Lifestyle adaptations ofRhizobiumfrom rhizosphere to symbiosis

By analyzing successive lifestyle stages of a modelRhizobium–legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N2-fixing bacteroids, and release from legume (pea) nodules. While only 27 genes are annotated asnifandfixinRhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 transfer RNAs, and 5 RNA features) are required for the competitive ability to nodulate pea and fix N2. Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signaling, N2fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism, and glutamine synthesis (GlnII). There are 17 separate lifestyle adaptations specific to rhizosphere growth and 23 to root colonization, distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of aRhizobium–legume symbiosis.

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Lifestyle adaptations of Rhizobium from rhizosphere to symbiosis

10.1101/2020.05.07.082560 ◽

2020 ◽

Cited By ~ 1

Author(s):

Rachel M. Wheatley ◽

Brandon L. Ford ◽

Li Li ◽

Samuel T. N. Aroney ◽

Hayley E. Knights ◽

...

Keyword(s):

Rhizobium Leguminosarum ◽

Root Colonization ◽

Glycogen Synthesis ◽

Rapid Expansion ◽

Metabolic Adaptation ◽

Nodule Formation ◽

Transposon Insertion ◽

Legume Symbiosis ◽

Transposon Insertion Sequencing ◽

Multiple Stages

AbstractBy analyzing successive lifestyle stages of a model Rhizobium-legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N2-fixing bacteroids and release from legume (pea) nodules. While only 27 genes are annotated as nif and fix in Rhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 tRNAs and 5 RNA features) are required for the competitive ability to nodulate pea and fix N2. Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signalling, N2 fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism and glutamine synthesis (GlnII). There are separate lifestyle adaptations specific to rhizosphere growth (17) and root colonization (23), distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of a Rhizobium-legume symbiosis.SignificanceRhizobia are soil-dwelling bacteria that form symbioses with legumes and provide biologically useable nitrogen as ammonium for the host plant. High-throughput DNA sequencing has led to a rapid expansion in publication of complete genomes for numerous rhizobia, but analysis of gene function increasingly lags gene discovery. Mariner-based transposon insertion sequencing (INSeq) has allowed us to characterize the fitness contribution of bacterial genes and determine those functionally important in a Rhizobium-legume symbiosis at multiple stages of development.

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The Use of Transposon Insertion Sequencing to Interrogate the Core Functional Genome of the Legume Symbiont Rhizobium leguminosarum

Frontiers in Microbiology ◽

10.3389/fmicb.2016.01873 ◽

2016 ◽

Vol 7 ◽

Cited By ~ 15

Author(s):

Benjamin J. Perry ◽

Mir S. Akter ◽

Christopher K. Yost

Keyword(s):

Rhizobium Leguminosarum ◽

The Core ◽

Transposon Insertion ◽

Transposon Insertion Sequencing ◽

Functional Genome

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Root colonization of faba bean (Vicia faba L.) and pea (Pisum sativum L.) by Rhizobium leguminosarum bv. viciae in the presence of nitrate-nitrogen

Canadian Journal of Microbiology ◽

10.1139/w01-113 ◽

2001 ◽

Vol 47 (12) ◽

pp. 1068-1074 ◽

Cited By ~ 3

Author(s):

Chantal J Beauchamp ◽

Joseph W Kloepper ◽

Joseph J Shaw ◽

François-P. Chalifour

Keyword(s):

