scholarly journals Induction of a Toxin-Antitoxin Gene Cassette under High Hydrostatic Pressure Enables Markerless Gene Disruption in the Hyperthermophilic Archaeon Pyrococcus yayanosii

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Qinghao Song ◽  
Zhen Li ◽  
Rouke Chen ◽  
Xiaopan Ma ◽  
Xiang Xiao ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
High Temperatures ◽  
High Hydrostatic Pressure ◽  
Gene Disruption ◽  
Inducible Promoter ◽  
Constitutive Promoter ◽  
Gene Encoding ◽  
Content Type ◽  
Genetic Tools ◽  
Pyrococcus Yayanosii

ABSTRACT The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)-inducible promoter (Phhp) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing (i) a counterselectable marker in which a gene encoding a putative toxin (virulence-associated protein C [PF0776 {VapC}]) controlled by the HHP-inducible promoter was used in conjunction with the gene encoding antitoxin PF0775 (VapB), which was fused to a constitutive promoter (PhmtB), and (ii) a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive promoter Pgdh. The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-α-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ1369 mutant decreased dramatically compared to that in the wild-type strain. The inducible toxin-antitoxin system developed in this study greatly increases the genetic tools available for use in hyperthermophiles. IMPORTANCE Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHP-inducible promoter was used to control the expression of a toxin. Compared to sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible toxin-antitoxin (TA) system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles.

scholarly journals Ribosome Reconstruction during Recovery from High-Hydrostatic-Pressure-Induced Injury in Bacillus subtilis

2019 ◽  
Vol 86 (1) ◽  
Author(s):  
Huyen Thi Minh Nguyen ◽  
Genki Akanuma ◽  
Tu Thi Minh Hoa ◽  
Yuji Nakai ◽  
Keitarou Kimura ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
Growth Arrest ◽  
Recovery Process ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Vegetative Cells ◽  
Content Type ◽  
Growth Recovery

ABSTRACT Vegetative cells of Bacillus subtilis can recover from injury after high-hydrostatic-pressure (HHP) treatment at 250 MPa. DNA microarray analysis revealed that substantial numbers of ribosomal genes and translation-related genes (e.g., translation initiation factors) were upregulated during the growth arrest phase after HHP treatment. The transcript levels of cold shock-responsive genes, whose products play key roles in efficient translation, and heat shock-responsive genes, whose products mediate correct protein folding or degrade misfolded proteins, were also upregulated. In contrast, the transcript level of hpf, whose product (Hpf) is involved in ribosome inactivation through the dimerization of 70S ribosomes, was downregulated during the growth arrest phase. Sucrose density gradient sedimentation analysis revealed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed. We also found that cell growth after HHP-induced injury was apparently inhibited by the addition of Mn2+ or Zn2+ to the recovery medium. Ribosome reconstruction in the HHP-injured cells was also significantly delayed in the presence of Mn2+ or Zn2+. Moreover, Zn2+, but not Mn2+, promoted dimer formation of 70S ribosomes in the HHP-injured cells. Disruption of the hpf gene suppressed the Zn2+-dependent accumulation of ribosome dimers, partially relieving the inhibitory effect of Zn2+ on the growth recovery of HHP-treated cells. In contrast, it was likely that Mn2+ prevented ribosome reconstruction without stimulating ribosome dimerization. Our results suggested that both Mn2+ and Zn2+ can prevent ribosome reconstruction, thereby delaying the growth recovery of HHP-injured B. subtilis cells. IMPORTANCE HHP treatment is used as a nonthermal processing technology in the food industry to inactivate bacteria while retaining high quality of foods under suppressed chemical reactions. However, some populations of bacterial cells may survive the inactivation. Although the survivors are in a transient nongrowing state due to HHP-induced injury, they can recover from the injury and then start growing, depending on the postprocessing conditions. The recovery process in terms of cellular components after the injury remains unclear. Transcriptome analysis using vegetative cells of Bacillus subtilis revealed that the translational machinery can preferentially be reconstructed after HHP treatment. We found that both Mn2+ and Zn2+ prolonged the growth-arrested stage of HHP-injured cells by delaying ribosome reconstruction. It is likely that ribosome reconstruction is crucial for the recovery of growth ability in HHP-injured cells. This study provides further understanding of the recovery process in HHP-injured B. subtilis cells.

