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1.
Microb Cell Fact ; 18(1): 127, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31345221

RESUMO

BACKGROUND: Our laboratory has constructed a Bacillus licheniformis strain that secretes alkaline protease (AprE) with excellent enzymatic properties. B. licheniformis is generally regarded as safe and has a high industrial exoenzyme secretion capacity, but the host retains some undomesticated characteristic that increase its competitiveness and survival, such as spore-formation, which increases the requirements and difficulties in industrial operations (e.g. sterilization and enzyme activity control). Furthermore, the influence of sporulation on alkaline protease production in B. licheniformis has not been elucidated in detail. RESULT: A series of asporogenic variants of the parent strain were constructed by individually knocking out the master regulator genes (spo0A, sigF and sigE) involved in sporulation. Most of the variants formed abortively disporic cells characterized by asymmetric septa at the poles and unable to survive incubation at 75 °C for 10 min. Two of them (ΔsigF and ΔsigE) exhibited superior characteristics in protease production, especially improving the expression of the aprE gene. Under the currently used fermentation conditions, the vegetative production phase of ΔsigF can be prolonged to 72 h, and the highest protease production of ΔsigF reached 29,494 ± 1053 U/mL, which was about 19.7% higher than that of the wild-type strain. CONCLUSION: We first constructed three key sporulation-deficient strain to investigate the effect of sporulation on alkaline protease synthesis. The sigF mutant retained important industrial properties such as facilitating the sterilization process, a prolonged stable phase of enzyme production and slower decreasing trend, which will be superior in energy conservation, simpler operations and target product controlling effect. In summary, the work provides a useful industrial host with preferable characteristics and a novel strategy to enhance the production of protease.


Assuntos
Bacillus licheniformis/enzimologia , Bacillus licheniformis/genética , Proteínas de Bactérias/biossíntese , Endopeptidases/biossíntese , Esporos Bacterianos/genética , Fermentação , Técnicas de Inativação de Genes , Teste de Complementação Genética , Deleção de Sequência
2.
Nucleic Acids Res ; 47(11): 5777-5791, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31216041

RESUMO

Utilization of non-AUG alternative translation start sites is most common in bacteria and viruses, but it has been also reported in other organisms. This phenomenon increases proteome complexity by allowing expression of multiple protein isoforms from a single gene. In Saccharomyces cerevisiae, a few described cases concern proteins that are translated from upstream near-cognate start codons as N-terminally extended variants that localize to mitochondria. Using bioinformatics tools, we provide compelling evidence that in yeast the potential for producing alternative protein isoforms by non-AUG translation initiation is much more prevalent than previously anticipated and may apply to as many as a few thousand proteins. Several hundreds of candidates are predicted to gain a mitochondrial targeting signal (MTS), generating an unrecognized pool of mitochondrial proteins. We confirmed mitochondrial localization of a subset of proteins previously not identified as mitochondrial, whose standard forms do not carry an MTS. Our data highlight the potential of non-canonical translation initiation in expanding the capacity of the mitochondrial proteome and possibly also other cellular features.


Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Códon de Iniciação/metabolismo , Biologia Computacional , Teste de Complementação Genética , Humanos , Mitocôndrias/genética , Iniciação Traducional da Cadeia Peptídica , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
Plant Mol Biol ; 100(6): 635-645, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31147815

RESUMO

KEY MESSAGE: Rice WSL6 is involved in chloroplast ribosome biogenesis and is essential for early chloroplast development. Construction of the genetic translation system is a prerequisite for chloroplast development in plants. However, the molecular mechanism underlying this process is largely unknown. Here, we isolated a white stripe leaf6 (wsl6) mutant in rice. The mutant seedlings displayed white-striped leaves that were more severe under low-temperature conditions. Transmission electron microscopy analysis showed that the wsl6 mutant was defective in early chloroplast development. Map-based cloning revealed that WSL6 encodes an Era-type guanosine-5'-triphosphate (GTP)-binding protein located in chloroplasts. Immunoblotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses demonstrated an absence of 70S ribosomes in wsl6 chloroplasts. Further research showed that WSL6 binds to the 16S ribosomal RNA (rRNA) subunit of chloroplast ribosome 30S. In summary, these results show that WSL6 is essential for chloroplast ribosome biogenesis during early chloroplast development in rice.


