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

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.

Download Full-text

Morphogenesis of root nodules in white clover. II. The effect of mutation in genes nod IJ of the microsymbiont upon the nodule structure

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1998.001 ◽

2014 ◽

Vol 67 (1) ◽

pp. 5-22

Author(s):

Barbara Łotocka ◽

Joanna Kopcińska ◽

Władysław Golinowski

Keyword(s):

White Clover ◽

Rhizobium Leguminosarum ◽

Root Nodules ◽

Single Cells ◽

Wild Strain ◽

Root Hairs ◽

Nodule Development ◽

Wild Type ◽

Delayed Reaction ◽

Infection Threads

Morphogenesis of ineffective root nodules initiated on the roots of white clover 'Astra' by the <em>Rhizobium leguminosarum</em> biovar. <em>trifolii</em> strains ANU261 (Tn5 insertion in nod 1 gene) and ANU262 (Tn5 insertion in nod J gene) was investigated. Following changes were observed, as compared to the wild-type nodulation: the exaggerated, not delayed reaction of root hairs; the delay in nodulation with the number of nodules the same as in plants inoculated with a wild strain; the formation and organization of the nodule primordium not changed in comparison with the wild-type nodules; infection threads abnormally branched and diffusing with bacteria deprived of light zone and enriched with storage material; infected cells of bacteroidal tissue abnormally strongly osmiophilic and only slightly vacuolated; symbiosomes with very narrowed peribacteroidal space, subject to premature degradation; abnormal accumulation of starch in the nodule tissues; nodule development blocked at the stage of laterally situated meristem and single nodule bundle; inhibition of divisions in the meristem and vacuolation of its cells; the appearance of single cells with colonies of saprophytic rhizobia embedded in the fibrillar matrix in the old, degraded regions of the bacteroidal tissue.

Download Full-text

Mutational Analyses of Open Reading Frames within thevraSROperon and Their Roles in the Cell Wall Stress Response ofStaphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01213-10 ◽

2011 ◽

Vol 55 (4) ◽

pp. 1391-1402 ◽

Cited By ~ 34

Author(s):

N. McCallum ◽

P. Stutzmann Meier ◽

R. Heusser ◽

B. Berger-Bächi

Keyword(s):

Signal Transduction ◽

Cell Wall ◽

Cell Envelope ◽

Wall Stress ◽

Integral Membrane Protein ◽

Luciferase Reporter ◽

Wild Type ◽

Luciferase Reporter Gene ◽

Cell Wall Stress ◽

Type Strains

ABSTRACTThe exposure ofStaphylococcus aureusto a broad range of cell wall-damaging agents triggers the induction of a cell wall stress stimulon (CWSS) controlled by the VraSR two-component system. ThevraSRgenes form part of the four-cistron autoregulatory operonorf1-yvqF-vraS-vraR. The markerless inactivation of each of the genes within this operon revealed thatorf1played no observable role in CWSS induction and had no influence on resistance phenotypes for any of the cell envelope stress-inducing agents tested. The remaining three genes were all essential for the induction of the CWSS, and mutants showed various degrees of increased susceptibility to cell wall-active antibiotics. Therefore, the role of YvqF inS. aureusappears to be opposite that in other Gram-positive bacteria, where YvqF homologs have all been shown to inhibit signal transduction. This role, as an activator rather than repressor of signal transduction, corresponds well with resistance phenotypes of ΔYvqF mutants, which were similar to those of ΔVraR mutants in which CWSS induction also was completely abolished. Resistance profiles of ΔVraS mutants differed phenotypically from those of ΔYvqF and ΔVraR mutants on many non-ß-lactam antibiotics. ΔVraS mutants still became more susceptible than wild-type strains at low antibiotic concentrations, but they retained larger subpopulations that were able to grow on higher antibiotic concentrations than ΔYvqF and ΔVraR mutants. Subpopulations of ΔVraS mutants could grow on even higher glycopeptide concentrations than wild-type strains. The expression of a highly sensitive CWSS-luciferase reporter gene fusion was up to 2.6-fold higher in a ΔVraS than a ΔVraR mutant, which could be linked to differences in their respective antibiotic resistance phenotypes. Bacterial two-hybrid analysis indicated that the integral membrane protein YvqF interacted directly with VraS but not VraR, suggesting that it plays an essential role in sensing the as-yet unknown trigger of CWSS induction.

Download Full-text

Lifestyle adaptations ofRhizobiumfrom rhizosphere to symbiosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2009094117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23823-23834

Author(s):

Rachel M. Wheatley ◽

Brandon L. Ford ◽

Li Li ◽

Samuel T. N. Aroney ◽

Hayley E. Knights ◽

...

Keyword(s):

Rhizobium Leguminosarum ◽

Root Colonization ◽

Cell Envelope ◽

Glycogen Synthesis ◽

Metabolic Adaptation ◽

Nodule Formation ◽

Transposon Insertion ◽

Legume Symbiosis ◽

Glutamine Synthesis ◽

Transposon Insertion Sequencing

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.

