scholarly journals Generation of Enhanced Competitive Root-Tip-Colonizing Pseudomonas Bacteria through Accelerated Evolution

2004 ◽  
Vol 186 (10) ◽  
pp. 3153-3159 ◽  
Author(s):  
Sandra de Weert ◽  
Linda C. Dekkers ◽  
Irene Kuiper ◽  
Guido V. Bloemberg ◽  
Ben J. J. Lugtenberg
Keyword(s):  
Selection Procedure ◽  
Root Tip ◽  
Wild Type ◽  
Enrichment Procedure ◽  
Accelerated Evolution ◽  
Colonization Ability ◽  
Mutant Bank ◽  
Flanking Regions ◽  
Spontaneous Oxidation ◽  
Better Than

ABSTRACT A recently published procedure to enrich for efficient competitive root tip colonizers (I. Kuiper, G. V. Bloemberg, and B. J. J. Lugtenberg, Mol. Plant-Microbe Interact. 14:1197-1205) after bacterization of seeds was applied to isolate efficient competitive root tip colonizers for both the dicotyledenous plant tomato and the monocotyledenous plant grass from a random Tn5luxAB mutant bank of the good root colonizer Pseudomonas fluorescens WCS365. Unexpectedly, the best-colonizing mutant, strain PCL1286, showed a strongly enhanced competitive root-tip-colonizing phenotype. Sequence analyses of the Tn5luxAB flanking regions showed that the transposon had inserted in a mutY homolog. This gene is involved in the repair of A · G mismatches caused by spontaneous oxidation of guanine. We hypothesized that, since the mutant is defective in repairing its mismatches, its cells harbor an increased number of mutations and therefore can adapt faster to the environment of the root system. To test this hypothesis, we constructed another mutY mutant and analyzed its competitive root tip colonization behavior prior to and after enrichment. As a control, a nonmutated wild type was subjected to the enrichment procedure. The results of these analyses showed (i) that the enrichment procedure did not alter the colonization ability of the wild type, (ii) that the new mutY mutant was strongly impaired in its colonization ability, but (iii) that after three enrichment cycles it colonized significantly better than its wild type. Therefore it is concluded that both the mutY mutation and the selection procedure are required to obtain an enhanced root-tip-colonizing mutant.

scholarly journals The sss Colonization Gene of the Tomato-Fusarium oxysporum f. sp. radicis-lycopersici Biocontrol Strain Pseudomonas fluorescens WCS365 Can Improve Root Colonization of Other Wild-type Pseudomonas spp. Bacteria

2000 ◽  
Vol 13 (11) ◽  
pp. 1177-1183 ◽  
Author(s):  
Linda C. Dekkers ◽  
Ine H. M. Mulders ◽  
Claartje C. Phoelich ◽  
Thomas F. C. Chin-A-Woeng ◽  
André H. M. Wijfjes ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Pseudomonas Fluorescens ◽  
Pathogenic Fungus ◽  
Cell Types ◽  
Disease Suppression ◽  
Systemic Resistance ◽  
Root Tip ◽  
Wild Type ◽  
Tomato Root ◽  
Colonization Ability

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a bio-control strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.

scholarly journals Increased Uptake of Putrescine in the Rhizosphere Inhibits Competitive Root Colonization by Pseudomonas fluorescens Strain WCS365

2001 ◽  
Vol 14 (9) ◽  
pp. 1096-1104 ◽  
Author(s):  
Irene Kuiper ◽  
Guido V. Bloemberg ◽  
Sadaf Noreen ◽  
Jane E. Thomas-Oates ◽  
Ben J. J. Lugtenberg
Keyword(s):  
Growth Rate ◽  
Pseudomonas Fluorescens ◽  
Root Exudate ◽  
Root Colonization ◽  
Wild Type ◽  
Tomato Root ◽  
Mutant Strains ◽  
Colonization Ability ◽  
Flanking Regions ◽  
Layer Chromatography

