In depth analysis of the mechanism of action of metal-dependent sigma factors: characterization of CorE2 from Myxococcus xanthus

Published online 6 March 2016 Nucleic Acids Research, 2016, Vol. 44, No. 12 5571–5584 doi: 10.1093/nar/gkw150 In depth analysis of the mechanism of action of metal-dependent sigma factors: characterization of CorE2 from Myxococcus xanthus Francisco Javier Marcos-Torres, Juana P´ erez, Nuria G´ omez-Santos, Aurelio Moraleda-Mu˜ noz and Jos´ eMu˜ noz-Dorado* Departamento de Microbiolog´ ıa, Facultad de Ciencias, Universidad de Granada, Avda. Fuentenueva s/n, E-18071 Granada, Spain Received July 30, 2015; Revised February 26, 2016; Accepted February 29, 2016 ABSTRACT Extracytoplasmic function sigma factors represent the third pillar of signal-transduction mechanisms in bacteria. The variety of stimuli they recognize and mechanisms of action they use have allowed their classification into more than 50 groups. We have characterized CorE2 from Myxococcus xanthus , which belongs to group ECF44 and upregulates the expression of two genes when it is activated by cadmium and zinc. Sigma factors of this group contain a Cys-rich domain (CRD) at the C terminus which is essential for detecting metals. Point mutations at the six Cys residues of the CRD have revealed the contribution of each residue to CorE2 activity. Some of them are essential, while others are either dispensable or their mutations only slightly affect the activity of the protein. However, importantly, mutation of Cys174 completely shifts the specificity of CorE2 from cadmium to copper, indicating that the Cys arrangement of the CRD determines the metal specificity. Moreover, the conserved CxC motif located between the ␴2 domain and the ␴4.2 region has also been found to be essential for activity. The results presented here contribute to our understanding of the mechanism of action of metal-dependent sigma factors and help to define new common features of the members of this group of regulators. INTRODUCTION Myxococcus xanthus is a soil ␦-proteobacterium of the group of myxobacteria used as a model to study multicellular behavior and differentiation due to its unique and complex life cycle. M. xanthus cells feed as coordinated groups until nutrients are depleted. Upon starvation they initiate a developmental program, during which cells must produce and respond to several signals in order to aggregate and differentiate into myxospores, which are resistant to a variety of adverse conditions (1,2). Bacteria adapt to environmental changes by using a large number of signal-transduction systems which connect extracellular inputs with the appropriate cellular responses. There are three main common and universally present signal-transduction mechanisms in bacteria: oneand two-component systems, and the extracytoplasmic function (ECF) sigma factors (3–6). Moreover, there is a fourth signal-transduction system less widespread among prokaryotes which involves Ser/Thr protein kinases and phosphatases (7,8). ECF sigma factors belong to group 4 of the ␴70 family of sigma factors (9). Members of this group are small proteins that contain only two of the four conserved domains found in sigma factors of groups 1 and 2, the ␴2 and the ␴4 domains. The ␴2 domain is essential for recognition of the −10 promoter sequences and coupling with the RNA polymerase core enzyme, while the ␴4.2 region (included in the ␴4 domain) is required for recognition of the −35 promoter regions (10). ECF sigma factors are abundant and diverse in bacterial genomes, especially in those with a complex life cycle (11). Many ECF sigma factors function with a cognate anti-sigma factor. Anti-sigma factors are usually membrane-anchored proteins, co-expressed with their cognate sigma factor, which contain the sensor domains of these signal-transduction systems. In absence of the right environmental stimulus, anti-sigma factors sequester their sigma factors in the membrane and block the expression of specific genes. When anti-sigma factors do detect these external signals, sigma factors are released, recruiting the RNA polymerase core enzyme and binding to DNA to initiate transcription of the genes required to respond to stimuli (6,12–14). *To whom correspondence should be addressed. Tel: +34 958243183; Fax: +34 958249486; Email: [email protected] Present address: Nuria G´ omez-Santos, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany. C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Downloaded from https://academic.oup.com/nar/article/44/12/5571/2457481 by Universidad de Granada - Biblioteca user on 31 July 2025 5572 Nucleic Acids Research, 2016, Vol. 44, No. 12 The mechanism of activation of ECF sigma factors, together with their sequence similarities, has allowed the classification of these transcriptional regulators into more than 50 groups (13). Even though the mechanism described above is the main mode of activation of ECF sigma factors, three other mechanisms have been reported for these regulators, in which anti-sigma factors do not participate. One of these other mechanisms is used by groups ECF32 and ECF39, which consists of direct