Pisum Sativum ◽

Vicia Faba ◽

Faba Bean ◽

Rhizobium Leguminosarum ◽

Root Colonization ◽

Nitrate Nitrogen ◽

Nodule Formation ◽

Pisum Sativum L ◽

Vicia Faba L ◽

Rhizobial Population

There is a lack of knowledge concerning the effect of nitratenitrogen (NO3N) at levels known to inhibit nodule formation and functioning on root colonization of dinitrogen-fixing legumes. Firstly, this study investigated potential differences between Rhizobium leguminosarum bv. viciae 175F9 and its bioluminescent-labeled strain 175F9.lux on root colonization of faba bean (Vicia faba L.) and pea (Pisum sativum L.). These two strains similarly colonized the roots of both hosts. Secondly, this study evaluated the effects of 0 and 10 mol·m3 NO3N on root colonization of faba bean and pea by strain 175F9.lux, over time. Averaged over both hosts and harvest dates, the presence of NO3N increased the rhizobial population and the root length colonized. In addition, our results showed that bioluminescence activity increased from 7 to 14 days after sowing and was not correlated to rhizobial population. Finally, to demonstrate that an increase in bioluminescence activity was not an indirect effect of nitrate on R. leguminosarum bv. viciae 175F9.lux, this study investigated the effects of increasing carbon (mannitol) and nitrogen (NO3N) concentrations on the rhizobial population and bioluminescence activity. The carbon source was more important than the nitrogen source to increase the rhizobial population and bioluminescence activity, which increased with increasing mannitol concentration, but not with increasing nitrate concentration. Results from this study demonstrated that NO3N increased rhizobial population, especially for faba bean, and the length of root colonized.Key words: nitrate, nitrogen, rhizosphere, rhizobacteria, luminescence

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Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope

Nature Communications ◽

10.1038/s41467-020-18301-2 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 2

Author(s):

Edward Geisinger ◽

Nadav J. Mortman ◽

Yunfei Dai ◽

Murat Cokol ◽

Sapna Syal ◽

...

Keyword(s):

Cell Wall ◽

Acinetobacter Baumannii ◽

Antibiotic Susceptibility ◽

Cell Envelope ◽

Specific Cell ◽

Cell Wall Assembly ◽

Transposon Insertion ◽

Wall Assembly ◽

Transposon Insertion Sequencing ◽

Susceptibility Profiles

AbstractA unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.

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Rhizobium leguminosarum bv. trifolii NodD2 Enhances Competitive Nodule Colonization in the Clover-Rhizobium Symbiosis

Applied and Environmental Microbiology ◽

10.1128/aem.01268-20 ◽

2020 ◽

Vol 86 (18) ◽

Cited By ~ 1

Author(s):

Shaun Ferguson ◽

Anthony S. Major ◽

John T. Sullivan ◽

Scott D. Bourke ◽

Simon J. Kelly ◽

...

Keyword(s):

Competitive Ability ◽

Rhizobium Leguminosarum ◽

Complex Signal ◽

Nodule Formation ◽

Pasture Production ◽

Content Type ◽

Multiple Copies ◽

Thread Formation ◽

Legume Symbiosis ◽

Signal Exchange

ABSTRACT Establishment of the symbiotic relationship that develops between rhizobia and their legume hosts is contingent upon an interkingdom signal exchange. In response to host legume flavonoids, NodD proteins from compatible rhizobia activate expression of nodulation genes that produce lipochitin oligosaccharide signaling molecules known as Nod factors. Root nodule formation commences upon legume recognition of compatible Nod factor. Rhizobium leguminosarum was previously considered to contain one copy of nodD; here, we show that some strains of the Trifolium (clover) microsymbiont R. leguminosarum bv. trifolii contain a second copy designated nodD2. nodD2 genes were present in 8 out of 13 strains of R. leguminosarum bv. trifolii, but were absent from the genomes of 16 R. leguminosarum bv. viciae strains. Analysis of single and double nodD1 and nodD2 mutants in R. leguminosarum bv. trifolii strain TA1 revealed that NodD2 was functional and enhanced nodule colonization competitiveness. However, NodD1 showed significantly greater capacity to induce nod gene expression and infection thread formation. Clover species are either annual or perennial and this phenological distinction is rarely crossed by individual R. leguminosarum bv. trifolii microsymbionts for effective symbiosis. Of 13 strains with genome sequences available, 7 of the 8 effective microsymbionts of perennial hosts contained nodD2, whereas the 3 microsymbionts of annual hosts did not. We hypothesize that NodD2 inducer recognition differs from NodD1, and NodD2 functions to enhance competition and effective symbiosis, which may discriminate in favor of perennial hosts. IMPORTANCE Establishment of the rhizobium-legume symbiosis requires a highly specific and complex signal exchange between both participants. Rhizobia perceive legume flavonoid compounds through LysR-type NodD regulators. Often, rhizobia encode multiple copies of nodD, which is one determinant of host specificity. In some species of rhizobia, the presence of multiple copies of NodD extends their symbiotic host-range. Here, we identified and characterized a second copy of nodD present in some strains of the clover microsymbiont Rhizobium leguminosarum bv. trifolii. The second nodD gene contributed to the competitive ability of the strain on white clover, an important forage legume. A screen for strains containing nodD2 could be utilized as one criterion to select strains with enhanced competitive ability for use as inoculants for pasture production.

