Root Colonization by Phenazine-1-Carboxamide-Producing Bacterium Pseudomonas chlororaphis PCL1391 Is Essential for Biocontrol of Tomato Foot and Root Rot

The phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of phenazine-1-carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce phenazine-1-carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.

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Flagella-Driven Chemotaxis Towards Exudate Components Is an Important Trait for Tomato Root Colonization by Pseudomonas fluorescens

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.11.1173 ◽

2002 ◽

Vol 15 (11) ◽

pp. 1173-1180 ◽

Cited By ~ 329

Author(s):

Sandra de Weert ◽

Hans Vermeiren ◽

Ine H. M. Mulders ◽

Irene Kuiper ◽

Nico Hendrickx ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Root Exudate ◽

Root Colonization ◽

Chemotactic Response ◽

Root Tip ◽

Wild Type ◽

Tomato Root ◽

Important Trait ◽

Colonizing Ability

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in non-sterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.

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Introduction of the phzH Gene of Pseudomonas chlororaphis PCL1391 Extends the Range of Biocontrol Ability of Phenazine-1-Carboxylic Acid-Producing Pseudomonas spp. Strains

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.8.1006 ◽

2001 ◽

Vol 14 (8) ◽

pp. 1006-1015 ◽

Cited By ~ 116

Author(s):

Thomas F. C. Chin-A-Woeng ◽

Jane E. Thomas-Oates ◽

Ben J. J. Lugtenberg ◽

Guido V. Bloemberg

Keyword(s):

Carboxylic Acid ◽

Root Rot ◽

Single Gene ◽

Pseudomonas Chlororaphis ◽

The Novel ◽

Bacterial Strains ◽

Biocontrol Activity ◽

Tac Promoter ◽

In Trans ◽

First Time

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. s radicislycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2–79 and P. aureofaciens 30–84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2–79 and P. aureofaciens 30–84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.

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Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization

Microbiology ◽

10.1099/mic.0.27362-0 ◽

2004 ◽

Vol 150 (11) ◽

pp. 3889-3897 ◽

Cited By ~ 80

Author(s):

Silvia Capdevila ◽

Francisco M. Martínez-Granero ◽

María Sánchez-Contreras ◽

Rafael Rivilla ◽

Marta Martín

Keyword(s):

Pseudomonas Fluorescens ◽

Root Colonization ◽

Root Tip ◽

Soil Bioremediation ◽

Rich Medium ◽

Wild Type ◽

Biotechnological Application ◽

Flagellar Filament ◽

Micro Organisms ◽

Biocontrol Strain

The ability of plant-associated micro-organisms to colonize and compete in the rhizosphere is specially relevant for the biotechnological application of micro-organisms as inoculants. Pseudomonads are one of the best root colonizers and they are widely used in plant-pathogen biocontrol and in soil bioremediation. This study analyses the motility mechanism of the well-known biocontrol strain Pseudomonas fluorescens F113. A 6·5 kb region involved in the flagellar filament synthesis, containing the fliC, flaG, fliD, fliS, fliT and fleQ genes and part of the fleS gene, was sequenced and mutants in this region were made. Several non-motile mutants affected in the fliC, fliS and fleQ genes, and a fliT mutant with reduced motility properties, were obtained. These mutants were completely displaced from the root tip when competing with the wild-type F113 strain, indicating that the wild-type motility properties are necessary for competitive root colonization. A mutant affected in the flaG gene had longer flagella, but the same motility and colonization properties as the wild-type. However, in rich medium or in the absence of iron limitation, it showed a higher motility, suggesting the possibility of improving competitive root colonization by manipulating the motility processes.

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Galactinol Is a Signaling Component of the Induced Systemic Resistance Caused by Pseudomonas chlororaphis O6 Root Colonization

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-12-1643 ◽

2008 ◽

Vol 21 (12) ◽

pp. 1643-1653 ◽

Cited By ~ 74

Author(s):

Mi Seong Kim ◽

Song Mi Cho ◽

Eun Young Kang ◽

Yang Ju Im ◽

Hoon Hwangbo ◽

...

