Cyanobacteria are oxygenic photosynthetic prokaryotes that

Transcription

1 Identification of the HetR Recognition Sequence Upstream of hetz in Anabaena sp. Strain PCC 7120 Ye Du, Yan Cai, Shengwei Hou, and Xudong Xu The State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China HetR is the master regulator of heterocyst differentiation in Anabaena sp. strain PCC 7120 and has been found to specifically bind to an inverted-repeat-containing region upstream of hetp, a heterocyst differentiation gene. However, no such invertedrepeat sequence can be found in promoters of other genes in the genome. hetz is a gene involved in early heterocyst differentiation. As shown with the gfp reporter gene, transcription from P hetz was correlated to the expression level of hetr and inhibition by RGSGR, the pentapeptide derived from the C terminus of PatS. As detected by electrophoretic mobility shift assay, a recombinant HetR showed specific binding to the region upstream of hetz, and the binding was inhibited by RGSGR. Tests of a series of the upstream fragments delimited the HetR-binding site to a 40-bp region that shows similarity to that upstream of hetp. The introduction of substitutions of bases conserved in the two HetR-binding sites showed that at least 12 bases are required for recognition by HetR. Deletion of a 51-bp region containing the HetR-binding site completely eliminated the transcription activity of P hetz. Based on the HetR recognition sequence of hetz, those upstream of hetr and pata are proposed. Cyanobacteria are oxygenic photosynthetic prokaryotes that are widely distributed in various aquatic and terrestrial environments (7). Under nitrogen-deficient conditions, some filamentous cyanobacteria can produce specialized cells, called heterocysts, to perform nitrogen fixation (34). Anabaena/Nostoc sp. strain PCC 7120 (here referred to as Anabaena 7120), often used in the study of heterocyst differentiation, responds to nitrogen stepdown by producing semiregularly spaced heterocysts along its filaments. Heterocyst pattern formation may be largely interpreted by an activator-inhibitor model (13, 30, 32). HetR is often referred to as the master regulator or activator of heterocyst differentiation (4, 20, 27), while RGSGR or RGSGR-containing peptides are considered to be the inhibitors that may diffuse from heterocysts to vegetative cells (27, 36). HetR has been shown to be a DNA-binding regulatory protein (16, 18, 20, 28). Purified recombinant HetR shows strong and specific binding to an inverted-repeat-containing sequence upstream of hetp (16), a gene required for heterocyst differentiation (12). However, the inverted-repeat sequence is not found in fragments upstream of hetr, hepa, pats (18), and pkne (28), to which HetR can bind. The structure of HetR from Fischerella sp. strain MV11, a member of subsection V of cyanobacteria, forms a homodimer that comprises a central DNA-binding domain with two helix-turn-helix motifs (N-terminal regions), two flap domains extending in opposite directions, and a hood domain over the central core (C-terminal region) (20). A hetr-null mutant of Anabaena 7120 is unable to initiate heterocyst differentiation (1, 3), whereas various point mutations in hetr abolish, reduce, or greatly enhance heterocyst differentiation (19, 25). RGSGR, the pentapeptide that promotes the decay of HetR in vegetative cells (27) and inhibits the binding of HetR to the DNA target (16), was identified at the C terminus of PatS (13 or 17 amino acids [aa]) (36) and also within HetN, a protein inhibitor of heterocyst differentiation (2, 6, 27). No direct evidence has shown that PatS and HetN or their derivatives carrying RGSGR can diffuse or be transported from heterocysts and proheterocysts to adjacent vegetative cells. However, RGSGR added to nitrogen-deficient medium can inhibit heterocyst formation in Anabaena 7120 (36). In a pata mutant that almost only forms terminally positioned heterocysts in a long filament (21), HetR-green fluorescent protein (GFP) forms a gradient of fluorescence in vegetative cells contiguous with heterocysts, and similar gradients can be generated, without induction of cell differentiation, in vegetative cells around the one ectopically producing PatS or HetN (27). Several lines of evidence indicated that RGSGR can directly interact with HetR (11). Located in the hetz-patu5-patu3 cluster, patu3 is involved in pattern formation, whereas hetz is required for heterocyst formation (39). hetz and patu5-patu3 are independently transcribed but also cotranscribed. patu5 and patu3 in Anabaena 7120 correspond to the 5= and 3= portions of patu in Nostoc punctiforme. The hetz-patu cluster, hetr, and pats-like genes have been found in all filamentous cyanobacteria whose genomes have been analyzed (no genomic sequence is yet available for an organism in subsection V) (29, 39). Because hetz and hetr are both among the core set genes of filamentous cyanobacteria (29) and involved in heterocyst differentiation in Anabaena 7120, the two genes may be related in function and associated in evolution. We report that HetR can specifically bind to a region upstream of the only transcriptional start point of hetz and directly controls expression from P hetz. Based on analyses of the conserved bases in the two HetR-binding regions, the HetR recognition sequence is proposed. MATERIALS AND METHODS General. Anabaena 7120 was cultured in BG11 medium (7) on a rotary shaker at 30 C with illumination of 30 microeinsteins m 2 s 1. Antibiot- Received 26 January 2012 Accepted 23 February 2012 Published ahead of print 2 March 2012 Address correspondence to Xudong Xu, xux@ihb.ac.cn. Supplemental material for this article may be found at Copyright 2012, American Society for Microbiology. All Rights Reserved. doi: /jb /12/$12.00 Journal of Bacteriology p jb.asm.org 2297

