Article Figures & Data
Figures
- FIG 1
E. coli RIC interactions investigated by BACTH. β-Galactosidase activities of E. coli DHM1 cell lysates separately expressing plasmids A to G, which were extracted from the BamHI library and cotransformed with the pKTN25-RIC (gray bars), pKTN25 empty plasmid (white bars), and pKTN25 fused to TorD (black bars). Each bar represents the mean value ± standard error from results of at least three independent cultures.
- FIG 2
E. coli RIC interacts with Dps. (A) Bacterial two-hybrid assay. The interaction of the RIC protein, linked to the C terminal (white bar) or N terminal (gray bar) of the T18-Cya domain and expressed from pUT18 or pUT18C, respectively, was evaluated in E. coli DHM1 cotransformed with pKTN25 containing a Dps linked to the N terminal of the T25-Cya domain. E. coli cells harboring simultaneously empty pKTN25 and pUT18/pUT18C vectors expressing ric fusions served as negative controls (black bars). (B and C) BiFC assays. Cells were cotransformed with vectors expressing either RICC-GFP (pMRBAD-link-C-GFP-RIC) or RIC-TruncatedC-GFP (pMRBAD-link-C-GFP-RICTrunc) with DpsN-GFP (pET11a-link-N-GFP-Dps). The inverse configurations were also included. (B) Cells expressing RICC-GFP and DpsN-GFP were analyzed by light microscopy (bright field, left upper panel) and fluorescence microscopy (right upper panel). Lower panels depict images of cotransformed cells with empty vectors. Images were acquired using a 100× objective, and a fluorescein isothiocyanate (FITC) filter was used for the acquisition of the fluorescence images. (C) Fluorescence quantification was performed using MetaMorph microscopy automation and image analysis software. Fluorescence values for the negative control (empty plasmid vectors) were normalized to 1. (D) Pulldown assays. Lane 1, cells expressing pET-28a-RIC(HisTag) (RIC protein linked to a N-terminal His-tagged RIC [∼30 kDa]) and pACYCDuet-1-Dps (nonlabeled Dps [∼18 kDa]). Lane 2, cells expressing pET-28a (empty vector) and pACYCDuet-1-Dps (nonlabeled Dps [∼18 kDa]). Protein fractions were eluted from the Ni-chelating column at 100 mM imidazole and analyzed by SDS-PAGE (upper panel) and Western blotting using the anti-E. coli Dps antibody (bottom panel). Values are means ± standard errors from at least three independent cultures analyzed in duplicate. ***, P < 0.0005 (one-way analysis of variance [ANOVA] multiple-comparison test).
- FIG 3
Growth of E. coli Δric, Δdps, and Δdps Δric strains under nitrosative and oxidative stress. E. coli wild-type, Δric, Δdps, and Δdps Δric strains were grown under anaerobic (A) and aerobic (B) conditions. At an OD600 of 0.3, cells were left untreated (No stress), treated with 250 μM spermine NONOate for 7 h (A), or treated with 4 mM H2O2 for 5 h (B). Error bars are ±standard deviations (SD) for experiments carried out at least three times.
- FIG 4
Aconitase and fumarase activity of E. coli Δric and Δdps mutant strains. Aconitase activity of E. coli wild-type, Δric, Δdps, and Δdps Δric strains grown aerobically to OD600 of 0.6 (A and D) or 2.0 (B). Fumarase activity of E. coli wild-type, Δric, Δdps, and Δdps Δric strains grown aerobically to an OD600 of 0.6 (C). Complementation experiments of aconitase activity (D) were performed using Δric and Δdps Δric strains transformed with pUC18, and with pUC18 encoding the RIC protein and the mutated Glu133Leu-RIC (a RIC protein where glutamate 133 underwent site-directed mutation by leucine [10]). Values are represented as normalized to the aconitase or fumarase activity of wild-type cells. Values are means (±standard errors) from at least two independent cultures analyzed in duplicate. *, P < 0.05; **, P < 0.005; ns, not significant (unpaired Student's t test).
