Figure 1.
CorE is involved in copper homeostasis.
(A) Copper up-regulation of corE. β-gal specific activity was determined in cell extracts of the WT (blue lines) and ΔcorE (red lines) strains (harboring the fusion corE-lacZ) from CTT agar plates containing no copper (open symbols) or 0.6 mM (closed symbols) copper sulfate. (B) Effect of copper on M. xanthus growth. WT (blue line) and ΔcorE (red line) strains were grown in the absence of the metal and diluted to an OD600 of 0.05 into fresh CTT liquid media containing the indicated copper concentrations. The OD600 was then monitored after 24 h of incubation. Error bars indicate standard deviations.
Figure 2.
(A) Regulation of cuoB and copB by CorE. Plasmids containing cuoB-lacZ (blue lines) and copB-lacZ (red lines) fusions were introduced into the WT (solid symbols) or the ΔcorE (open symbols) backgrounds, and incubated on CTT agar plates containing 0.6 mM CuSO4. β-gal specific activity was determined in cell extracts harvested at the indicated times. The same approach reported above was followed to study the regulation of MXAN_3427 (B) and copA (C and D) by CorE, although 0.3 mM CuSO4 was used to get an optimal difference in the copA expression levels between the WT and the ΔcorE strains at early times (panels C and D). The dashed arrow from panel C to D indicates that in panel D only the indicated part of panel C is shown. Please note the difference in the scale in each panel, and the different time course of panel D. Error bars indicate standard deviations.
Figure 3.
cuoB is only up-regulated by copper and other divalent metals.
(A) The WT strain harboring the cuoB-lacZ fusion was spotted onto CTT agar plates containing metals or oxidants at the concentrations indicated above each picture. Plates also contained 5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside to qualitatively monitor β-gal activity (blue color development). Pictures were taken after 48 h of incubation. (B) Up-regulation of cuoB by Cd2+. The WT (continuous line) and the ΔcorE strains (dashed line) harboring the cuoB-lacZ fusion were incubated on CTT agar plates containing 0.1 mM Cd(NO3)2. β-gal specific activity was determined in cell extracts harvested at the indicated times. (C) Up-regulation of cuoB by Zn2+. The approach followed was the same as the one reported in panel B. The concentration of metal used was 0.4 mM Zn(NO3)2. Error bars indicate standard deviations.
Figure 4.
Searching for the CorE anti-σ factor.
(A and B) Quantification of β-gal activity (cuoB expression) in strains where corE was cloned under the control of the oar promoter (red lines) or its own promoter (blue lines). Activities were determined in the absence of metal (A) or in the presence of 0.3 mM copper (B). Note the difference in the scales of the two panels. Error bars indicate standard deviations. (C and D) Western blot analyses to confirm the over-production of CorE in the absence (C) or in the presence (D) of 0.3 mM copper in the strains harboring the gene corE cloned under the oar promoter (lane 1) or its own promoter (lane 2). Proteins were collected at 2 h after copper addition. The band of equal intensity in all the lanes corresponds to an unidentified M. xanthus protein that reacts with the anti-His tag antibody used in the assay. The intensity of this band, which does not change in the conditions tested, has been used to standardize the amount of protein loaded in each lane.
Figure 5.
CorE needs copper and BCS to bind DNA.
Electrophoretic mobility shift assay with purified hCorE and a radiolabeled DNA fragment containing the copB promoter was carried out with the additives indicated in each lane. Details are given in Materials and Methods.
Figure 6.
Activation and inactivation of CorE.
(A) CorE is not degraded. M. xanthus cells harboring the hCorE protein were harvested at the times (h) indicated above each lane after the addition of 0.3 mM copper sulfate and analyzed by Western blot using an anti-His tag antibody. (B) cuoB is not up-regulated by any of the additives indicated in the panel. (C) CorE is activated and inactivated by the redox state of copper. cuoB expression was analyzed in the presence of different additives that modify the redox state of copper, that mimic Cu(I), or that chelate copper in any of its two redox states. For panel B and C, M. xanthus cells harboring the fusion cuoB-lacZ were incubated on CTT agar plates containing the additives indicated. Samples were harvested at different times and β-gal specific activity was determined. Error bars indicate standard deviations.
