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SUMMARY: Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium‐mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene‐editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1‐VIR2 virulence helper plasmid to improve T‐DNA delivery, and/or expressing a fusion protein between a GROWTH‐REGULATING FACTOR (GRF) and GRF‐INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1‐VIR2 helper vector with no effect on event quality regarding T‐DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5‐ to 6.5‐fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR‐/Cas9‐mediated gene editing. Our results demonstrate how a GRF‐GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene‐editing applications and molecular biology studies in maize. [ABSTRACT FROM AUTHOR]

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The essential yeast protein GPN‐loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy‐terminal 106‐amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin‐like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild‐type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6‐azauracil, mycophenolic acid or 1,10‐phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent‐polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high‐copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII‐independent and previously unrecognized role in translation initiation. [ABSTRACT FROM AUTHOR]

ABSTRACT In an attempt to identify novel bacterial species, microbiologists have examined a wide range of environmental niches. We describe the serendipitous discovery of a novel gram-negative bacterial species from a different type of extreme niche: a purchased vial of antibiotic. The vial of antibiotic hygromycin B was found to be factory contaminated with a bacterial species, which we designate Pseudomonas hygromyciniae sp. nov. The proposed novel species belongs to the P. fluorescens complex and is most closely related to P. brenneri, P. proteolytica, and P. fluorescens. The type strain Pseudomonas hygromyciniae sp. nov. strain SDM007T (SDM007T) harbors a novel 250 kb megaplasmid which confers resistance to hygromycin B and contains numerous other genes predicted to encode replication and conjugation machinery. SDM007T grows in hygromycin concentrations of up to 5 mg/mL but does not use the antibiotic as a carbon or nitrogen source. While unable to grow at 37°C ruling out its ability to infect humans, it grows and survives at temperatures between 4 and 30°C. SDM007T can infect plants, as demonstrated by the lettuce leaf model, and is highly virulent in the Galleria mellonella infection model but is unable to infect mammalian A549 cells. These findings indicate that commercially manufactured antibiotics represent another extreme environment that may support the growth of novel bacterial species. IMPORTANCE Physical and biological stresses in extreme environments may select for bacteria not found in conventional environments providing researchers with the opportunity to not only discover novel species but to uncover new enzymes, biomolecules, and biochemical pathways. This strategy has been successful in harsh niches such as hot springs, deep ocean trenches, and hypersaline brine pools. Bacteria belonging to the Pseudomonas species are often found to survive in these unusual environments, making them relevant to healthcare, food, and manufacturing industries. Their ability to survive in a variety of environments is mainly due to the high genotypic and phenotypic diversity displayed by this genus. In this study, we discovered a novel Pseudomonas sp. from a desiccated environment of a sealed antibiotic bottle that was considered sterile. A close genetic relationship with its phylogenetic neighbors reiterated the need to use not just DNA-based tools but also biochemical characteristics to accurately classify this organism.

Fusarium wilt of bananas (FWB) is seriously affecting the sustainable development of the banana industry and is caused by the devastating soil-borne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Biological control is a promising strategy for controlling Fusarium wilt in bananas. We previously identified Streptomyces hygroscopicus subsp. hygroscopicus 5-4 with strong antifungal activity against the FWB. The most possible antimicrobial mechanism of strain 5-4 was explored using the metabolomics approach, light microscopy imaging, and transmission electron microscopy (TEM). The membrane integrity and ultrastructure of Foc TR4 was damaged after extract treatment, which was supported by the degradation of mycelium, soluble protein content, extracellular reducing sugar content, NADH oxidase activity, malondialdehyde content, mitochondrial membrane potential, and mitochondrial respiratory chain complex enzyme activity. The extracts of strain 5-4 cultivated at different times were characterized by a liquid chromatography-mass spectrometer (LC-MS). 647 known metabolites were detected in the extracts of strains 5-4. Hygromycin B, gluten exorphin B4, torvoside G, (z)-8-tetradecenal, piperitoside, sarmentosin, pubescenol, and other compounds were the main differential metabolites on fermentation culture for 7 days. Compared with strain 5-4 extracts, hygromycin B inhibited the mycelial growth of Foc TR4, and the EC50 concentration was 7.4µg/mL. These results showed that strain 5-4 could destroy the cell membrane of Foc TR4 to inhibit the mycelial growth, and hygromycin B may be the key antimicrobial active metabolite. Streptomyces hygroscopicus subsp. hygroscopicus 5-4 might be a promising candidate strain to control the FWB and provide a scientific basis for the practical application of hygromycin B as a biological control agent. [ABSTRACT FROM AUTHOR]