2 Du et al. ics were added to the medium as required (22). Plasmids were introduced into Anabaena 7120 by conjugation (9). Bright-field and fluorescence photomicrographs were taken as described by Wang and Xu (31). Plasmid construction. Plasmid construction processes described briefly below are detailed in Table S1 in the supplemental material. The accuracy of cloned PCR fragments was confirmed by sequencing. (i) The plasmid used to inactivate hetr. The hetr::c.ce2 fragment was generated by cloning a PCR fragment containing hetr and inserting C.CE2 (2) into the ClaI site within hetr. The fragment carrying hetr:: C.CE2 was then excised from the T-vector and cloned into the sacbcontaining vector prl277 (1), producing phb2458. (ii) The plasmid used to express the HetR-GFP fusion protein in Anabaena The hetr-gfp translational fusion gene was generated by overlap PCR (17) using a PCR fragment carrying hetr with a promoter from Anabaena 7120 and that carrying gfp from pgfpe (24). The PCR fragment carrying P hetr -hetr-gfp was cloned into pmd18-t, and a spectinomycin resistance marker was inserted downstream of hetr-gfp.p hetr - hetr-gfp with the spectinomycin resistance marker was recloned into the pdu1-based vector prl25c (33), producing phb4024. (iii) Plasmids used to produce EF-Ts HetR or EF-Ts in E. coli. The tsf-hetr translational fusion gene (encoding EF-Ts HetR) was generated by overlap PCR using a PCR fragment carrying tsf from Escherichia coli BL21(DE3) and that carrying hetr from Anabaena PCR fragments carrying tsf-hetr or tsf were cloned into pmd18-t and then recloned into pet21b, producing phb3746 or phb3831, respectively. (iv) Plasmids used to probe the transcription of hetz. DNA fragments to be tested were generated by PCR, cloned into pmd18-t, and then recloned into the SmaI-PstI site upstream of gfp in phb912 (31). (v) The plasmid used to overexpress hetr from P psba in the hetf mutant. A PCR fragment containing hetr was cloned in pmd18-t. A kanamycin resistance cassette, C.K2, excised from prl446 (from C. P. Wolk, GenBank accession number EU ) was inserted upstream of P psba in prl439 (8), while the hetr-containing fragment were recloned downstream of P psba. The C.K2-P psba -hetr fragment was cloned into RSF1010-derived plasmid prl59eh (2), producing phb3271. (vi) The plasmid used to test the function of tsf-hetr in Anabaena 7120 hetr::c.ce2. The tsf-hetr fusion gene was positioned downstream of cloned P hetr, and the fragment carrying P hetr -tsfhetr was cloned into prl25c, producing phb4024. Construction of Anabaena 7120 strains. Plasmid phb2458, described in Table S1 in the supplemental material, was introduced into the wild type to inactivate hetr. Homologous double-crossover recombinants were generated based on positive selection with sacb (5). The complete segregation of the hetr::c.ce2 mutant was confirmed by PCR. The hetf mutant was obtained by screening transposon-generated mutants (22), and the insertion site was localized by sequencing inverse PCR products as described in reference 22. Strains expressing gfp from the cloned fragments were generated by introducing the phb912-derived plasmids listed in Table S1 into Anabaena 7120 or mutants. All Anabaena strains are listed in Table 1. Electrophoretic mobility shift assays (EMSAs). EF-Ts HetR was overproduced in E. coli BL21(DE3) (phb3746) induced with 1 mm IPTG (isopropyl- -D-thiogalactopyranoside) for 4 h. The E. coli cells were washed with TS buffer (20 mm Tris-HCl, ph 8.0, 100 mm NaCl), resuspended in binding buffer (TS buffer plus 20% glycerol), and broken with a French press at 6 MPa followed by sonication for 2 min. The cell lysate was centrifuged at 13,360 g at 4 C for 20 min, and EF-Ts HetR was purified from the supernatant by using the HiTrap heparin column (GE Healthcare) as described by the manufacturer. EF-Ts HetR was eluted from the column with the elution buffer (20 mm Tris-HCl, ph 8.0, 200 mm NaCl, 20% glycerol) and desalted using a HiTrap desalting column. His 6 -tagged EF-Ts was overexpressed in E. coli BL21(DE3) (phb3831) induced with IPTG, purified from total soluble proteins using the His Bind purification kit (Novagen) and desalted using the HiTrap desalting column according to the manufacturers instructions. The cleavage of EF-Ts HetR ( 95 g) by ProTEV Plus (10 U; Promega) was performed at 25 C for 1 and 2 h. The