Glutathione Plays a Fundamental Role in Growth and Symbiotic Capacity of Sinorhizobium meliloti

ABSTRACT Rhizobia form a symbiotic relationship with plants of the legume family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We have examined the importance of glutathione (GSH) during free-living growth and symbiosis of Sinorhizobium meliloti. An S. meliloti mutant strain (SmgshA) which is unable to synthesize GSH due to a gene disruption in gshA, encoding the enzyme for the first step in the biosynthesis of GSH, was unable to grow under nonstress conditions, precluding any nodulation. In contrast, an S. meliloti strain (SmgshB) with gshB, encoding the enzyme involved in the second step in GSH synthesis, deleted was able to grow, indicating that γ-glutamylcysteine, the dipeptide intermediate, can partially substitute for GSH. However, the SmgshB strain showed a delayed-nodulation phenotype coupled to a 75% reduction in the nitrogen fixation capacity. This phenotype was linked to abnormal nodule development. Both the SmgshA and SmgshB mutant strains exhibited higher catalase activity than the wild-type S. meliloti strain, suggesting that both mutant strains are under oxidative stress. Taken together, these results show that GSH plays a critical role in the growth of S. meliloti and during its interaction with the plant partner.

Download Full-text

Malic Enzyme Cofactor and Domain Requirements for Symbiotic N2 Fixation by Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.01425-06 ◽

2006 ◽

Vol 189 (1) ◽

pp. 160-168 ◽

Cited By ~ 11

Author(s):

Michael J. Mitsch ◽

Alison Cowie ◽

Turlough M. Finan

Keyword(s):

Amino Acid ◽

Malic Enzyme ◽

Sinorhizobium Meliloti ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

High Ratio ◽

Terminal Region ◽

Wild Type ◽

Mutant Strains ◽

Alfalfa Root

ABSTRACT The NAD+-dependent malic enzyme (DME) and the NADP+-dependent malic enzyme (TME) of Sinorhizobium meliloti are representatives of a distinct class of malic enzymes that contain a 440-amino-acid N-terminal region homologous to other malic enzymes and a 330-amino-acid C-terminal region with similarity to phosphotransacetylase enzymes (PTA). We have shown previously that dme mutants of S. meliloti fail to fix N2 (Fix−) in alfalfa root nodules, whereas tme mutants are unimpaired in their N2-fixing ability (Fix+). Here we report that the amount of DME protein in bacteroids is 10 times greater than that of TME. We therefore investigated whether increased TME activity in nodules would allow TME to function in place of DME. The tme gene was placed under the control of the dme promoter, and despite elevated levels of TME within bacteroids, no symbiotic nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme from the tme promoter resulted in a large reduction in DME activity and symbiotic N2 fixation. Hence, TME cannot replace the symbiotic requirement for DME. In further experiments we investigated the DME PTA-like domain and showed that it is not required for N2 fixation. Thus, expression of a DME C-terminal deletion derivative or the Escherichia coli NAD+-dependent malic enzyme (sfcA), both of which lack the PTA-like region, restored wild-type N2 fixation to a dme mutant. Our results have defined the symbiotic requirements for malic enzyme and raise the possibility that a constant high ratio of NADPH + H+ to NADP in nitrogen-fixing bacteroids prevents TME from functioning in N2-fixing bacteroids.

Download Full-text

Regulation of Phosphate Assimilation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽

10.1093/genetics/148.4.1689 ◽

1998 ◽

Vol 148 (4) ◽

pp. 1689-1700 ◽

Cited By ~ 5

Author(s):

Sylvie D Bardin ◽

Turlough M Finan

Keyword(s):

Escherichia Coli ◽

Transport System ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Phosphate Transport ◽

