Browsing by Author "Namrata Gupta"

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A constitutively expressed pair of rpoE2-chrR2 in azospirillum brasilense Sp7 is required for survival under antibiotic and oxidative stress
(2013) Namrata Gupta; Santosh Kumar; Mukti Nath Mishra; Anil Kumar Tripathi
Extracytoplasmic function (ECF) sigma factors (σE) are known to bring about changes in gene expression to enable bacteria to adapt to different stresses. The Azospirillum brasilense Sp245 genome harbours nine genes encoding σE, of which two are adjacent to the genes encoding ChrR-type zinc-binding anti-sigma (ZAS) factors. We describe here the role and regulation of a new pair of rpoE-chrR, which was found in the genome of A. brasilense Sp7 in addition to the previously described rpoE-chrR pair (designated rpoE1-chrR1). The rpoE2-chrR2 pair is also cotranscribed, and their products show protein-protein interaction. The"10 and"35 promoter elements of rpoE2-chrR2 and rpoE1-chrR1 were similar but not identical. Unlike the promoter of rpoE1-chrR1, the rpoE2-chrR2 promoter was neither autoregulated nor induced by oxidative stress. Inactivation of chrR2 or overexpression of rpoE2 in A. brasilense Sp7 resulted in an overproduction of carotenoids. It also conferred resistance to oxidative stresses and antibiotics. By controlling the synthesis of carotenoids, initiation and elongation of translation, protein folding and purine biosynthesis, RpoE2 seems to play a crucial role in preventing and repairing the cellular damage caused by oxidative stress. Lack of autoregulation and constitutive expression of rpoE2-chrR2 suggest that RpoE2-ChrR2 may provide a rapid mechanism to cope with oxidative stress, wherein singlet oxygen (1O2)-mediated dissociation of the RpoE2-ChrR2 complex might release RpoE2 to drive the expression of its target genes. © 2013 SGM.
An extracytoplasmic function sigma factor cotranscribed with its cognate anti-sigma factor confers tolerance to NaCl, ethanol and methylene blue in Azospirillum brasilense Sp7
(2011) Mukti Nath Mishra; Santosh Kumar; Namrata Gupta; Simarjot Kaur; Ankush Gupta; Anil K. Tripathi
Azospirillum brasilense, a plant-growth-promoting rhizobacterium, is exposed to changes in its abiotic environment, including fluctuations in temperature, salinity, osmolarity, oxygen concentration and nutrient concentration, in the rhizosphere and in the soil. Since extracytoplasmic function (ECF) sigma factors play an important role in stress adaptation, we analysed the role of ECF sigma factor (also known as RpoE or σ E) in abiotic stress tolerance in A. brasilense. An in-frame rpoE deletion mutant of A. brasilense Sp7 was carotenoidless and slowgrowing, and was sensitive to salt, ethanol and methylene blue stress. Expression of rpoE in the rpoE deletion mutant complemented the defects in growth, carotenoid biosynthesis and sensitivity to different stresses. Based on data from reverse transcriptase-PCR, a two-hybrid assay and a pull-down assay, we present evidence that rpoE is cotranscribed with chrR and the proteins synthesized from these two overlapping genes interact with each other. Identification of the transcription start site by 5′ rapid amplification of cDNA ends showed that the rpoE-chrR operon was transcribed by two promoters. The proximal promoter was less active than the distal promoter, whose consensus sequence was characteristic of RpoE-dependent promoters found in alphaproteobacteria. Whereas the proximal promoter was RpoE-independent and constitutively expressed, the distal promoter was RpoE-dependent and strongly induced in response to stationary phase and elevated levels of ethanol, salt, heat and methylene blue. This study shows the involvement of RpoE in controlling carotenoid synthesis as well as in tolerance to some abiotic stresses in A. brasilense, which might be critical in the adaptation, survival and proliferation of this rhizobacterium in the soil and rhizosphere under stressful conditions. © 2011 SGM.
Cross-talk between cognate and noncognate rpoe sigma factors and Zn 2+-binding anti-sigma factors regulates photooxidative stress response in azospirillum brasilense
(2014) Namrata Gupta; Ankush Gupta; Santosh Kumar; Rajeev Mishra; Chhaya Singh; Anil Kumar Tripathi
Aims: Azospirillum brasilense harbors two redox-sensitive Zinc-binding anti-sigma (ZAS) factors (ChrR1 and ChrR2), which negatively regulate the activity of their cognate extra-cytoplasmic function (ECF) σ factors (RpoE1 and RpoE2) by occluding their binding to the core enzyme. Both pairs of RpoE-ChrR control responses to photooxidative stress. The aim of this study was to investigate whether the two RpoE-ChrR pairs cross-talk while responding to the stress. Results: In silico analysis showed a high sequence similarity between ChrR1 and ChrR2 proteins, but differences in redox sensitivity. Using in silico and in vitro methods of protein-protein interaction, we have shown that both ChrR1 and ChrR2 proteins physically bind to their noncognate RpoE proteins. Restoration of the phenotypes of chrR1::Tn5 and chrR2::Km mutants related to carotenoid biosynthesis and photooxidative stress tolerance by expressing chrR1 or chrR2 provided in vivo evidence for the cross-talk. In addition, up- or down-regulation of several identical proteins by expressing chrR1 or chrR2 in the chrR1::Tn5 mutant provided another in vivo evidence for the cross-talk. Innovation: Although multiple redox-sensitive ZAS anti-σ factors occur in some Gram-positive bacteria, no cross-talk is reported among them. We report here, for the first time, that the two ZAS anti-σ factors of A. brasilense also interact with their noncognate σ factors and affect gene expression. Conclusion: The two redox-sensitive ZAS anti-σ factors in A. brasilense may interact with their cognate as well as noncognate ECF σ factors to play an important role in redox homeostasis by facilitating recovery from the oxidative stress. Antioxid. Redox Signal. 20, 42-59. © Copyright 2014, Mary Ann Liebert, Inc. 2014.