reaction mixture was then centrifuged at 13,360 g at 4 C for 20 min, and the supernatant solution was used for the EMSA. DNA fragments F1 to F5 were generated by PCR using primer pairs PhetZ-1/PhetZ-2, PhetZ-3/PhetZ-4, PhetZ-5/PhetZ-6, PhetZ-7/PhetZ-8, and PhetZ-9/PhetZ-10, respectively, F4L was generated by using primer pair PhetZ-4-up/PhetZ-4-down, F4-1 was generated by using primer pair PhetZ-win1-up/PhetZ-4-down, and F4-4 was generated by using primer pair PhetZ-4-up/PhetZ-win4-down. F4-2 was generated by overlap PCR (17) using primers PhetZ-4-up/PhetZ-win2-up and PhetZ-win2- down/phetz-4-down, and F4-3 was generated by using primers PhetZ-4- up/phetz-win3-up and PhetZ-win3-down/PhetZ-4-down. Biotin labeling of these DNA fragments was performed by second-round PCR using primers Biotin-oligo-1/oligo-2, which match the 5= end sequences of primers used in the first-round PCR. PCR products were purified after agarose gel electrophoresis. Small fragments win4 and win4-1 through win4-5, used as competitors for F4L, were prepared by annealing complementary oligonucleotide pairs PhetZ-Win4-for/PhetZ-Win4-rev and PhetZ-Win4-1-for/PhetZ-Win4-1-rev through PhetZ-Win4-5-for/ PhetZ-Win4-5-rev, respectively. The biotin-labeled 40-bp fragment and 30-bp fragments without region a, b, c, or d (see Results) were prepared by annealing complementary oligonucleotide pairs Bio-PhetZ-f/PhetZ-r, and Bio-PhetZ-1-f/PhetZ-1-r through Bio-PhetZ-4-f/PhetZ-4-r, respectively. The 193-bp fragments with substitution groups i through vii were generated by PCR using primer pairs PM-f/c-1-r, PM-f/c-2-r, PM-f/c-3-r, PM-f/c-4-r, PM-f/c-5-r, PM-f/c-6-r, and PM-f/c-7-r, respectively. The first five fragments were reamplified using primers PM-b-f/PM-b-r, while the 6th and 7th fragments were reamplified using PM-b-f/c-6b-r and PMb-f/c-7b-r, respectively. Biotin labeling of these 7 fragments were performed by PCR using primer pair bio-f/bio-r. A ca. 190-bp fragment within rbp1 of Synechocystis sp. strain PCC 6803 generated by PCR using primer pair rbp1-1/rbp1-2 was used as the nonspecific competitor in EMSA. The oligonucleotides are listed in the Table S1 in the supplemental material. About 44 nm (or as indicated) biotin-labeled DNA fragments with or without unlabeled specific or nonspecific competitor DNA fragments (40-fold excess or as indicated) was incubated with 1 M EF-Ts HetR or EF-Ts or 1.5 M ProTEV Plus protease-cleaved EF-Ts HetR in 20 l of EMSA binding buffer containing 4 mm Tris-HCl (ph 7.5), 12 mm HEPES (ph 7.5), 12% glycerol, 50 mm NaCl, 10 mm MgCl 2, 0.1 g bovine serum albumin (BSA), and 0.5 g poly(di-dc). The mixture was incubated at 25 C for 20 min and separated by electrophoresis with 4% nondenaturing polyacrylamide gel at 4 C. Labeled DNA fragments were then transferred onto Hybond-N membrane and visualized as previously described (15). RACE. The transcriptional start point of all0097 was determined by rapid amplification of cdna ends (RACE) as described by Zhang et al. (39), but primers all and all (see Table S1 in the supplemental material) were used in the first and second rounds of PCR, respectively. RESULTS Responses of P hetz to HetR and RGSGR in vegetative cells. In a previous report (39), P hetz -gfp showed no expression in a hetr:: Tn mutant of Anabaena 7120, suggesting that the transcription of hetz depends on HetR in both vegetative cells and heterocysts. We have now tested whether overexpression of hetr in the presence of fixed nitrogen could elicit overexpression of hetz or other genes in vegetative cells. Because overexpressed hetr induces heterocyst formation in wild-type Anabaena, even in the presence of fixed nitrogen (4), and the expression of P hetz -gfp in vegetative cells could be affected by heterocysts, we used a hetf:: Tn mutant (Table 1) to test the effect of hetr on the expression of 15 genes previously reported to be upregulated in response 2298 jb.asm.org Journal of Bacteriology

3 Direct Regulation of hetz by HetR TABLE 1 Anabaena strains Strain/genotype a Derivation/relevant characteristics b Reference or source Anabaena sp. PCC 7120 Wild type FACHB c DRHB213 Nm r, pata::c.k2d with neomycin resistance cassette C.K2d inserted into the 39 EcoRV site of pata, downstream of which is a gene in the opposite direction; C.K2d is essentially the same as C.K2 (10) except for a 244-bp deletion downstream of the neomycin resistance gene DRHB213 (phb1123) Nm r Sp r, phb1123 carrying P hetr -gfp in phb912 (31) introduced into the pata::c.k2d mutant DRHB213 (phb1465) Sp r Nm r, phb1465 carrying P hetz -gfp in the pdu1-based high-copy-no. 39 plasmid phb912, introduced into the pata::c.k2d mutant DRHB213 (phb3103) Nm r Sp r, phb3103 carrying P all0097 (chromosomal DNA bp ) upstream of gfp in phb912 introduced into the pata::c.k2d mutant DRHB213 (phb3706) Nm r Sp r, phb3706 carrying P hetr -hetr-gfp encoding HetR-GFP in prl25c introduced into the pata::c.k2d mutant DRHB214 Nm r, hetz::c.k2 with neomycin resistance cassette C.K2 inserted into the 39 EcoRV site of hetz, downstream of which is patu3, which can be independently transcribed DRHB214 (phb1465) Sp