Assuntos
Cloroplastos/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Clonagem Molecular , Proteínas de Ligação ao GTP/fisiologia , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Microscopia Eletrônica de Transmissão , Mutação , Oryza/fisiologia , Fenótipo , Pigmentação , Proteínas de Plantas/fisiologia , Biossíntese de Proteínas , RNA Ribossômico 16S/genética , Temperatura Ambiente
4.
Plant Mol Biol ; 100(6): 647-658, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154655

RESUMO

KEY MESSAGE: Found a trans-splicing of PHYTOENE SYNTHASE 1 alters tomato fruit color by map-based cloning, functional complementation and RACE providing an insight into fruit color development. Color is an important fruit quality trait and a major determinant of the economic value of tomato (Solanum lycopersicum). Fruit color inheritance in a yellow-fruited cherry tomato (cv. No. 22), named yellow-fruited tomato 2 (yft2), was shown to be controlled by a single recessive gene, YFT2. The YFT2 gene was mapped in a 95.7 kb region on chromosome 3, and the candidate gene, PHYTOENE SYNTHASE 1 (PSY1), was confirmed by functional complementation analysis. Constitutive over expression of PSY1 in yft2 increased the accumulation of carotenoids and resulted in a red fruit color, while no causal mutation was detected in the YFT2 allele of yft2, compared with red-fruited SL1995 cherry tomato or cultivated variety (cv. M82). Expression of YFT2 3' region in yft2 was significantly lower than in SL1995, and further studies revealed a difference in YFT2 post-transcriptional processing in yft2 compared with SL1995 and cv. M82, resulting in a longer YFT2 transcript. The alternatively trans-spliced allele of YFT2 in yft2 is predicted to encode a novel LT-YFT2 protein of 432 amino acid (AA) residues, compared to the 412 AA YFT2 protein of SL1995. The trans-spliced event also resulted in significantly down regulated expression of YFT2 in yft2 tomato, and the YFT2 allele suppressed expression of the downstream genes involved in the carotenoid biosynthesis pathway and carotenoids synthesis by a mechanism of the feed-forward regulation. In conclusion, we found that trans-splicing of YFT2 alters tomato fruit color, providing new insights into fruit color development.


Assuntos
Lycopersicon esculentum/metabolismo , Pigmentação/genética , Proteínas de Plantas/metabolismo , Processamento Alternativo , Carotenoides/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Cor , DNA Complementar/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Recessivos , Teste de Complementação Genética , Genótipo , Lycopersicon esculentum/genética , Mutação , Proteínas de Plantas/genética , Trans-Splicing
5.
Int J Mol Sci ; 20(9)2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083521

RESUMO

ADP-ribosylation factor-guanine nucleotide exchange factors (ARF-GEFs) act as key regulators of vesicle trafficking in all eukaryotes. In Arabidopsis, there are eight ARF-GEFs, including three members of the GBF1 subfamily and five members of the BIG subfamily. These ARF-GEFs have different subcellular localizations and regulate different trafficking pathways. Until now, the roles of these BIG-subfamily ARF-GEFs have not been fully revealed. Here, analysis of the BIGs expression patterns showed that BIG3 and BIG5 have similar expression patterns. big5-1 displayed a dwarf growth and big3-1 big5-1 double mutant showed more severe defects, indicating functional redundancy between BIG3 and BIG5. Moreover, both big5-1 and big3-1 big5-1 exhibited a reduced sensitivity to Brassinosteroid (BR) treatment. Brefeldin A (BFA)-induced BR receptor Brassinosteroid insensitive 1 (BRI1) aggregation was reduced in big5-1 mutant, indicating that the action of BIG5 is required for BRI1 recycling. Furthermore, BR-induced dephosphorylation of transcription factor BZR1 was decreased in big3-1 big5-1 double mutants. The introduction of the gain-of-function of BZR1 mutant BZR1-1D in big3-1 big5-1 mutants can partially rescue the big3-1 big5-1 growth defects. Our findings revealed that BIG5 functions redundantly with BIG3 in plant growth and gravitropism, and BIG5 participates in BR signal transduction pathway through regulating BRI1 trafficking.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Gravitropismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Desenvolvimento Vegetal , Proteínas Quinases/metabolismo , Proteínas de Arabidopsis/genética , Brassinosteroides/farmacologia , Teste de Complementação Genética , Gravitropismo/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Inflorescência/efeitos dos fármacos , Inflorescência/crescimento & desenvolvimento , Mutação/genética , Fenótipo , Desenvolvimento Vegetal/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
6.
PLoS Comput Biol ; 15(5): e1006802, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31120875

