation. The double deletion mutant of A. nidulans resulted in a 10-fold increased psilocybin production. Process optimization based on respiratory activity measurements led to a final psilocybin titre of 267 mg/L in batch cultures with a space-time-yield of 3.7 mg/L/h. These results demonstrate the suitability of our engineered A. nidulans to serve as a production strain for psilocybin and other tryptamine-derived pharmaceuticals.

Deciphering FUR-Regulated Gene Networks in Klebsiella pneumoniae Using FUR Knockout mutant

Klebsiella pneumoniae is a nosocomial pathogen causing life-threatening infections worldwide. It possesses many virulence properties and the ferric uptake regulator (FUR) is the transcription factor that regulates the expression of various genes in K. pneumoniae. FUR binds to a conserved 19bp sequence, called the FUR box, for either positive or negative regulation of the gene. FUR alters gene expression according to iron availability and also participates in virulence, colonization & toxin secretion in K. pneumoniae. FUR deletion mutant is an interesting model to understand the virulence factors. In the present study, we identified the genes containing putative FUR boxes (>50% sequence homology) from the genome of K. pneumoniae M33. FUR deletion mutant for K. pneumoniae M33 was created using lambda red recombinase system using the plasmids- pACBSR-hyg, pMDIAI, and pFLP-hyg. The mutant (M33∆FUR) was characterized using various assays like CAS assay, string test, crystal violet assay to access siderophore production, capsule synthesis and biofilm formation respectively. FUR box was present upstream of 18 genes with distinct functions. In the FUR knockout mutant, M33∆FUR an increase in expression of iron transport and siderophore related genes feoC, fhuA, fepB and fes was observed whereas genes feoA, cirA, fecA, fepA and entC were down-regulated. Genes related to biofilm (fimA and mrkD) were downregulated whereas genes related to capsular polysaccharide (rcsA and rcsB) were upregulated upon FUR deletion. Among other genes murG and sucA were downregulated and priB and srlB were upregulated upon FUR deletion. This study shows that FUR regulates many genes involved in virulence, either positively or negatively.

Genetic analysis of a family with hereditary hemorrhagic telangiectasia caused by a novel frameshift deletion mutation of the endoglin (ENG) gene

Genetic analysis of a family with hereditary hemorrhagic telangiectasia caused by a novel frameshift deletion mutation of the endoglin (ENG) gene

Impact of ERG6 Gene Deletion on Membrane Composition and Properties in the Pathogenic Yeast Candida glabrata.

The ERG6 gene is crucial for the biosynthesis of ergosterol, a key component of yeast cell membranes. Our study examines the impact of ERG6 gene deletion on the membrane composition and physicochemical properties of the pathogenic yeast Candida glabrata. Specifically, we investigated changes in selected sterol content, phospholipid composition, transmembrane potential, and PDR16 gene activity. Sterol levels were measured using high-performance liquid chromatography, the phospholipid profile was analysed via thin-layer chromatography, transmembrane potential was assessed with fluorescence spectroscopy, and gene expression levels were determined by quantitative PCR. Our findings revealed a depletion of ergosterol, increased zymosterol and eburicol content, an increased phosphatidylcholine and a reduced phosphatidylethanolamine content in the Δerg6 strain compared to the wt. Additionally, the Δerg6 strain exhibited membrane hyperpolarization without changes in PDR16 expression. Furthermore, the Δerg6 strain showed increased sensitivity to the antifungals myriocin and aureobasidine A. These results suggest that ERG6 gene deletion leads to significant alterations in membrane composition and may activates an alternative ergosterol synthesis pathway in the C. glabrata Δerg6 deletion mutant.