r Nm r, phb1465 carrying P hetz -gfp in phb912 introduced into the hetz:: 39 C.K2 mutant DRHB2458 Em r, hetr::c.ce2 with chloramphenicol/erythromycin resistance cassette C.CE2 (2) inserted into the ClaI site of hetr, downstream of which is a gene in the opposite direction DRHB2458 (phb4024) Em r Sp r, phb4024 carrying P hetr -tsf-hetr in prl25c introduced into the hetr::c.ce2 mutant hetf::tn5-1087b Em r,tn5-1087b inserted within hetf 310 bp away from its 3= terminus 22 hetf::tn5-1087b (phb1123) Em r Sp r, phb1123 carrying P hetr -gfp in phb912 introduced into the hetf mutant hetf::tn5-1087b (phb1127) Em r Sp r, phb1127 carrying P pata -gfp in phb912 introduced into the hetf mutant hetf::tn5-1087b (phb1465) Em r Sp r, phb1465 carrying P hetz -gfp in phb912 introduced into the hetf mutant hetf::tn5-1087b (phb3271, phb1123) Em r Sp r, phb1123 carrying P hetr -gfp in phb912 introduced into hetf::tn5-1087b (phb3271) d hetf::tn5-1087b (phb3271, phb1127) Em r Sp r, phb1127 carrying P pata -gfp in phb912 introduced into hetf::tn5-1087b (phb3271) hetf::tn5-1087b (phb3271, phb1465) Em r Nm r Sp r, phb1465 carrying P hetz -gfp in phb912 introduced into hetf:: Tn5-1087b (phb3271) WT (phb1465) Sp r, phb1465 carrying P hetz - 39 WT (phb3103) Sp r, phb3103 carrying P all0097 (chromosomal bp ) upstream of WT (phb3106) Sp r, phb3106 carrying P hetz -1 (chromosomal bp ) upstream of WT (phb3107) Sp r, phb3107 carrying P hetz -2 (chromosomal bp ) upstream of WT (phb3108) Sp r, phb3108 carrying P hetz -3 (chromosomal bp ) upstream of WT (phb3109) Sp r, phb3109 carrying P hetz -4 (chromosomal bp ) upstream of WT (phb3110) Sp r, phb3110 carrying P hetz -5 (chromosomal bp ) upstream of WT (phb3224) Sp r, phb3224 carrying P hetz -6 (chromosomal bp ) upstream of WT (phb4067) Sp r, phb4067 carrying P hetz -5-1 (chromosomal bp , with bp deleted) upstream of gfp in phb912 introduced into Anabaena 7120 WT (phb4114) Sp r, phb4114 carrying P hetz -8 (chromosomal bp ) upstream of WT (phb4115) Sp r, phb4115 carrying P hetz -7 (chromosomal bp ) upstream of a DRHB (number) refers to a product of double homologous recombination between plasmid phb (number) and the Anabaena 7120 genome; the plasmids are described in detail in Table S1 in the supplemental material. WT, wild type. b Ap, ampicillin; Em, erythromycin; Km, kanamycin; Sm, streptomycin; Sp, spectinomycin. Unless stated otherwise, the template for PCRs was Anabaena 7120 genomic DNA. c FACHB, Freshwater Algal Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences. d The plasmid phb3271 carries P psba -hetr. May 2012 Volume 194 Number 9 jb.asm.org 2299

4 Du et al. to nitrogen deprivation, namely, coxbii, devb, hepb, hepk, hetn, hetr, hetz, hgld, hgle, nifb, pata, patb, pats, sula, and xisa (31, 39). In a hetf-null mutant, heterocyst differentiation is not initiated, but HetR is accumulated in greater quantities than in the wild type (26, 35). The hetf-minus background precludes the detection of promoters that require both HetR and HetF for expres- FIG 2 Bright-field (I and III) and GFP fluorescence (II and IV) images showing inhibition of the expression of PhetZ-gfp by RGSGR in the pata::c.k2d or hetz::c.k2 mutant. The pata and hetz mutants are denoted DRHB213 and DRHB214 in Table 1, respectively. Arrowheads point to heterocysts. (A) Elimination of the gradient of GFP by RGSGR in pata::c.k2d carrying PhetZ-gfp. Rows: 1, filaments induced to form heterocysts in BG11o (BG11 minus nitrate) without RGSGR; 2, 24 h after nitrogen step-down in the presence of 1 M RGSGR (note inhibition of heterocyst formation); 3, 24 h after addition of RGSGR to inhibit gradients of GFP fluorescence in filaments that had formed the terminal heterocysts. (B) Inhibition of the expression of PhetZ-gfp in hetz::c.k2. Rows: 1, filaments grown in BG11; 2, 48 h after addition of RGSGR; 3, 48 h after removal of RGSGR. (C) Expression of PhetZ-gfp in the wild-type strain (WT) grown in BG11 (III and IV) or at 24 h after nitrogen step-down (I and II) without RGSGR jb.asm.org Journal of Bacteriology FIG 1 Bright-field (I) and GFP fluorescence (II) images showing the expression of PhetZ-gfp, PpatA-gfp, and PhetR-gfp in Anabaena 7120 hetf::tn5-1087b with or without PpsbA-hetR in the presence of fixed nitrogen (BG11). sion. In response to the overexpression of hetr from the strong PpsbA promoter (8), only PhetZ showed greatly increased activity, driving the expression of GFP in vegetative cells, whereas all the other promoters were unaffected (PpatA and PhetR are shown in Fig. 1; other data not shown). As noted above, a HetR-GFP translational fusion exhibits a gradient of fluorescence in vegetative cells near terminal heterocysts in a pata mutant (27), which is reproducible under our conditions (see Fig. S1 in the supplemental material). If the expression from PhetZ is correlated to the amount of HetR, the expression of PhetZ-gfp in this mutant should result in a similar gradient. Indeed, PhetZ-gfp produced a gradient of GFP decreasing toward terminal heterocysts in most filaments of the pata::c.k2d mutant, whereas the transcriptional fusion PhetR-gfp produced no such gradient (see Fig. S2). The binding of HetR to DNA can be inhibited by the pentapeptide RGSGR, which is found at the C terminus of PatS (36) and internally in HetN (27). To test whether the expression of PhetZ-gfp in Anabaena strains could be inhibited by exogenous RGSGR, we induced heterocyst differentiation in the pata mutant carrying PhetZ-gfp for 24 h, by which time a gradient of GFP had been formed in proximity to heterocysts (Fig. 2A, row 1), and then added 1 M RGSGR to the medium. Twenty-four hours later, the pentapeptide fully or partially inhibited the GFP fluorescence in vegetative cells in different filaments, and no GFP gradient was

