Roles of DctA and DctB in Signal Detection by the Dicarboxylic Acid Transport System of Rhizobium leguminosarum

ABSTRACT The dctA gene, coding for the dicarboxylate transport protein, has an inducible promoter dependent on activation by the two-component sensor-regulator pair DctB and DctD. LacZ fusion analysis indicates that there is a single promoter for dctB anddctD. The dctA promoter is also induced by nitrogen limitation, an effect that requires DctB-DctD and NtrC. DctB alone is able to detect dicarboxylates in the absence of DctA and initiate transcription via DctD. However, DctA modifies signal detection by DctB such that in the absence of DctA, the ligand specificity of DctB is broader. dctAp also responds to heterologous induction by osmotic stress in the absence of DctA. This effect requires both DctB and DctD. A transposon insertion in thedctA-dctB intergenic region (dctA101) which locks transcription of dctA at a constitutive level independent of DctB-DctD results in improper signalling by DctB-DctD. Strain RU150, which carries this insertion, is defective in nitrogen fixation (Fix−) and grows very poorly on ammonia as a nitrogen source whenever the DctB-DctD signalling circuit is activated by the presence of a dicarboxylate ligand. Mutation ofdctB or dctD in strain RU150 reinstates normal growth on dicarboxylates. This suggests that DctD-P improperly regulates a heterologous nitrogen-sensing operon. Increased expression of DctA, either via a plasmid or by chromosomal duplication, restores control of DctB-DctD and allows strain RU150 to grow on ammonia in the presence of a dicarboxylate. Thus, while DctB is a sensor for dicarboxylates in its own right, it is regulated by DctA. The absence of DctA allows DctB and DctD to become promiscuous with regard to signal detection and cross talk with other operons. This indicates that DctA contributes significantly to the signalling specificity of DctB-DctD and attenuates cross talk with other operons.

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Two of the three groEL homologues in Rhizobium leguminosarum are dispensable for normal growth

Archives of Microbiology ◽

10.1007/s00203-005-0768-7 ◽

2005 ◽

Vol 183 (4) ◽

pp. 253-265 ◽

Cited By ~ 35

Author(s):

F. Rodríguez-Quiñones ◽

M. Maguire ◽

E. J. Wallington ◽

Phillip S. Gould ◽

V. Yerko ◽

...

Keyword(s):

Rhizobium Leguminosarum ◽

Normal Growth

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The maize heterotrimeric G protein β subunit controls shoot meristem development and immune responses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1917577116 ◽

2019 ◽

Vol 117 (3) ◽

pp. 1799-1805 ◽

Cited By ~ 9

Author(s):

Qingyu Wu ◽

Fang Xu ◽

Lei Liu ◽

Si Nian Char ◽

Yezhang Ding ◽

...

Keyword(s):

G Protein ◽

Cross Talk ◽

Normal Growth ◽

Shoot Meristem ◽

Β Subunit ◽

Subunit Gene ◽

Α Subunit ◽

Knock Out ◽

Meristem Size ◽

Meristem Development

Heterotrimeric G proteins are important transducers of receptor signaling, functioning in plants with CLAVATA receptors in controlling shoot meristem size and with pathogen-associated molecular pattern receptors in basal immunity. However, whether specific members of the heterotrimeric complex potentiate cross-talk between development and defense, and the extent to which these functions are conserved across species, have not yet been addressed. Here we used CRISPR/Cas9 to knock out the maize G protein β subunit gene (Gβ) and found that the mutants are lethal, differing from those in Arabidopsis, in which homologous mutants have normal growth and fertility. We show that lethality is caused not by a specific developmental arrest, but by autoimmunity. We used a genetic diversity screen to suppress the lethal Gβ phenotype and also identified a maize Gβ allele with weak autoimmune responses but strong development phenotypes. Using these tools, we show that Gβ controls meristem size in maize, acting epistatically with G protein α subunit gene (Gα), suggesting that Gβ and Gα function in a common signaling complex. Furthermore, we used an association study to show that natural variation in Gβ influences maize kernel row number, an important agronomic trait. Our results demonstrate the dual role of Gβ in immunity and development in a cereal crop and suggest that it functions in cross-talk between these competing signaling networks. Therefore, modification of Gβ has the potential to optimize the trade-off between growth and defense signaling to improve agronomic production.

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An uncharacterized gene coding a conserved lytic transglycosylase domain (RL4716) is required for proper cell envelope function in Rhizobium leguminosarum

FEMS Microbiology Letters ◽

10.1093/femsle/fnx035 ◽

2017 ◽

Vol 364 (5) ◽

Cited By ~ 2

Author(s):

Kara D. Neudorf ◽

Christopher K. Yost

Keyword(s):

Rhizobium Leguminosarum ◽

Cell Envelope ◽

Envelope Function ◽

Lytic Transglycosylase ◽

Uncharacterized Gene ◽

Gene Coding

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Inducible Expression of a Phytolacca heterotepala Ribosome-Inactivating Protein Leads to Enhanced Resistance Against Major Fungal Pathogens in Tobacco

Phytopathology ◽

10.1094/phyto-95-0206 ◽

2005 ◽

Vol 95 (2) ◽

pp. 206-215 ◽

Cited By ~ 36

Author(s):

Giandomenico Corrado ◽

Pasquale Delli Bovi ◽

Rosalia Ciliento ◽

Luciano Gaudio ◽

Antimo Di Maro ◽

...