Zinc metalloprotease FgM35, which targets the wheat zinc-binding protein TaZnBP, contributes to the virulence of Fusarium graminearum

Metalloproteinases are ubiquitous in organisms. Most metalloproteinases secreted by pathogenic microorganisms are also called virulence factors, because they degrade proteins in the external tissues of the host, thereby reducing the host’s immunity and increasing its susceptibility to disease. Zinc metalloproteinase is one of the most common metalloproteinases. In our report, we studied the biological function of zinc metalloprotease FgM35 in Fusarium graminearum and the pathogen–host interaction during infection. We found that the asexual and sexual reproduction of the deletion mutant ΔFgM35 were affected, as well as the tolerance of F. graminearum to metal stress. In addition, deletion of FgM35 reduced the virulence of F. graminearum. The wheat target TaZnBP was screened using a wheat yeast cDNA library, and the interaction between FgM35 and TaZnBP was verified by HADDOCK molecular docking, yeast two-hybrid, Bi-FC, Luc, and Co-IP assays. The contribution of TaZnBP to plant immunity was also demonstrated. In summary, our work revealed the indispensable role of FgM35 in the reproductive process and the pathogenicity of F. graminearum, and it identified the interaction between FgM35 and TaZnBP as well as the function of TaZnBP. This provides a theoretical basis for further study of the function of metalloproteinases in pathogen–host interactions.

Sugar alcohol degradation in Archaea: uptake and degradation of mannitol and sorbitol in Haloarcula hispanica

The halophilic archaeon Haloarcula hispanica utilizes the sugar alcohols mannitol and sorbitol as carbon and energy sources. Genes, enzymes, and transcriptional regulators involved in uptake and degradation of these sugar alcohols were identified by growth experiments with deletion mutants and enzyme characterization. It is shown that both mannitol and sorbitol are taken up via a single ABC transporter of the CUT1 transporter family. Then, mannitol and sorbitol are oxidized to fructose by two distinct dehydrogenases. Fructose is further phosphorylated to fructose-1-phosphate by a haloarchaeal ketohexokinase, providing the first evidence for a physiological function of ketohexokinase in prokaryotes. Finally, fructose-1-phosphate is phosphorylated via fructose-1-phosphate kinase to fructose-1,6-bisphosphate, which is cleaved to triosephosphates by a Class I fructose-1,6-bisphosphate aldolase. Two distinct transcriptional regulators, acting as activators, have been identified: an IclR-like regulator involved in activating genes for sugar alcohol uptake and oxidation to fructose, and a GfcR-like regulator that likely activates genes involved in the degradation of fructose to pyruvate. This is the first comprehensive analysis of a sugar alcohol degradation pathway in Archaea.

The Yeast Ribosomal Protein Rpl1b Is Not Required for Respiration

Previously, Segev and Gerst found that mutants in any of the four ribosomal protein genes rpl1b, rpl2b, rps11a, or rps26b had a petite phenotype—i.e., the mutants were deficient in respiration. Strikingly, mutants of their paralogs rpl1a, rpl2a, rps11b, and rps26a were grande—i.e., competent for respiration. It is remarkable that these paralogs should have opposite phenotypes, because three of the paralog pairs (Rpl1a/Rpl1b, Rpl2a/Rpl2b, Rps11a/Rps11b) are 100% identical to each other in terms of their amino acid sequences, while Rps26a and Rps26b differ in 2 amino acids out of 119. However, while attempting to use this paralog-specific petite phenotype in an unrelated experiment, I found that the rpl1b, rpl2b, rps11a, and rps26b deletion mutants are competent for respiration, contrary to the findings of Segev and Gerst.

CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes.

Focal Segmental Glomerulosclerosis (FSGS) is a histologic lesion caused by a variety of injurious stimuli that lead to dysfunction/loss of glomerular visceral epithelial cells (i.e. podocytes). Pathogenic mutations in CRB2, encoding the type 1 transmembrane protein Crumb 2 Homolog Protein, have been shown to cause early-onset corticosteroid-resistant nephrotic syndrome (SRNS)/FSGS. Here, we identified a 2-generation East Asian kindred (DUK40595) with biopsy-proven SRNS/FSGS caused by a compound heterozygous mutation in CRB2 comprised of the previously described truncating mutation p.Gly1036_Alafs*43 and a rare 9-bp deletion mutation p.Leu1074_Asp1076del. Because compound heterozygous mutations involving the truncating p.Gly1036_Alafs*43 variant have been associated with reduced CRB2 expression in podocytes and autosomal recessive SRNS/FSGS, we sought to define the pathogenic effects of CRB2 deficiency in podocytes. We show that CRB2 knockdown induces YAP activity and target gene expression in podocytes. It upregulates YAP-mediated mechanosignaling and increases the density of focal adhesion and F-actin. Using Elastic Resonator Interference Stress Microscopy (ERISM), we demonstrate that CRB2 knockdown also enhances podocyte contractility in a substrate stiffness-dependent manner. The knockdown effect decreases with increasing substrate stiffness, indicating impaired mechanosensing in CRB2 knockdown cells at low substrate stiffness. While the mechanical activation of CRB2 knockdown cells is associated with increased YAP activity, the enhanced cell contractility is not significantly reduced by the selective YAP inhibitors K-975 and verteporfin, suggesting that multiple pathways may be involved in mechanosignaling downstream of CRB2. Taken together, these studies provide the first evidence that CRB2 deficiency may impair podocyte mechanotransduction via disruption of YAP signaling in podocytes.