5 Direct Regulation of hetz by HetR FIG 3 Preparation of EF-Ts HetR fusion protein that retains the function of HetR. (A) Complementation of Anabaena 7120 hetr::c.ce2 with the EF-Ts HetR fusion gene. The hetr mutant is denoted DRHB2458 in Table 1. The hetr::c.ce2 mutant with or without phb4024 carrying the fusion gene was deprived of nitrogen for 24 h. Arrows point to heterocysts. (B) Purification of EF-Ts HetR from E. coli. EF-Ts HetR was induced in E. coli BL21(DE3) (phb3746) and purified from total soluble proteins by using a heparin column. Lane 1, soluble proteins extracted from E. coli cells induced by IPTG for 3 h; lanes 2 to 4, fractions eluted from the heparin column; lane 5, EF-Ts HetR after desalting. evident (Fig. 2A, row 3). If RGSGR was added at the beginning of nitrogen step-down, neither heterocysts nor GFP gradients could be formed in filaments (Fig. 2A, row 2). Also, we wondered if the expression from P hetz is responsive to the addition of RGSGR in filaments not induced for heterocyst formation. In BG11 medium, which contains fixed nitrogen, the expression of P hetz -gfp was intensified in the hetz::c.k2 mutant relative to the wild type (39), was strongly diminished by RGSGR, and was restored upon removal of RGSGR from the medium (Fig. 2B). Representing a control to Fig. 2A and B, Fig. 2C shows the expression of P hetz -gfp in the wild type without RGSGR before and after nitrogen stepdown. Specific binding of an EF-Ts HetR fusion protein to a region upstream of hetz. The evidence described above suggested that HetR may directly regulate transcription of hetz. Therefore, we sought to determine whether HetR binds upstream from the gene and, if so, where the binding site is. To promote solubilization of HetR in Escherichia coli, we joined HetR to elongation factor EF-Ts (14) with a linker peptide (ENLYFQGQF [underlining represents the tobacco etch virus {TEV} protease recognition site]). The fusion, encoded in plasmid phb4024 (see Table S1 in the supplemental material), complemented the hetr::c.ce2 mutant of Anabaena 7120 (Table 1), restoring heterocyst formation (Fig. 3A). Therefore, we consider the fusion protein to be a suitable surrogate for HetR in electrophoretic mobility shift assay (EMSA) experiments. The fusion was overexpressed in E. coli as inclusion bodies and in a soluble form and purified from total soluble proteins (Fig. 3B). The EF-Ts HetR fusion protein was used in EMSAs with a series of 200-bp PCR fragments derived from the regions upstream from hetz (primers are described in Materials and Methods and in Table S1 in the supplemental material). Of the five tested fragments F1 to F5 (Fig. 4A), F3 (chromosomal bp to ) and F4 (chromosomal bp to ) showed strong binding to the fusion protein (Fig. 4B, panel I). The binding of EF-Ts HetR to F3 could be eliminated by adding 40-fold excess nonspecific competitor (a ca.190-bp fragment within rbp1 of Synechocystis sp. PCC 6803), whereas the binding to F4, although reduced, remained strong. The EF-Ts HetR F4 binding, however, could be eliminated by addition of nonlabeled F4 as the specific competitor and was greatly reduced by the addition of RGSGR. These results indicated that the fusion protein can bind specifically to a region upstream of hetz. We tested whether EF-Ts could bind to these fragments but found no binding activity (see Fig. S3 in the supplemental material). In addition, we used ProTEV Plus to excise HetR from the fusion protein and determined that the excised HetR bound F4 specifically (see Fig. S4). To localize further the site of HetR binding in F4, we generated a series of 50-bp deletions in F4 by PCR (Fig. 4A) and assessed the binding of EF-Ts HetR to the resulting PCR fragments. EF- Ts HetR bound to F4, a slightly larger fragment (F4L), and F4-1 through F4-4 (Fig. 4C, panel I). The binding to F4-4 was weaker than that to F4-1 through F4-3 and could be eliminated by nonspecific competition. Based on the sequence of the 51-bp region (chromosomal bp to ) deleted from F4-4, we designed a series of DNA fragments, including the full-length fragment win4 and fragments with 10- or 11-bp deletions, named win4-1 to win4-5 (Fig. 4A). The synthetic DNA fragments were used as the competitors to F4L for binding to EF-Ts HetR (Fig. 4C, panel II). Fragments win4 and win4-1 reduced the binding of EF-Ts HetR to F4L. Fragments win4-2 through win4-5 showed no such effect. The 40-bp region deleted from win4-2 through