Keyword(s):

Fungal Pathogens ◽

Inducible Promoter ◽

Antimicrobial Activities ◽

Leaf Damage ◽

Ribosome Inactivating Proteins ◽

Gene Coding ◽

Transgenic Lines ◽

New Gene ◽

Polygalacturonase Inhibiting Protein ◽

I Gene

Plant genetic engineering has long been considered a valuable tool to fight fungal pathogens because it would limit the economically costly and environmentally undesirable chemical methods of disease control. Ribosome-inactivating proteins (RIPs) are potentially useful for plant defense considering their antiviral and antimicrobial activities but their use is limited by their cytotoxic activity. A new gene coding for an RIP isolated from leaves of Phytolacca heterotepala was expressed in tobacco under the control of the wound-inducible promoter of the bean polygalacturonase-inhibiting protein I gene to increase resistance against different fungal pathogens, because an individual RIP isolated from P. heterotepala showed direct antifungal toxicity. Phenotypically normal transgenic lines infected with Alternaria alternata and Botrytis cinerea showed a significant reduction of leaf damage while reverse transcription-polymerase chain reaction and western analysis indicated the expression of the RIP transgene upon wounding and pathogen attack. This work demonstrates that use of a wound-inducible promoter is useful to limit the accumulation of antimicrobial phytotoxic proteins only in infected areas and that the controlled expression of the PhRIP I gene can be very effective to control fungal pathogens with different phytopathogenic actions.

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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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Inactivation of a Cold-Induced Putative RNA Helicase Gene of Listeria monocytogenes Is Accompanied by Failure To Grow at Low Temperatures but Does Not Affect Freeze-Thaw Tolerance

Journal of Food Protection ◽

10.4315/0362-028x-73.8.1474 ◽

2010 ◽

Vol 73 (8) ◽

pp. 1474-1479 ◽

Cited By ~ 12

Author(s):

REHA O. AZIZOGLU ◽

S. KATHARIOU

Keyword(s):

Listeria Monocytogenes ◽

Normal Growth ◽

Rna Helicase ◽

Soft Agar ◽

Freezing And Thawing ◽

Freeze Thaw ◽

Transposon Insertion ◽

Helicase Gene ◽

Repeated Freezing ◽

Cold Sensitive

Freeze-thaw tolerance (cryotolerance) of Listeria monocytogenes is markedly influenced by temperature of growth of the bacteria, and may involve responses to low-temperature stresses encountered during freezing and thawing. A cold-sensitive mariner-based transposon mutant of L. monocytogenes F2365 was found to harbor a single insertion in LMOf2365_1746, encoding a putative RNA helicase, and earlier shown by other investigators to be induced during 4°C growth of L. monocytogenes. The mutant had normal growth at 37°C but completely failed to grow at either 4 or 10°C, and had impaired growth and reduced swarming on soft agar at 25°C. However, the mutation had no discernible influence on the ability of the bacteria to tolerate repeated freezing and thawing after growth at either 25 or 37°C. The findings suggest that the transposon insertion in the putative helicase gene, in spite of the severely cold-sensitive phenotype that accompanies it, does not affect the ability of the bacteria to cope with cold-related stresses encountered during repeated freezing and thawing.

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The Rhizobium GstI Protein Reduces the NH4+ Assimilation Capacity of Rhizobium leguminosarum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.7.823 ◽

2001 ◽

Vol 14 (7) ◽

pp. 823-831 ◽

Cited By ~ 7

Author(s):

Rosarita Tatè ◽

Luigi Mandrich ◽

Maria R. Spinosa ◽

Anna Riccio ◽

Alessandro Lamberti ◽

...

Keyword(s):

Glutamine Synthetase ◽

Rhizobium Leguminosarum ◽

Inducible Promoter ◽

Complete Loss ◽

Host Cells ◽

Single Amino Acid ◽

Assimilation Capacity ◽

Translational Inhibitor ◽

Glutamine Synthetase I ◽

N Status

We show that the protein encoded by the glutamine synthetase translational inhibitor (gstI) gene reduces the NH4+ assimilation capacity of Rhizobium leguminosarum. In this organism, gstI expression is regulated by the ntr system, including the PII protein, as a function of the nitrogen (N) status of the cells. The GstI protein, when expressed from an inducible promoter, inhibits glutamine synthetase II (glnII) expression under all N conditions tested. The induction of gstI affects the growth of a glutamine synthetase I (glnA-) strain and a single amino acid substitution (W48D) results in the complete loss of GstI function. During symbiosis, gstI is expressed in young differentiating symbiosomes (SBs) but not in differentiated N2-fixing SBs. In young SBs, the PII protein modulates the transcription of NtrC-regulated genes such as gstI and glnII. The evidence presented herein strengthens the idea that the endocytosis of bacteria inside the cytoplasm of the host cells is a key step in the regulation of NH4+ metabolism.

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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.

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mariner-Based Transposon Mutagenesis of Rickettsia prowazekii

Applied and Environmental Microbiology ◽

10.1128/aem.01727-07 ◽

2007 ◽

Vol 73 (20) ◽

pp. 6644-6649 ◽

Cited By ~ 50

Author(s):

Zhi-Mei Liu ◽

Aimee M. Tucker ◽

Lonnie O. Driskell ◽

David O. Wood

Keyword(s):

Fluorescent Protein ◽

Rickettsia Prowazekii ◽

Obligate Intracellular ◽

Transposon Insertion ◽

Gene Coding ◽

Epidemic Typhus ◽

Intergenic Regions ◽

Rifampin Resistance ◽

Green Fluorescent ◽

Random Transpositions

ABSTRACT Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFPUV) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFPUV were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFPUV fluorescence.

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