transcription of the sigma factor (15,16). A hypothetical phosphorelay involving a Ser/Thr protein kinase co-transcribed with the sigma factor has been postulated for groups ECF43 and ECFSTK1– 4(5,17). Finally, some ECF sigma factors contain a Cterminal extension responsible for the modulation of their own activity. To date only four groups have been described with C-terminal extensions: ECF41, ECF42, ECF01-Gob and ECF44 (5,6,17,18). Myxococcus xanthus CorE is the founding member and the only characterized sigma factor of the group ECF44. This sigma factor confers copper resistance to M. xanthus by regulating the expression of the P1B-type ATPases CopA and CopB, and the multicopper oxidase CuoB (14,19–21). In contrast to most ECF sigma factors, CorE only partially regulates its own expression, and its activation state does not depend on an anti-sigma factor. CorE-regulated genes show a peak of expression at 2 h after copper addition that rapidly decreases due to CorE inactivation. It has been proposed that Cu(II) activates CorE, allowing DNA-binding, whereas Cu(I) inactivates the sigma factor preventing DNA binding. A conserved C-terminal Cys-rich domain (CRD) with 38 residues in CorE controls the activation and inactivation mediated by copper of this ECF sigma factor. Point mutations at each Cys residue of the CRD have revealed that certain key residues play a role in CorE activation and/or inactivation (14). We have identified a second member of the ECF44 group in the M. xanthus genome, which has been named corE2 (MXAN 5263). In the present work we dissect the mechanism of action of CorE2, which is activated by cadmium and zinc, and regulates the expression of at least a cation efflux pump and a glyoxal oxidase and Kelch domain containing protein. We have compared the metal responses and specificities of CorE and CorE2, and have performed an in-depth investigation of their CRD Cys arrangement to understand their differences. We have found that a change in just one Cys residue of the CRD of CorE2 shifts the response of the sigma factor from cadmium to copper. Furthermore, we have demonstrated that a CxC motif located between the ␴2 domain and the ␴4.2 region, conserved in all the CorE-like ECF sigma factors, is essential for the activity of CorE2. MATERIALS AND METHODS Bacterial strains, plasmids and growth conditions Genotypes of M. xanthus and Escherichia coli strains, plasmids and oligonucleotides used in this study are listed in Supplementary Tables S1, 2 and 3, respectively. E. coli strains were grown in lysogenic broth (LB) (22)at37 ◦C. Agar plates contained 1.5% Bacto-agar (Difco), which were supplemented with 40 ␮g/ml X-gal (5-bromo-4-chloro3-indolyl-␤-D-galactopyranoside), kanamycin (25 ␮g/ml) and/or tetracycline (25 ␮g/ml) when necessary. M. xanthus strains were grown in CTT medium (23)at30 ◦C with vigorous shaking (300 rpm). CTT agar plates (1.5% agar) were supplemented with X-gal (100 ␮g/ml), galactose (10 mg/ml), kanamycin (80 ␮g/ml) and/or tetracycline (15 ␮g/ml). When needed, different metals were also added to the medium at the concentrations indicated in each figure. To induce development, starvation medium CF (23)was used. Cells exponentially growing to approximately 3.0 × 108cells/ml (optical density at 600 nm [OD600]of1)were concentrated and resuspended to an OD600 of 15 in TM buffer (10 mM Tris–HCl [pH 7.6], 1 mM MgSO4). Ten microliter drops were spotted onto CF agar plates supplemented with the metals indicated in the figures and/or X-gal (100 ␮g/ml) and incubated at 30◦C. Fruiting bodies were observed with an Olympus dissecting microscope. Nucleic acid manipulations Routine molecular biology techniques were used for nucleic acid manipulations (22). The various plasmids were introduced into E. coli by heat-shock transformation and into M. xanthus by electroporation (24). Total RNA was extracted from M. xanthus with the High Pure RNA Isolation kit provided by Roche. Samples were then treated with DNAse I (Sigma) to ensure removal of chromosomal DNA. Complementary DNA (cDNA) was obtained by reverse transcription (SuperScript III reverse transcriptase, Life Technologies) from the RNA template using the primer 65RT, which anneals to the gene MXAN 5265 (Supplementary Table S3). A polymerase chain reaction (PCR) was then performed with the primers listed in Supplementary Table S3, using total RNA or cDNA as a template. Construction of in-frame deletion mutants The in-frame deletion mutants used in this study were obtained as previously reported (21). To generate the corresponding plasmids (listed in Supplementary Table S2), sequences upstream and downstream of the M. xanthus regions to be deleted were amplified by PCR with wild-type (WT) chromosomal DNA as a template, the primers listed in Supplementary Table S3, and the high-fidelity DNApolymerase PrimeSTAR HS (Takara). The PCR products were digested and ligated to vector pBJ113 (25), which had previously been digested with the same restriction enzymes to obtain the desired plasmids (Supplementary Table S2). The resulting plasmids were introduced into M. xanthus strains by electroporation. Kanamycin resistant (KmR) merodiploids were selected from CTT agar plates supplemented with this antibiotic and analyzed by Southern blot hybridization to corroborate the proper recombination events. Positive strains were grown on CTT agar plates without kanamycin and containing 1% galactose, favoring the loss of the plasmid by a second homologous recombination. Southern blot analysis was used to screen kanamycinsensitive (KmS) and galactose-resistant (GalR) colonies for the loss of the WT allele. Downloaded from https://academic.oup.com/nar/article/44/12/5571/2457481 by Universidad de Granada - Biblioteca user on 31 July 2025 Nucleic Acids Research, 2016, Vol. 44, No. 12 5573 Construction of strains harboring lacZ fusions and ␤- galactosidase assays Plasmids harboring lacZ fusions (Supplementary Table S2) were constructed as previously reported (20). In summary, to generate the corresponding plasmids, PCR was performed using chromosomal DNA of M. xanthus as a template and the primers listed in Supplementary Table S3. PCR products were then digested with their respective restriction enzymes and ligated into vector pKY481 (26) digested with the same enzymes to generate transcriptional lacZ fusions. M. xanthus strains were electroporated with these plasmids to generate the desired strains. The resultant KmRrecombinant strains (Supplementary Table S1) were confirmed by Southern blot analysis. For qualitative ␤-galactosidase activity analyses, cells were concentrated to an OD600 of 15 and spotted onto CTT or CF agar plates containing 100 ␮g/ml X-gal and the additives indicated in each figure. For quantitative analysis, cells grown on CTT liquid medium were concentrated at an OD600 of 15 and spotted onto CTT or CF agar plates. ␤-galactosidase-specific activity was determined in cell extracts obtained at various time points by sonication as previously reported (21), and is expressed as nmol of onitrophenol produced per min and mg of protein. All extracts were assayed in triplicate, and values were averaged from three independent measurements. Site-directed mutagenesis Single amino acid substitutions in CorE and CorE2 were performed using the QuikChange II site-directed mutagenesis kit (Agilent) as recommended by the manufacturer. Plasmids pNG00 and pMT00 (Supplementary Table S2), containing the WT corE and corE2 sequences, respectively, were used as templates. The oligonucleotides used as primers (listed in Supplementary Table S3) were designed using the QuikChange Primer Design Program (http:// www.genomics.agilent.com/). The amplified plasmids were digested with DpnI and transformed into E. coli to obtain the mutant plasmids listed in Supplementary Table S2. All plasmids were sequenced to confirm the presence of the desired mutations and the absence of unwanted mutations, and were introduced by electroporation into M. xanthus JM51EBZY (cuoB-lacZ-corE) for mutations in CorE, and into M. xanthus JM52IF3ZY5 (5265-lacZ-corE2)for point mutations in CorE2. The mutant strains obtained are listed in Supplementary Table S1. Plasmid pMT00 was also electroporated in JM52IF3ZY5 to construct the strain JM52SDM00, which was used as a control for CorE2 mutation analyses. All TetRand KmRrecombinants were analyzed by Southern blot. As in previous studies for the CorE mutation, strain JM00BZY was used as a control (14). Studies on the stability of the proteins harboring point mutations In order to determine whether the proteins harboring point mutations of CorE and CorE2 were stable in M. xanthus, genes encoding them and the WT genes were cloned with an N-terminal S tag and under control of the constitutive oar promoter (27). A 781-bp fragment upstream of the oar gene (MXAN 1450) was amplified by PCR, using the appropriate oligonucleotide pair listed in Supplementary Table S3 as primers. The PCR product was introduced inframe with the coding sequence of the S tag present in the pRSFDuet-1 vector (Novagen). The resulting plasmid was named pPOar-S and used to amplify the oar promoter fused to the S tag. Next, the WT corE and corE2 genes were amplified from the M. xanthus chromosomal DNA, while the corresponding plasmids (Supplementary Table S2) were used to amplify corE and corE2 genes harboring point mutations and were cloned in-frame with the S-tagged oar promoter in the pBJ113 vector (25). The resulting plasmids were introduced by electroporation into a corE (those containing the WT corE or point mutations in corE)orcorE2 M. xanthus strain (those containing the WT corE2 or point mutations in corE2). Several kanamycin-resistant (KmR) colonies were analyzed by PCR to confirm the proper recombination event. To detect the S-tagged proteins, the different strains were grown for 24 h in CTT supplemented with 200 ␮M of ZnNO3, after which the cells were harvested in TM buffer containing a protease inhibitor cocktail (Promega) and disrupted by sonication. The sonicated samples were centrifuged and the protein concentration of the supernatants was determined by the Bio-Rad protein assay kit using bovine serum albumin as standard. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto a membrane of PVDF. S-tagged proteins were detected using an S-protein HRP Conjugate (Novagen) antibody, which is conjugated with horseradish peroxidase, using 1-Step Ultra TMB Blotting Solution (Pierce) as the substrate, following the instructions specified by the manufacturer. Bioinformatic analysis The list of CorE-like ECF sigma factors was updated, as previously reported (14), by BLASTP analysis of all genome and protein sequences deposited in the database of the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/genome/browse/). Protein sequence alignments were performed using the ClustalW program (28), and a graphic representation of the results was generated with ESPript.cgi Version 3.0 (29)(http://espript.ibcp. fr/ESPript/cgi-bin/ESPript.cgi). The domain architecture of proteins was analyzed against the Pfam database (30). RESULTS CorE-like ECF sigma factors in myxobacteria A BLASTP analysis in search of CorE-like ECF sigma factors resulted in the identification of 67 of these regulators in bacteria, 17 of which are found in species of the order Myxococcales (Supplementary Figure S1). Interestingly, all the myxobacterial genomes so far sequenced encode at least one CorE-like sigma factor, with the exception of several species of the genus Anaeromyxobacter (where one is only found in the strain Fw109) and Haliangium ochraceum.TheMyxococcus stipitatus genome harbors three sigma factors of this type (Supplementary Figure S1). In the case of M. xanthus, in addition to two complete CorE-like sigma factors, Downloaded from https://academic.oup.com/nar/article/44/12/5571/2457481 by Universidad de Granada - Biblioteca user on 31 July 2025 5574 Nucleic Acids Research, 2016, Vol. 44, No. 12 Figure 1. The Myxococcus xanthus genome contains three genes with conserved C-terminal CRD domains. (A) Domain architecture of the three proteins with a CRD domain. CorE and CorE2 have the ␴2 domain (Sigma70 r2, PF04542) and the ␴4.2 region (sigma70 r4 2, PF08281) typical of ECF sigma factors. MXAN 0974 shows sequence similarities of 40.4% with CorE and 36.5% with CorE2 within the ␴4.2 region. (B)Sequence alignment of the three CRDs showing the conserved Cys residues. Numbers indicate the position of the first and last residue of the sequences shown for each protein. Identical residues in two proteins are written in red and highlighted in yellow, and those that are identical in the three proteins are written in white and highlighted in red. (C) Genetic environment of the three genes with a CRD (represented in red). Genes of these regions that encode proteins associated with metals are drawn following the color code indicated at the bottom of the panel. a third gene (MXAN 0974) is present which encodes a protein with a region resembling that of ␴4.2 of sigma factors andwithaCRD(Figure1A and B). However, this protein is not expected to function as an ECF sigma factor because it lacks the ␴2 domain. The paralog of CorE found in M. xanthus has been designated as CorE2 (MXAN 5263), and in this report we have focused on its characterization. Analysis of the genetic context of corE2 has revealed a gene upstream encoding a hypothetical lipoprotein (MXAN 5262) which has been predicted to be the cognate anti-sigma factor of CorE2 (12). Moreover, downstream of corE2 there are two genes which encode proteins associated with metals. MXAN 5264 (with Pfam PF01545) is similar to cation efflux systems, such as CzcD of Cupriavidus metalidurans (31), while MXAN 5265 corresponds to a metalloenzyme with a Kelch domain and a glyoxal oxidase domain (PF07250 and PF01344, respectively), which resembles the developmental protein FbfB of Stigmatella aurantiaca (32)(Figure1C). Genes encoding proteins involved in metal homeostasis and detoxification are also found in the proximity of corE and MXAN 0974 (Figure 1C). This observation, along with the presence of a CRD, suggests that CorE2, like CorE, might be metal-responsive. Genes of the corE2 region, but not corE2, are upregulated by metals To analyze the metal response of genes in the corE2 region, four genes (MXAN 5262,corE2,MXAN 5264 and MXAN 5265) were tested for metal regulation. Plasmids containing transcriptional fusions between these four genes and E. coli lacZ (Figure 2A) were electroporated into the WT strain of M. xanthus. The resulting KmRstrains JM52ZY2 (5262-lacZ), JM52ZY3 (corE2-lacZ), JM52ZY4 (5264-lacZ) and JM52ZY5 (5265-lacZ) (Supplementary Table S1) were confirmed by Southern blot. The strains containing the fusions, and the WT strain as a negative control, were spotted onto CTT (growth) and CF (development) agar plates containing 100 ␮g/ml of X-gal and several metals to qualitatively screen for gene expression. Surprisingly, none of the four genes was upregulated by copper (Figure 2B), as happened with CorE-regulated genes (14). However, two genes downstream of corE2 were upregulated in the presence of cadmium and zinc (Figure 2B). Both genes exhibit an expression profile with cadmium in which a plateau is reached several hours after addition of the metal (Figure 2C). In contrast, the expression of these two genes rapidly increases after the addition of zinc, exhibiting a maximum at 2 h. Thereafter the