Download Full-text

A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria

Microbiology ◽

10.1099/mic.0.28311-0 ◽

2005 ◽

Vol 151 (10) ◽

pp. 3249-3256 ◽

Cited By ~ 43

Author(s):

R. Karunakaran ◽

T. H. Mauchline ◽

A. H. F. Hosie ◽

P. S. Poole

Keyword(s):

Rhizobium Leguminosarum ◽

Nodule Formation ◽

Broad Host Range ◽

Gram Negative Bacteria ◽

Acid Transport ◽

Promoter Regions ◽

Gram Negative ◽

Infection Threads ◽

Promoter Probe ◽

Made In

A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfpmut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT.3, dsRedT.4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA p) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

Download Full-text

Investigation of a non-nodulating cultivar of Pisum sativum

Canadian Journal of Botany ◽

10.1139/b76-175 ◽

1976 ◽

Vol 54 (14) ◽

pp. 1633-1636 ◽

Cited By ~ 23

Author(s):

T. L. Degenhardt ◽

T. A. Larue ◽

E. A. Paul

Keyword(s):

Pisum Sativum ◽

Rhizobium Leguminosarum ◽

Root Hairs ◽

Infection Process ◽

Nodule Formation ◽

Temperature Sensitive ◽

Infection Threads

A non-nodulating cultivar of Pisum sativum cv. Afghanistan was studied to characterize the nature and location of the non-nodulating factor. Nodule formation was not temperature sensitive. Rhizobium leguminosarum could exist in the rhizosphere. Root secretions did not decrease nodulation in adjacent normal plants, nor did the proximity of normal plants promote nodulation. Infection threads formed in the root hairs, but nodules were not formed. The infection process apparently aborted, resulting in the formation of swellings on areas of the root where nodulation would normally occur. Grafting experiments indicate that the factor preventing nodulation is in the root and is not translocated from the cotyledon or plant top.

Download Full-text

Altered Envelope Structure and Nanomechanical Properties of a C-Terminal Protease A-Deficient Rhizobium leguminosarum

Microorganisms ◽

10.3390/microorganisms8091421 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1421

Author(s):

Dong Jun ◽

Ubong Idem ◽

Tanya E. S. Dahms

Keyword(s):

Outer Membrane ◽

Creep Deformation ◽

High Performance ◽

Rhizobium Leguminosarum ◽

Cell Envelope ◽

Time Dependent ◽

Wild Type ◽

Force Microscopy ◽

Viscoelastic Parameters ◽

Transmission Electron

(1) Background: Many factors can impact bacterial mechanical properties, which play an important role in survival and adaptation. This study characterizes the ultrastructural phenotype, elastic and viscoelastic properties of Rhizobium leguminosarum bv. viciae 3841 and the C-terminal protease A (ctpA) null mutant strain predicted to have a compromised cell envelope; (2) Methods: To probe the cell envelope, we used transmission electron microscopy (TEM), high performance liquid chromatography (HPLC), mass spectrometry (MS), atomic force microscopy (AFM) force spectroscopy, and time-dependent AFM creep deformation; (3) Results: TEM images show a compromised and often detached outer membrane for the ctpA mutant. Muropeptide characterization by HPLC and MS showed an increase in peptidoglycan dimeric peptide (GlcNAc-MurNAc-Ala-Glu-meso-DAP-Ala-meso-DAP-Glu-Ala-MurNAc-GlcNAc) for the ctpA mutant, indicative of increased crosslinking. The ctpA mutant had significantly larger spring constants than wild type under all hydrated conditions, attributable to more highly crosslinked peptidoglycan. Time-dependent AFM creep deformation for both the wild type and ctpA mutant was indicative of a viscoelastic cell envelope, with best fit to the four-element Burgers model and generating values for viscoelastic parameters k1, k2, η1, and η2; (4) Conclusions: The viscoelastic response of the ctpA mutant is consistent with both its compromised outer membrane (TEM) and fortified peptidoglycan layer (HPLC/MS).

Download Full-text

Homology of genes for exopolysaccharide synthesis in Rhizobium leguminosarum and effect of cloned exo genes on nodule formation.

Acta Biochimica Polonica ◽

10.18388/abp.1993_4787 ◽

1993 ◽

Vol 40 (4) ◽

pp. 477-482

Author(s):

A Skorupska ◽

M Deryło

Keyword(s):

Rhizobium Leguminosarum ◽

The Other ◽

Nodule Formation ◽

Nitrogen Fixing ◽

Wild Type ◽

Bamhi Fragment ◽

Other Hand ◽

Indeterminate Nodules ◽

Total Dna ◽

Rhizobium Sp

A 5.4 kb BamHI fragment of R. leguminosarum bv. trifolii TA1 was found to carry genes involved in exopolysaccharide synthesis (exo genes). This fragment was strongly hybridized to the total DNA from R. l. bv. viciae and bv. phaseoli digested with EcoRI. No homology was found with total DNA of R. meliloti and Rhizobium sp. NGR 234. The exo genes from R. l. bv. trifolii TA1 conjugally introduced into R. l. bv. viciae 1302 considerably affected the symbiosis: the nodules induced on vetch were abortive and did not fix nitrogen. On the other hand, Phaseolus beans infected with R. l. bv. phaseoli harbouring R. l. bv. trifolii exo genes formed the nitrogen-fixing nodules. It can be concluded that additional copies of exo genes introduced into wild type Rhizobium leguminosarum strains can disturb the synthesis of acidic exopolysaccharides and affect symbiosis of the plants forming indeterminate nodules, but do not affect symbiosis of the plants forming the determinate nodules.