Meta-topolin (mT) is a novel aromatic cytokinin that stimulates morphogenesis and is an alternative source of cytokinins frequently employed in regeneration systems. Hence, the present research explored the prospect of mT for improving regeneration and genetic transformation efficiency. Cotyledonary node explants were cultured on optimum plant growth regulator medium incorporated with mT for enhanced shoot induction (93.6%, 2.0 mg l−1 BA and 0.9 mg l−1mT), elongation (89.3%, 2.0 mg l−1 GA3 and 0.9 mg l−1mT), and rooting (90.3%, 0.9 mg l−1 IBA and 0.9 mg l−1mT) respectively. The plant transformation study was carried out through Agrobacterium-mediated transformation using the pCAMBIA1301 vector containing the LB4404 strain for standardizing transformation strategies. Transformed shoots and rooting were determined in two stages using 10 and 6 mg l−1 hygromycin B for exterminating chimeric explants. After co-cultivation, explants were cultured at the optimal concentration of 0.9 mg l−1mT, 2.0 mg l−1 BA, 2.0 mg l−1 GA3, and 0.9 mg l−1 IBA for enhanced transformation efficiency (27.3%), corresponding to transformed regeneration without mT (18.6%) occurring with less efficiency. The existence of transgenes in the radish genome was ascertained by the GUS assay, PCR, RT-PCR, and qRT-PCR. Overall, our investigation demonstrated that including mT increases regeneration and enhances transformation efficiency in radish. Therefore, diverse radish varieties could use a designed transformation strategy to acquire essential traits. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, is arguably the most applicable synthetic genome-wide alteration to date. The SCRaMbLE system offers the capability to perform rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting long-read nanopore sequencing, we show that all structural variations are due to recombination between loxP sites, with no off-target effects. We also highlight a phenomenon imposed on SCRaMbLE termed "essential raft", where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to essentiality restrictions. Despite this, SCRaMbLE was able to explore the genomic space and produce alternative structural compositions that resulted in an increased hygromycin B resistance in the synII strain. We show that among the rearrangements generated via SCRaMbLE, deletions of YBR219C and YBR220C contribute to hygromycin B resistance phenotypes. However, the hygromycin B resistance provided by SCRaMbLEd genomes showed significant improvement when compared to corresponding single deletions, demonstrating the importance of the complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its successors will be an invaluable tool to predict and evaluate the emergence of antibiotic resistance in yeast. [ABSTRACT FROM AUTHOR]

Objectives: To determine the effect of sea salt on the resistance of Trichoderma harzianum LZDX-32-08 to hygromycin B and speculate the possible mechanisms involved via transcriptome analysis. Results: Sea salt addition in media to simulate marine environment significantly increased the tolerance of marine-derived fungus Trichoderma harzianum LZDX-32-08 to hygromycin B from 40 to 500 μg/ml. Meanwhile, sea salt addition also elicited the hygromycin B resistance of 5 other marine or terrestrial fungi. Transcriptomic analyses of T. harzianum cultivated on PDA, PDA supplemented with sea salt and PDA with both sea salt and hygromycin B revealed that genes coding for P-type ATPases, multidrug resistance related transporters and acetyltransferases were up-regulated, while genes coding for Ca2+/H+ antiporter and 1,3-glucosidase were down-regulated, indicating probable increased efflux and inactivation of hygromycin B as well as enhanced biofilm formation, which could jointly contribute to the drug resistance. Conclusions: Marine environment or high ion concentration in the environment could be an importance inducer for antifungal resistance. Possible mechanisms and related key genes were proposed for understanding the molecular basis and overcoming this resistance. [ABSTRACT FROM AUTHOR]

Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand-RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.

