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 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 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 Salt-Induced Stress Stimulates a Lipoteichoic Acid-Specific Three-Component Glycosylation System inStaphylococcus aureus

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Kelvin Kho ◽  
Timothy C. Meredith
Keyword(s):  
Staphylococcus Aureus ◽  
Sigma Factor ◽  
Cell Envelope ◽  
Normal Growth ◽  
Lipoteichoic Acid ◽  
Growth Conditions ◽  
Growth Media ◽  
Alternative Sigma Factor ◽  
Content Type

ABSTRACTLipoteichoic acid (LTA) inStaphylococcus aureusis a poly-glycerophosphate polymer anchored to the outer surface of the cell membrane. LTA has numerous roles in cell envelope physiology, including regulating cell autolysis, coordinating cell division, and adapting to environmental growth conditions. LTA is often further modified with substituents, includingd-alanine and glycosyl groups, to alter cellular function. While the genetic determinants ofd-alanylation have been largely defined, the route of LTA glycosylation and its role in cell envelope physiology have remained unknown, in part due to the low levels of basal LTA glycosylation inS. aureus. We demonstrate here thatS. aureusutilizes a membrane-associated three-component glycosylation system composed of an undecaprenol (Und)N-acetylglucosamine (GlcNAc) charging enzyme (CsbB; SAOUHSC_00713), a putative flippase to transport loaded substrate to the outside surface of the cell (GtcA; SAOUHSC_02722), and finally an LTA-specific glycosyltransferase that adds α-GlcNAc moieties to LTA (YfhO; SAOUHSC_01213). We demonstrate that this system is specific for LTA with no cross recognition of the structurally similar polyribitol phosphate containing wall teichoic acids. We show that while wild-typeS. aureusLTA has only a trace of GlcNAcylated LTA under normal growth conditions, amounts are raised upon either overexpressing CsbB, reducing endogenousd-alanylation activity, expressing the cell envelope stress responsive alternative sigma factor SigB, or by exposure to environmental stress-inducing culture conditions, including growth media containing high levels of sodium chloride.IMPORTANCEThe role of glycosylation in the structure and function ofStaphylococcus aureuslipoteichoic acid (LTA) is largely unknown. By defining key components of the LTA three-component glycosylation pathway and uncovering stress-induced regulation by the alternative sigma factor SigB, the role ofN-acetylglucosamine tailoring during adaptation to environmental stresses can now be elucidated. As thedltand glycosylation pathways compete for the same sites on LTA and induction of glycosylation results in decreasedd-alanylation, the interplay between the two modification systems holds implications for resistance to antibiotics and antimicrobial peptides.

scholarly journals Examining the Effects of the Nitrogen Environment on Growth and N2-Fixation of Endophytic Herbaspirillum seropedicae in Maize Seedlings by Applying 11C Radiotracing

2021 ◽  
Vol 9 (8) ◽  
pp. 1582
Author(s):  
Spenser Waller ◽  
Stacy L. Wilder ◽  
Michael J. Schueller ◽  
Alexandra B. Housh ◽  
Stephanie Scott ◽  
...  
Keyword(s):  
Plant Growth ◽  
Root Colonization ◽  
Growth Promotion ◽  
Nitrogenase Activity ◽  
Growth Conditions ◽  
Significant Inhibition ◽  
Cereal Crops ◽  
Herbaspirillum Seropedicae ◽  
Promote Plant Growth ◽  
Biological Nitrogen

