Role of the extracytoplasmic function sigma factor RpoE4 in oxidative and osmotic stress responses in Rhizobium etli

Abstract

The aims of this study were to functionally characterize and analyze the transcriptional regulation and transcriptome of the Rhizobium etli rpoE4 gene. An R. etli rpoE4 mutant was sensitive to oxidative, saline, and osmotic stresses. Using transcriptional fusions, we determined that RpoE4 controls its own transcription and that it is negatively regulated by rseF (regulator of sigma rpoE4; CH03274), which is cotranscribed with rpoE4. rpoE4 expression was induced not only after oxidative, saline, and osmotic shocks, but also under microaerobic and stationary-phase growth conditions. The transcriptome analyses of an rpoE4 mutant and an rpoE4-overexpressing strain revealed that the RpoE4 extracytoplasmic function sigma factor regulates about 98 genes; 50 of them have the rpoE4 promoter motifs in the upstream regulatory regions. Interestingly, 16 of 38 genes upregulated in the rpoE4-overexpressing strain encode unknown putative cell envelope proteins. Other genes controlled by RpoE4 include rpoH2, CH00462, CH02434, CH03474, and xthA1, which encode proteins involved in the stress response (a heat shock sigma factor, a putative Mn-catalase, an alkylation DNA repair protein, pyridoxine phosphate oxidase, and exonuclease III, respectively), as well as several genes, such as CH01253, CH03555, and PF00247, encoding putative proteins involved in cell envelope biogenesis (a putative peptidoglycan binding protein, a cell wall degradation protein, and phospholipase D, respectively). These results suggest that rpoE4 has a relevant function in cell envelope biogenesis and that it plays a role as a general regulator in the responses to several kinds of stress.

FIG. 1.

Roles of the tcrX and rpoE genes of R. etli in responses to oxidative and osmotic stresses. The viabilities of the CFNXE1Sp (rpoE1 Sp), CFNXE2Sp (rpoE2 Sp), CFNXE3Sp (rpoE3 Sp), CFNXE4Sp (rpoE4 Sp), CFNXΔ3274Sp (rseF Sp), CFNXΔ3274lox (rseF lox), and CFNXTXSp (tcrX Sp) strains relative to that of wild-type strain CE3 in the presence of H₂O₂ (A), methyl viologen (B), NaCl (C), and sucrose (D) are expressed as the surviving fractions of the mutant strain populations divided by the surviving fraction of the CE3 population. The surviving fraction was calculated as the number of viable cells after treatment with or in the presence of the compound, divided by the number of viable cells in the absence of stress. The surviving fractions of the CE3 populations in the presence of H₂O₂, methyl viologen, NaCl, and sucrose were 0.02, 0.92, 0.93, and 0.93, respectively. The data presented are the averages of results from at least three independent experiments. For H₂O₂ treatment, exponential-phase cultures (OD₆₀₀, ∼0.3) were incubated with 5 mM H₂O₂ for 45 min. For the other treatments, the bacteria were grown in PY medium containing methyl viologen (40 μM), NaCl (80 mM), or sucrose (15%, wt/vol).

FIG. 2.

Physical organization of R. etli rseF-rpoE4 and tcrX genes and expression under different stress conditions. (A) Genomic organization of the rseF-rpoE4 and tcrX region. The fragment cloned upstream of uidA into pBBMCS53 is indicated by the lower diagram. (B and C) Levels of expression of the rpoE4-uidA (B) and trcX-uidA (C) transcriptional fusions under oxidative, saline, or osmotic stress. Exponential-phase cultures (OD₆₀₀, ∼0.3) were grown on MM, and CE3/pJMS24 and CE3/pJMS25 strains were incubated with 1 mM H₂O₂, 100 mM NaCl, 10% (wt/vol) sucrose (Suc), or 40 μM methyl viologen (PQ) for 45 min. Specific activity (Sp Act) was determined by using β-glucuronidase activity. The data shown are the averages of results from at least three independent experiments, and the vertical bars represent the standard deviations. −, no stress treatment.

FIG. 3.

The R. etli rpoE4 gene is autoregulated and controls the expression of the rpoE4, trcX, and rpoH2 genes under different conditions. The rpoE4-uidA (A), trcX-uidA (B), and rpoH2-uidA (C) transcriptional fusions were expressed in the wild-type (wt), rpoE4::Sp (E4), and ΔrseF::lox (rse) genetic backgrounds. The strains containing pJMS24, pJMS25, or pGUSprpoH2 were grown in MM under aerobic (Aer) or microaerobic (MA) conditions, and samples were collected at exponential (Exp; OD₆₀₀, ∼0.3) and stationary (Sta; OD₆₀₀, ∼0.8) growth phases. Specific activity (Sp Act) was determined by using β-glucuronidase activity. The data shown are the averages of results from at least three independent experiments, and the vertical bars represent the standard deviations.

FIG. 4.

R. elti rpoE4 motifs located in the regulatory regions of rpoE4-regulated genes. (A) Alignment of nucleotide sequences of upstream regions of rpoE4-regulated genes (motifs are shown in boldface, uppercase letters and are underlined); (B) WebLogo of the rpoE4 consensus motifs; (C) alignment of R. etli rpoE4 consensus motifs with promoter sequences of S. meliloti rpoE2, C. crescentus sigT, M. ex torquens AM1 phyR, M. tuberculosis sigH, E. coli rpoE, S. coelicolor sigR, R. sphaeroides rpoE, B. subtilis sigW, and P. aeruginosa algU (4, 5, 17, 22, 34, 39, 41, 48).