To investigate the inhibitory concentration of hygromycin B (hygromycin B) on the mycelium and protoplasts of Trametes gallica lg-9, JM-70S3, JM70S8. Wild type Trametes gallica lg-9 and high yield strains secreting atypical laccase by ultraviolet mutagenesis were used to investigate the inhibitory concentration of hygromycin Bon its bacteria and protoplasts Trametes gallica JM-70S3 and JM-70S8 were the experimental strains. Using hygromycin B resistance gene as screening marker, Agar dilution method was used to study the inhibitory concentration of strain lg-9, JM-70S3, JM-70S8 and its protoplasts. We obtained hygromycin B against lg-9J. The minimum inhibitory concentrations of M-70S3 and JM-70S8 against protoplasts were 49 µg/mL and 55 µg/mL, respectively. The minimum inhibitory concentrations of hygromycin Bon protoplasts were 35 µg/mL. Hygromycin B at the concentration of 55 µg/mL could inhibit the bacteria of Hypoderma vulgaris, and the protoplast could achieve the bacteriostatic effect when the concentration of hygromycin B was 35 µg/mL. [ABSTRACT FROM AUTHOR]

In order to decipher the hygromycin B tolerance and resistance mechanisms of Nannochloropsis oceanica, the transcriptome profiles of a transgenic strain carrying a randomly integrated hygromycin B resistant gene, a hygromycin B-adaptive strain and a wild type strain of N. oceanica were compared by transcriptome sequencing (RNA-seq) without referring to a high quality genome sequence. The results showed that the adaptive strain adapts to the hygromycin B existing environments mainly by intensifying the expressions of the efflux pump ABC and MFS superfamily transporter genes, thus reducing the intracellular concentration of hygromycin B. The transgenic strain obtains the hygromycin B resistance ability solely by expressing exogenous resistance gene. Accordingly, the screening and maintenance of N. oceanica transformants should be carried out at an antibiotics concentration higher than the adaptive threshold. Our findings can help the genetic modification of N. oceanica with the application of hygromycin B. [ABSTRACT FROM AUTHOR]

In eukaryotic organisms, stress-activated mitogen-activated protein kinases (MAPK) play crucial roles in transmitting environmental signals to regulate gene expression for cellular stress adaptation. Here we report that, in the fission yeast Schizosaccharomyces pombe, Spc1/Sty1 MAPK and the Atf1 transcription factor regulate the stress-induced expression of Pmp3, a ubiquitous small membrane protein implicated in the modulation of the plasma membrane potential. The pmp3 null mutant, as well as the spc1 and atf1 mutants, is hypersensitive to the cationic antibiotic hygromycin B. Transcriptional regulation of the Pmp3-like genes by the stress-activated MAPK may also be conserved in other eukaryotes, including plants. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular‐biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7′′‐phosphotransferase‐Ia [APH(7′′)‐Ia] from Streptomyces hygroscopicus and aminoglycoside 4‐phosphotransferase‐Ia [APH(4)‐Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7′′ and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)‐Ia was reported as a ternary complex with HygB and 5′‐adenylyl‐β,γ‐imidodiphosphate (AMP‐PNP). To investigate the differences in the substrate‐recognition mechanism between APH(7′′)‐Ia and APH(4)‐Ia, the crystal structure of APH(7′′)‐Ia complexed with HygB is reported. The overall structure of APH(7′′)‐Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)‐Ia, and consists of an N‐terminal lobe (N‐lobe) and a C‐terminal lobe (C‐lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7′′)‐Ia and APH(4)‐Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C‐lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7′′)‐Ia and APH(4)‐Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes. [ABSTRACT FROM AUTHOR]

Volvox carteri is an excellent model for investigating the evolution of multicellularity and cell differentiation, and the rate of future progress with this system will depend on improved molecular genetic tools. Several selectable markers for nuclear transformation of V. carteri have been developed, including the nitrate reductase (nitA) gene, but it would be useful to have additional markers to multiplex transgenes in this species. To further facilitate molecular genetic analyses of V. carteri , we developed two new selectable markers that provide rapid, easily selected, and stable resistance to the antibiotics hygromycin and blasticidin. We generated constructs with Volvox-specific regulatory sequences and codon-optimized hygromycin (VcHyg) and blasticidin (VcBlast) resistance genes from Coccidioides posadasii and Bacillus cereus , respectively. With these constructs, transformants were obtained via biolistic bombardment at rates of 0.5–13 per million target cells bombarded. Antibiotic-resistant survivors were readily isolated 7 days post bombardment. VcHyg and VcBlast transgenes and transcripts were detected in transformants. Co-transformation rates using the VcHyg or VcBlast markers with unselected genes were comparable to those obtained with nitA. These results indicate that the pVcHyg and pVcBlast plasmids are highly efficient and convenient for transforming and co-transforming a broad range of V. carteri strains. [ABSTRACT FROM AUTHOR]

Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, is arguably the most applicable synthetic genome-wide alteration to date. The SCRaMbLE system offers the capability to perform rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting long-read nanopore sequencing, we show that all structural variations are due to recombination between loxP sites, with no off-target effects. We also highlight a phenomenon imposed on SCRaMbLE termed “essential raft”, where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to essentiality restrictions. Despite this, SCRaMbLE was able to explore the genomic space and produce alternative structural compositions that resulted in an increased hygromycin B resistance in the synII strain. We show that among the rearrangements generated via SCRaMbLE, deletions of YBR219C and YBR220C contribute to hygromycin B resistance phenotypes. However, the hygromycin B resistance provided by SCRaMbLEd genomes showed significant improvement when compared to corresponding single deletions, demonstrating the importance of the complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its successors will be an invaluable tool to predict and evaluate the emergence of antibiotic resistance in yeast.

Eggplant is an agronomically important non-tuberous commercial crop belongs to Solanaceae family grown primarily for its large oval fruit. Herein, we have formulated an efficient in planta transformation method for brinjal by employing thiol compounds as antioxidants to eradicate wound stress. The seeds were initially infected with Agrobacterium tumefaciens EHA 105 harbouring pCAMBIA1301 plasmid and co-cultivated with different concentrations and combinations of antioxidants. The thiol compounds used in the present study were L-cysteine, Sodium thiosulfate and dithiothreitol. After cocultivation, the transformed seedlings were selected with 4.0 mg l-1 Hygromycin B. The survived seedlings were analysed by GUS and PCR assay for the presence of transformed genes. The integration of transgene in the plant genome was also confirmed by Southern blot hybridization. Among the different concentrations and combinations tested, collective supplementation of 400 mg l-1 L-cysteine, 250 mg l-1 STS and 150 mg l-1 DTT enhanced the viability of seedlings by reducing primary and secondary responses of injured plants. With the aid of thiol compounds, the formulated protocol attained the highest transformation efficiency of 53% in brinjal cv. Pusa Kranti. [ABSTRACT FROM AUTHOR]

Aims Agrobacterium tumefaciens-mediated transformation (At MT) is an effective method for generation of entomopathogenic Beauveria bassiana transformants. However, some strains grow on the selective medium containing hygromycin B (HygB), which reduces the selection efficiency of the putative transformants. In this work, a relationship between HygB resistance gene promoter and At MT efficiency was investigated to improve the transformant selection. Methods and Results Ten B. bassiana isolates were grown on 800 μg ml−1 HygB medium, but only JEF-006, -007 and -013 showed susceptibility to the antibiotics. Particularly, JEF-007 showed the most dose-dependent susceptibility. Two different Ti-Plasmids, pCeg ( gpdA promoter based) and pCambia-egfp (Ca MV 35S promoter based), were constructed to evaluate the promoters on the expression of HygB resistance gene ( hph) at 100, 150 and 200 μg ml−1 HygB medium. Eight days after the transformation, wild type, At MT/ pCeg and At MT/ pCambia-egfp colonies were observed on 100 μg ml−1 HygB, but significantly larger numbers of colonies were counted on At MT/ pCeg plates. At higher HygB concentration (150 μg ml−1), only At MT/ pCeg colonies were further observed, but very few colonies were observed on the wild type and At MT/ pCambia-egfp plates. Putative transformants were subjected to PCR, RT- PCR and qRT- PCR to investigate the T- DNA insertion rate and gene expression level. Consequently, >80% of colonies showed successful At MT transformation, and the hph expression level in At MT/ pCeg colonies was higher than that of At MT/ pCambia-egfp colonies. Conclusions In the HygB-susceptible B. bassiana JEF-007, gpdA promoter works better than Ca MV 35S promoter in the expression of HygB resistance gene at 150 μg ml−1 HygB, consequently improving the selection efficiency of putative transformants. Significance and Impact of the Study These results provide useful information for determining At MT effectiveness in B. bassiana isolates, particularly antibiotic susceptibility and the role of promoters. [ABSTRACT FROM AUTHOR]