Serine protease RAYM_01812 (SspA) inhibits complement-mediated killing and monocyte chemotaxis and contributes to virulence of Riemerella anatipestifer in ducks

ABSTRACT Riemerella anatipestifer (RA) is a significant poultry pathogen causing acute septicemia and inflammation. The function of protease RAYM_01812, responsible for gelatin degradation, is unexplored in RA pathogenesis. To elucidate its role, we generated a deletion mutant ΔRAYM_01812 (ΔRAYM) and complementary CΔRAYM_01812 (CΔRAYM) strain and revealed the protease’s role in extracellular gelatinase activity. By expressing full-length 76 kDa RAYM_01812 protein without signal peptide as well as seven partial structural domains fragments, we evidence that the N-terminal propeptide acts as an enzymatic activity inhibitor and it gets cleaved at A112. Also, we show that the β-fold sheet domain is necessary for enhancing the enzymatic protease activity. Sequential auto-proteolysis forms a stable 40 kDa enzyme. Then, testing the strains in duck sera indicated that the absence or presence of RAYM_01812 results in reduced or enhanced bacterial survival, respectively. Furthermore, we found that the protease is able to cleave IgY antibodies as well as the complement factors C3a and C5a, that the protease reduces C3a- or C5a-mediated monocyte chemotaxis, and results in enhanced membrane attack complex (MAC) formation on the surface of ΔRAYM compared to CΔRAYM. This suggests that RAYM_01812 plays a crucial role in protecting against the serum complement-mediated bactericidal effect through inhibiting MAC formation and monocyte chemotaxis. Animal infection assays showed a 1090-fold reduced virulence of ΔRAYM compared to RA-YM, evidenced by decreased tissue loading and weaker histopathological changes. In conclusion, RAYM_01812 acts as a vital virulence factor, enabling host innate immune defence escape through complement killing evasion and monocyte chemotaxis inhibition.

Identification and characterization of LuxR solo homolog PplR in pathogenic Pseudomonas plecoglossicida NB2011

Pseudomonas plecoglossicida is a causative agent of visceral granulomas in large yellow croaker (Larimichthys crocea). Quorum sensing (QS) is widely involved in imparting virulence to pathogenic bacteria; however, it has not been studied in P. plecoglossicida. In this study, we annotated a LuxR family transcriptional regulator in P. plecoglossicida NB2011 and designated as PplR. We aligned the protein sequence by BlastP and Clustal X2, monitored the N-acyl-homoserine lactone (AHL) signal production through cross-feeding bioassay and HC-MS/MS; investigated exogenous AHL signal binding by recombinant expression and thin layer chromatography; constructed a deletion mutant of the target gene by method of double homologous recombination; sequenced the transcript RNA and analyzed the data; additionally, characterized phenotypes of wild type and mutant strain. The LuxR homolog PplR was found to share high similarity with PpoR—the LuxR solo of Pseudomonas putida—without a cognate LuxI. The wild-type strain did not produce any AHL signals and the recombinant LuxR protein was found to bind C6-L-homoserine lactone (C6-HSL), C8-HSL, 3-oxo-C10-HSL, and 3-oxo-C12-HSL. RNA-seq analysis indicated 84 differentially expressed genes—5 upregulated and 79 downregulated—mainly enriched in gene ontology terms, such as flagella-dependent motility, integral component of membrane, DNA binding and transcription, and metal ion binding, suggesting that PplR is a master transcription regulator. The mutant strain showed attenuated biofilm-forming ability and stress resistance, and the data support a role for PplR in the regulation of these traits in P. plecoglossicida NB2011 independent of the presence of AHL signals. This is the first study to provide QS-related information on P. plecoglossicida.