expression levels slightly decrease, remaining quite high for an extended period (Figure 2D). None of the metals tested was able to affect corE2 and MXAN 5262 expression (Figure 2B). However, an upregulation of the four genes was observed during development in the absence of metals (Figure 2E), although at different levels (Figure 2F), indicating regulation of all these genes during fruiting-body formation. Due to their proximity and orientation, and the dual expression profile of the genes in the cluster, co-expression of these four genes was examined under two different conditions: growth on CTT agar plates with 0.1 mM cadmium and development on CF agar plates with no metals added. Total RNA was extracted after 48 h of incubation under these two conditions, and it was used as a template for synthesis of cDNA using primer 65RT (Supplementary Figure S2). Using the two cDNAs as templates and the strategy depicted in Supplementary Figure S2A, it was found that two differentially regulated operons are found in the corE2 region, one containing the four genes (MXAN 5262 through MXAN 5265), which is expressed during development (Supplementary Figure S2B), and a second one, containing only two genes (MXAN 5264 and MXAN 5265), which is upregulated by cadmium (Supplementary Figure Downloaded from https://academic.oup.com/nar/article/44/12/5571/2457481 by Universidad de Granada - Biblioteca user on 31 July 2025 Nucleic Acids Research, 2016, Vol. 44, No. 12 5575 Figure 2. Expression of genes located in the proximity of corE2.(A) Schematic representation of the corE2 context. Red arrows indicate Escherichia coli lacZ transcriptional fusions. (B) Qualitative analysis of lacZ fusions for genes of the corE2 region in the presence of various metals. Cells harboring fusions between genes of the corE2 cluster and lacZ were inoculated on CTT medium (growth) containing X-gal and different metals. Pictures were taken after 96 h of incubation. (Cand D) Quantification of ␤-galactosidase-specific activity of the four fusions with lacZ. Cells were grown on CTT without metals (orange lines) or containing either 0.1 mM cadmium (C, red lines) or 0.5 mM zinc (D, green lines), and samples were harvested at the time points indicated in each figure. Specific activity was determined as indicated in ‘Materials and Methods’ section. (E) Qualitative analysis of gene expression of the corE2 cluster during development. Cells were spotted onto both CTT agar plates (growth) and CF agar plates (development) containing X-gal and no metals. Pictures were taken after 96 h of incubation. Bar in panels B and E represents 0.5 mm. (F) Quantification of ␤-galactosidase-specific activity during growth (orange lines) and development (blue lines) in the absence of metals. Cells of each strain were spotted onto CF agar plates, and at the time points indicated in the figures they were harvested and analyzed for ␤-galactosidase activity. Error bars in panels C, D and F indicate standard deviations. Extracts were assayed in triplicate, and values were averaged from three independent measurements. Please note that the graphs have different scales. S2C). These data are in good agreement with the data presented in Figure 2. The mutant corE2 is more sensitive to cadmium and zinc than the WT strain, and exhibits delay in development To determine whether CorE2 is involved in metal detoxification, an in-frame deletion mutant (corE2) was constructed, lacking the essential ␴2 domain of the sigma factor (Figure 3A), and the phenotype of the mutant was analyzed for metal sensitivity. When the mutant corE2 and the WT strain were cultured in liquid CTT medium, no significant difference in growth was observed between the two strains when several concentrations of zinc or cadmium were tested (Supplementary Figure S3). Since metal homeostasis mechanisms require preadaptation to be fully active (33), WT and corE2 strains were first grown for 24 h in CTT medium containing either 10 ␮M cadmium or 0.2 mM zinc. Cells were then transferred to CTT liquid cultures containing varying concentrations of metals. As shown in Figure 3B, zinc tolerance was reduced in the corE2 preadapted cells. In contrast, cadmium tolerance was only slightly reduced in the mutant (Figure 3C). These data indicate that CorE2 is involved in conferring resistance to some metals, and suggest that this sigma factor could be regulating the expression of the two genes located downstream of itself, which are upregulated by these two metals. Downloaded from https://academic.oup.com/nar/article/44/12/5571/2457481 by Universidad de Granada - Biblioteca user on 31 July 2025 5576 Nucleic Acids Research, 2016, Vol. 44, No. 12 Figure 3. Phenotype of the corE2 mutant. (A) Domains present in CorE2 in the WT strain and in the mutants corE2 and corE2CRD.