win4-5, chromosomal bp to , shows significant similarity to a region upstream of hetp, overlapping the reported inverted-repeat-containing sequence. As shown in Fig. 4D, over the 40-bp region, at least 24 bases are identical between them. Higa and Callahan (16) reported that the binding of HetR to P hetp requires the region containing a 17-bp inverted repeat. We then used the synthetic 40-bp double-stranded DNA or 30 bp without each of 10-bp sequences (a to d in Fig. 4D) to perform EMSA with recombinant HetR (Fig. 4E, panel I). The lack of region d reduced the binding of HetR to the target DNA, while the lack of any of the 3 other regions (a to c) almost abolished the binding. We also generated 193-bp DNA fragments carrying mutated HetR-binding regions. In these fragments, the 24 conserved bases were replaced in 7 groups, indicated as i to vii in Fig. 4D. In the presence of nonspecific competitor DNA, binding of recombinant HetR to these fragments was reduced by substitutions located within GGGTCTAgCCCagCA (where lowercase letters indicate nonconserved bases) (Fig. 4E, panel II) but not by substitution groups i, ii, and vii. These results indicated that GGGTCTAgCCCagCA should be the core sequence required for recognition of P hetz by HetR. Similar sequences can be found upstream of hetr (GGGTGCAGCCC AAAA, chromosomal bp to ), pata (GGCTCAAA CCCATCA, chromosomal bp to ), and hepa (GG GCAATGCCCACCA, chromosomal bp to ). A search of the genome with GGGCAATGCCCACCA (upstream of May 2012 Volume 194 Number 9 jb.asm.org 2301

6 Du et al. FIG 4 Delimitation of the region required for the binding of HetR to the hetz promoter. Primers used to generate the DNA fragments by PCR or annealing are listed in Table S1 in the supplemental material. A ca. 190-bp fragment within rbp1 of Synechocystis PCC 6803 was used as the nonspecific competitor. (A) A schematic diagram showing nested constructs used to delimit the HetR-binding site. tsp, transcriptional start point. (B) EMSA detecting the binding of EF-Ts HetR to regions upstream of hetz showing the specific binding to F4, a 189-bp fragment. Panel I shows EMSA results for fragments F1 to F5. Lanes 1, 44 nm biotin-labeled fragment as indicated; lanes 2, 44 nm biotin-labeled fragment with 1 M EF-Ts HetR; lanes 3, 44 nm biotin-labeled fragment with 1 M EF-Ts HetR and 1.6 M nonspecific DNA. Panel II shows inhibition of the binding of EF-Ts HetR by unlabeled F4 or RGSGR. Lane 1, 44 nm biotin-labeled F4 with 1 M EF-Ts HetR; lane 2, 44 nm biotin-labeled F4 and 1 M EF-Ts HetR supplemented with 20-fold excess nonspecific DNA; lane 3, the same as lane 2 but with 40-fold excess nonspecific DNA; lane 4, 44 nm biotin-labeled F4 and 1 M EF-Ts HetR supplemented with 20-fold excess nonlabeled F4; lane 5, the same as lane 4 but with 40-fold excess nonlabeled F4; lane 6, 44 nm biotin-labeled F4 with 1 M EF-Ts HetR supplemented with 10 M RGSGR. (C) EMSA delimiting the HetR-binding site to a 40-bp region (overlapping the deletions in win4-2 to win4-5) in F4. Panel I shows EMSA results with fragments F4L and F4-1 to F4-4 showing the requirement of the region deleted from F4-4 for recognition by EF-Ts HetR. Lanes 1, 44 nm biotin-labeled fragment as indicated; lanes 2, 44 nm biotin-labeled fragment with 1 M EF-Ts HetR; lanes 3, 44 nm biotin-labeled fragment with 1 M EF-Ts HetR and 40-fold excess nonspecific DNA. Panel II shows EMSA results showing the inhibition of the binding of EF-Ts HetR to F4L by synthetic fragments win4 and win4-1 but not by or only weakly by win4-2 to win4-5. About 30 nm F4L and 1 M EF-Ts HetR, supplemented with 50-fold excess nonlabeled synthetic fragments, were used. (D) An alignment of 51-bp sequences containing the HetR-binding site of P hetz and that of P hetp. Identical bases are shown in boldface. The inverted-repeat structure of P hetp is indicated by arrows. a to d represent regions being deleted in synthetic 40-bp biotin-labeled fragments. i to vii represent substitution groups being introduced into a 193-bp fragment. (E) EMSA results showing the HetR recognition site in the 40-bp region upstream of hetz. Panel I shows binding of EF-Ts HetR to synthetic biotin-labeled fragments without (K; 40 bp) or with (30 bp) a 10-bp deletion (-a to -d). Lane 1, 1.5 nm biotin-labeled fragment; lane 2, 1.5 nm biotin-labeled fragment with 1.5 M EF-Ts HetR. Panel II shows binding of EF-Ts HetR to a 193-bp fragment without (K) or with group substitutions (i to vii) in the 40-bp region. The reaction mixture contained 80 nm biotin-labeled 193-bp fragment, 1.5 M EF-Ts HetR, and 20-fold nonspecific DNA. hepa) resulted in a total of 29 copies (see Table S2 in the supplemental material). cis elements that regulate the expression from P hetz. To identify possible cis-localized elements that may regulate hetz, we tested a series of DNA fragments upstream of or overlapping the 5= end of hetz (Fig. 5A) with the gfp reporter gene in pdu1-based plasmid phb912 (31). Fragments P hetz -1 to P hetz -5, with 3= ends all located 4 bp downstream of the transcriptional start point (chromosomal bp ) of hetz (39), are trimmed at their 