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BacA Is Essential for Bacteroid Development in Nodules of Galegoid, but not Phaseoloid, Legumes

Journal of Bacteriology ◽

10.1128/jb.00020-10 ◽

2010 ◽

Vol 192 (11) ◽

pp. 2920-2928 ◽

Cited By ~ 42

Author(s):

Ramakrishnan Karunakaran ◽

Andreas F. Haag ◽

Alison K. East ◽

Vinoy K. Ramachandran ◽

Jurgen Prell ◽

...

Keyword(s):

Rhizobium Leguminosarum ◽

Cell Envelope ◽

Integral Membrane Protein ◽

Nodule Formation ◽

Wild Type ◽

Free Living ◽

Indeterminate Nodule ◽

Living Culture ◽

Infection Threads ◽

Membrane Components

ABSTRACT BacA is an integral membrane protein, the mutation of which leads to increased resistance to the antimicrobial peptides bleomycin and Bac71-35 and a greater sensitivity to SDS and vancomycin in Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli, and Rhizobium etli. The growth of Rhizobium strains on dicarboxylates as a sole carbon source was impaired in bacA mutants but was overcome by elevating the calcium level. While bacA mutants elicited indeterminate nodule formation on peas, which belong to the galegoid tribe of legumes, bacteria lysed after release from infection threads and mature bacteroids were not formed. Microarray analysis revealed almost no change in a bacA mutant of R. leguminosarum bv. viciae in free-living culture. In contrast, 45 genes were more-than 3-fold upregulated in a bacA mutant isolated from pea nodules. Almost half of these genes code for cell membrane components, suggesting that BacA is crucial to alterations that occur in the cell envelope during bacteroid development. In stark contrast, bacA mutants of R. leguminosarum bv. phaseoli and R. etli elicited the formation of normal determinate nodules on their bean host, which belongs to the phaseoloid tribe of legumes. Bacteroids from these nodules were indistinguishable from the wild type in morphology and nitrogen fixation. Thus, while bacA mutants of bacteria that infect galegoid or phaseoloid legumes have similar phenotypes in free-living culture, BacA is essential only for bacteroid development in indeterminate galegoid nodules.

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Faculty Opinions recommendation of Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736413515.793564769 ◽

2019 ◽

Author(s):

Martin Marinus

Keyword(s):

Intestinal Colonization ◽

Transposon Insertion ◽

Transposon Insertion Sequencing

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Reproducible and accessible analysis of transposon insertion sequencing in Galaxy for qualitative essentiality analyses

BMC Microbiology ◽

10.1186/s12866-021-02184-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Delphine Larivière ◽

Laura Wickham ◽

Kenneth Keiler ◽

Anton Nekrutenko ◽

Keyword(s):

Data Analysis ◽

Promoter Sequence ◽

Entire Genome ◽

Link Type ◽

Transposon Insertion ◽

Control Procedures ◽

Reproducible Analysis ◽

Using Data ◽

Transposon Insertion Sequencing ◽

The Impact

Abstract Background Significant progress has been made in advancing and standardizing tools for human genomic and biomedical research. Yet, the field of next-generation sequencing (NGS) analysis for microorganisms (including multiple pathogens) remains fragmented, lacks accessible and reusable tools, is hindered by local computational resource limitations, and does not offer widely accepted standards. One such “problem areas” is the analysis of Transposon Insertion Sequencing (TIS) data. TIS allows probing of almost the entire genome of a microorganism by introducing random insertions of transposon-derived constructs. The impact of the insertions on the survival and growth under specific conditions provides precise information about genes affecting specific phenotypic characteristics. A wide array of tools has been developed to analyze TIS data. Among the variety of options available, it is often difficult to identify which one can provide a reliable and reproducible analysis. Results Here we sought to understand the challenges and propose reliable practices for the analysis of TIS experiments. Using data from two recent TIS studies, we have developed a series of workflows that include multiple tools for data de-multiplexing, promoter sequence identification, transposon flank alignment, and read count repartition across the genome. Particular attention was paid to quality control procedures, such as determining the optimal tool parameters for the analysis and removal of contamination. Conclusions Our work provides an assessment of the currently available tools for TIS data analysis. It offers ready to use workflows that can be invoked by anyone in the world using our public Galaxy platform (https://usegalaxy.org). To lower the entry barriers, we have also developed interactive tutorials explaining details of TIS data analysis procedures at https://bit.ly/gxy-tis.

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