RESUMO

Recent large cancer studies have measured somatic alterations in an unprecedented number of tumours. These large datasets allow the identification of cancer-related sets of genetic alterations by identifying relevant combinatorial patterns. Among such patterns, mutual exclusivity has been employed by several recent methods that have shown its effectiveness in characterizing gene sets associated to cancer. Mutual exclusivity arises because of the complementarity, at the functional level, of alterations in genes which are part of a group (e.g., a pathway) performing a given function. The availability of quantitative target profiles, from genetic perturbations or from clinical phenotypes, provides additional information that can be leveraged to improve the identification of cancer related gene sets by discovering groups with complementary functional associations with such targets. In this work we study the problem of finding groups of mutually exclusive alterations associated with a quantitative (functional) target. We propose a combinatorial formulation for the problem, and prove that the associated computational problem is computationally hard. We design two algorithms to solve the problem and implement them in our tool UNCOVER. We provide analytic evidence of the effectiveness of UNCOVER in finding high-quality solutions and show experimentally that UNCOVER finds sets of alterations significantly associated with functional targets in a variety of scenarios. In particular, we show that our algorithms find sets which are better than the ones obtained by the state-of-the-art method, even when sets are evaluated using the statistical score employed by the latter. In addition, our algorithms are much faster than the state-of-the-art, allowing the analysis of large datasets of thousands of target profiles from cancer cell lines. We show that on two such datasets, one from project Achilles and one from the Genomics of Drug Sensitivity in Cancer project, UNCOVER identifies several significant gene sets with complementary functional associations with targets. Software available at: https://github.com/VandinLab/UNCOVER.


Assuntos
Biologia Computacional/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Algoritmos , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Teste de Complementação Genética/métodos , Genômica/métodos , Humanos , Mutação , Software
7.
Cell Mol Life Sci ; 76(20): 4131-4144, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31053883

RESUMO

ABCB6 belongs to the family of ATP-binding cassette (ABC) transporters, which transport various molecules across extra- and intra-cellular membranes, bearing significant impact on human disease and pharmacology. Although mutations in the ABCB6 gene have been linked to a variety of pathophysiological conditions ranging from transfusion incompatibility to pigmentation defects, its precise cellular localization and function is not understood. In particular, the intracellular localization of ABCB6 has been a matter of debate, with conflicting reports suggesting mitochondrial or endolysosomal expression. ABCB6 shows significant sequence identity to HMT-1 (heavy metal tolerance factor 1) proteins, whose evolutionarily conserved role is to confer tolerance to heavy metals through the intracellular sequestration of metal complexes. Here, we show that the cadmium-sensitive phenotype of Schizosaccharomyces pombe and Caenorhabditis elegans strains defective for HMT-1 is rescued by the human ABCB6 protein. Overexpression of ABCB6 conferred tolerance to cadmium and As(III) (As2O3), but not to As(V) (Na2HAsO4), Sb(V), Hg(II), or Zn(II). Inactivating mutations of ABCB6 abolished vacuolar sequestration of cadmium, effectively suppressing the cadmium tolerance phenotype. Modulation of ABCB6 expression levels in human glioblastoma cells resulted in a concomitant change in cadmium sensitivity. Our findings reveal ABCB6 as a functional homologue of the HMT-1 proteins, linking endolysosomal ABCB6 to the highly conserved mechanism of intracellular cadmium detoxification.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Cádmio/toxicidade , Proteínas de Caenorhabditis elegans/genética , Inativação Metabólica/genética , Poluentes Químicos da Água/toxicidade , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antimônio/toxicidade , Arseniatos/toxicidade , Trióxido de Arsênio/toxicidade , Cádmio/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Expressão Gênica , Teste de Complementação Genética , Células HeLa , Humanos , Mercúrio/toxicidade , Mutação , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Poluentes Químicos da Água/metabolismo , Zinco/toxicidade
8.
Curr Microbiol ; 76(7): 879-887, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31089795