5= ends to different lengths. All of these fragments promoted the expression of gfp in Anabaena 7120 (Fig. 5B), suggesting that P hetz -5, chromosomal bp to , suffices to drive the expression of hetz.p hetz -1 to P hetz -3 showed stronger expression of GFP than did P hetz -5. However, P hetz -6, the region of P hetz -3 upstream of P hetz -5, showed no promoter activity. P hetz -5-1, which is the same as P hetz -5 except that it lacks the 51-bp region shown in Fig. 4D, failed to promote the expression of gfp. Upstream from hetz and transcribed in the opposite direction is all0097 (Fig. 5A). Please note that all0097 (chromosomal bp to ) is a reannotation of asl0097 ( to ) in Cyanobase ( (39). The transcriptional start point of all0097 was located at chromosomal bp by RACE. In contrast to P hetz,p all0097 showed slightly stronger expression in vegetative cells than in heterocysts in the wild type (Fig. 5B) and produced no gradient of GFP near the terminal heterocysts in a pata mutant (see Fig. S5 in the supplemental material). To test if the expression from P hetz can be affected by the antisense transcription from P all0097, we generated P hetz -7, a fragment without transcription from P all0097, and P hetz -8, a longer fragment with transcription from P all0097 (Fig. 5B). As shown with GFP fluorescence, expression from P hetz -7 in both vegetative cells and heterocysts was stronger than that from P hetz -8. Without the antisense transcription from P all0097,p hetz -1 through P hetz -5 also showed expression levels remarkably higher than that of P hetz -8. DISCUSSION HetR is the master regulator of heterocyst differentiation, and as a transcriptional regulator, it directly regulates some genes involved in heterocyst differentiation or pattern formation (16, 18, 28). Therefore, identification of the HetR recognition sequence is important to understanding the mechanism of gene regulation by HetR and that of heterocyst differentiation. A 17-bp invertedrepeat-containing sequence has been identified to be involved in the binding of HetR upstream of hetp (16). In this study, we identified the HetR-binding site of hetz, and by comparing the two sites and introducing mutations to the binding target sequence, we further defined the sequence required for recognition by HetR jb.asm.org Journal of Bacteriology

7 Direct Regulation of hetz by HetR FIG 4 continued The dependence of P hetz activity on the function of hetr was reported in our previous work (39). Here, we found that overexpression of hetr in vegetative cells of a hetf-null mutant promoted the overexpression of P hetz -gfp. The inability to promote the overexpression of P hetr -gfp was probably due to the hetf-minus background. Transcriptional autoregulation of hetr during heterocyst differentiation requires hetf (26). The regulation of hetz by HetR, as we have shown, is independent of hetf. Moreover, RGSGR, which inhibits the binding of HetR to a fragment upstream of hetr (18) and hetp (16), greatly inhibited the expression of P hetz -gfp in vegetative cells in the presence and absence of fixed nitrogen. In addition, Risser and Callahan (27) showed that in a pata mutant, fluorescence of HetR-GFP diminishes near terminal heterocysts, and we observed that in a pata mutant, the expression of P hetz -gfp resulted in GFP fluorescence decreasing toward terminal heterocysts. All of these lines of evidence support the premise that HetR May 2012 Volume 194 Number 9 jb.asm.org 2303

8 Du et al. FIG 5 Regions required for the expression or modulation of the expression of hetz as shown with a gfp reporter gene. (A) DNA fragments used to drive the expression of gfp.p hetz -1 through P hetz -5, P hetz -7, and P hetz -8 all possess the HetR-binding site. P hetz -5-1 is P hetz -5 without the 51-bp region shown in Fig. 4D. P hetz -7 has the same 5= end that P hetz -3 and P hetz -8 have; its 3= end (chromosomal bp ) lies 47 bp beyond the transcriptional start point (chromosomal bp ) of P all0097. Transcription from P all0097 is in the direction opposite that of P hetz fragments. Plasmids carrying the DNA fragments upstream of gfp and Anabaena 7120 strains carrying these plasmids are described in Table S1 in the supplemental material. (B) Bright-field (I) and GFP fluorescence (II) images showing the expression of gfp from the upstream DNA fragments at 24 h after nitrogen step-down. Arrows point to heterocysts. regulates hetz and that the expression from P hetz is correlated to the amount of HetR. We also identified the HetR-binding site by a series of electrophoretic mobility shift assays. In vitro, 1 M recombinant HetR showed strong and specific binding to a region 5= from hetz. The HetR-binding site was first localized to a 189-bp fragment (F4) upstream of the transcriptional start point of hetz and then to a 51-bp region (the deletion from F4-4). Deletion of each quartile of 2304 jb.asm.org Journal of Bacteriology

