scholarly journals Essential Genes forIn VitroGrowth of the Endophyte Herbaspirillum seropedicae SmR1 as Revealed by Transposon Insertion Site Sequencing

2016 ◽  
Vol 82 (22) ◽  
pp. 6664-6671 ◽  
Author(s):  
Federico Rosconi ◽  
Stefan P. W. de Vries ◽  
Abiyad Baig ◽  
Elena Fabiano ◽  
Andrew J. Grant
Keyword(s):  
Plant Growth ◽  
Massively Parallel Sequencing ◽  
Focal Point ◽  
Insertion Site ◽  
Essential Genes ◽  
Plant Interactions ◽  
Herbaspirillum Seropedicae ◽  
Content Type ◽  
Functional Genomic Analysis ◽  
Genetic Mechanisms

ABSTRACTThe interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil.Herbaspirillum seropedicaestrain SmR1, belonging to the betaproteobacteria, is an endophyte that colonizes crops, including rice, maize, sugarcane, and sorghum. Different approaches have revealed genes and pathways regulated during the interactions ofH. seropedicaewith its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools. Here we successfully employed a combination ofin vivohigh-densitymarinerTn mutagenesis and targeted Tn insertion site sequencing (Tn-seq) inH. seropedicaeSmR1. The analysis of multiple gene-saturating Tn libraries revealed that 395 genes are essential for the growth ofH. seropedicaeSmR1 in tryptone-yeast extract medium. A comparative analysis with the Database of Essential Genes (DEG) showed that 25 genes are uniquely essential inH. seropedicaeSmR1. The Tn mutagenesis protocol developed and the gene-saturating Tn libraries generated will facilitate elucidation of the genetic mechanisms of theH. seropedicaeendophytic lifestyle.IMPORTANCEA focal point in the study of endophytes is the development of effective biofertilizers that could help to reduce the input of agrochemicals in croplands. Besides the ability to promote plant growth, a good biofertilizer should be successful in colonizing its host and competing against the native microbiota. By using a systematic Tn-based gene-inactivation strategy and massively parallel sequencing of Tn insertion sites (Tn-seq), it is possible to study the fitness of thousands of Tn mutants in a single experiment. We have applied the combination of these techniques to the plant-growth-promoting endophyteHerbaspirillum seropedicaeSmR1. The Tn mutant libraries generated will enable studies into the genetic mechanisms ofH. seropedicae-plant interactions. The approach that we have taken is applicable to other plant-interacting bacteria.

scholarly journals Importance of Poly-3-Hydroxybutyrate Metabolism to the Ability ofHerbaspirillum seropedicaeTo Promote Plant Growth

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Luis Paulo Silveira Alves ◽  
Fernanda Plucani do Amaral ◽  
Daewon Kim ◽  
Maritza Todo Bom ◽  
Manuel Piñero Gavídia ◽  
...  
Keyword(s):  
Plant Growth ◽  
Root Colonization ◽  
Normal Growth ◽  
Setaria Viridis ◽  
Low Oxygen ◽  
Herbaspirillum Seropedicae ◽  
Content Type ◽  
Stimulate Plant Growth ◽  
Microaerobic Conditions