(B) Cells of the WT strain (blue line) and the corE2 mutant (red line) were grown for 24 h in CTT containing 0.2 mM zinc and then diluted to an OD600 of 0.05 in CTT medium containing 0, 0.2, 0.3 and 0.4 mM zinc. OD600 was determined after 32 h of incubation. (C) As in panel B, cells were grown for 24 h in 10 ␮M cadmium and then diluted to an OD600 of 0.05 in CTT medium containing 0, 10, 25 and 40 ␮M cadmium. OD600 was determined after 32 h of incubation. Error bars in panels B and C indicate standard deviations. Values are averages of three experiments. (D) Fruiting body formation of the WT strain and the corE2 mutant in the absence of metals. (E) Fruiting body formation of the WT strain and the corE2 mutant on CF medium with 200 ␮M zinc. (F) Fruiting body formation of the WT strain and the corE2 mutant on CF medium with 20 ␮M cadmium. Pictures in panels D, E, and F were taken at the time indicated. Bar in panels D, E, and F represents 1 mm. As the four genes of the operon are induced during development, it was also tested whether the corE2 mutant exhibits developmental defects. When dropped onto CF starvation medium the corE2 mutant exhibited a clear delay in development, although normal fruiting bodies were observed at 72 h of incubation (Figure 3D). This delay in development indicates that this sigma factor must be regulating the expression of some genes required for the proper timing of fruiting body formation. When development was analyzed in the presence of zinc, a slightly longer delay in fruiting body formation was observed in both the WT strain and the mutant (Figure 3E). In contrast, fruiting bodies were not observed in the mutant strain spotted on CF medium containing 20 ␮M cadmium even after 72 h of incubation, while the WT strain developed almost normally at 48 h (Figure 3F). These results corroborate that CorE2 plays a role in metal detoxification as well as in development. Downloaded from https://academic.oup.com/nar/article/44/12/5571/2457481 by Universidad de Granada - Biblioteca user on 31 July 2025 Nucleic Acids Research, 2016, Vol. 44, No. 12 5577 CorE2 is controlling the cadmiumand zinc-dependent expression of genes MXAN 5264 and MXAN 5265 As we have demonstrated, genes MXAN 5264 and MXAN 5265 form an operon which is upregulated by cadmium and zinc (Figure 2and Supplementary Figure S2). To elucidate whether CorE2 is responsible for the metal-dependent expression of this operon, the corE2 mutant was used as genetic background to introduce the transcriptional fusion 5265-lacZ, to obtain the strain JM52IF3ZY5 (5265-lacZ-corE2). This strain was used to compare the upregulation of the gene MXAN 5265 by cadmium and zinc to the WT strain. As shown in Figure 4A and B, lack of a functional CorE2 results in the absence of both cadmiumand zinc-dependent expression of the gene MXAN 5265, demonstrating that the metal induction of this operon is controlled by this sigma factor. It remains to be elucidated why the phenotype of the corE2 mutant during growth is more dramatic with zinc than with cadmium (Figure 3), whereas genes regulated by this sigma factor exhibit higher expression levels with cadmium (Figure 2). However, one explanation could be that the proteins responsible for metal detoxification regulated by CorE2 exhibit a higher affinity for zinc than for cadmium. As the four genes are induced during development in the absence of metal, it was also tested whether CorE2 was also responsible for this expression. The strains 5265-lacZ- corE2 and the WT harboring the same lacZ fusion were plated onto CF agar without metals. As shown in Figure 4C, CorE2 is not responsible for the expression of gene MXAN 5265 during development. This result supports the notion of a complex regulation of this operon, with several transcriptional regulators involved. Investigating the mode of action of CorE-like ECF sigma factors In this section, the mechanism of action of CorE2 will be compared with that reported for CorE, in order to identify common features for the whole ECF44 group of sigma factors and also the peculiarities of each particular regulator. CorE2 does not regulate its own expression. As shown above (Figure 2and Supplementary Figure S2), corE2 is part of an operon that is only induced during development, but not by metals. Moreover, the expression of the gene MXAN 5265 during development, representing the operon, does not depend on CorE2, indicating that CorE2 does not regulate its own expression, in contrast to what has been reported for other ECF sigma factors, including CorE (9,14). However, due to the low expression levels of corE2, ␤-galactosidase activity was directly quantified in the strain JM52ZY3 (corE2-lacZ) during development, both in the presence and in the absence of 7.5 ␮M cadmium. As shown in Figure 5A, corE2 expression is not upregulated by cadmium, as no significant difference between the two conditions was observed. Furthermore, to support these data, the strain JM52IF3ZY3 (corE2-lacZ-corE2) was constructed introducing the corE2-lacZ transcriptional fusion into the corE2 mutant. Quantitative and qualitative analyses of this strain during development with and without cadmium rendered similar expression patterns to those of the WT Figure 4. CorE2 regulation of gene expression. (Aand B) Expression of the gene MXAN 5265 in the WT strain and the mutants corE2 and corE2CRD. The strains were incubated on CTT agar plates containing either 0.1 mM cadmium (A) or 0.5 mM zinc (B), and X-gal to monitor ␤-galactosidase