Herbaspirillum seropedicae, as an endophyte and prolific root colonizer of numerous cereal crops, occupies an important ecological niche in agriculture because of its ability to promote plant growth and potentially improve crop yield. More importantly, there exists the untapped potential to harness its ability, as a diazotroph, to fix atmospheric N2 as an alternative nitrogen resource to synthetic fertilizers. While mechanisms for plant growth promotion remain controversial, especially in cereal crops, one irrefutable fact is these microorganisms rely heavily on plant-borne carbon as their main energy source in support of their own growth and biological functions. Biological nitrogen fixation (BNF), a microbial function that is reliant on nitrogenase enzyme activity, is extremely sensitive to the localized nitrogen environment of the microorganism. However, whether internal root colonization can serve to shield the microorganisms and de-sensitize nitrogenase activity to changes in the soil nitrogen status remains unanswered. We used RAM10, a GFP-reporting strain of H. seropedicae, and administered radioactive 11CO2 tracer to intact 3-week-old maize leaves and followed 11C-photosynthates to sites within intact roots where actively fluorescing microbial colonies assimilated the tracer. We examined the influence of administering either 1 mM or 10 mM nitrate during plant growth on microbial demands for plant-borne 11C. Nitrogenase activity was also examined under the same growth conditions using the acetylene reduction assay. We found that plant growth under low nitrate resulted in higher nitrogenase activity as well as higher microbial demands for plant-borne carbon than plant growth under high nitrate. However, carbon availability was significantly diminished under low nitrate growth due to reduced host CO2 fixation and reduced allocation of carbon resources to the roots. This response of the host caused significant inhibition of microbial growth. In summary, internal root colonization did little to shield these endophytic microorganisms from the nitrogen environment.

scholarly journals Potassium Uptake Modulates Staphylococcus aureus Metabolism

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Casey M. Gries ◽  
Marat R. Sadykov ◽  
Logan L. Bulock ◽  
Sujata S. Chaudhari ◽  
Vinai C. Thomas ◽  
...  
Keyword(s):  
Carbon Flux ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Normal Growth ◽  
Cytoplasmic Ph ◽  
Carbon Utilization ◽  
Content Type ◽  
Alkaline Conditions ◽  
Green Fluorescent

ABSTRACT Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization. As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K+) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K+ uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K+ deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K+ uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization.

scholarly journals Increase in Campylobacter jejuni Invasion of Intestinal Epithelial Cells under Low-Oxygen Coculture Conditions That Reflect theIn VivoEnvironment

2012 ◽  
Vol 80 (5) ◽  
pp. 1690-1698 ◽  
Author(s):  
Dominic C. Mills ◽  
Ozan Gundogdu ◽  
Abdi Elmi ◽  
Mona Bajaj-Elliott ◽  
Peter W. Taylor ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Model System ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Low Oxygen ◽  
Aerobic Conditions ◽  
Content Type ◽  
Microaerobic Conditions

ABSTRACTCampylobacter jejuniinfection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. WhileC. jejuniinfection can be reproducedin vitrousing intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performingin vitroassays under atmospheric oxygen conditions neither is optimal for microaerophilicC. jejuninor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing anin vitrosystem that closely mimicsin vivoconditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellularC. jejuni11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotileC. jejuni11168HrpoNmutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent thein vivosituation and will allow novel insights intoC. jejunipathogenic mechanisms.

scholarly journals Duckweed roots are dispensable and are on a trajectory toward vestigiality

2022 ◽  
Author(s):  
Anthony Bishopp ◽  
Alexander Ware ◽  
Dylan H Jones ◽  
Paulina Flis ◽  
Kellie E Smith ◽  
...  
Keyword(s):  
Plant Growth ◽  
Lemna Minor ◽  
Normal Growth ◽  
Root Anatomy ◽  
Pistia Stratiotes ◽  
Complex Root ◽  
Spirodela Polyrhiza ◽  
Mineral Profile ◽  
Root Excision

Duckweeds are morphologically simplified, free floating aquatic monocots comprising both rooted and rootless genera. This has led to the idea that roots in these species may be vestigial, but empirical evidence supporting this is lacking. Here we show that duckweed roots are no longer required for their ancestral role of nutrient uptake. Comparative analyses of nearly all rooted duckweed species revealed a highly reduced anatomy, with greater simplification in the more recently diverged genus Lemna. A series of root excision experiments demonstrated that roots are dispensable for normal growth in Spirodela polyrhiza and Lemna minor. Furthermore, ionomic analyses of fronds in these two species showed little difference in the elemental composition of plants in rooted versus root-excised samples. In comparison, another free-floating member of the Araceae, Pistia stratiotes, which colonized the aquatic environment independently of duckweeds, has retained a more complex root anatomy. Whilst Pistia roots were not absolutely required for growth, their removal inhibited plant growth and resulted in a broad change in the mineral profile of aerial tissues. Collectively, these observations suggest that duckweeds and Pistia may be different stages along a trajectory towards root vestigialisation Given this, along with the striking diversity of root phenotypes, culminating in total loss in the most derived species, we propose that duckweed roots are a powerful system with which to understand organ loss and vestigiality.