RESUMO

The goal of this study was to elucidate the role of the outer membrane protein A (ompA) gene of Xanthomonas axonopodis pv. glycines in bacterial pustule pathogenesis of soybean. An ompA mutant of X. axonopodis pv. glycines KU-P-SW005 was shown to significantly decrease cellulase, pectate lyase, and polysaccharide production. The production of these proteins in the ompA mutant was approximately five times lower than that of the wildtype. The ompA mutant also exhibited modified biofilm development. More importantly, the mutant reduced disease severity to the soybean. Ten days after inoculation, the virulence rating of the susceptible soybean cv. SJ4 inoculated with the ompA mutant was 11.23%, compared with 87.98% for the complemented ompA mutant. Production of cellulase, pectate lyase, polysaccharide was restored, biofilm, and pustule numbers were restored in the complemented ompA mutant that did not differ from the wild type. Taken together, these data suggest that OmpA-mediated invasion plays an important role in protein secretion during pathogenesis to soybean.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Doenças das Plantas/microbiologia , Soja/microbiologia , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/patogenicidade , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Celulase/metabolismo , Teste de Complementação Genética , Mutação , Folhas de Planta/microbiologia , Polissacarídeo-Liase/metabolismo , Polissacarídeos Bacterianos/metabolismo , Virulência/genética
9.
Mol Biol (Mosk) ; 53(2): 274-281, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31099777

RESUMO

Expression of Saccharomyces cerevisiae proteasomal genes is regulated in a coordinated manner by a system that includes the ScRpn4 transcription factor and its binding site known as PACE. Earlier we showed that, Rpn4-like proteins from the biotechnologically important yeast species Komagataella pfaffii (Pichia pastoris), Yarrowia lipolytica, and Debaryomyces hansenii are capable of complementing the RPN4 deletion in S. cerevisiae in spite of their low structural similarity to ScRpn4. The opportunistic yeast pathogen Candida glabrata has a gene coding for a Rpn4-like protein, which has not been characterized experimentally yet. The С. glabrata ortholog ScRpn4 was expressed heterologously and found to restore the stress resistance and expression of proteasomal genes in a mutant S. cerevisiae strain with a RPN4 deletion. This complementation required the unique N-terminal region of CgRpn4. The results indicate that CgRpn4 acts as a transcriptional activator of proteasomal genes. The S. cerevisiae model can be used for further structural and functional analyses of CgRpn4.


Assuntos
Candida glabrata/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Deleção de Genes , Teste de Complementação Genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/metabolismo
10.
Fungal Biol ; 123(5): 423-430, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31053331

RESUMO

Plant pathogens employ effectors as molecular weapons to manipulate host immunity and facilitate colonization. Fusarium oxysporum f. sp. cubense is the agent of wilt disease in banana plantlets and four races of the pathogen have been identified based on the cultivar specificity. A total of 9 SIX genes have been detected in the genome of Foc TR4 and 6 genes detected in Foc1. Among these SIX genes, SIX2 and SIX8 are only detected in Foc TR4, not identified in Foc1. Expression profiles analysis revealed that SIX genes of Foc TR4 are highly induced after inoculation to Cavendish banana plantlets. Virulence analysis of the SIX2 and SIX8 knock-out mutants showed that SIX8 is required for the virulence of Foc TR4 while SIX2 has no obvious functions. Over expression of SIX8-FLAG proteins in the SIX8 knock-out mutant partly restored the virulence. Western blot analysis suggested that SIX8 could be secreted into the extracellular space and a signal peptide resided the N-terminal polypeptide sequence. This study provides some clues for further research on mechanism of SIX8 in regulating virulence of Foc TR4.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/patogenicidade , Musa/microbiologia , Doenças das Plantas/microbiologia , Fatores de Virulência/metabolismo , Proteínas Fúngicas/genética , Fusarium/genética , Fusarium/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Teste de Complementação Genética , Fatores de Virulência/genética
11.
Appl Microbiol Biotechnol ; 103(11): 4539-4548, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997553