ABSTRACTHerbaspirillum seropedicaeis an endophytic bacterium that establishes an association with a variety of plants, such as rice, corn, and sugarcane, and can significantly increase plant growth.H. seropedicaeproduces polyhydroxybutyrate (PHB), stored in the form of insoluble granules. Little information is available on the possible role of PHB in bacterial root colonization or in plant growth promotion. To investigate whether PHB is important for the association ofH. seropedicaewith plants, we inoculated roots ofSetaria viridiswithH. seropedicaestrain SmR1 and mutants defective in PHB production (ΔphaP1, ΔphaP1ΔphaP2, ΔphaC1, and ΔphaR) or mobilization (ΔphaZ1ΔphaZ2). The strains producing large amounts of PHB colonized roots, significantly increasing root area and the number of lateral roots compared to those of PHB-negative strains.H. seropedicaegrows under microaerobic conditions, which can be found in the rhizosphere. When grown under low-oxygen conditions, only the parental strain and ΔphaP2mutant exhibited normal growth. The lack of normal growth under low oxygen correlated with the inability to stimulate plant growth, although there was no effect on the level of root colonization. The data suggest that PHB is produced in the root rhizosphere and plays a role in maintaining normal metabolism under microaerobic conditions. To confirm this, we screened for green fluorescent protein (GFP) expression under the control of theH. seropedicaepromoters of the PHA synthase and PHA depolymerase genes in the rhizosphere. PHB synthesis is active on the root surface and later PHB depolymerase expression is activated.IMPORTANCEThe application of bacteria as plant growth promoters is a sustainable alternative to mitigate the use of chemical fertilization in agriculture, reducing negative economic and environmental impacts. Several plant growth-promoting bacteria synthesize and accumulate the intracellular polymer polyhydroxybutyrate (PHB). However, the role of PHB in plant-bacterium interactions is poorly understood. In this study, applying the C4 model grassSetaria viridisand several mutants in the PHB metabolism of the endophyteHerbaspirillum seropedicaeyielded new findings on the importance of PHB for bacterial colonization ofS. viridisroots. Taken together, the results show that deletion of genes involved in the synthesis and degradation of PHB reduced the ability of the bacteria to enhance plant growth but with little effect on overall root colonization. The data suggest that PHB metabolism likely plays an important role in supporting specific metabolic routes utilized by the bacteria to stimulate plant growth.

scholarly journals Genome-Wide Saturation Mutagenesis of Burkholderia pseudomallei K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Madeleine G. Moule ◽  
Claudia M. Hemsley ◽  
Qihui Seet ◽  
José Afonso Guerra-Assunção ◽  
Jiali Lim ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Treatment Options ◽  
Insertion Site ◽  
Essential Genes ◽  
Natural Resistance ◽  
Saturation Mutagenesis ◽  
Lethal Gene ◽  
Content Type ◽  
Genome Wide ◽  
A Genome

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.

scholarly journals Diverse Bacterial Genes Modulate Plant Root Association by Beneficial Bacteria

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e03078-20
Author(s):  
Fernanda Plucani do Amaral ◽  
Thalita Regina Tuleski ◽  
Vania Carla Silva Pankievicz ◽  
Ryan A. Melnyk ◽  
Adam P. Arkin ◽  
...  
Keyword(s):  
Plant Growth ◽  
Root Colonization ◽  
Transposon Mutagenesis ◽  
Plant Growth Promoting Bacteria ◽  
Setaria Viridis ◽  
Herbaspirillum Seropedicae ◽  
Content Type ◽  
Plant Growth Promoting ◽  
Growth Promoting ◽  
Bacterial Genes

ABSTRACTThe plant rhizosphere harbors a diverse population of microorganisms, including beneficial plant growth-promoting bacteria (PGPB), that colonize plant roots and enhance growth and productivity. In order to specifically define bacterial traits that contribute to this beneficial interaction, we used high-throughput transposon mutagenesis sequencing (TnSeq) in two model root-bacterium systems associated with Setaria viridis: Azoarcus olearius DQS4T and Herbaspirillum seropedicae SmR1. This approach identified ∼100 significant genes for each bacterium that appeared to confer a competitive advantage for root colonization. Most of the genes identified specifically in A. olearius encoded metabolism functions, whereas genes identified in H. seropedicae were motility related, suggesting that each strain requires unique functions for competitive root colonization. Genes were experimentally validated by site-directed mutagenesis, followed by inoculation of the mutated bacteria onto S. viridis roots individually, as well as in competition with the wild-type strain. The results identify key bacterial functions involved in iron uptake, polyhydroxybutyrate metabolism, and regulation of aromatic metabolism as important for root colonization. The hope is that by improving our understanding of the molecular mechanisms used by PGPB to colonize plants, we can increase the adoption of these bacteria in agriculture to improve the sustainability of modern cropping systems.IMPORTANCE There is growing interest in the use of associative, plant growth-promoting bacteria (PGPB) as biofertilizers to serve as a sustainable alternative for agriculture application. While a variety of mechanisms have been proposed to explain bacterial plant growth promotion, the molecular details of this process remain unclear. The current research supports the idea that PGPB use in agriculture will be promoted by gaining more knowledge as to how these bacteria colonize plants, promote growth, and do so consistently. Specifically, the research seeks to identify those bacterial genes involved in the ability of two, PGPB strains, Azoarcus olearius and Herbaspirillum seropedicae, to colonize the roots of the C4 model grass Setaria viridis. Applying a transposon mutagenesis (TnSeq) approach, we assigned phenotypes and function to genes that affect bacterial competitiveness during root colonization. The results suggest that each bacterial strain requires unique functions for root colonization but also suggests that a few, critical functions are needed by both bacteria, pointing to some common mechanisms. The hope is that such information can be exploited to improve the use and performance of PGPB in agriculture.