Wild Type ◽

Pi Transport ◽

Regulatory Systems ◽

Mutant Strains ◽

Insertion Mutations

Abstract We report the isolation of phoB and phoU mutants of the bacterium Rhizobium (Sinorhizobium) meliloti. These mutants form N2-fixing nodules on the roots of alfalfa plants. R. meliloti mutants defective in the phoCDET (ndvF) encoded phosphate transport system grow slowly in media containing 2 mm Pi, and form nodules which fail to fix nitrogen (Fix−). We show that the transfer of phoB or phoU insertion mutations into phoC mutant strains restores the ability of these mutants to: (i) form normal N2-fixing root-nodules, and (ii) grow like the wild type in media containing 2 mm Pi. We also show that expression of the alternate orfA pit encoded Pi transport system is negatively regulated by the phoB gene product, whereas phoB is required for phoCDET expression. We suggest that in R. meliloti cells growing under Pi limiting conditions, PhoB protein activates phoCDET transcription and represses orfA pit transcription. Our results suggest that there are major differences between the Escherichia coli and R. meliloti phosphate regulatory systems.

Download Full-text

Role of the Sinorhizobium meliloti Global Regulator Hfq in Gene Regulation and Symbiosis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-4-0355 ◽

2010 ◽

Vol 23 (4) ◽

pp. 355-365 ◽

Cited By ~ 42

Author(s):

Mengsheng Gao ◽

Melanie J. Barnett ◽

Sharon R. Long ◽

Max Teplitski

Keyword(s):

Sinorhizobium Meliloti ◽

Rna Binding ◽

Nodule Development ◽

Wild Type ◽

Global Regulator ◽

Free Living ◽

Symbiotic Efficiency ◽

Cellular Processes ◽

Nitrogen Containing Compounds

The RNA-binding protein Hfq is a global regulator which controls diverse cellular processes in bacteria. To begin understanding the role of Hfq in the Sinorhizobium meliloti–Medicago truncatula nitrogen-fixing symbiosis, we defined free-living and symbiotic phenotypes of an hfq mutant. Over 500 transcripts were differentially accumulated in the hfq mutant of S. meliloti Rm1021 when grown in a shaking culture. Consistent with transcriptome-wide changes, the hfq mutant displayed dramatic alterations in metabolism of nitrogen-containing compounds, even though its carbon source utilization profiles were nearly identical to the wild type. The hfq mutant had reduced motility and was impaired for growth at alkaline pH. A deletion of hfq resulted in a reduced symbiotic efficiency, although the mutant was still able to initiate nodule development and differentiate into bacteroids.

Download Full-text

Functional diversity of five homologous Cu+-ATPases present in Sinorhizobium meliloti

Microbiology ◽

10.1099/mic.0.079137-0 ◽

2014 ◽

Vol 160 (6) ◽

pp. 1237-1251 ◽

Cited By ~ 16

Author(s):

Sarju J. Patel ◽

Teresita Padilla-Benavides ◽

Jessica M. Collins ◽

José M. Argüello

Keyword(s):

Sinorhizobium Meliloti ◽

Nitrosative Stress ◽

Nodule Development ◽

Symbiotic Bacteria ◽

Redox Stress ◽

Free Living ◽

Mutant Strains ◽

Microbe Interactions ◽

Differential Gene ◽

Host Microbe Interactions

Copper is an important element in host–microbe interactions, acting both as a catalyst in enzymes and as a potential toxin. Cu+-ATPases drive cytoplasmic Cu+ efflux and protect bacteria against metal overload. Many pathogenic and symbiotic bacteria contain multiple Cu+-ATPase genes within particular genetic environments, suggesting alternative roles for each resulting protein. This hypothesis was tested by characterizing five homologous Cu+-ATPases present in the symbiotic organism Sinorhizobium meliloti. Mutation of each gene led to different phenotypes and abnormal nodule development in the alfalfa host. Distinct responses were detected in free-living S. meliloti mutant strains exposed to metal and redox stresses. Differential gene expression was detected under Cu+, oxygen or nitrosative stress. These observations suggest that CopA1a maintains the cytoplasmic Cu+ quota and its expression is controlled by Cu+ levels. CopA1b is also regulated by Cu+ concentrations and is required during symbiosis for bacteroid maturation. CopA2-like proteins, FixI1 and FixI2, are necessary for the assembly of two different cytochrome c oxidases at different stages of bacterial life. CopA3 is a phylogenetically distinct Cu+-ATPase that does not contribute to Cu+ tolerance. It is regulated by redox stress and required during symbiosis. We postulated a model where non-redundant homologous Cu+-ATPases, operating under distinct regulation, transport Cu+ to different target proteins.