RESUMO

The MtrA-MtrB two-component regulatory system is highly conserved in Actinobacteria and plays crucial roles in cell cycle progression, cell morphology, antibiotic resistance, and osmoprotection. Previously, we revealed that the MtrA protein of Saccharopolyspora erythraea E3 strain (a high erythromycin-producing strain) had a two amino acid (H197 and V198) deletion in the DNA recognition helices of the C-terminal domain compared to the wild type S. erythraea strain NRRL2338. Here, we identified mepA (encoding a membrane protein related to metalloendopeptidases) as an MtrA target gene, and found that deleting the two amino acids in MtrA (MtrAdel) resulted in the loss of its DNA-binding activity for the mepA gene. The mutant MtrAdel lost its regulatory activity and affected various physiological functions consistent with mtrA deletion, including increased erythromycin biosynthesis, enhanced antibiotic resistance, deregulated osmoprotection, and improved transport of substances. The introduction of the wild type mtrA gene into the S. erythraea E3 strain with the mtrAdel gene decreased the erythromycin yield by approximately 50%, confirming that MtrA repressed erythromycin production. These findings demonstrate that MtrA is an important pleiotropic regulator of erythromycin biosynthesis, antibiotic resistance, osmoprotection, and substance transport in S. erythraea and provide new insights for improving erythromycin production. Future studies linking the molecular effects of MtrA to these phenotypes will improve our understanding of the MtrA-MtrB two-component regulatory system in Actinobacteria.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eritromicina/biossíntese , Saccharopolyspora/enzimologia , Saccharopolyspora/metabolismo , Deleção de Sequência , Transporte Biológico , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fenótipo , Saccharopolyspora/crescimento & desenvolvimento
12.
G3 (Bethesda) ; 9(5): 1581-1595, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30948422

RESUMO

A large portion of the Drosophila melanogaster genome is contained within heterochromatic regions of chromosomes, predominantly at centromeres and telomeres. The remaining euchromatic portions of the genome have been extensively characterized with respect to gene organization, function and regulation. However, it has been difficult to derive similar data for sequences within centromeric (centric) heterochromatin because these regions have not been as amenable to analysis by standard genetic and molecular tools. Here we present an updated genetic and molecular analysis of chromosome 3L centric heterochromatin (3L Het). We have generated and characterized a number of new, overlapping deficiencies (Dfs) which remove regions of 3L Het. These Dfs were critically important reagents in our subsequent genetic analysis for the isolation and characterization of lethal point mutations in the region. The assignment of these mutations to genetically-defined essential loci was followed by matching them to gene models derived from genome sequence data: this was done by using molecular mapping plus sequence analysis of mutant alleles, thereby aligning genetic and physical maps of the region. We also identified putative essential gene sequences in 3L Het by using RNA interference to target candidate gene sequences. We report that at least 25, or just under 2/3 of loci in 3L Het, are essential for viability and/or fertility. This work contributes to the functional annotation of centric heterochromatin in Drosophila, and the genetic and molecular tools generated should help to provide important insights into the organization and functions of gene sequences in 3L Het.


Assuntos
Centrômero/genética , Cromossomos de Insetos , Drosophila melanogaster/genética , Genes Essenciais , Genômica , Heterocromatina/genética , Animais , Mapeamento Cromossômico , Técnicas de Silenciamento de Genes , Genes Letais , Teste de Complementação Genética , Testes Genéticos , Genômica/métodos , Genótipo , Modelos Genéticos , Mutagênese/efeitos da radiação , Mutação , Interferência de RNA , Raios X
13.
Food Microbiol ; 82: 482-488, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31027809

RESUMO

Shiga toxin-producing Escherichia coli (STEC) is one of the most common causal agents of foodborne illness linked to fresh leafy vegetables. Here, we investigated the impact of spinach-associated microorganisms on proliferation and biofilm formation of STEC O157:H7 on stainless steel surfaces at temperatures related to produce production and postharvest processing environments. Although a proliferation of inoculated pathogen cells in spinach leaf wash water was detected at all temperatures examined, the impact of spinach-associated microorganisms on the proliferation of E. coli O157:H7 was observed at 10 °C and 26 °C, but not at 4 °C. The inhibition of E. coli O157:H7 growth by spinach-associated microorganisms indicated a competition between the pathogen and spinach indigenous microflora. A significant decrease of the pathogen population in mixed biofilms was observed only at 26 °C for curli-deficient strain MQC43, but not for curli-expressing strain MQC57. Deletion of curli genes in a curli-expressing strain resulted in a phenotype similar to that of MQC43 in mixed biofilms; however, this deficiency was rescued when curli biogenesis was restored in the curli-deletion mutant strain. Our data support that curli confer E. coli O157:H7 a competitive trait in mixed biofilms, presumably through the interaction between STEC and the biofilm-proficient microorganisms associated with spinach leaves.