scholarly journals ResDE Two-Component Regulatory System Mediates Oxygen Limitation-Induced Biofilm Formation byBacillus amyloliquefaciensSQR9

2018 ◽  
Vol 84 (8) ◽  
pp. e02744-17 ◽  
Author(s):  
Xuan Zhou ◽  
Nan Zhang ◽  
Liming Xia ◽  
Qing Li ◽  
Jiahui Shao ◽  
...  
Keyword(s):  
Plant Growth ◽  
Biofilm Formation ◽  
Root Colonization ◽  
Oxygen Deficiency ◽  
Regulatory System ◽  
Regulatory Function ◽  
Content Type ◽  
Environmental Signals ◽  
Link Type ◽  
Two Component

ABSTRACTEfficient biofilm formation and root colonization capabilities facilitate the ability of beneficial plant rhizobacteria to promote plant growth and antagonize soilborne pathogens. Biofilm formation by plant-beneficialBacillusstrains is triggered by environmental cues, including oxygen deficiency, but the pathways that sense these environmental signals and regulate biofilm formation have not been thoroughly elucidated. In this study, we showed that the ResDE two-component regulatory system in the plant growth-promoting rhizobacteriumBacillus amyloliquefaciensstrain SQR9 senses the oxygen deficiency signal and regulates biofilm formation. ResE is activated by sensing the oxygen limitation-induced reduction of the NAD+/NADH pool through its PAS domain, stimulating its kinase activity, and resulting in the transfer of a phosphoryl group to ResD. The phosphorylated ResD directly binds to the promoter regions of theqoxABCDandctaCDEFoperons to improve the biosynthesis of terminal oxidases, which can interact with KinB to activate biofilm formation. These results not only revealed the novel regulatory function of the ResDE two-component system but also contributed to the understanding of the complicated regulatory network governingBacillusbiofilm formation. This research may help to enhance the root colonization and the plant-beneficial efficiency of SQR9 and otherBacillusrhizobacteria used in agriculture.IMPORTANCEBacillusspp. are widely used as bioinoculants for plant growth promotion and disease suppression. The exertion of their plant-beneficial functions is largely dependent on their root colonization, which is closely related to their biofilm formation capabilities. On the other hand,Bacillusis the model bacterium for biofilm study, and the process and molecular network of biofilm formation are well characterized (B. Mielich-Süss and D. Lopez, Environ Microbiol 17:555–565, 2015,https://doi.org/10.1111/1462-2920.12527; L. S. Cairns, L. Hobley, and N. R. Stanley-Wall, Mol Microbiol 93:587–598, 2014,https://doi.org/10.1111/mmi.12697; H. Vlamakis, C. Aguilar, R. Losick, and R. Kolter, Genes Dev 22:945–953, 2008,https://doi.org/10.1101/gad.1645008; S. S. Branda, A. Vik, L. Friedman, and R. Kolter, Trends Microbiol 13:20–26, 2005,https://doi.org/10.1016/j.tim.2004.11.006; C. Aguilar, H. Vlamakis, R. Losick, and R. Kolter, Curr Opin Microbiol 10:638–643, 2007,https://doi.org/10.1016/j.mib.2007.09.006; S. S. Branda, J. E. González-Pastor, S. Ben-Yehuda, R. Losick, and R. Kolter, Proc Natl Acad Sci U S A 98:11621–11626, 2001,https://doi.org/10.1073/pnas.191384198). However, the identification and sensing of environmental signals triggeringBacillusbiofilm formation need further research. Here, we report that the oxygen deficiency signal inducingBacillusbiofilm formation is sensed by the ResDE two-component regulatory system. Our results not only revealed the novel regulatory function of the ResDE two-component regulatory system but also identified the sensing system of a biofilm-triggering signal. This knowledge can help to enhance the biofilm formation and root colonization of plant-beneficialBacillusstrains and also provide new insights of bacterial biofilm formation regulation.

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.

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