Keyword(s):

Root Colonization ◽

Induced Systemic Resistance ◽

Systemic Resistance ◽

Suppressive Subtractive Hybridization ◽

Transcriptional Level ◽

Hybridization Technique ◽

Pseudomonas Chlororaphis ◽

Wild Type ◽

Water Control ◽

Tobacco Plants

Root colonization by Pseudomonas chlororaphis O6 in cucumber elicited an induced systemic resistance (ISR) against Corynespora cassiicola. In order to gain insight into O6-mediated ISR, a suppressive subtractive hybridization technique was applied and resulted in the isolation of a cucumber galactinol synthase (CsGolS1) gene. The transcriptional level of CsGolS1 and the resultant galactinol content showed an increase several hours earlier under O6 treatment than in the water control plants following C. cassiicola challenge, whereas no difference was detected in the plants without a pathogen challenge. The CsGolS1-overexpressing transgenic tobacco plants demonstrated constitutive resistance against the pathogens Botrytis cinerea and Erwinia carotovora, and they also showed an increased accumulation in galactinol content. Pharmaceutical application of galactinol enhanced the resistance against pathogen infection and stimulated the accumulation of defense-related gene transcripts such as PR1a, PR1b, and NtACS1 in wild-type tobacco plants. Both the CsGolS1-overexpressing transgenic plants and the galactinol-treated wild-type tobacco plants also demonstrated an increased tolerance to drought and high salinity stresses.

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Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽

10.3390/pathogens10010054 ◽

2021 ◽

Vol 10 (1) ◽

pp. 54

Author(s):

Christine Landlinger ◽

Lenka Tisakova ◽

Vera Oberbauer ◽

Timo Schwebs ◽

Abbas Muhammad ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Bactericidal Activity ◽

High Selectivity ◽

Ex Vivo ◽

Vaginal Microbiome ◽

Wild Type ◽

Wild Type Enzyme ◽

Promising Alternative ◽

First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

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Seed morphology uncovers 1500 years of vine agrobiodiversity before the advent of the Champagne wine

Scientific Reports ◽

10.1038/s41598-021-81787-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Vincent Bonhomme ◽

Jean-Frédéric Terral ◽

Véronique Zech-Matterne ◽

Sarah Ivorra ◽

Thierry Lacombe ◽

...

Keyword(s):

Middle Ages ◽

Climatic Changes ◽

Seed Morphology ◽

Crucial Aspect ◽

Wild Type ◽

Grape Seeds ◽

The Social ◽

Reference Collection ◽

Grape Varieties ◽

First Time

AbstractA crucial aspect of viticulture is finally unveiled as the historical dynamics of its agrobiodiversity are described in the Champagne region for the first time. Outline analyses were carried out to compare the morphology of archaeological grape seeds from Troyes and Reims (first c. AD to fifteenth c. AD) with that of a reference collection of modern seeds, including wild vines and traditional grape varieties, believed to be ancient and characteristic of the French vine heritage. This allows us to document the chronological dynamics of the use of the wild Vitis type and of the diversity of the varieties used, based on morphological disparity. After showing the existence of morphological types corresponding to geographical groups, we highlight a geochronological dynamic. Our results show that the wild type is used throughout the series, up to the Middle Ages. In addition, domestic forms, morphologically related to southern varietal groups, are very early involved in the Champagne grape agrodiversity. The groups corresponding to the typical grape varieties of today do not appear until the second millennium. These previously unsuspected dynamics are discussed in light of the social, societal and climatic changes documented for the period.

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A Novel and Efficient High-Yield Method for Preparing Bacterial Ghosts

Toxins ◽

10.3390/toxins13060420 ◽

2021 ◽

Vol 13 (6) ◽

pp. 420

Author(s):

Yi Ma ◽

Liu Cui ◽

Meng Wang ◽

Qiuli Sun ◽

Kaisheng Liu ◽

...