activity. Pictures were taken at the time points indicated at the top of each picture. Numbers below each picture indicate ␤- galactosidase-specific activity obtained for each strain. (C) Expression of MXAN 5265 during development in the WT strain and the corE2 mutant. Cells were spotted onto CF agar plates without metals. As in panels A and B, the time at which each picture was taken is shown above it, and numbers below the pictures indicate ␤-galactosidase-specific activity at that time point. Values are average of three experiments. Bar represents 0.5 cm. Downloaded from https://academic.oup.com/nar/article/44/12/5571/2457481 by Universidad de Granada - Biblioteca user on 31 July 2025 5578 Nucleic Acids Research, 2016, Vol. 44, No. 12 Figure 5. corE2 auto-regulation. (A) Quantitative analysis of ␤- galactosidase-specific activity of the corE2-lacZ fusion in the WT (red line) and the corE2 (blue line) strains during development without metals (solid lines) and with 7.5 ␮M cadmium (dashed lines). Experiments were carried out in triplicate, and error bars indicate standard deviations of three measurements. (Band C) Qualitative analysis of ␤-galactosidase activity of both strains on CF agar plates with X-gal and no metals (B), and plates supplemented with X-gal and 7.5 ␮M cadmium (C). Bar represents 0.5 cm. strain (Figure 5). These data all support the conclusion that the expression of corE2 is not auto-regulated. MXAN 5262 does not function as an anti-sigma factor for CorE2. Even though it has been postulated that the activity of CorE-like sigma factors is only regulated by their own CRD (13,14), it has been proposed that a membrane lipoprotein encoded by a gene co-expressed with corE2 might function as an anti-sigma factor for CorE2 (12). If the cognate anti-sigma factor of an ECF sigma factor is knocked out, genes regulated by such an ECF sigma factor would be expressed even in the absence of the stimulus (14). In order to test whether MXAN 5262 acts as the cognate anti-sigma factor for CorE2, a new Figure 6. The lipoprotein encoded by the gene MXAN 5262 does not function as the anti-sigma factor of CorE2. The WT and 5262 strains harboring the 5265-lacZ fusion were spotted onto CTT agar plates without metals (A) and containing 0.1 mM cadmium (B). For qualitative assays, plates also contain X-gal to monitor ␤-galactosidase activity. (C) Quantitative analysis of the cadmium-dependent expression of the CorE2 regulated gene MXAN 5265 in the WT and 5262 genetic backgrounds. The WT strain (blue lines) and the 5262 mutant (red lines) were incubated on CTT agar plates without metals (dashed lines) and containing 0.1 mM cadmium (continuous lines). ␤-galactosidase-specific activity was determined in cell extracts harvested at the time points indicated in the figure. Values are averages of three experiments. Error bars indicate standard deviations. Bar represents 0.5 cm. in-frame deletion strain, JM52IF2 (5262), was obtained. Next, the fusion 5265-lacZ was introduced into this mutant. When ␤-galactosidase activity was determined in the strain JM52IF2ZY5 (5265-lacZ-5262), both qualitatively and quantitatively, and compared with that of the WT cells carrying the 5265-lacZ fusion, it was observed that deletion of MXAN 5262 does not increase CorE2 activity, even in the absence of cadmium (Figure 6A and C), demonstrating that this membrane protein does not function as a Downloaded from https://academic.oup.com/nar/article/44/12/5571/2457481 by Universidad de Granada - Biblioteca user on 31 July 2025 Nucleic Acids Research, 2016, Vol. 44, No. 12 5579 cognate anti-sigma factor for CorE2. However, deletion of the gene MXAN 5262 delays the upregulation of the gene MXAN 5265 in the presence of cadmium (Figure 6Band C), indicating that this lipoprotein must somehow participate in the same signal-transduction pathway as CorE2. CRD is essential for the activity of all CorE-like ECF sigma factors. As a member of group ECF44, CorE2 contains a CRD consisting of 28 residues (as opposed to the 38 amino acids of the CRD of CorE). It also contains 6 Cys, but in a different arrangement from that of CorE (Figure 1B). To test whether the CorE2 CRD is essential for CorE2 activity, an in-frame deletion mutant, JM52IF3DCRD (corE2CRD), was generated in which most of the CRD was deleted (Figure 3A). This mutant was used to electroporate the fusion 5265-lacZ to obtain the strain JM52IF3DCRDZY5 (5265-lacZ-corE2CRD). Although CorE2CRD harbors the essential ␴2 domain and ␴4.2 region of ECF sigma factors, it remains inactive even in the presence of 0.1 mM cadmium, as shown by the lack of ␤-galactosidase activity in the strain JM52IF3DCRDZY5 (Figure 4A). Since the CRD of CorE is also required for activity (14), it is plausible to postulate that all the sigma factors of the group ECF44 exhibit a similar mechanism of action. In view of the differences between the Cys arrangement in the CRD domain of CorE2 and CorE, the metal activation by each sigma factor, and the expression profile of genes regulated by each regulator, it is plausible to expect each Cys of the CRD of CorE2 to play a different role in CorE2 activity from the