scholarly journals Identification of Host Adaptation Genes in Extraintestinal Pathogenic Escherichia coli during Infection in Different Hosts

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Haobo Zhang ◽  
Xuhua Chen ◽  
Lisa K. Nolan ◽  
Wei Zhang ◽  
Ganwu Li
Keyword(s):  
Escherichia Coli ◽  
Host Specificity ◽  
Host Species ◽  
Insertion Site ◽  
Systemic Infection ◽  
Gene Clusters ◽  
Essential Genes ◽  
Species Barrier ◽  
Content Type ◽  
Systemic Infections

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) is an important human and animal pathogen. Despite the apparent similarities in their known virulence attributes, some ExPEC strains can cross the host species barrier and present a zoonotic potential, whereas other strains exhibit host specificity, suggesting the existence of unknown mechanisms that remain to be identified. We applied a transposon-directed insertion site sequencing (TraDIS) strategy to investigate the ExPEC XM strain, which is capable of crossing the host species barrier, and to screen for virulence-essential genes in both mammalian (mouse) and avian (duck) models of E. coli-related septicemia. We identified 151 genes essential for systemic infection in both mammalian and avian models, 97 required only in the mammalian model, and 280 required only in the avian model. Ten genes/gene clusters were selected for further validation, and their contributions to ExPEC virulence in both mammalian and avian models or mammalian- or avian-only models were confirmed by animal tests. This represents the first comprehensive genome-wide analysis of virulence-essential genes required for systemic infections in two different host species and provides a further comprehensive understanding of ExPEC-related virulence, host specificity, and adaptation.

scholarly journals Complete Genome Sequences of Two Plant Growth-Inhibiting Bacteria, Acinetobacter ursingii M3 and Asticcacaulis excentricus M6, Isolated from Duckweed (Lemna minor)

2018 ◽  
Vol 7 (12) ◽  
Author(s):  
Hidehiro Ishizawa ◽  
Masashi Kuroda ◽  
Daisuke Inoue ◽  
Michihiko Ike
Keyword(s):  
Plant Growth ◽  
Complete Genome ◽  
Lemna Minor ◽  
Genome Sequences ◽  
Content Type ◽  
Pacbio Rs Ii ◽  
Growth Inhibiting

Acinetobacter ursingii M3 and Asticcacaulis excentricus M6 are plant growth-inhibiting bacteria that reduce the yield of the duckweed Lemna minor. We report here the complete genome sequences of A. ursingii M3 and A. excentricus M6, sequenced using the PacBio RS II platform.

scholarly journals Genetic Mechanisms behind the Spread of Reduced Susceptibility to Azithromycin in Shigella Strains Isolated from Men Who Have Sex with Men in Québec, Canada

2018 ◽  
Vol 63 (2) ◽  
pp. e01679-18 ◽  
Author(s):  
Khadidja Yousfi ◽  
Christiane Gaudreau ◽  
Pierre A. Pilon ◽  
Brigitte Lefebvre ◽  
Matthew Walker ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Shigella Flexneri ◽  
Complete Sequence ◽  
Shigella Sonnei ◽  
Content Type ◽  
Genetic Mechanisms ◽  
Sex With Men

ABSTRACT We analyzed 254 Shigella species isolates collected in Québec, Canada, during 2013 and 2014. Overall, 23.6% of isolates showed reduced susceptibility to azithromycin (RSA) encoded by mphA (11.6%), ermB (1.7%), or both genes (86.7%). Shigella strains with RSA were mostly isolated from men who have sex with men (68.8% or higher) from the Montreal region. A complete sequence analysis of six selected plasmids from Shigella sonnei and different serotypes of Shigella flexneri emphasized the role of IS26 in the dissemination of RSA.