- FIG 5
Iron storage proteins Bfr and FtnA do not interact with RIC. (A) The possible interaction between RIC and Bfr or FtnA was analyzed by fluorescence microscopy and quantified by MetaMorph microscopy automation and image analysis software. Cells were cotransformed with vectors expressing RICC-GFP (pMRBAD-C-GFP-RIC) and BfrN-GFP (pET11a-N-GFP-Bfr) or FtnAN-GFP (pET11a-N-GFP-FtnA). The inverse conformations were also examined. Fluorescence values for negative control (empty plasmid vectors) were normalized to 1. Values are means ± standard errors from at least three independent cultures analyzed in duplicate. ns, not significant (one-way ANOVA multiple-comparison test). (B) E. coli cells of single (Δric, Δbfr, and ΔftnA) and double mutant strains (Δbfr Δric and ΔftnA Δric) were grown aerobically, collected at an OD of 0.6, and the aconitase activity was determined. Values are represented as normalized to the aconitase activity of wild-type cells. Values are means ± standard errors from at least two independent cultures analyzed in duplicate. *, P < 0.05; ns, not significant (unpaired Student's t test).
- FIG 6
Intracellular ROS content of Δdps and Δric mutant strains and the effect of combining the Δhpx and Δric mutations on aconitase activity. (A) The E. coli wild-type and isogenic Δric, Δdps, and Δdps Δric mutant strains were grown to OD600 of 0.6, and the intracellular ROS levels were measured by incubation of cell suspensions with 10 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA) for 2 h. Fluorescence intensity was normalized according to the final OD (FI/OD). (B) Aconitase activity of the E. coli wild-type, Δric, Δhpx (ΔahpCF ΔkatE ΔkatG), and Δhpx Δric strains grown aerobically to the log phase (OD600 = 0.6). Values are represented as normalized to the aconitase activity of wild-type cells. Values are means (±standard errors) from at least two independent cultures analyzed in duplicate. ***, P < 0.0005; ns, not significant (unpaired Student's t test).
Tables
- TABLE 1
Strains and plasmids used in this study
E. coli strain or plasmid Descriptiona Source or reference Strains DHM1 F′ cya-854 recA1 endA1 gyrA96 (Nalr) thi1 hsdR17 spoT1 rfbD1 glnV44(AS) Euromedex Wild type K-12 ATCC 23716 ATCC Δric strain K-12 Δric::cat 36 Δdps strain JS091 Δdps::kan 37 Δbfr strain JW3298 Δbfr::kan 37 ΔftnA strain MC4100 ΔftnA::spc 28 Δdps Δric strain K-12 Δdps::kan Δric::cat This study Δbfr Δric strain K-12 Δbfr::kan Δric::cat This study ΔftnA Δric strain K-12 ΔftnA::spc Δric::cat This study MG1655 F− WT 38 SJ90 BW25113 Δric::cat 39 LC106 (hpx) ΔahpCF ′kan::′ahpF Δ(katG17::Tn10)1 Δ(katE12::Tn10)1 26 Δhpx Δric strain LC106 Δric1::cat This study XL2-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB+ lacIqZΔM15 Tn10 (Tetr)] Agilent BL21Gold(DE3) E. coli B F− ompT hsdS(rB− mB−) dcm+ Tetr gal λ(DE3) endA Hte Agilent Plasmids pUT18/pUT18C Vector that allows construction of in-frame fusions at the N terminal/C terminal of T18 fragment (amino acids 225–399 of CyaA) 17 pKT25/pKNT25 Vector that allows construction of in-frame fusions at the N terminal/C terminal of T25 fragment (amino acids 1–224 of CyaA) 17 pUT18/pUT18C-RIC RIC fused to T18 fragment in N/C terminal This study pKT25/pKNT25-RIC RIC fused to T25 fragment in N/C terminal This study pUT18/pUT18C-Dps Dps fused to T18 fragment in N/C terminal This study pKT25/pKNT25-Dps Dps fused to T25 fragment in N/C terminal This study pUT18-Zip Leucine zipper fused to T18 fragment in the N terminal 17 pKT25-Zip Leucine zipper fused to T25 fragment in the C terminal 17 pUT18-TorD TorD fused to T18 fragment in N terminal 18 pKT25-TorD TorD fused to T25 fragment in C terminal 18 BamHI pUT18 plasmid that contains chromosomal fragments obtained by partial digestion of the MC4100 chromosomal DNA with Sau3A1 and cloned into the BamHI site 18 pUC18 Expression vector ATCC pUC18-RIC Vector for expression of RIC 4 pUC18-RIC-Glu133Leu Vector for expression of RIC-Glu133Leu 10 pET11a-link-GFP Vector for expression of fusions with N-terminal fragment of GFP 33 pMRBAD-link-GFP Vector for expression of fusions with C-terminal fragment of GFP 33 pET11a-RIC-GFP RIC fused to N-terminal GFP fragment This study pMRBAD-RIC-GFP RIC fused to C-terminal GFP fragment This study pET11a-Dps-GFP Dps fused to N-terminal GFP fragment This study pMRBAD-Dps-GFP Dps fused to C-terminal GFP fragment This study pET11a-Bfr-GFP Bfr fused to N-terminal GFP fragment This study pMRBAD-Bfr-GFP Bfr fused to C-terminal GFP fragment This study pET11a-FtnA-GFP FtnA fused to N-terminal GFP fragment This study pMRBAD-FtnA-GFP FtnA fused to C-terminal GFP fragment This study pET11a-RICTrunc-GFP Truncated RIC fused to N-terminal GFP fragment This study pMRBAD-RICTrunc-GFP Truncated RIC fused to C-terminal GFP fragment This study pET-28a Expression vector Novagen pET-28a-RIC(HisTag) Vector for expression of N-terminal poly-His-tagged RIC This study pACYCDuet-1-Dps Vector for expression of Dps This study ↵a WT, wild type; GFP, green fluorescent protein.
