Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Tue, 11 Dec 2018 15:02:10 A constitutively expressed pair of rpoE2–chrR2 in Azospirillum brasilense Sp7 is required for survival under antibiotic and oxidative stress Namrata Gupta, Santosh Kumar, Mukti Nath Mishra and Anil Kumar Tripathi Correspondence Anil Kumar Tripathi tripathianil@rediffmail.com Received 5 July 2012 Revised 21 September 2012 Accepted 4 October 2012 School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, India Extracytoplasmic function (ECF) sigma factors (s E ) 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 s E , 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 ( 1 O 2 )-mediated dissociation of the RpoE2–ChrR2 complex might release RpoE2 to drive the expression of its target genes. INTRODUCTION Azospirillum brasilense, a Gram-negative, plant-growth-pro- moting a-proteobacterium of the family Rhodospirillaceae, lives in association with the rhizosphere of a large number of non-leguminous plants and brings about plant growth promotion via the production of phytohormones and siderophores (Baldani et al., 1979; Steenhoudt & Vanderleyden, 2000). Bacteria inhabiting the soil and rhizo- sphere frequently encounter fluctuations in osmolarity, temperature, pH, nutrients, reactive oxygen species and other abiotic factors. They have evolved mechanisms to respond and adapt to such environmental contingencies (Park et al., 2008; Steenhoudt & Vanderleyden, 2000). This is achieved by the reversible association of different s factors with bacterial RNA polymerase to alter the pattern of gene expression by changing the affinity and specificity of RNA polymerase for different promoters (Helmann, 2002). The extracytoplasmic function (ECF) sigma factors (s E ) belong to the largest and the most diverse subfamily of s factors, which is involved in a wide range of stress adaptations (Alvarez-Martinez et al., 2007; Anthony et al., 2005; Bang et al., 2005; Firoved et al., 2002; Martı´nez- Salazar et al., 2009; Rowley et al., 2006; Schurr et al., 1996). The activity of an ECF sigma factor is often regulated by its association with an anti-sigma factor that receives diverse signals via different mechanisms (Helmann, 2002). After receiving a stimulus, the anti-sigma factor is released from the sigma factor, which can bind together with the core enzyme to the promoter regions of specific genes to drive their transcription. Most often, ECF sigma factor-encoding genes are organized in an operon along with an anti-sigma factor-encoding gene located downstream (Brown & Hughes, 1995; Mishra et al., 2011; Staron´ et al., 2009). Most of the anti-sigma factors possess a well-conserved N- terminal anti-sigma domain (ASD), which is involved in the sequestering of the ECF sigma factor to prevent it from binding to the RNA polymerase core enzyme. The anti- sigma factors, such as RseA of Escherichia coli, are integral cytoplasmic membrane proteins with a C-terminal extra- cytoplasmic sensory domain and an N-terminal intracel- lular inhibitory domain that binds and inhibits the cognate Abbreviations: ASD, anti-sigma domain; CLD, cupin-like domain; ECF, extracytoplasmic function; Ni-NTA, nickel-nitrilotriacetic acid; 1 O 2 , singlet oxygen; RACE, random amplification of cDNA ends; TSS, transcription start site; ZAS, zinc-binding anti-sigma. Five supplementary figures and a supplementary table are available with the online version of this paper. Microbiology (2013), 159, 205–218 DOI 10.1099/mic.0.061937-0 061937 G 2013 SGM Printed in Great Britain 205