&amp;lt;italic&amp;gt;Sinorhizobium meliloti&amp;lt;/italic&amp;gt; NtrX interacts with different regions of the &amp;lt;italic&amp;gt;visN&amp;lt;/italic&amp;gt; promoter

Huang

et al. 2020

Preprint

Self Cite

In α-proteobacteria, the CtrA signaling pathway regulates cell cycle progression. A species whose cell duplication is associated with CtrA stability is affected by the response regulator NtrX. However, the function of NtrX acting on the cell cycle regulation in bacteria remains unclear. Here, we report that NtrX controls transcription of the CtrA system genes involved in cell cycle regulation in a legume symbiont, Sinorhizobium meliloti. Three groups of ntrX mutants showed the similar cell cycle defects, such as slow growth, abnormal shapes, and irregular genomic DNA accumulation. Expression of the CtrA signaling pathway genes including ctrA, gcrA, dnaA, divL and cpdR1, is differentially regulated by the phosphorylated NtrX protein. The regulation is achieved through direct protein-DNA interactions. The 53rd aspartate residue known as the conserved phosporylation site and located in the receiver domain of NtrX, is required for S. meliloti cell cycle regulation. Interestingly, expression of S. meliloti ntrX derivatives in Caulobacter and Agrobacterium strains showed distinct defects of cell duplication and growth, suggesting that NtrX plays different roles in cell cycle regulation in these bacteria. Our findings demonstrate that NtrX is an upstream transcriptional regulator of the CtrA signaling pathway in S. meliloti, which could be associated with nitrogen nutrient response.

“…EMSA was performed as described by Zeng (35). LightShift™ Chemiluminescent EMSA Kit (ThermoFisher) was applied in the assays.…”

Section: Methodsmentioning

confidence: 99%

“…ChIP was performed as described by Pini (32). Anti-NtrX antibodies prepared by Wenyuange (Shanghai) were used for ChIP assays (35).…”

Section: Chromatin Immunoprecipitation (Chip)mentioning

confidence: 99%

Section: Western Blottingmentioning

confidence: 99%

Section: Regulation Of Protein Levels Of Ctra and Gcra By Ntrx In S Melilotimentioning

confidence: 99%

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Direct regulation of cell cycle regulatory gene expression by NtrX to promoteSinorhizobium meliloticell division

Huang

et al. 2020

Preprint

Self Cite

“…Samples were separated by a Phos-Tag Synchronized S. meliloti cells (Sm1021, SmLL1, Sm1021/pntrX and Sm1021/pntrX D53E ) were subcultured in LB/MC broth containing 1 mM IPTG or not for 1 to 3 h. The cells from 1 ml culture were pelleted, resuspended in the buffer of 10 mM Tris-Cl, pH 7.5 and 4% SDS, incubated at room temperature for 5 min, mixed the loading dye, boiled for 10 min, and then loaded into the wells of Phos-tag™ acrylamide gels. Western blots were performed as described as Tang (41), with rabbit anti-NtrX (1:10000) antibodies (36). Chemiluminescent detection was performed using an ECL fluorescence colorimetric kit (Tiangen) and fluorescent signals were visualized using a Bio-Rad Gel Doc XR.…”

Section: Rna Extraction Purification and Qrt-pcrmentioning

confidence: 99%

Direct regulation of cell cycle regulatory gene expression by NtrX to promote Sinorhizobium meliloti cell division

Luo

Zhang

et al. 2021

Preprint

Self Cite

Cell division of the alfalfa symbiont, Sinorhizobium meliloti, is regulated by the CtrA signaling network. The gene expression of regulatory proteins in the network is affected by nutrient signaling. In this study, we found that NtrX, one of the regulators of nitrogen metabolic response, can directly regulate the expression of several regulatory genes from the CtrA signaling network. Three sets of S. meliloti ntrX mutants, including the plasmid insertion strain, the depletion strain and the substitution of the 53rd aspartate (ntrXD53E) from a plasmid in the wild-type strain (Sm1021), showed similar cell division defects, such as slow growth, abnormal morphology of partial cells and delayed DNA synthesis. Transcript quantitative evaluation indicated that the transcription of genes such as ctrA and gcrA was up-regulated, while the transcription of genes such as dnaA and ftsZl was down-regulated in the insertion mutant and the strain of Sm1021 expressing ntrXD53E. Correspondingly, inducible transcription of ntrX activates the expression of dnaA and ftsZ1, but represses ctrA and gcrA in the depletion strain. The expression levels of CtrA and GcrA were confirmed by western blotting, which were consistent with the transcription data. The transcriptional regulation of these genes requires phosphorylation of the conserved 53rd aspartate in the NtrX protein. The NtrX protein binds directly to the promoter regions of ctrA, gcrA, dnaA and ftsZ1 by recognizing the characteristic sequence CAAN2-5TTG Our findings reveal that NtrX is a novel transcriptional regulator of the CtrA signaling pathway genes, and positively affects bacterial cell division, associated with nitrogen metabolism.IMPORTANCESinorhizobium meliloti infects the host alfalfa and induces formation of nitrogen-fixing nodules. Proliferation of rhizobia in plant tissues and cells is strictly controlled in the early stage of symbiotic interactions. However, the control mechanism is not very clear. Cell division of S. meliloti in the free-living state is regulated by the CtrA signaling network, but molecular mechanisms by which the CtrA system is associated with environmental nutrient signals (e.g., ammonia nitrogen) need to be further explored. This study demonstrates that NtrX, a regulator of nitrogen metabolism, required for symbiotic nodulation and nitrogen fixation by S. meliloti 1021, can act as a transcriptional regulator of the CtrA signaling system. It may link nitrogen signaling to cell cycle regulation in Rhizobium species.

Transcription Regulation of Cell Cycle Regulatory Genes Mediated by NtrX to Affect Sinorhizobium meliloti Cell Division

Zheng

et al. 2022

Genes

Self Cite

The cell division of the alfalfa symbiont, Sinorhizobium meliloti, is dictated by a cell cycle regulatory pathway containing the key transcription factors CtrA, GcrA, and DnaA. In this study, we found that NtrX, one of the regulators of nitrogen metabolism, can directly regulate the expression of ctrA, gcrA, and dnaA from the cell cycle pathway. Three sets of S. meliloti ntrX mutants showed similar cell division defects, such as slow growth, abnormal morphology of some cells, and delayed DNA synthesis. Transcription of ctrA and gcrA was upregulated, whereas the transcription of dnaA and ftsZ1 was downregulated in the insertion mutant and the strain of Sm1021 expressing ntrXD53E. Correspondingly, the inducible transcription of ntrX activates the expression of dnaA and ftsZ1, but represses ctrA and gcrA in the depletion strain. The expression levels of CtrA and GcrA were confirmed by Western blotting. The transcription regulation of these genes requires phosphorylation of the conserved 53rd aspartate in the NtrX protein that binds directly to the promoter regions of ctrA, gcrA, dnaA, and ftsZ1 by recognizing the characteristic sequence CAAN2-5TTG. Our findings suggest that NtrX affects S. meliloti cell division by regulating the transcription of the key cell cycle regulatory genes.