Sequence analysis of the chromosomal Tn5lacZ flanking regions of the Pseudomonas fluorescens WCS365 competitive root colonization mutant PCL1206 showed that the Tn5lacZ is inserted between genes homologous to bioA and potF. The latter gene is the first gene of the potF1F2GHI operon, which codes for a putrescine transport system in Escherichia coli. The position of the Tn5lacZ suggests an effect on the expression of the pot operon. A mutation in the potF1 gene as constructed in PCL1270, however, had no effect on competitive root colonization. The rate of uptake of [1,4-14C]putrescine by cells of mutant PCL1206 appeared to be increased, whereas cells of strain PCL1270 were strongly impaired in the uptake of putrescine. Dansylation of tomato root exudate and subsequent thin-layer chromatography showed the presence of a component with the same Rf value as dansyl-putrescine, which was identified as dansyl-putrescine by mass spectrometric analyses. Other polyamines such as spermine and spermidine were not detected in the root exudate. Growth of mutant strains, either alone or in competition with the wild type, was tested in media containing putrescine, spermine, or spermidine as the sole nitrogen source. The results show that mutant PCL1206 is strongly impaired in growth on putrescine and slightly impaired on spermine and spermidine. The presence of the polyamines had a similar effect on the growth rate of strain PCL1270 in the presence of putrescine but a less severe effect in the presence of spermine and spermidine. We conclude that an increased rate of putrescine uptake has a bacteriostatic effect on Pseudomonas spp. cells. We have shown that putrescine is an important tomato root exudate component and that root-colonizing pseudomonads must carefully regulate their rate of uptake because increased uptake causes a decreased growth rate and, therefore, a decreased competitive colonization ability.

scholarly journals Putative Surface Proteins Encoded within a Novel Transferable Locus Confer a High-Biofilm Phenotype to Enterococcus faecalis

2006 ◽  
Vol 188 (6) ◽  
pp. 2063-2072 ◽  
Author(s):  
Preeti M. Tendolkar ◽  
Arto S. Baghdayan ◽  
Nathan Shankar
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Insertional Mutagenesis ◽  
Surface Proteins ◽  
Conjugative Plasmid ◽  
Sequence Information ◽  
Wild Type ◽  
Abiotic Surfaces ◽  
Mutant Bank

ABSTRACT Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.

scholarly journals Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters

2007 ◽  
Vol 73 (18) ◽  
pp. 5711-5715 ◽  
Author(s):  
Sung Kuk Lee ◽  
Howard H. Chou ◽  
Brian F. Pfleger ◽  
Jack D. Newman ◽  
Yasuo Yoshikuni ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Cross Talk ◽  
Biological Systems ◽  
Wild Type ◽  
Dimerization Domain ◽  
Expression Levels ◽  
Inducible Promoters ◽  
C Terminus ◽  
Increased Sensitivity ◽  
Better Than

ABSTRACT Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (PBAD) system that is more compatible with IPTG (isopropyl-β-d-1-thiogalactopyranoside) induction of a lactose-inducible (Plac) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.

scholarly journals Strigolactones Control Root System Architecture and Tip Anatomy in Solanum lycopersicum L. Plants under P Starvation

Plants ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 612 ◽  
Author(s):  
Veronica Santoro ◽  
Michela Schiavon ◽  
Francesco Gresta ◽  
Andrea Ertani ◽  
Francesca Cardinale ◽  
...  
Keyword(s):  
Solanum Lycopersicum ◽  
Lateral Root ◽  
Primary Root ◽  
Root Traits ◽  
Root System Architecture ◽  
Root Tip ◽  
Root Number ◽  
Wild Type ◽  
Solanum Lycopersicum L ◽  
P Supply

The hormones strigolactones accumulate in plant roots under phosphorus (P) shortage, inducing variations in plant phenotype. In this study, we aimed at understanding whether strigolactones control morphological and anatomical changes in tomato (Solanum lycopersicum L.) roots under varying P supply. Root traits were evaluated in wild-type seedlings grown in high vs. low P, with or without exogenous strigolactones, and in wild-type and strigolactone-depleted plants grown first under high vs. no P, and then under high vs. no P after acclimation on low P. Exogenous strigolactones stimulated primary root and lateral root number under low P. Root growth was reduced in strigolactone-depleted plants maintained under continuous P deprivation. Total root and root hair length, lateral root number and root tip anatomy were impaired by low strigolactone biosynthesis in plants grown under low P or transferred from low to no P. Under adequate P conditions, root traits of strigolactone-depleted and wild-type plants were similar. Concluding, our results indicate that strigolactones (i) control macro- and microscopic changes of root in tomato depending on P supply; and (ii) do not affect root traits significantly when plants are supplemented with adequate P, but are needed for acclimation to no P and typical responses to low P.

scholarly journals Nitrate Induction of Primary Root Growth Requires Cytokinin Signaling in Arabidopsis thaliana