scholarly journals Synergistic Effects of High Hydrostatic Pressure, Mild Heating, and Amino Acids on Germination and Inactivation of Clostridium sporogenes Spores

2012 ◽  
Vol 78 (23) ◽  
pp. 8202-8207 ◽  
Author(s):  
Takateru Ishimori ◽  
Katsutoshi Takahashi ◽  
Masato Goto ◽  
Suguru Nakagawa ◽  
Yoshiaki Kasai ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hydrostatic Pressure ◽  
Vegetative Growth ◽  
High Hydrostatic Pressure ◽  
Synergistic Effects ◽  
Clostridium Sporogenes ◽  
Spore Viability ◽  
Content Type

ABSTRACTThe synergistic effects of high hydrostatic pressure (HHP), mild heating, and amino acids on the germination ofClostridium sporogenesspores were examined by determining the number of surviving spores that returned to vegetative growth after pasteurization following these treatments. Pressurization at 200 MPa at a temperature higher than 40°C and treatment with some of the 19l-amino acids at 10 mM or higher synergistically facilitated germination. When one of these factors was omitted, the level of germination was insignificant. Pressures of 100 and 400 MPa were less effective than 200 MPa. The spores were effectively inactivated by between 1.8 and 4.8 logs by pasteurization at 80°C after pressurization at 200 MPa at 45°C for 120 min with one of the amino acids with moderate hydrophobicity, such as Leu, Phe, Cys Met, Ala, Gly, or Ser. However, other amino acids showed poor inactivation effects of less than 0.9 logs. Spores in solutions containing 80 mM of either Leu, Phe, Cys, Met, Ala, Gly, or Ser were successfully inactivated by pasteurization by more than 5.4 logs after pressurization at 200 MPa at 70°C for 15 to 120 min. Ala and Met reduced the spore viability by 2.8 and 1.8 logs, respectively, by pasteurization at a concentration of 1 mM under 200 MPa at 70°C. These results indicate that germination of the spores is facilitated by a combination of high hydrostatic pressure, mild heating, and amino acids.

scholarly journals Targeting the Nonmevalonate Pathway inBurkholderia cenocepaciaIncreases Susceptibility to Certain β-Lactam Antibiotics

2018 ◽  
Vol 62 (5) ◽  
pp. e02607-17 ◽  
Author(s):  
Andrea Sass ◽  
Annelien Everaert ◽  
Heleen Van Acker ◽  
Freija Van den Driessche ◽  
Tom Coenye
Keyword(s):  
Inducible Promoter ◽  
Burkholderia Cenocepacia ◽  
Growth Defect ◽  
Constitutive Promoter ◽  
Content Type ◽  
Nonmevalonate Pathway ◽  
In Trans ◽  
Antibacterial Chemotherapy ◽  
Novel Target ◽  
Increased Susceptibility

ABSTRACTThe nonmevalonate pathway is the sole pathway for isoprenoid biosynthesis inBurkholderia cenocepaciaand is possibly a novel target for the development of antibacterial chemotherapy. The goals of the present study were to evaluate the essentiality ofdxr, the second gene of the nonmevalonate pathway, inB. cenocepaciaand to determine whether interfering with the nonmevalonate pathway increases susceptibility toward antibiotics. To this end, a rhamnose-inducible conditionaldxrknockdown mutant ofB. cenocepaciastrain K56-2 (B. cenocepaciaK56-2dxr) was constructed, using a plasmid which enables the delivery of a rhamnose-inducible promoter in the chromosome. Expression ofdxris essential for bacterial growth; the growth defect observed in thedxrmutant could be complemented by expressingdxr in transunder the control of a constitutive promoter, but not by providing 2-C-methyl-d-erythritol-4-phosphate, the reaction product of DXR (1-deoxy-d-xylulose 5-phosphate reductoisomerase).B. cenocepaciaK56-2dxrshowed markedly increased susceptibility to the β-lactam antibiotics aztreonam, ceftazidime, and cefotaxime, while susceptibility to other antibiotics was not (or was much less) affected; this increased susceptibility could also be complemented byin transexpression ofdxr. A similarly increased susceptibility was observed when antibiotics were combined with FR900098, a known DXR inhibitor. Our data confirm that the nonmevalonate pathway is essential inB. cenocepaciaand suggest that combining potent DXR inhibitors with selected β-lactam antibiotics is a useful strategy to combatB. cenocepaciainfections.