Download Full-text

Sinorhizobium meliloti Mutants Lacking Phosphotransferase System Enzyme HPr or EIIA Are Altered in Diverse Processes, Including Carbon Metabolism, Cobalt Requirements, and Succinoglycan Production

Journal of Bacteriology ◽

10.1128/jb.01917-07 ◽

2008 ◽

Vol 190 (8) ◽

pp. 2947-2956 ◽

Cited By ~ 32

Author(s):

Catalina Arango Pinedo ◽

Ryan M. Bringhurst ◽

Daniel J. Gage

Keyword(s):

Carbon Metabolism ◽

Sinorhizobium Meliloti ◽

Carbon Sources ◽

Deletion Mutants ◽

Symbiotic Relationship ◽

Wild Type ◽

Phosphotransferase System ◽

Carbon Utilization ◽

Carbon Regulation ◽

Extreme Sensitivity

ABSTRACT Sinorhizobium meliloti is a member of the Alphaproteobacteria that fixes nitrogen when it is in a symbiotic relationship. Genes for an incomplete phosphotransferase system (PTS) have been found in the genome of S. meliloti. The genes present code for Hpr and ManX (an EIIAMan-type enzyme). HPr and EIIA regulate carbon utilization in other bacteria. hpr and manX in-frame deletion mutants exhibited altered carbon metabolism and other phenotypes. Loss of HPr resulted in partial relief of succinate-mediated catabolite repression, extreme sensitivity to cobalt limitation, rapid die-off during stationary phase, and altered succinoglycan production. Loss of ManX decreased expression of melA-agp and lac, the operons needed for utilization of α- and β-galactosides, slowed growth on diverse carbon sources, and enhanced accumulation of high-molecular-weight succinoglycan. A strain with both hpr and manX deletions exhibited phenotypes similar to those of the strain with a single hpr deletion. Despite these strong phenotypes, deletion mutants exhibited wild-type nodulation and nitrogen fixation when they were inoculated onto Medicago sativa. The results show that HPr and ManX (EIIAMan) are involved in more than carbon regulation in S. meliloti and suggest that the phenotypes observed occur due to activity of HPr or one of its phosphorylated forms.

Download Full-text

MtBZR1 Plays an Important Role in Nodule Development in Medicago truncatula

International Journal of Molecular Sciences ◽

10.3390/ijms20122941 ◽

2019 ◽

Vol 20 (12) ◽

pp. 2941

Author(s):

Can Cui ◽

Hongfeng Wang ◽

Limei Hong ◽

Yiteng Xu ◽

Yang Zhao ◽

...

Keyword(s):

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Normal Growth ◽

Dry Mass ◽

Growth Conditions ◽

Nodule Development ◽

Functional Study ◽

Loss Of Function ◽

Wild Type

Brassinosteroid (BR) is an essential hormone in plant growth and development. The BR signaling pathway was extensively studied, in which BRASSINAZOLE RESISTANT 1 (BZR1) functions as a key regulator. Here, we carried out a functional study of the homolog of BZR1 in Medicago truncatula R108, whose expression was induced in nodules upon Sinorhizobium meliloti 1021 inoculation. We identified a loss-of-function mutant mtbzr1-1 and generated 35S:MtBZR1 transgenic lines for further analysis at the genetic level. Both the mutant and the overexpression lines of MtBZR1 showed no obvious phenotypic changes under normal growth conditions. After S. meliloti 1021 inoculation, however, the shoot and root dry mass was reduced in mtbzr1-1 compared with the wild type, caused by partially impaired nodule development. The transcriptomic analysis identified 1319 differentially expressed genes in mtbzr1-1 compared with wild type, many of which are involved in nodule development and secondary metabolite biosynthesis. Our results demonstrate the role of MtBZR1 in nodule development in M. truncatula, shedding light on the potential role of BR in legume–rhizobium symbiosis.