Assuntos
Biofilmes , Proteínas de Escherichia coli/fisiologia , Microbiologia de Alimentos , Escherichia coli Shiga Toxigênica/fisiologia , Spinacia oleracea/microbiologia , Aderência Bacteriana/genética , Biofilmes/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/fisiologia , Proteínas de Escherichia coli/genética , Teste de Complementação Genética , Interações Microbianas , Microbiota/fisiologia , Mutação , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/crescimento & desenvolvimento , Aço Inoxidável , Temperatura Ambiente
14.
Mol Biotechnol ; 61(6): 442-450, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980224

RESUMO

Soil salinity imposes a serious threat to the productivity of agricultural crops. Among several other transporters, high-affinity K+ transporter (HKT)'s play an important role in reducing the phytotoxicity of Na+. Expression of Eutrema salsugineum (a halophyte) HKT1;2 is induced upon salt exposure. To elucidate the role of its promoter, we compared the sequences of HKT1;2 promoters from E. salsugineum (1822 bp) and E. botschantzevii (1811 bp) with Arabidopsis thaliana HKT1;1 (846 bp) promoter. In silico analysis predicted several cis-acting regulatory elements (GT-1 elements, core motifs of DRE/CRT, MYC/MYB-recognition sites and ACGT elements). Activities of the three promoters were analyzed by measuring HKT1;1 and/or HKT1;2 transcript level in the Athkt1;1 mutant plants. NaCl tolerance of the transgenics was also assessed. Our results depicted that expressing either AtHKT1;1 or EsHKT1;2 coding regions under the control of AtHKT1;1 promoter, almost reversed the hypersensitivity of the mutant for salt, on contrarily, when AtHKT1;1 coding sequence expressed under either Es or EbHKT1;2 promoters did not. Changes in shoot Na+/K+ concentrations under salt exposure is significantly consistent with the complementation ability of the mutant. The transcript concentration for genes under the control of either of Eutrema promoters, at control level was very less. This may suggest that either an important upstream response motif is missed or that A. thaliana misses a transcriptional regulator that is essential for salt-inducible HKT1 expression in Eutrema.


Assuntos
Arabidopsis/genética , Brassicaceae/genética , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tolerância ao Sal/genética , Simportadores/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Sequência de Bases , Brassicaceae/efeitos dos fármacos , Brassicaceae/crescimento & desenvolvimento , Brassicaceae/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Teste de Complementação Genética , Transporte de Íons/efeitos dos fármacos , Mutação , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Potássio/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Sódio/metabolismo , Cloreto de Sódio/farmacologia , Especificidade da Espécie , Estresse Fisiológico/genética , Simportadores/metabolismo
15.
Nature ; 568(7751): 259-263, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30944473

RESUMO

The genetic compensation response (GCR) has recently been proposed as a possible explanation for the phenotypic discrepancies between gene-knockout and gene-knockdown1,2; however, the underlying molecular mechanism of the GCR remains uncharacterized. Here, using zebrafish knockdown and knockout models of the capn3a and nid1a genes, we show that mRNA bearing a premature termination codon (PTC) promptly triggers a GCR that involves Upf3a and components of the COMPASS complex. Unlike capn3a-knockdown embryos, which have small livers, and nid1a-knockdown embryos, which have short body lengths2, capn3a-null and nid1a-null mutants appear normal. These phenotypic differences have been attributed to the upregulation of other genes in the same families. By analysing six uniquely designed transgenes, we demonstrate that the GCR is dependent on both the presence of a PTC and the nucleotide sequence of the transgene mRNA, which is homologous to the compensatory endogenous genes. We show that upf3a (a member of the nonsense-mediated mRNA decay pathway) and components of the COMPASS complex including wdr5 function in GCR. Furthermore, we demonstrate that the GCR is accompanied by an enhancement of histone H3 Lys4 trimethylation (H3K4me3) at the transcription start site regions of the compensatory genes. These findings provide a potential mechanistic basis for the GCR, and may help lead to the development of therapeutic strategies that treat missense mutations associated with genetic disorders by either creating a PTC in the mutated gene or introducing a transgene containing a PTC to trigger a GCR.