Download Full-text

Role of Cellulose Fibrils and Exopolysaccharides of Rhizobium leguminosarum in Attachment to and Infection of Vicia sativa Root Hairs

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0533 ◽

2005 ◽

Vol 18 (6) ◽

pp. 533-538 ◽

Cited By ~ 71

Author(s):

M. C. Laus ◽

A. A. N. van Brussel ◽

J. W. Kijne

Keyword(s):

Root Hair ◽

Rhizobium Leguminosarum ◽

Developmental Stages ◽

Cortical Cell ◽

Root Hairs ◽

Infection Thread ◽

Vicia Sativa ◽

Wild Type ◽

Infection Threads ◽

Cellulose Fibrils

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V. sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.

Download Full-text

Regulation of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum by the VisN/R-Rem cascade

Microbiology ◽

10.1099/mic.0.035386-0 ◽

2010 ◽

Vol 156 (6) ◽

pp. 1673-1685 ◽

Cited By ~ 25

Author(s):

Dinah D. Tambalo ◽

Kate L. Del Bel ◽

Denise E. Bustard ◽

Paige R. Greenwood ◽

Audrey E. Steedman ◽

...

Keyword(s):

Gene Cluster ◽

Rhizobium Leguminosarum ◽

Flagellar Motility ◽

Growth Phases ◽

Wild Type ◽

Free Living ◽

Regulatory Cascade ◽

Genes Encoding ◽

And Function ◽

Free Living Conditions

In this paper, we describe the regulatory roles of VisN, VisR and Rem in the expression of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum biovar viciae strains VF39SM and 3841. Individual mutations in the genes encoding these proteins resulted in a loss of motility and an absence of flagella, indicating that these regulatory genes are essential for flagellar synthesis and function. Transcriptional experiments involving gusA–gene fusions in wild-type and mutant backgrounds were performed to identify the genes under VisN/R and Rem regulation. Results showed that the chemotaxis and motility genes of R. leguminosarum could be separated into two groups: one group under VisN/R-Rem regulation and another group that is independent of this regulation. VisN and VisR regulate the expression of rem, while Rem positively regulates the expression of flaA, flaB, flaC, flaD, motA, motB, che1 and mcpD. All of these genes except mcpD are located within the main motility and chemotaxis gene cluster of R. leguminosarum. Other chemotaxis and motility genes, which are found outside of the main motility gene cluster (che2 operon, flaH for VF39SM, and flaG) or are plasmid-borne (flaE and mcpC), are not part of the VisN/R-Rem regulatory cascade. In addition, all genes exhibited the same regulation pattern in 3841 and in VF39SM, except flaE and flaH. flaE is not regulated by VisN/R-Rem in 3841 but it is repressed by Rem in VF39SM. flaH is under VisN/R-Rem regulation in 3841, but not in VF39SM. A kinetics experiment demonstrated that a subset of the flagellar genes is continuously expressed in all growth phases, indicating the importance of continuous motility for R. leguminosarum under free-living conditions. On the other hand, motility is repressed under symbiotic conditions. Nodulation experiments showed that the transcriptional activators VisN and Rem are dramatically downregulated in the nodules, suggesting that the symbiotic downregulation of motility-related genes could be mediated by repressing the expression of VisN/R and Rem.

Download Full-text

Gene Products of the hupGHIJ Operon Are Involved in Maturation of the Iron-Sulfur Subunit of the [NiFe] Hydrogenase from Rhizobium leguminosarum bv. viciae

Journal of Bacteriology ◽

10.1128/jb.187.20.7018-7026.2005 ◽

2005 ◽

Vol 187 (20) ◽

pp. 7018-7026 ◽

Cited By ~ 34

Author(s):

Hamid Manyani ◽

Luis Rey ◽

José M. Palacios ◽

Juan Imperial ◽

Tomás Ruiz-Argüeso

Keyword(s):

Rhizobium Leguminosarum ◽

Hydrogenase Activity ◽

Gene Products ◽

Structural Genes ◽

Wild Type ◽

Free Living ◽

Iron Sulfur ◽

Specific Antisera ◽

Mutant Strains ◽

Isogenic Mutants

ABSTRACT In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions (ΔhupG, ΔhupH, ΔhupI, and ΔhupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The ΔhupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2. Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the ΔhupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from ΔhupH, ΔhupI, and ΔhupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the ΔhupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.

Download Full-text