scholarly journals Use of High Hydrostatic Pressure To Inactivate Escherichia coli O157:H7 and Salmonella enterica Internalized within and Adhered to Preharvest Contaminated Green Onions

2012 ◽  
Vol 78 (6) ◽  
pp. 2063-2065 ◽  
Author(s):  
Hudaa Neetoo ◽  
Yingjian Lu ◽  
Changqing Wu ◽  
Haiqiang Chen
Keyword(s):  
Escherichia Coli ◽  
Hydrostatic Pressure ◽  
Plant Growth ◽  
High Hydrostatic Pressure ◽  
Salmonella Enterica ◽  
Escherichia Coli O157 ◽  
Pressure Treatment ◽  
Content Type ◽  
Stems And Leaves

ABSTRACTGreen onions grown in soil and hydroponic medium contaminated withEscherichia coliO157:H7 andSalmonellawere found to take up the pathogens in their roots, bulbs, stems, and leaves. Pressure treatment at 400 to 500 MPa for 2 min at 20 to 40°C eliminated both pathogens that were internalized within green onions during plant growth.

scholarly journals Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

2013 ◽  
Vol 12 (7) ◽  
pp. 990-997 ◽  
Author(s):  
Asaha Suzuki ◽  
Takahiro Mochizuki ◽  
Satoshi Uemura ◽  
Toshiki Hiraki ◽  
Fumiyoshi Abe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Pressure ◽  
Hydrostatic Pressure ◽  
Ubiquitin Ligase ◽  
High Hydrostatic Pressure ◽  
Intracellular Localization ◽  
Normal Growth ◽  
Growth Conditions ◽  
Content Type ◽  
Genes Encoding

ABSTRACT Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1 K29R-K31R -GFP remained. The HPG1-1 (Rsp5 P514T ) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1 , rsp5-ww2 , and bul1 Δ bul2 Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.

scholarly journals Stress-Induced Evolution of Heat Resistance and Resuscitation Speed in Escherichia coli O157:H7 ATCC 43888

2016 ◽  
Vol 82 (22) ◽  
pp. 6656-6663 ◽  
Author(s):  
Elisa Gayán ◽  
Alexander Cambré ◽  
Chris W. Michiels ◽  
Abram Aertsen
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Hydrostatic Pressure ◽  
Foodborne Pathogens ◽  
High Hydrostatic Pressure ◽  
Cross Resistance ◽  
Content Type ◽  
E Coli ◽  
Mild Heat ◽  
Mutant Strains

ABSTRACTThe development of resistance in foodborne pathogens to food preservation techniques is an issue of increasing concern, especially in minimally processed foods where safety relies on hurdle technology. In this context, mild heat can be used in combination with so-called nonthermal processes, such as high hydrostatic pressure (HHP), at lower individual intensities to better retain the quality of the food. However, mild stresses may increase the risk of (cross-)resistance development in the surviving population, which in turn might compromise food safety. In this investigation, we examined the evolution ofEscherichia coliO157:H7 strain ATCC 43888 after recurrent exposure to progressively intensifying mild heat shocks (from 54.0°C to 60.0°C in 0.5°C increments) with intermittent resuscitation and growth of survivors. As such, mutant strains were obtained after 10 cycles of selection with ca. 106-fold higher heat resistance than that for the parental strain at 58.0°C, although this resistance did not extend to temperatures exceeding 60.0°C. Moreover, these mutant strains typically displayed cross-resistance against HHP shock and displayed signs of enhanced RpoS and RpoH activity. Interestingly, additional cycles of selection maintaining the intensity of the heat shock constant (58.5°C) selected for mutant strains in which resuscitation speed, rather than resistance, appeared to be increased. Therefore, it seems that resistance and resuscitation speed are rapidly evolvable traits inE. coliATCC 43888 that can compromise food safety.IMPORTANCEIn this investigation, we demonstrated thatEscherichia coliO157:H7 ATCC 43888 rapidly acquires resistance to mild heat exposure, with this resistance yielding cross-protection to high hydrostatic pressure treatment. In addition, mutants ofE. coliATCC 43888 in which resuscitation speed, rather than resistance, appeared to be improved were selected. As such, both resistance and resuscitation speed seem to be rapidly evolvable traits that can compromise the control of foodborne pathogens in minimal processing strategies, which rely on the efficacy of combined mild preservation stresses for food safety.