Download Full-text

A Positive Role for Rhizobitoxine in Rhizobium-legume Symbiosis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.12.1082 ◽

1999 ◽

Vol 12 (12) ◽

pp. 1082-1089 ◽

Cited By ~ 47

Author(s):

Samuel Duodu ◽

T. V. Bhuvaneswari ◽

Thomas J. W. Stokkermans ◽

N. Kent Peters

Keyword(s):

Ethylene Biosynthesis ◽

Nodule Development ◽

Ethylene Inhibitors ◽

Wild Type ◽

Positive Role ◽

Rate Limiting Step ◽

Mutant Strains ◽

Legume Symbiosis ◽

Exogenous Ethylene ◽

Rate Limiting

Although Bradyrhizobium elkanii is a mutualistic symbiont of legumes, it synthesizes a phytotoxin, rhizobitoxine, that causes chlorosis on a variety of legume hosts, giving a pathogenic character to these interactions. No positive role for rhizobitoxine has been previously demonstrated. Interestingly, rhizobitoxine inhibits the rate-limiting step for ethylene biosynthesis, a plant hormone known to inhibit or down-regulate nodule development. We hypothesized that rhizobitoxine plays a positive role in nodule development through its inhibition of ethylene biosynthesis. To test this hypothesis, host plants of B. elkanii were screened for a differential nodulation response to the wild-type and rhizobitoxine mutant strains. In Vigna radiata (mungbean), the rhizobitoxine mutant strains induced many aborted nodules arrested at all stages of pre-emergent and post-emergent development and formed significantly fewer mature nodules than the wild type. Experiments revealed that nodulation of mungbean plants is sensitive to exogenous ethylene, and that the ethylene inhibitors aminoethoxyvinylglycine and Co2+ were able to partially restore a wild-type nodulation pattern to the rhizobitoxine mutants. This is the first demonstration of a nodulation phenotype of the rhizobitoxine mutants and suggests that rhizobitoxine plays a positive and necessary role in Rhizobium-legume symbiosis through its inhibition of ethylene biosynthesis.

Download Full-text

HPrK Regulates Succinate-Mediated Catabolite Repression in the Gram-Negative Symbiont Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.01115-08 ◽

2008 ◽

Vol 191 (1) ◽

pp. 298-309 ◽

Cited By ~ 24

Author(s):

Catalina Arango Pinedo ◽

Daniel J. Gage

Keyword(s):

Catabolite Repression ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Phosphotransferase System ◽

Free Living ◽

Gene Encoding ◽

Common Component ◽

Gram Negative ◽

Gram Positive Bacteria ◽

Living Organisms

ABSTRACT The HPrK kinase/phosphatase is a common component of the phosphotransferase system (PTS) of gram-positive bacteria and regulates catabolite repression through phosphorylation/dephosphorylation of its substrate, the PTS protein HPr, at a conserved serine residue. Phosphorylation of HPr by HPrK also affects additional phosphorylation of HPr by the PTS enzyme EI at a conserved histidine residue. Sinorhizobium meliloti can live as symbionts inside legume root nodules or as free-living organisms and is one of the relatively rare gram-negative bacteria known to have a gene encoding HPrK. We have constructed S. meliloti mutants that lack HPrK or that lack key amino acids in HPr that are likely phosphorylated by HPrK and EI. Deletion of hprK in S. meliloti enhanced catabolite repression caused by succinate, as did an S53A substitution in HPr. Introduction of an H22A substitution into HPr alleviated the strong catabolite repression phenotypes of strains carrying ΔhprK or hpr(S53A) mutations, demonstrating that HPr-His22-P is needed for strong catabolite repression. Furthermore, strains with a hpr(H22A) allele exhibited relaxed catabolite repression. These results suggest that HPrK phosphorylates HPr at the serine-53 residue, that HPr-Ser53-P inhibits phosphorylation at the histidine-22 residue, and that HPr-His22-P enhances catabolite repression in the presence of succinate. Additional experiments show that ΔhprK mutants overproduce exopolysaccharides and form nodules that do not fix nitrogen.