Background: Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain. Results: We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the "Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells. Conclusions: These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products. [ABSTRACT FROM AUTHOR]

Current studies have shown that centromere protein F (CENPF) was overexpressed in hepatocellular carcinoma (HCC) and might be involved in the pathogenesis of HCC. Specifically, due to the very large molecular weight (358 kDa) of CENPF full length protein, only CENPF knock-down, but not overexpression models, were applied currently to explore the carcinogenicity of CENPF in HCC. Whether CENPF overexpression is a cause or an effect in HCC remains to be illustrated. We aimed to establish a CENPF overexpression cell model using CRISPR/dCas9 synergistic activation mediator (SAM) system with lentiMPHv2 and lentiSAMv2 vectors to explore the role of CENPF overexpression in HCC. Single guide RNAs (sgRNAs) that specifically identify the transcription initiation site of CENPF gene were synthesized and inserted into the lentiSAMv2 plasmid. Huh-7 and HCCLM3 cells were first transduced with lentiMPHv2 and then selected with hygromycin B. The cells were then transduced with lentiSAMv2 carrying specific sgRNA for CENPF gene, followed by blasticidin S selection. The mRNA and protein detection results of Huh-7 and HCCLM3 cells screened by hygromycin B and blasticidin S showed that the endogenous overexpression of CENPF can be induced by sgRNA1 and sgRNA4, especially by sgRNA4. By using the CRISPR/dCas9 technique, stable cell models with overexpressed CENPF were successfully constructed to explore the role of CENPF in tumorigenesis, which provides a reference for the construction of cell models overexpressing large molecular weight protein.

In an attempt to identify novel bacterial species, microbiologists have examined a wide range of environmental niches. We describe the serendipitous discovery of a novel gram-negative bacterial species from a different type of extreme niche: a purchased vial of antibiotic. The vial of antibiotic hygromycin B was found to be factory contaminated with a bacterial species, which we designate Pseudomonas hygromyciniae sp. nov. The proposed novel species belongs to the P. fluorescens complex and is most closely related to P. brenneri , P. proteolytica , and P. fluorescens . The type strain Pseudomonas hygromyciniae sp. nov. strain SDM007 T (SDM007 T ) harbors a novel 250 kb megaplasmid which confers resistance to hygromycin B and contains numerous other genes predicted to encode replication and conjugation machinery. SDM007 T grows in hygromycin concentrations of up to 5 mg/mL but does not use the antibiotic as a carbon or nitrogen source. While unable to grow at 37°C ruling out its ability to infect humans, it grows and survives at temperatures between 4 and 30°C. SDM007 T can infect plants, as demonstrated by the lettuce leaf model, and is highly virulent in the Galleria mellonella infection model but is unable to infect mammalian A549 cells. These findings indicate that commercially manufactured antibiotics represent another extreme environment that may support the growth of novel bacterial species. IMPORTANCE Physical and biological stresses in extreme environments may select for bacteria not found in conventional environments providing researchers with the opportunity to not only discover novel species but to uncover new enzymes, biomolecules, and biochemical pathways. This strategy has been successful in harsh niches such as hot springs, deep ocean trenches, and hypersaline brine pools. Bacteria belonging to the Pseudomonas species are often found to survive in these unusual environments, making them relevant to healthcare, food, and manufacturing industries. Their ability to survive in a variety of environments is mainly due to the high genotypic and phenotypic diversity displayed by this genus. In this study, we discovered a novel Pseudomonas sp. from a desiccated environment of a sealed antibiotic bottle that was considered sterile. A close genetic relationship with its phylogenetic neighbors reiterated the need to use not just DNA-based tools but also biochemical characteristics to accurately classify this organism.