2019 ◽  
Vol 61 (2) ◽  
pp. 342-352 ◽  
Author(s):  
Pamela A Naulin ◽  
Grace I Armijo ◽  
Andrea S Vega ◽  
Karem P Tamayo ◽  
Diana E Gras ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Division ◽  
Root Growth ◽  
Primary Root ◽  
Growth Arrest ◽  
Root Tip ◽  
Wild Type ◽  
Cytokinin Signaling ◽  
Control Growth ◽  
Primary Root Growth

Abstract Nitrate can act as a potent signal to control growth and development in plants. In this study, we show that nitrate is able to stimulate primary root growth via increased meristem activity and cytokinin signaling. Cytokinin perception and biosynthesis mutants displayed shorter roots as compared with wild-type plants when grown with nitrate as the only nitrogen source. Histological analysis of the root tip revealed decreased cell division and elongation in the cytokinin receptor double mutant ahk2/ahk4 as compared with wild-type plants under a sufficient nitrate regime. Interestingly, a nitrate-dependent root growth arrest was observed between days 5 and 6 after sowing. Wild-type plants were able to recover from this growth arrest, while cytokinin signaling or biosynthesis mutants were not. Transcriptome analysis revealed significant changes in gene expression after, but not before, this transition in contrasting genotypes and nitrate regimes. We identified genes involved in both cell division and elongation as potentially important for primary root growth in response to nitrate. Our results provide evidence linking nitrate and cytokinin signaling for the control of primary root growth in Arabidopsis thaliana.

scholarly journals A Mutation in the luxS Gene Influences Listeria monocytogenes Biofilm Formation

2006 ◽  
Vol 72 (8) ◽  
pp. 5653-5658 ◽  
Author(s):  
Shlomo Sela ◽  
Shmulik Frank ◽  
Eddy Belausov ◽  
Riky Pinto
Keyword(s):  
Listeria Monocytogenes ◽  
Biofilm Formation ◽  
Parent Strain ◽  
Vibrio Harveyi ◽  
Reporter Strain ◽  
Luxs Gene ◽  
Wild Type ◽  
Autoinducer 2 ◽  
Wild Type Phenotype ◽  
Better Than

ABSTRACT Using a Vibrio harveyi reporter strain, we demonstrated that Listeria monocytogenes secretes a functional autoinducer 2 (AI-2)-like signal. A luxS-deficient mutant produced a denser biofilm and attached to a glass surface 19-fold better than the parent strain. Exogenous AI-2 failed to restore the wild-type phenotype to the mutant. It seems that an intact luxS gene is associated with repression of components required for attachment and biofilm formation.

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

2000 ◽  
Vol 13 (12) ◽  
pp. 1340-1345 ◽  
Author(s):  
Thomas F. C. Chin-A-Woeng ◽  
Guido V. Bloemberg ◽  
Ine H. M. Mulders ◽  
Linda C. Dekkers ◽  
Ben J. J. Lugtenberg
Keyword(s):  
Root Rot ◽  
Root Colonization ◽  
Root Tip ◽  
Pseudomonas Chlororaphis ◽  
Wild Type ◽  
Animal Tissues ◽  
Tomato Seedling ◽  
Antifungal Metabolites ◽  
Seedling Inoculation ◽  
First Time

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.

scholarly journals Equal Force Recovery in Dysferlin-Deficient and Wild-Type Muscles Following Saponin Exposure

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Piming Zhao ◽  
Li Xu ◽  
Younss Ait-Mou ◽  
Pieter P. de Tombe ◽  
Renzhi Han
Keyword(s):  
Muscular Dystrophy ◽  
Extensor Digitorum Longus ◽  
Membrane Damage ◽  
Force Production ◽  
Slow Muscle ◽  
Wild Type ◽  
Tetanic Force ◽  
Induced Membrane ◽  
Force Recovery ◽  
Better Than

Dysferlin plays an important role in repairing membrane damage elicited by laser irradiation, and dysferlin deficiency causes muscular dystrophy and associated cardiomyopathy. Proteins such as perforin, complement component C9, and bacteria-derived cytolysins, as well as the natural detergent saponin, can form large pores on the cell membrane via complexation with cholesterol. However, it is not clear whether dysferlin plays a role in repairing membrane damage induced by pore-forming reagents. In this study, we observed that dysferlin-deficient muscles recovered the tetanic force production to the same extent as their WT counterparts following a 5-min saponin exposure (50 μg/mL). Interestingly, the slow soleus muscles recovered significantly better than the fastextensor digitorum longus(EDL) muscles. Our data suggest that dysferlin is unlikely involved in repairing saponin-induced membrane damage and that the slow muscle is more efficient than the fast muscle in repairing such damage.

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