Molecular cloning and genetic organization of C4-dicarboxylate transport genes from Rhizobium leguminosarum.

ABSTRACT Rhizobium leguminosarum bv. viciae forms nitrogen-fixing nodules on several legumes, including pea (Pisum sativum) and vetch (Vicia cracca), and has been widely used as a model to study nodule biochemistry. To understand the complex biochemical and developmental changes undergone by R. leguminosarum bv. viciae during bacteroid development, microarray experiments were first performed with cultured bacteria grown on a variety of carbon substrates (glucose, pyruvate, succinate, inositol, acetate, and acetoacetate) and then compared to bacteroids. Bacteroid metabolism is essentially that of dicarboxylate-grown cells (i.e., induction of dicarboxylate transport, gluconeogenesis and alanine synthesis, and repression of sugar utilization). The decarboxylating arm of the tricarboxylic acid cycle is highly induced, as is γ-aminobutyrate metabolism, particularly in bacteroids from early (7-day) nodules. To investigate bacteroid development, gene expression in bacteroids was analyzed at 7, 15, and 21 days postinoculation of peas. This revealed that bacterial rRNA isolated from pea, but not vetch, is extensively processed in mature bacteroids. In early development (7 days), there were large changes in the expression of regulators, exported and cell surface molecules, multidrug exporters, and heat and cold shock proteins. fix genes were induced early but continued to increase in mature bacteroids, while nif genes were induced strongly in older bacteroids. Mutation of 37 genes that were strongly upregulated in mature bacteroids revealed that none were essential for nitrogen fixation. However, screening of 3,072 mini-Tn5 mutants on peas revealed previously uncharacterized genes essential for nitrogen fixation. These encoded a potential magnesium transporter, an AAA domain protein, and proteins involved in cytochrome synthesis.

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Inactivation and Regulation of the Aerobic C4-Dicarboxylate Transport (dctA) Gene ofEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.181.18.5624-5635.1999 ◽

1999 ◽

Vol 181 (18) ◽

pp. 5624-5635 ◽

Cited By ~ 72

Author(s):

Suzanne J. Davies ◽

Paul Golby ◽

Davood Omrani ◽

Susan A. Broad ◽

Vikki L. Harrington ◽

...

Keyword(s):

Catabolite Repression ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Northern Blot Analysis ◽

Rhodobacter Capsulatus ◽

Transcriptional Start Site ◽

Receptor Protein ◽

Growth Defect ◽

E Coli ◽

Dicarboxylate Transport

ABSTRACT The gene (dctA) encoding the aerobic C4-dicarboxylate transporter (DctA) of Escherichia coli was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the dctA gene: f428 at 79.3 min and theo157a-o424-o328 (or orfQMP) operon at 79.9 min. The f428 gene encodes a homologue of theSinorhizobium meliloti and Rhizobium leguminosarum H+/C4-dicarboxylate symporter, DctA, whereas the orfQMP operon encodes homologues of the aerobic periplasmic-binding protein- dependent C4-dicarboxylate transport system (DctQ, DctM, and DctP) ofRhodobacter capsulatus. To determine which, if either, of these loci specify the E. coli DctA system, the chromosomalf428 and orfM genes were inactivated by inserting Spr or Apr cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in thef428 mutant and succinate transport was ∼10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the f428 mutant were complemented by transformation with an f428-containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to thedctA gene and indicate that the orfQMP products play no role in C4-dicarboxylate transport. Regulation studies with a dctA-lacZ (f428-lacZ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C4-dicarboxylates and citrate. Interestingly, in a dctA mutant, expression ofdctA is constitutive with respect to C4-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic dctA transcript and confirmed thatdctA is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.

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Study of the genetic organization of the strain Rhizobium leguminosarum bv. trifolii forming a symbiosis with clover Trifolium ambiguum

Abstract book of the 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology" PLAMIC2020 ◽

10.28983/plamic2020.014 ◽

2020 ◽

Author(s):

T. S. Aksenova ◽

O. P. Onishchuk ◽

O. N. Kurchak ◽

E. E. Andronov ◽

N. A. Provorov

Keyword(s):

Host Specificity ◽

Rhizobium Leguminosarum ◽

Trifolium Ambiguum ◽

Genetic Organization

R. leguminosarum bv. trifolii strains are characterized by narrow host specificity. We have identified a strain that forms nodules on several types of clover and studied the genetic organization of its symbiotic region.

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

Journal of Bacteriology ◽

10.1128/jb.180.10.2660-2669.1998 ◽

1998 ◽

Vol 180 (10) ◽

pp. 2660-2669 ◽

Cited By ~ 48

Author(s):

Colm J. Reid ◽

Philip S. Poole

Keyword(s):

Signal Detection ◽

Cross Talk ◽

Rhizobium Leguminosarum ◽

Normal Growth ◽

Inducible Promoter ◽

Ligand Specificity ◽

Dicarboxylate Transport ◽

Transposon Insertion ◽

Gene Coding ◽

Fusion Analysis

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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