title: An extracytoplasmic function sigma factor cotranscribed with its cognate anti-sigma factor confers tolerance to NaCl, ethanol and methylene blue in Azospirillum brasilense Sp7.
abstract: 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 extra-cytoplasmic 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 slow-growing, 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.
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, India.
authors.forename: Mukti Nath
authors.initials: MN
authors.lastname: Mishra
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, India.
authors.forename: Santosh
authors.initials: S
authors.lastname: Kumar
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, India.
authors.forename: Namrata
authors.initials: N
authors.lastname: Gupta
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, India.
authors.forename: Simarjot
authors.initials: S
authors.lastname: Kaur
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, India.
authors.forename: Ankush
authors.initials: A
authors.lastname: Gupta
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, India.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Tripathi
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000633:toxicity*; D015806:Azospirillum brasilense / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000431:Ethanol / Q000378:metabolism / Q000633:toxicity; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008751:Methylene Blue / Q000378:metabolism / Q000633:toxicity; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics; D012808:Sigma Factor / Q000096:biosynthesis* / Q000235:genetics; D012965:Sodium Chloride / Q000378:metabolism / Q000633:toxicity; D013312:Stress, Physiological; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
chemical_list: D000900:Anti-Bacterial Agents; D012097:Repressor Proteins; D012808:Sigma Factor; C063202:sporulation-specific sigma factors; D000431:Ethanol; D012965:Sodium Chloride; D008751:Methylene Blue