Digging deep? Digging more? A research on the influence of corporate CSR theme consistency strategy

2018 ◽  
Vol 1 (1) ◽  
pp. 76-93 ◽  
Author(s):  
Yang Zhang ◽  
Xuhui Wang ◽  
Yingying Shen
Keyword(s):  
Communication Channel ◽  
Information Dissemination ◽  
Focal Point ◽  
Social Programs ◽  
Business Practices ◽  
Content Type ◽  
Moderation Effect ◽  
Corporate Social ◽  
And Behavior ◽  
Selection Of

PurposeAs the focal point of both academic studies and business practices, the theme strategy of corporate social responsibility (CSR) arouses wide attention. However, extant studies concentrate more on the selection of the theme of CSR activities, such as the fitness between CSR activities and the core business, thus largely neglecting the consistency of the theme. The purpose of this paper is to determine whether the enterprise should adopt a consistent theme strategy or should participate in different social programs, and how do customers response to the lack of studies in different theme-consistent strategies.Design/methodology/approachIn this paper, two progressive experiments are performed. The purpose of study 1 is to examine the influence of theme consistency on consumers’ CSR association and how consumers’ attribution to corporation motivation mediates such impacts. The purpose for study 2 is to examine whether information dissemination channels and cooperation with public organization could affect the influence of theme consistency strategy.FindingsThe significant influences of theme consistency on consumer CSR association was demonstrated, and consumer’s perceived motivation of CSR was found to play the mediation role. Moreover, the moderation effect of the communication channel of CSR information was found to be important to strengthen the influence of the theme-consistent strategy.Originality/valueThis paper not only demonstrates the influence of theme consistency, but also explains how theme consistency influences consumers’ attitude and behavior. It enriches the study on the antecedent variables of consumers’ attribution to corporate motivation.

scholarly journals Bacillus velezensisWall Teichoic Acids Are Required for Biofilm Formation and Root Colonization

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Zhihui Xu ◽  
Huihui Zhang ◽  
Xinli Sun ◽  
Yan Liu ◽  
Wuxia Yan ◽  
...  
Keyword(s):  
Plant Growth ◽  
Biofilm Formation ◽  
Root Colonization ◽  
Molecular Networks ◽  
Model Organisms ◽  
Bacillus Velezensis ◽  
Content Type ◽  
Plant Growth Promoting ◽  
Colonization Process ◽  
Growth Promoting

ABSTRACTRhizosphere colonization by plant growth-promoting rhizobacteria (PGPR) along plant roots facilitates the ability of PGPR to promote plant growth and health. Thus, an understanding of the molecular mechanisms of the root colonization process by plant-beneficialBacillusstrains is essential for the use of these strains in agriculture. Here, we observed that ansfpgene mutant of the plant growth-promoting rhizobacteriumBacillus velezensisSQR9 was unable to form normal biofilm architecture, and differential protein expression was observed by proteomic analysis. A minor wall teichoic acid (WTA) biosynthetic protein, GgaA, was decreased over 4-fold in the Δsfpmutant, and impairment of theggaAgene postponed biofilm formation and decreased cucumber root colonization capabilities. In addition, we provide evidence that the major WTA biosynthetic enzyme GtaB is involved in both biofilm formation and root colonization. The deficiency in biofilm formation of the ΔgtaBmutant may be due to an absence of UDP-glucose, which is necessary for the synthesis of biofilm matrix exopolysaccharides (EPS). These observations provide insights into the root colonization process by a plant-beneficialBacillusstrain, which will help improve its application as a biofertilizer.IMPORTANCEBacillus velezensisis a Gram-positive plant-beneficial bacterium which is widely used in agriculture. Additionally,Bacillusspp. are some of the model organisms used in the study of biofilms, and as such, the molecular networks and regulation systems of biofilm formation are well characterized. However, the molecular processes involved in root colonization by plant-beneficialBacillusstrains remain largely unknown. Here, we showed that WTAs play important roles in the plant root colonization process. The loss of thegtaBgene affects the ability ofB. velezensisSQR9 to sense plant polysaccharides, which are important environmental cues that trigger biofilm formation and colonization in the rhizosphere. This knowledge provides new insights into theBacillusroot colonization process and can help improve our understanding of plant-rhizobacterium interactions.

scholarly journals Biochemical and Genetic Bases of Indole-3-Acetic Acid (Auxin Phytohormone) Degradation by the Plant-Growth-Promoting Rhizobacterium Paraburkholderia phytofirmans PsJN