9 Direct Regulation of hetz by HetR the 3=-end 40-bp sequence from the 51-bp fragment significantly reduced its effectiveness as a specific competitor in the F-4L/HetR binding assay. We further defined the sequence required for HetR recognition by binding of recombinant HetR to synthetic 40- or 30-bp double-stranded DNA or to 193-bp target DNA with substitutions in the 40-bp region, and both indicated that the core recognition sequence should be within GGGTCTAGCCCAGCA. The results produced with short synthetic fragments as the competitor or directly as labeled binding target and those with 193-bp labeled fragments bearing substitutions were partially different from each other. Notably, the 3=-most 10 bases, TAGAGAAACA, required for the synthetic DNA competitor to reduce the binding of HetR to F-4L, showed a partial effect on binding to the synthetic labeled fragment and no effect on binding to the 193-bp fragment. This region is probably not essential for recognition by HetR but enhances the binding of HetR to the core recognition sequence. Even though P hetp and P hetz both possess a HetR-binding site, the transcription of hetz depends strictly on the presence of HetR, whereas hetp has multiple transcriptional start points, including the one controlled by HetR (16). The lack of either HetR (39) or a HetR-binding site (Fig. 5B) completely abolished the expression from P hetz. For this reason, P hetz -gfp may be a better biosensor to show the concentrations of HetR and RGSGR in Anabaena cells than P hetp -gfp. In addition to P hetz and P hetp, fragments upstream of hetr, hepa, pats (18), and pkne (28) have been shown to be bound by recombinant HetR. Sequences similar to the core HetR recognition sequence could be found upstream of hetr, pata, and hepa but not pats and pkne. The potential HetR recognition site upstream of hetr is not within the tested fragments (18) but upstream of the 271 transcriptional start point whose activity is inhibited by HetN and PatS (see Fig. S6 in the supplemental material) (23). That of pata is upstream of the 305 transcriptional start point (37) but oriented in the direction opposite that of the transcription (see Fig. S6). Because the tertiary structure of the HetR dimer is symmetric (20), whether and how the orientation of the HetR-binding site affects the expression of a gene remains to be investigated. That of hepa is 622 bp upstream of the reported transcriptional start point (40). However, according to a promoter analysis using luxab as the reporter (40), the region containing the potential HetR-binding site is not required for the transcription activity of hepa. Multiple alignments of (potential) HetR-binding regions of hetz, hetp, hetr, and pata resulted in the consensus recognition sequence GGGTC-A-CCCA-CA (see Fig. S7) in Anabaena 7120, which is consistent with the core recognition sequence GGGTCTAgCCCagCA derived from our experimental analyses. Upon nitrogen step-down, HetR is accumulated in cells developing toward heterocysts. Hypothetically, the affinity of HetR to different promoters, which may largely depend on the target sequences, can be important for determining when and how strongly the genes are expressed. It has been pointed out that genes predicted to encode HetR and small peptides with C-terminal RGSGR are found in all filamentous cyanobacteria with sequenced genomes (38). Moreover, hetr-like genes from non-heterocyst-forming species and a patslike gene from one such species function in heterocyst formation in Anabaena 7120 (38). Available data (see Table S3 in the supplemental material) concerning the distribution of HetZ- and HetPlike proteins in filamentous cyanobacteria suggest that both are found in heterocyst-forming and non-heterocyst-forming species, but hetp-like genes are missing in some of the latter type. The regulation of hetz and hetp by HetR/RGSGR might have originated in filamentous cyanobacteria long before the emergence of heterocysts. Generally, the HetR-binding sites of hetz and hetp appear to be highly conserved in a number of heterocyst-forming species but much less conserved in species that do not form heterocysts (see Fig. S8 and S9). However, even in Cylindrospermopsis raciborskii, a heterocyst-forming species, the presumed HetRbinding site of the most probable hetz gene is much less conserved than those of Anabaena/Nostoc species. Similarly, the presumed HetR-binding sites of the most probable hetp genes in Nostoc azollae and Cylindrospermopsis raciborskii are much less conserved than those of other Anabaena/Nostoc species. Perhaps recognition sites for HetR proteins changed during evolution. Also, in addition to the type of recognition sequence reported in this paper, there may be other types of HetR recognition sequences, like those upstream of pats and pkne, to be identified. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grants and ) and the State Key Basic Research Development Program of China (grant 2008CB418001). We thank an anonymous reviewer for pointing out the potential HetR recognition sequences upstream of hetr, pata, and hepa. REFERENCES 1. Black TA, Cai Y, Wolk CP Spatial expression and autoregulation of hetr, a gene involved in the control of heterocyst development in Anabaena. Mol. 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