scholarly journals Isolation of Highly Heat-Resistant Listeria monocytogenes Variants by Use of a Kinetic Modeling-Based Sampling Scheme

2011 ◽  
Vol 77 (8) ◽  
pp. 2617-2624 ◽  
Author(s):  
Ineke K. H. Van Boeijen ◽  
Christof Francke ◽  
Roy Moezelaar ◽  
Tjakko Abee ◽  
Marcel H. Zwietering
Keyword(s):  
Listeria Monocytogenes ◽  
Hydrostatic Pressure ◽  
Kinetic Modeling ◽  
High Resistance ◽  
High Hydrostatic Pressure ◽  
Heat Inactivation ◽  
Inactivation Kinetics ◽  
Wild Type ◽  
Content Type ◽  
Heat Resistant

ABSTRACTStable high-hydrostatic-pressure (HHP)-resistantListeria monocytogenesLO28 variants were previously isolated and characterized. These HHP variants were also more resistant to heat. In addition, nonlinear heat inactivation kinetics pointed toward the existence of heat-resistant variants, although these could not be isolated so far. In this study, we used kinetic modeling of inactivation curves of two isolated HHP variants and their wild type, and this revealed that the probability of finding resistant variants should depend on the nature of the inactivation treatment and the time of exposure. At specific heat and HHP conditions, resistant LO28 and EGDe variants were indeed isolated. Resistant LO28 variants were even isolated after a heat inactivation at 72°C in milk, and these variants showed high resistance to standard pasteurization conditions. The increased resistance of part of the isolated LO28 and EGDe variants was due to mutations in theirctsRgenes. For the variants whosectsRgenes and upstream regions were not altered, the mechanisms leading to increased resistance remain to be elucidated. This research showed the strength of kinetic modeling in unraveling the causes of nonlinear inactivation and facilitating the isolation of heat-resistantL. monocytogenesvariants.

scholarly journals Involvement of Trichoderma Trichothecenes in the Biocontrol Activity and Induction of Plant Defense-Related Genes

2012 ◽  
Vol 78 (14) ◽  
pp. 4856-4868 ◽  
Author(s):  
M. G. Malmierca ◽  
R. E. Cardoza ◽  
N. J. Alexander ◽  
S. P. McCormick ◽  
R. Hermosa ◽  
...  
Keyword(s):  
Plant Defense ◽  
Gene Disruption ◽  
Biosynthetic Pathway ◽  
Wild Type Strain ◽  
Gene Encoding ◽  
P450 Monooxygenase ◽  
Content Type ◽  
Tomato Seedlings ◽  
Biocontrol Activity ◽  
Genes Encoding

ABSTRACTTrichodermaspecies produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to theirFusariumorthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of theFusarium triclusters. Here we describe the isolation ofTrichoderma arundinaceumIBT 40837 transformants which have a disrupted or silencedtri4gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect oftri4gene disruption and silencing on the expression of othertrigenes. Our results indicate that thetri4gene disruption resulted in a reduced antifungal activity againstBotrytis cinereaandRhizoctonia solaniand also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways againstB. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization ofTrichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction ofT. arundinaceumwithB. cinereaorR. solaniand with tomato seedlings on the expressions of thetrigenes was studied.

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