Figure 7.
Silver and TTM prevent CorE from binding to DNA.
An electrophoretic mobility shift assay was carried out as described in Figure 5, with the additives indicated in each lane.
Figure 8.
Expression of cuoB in strains harboring point mutations in the CRD region of CorE.
The mutated Cys are indicated in each panel. Cells were incubated on CTT agar plates containing 0.3 mM copper (continuous lines) or without metal (dashed lines), and samples were harvested at different times to determine β-gal specific activity. Note that the scale in panel F is different from that used in the other panels. Error bars indicate standard deviations.
Figure 9.
ECF σ factors with CRD in other bacteria.
(A) Multiple sequence alignment of the CRD of the 21 ECF σ factors found in prokaryotes. The four invariant Cys are marked on a red background with letters in white. Residues conserved in at least 11 proteins are shown on a green background. The functionally similar amino acids are highlighted in yellow. The number of each gene in the corresponding genome is shown in parenthesis. The codes for the strains, gene identifiers in the genomes (in parentheses), and accession numbers for the proteins are as follows: M. xanthus (MXAN_3426): Myxococcus xanthus DK 1622, YP_631623.1; S. aurantiaca (STIAU_8272): Stigmatella aurantiaca DW4/3-1, ZP_01463028.1; R. palustris (Rpa1_1094): Rhodopseudomonas palustris TIE-1, YP_001990117.1; A. capsulatum (ACP_1212): Acidobacterium capsulatum ATCC 51196, YP_002754310.1; V. spinosum (010100010715): Verrucomicrobium spinosum DSM 4136, ZP_02927111.1; Anaeromyxobacter (Anae109_4009): Anaeromyxobacter sp. Fw109-5, YP_001381261.1; O. terrae (Oter_1731): Opitutus terrae PB90-1, YP_001818615.1; M. xanthus (MXAN_5263): Myxococcus xanthus DK 1622, YP_633415.1; Roseovarius sp. 217 (ROS217_03265): Roseovarius sp. 217, ZP_01038131.1; Roseovarius sp.TM1035 (RTM1035_00365): Roseovarius sp. TM1035, ZP_01881487.1; R. litoralis (RLO149_0305): Roseobacter litoralis Och 149, ZP_02143093.1; Roseovarius sp.217 (ROS217_03325): Roseovarius sp. 217 ZP_01038143.1; C. Nitrospira defluvii (NIDE1902): Candidatus Nitrospira defluvii, YP_003797553.1; Roseovarius sp.TM1035 (RTM1035_00375): Roseovarius sp. TM1035, ZP_01881489.1; Roseovarius sp. 217 (ROS217_03275): Roseovarius sp. 217, ZP_01038133.1; Mesorhizobium (Meso_3863): Mesorhizobium sp. BNC1, YP_676395.1; P. zucineum (PHZ_p0174): Phenylobacterium zucineum HLK1, YP_002128692.1; S. cellulosum (sce8251): Sorangium cellulosum So ce 56,YP_001618901.1; Acidobacterium sp. SP1PR4 (AciPR4DRAFT_0832): Acidobacterium sp. SP1PR4, ZP_07648663.1; Acidobacterium sp.MP5ACTX8 (A9ciX8DRAFT_2097): Acidobacterium sp. MP5ACTX8, ZP_07030792.1; Acidobacterium sp.MP5ACTZ9 (AciX9DRAFT_0224): Acidobacterium sp. MP5ACTX9, ZP_07062224.1. (B) Synteny in the genomic regions where some of the EFC σ factors with CRD are encoded. Genes shown in the same color (except black genes) in the different strains encode proteins with high homology.