Assuntos
Códon sem Sentido/genética , Teste de Complementação Genética , Complexos Multiproteicos/metabolismo , RNA Mensageiro/genética , Peixe-Zebra/genética , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Deleção de Genes , Células HCT116 , Histonas/metabolismo , Humanos , Complexos Multiproteicos/química , Degradação do RNAm Mediada por Códon sem Sentido , Organismos Geneticamente Modificados , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
16.
MBio ; 10(2)2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862746

RESUMO

Clostridioides difficile infection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two major C. difficile toxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified a C. difficile regulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, and rstA transcription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds the rstA promoter via the predicted DNA-binding domain. Through mutational analysis of the rstA promoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genes tcdA and tcdB, as well as the promoters for the sigD and tcdR genes, which encode regulators of toxin gene expression. Complementation analyses with the Clostridium perfringens RstA ortholog and a multispecies chimeric RstA protein revealed that the C. difficile C-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCE Clostridioides difficile is an anaerobic, gastrointestinal pathogen of humans and other mammals. C. difficile produces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link between C. difficile sporulation and toxin production. Further, our data suggest that C. difficile toxin production is regulated through a direct, species-specific sensing mechanism.


Assuntos
Proteínas de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Clostridium difficile/genética , Clostridium difficile/fisiologia , Enterotoxinas/biossíntese , Regulação Bacteriana da Expressão Gênica , Locomoção , Proteínas Repressoras/metabolismo , Clostridium perfringens/genética , Análise Mutacional de DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Teste de Complementação Genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética
17.
Nat Commun ; 10(1): 1360, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30911012

RESUMO

TonB-dependent transporters (TBDTs) are ubiquitous outer membrane ß-barrel proteins that import nutrients and bacteriocins across the outer membrane in a proton motive force-dependent manner, by directly connecting to the ExbB/ExbD/TonB system in the inner membrane. Here, we show that the TBDT Oar in Myxococcus xanthus is required for secretion of a protein, protease PopC, to the extracellular milieu. PopC accumulates in the periplasm before secretion across the outer membrane, and the proton motive force has a role in secretion to the extracellular milieu. Reconstitution experiments in Escherichia coli demonstrate that secretion of PopC across the outer membrane not only depends on Oar but also on the ExbB/ExbD/TonB system. Our results indicate that TBDTs and the ExbB/ExbD/TonB system may have roles not only in import processes but also in secretion of proteins.


Assuntos
Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/genética , Myxococcus xanthus/genética , Peptídeo Hidrolases/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana/metabolismo , Myxococcus xanthus/classificação , Myxococcus xanthus/metabolismo , Peptídeo Hidrolases/metabolismo , Periplasma/metabolismo , Filogenia , Força Próton-Motriz
18.
J Biosci Bioeng ; 128(1): 33-38, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30711353

RESUMO

In this work, we analyzed several genes participating in the rearrangement pathway for xylulose 5-phosphate (Xu5P) in the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii). P. pastoris has two set of genes for fructose-1,6-bisphosphate aldolase (FBA1 and FBA2) and transaldolase (TAL1 and TAL2), although there are single-copy genes for fructose-1,6-bisphosphatase (FBP1) and transketolase (TKL1), respectively. Expressions of FBP1 and TAL2 were upregulated by non-fermentative carbon sources, especially methanol was the best inducer for them, and FBA2 was induced only by methanol. On the other hand, FBA1, TAL1 and TKL1 showed constitutive expression. Strain fbp1Δ showed severe growth defect on methanol and non-fermentable carbon sources, and growth rate of strain fba2Δ in methanol medium was slightly decreased. Moreover, Fba2p and Tal2p possessed peroxisome targeting signal type 1 (PTS1), and EGFP-Fba2p and EGFP-Tal2p were found to be localized in peroxisomes. From these findings, it was suggested that Fba2p, Fbp1p and Tal2p participate in the rearrangement pathway for Xu5P in peroxisomes, and that the altered Calvin cycle and non-oxidative pentose phosphate pathway involving Tal2p function in a complementary manner.