The secreted protein FonCHRD is essential for vegetative growth, asexual reproduction, and pathogenicity in watermelon Fusarium wilt fungus

Fungal pathogens often secrete numerous effectors to interfere with and/or suppress plant immunity to promote their infection. Watermelon Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. niveum (Fon), is one of the devastating diseases that severely affect the watermelon industry. Here, we report the function of a candidate effector protein, FonCHRD, in Fon. FonCHRD harbors a chordin (CHRD) domain of unknown function and has a signal peptide with secretion activity. FonCHRD shows a relatively high expression level in Fon marcoconidia and is inducible by watermelon root tissues. Phenotypic analysis of the targeted deletion mutant revealed that FonCHRD plays roles in vegetative growth, asexual reproduction, and conidial morphology of Fon, while it is not involved in spore germination as well as cell wall, oxidative and salt stress responses. Deletion of FonCHRD impaired the ability to colonize and spread within host plants, significantly reducing its virulence on watermelon. FonCHRD is distributed across multiple compartments of plant cells but can target to the apoplast space in plants. FonCHRD inhibits the INF1- and Bcl2-associated X protein-triggered cell death and defense gene expression in transiently expressed Nicotiana benthamiana leaves. These findings suggest that FonCHRD is essential for Fon pathogenicity by modulating invasive growth and spreading abilities as well as by suppressing plant immune responses.

Towards the genomic sequence code of DNA fragility for machine learning.

Genomic DNA breakages and the subsequent insertion and deletion mutations are important contributors to genome instability and linked diseases. Unlike the research in point mutations, the relationship between DNA sequence context and the propensity for strand breaks remains elusive. Here, by analyzing the differences and commonalities across myriads of genomic breakage datasets, we extract the sequence-linked rules and patterns behind DNA fragility. We show the overall deconvolution of the sequence influence into short-, mid- and long-range effects, and the stressor-dependent differences in defining the range and compositional effects on DNA fragility. We summarize and release our feature compendium as a library that can be seamlessly incorporated into genomic machine learning procedures, where DNA fragility is of concern, and train a generalized DNA fragility model on cancer-associated breakages. Structural variants (SVs) tend to stabilize regions in which they emerge, with the effect most pronounced for pathogenic SVs. In contrast, the effects of chromothripsis are seen across regions less prone to breakages. We find that viral integration may bring genome fragility, particularly for cancer-associated viruses. Overall, this work offers novel insights into the genomic sequence basis of DNA fragility and presents a powerful machine learning resource to further enhance our understanding of genome (in)stability and evolution.

Examination of yield, bacteriolytic activity and cold storage of linker deletion mutants based on endolysin S6_ORF93 derived from Staphylococcus giant bacteriophage S6.

Methicillin-resistant Staphylococcus spp. present challenges in clinical and veterinary settings because effective antimicrobial agents are limited. Phage-encoded peptidoglycan-degrading enzyme, endolysin, is expected to be a novel antimicrobial agent. The enzymatic activity has recently been shown to be influenced by the linker between functional domains in the enzyme. S6_ORF93 (ORF93) is one of the endolysins derived from previously isolated Staphylococcus giant phage S6. The ORF93 was speculated to have a catalytic and peptidoglycan-binding domain with a long linker. In this study, we examined the influence of linker shortening on the characteristics of ORF93. We produce wild-type ORF93 and the linker deletion mutants using an Escherichia coli expression system. These mutants were designated as ORF93-Δ05, ORF93-Δ10, ORF93-Δ15, and ORF93-Δ20, from which 5, 10, 15, and 20 amino acids were removed from the linker, respectively. Except for the ORF93-Δ20, ORF93 and its mutants were expressed as soluble proteins. Moreover, ORF93-Δ15 showed the highest yield and bacteriolytic activity, while the antimicrobial spectrum was homologous. The cold storage experiment showed a slight effect by the linker deletion. According to our results and other studies, linker investigations are crucial in endolysin development.

