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An Extracytoplasmic Function Sigma Factor-Dependent Periplasmic Dai et al. BMC Microbiology (2015) 15:34 DOI 10.1186/s12866-015-0357-0 RESEARCH ARTICLE Open Access An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis Jingcheng Dai1,2, Hehong Wei1,2, Chunyuan Tian3, Fredrick Heath Damron4, Jizhong Zhou5* and Dongru Qiu1,2* Abstract Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2,are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses. Keywords: Periplasmic glutathione peroxidase, Shewanella, ECF sigma factor, Oxidative stress response Background to electrodes [3,4]. Shewanella species harbor a variety of The γ-proteobacteria Shewanella species have two outer membrane and periplasmic c-type cytochrome genes hallmark traits, respiratory versatility and psychrophily expressed for respiration under different environmental [1,2]. Respiratory versatility is characterized by their conditions. Bacterial gene expression is regulated by a series ability to utilize a series of organic and inorganic of transcriptional factors including alternative sigma factors electron acceptors, particularly metals and metalloids (σS). Sigma factors are a component of bacterial RNA of Fe(III), Mn(IV), Ur(VI) and the direct electron transfer polymerase (RNAP) and determine promoter selectivity of the holoenzyme, thus playing a central role in the regulation of gene expression. Bacteria usually have * Correspondence: [email protected]; [email protected] 5Institute for Environmental Genomics and Department of Botany and one housekeeping σ factor (RpoD) and a variable Microbiology, The University of Oklahoma, Stephenson Research and number of alternative σ factors that possess different Technology Center, 101 David L. Boren Blvd, Norman OK 73019, USA promoter-recognition properties [5]. The number of 1Institute of Hydrobiology, Chinese Academy of Sciences, 7 South Donghu Road, Wuchang District, Wuhan 430072, China alternative σ factors highly varies among bacteria and may Full list of author information is available at the end of the article © 2015 Dai et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dai et al. BMC Microbiology (2015) 15:34 Page 2 of 12 be related to their specific habitat, metabolisms, and as the RpoE2 dependent periplasmic glutathione peroxidase development [5-9]. Extracytoplasmic function (ECF) σ fac- that facilitates resistance to oxidative stress. Understanding tors are highly regulated factors that control expression of the regulation of RpoE and RpoE2 and the genes they con- genes and constitute the third pillar of bacterial signal trans- trol can help explain the ability of S. oneidensis to survive duction after the one-component and two-component against environmental stress. systems [9]. Most ECF σs are sequestered by an anti-sigma factor, which can be deactivated by proteolysis, conform- Results ational change, partner switching (including mimicry) or RpoE ECF sigma factors and anti-sigma factor genes in other unknown mechanisms to release the ECF sigma factor S. oneidensis MR-1 from being sequestered [9]. Once the ECF sigma factor is The homologues for the E. coli primary σ factor, RpoD, released it can then activate of transcription of regulon and five out of six alternative σ factors RpoN, RpoS, genes throughout the genome. ECF sigma factor RpoE and RpoH, RpoE, and FliA (RpoF), are present in all the its regulators have been extensively studied in E. coli sequenced genomes of Shewanella (data not shown). [10-17], Pseudomonas aeruginosa [18-22] and Bacillus Several Shewanella strains such as S. baltica OS155 and subtilis [6,7]. RpoE regulates a series of extracytoplasmic S. putrefaciens W3-18-1 also contain another FliA for functions, including synthesis of envelope proteins, outer lateral flagella [38]. However, the FecR (anti-sigma membrane protein (OMP) modification, cell envelope struc- factor)-FecI (sigma factor)-FecA (ferric citrate receptor) ture and cell division in E. coli [23]. The RpoE counterpart iron-starvation signaling system is absent in most of the AlgU/T controls the production of a series of pathogenic sequenced Shewanella strains, except for a few S. baltica factors, lipoproteins, and the extracellular polysaccharide strains. There are five ECF-like σ factors, encoded by alginate in P. aeruginosa which causes the mortality and SO_1342, SO_1986 SO_3096, SO_3551, and SO_3840, morbidity of patients with cystic fibrosis [24-26]. found in S. oneidensis MR-1. SO_1342 was identified as The sigma factors of Shewanella have remained relatively the orthologue for rpoE (σE)ofE. coli and algU/T of P. uncharacterized. The genome of Shewanella oneidensis aeruginosa based on the high sequence similarity and MR-1 encodes 10 sigma factors (RpoD, RpoH, RpoS, RpoN, the well-conserved gene cluster of rpoE-rseA-rseB-rseC FliA, and five ECF sigma factors RpoE, RpoE2, SO_3551 and flanking genes (Figure 1). SO_1986 (designated (ECF-like), SO_3096 (ECF-like) and SO_3840 (ECF-like). rpoE2 hereafter) encodes the orthologue for RpoE of Sigma32 (RpoH) is the heat shock response sigma factor the photosynthetic α-proteobacterium Rhodobacter and it has been shown that heat shock activates expression sphaeroides, and the downstream locus SO_1985 encodes of 323 genes and represses expression of 286 genes [27,28]. the putative cognate anti-σ factor homologous to ChrR In S. violacea strain DSS12, three RpoE-like sigma factors [39]. We further characterized the cellular functions of have been identified [29,30]. Numerous transcriptomic rpoE (σE)andrpoE2 experimentally and computationally. studies have shown Shewanella can modulate gene expres- sion in response to its environmental signals [29-37]. To RpoE and RpoE2 of S. oneidensis are responsible for shed light on the role of two of the RpoE sigma factors of S. diverse stress responses oneidensis MR-1, comparative studies were conducted in In order to characterize the roles of each of the RpoE this study. Deletion mutants were generated and utilized to sigma factors, the rpoE and rpoE2 genes were deleted ascertain the specific functions of each RpoE sigma from strain MR-1. The rpoE and rpoE2 mutant strains had factor and the two sigma factors dependent genes no observable growth defects in rich media (Figure 2A). were identified. RpoE was required for growth at cold We examined the role of the rpoE sigma factor genes in and high temperatures, in minimal media, and in high growth under the stress conditions. The rpoE mutant salt environments. Unlike RpoE, RpoE2 is responsible displayed a severe growth defect when cultured in the for resistance to oxidative stress. PgpD was identified minimal medium, but no growth defect was observed for Figure 1 The gene clusters of rpoE-rseA-rseB-rseC and rpoE2-ChrR and the flanking loci on the chromosome of the S. oneidensis MR-1 strains. The conserved gene cluster rpoE-rseA-rseB-rseC and the flanking genes are also found in the genomes of Escherichia coli and Pseudomonas aeruginosa. Dai et al. BMC Microbiology (2015) 15:34 Page 3 of 12 Figure 2 (See legend on next page.) Dai et al. BMC Microbiology (2015) 15:34 Page 4 of 12 (See figure on previous page.) Figure 2 The rpoE mutant had growth defects when cultured in minimal media, high salinity, and high or low temperature. Bacterial growth, as measured by OD600, are show for the strains growing in various conditions: A) Rich medium (LB broth); B) Nutrient-poor environment (the
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