Assuntos
Frutose-Bifosfato Aldolase/genética , Metanol/metabolismo , Pentosefosfatos/metabolismo , Pichia , Transaldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Redes e Vias Metabólicas/genética , Peroxissomos/genética , Peroxissomos/metabolismo , Pichia/enzimologia , Pichia/genética , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Transaldolase/metabolismo , Transcetolase/genética , Transcetolase/metabolismo
19.
mSphere ; 4(1)2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700512

RESUMO

Copper (Cu) is an essential trace element in all organisms, and Cu acquisition during periods of starvation is important for cell survival and proliferation. Although the Cu starvation-responsive transcription factor Mac1 as well as its targeted Cu transporters have been identified in Aspergillus fumigatus, the molecular mechanisms of Mac1-mediated Cu acquisition have not yet been investigated in Aspergillus We demonstrated that Mac1 and its regulated Cu transporters are required for growth and conidiophore development during Cu starvation in Aspergillus nidulans Moreover, A. nidulans Mac1 (AnMac1) showed highly functional conservation with the A. fumigatus homolog but not with homologs in Saccharomyces cerevisiae and Schizosaccharomyces pombe Molecular characterization of Mac1 in A. nidulans demonstrated that the "Cu fist" motif (i.e., residues 1 through 40) harboring Cys, RGHR, and GRP residues is required for the Mac1-mediated low-Cu response but not the Cys-rich motifs REP-I and REP-II. Notably, overexpression of either the CtrA2 Cu transporter or the CtrC Cu transporter individually was unable to functionally rescue the defects in the AnMac1 deletion strain, implying that Cu uptake might require both CtrA2 and CtrC during Cu starvation, which is different from results seen with A. fumigatus Findings in this study further suggest that the conserved Mac1-mediated Cu uptake machinery in A. fumigatus and A. nidulans is also species specific.IMPORTANCE Copper is an essential cofactor of enzymes during a variety of biochemical processes. Therefore, Cu acquisition plays critical roles in cell survival and proliferation, especially during Cu starvation. Knowledge of the key motif(s) by which the low-Cu-responsive transcription factor Mac1 senses Cu is important for understanding how Cu uptake is controlled. Findings in this study demonstrated that the Cu fist motif, but not Cys-rich motifs, is essential for Mac1-mediated Cu uptake in Aspergillus In addition, Cu transporters CtrA2 and CtrC are both required for Mac1-mediated Cu uptake during Cu starvation in A. nidulans, indicating that species-specific machinery exists for Cu acquisition in Aspergillus.


Assuntos
Aspergillus nidulans/enzimologia , Aspergillus nidulans/crescimento & desenvolvimento , Cobre/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Análise Mutacional de DNA , Deleção de Genes , Expressão Gênica , Teste de Complementação Genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação Puntual , Homologia de Sequência , Oligoelementos/metabolismo
20.
Microb Pathog ; 129: 1-6, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30703474

RESUMO

To persist in the host, Salmonella is known to facultatively parasitize cells to escape the immune response. Intracellular Salmonella enterica can replicate using effector proteins translocated across the Salmonella-containing vacuolar membrane via a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). One of these factors, Salmonella secreted factor L (SseL), is a deubiquitinase that contributes to the virulence of Salmonella Typhimurium in mice by inhibiting the cellular NF-κB inflammatory pathway. However, the nature of its effect on the NF-κB pathway is controversial, and little research has been performed in other animal models. In this study, the SseL of Salmonella Pullorum was studied, and chickens were used as an infection model. An sseL gene deletion strain, a complementation strain and a eukaryotic expression plasmid were used to clarify the means by which SseL regulates Salmonella virulence and the cellular inflammatory response. SseL significantly enhanced the virulence of Salmonella Pullorum in chickens and suppressed activation of the cellular NF-κB pathway, thus inhibiting cellular inflammatory cytokine expression.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , NF-kappa B/antagonistas & inibidores , Salmonelose Animal/patologia , Salmonella enterica/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Galinhas , Deleção de Genes , Teste de Complementação Genética , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/patologia , Salmonelose Animal/microbiologia , Virulência , Fatores de Virulência/genética
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