Evolution and maintenance of a large multidrug-resistant plasmid in a Salmonella enterica Typhimurium host under differing antibiotic selection pressures.

The dissemination of antibiotic resistance genes (ARGs) through plasmids is a major mechanism for the development of bacterial antimicrobial resistance. The adaptation and evolution mechanisms of multidrug-resistant (MDR) plasmids with their hosts are not fully understood. Herein, we conducted experimental evolution of a 244 kb MDR plasmid (pJXP9) under various conditions including no antibiotics and mono- or combinational drug treatments of colistin (CS), cefotaxime (CTX), and ciprofloxacin (CIP). Our results showed that long-term with or without positive selections for pJXP9, spanning approximately 600 generations, led to modifications of the plasmid-encoded MDR and conjugative transfer regions. These modifications could mitigate the fitness cost of plasmid carriage and enhance plasmid maintenance. The extent of plasmid modifications and the evolution of plasmid-encoded antibiotic resistance depended on treatment type, particularly the drug class and duration of exposure. Interestingly, prolonged exposure to mono- and combinational drugs of CS and CIP resulted in a substantial loss of the plasmid-encoded MDR region and antibiotic resistance, comparable to the selection condition without antibiotic. By contrast, combinational treatment with CTX contributed to the maintenance of the MDR region over a long period of time. Furthermore, drug selection was able to maintain and even amplify the corresponding plasmid-encoded ARGs, with co-selection of ARGs in the adjacent regions. In addition, parallel mutations in chromosomal arcA were also found to be associated with pJXP9 plasmid carriage among endpoint-evolved clones from diverse treatments. Meanwhile, arcA deletion improved the persistence of pJXP9 plasmid without drugs. Overall, our findings indicated that plasmid-borne MDR region deletion and chromosomal arcA inactivation mutation jointly contributed to co-adaptation and co-evolution between MDR IncHI2 plasmid and Salmonella Typhimurium under different drug selection pressure.IMPORTANCEThe plasmid-mediated dissemination of antibiotic resistance genes has become a significant concern for human health, even though the carriage of multidrug-resistant (MDR) plasmids is frequently associated with fitness costs for the bacterial host. However, the mechanisms by which MDR plasmids and bacterial pairs evolve plasmid-mediated antibiotic resistance in the presence of antibiotic selections are not fully understood. Herein, we conducted an experimental evolution of a large multidrug-resistant plasmid in a Salmonella enterica Typhimurium host under single and combinatorial drug selection pressures. Our results show the adaptive evolution of plasmid-encoded antibiotic resistance through alterations of the MDR region in the plasmid, in particular substantial loss of the MDR region, in response to different positive selections, especially mono- and combinational drugs of colistin and ciprofloxacin. In addition, strong parallel mutations in chromosomal arcA were associated with pJXP9 carriage in Salmonella Typhimurium from diverse treatments. Our results thus highlight promoting the loss of the plasmid's MDR region could offer an alternative approach for combating plasmid-encoded antibiotic resistance.

Differential expression of the yfj operon in a Bacillus subtilis biofilm.