Download Full-text

Comparison of the symbiotic and competition phenotypes of Sinorhizobium meliloti PHB synthesis and degradation pathway mutants

Canadian Journal of Microbiology ◽

10.1139/w05-042 ◽

2005 ◽

Vol 51 (7) ◽

pp. 599-604 ◽

Cited By ~ 20

Author(s):

P Aneja ◽

A Zachertowska ◽

T C Charles

Keyword(s):

Sinorhizobium Meliloti ◽

Degradation Pathway ◽

Nodule Occupancy ◽

Wild Type ◽

Term Survival ◽

Mutant Strains ◽

Key Factor ◽

Carbon Excess ◽

Synthesis And Degradation

The competitive abilities of Sinorhizobium meliloti mutant strains containing lesions in the PHB synthesis (phbC) and degradation (bdhA) pathways were compared. While the bdhA mutant showed no noticeable symbiotic defects on alfalfa host plants when inoculated alone, in mixed inoculation experiments it was found to be less competitive than the wild type for nodule occupancy. Long-term survival of the bdhA mutant on a carbon-limiting medium was not affected. However, when subjected to competition with the wild-type strain in periodic subculturing through alternating carbon-limiting and carbon-excess conditions, the bdhA mutant performed poorly. A more severe defect in competition for growth and nodule occupancy was observed with a mutant unable to synthesize PHB (phbC). These results indicate that the ability to efficiently deposit cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability, whereas the ability to use these stores is less important.Key words: Sinorhizobium meliloti, PHB metabolism, competition.

Download Full-text

Starch content and activities of starch-metabolizing enzymes in effective and ineffective root nodules of soybean

Canadian Journal of Botany ◽

10.1139/b91-094 ◽

1991 ◽

Vol 69 (4) ◽

pp. 697-701 ◽

Cited By ~ 15

Author(s):

Sharon I. Forrest ◽

Desh Pal S. Verma ◽

Rajinder S. Dhindsa

Keyword(s):

Nitrogen Fixation ◽

Starch Content ◽

Root Nodules ◽

Starch Synthase ◽

Nodule Development ◽

Wild Type ◽

Starch Phosphorylase ◽

High Concentration ◽

Key Words Nitrogen Fixation ◽

Glycine Max L

Starch content and activities of some enzymes of starch metabolism were determined in wild-type, N2-fixing (fix+) nodules and in two non-N2-fixing (fix−) nodules induced by Bradyrhizobium japonicum mutant strains, T5-95 and T8-1, on soybean (Glycine max L.) roots. The T5-95 nodules are similar to wild type in ultrastructure, but the T8-1 nodules are different in that the bacteroids are not released from the infection thread. After initial accumulation to relatively high concentration, starch was depleted during nitrogen fixation in fix+ nodules. However, in fix− nodules, the accumulated starch was not metabolized. The activity of starch-bound starch synthase (EC 2.4.1.21) declined in fix+ nodules but remained high in fix− nodules. The activity of α-amylase (EC 3.2.1.1) was only slightly higher than wild type in T5-95 but was four times higher than wild type in T8-1 nodules. The activity of starch phosphorylase (EC 2.4.1.1) increased in all nodule types from 14 to 21 days postinfection. A positive correlation was observed between the capacity of nodules to fix N2 and their capacity to degrade starch. Collectively, these results support the concept that starch accumulated during early stages of nodule development is metabolized to supply energy for nitrogen fixation and to meet the metabolic demands of bacteroids. Key words: nitrogen fixation, starch content, effective and ineffective nodules, starch synthase, starch phosphorylase, α-amylase.

Download Full-text