one reported for CorE (14). To analyze which Cys are responsible for CorE2 activity, all six residues were mutated individually to Ala by site-directed mutagenesis (Figure 7A). Each mutated corE2 was then introduced into the JM52IF3ZY5 strain (5265-lacZ-corE2)toevaluate the expression profile of the gene MXAN 5265 in the presence and absence of cadmium. Moreover, the WT corE2 gene was also electroporated into the strain JM52IF3ZY5 (strain JM52SDM00) to allow a reliable comparison between expression of MXAN 5265 by the WT protein and the six mutant proteins. Although the strain JM52SDM00 will be considered as a WT in these experiments, it should be remembered that it is not the same WT strain used in the previous experiments, so the results that we may obtain with JM52SDM00 will not necessarily be identical to those shown in Figures 2–6. The analysis revealed that the expression profile in the mutant C183A was nearly identicaltothatoftheWTcorE2’ strain (Figure 7B), indicating that this residue is not crucial for CorE2 activity. Some upregulation by cadmium still remained in the C173A and C181A mutants. However, the maximum expression levels achieved in these two mutants were only about 15% of that of the WT strain (Figure 7C). These results indicate that Cys173 and 181 are important, although not essential, for CorE2 activation by cadmium. Interestingly, when Cys174 was mutated to Ala, there was a constitutive expression of MXAN 5265 in the absence of cadmium, which did not significantly change in the presence of this metal (Figure 7D). These data indicate that Cys174 is involved in both activation of CorE2 in the presence of cadmium and inactivation when the metal is absent. Interestingly, Cys169 and Cys178 were revealed to be essential for CorE2 activity, because no expression was observed in both mutants even in the presence of cadmium (Figure 7E). To rule out the possibility that the lack of activity of some point-mutated proteins might be due to instability, all the mutated genes were cloned under control of the constitutive oar promoter and fused at the N-terminal region to an S tag, as described in ‘Materials and Methods’ section. Plasmids containing the hybrid genes were electroporated into M. xanthus corE2, and the different strains thus obtained (Supplementary Table S1) were analyzed to investigate whether the mutated proteins are produced and soluble. The results obtained have revealed that all the CorE2 point-mutated proteins are stable (Supplementary Figure S4). Cys arrangement in the CRD of metal-dependent sigma factors determines the metal specificity. As CRDs have been postulated to be the metal-recognition site of CorE-like ECF sigma factors (14 and data shown above), differences between the CRDs of CorE and CorE2 were analyzed for residues that might be responsible for recognition of copper by CorE and of cadmium by CorE2. Even though the Cys motif is well conserved within the ECF44 group of sigma factors, there is one Cys (Cys174 in CorE2) that represents a key difference between the two characterized sigma factors within this group. The CRD of CorE lacks this Cys and an Ala is found in this position (Figures 1Band8A). As shown above, cadmium recognition was impaired in the CorE2 C174A mutant (Figure 7D), so it was plausible to think that this mutant sigma factor might respond to copper, in the same way as CorE. To test this possibility, the CorE2 C174A mutant was assayed for copper upregulation of the gene MXAN 5265. Interestingly, the mutant C174A, which does not respond to cadmium, exhibits a change in metal specificity, and the expression of MXAN 5265 reaches quite high levels in the presence of copper (Figure 8B). When other metals were tested, it was observed that this mutated CorE2 is only activated by copper (Supplementary Figure S5A). It remains to be elucidated whether CorE2 C174A responds to Cu(I) or Cu(II). According to these data, a change of just one residue in the CRD of CorE2 is sufficient to completely shift the metal specificity of the sigma factor from cadmium to copper. To learn more about the significance of the residue located in position 174 in CorE2 in metal recognition, CorE CRD was also mutated, in this case to replace Ala185 with a Cys, in order to make the CorE CRD more similar to that of CorE2 (Figures 1Band8A). As shown in Figure 8C, the CorE A185C mutant was not significantly impaired in terms of copper induction of cuoB (one of the genes regulated by this sigma factor). However, upregulation of cuoB expression in the presence of cadmium was three times higher in the mutant A185C than in the WT strain (compare continuous lines in Figure 8C), indicating that this mutant likely binds both metals. The effect observed with cadmium was not obtained with any of the other metals tested (Supplementary Figure S5B). As the mutated CorE A185C is also stable (Supplementary Figure S4B), these results confirm that differences in only one residue in the Cys arrangement of the CRD of sigma factors of the group ECF44 afDownloaded from https://academic.oup.com/nar/article/44/12/5571/2457481 by Universidad de Granada - Biblioteca user on 31 July 2025