Type VII protein secretion systems play an important role in the survival and virulence of pathogens and in the competition among some microbes. Potential polymorphic toxin substrates of the type VII secretion system (T7SS) in Bacillus subtilis are important for competition in the context of biofilm communities. Within a biofilm, there is significant physiological heterogeneity as cells within the population take on differential cell fates. Which cells express and deploy the various T7SS substrates is still unknown. To identify which cells express at least one of the T7SS substrates, we investigated the yfj operon. The yfjABCDEF operon encodes at least one predicted T7SS substrate. Starting with an in silico analysis of the yfj operon promoter region, we identified potential regulatory sequences. Using a yfj promoter-reporter fusion, we then identified several regulators that impact expression of the operon, including a regulator of biofilm formation, DegU. In a degU deletion mutant, yfj expression is completely abolished. Mutation of predicted DegU binding sites also results in a significant reduction in yfj reporter levels. Further analysis of yfj regulation reveals that deletion of spo0A has the opposite effect of the degU deletion. Following the yfj reporter by microscopy of cells harvested from biofilms, we find that the yfj operon is expressed specifically in the subset of cells undergoing sporulation. Together, our results define cells entering sporulation as the subpopulation most likely to express products of the yfj operon in B. subtilis.IMPORTANCEDifferential expression of genes in a bacterial community allows for the division of labor among cells in the community. The toxin substrates of the type VII secretions system (T7SS) are known to be active in Bacillus subtilis biofilm communities. This work describes the expression of one of the T7SS-associated operons, the yfj operon, which encodes the YFJ toxin, in the sporulating subpopulation within a biofilm. The evidence that the YFJ toxin may be deployed specifically in cells at the early stages of sporulation provides a potential role for deployment of T7SS in community-associated activities, such as cannibalism.

Diversity of transactivation regions of DMRT1 in vertebrates

BackgroundDoublesex and mab-3 related transcription factor (DMRT) 1, commonly found in all vertebrates, regulates the transcription of genes involved in the masculinization and maintenance of gonadal somatic cells and/or germline cell development. DMRT1 has a DNA-binding domain called the DM domain and a transcription regulatory region. Unlike the former, there is little knowledge about the latter transcription regulatory region. This study aimed to identify the transcription activation regions of DMRT1 from four species: humans and mice (mammals), leopard geckos (reptiles), and medaka (teleost fish), adding perspectives on evolutionary conservation and diversity.Methods and resultsFor each species, several expression plasmids of deletion mutants were constructed, and the resultant plasmid and a DMRT1-driven luciferase reporter were co-transfected into cultured cells to measure transactivation ability. The key point of this analysis is that the transactivation ability was normalized by quantifying the expression levels of DMRT1 variants using the HiBiT tag. As a result, two to three transactivation regions were suggested to exist in the C-terminal region of the DM domain in all four species. Among seven regions in DMRT1, the fourth region from the N-terminus contributed to transactivation common to the four species, and the sixth and seventh regions on the C-terminal side differed depending on the species.ConclusionsThese findings indicated that the regions involved in the transactivation ability of DMRT1 could subtly change during evolution, indicating diversity in transactivation domains.

Candida albicans pathways that protect against organic peroxides and lipid peroxidation.

Human fungal pathogens must survive diverse reactive oxygen species (ROS) produced by host immune cells that can oxidize a range of cellular molecules including proteins, lipids, and DNA. Formation of lipid radicals can be especially damaging, as it leads to a chain reaction of lipid peroxidation that causes widespread damage to the plasma membrane. Most previous studies on antioxidant pathways in fungal pathogens have been conducted with hydrogen peroxide, so the pathways used to combat organic peroxides and lipid peroxidation are not well understood. The most well-known peroxidase in Candida albicans, catalase, can only act on hydrogen peroxide. We therefore characterized a family of four glutathione peroxidases (GPxs) that were predicted to play an important role in reducing organic peroxides. One of the GPxs, Gpx3 is also known to activate the Cap1 transcription factor that plays the major role in inducing antioxidant genes in response to ROS. Surprisingly, we found that the only measurable role of the GPxs is activation of Cap1 and did not find a significant role for GPxs in the direct detoxification of peroxides. Furthermore, a CAP1 deletion mutant strain was highly sensitive to organic peroxides and oxidized lipids, indicating an important role for antioxidant genes upregulated by Cap1 in protecting cells from organic peroxides. We identified GLR1 (Glutathione reductase), a gene upregulated by Cap1, as important for protecting cells from oxidized lipids, implicating glutathione utilizing enzymes in the protection against lipid peroxidation. Furthermore, an RNA-sequencing study in C. albicans showed upregulation of a diverse set of antioxidant genes and protein damage pathways in response to organic peroxides. Overall, our results identify novel mechanisms by which C. albicans responds to oxidative stress resistance which open new avenues for understanding how fungal pathogens resist ROS in the host.