Keyword(s):

Large Scale ◽

Expression System ◽

High Yield ◽

Wild Type ◽

High Quality ◽

Efficient Protection ◽

Bacterial Ghosts ◽

Cell Envelopes ◽

Extracellular Structures ◽

First Time

Bacterial ghosts (BGs) are empty cell envelopes possessing native extracellular structures without a cytoplasm and genetic materials. BGs are proposed to have significant prospects in biomedical research as vaccines or delivery carriers. The applications of BGs are often limited by inefficient bacterial lysis and a low yield. To solve these problems, we compared the lysis efficiency of the wild-type protein E (EW) from phage ΦX174 and the screened mutant protein E (EM) in the Escherichia coli BL21(DE3) strain. The results show that the lysis efficiency mediated by protein EM was improved. The implementation of the pLysS plasmid allowed nearly 100% lysis efficiency, with a high initial cell density as high as OD600 = 2.0, which was higher compared to the commonly used BG preparation method. The results of Western blot analysis and immunofluorescence indicate that the expression level of protein EM was significantly higher than that of the non-pLysS plasmid. High-quality BGs were observed by SEM and TEM. To verify the applicability of this method in other bacteria, the T7 RNA polymerase expression system was successfully constructed in Salmonella enterica (S. Enterica, SE). A pET vector containing EM and pLysS were introduced to obtain high-quality SE ghosts which could provide efficient protection for humans and animals. This paper describes a novel and commonly used method to produce high-quality BGs on a large scale for the first time.

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Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

RSC Advances ◽

10.1039/c5ra15220h ◽

2015 ◽

Vol 5 (93) ◽

pp. 76040-76047 ◽

Cited By ~ 11

Author(s):

Zhenya Chen ◽

Ye Li ◽

Yue Feng ◽

Liang Chen ◽

Qipeng Yuan

Keyword(s):

Active Site ◽

Simulation Analysis ◽

Docking Simulation ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Proteus Vulgaris ◽

Molecular Docking Simulation ◽

Activity Enhancement ◽

First Time

Arg660 was found as a new active site and Asn795Ala and Trp818Ala mutants showed higher activities than the wild type based on molecular docking simulation analysis for the first time.

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Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

Circulation Research ◽

10.1161/res.109.suppl_1.ap283 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Allen M Andres ◽

Chengqun Huang ◽

Eric P Ratliff ◽

Genaro Hernandez ◽

Pamela Lee ◽

...

Keyword(s):

Ex Vivo ◽

Wild Type ◽

Simulated Ischemia ◽

Selective Targeting ◽

Cardioprotective Effects ◽

Mitochondrial Turnover ◽

First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

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A GacS deficiency does not affectPseudomonas chlororaphisPA23 fitness when growing on canola, in aged batch culture or as a biofilm

Canadian Journal of Microbiology ◽

10.1139/w06-079 ◽

2006 ◽

Vol 52 (12) ◽

pp. 1177-1188 ◽

Cited By ~ 37

Author(s):

N Poritsanos ◽

C Selin ◽

W G.D Fernando ◽

S Nakkeeran ◽

T.R. de Kievit

Keyword(s):

Antifungal Activity ◽

Batch Culture ◽

Hydrogen Cyanide ◽

Fungal Pathogen ◽

Biocontrol Agent ◽

Antifungal Compounds ◽

Pseudomonas Chlororaphis ◽

Wild Type ◽

Biocontrol Activity ◽

Competition Assays

Pseudomonas chlororaphis PA23 is a biocontrol agent that protects against the fungal pathogen Sclerotinia sclerotiorum. Employing transposon mutagenesis, we isolated a gacS mutant that no longer exhibited antifungal activity. Pseudomonas chlororaphis PA23 was previously reported to produce the nonvolatile antibiotics phenazine 1-carboxylic acid and 2-hydroxyphenazine. We report here that PA23 produces additional compounds, including protease, lipase, hydrogen cyanide, and siderophores, that may contribute to its biocontrol ability. In the gacS mutant background, generation of these products was markedly reduced or delayed with the exception of siderophores, which were elevated. Not surprisingly, this mutant was unable to protect canola from disease incited by S. sclerotiorum. The gacS mutant was able to sustain itself in the canola phyllosphere, therefore, the loss of biocontrol activity can be attributed to a reduced production of antifungal compounds and not a declining population size. Competition assays between the mutant and wild type revealed equivalent fitness in aged batch culture; consequently, the gacS mutation did not impart a growth advantage in the stationary phase phenotype. Under minimal nutrient conditions, the gacS-deficient strain produced a tenfold less biofilm than the wild type. However, no difference was observed in the ability of the mutant biofilm to protect cells from lethal antibiotic challenge.Key words: Pseudomonas, biocontrol, gacS, fitness, biofilms.

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