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Raúl Donoso ◽  
Pablo Leiva-Novoa ◽  
Ana Zúñiga ◽  
Tania Timmermann ◽  
Gonzalo Recabarren-Gajardo ◽  
...  
Keyword(s):  
Energy Source ◽  
Gene Regulation ◽  
Acetic Acid ◽  
Plant Growth ◽  
Plant Hormone ◽  
Content Type ◽  
Gene Functions ◽  
Plant Growth Promoting ◽  
Growth Promoting ◽  
Indole 3 Acetic Acid

ABSTRACT Several bacteria use the plant hormone indole-3-acetic acid (IAA) as a sole carbon and energy source. A cluster of genes (named iac) encoding IAA degradation has been reported in Pseudomonas putida 1290, but the functions of these genes are not completely understood. The plant-growth-promoting rhizobacterium Paraburkholderia phytofirmans PsJN harbors iac gene homologues in its genome, but with a different gene organization and context than those of P. putida 1290. The iac gene functions enable P. phytofirmans to use IAA as a sole carbon and energy source. Employing a heterologous expression system approach, P. phytofirmans iac genes with previously undescribed functions were associated with specific biochemical steps. In addition, two uncharacterized genes, previously unreported in P. putida and found to be related to major facilitator and tautomerase superfamilies, are involved in removal of an IAA metabolite called dioxindole-3-acetate. Similar to the case in strain 1290, IAA degradation proceeds through catechol as intermediate, which is subsequently degraded by ortho-ring cleavage. A putative two-component regulatory system and a LysR-type regulator, which apparently respond to IAA and dioxindole-3-acetate, respectively, are involved in iac gene regulation in P. phytofirmans. These results provide new insights about unknown gene functions and complex regulatory mechanisms in IAA bacterial catabolism. IMPORTANCE This study describes indole-3-acetic acid (auxin phytohormone) degradation in the well-known betaproteobacterium P. phytofirmans PsJN and comprises a complete description of genes, some of them with previously unreported functions, and the general basis of their gene regulation. This work contributes to the understanding of how beneficial bacteria interact with plants, helping them to grow and/or to resist environmental stresses, through a complex set of molecular signals, in this case through degradation of a highly relevant plant hormone.

scholarly journals Genome Sequencing of Pantoea agglomerans C1 Provides Insights into Molecular and Genetic Mechanisms of Plant Growth-Promotion and Tolerance to Heavy Metals

2020 ◽  
Vol 8 (2) ◽  
pp. 153 ◽  
Author(s):  
Francesca Luziatelli ◽  
Anna Grazia Ficca ◽  
Mariateresa Cardarelli ◽  
Francesca Melini ◽  
Andrea Cavalieri ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Plant Growth ◽  
Plant Growth Promotion ◽  
Plant Pathogens ◽  
Growth Promotion ◽  
Pantoea Agglomerans ◽  
Toxic Heavy Metals ◽  
Circular Chromosome ◽  
Plant Growth Promoting ◽  
Genetic Mechanisms

Distinctive strains of Pantoea are used as soil inoculants for their ability to promote plant growth. Pantoea agglomerans strain C1, previously isolated from the phyllosphere of lettuce, can produce indole-3-acetic acid (IAA), solubilize phosphate, and inhibit plant pathogens, such as Erwinia amylovora. In this paper, the complete genome sequence of strain C1 is reported. In addition, experimental evidence is provided on how the strain tolerates arsenate As (V) up to 100 mM, and on how secreted metabolites like IAA and siderophores act as biostimulants in tomato cuttings. The strain has a circular chromosome and two prophages for a total genome of 4,846,925-bp, with a DNA G+C content of 55.2%. Genes related to plant growth promotion and biocontrol activity, such as those associated with IAA and spermidine synthesis, solubilization of inorganic phosphate, acquisition of ferrous iron, and production of volatile organic compounds, siderophores and GABA, were found in the genome of strain C1. Genome analysis also provided better understanding of the mechanisms underlying strain resistance to multiple toxic heavy metals and transmission of these genes by horizontal gene transfer. Findings suggested that strain C1 exhibits high biotechnological potential as plant growth-promoting bacterium in heavy metal polluted soils.

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