Fusarium wilt of bananas (FWB) is seriously affecting the sustainable development of the banana industry and is caused by the devastating soil-borne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Biological control is a promising strategy for controlling Fusarium wilt in bananas. We previously identified Streptomyces hygroscopicus subsp. hygroscopicus 5-4 with strong antifungal activity against the FWB. The most possible antimicrobial mechanism of strain 5-4 was explored using the metabolomics approach, light microscopy imaging, and transmission electron microscopy (TEM). The membrane integrity and ultrastructure of Foc TR4 was damaged after extract treatment, which was supported by the degradation of mycelium, soluble protein content, extracellular reducing sugar content, NADH oxidase activity, malondialdehyde content, mitochondrial membrane potential, and mitochondrial respiratory chain complex enzyme activity. The extracts of strain 5-4 cultivated at different times were characterized by a liquid chromatography-mass spectrometer (LC-MS). 647 known metabolites were detected in the extracts of strains 5-4. Hygromycin B, gluten exorphin B4, torvoside G, (z)-8-tetradecenal, piperitoside, sarmentosin, pubescenol, and other compounds were the main differential metabolites on fermentation culture for 7 days. Compared with strain 5-4 extracts, hygromycin B inhibited the mycelial growth of Foc TR4, and the EC 50 concentration was 7.4 μg/mL. These results showed that strain 5-4 could destroy the cell membrane of Foc TR4 to inhibit the mycelial growth, and hygromycin B may be the key antimicrobial active metabolite. Streptomyces hygroscopicus subsp. hygroscopicus 5-4 might be a promising candidate strain to control the FWB and provide a scientific basis for the practical application of hygromycin B as a biological control agent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JP declared a shared affiliation with the authors to the handling editor at the time of review., (Copyright © 2023 Yun, Jing, Zang, Zhou, Li, Zhao, Wang and Xie.)

Cotyledons from 6-day-old Lotus corniculatus cv. Bokor seedlings, transversally cut into two halves, were capa­ble of regenerating buds without intervening callus formation. The explants were co-cultivated with the Agrobacterium tumefaciens LBA4404/pTOK233 superbinary vector carrying the uidA-intron gene and the genes hpt and nptII. They were cultured for 14 days on a regeneration medium, then subjected to a stepwise hygromycin B selection procedure consisting of gradually increasing antibiotic concentrations (5-15 mg L-1) over 21 weeks. Transformed shoots were obtained within 5 months after co-cultivation. Out of 124 initially co-cultivated explants, 52 (42%) plants survived hygromycin B selection. The presence of transgenes in regenerated plants was verified by β-glucuronidase histochemical assays and PCR analysis for the presence of uidA gene sequences. Hygromycin B-resistant and PCR-positive T0 plants were cultured in the greenhouse to produce flowers and seeds. The obtained data demonstrate that the reported transformation protocol could be useful for introducing agriculturally important genes into the new L. corniculatus cultivar Bokor.

An Agrobacterium -mediated genetic transformation system for the rice false smut fungus Ustilaginoidea virens was developed using conidia as recipients. A binary vector, pCAMBIA1301-P gpdA - GUS -T trpC , was constructed. The gpdA promoter (P gpdA ) from Aspergillus nidulans was used to drive the expression of the β-glucuronidase (GUS) gene which enabled GUS activity visualization. The conidia transformation efficiency reached approximately 110 to 250 transformants per 1 × 10 5 conidia. Based on the analysis made on five successive generations of subcultures and PCR, the pCAMBIA1301-GUS cassette had integrated into the genomes of all transformants and clearly showed mitotic stability. The novel reporter vector constructed will promote the functional characterization of genes and the construction of genetically engineered strains of this important fungus. [ABSTRACT FROM AUTHOR]

Botryosphaeria dothidea is a severe causal agent of die-back and cankers of many woody plants and causes great losses in many regions. The pathogenic mechanism of this pathogen has not been well explored due to lack of mutants and genetic information. In this study, we developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for B. dothidea protoplasts using vector pBHt2 containing the hph gene as a selection marker under the control of trp C promoter. Using this protocol we successfully generated the B. dothidea transformants with efficiency about 23 transformants per 10 protoplasts. This is the first report of genetic transformation of B. dothidea via ATMT and this protocol provides an effective tool for B. dothidea genome manipulation, gene identification and functional analysis. [ABSTRACT FROM AUTHOR]