- TABLE 2
Oligonucleotides used in this study
Primer purpose and name Sequence Construction of plasmids used in BACTH ric_Fw GAGGTGTCGACTATGGCTTATC ric_Rv CTTTTAGGATCCTCACCCGCC dps_Fw GTTAATTACTGGGATCCAACATCAAGAGG dps_Rv TCCTGTCAGGTACCCGCTTTTATC T18_Fw CATTAGGCACCCCAGGCTTTAC T18_Rv GAGCGATTTTCCACAACAAGTC T18C_Fw CATACGGCGTGGCGGGGAAAAG T18C_Rv AGCGGGTGTTGGCGGGTGTCG T25_Fw ATGCCGCCGGTATTCCACTG T25_Rv CGGGCCTCTTCGCTATTACG NT25_Fw CACCCCAGGCTTTACACTTTATGC NT25_Rv CAATGTGGCGTTTTTTTCCTTCG Construction of plasmids used in BiFC ric_xhoFw GAATGAGGTCTCGAGTATGGCTTATC ric_bamRv GCGCAATGGGATCCAGCTTTTAGA ric_ncoFw GAGGTATCAGCCATGGCTTATCG ric_aatRv CCAGCTTTTAGACGTCTCACCC dps_xhoFw CGTTAATTACTCGAGCATAACATCAAG dps_bamRv GTACTAAGGATCCGCACCATCAGC dps_sphFw CAAGAGGATATGCATGCATGAGTACCGCTA dps_aatRv CATCAGCGATGGGACGTCTCGATGTTAG bfr_xhoFw GAGTGGAAGCGCTCGAGTCAAAAAATG bfr_bamRv GGAGGGTTCTGGATCCCGACACG bfr_ncoFw GAAGGAGTCAAACCATGGAAGGTGATAC bfr_aatRv CGGACGTCCCTTCTTCGCGGATC truncric_xhoFw CTTTAAGAAGGCTCGAGACATATGGCTG truncric_ncoFw GGAGATATACCCATGGCTGAACAAC ftna_xhoFw CAAATATAACCTTTCTCGAGCACTATC ftna_bamRv TGAAACGGATCCAGTAAACCTGC ftna_ncoFw GAGCACTACCATGGTGAAACCAGAAAT ftna_aatRv CGGAGAGGACGTCTTTTGTGTGTC Construction of plasmids used for protein expression pric_ndeFw AAGAATGAGGTATCACATATGGCTTATCGC pric_ecoriRv GGCTGTTTATTGGTAAGAATTCGGCTGCTG pdps_ndeFw GAGGATATGAACATATGAGTACCGC pdps_kpnRV GTACTAAAGTTCGGTACCATCAGCG Double mutant construction confirmation Conf_dps_Fw CAGAATAGCGGAACACATAGC Conf_dps_Rv GATGCACTAAATAAGTGCGTTG Conf_bfr_Fw CTCTTCAAAGAGTGGAAGCG Conf_bfr_Rv GATCTCTTATTAACCGGGAGG Conf_ftnA_Fw CAAATTATAGTGACGCCACAG Conf_ftnA_Rv ACCGATCAGAGTAAGATTTGC Conf_ric_Fw AAGAATGAGGTATCACATATGGCTTATCGC Conf_ric_Rv GGCTGTTTATTGGTAAGAATTCGGCTGCTG
