Sycrp2 Is Essential for Twitching Motility in theCyanobacterium Synechocystis sp. Strain PCC 6803

Wei-Yu Song,a Sha-Sha Zang,a Zheng-Ke Li,a Guo-Zheng Dai,a Ke Liu,a Min Chen,b Bao-Sheng Qiua

aSchool of Life Sciences and Hubei Key Laboratory of Genetic Regulation and Integrative Biology, CentralChina Normal University, Wuhan, Hubei, People's Republic of China

bSchool of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia

ABSTRACT Two cAMP receptor proteins (CRPs), Sycrp1 (encoded by sll1371) andSycrp2 (encoded by sll1924), exist in the cyanobacterium Synechocystis sp. strain PCC6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMPand is involved in motility by regulating the formation of pili. However, the functionof Sycrp2 remains unknown. Here, we report that sycrp2 disruption results in theloss of motility of Synechocystis sp. PCC 6803, and that the phenotype can be recov-ered by reintroducing the sycrp2 gene into the genome of sycrp2-disrupted mutants.Electron microscopy showed that the sycrp2-disrupted mutant lost the pilus appara-tus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcriptlevel of the pilA9-pilA11 operon (essential for cell motility and regulated by thecAMP receptor protein Sycrp1) was markedly decreased in sycrp2-disrupted mutantscompared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pull-down assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimerand that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gelmobility shift assays revealed that Sycrp1 specifically binds to the upstream regionof pilA9. Together, these findings indicate that in Synechocystis sp. PCC 6803, Sycrp2regulates the formation of pili and cell motility by interacting with Sycrp1.

IMPORTANCE cAMP receptor proteins (CRPs) are widely distributed in cyanobacteriaand play important roles in regulating gene expression. Although many cyanobacte-rial species have two cAMP receptor-like proteins, the functional links between themare unknown. Here, we found that Sycrp2 in the cyanobacterium Synechocystis sp.strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, aknown cAMP receptor protein involved with twitching motility. Our findings indicatethat the two cAMP receptor-like proteins in cyanobacteria do not have functional re-dundancy but rather work together.

KEYWORDS cAMP receptor protein, cyanobacterium, phototactic movement,twitching motility

Cyanobacteria are among the most diverse and ancient oxygen-evolving photosyn-thetic prokaryotic phyla, inhabiting almost every terrestrial and aquatic habitat on

Earth. Many have developed various motility strategies to navigate toward or awayfrom stress environments. Twitching motility, mediated by type IV pili, occurs in a widerange of Gram-negative bacteria, including cyanobacteria (1, 2). It is known that theextension, tethering, and then retraction activities of type IV pili are required fortwitching motility (3). Type IV pili are assembled into a highly ordered complex at theinner membrane, and span the murein layer and the outer membrane. The type IV pilussystem contains PilQ, PilN, PilO, PilP, PilM, PilC, PilB1, and PilT proteins (4–7). PilQ is amultimeric outer membrane protein that forms gated pores through which the pili are

Received 19 July 2018 Accepted 10 August2018

Accepted manuscript posted online 13August 2018

Citation Song W-Y, Zang S-S, Li Z-K, Dai G-Z,Liu K, Chen M, Qiu B-S. 2018. Sycrp2 is essentialfor twitching motility in the cyanobacteriumSynechocystis sp. strain PCC 6803. J Bacteriol200:e00436-18. https://doi.org/10.1128/JB.00436-18.

Editor Conrad W. Mullineaux, Queen MaryUniversity of London

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Bao-Sheng Qiu,bsqiu@mail.ccnu.edu.cn.

RESEARCH ARTICLE

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extruded (8, 9). Pilus extension and retraction are powered by PilB and PilT ATPases,respectively (4, 10–12).

Many cyanobacteria use type IV pilus-dependent twitching motility for phototacticmovement, which allows cyanobacterial cells to translocate themselves into optimallight conditions (13). Genes involved in positive phototactic movement have beenextensively studied in the model cyanobacterium Synechocystis sp. strain PCC 6803(here Synechocystis 6803). These genes are divided into two groups, as follows: (i) genesinvolved in pilus biogenesis and motility, such as pilA1, pilA9, pilA10, pilA11, and otherassembled proteins mentioned above (10, 13–17), and (ii) genes involved in signaltransduction for pilus assembly and phototaxis, such as taxD1 (pixJ1) and slr1694, whichboth encode proteins containing a light-sensing domain (10, 17–20). Adenylate cyclaseSlr1991 and the cyclic AMP (cAMP) receptor protein Sycrp1 belong to group II and areessential for motility in Synechocystis 6803 (21, 22). cAMP receptor proteins (CRPs) areinvolved in regulating the transcription of genes in catabolism and other processes inGram-negative bacteria (23). The larger NH2-terminal domain of CRPs contains a cAMPbinding site, and the COOH-terminal domain of CRPs has a helix-turn-helix domain forDNA binding (24). The crystal structure of CRPs has been well characterized in Esche-richia coli (25). The CRP-cAMP complex regulates the transcription of genes dependentupon binding of the complex to the promoter region (23). In Synechocystis 6803, thereare two CRPs, Sycrp1 (Sll1371) and Sycrp2 (Sll1924), which contain a domain homolo-gous to the cAMP binding domain of bacterial CRPs (26). Previous studies reported thatSycrp1 binds to cAMP and is essential for the motility of Synechocystis 6803 but thatSycrp2 does not bind cAMP (21, 26). DNA microarray results showed that the expressionof two gene clusters (slr1667-slr1668 and pilA9-pilA10-pilA11-slr2018), which are essen-tial for motility in Synechocystis 6803, was downregulated in Sycrp1-disrupted mutantscompared with that in the wild-type strain (27). Gel mobility shift assay analysisindicated that Sycrp1 directly binds to the upstream region of slr1667 (27). However, thefunction of Sycrp2 in Synechocystis 6803 remains unclear.

In this study, Sycrp2 was disrupted in Synechocystis 6803, resulting in the loss of motilityand piliation. Compared with those in the wild-type strain, the transcripts of three prepilinencoding genes (pilA9-pilA10-pilA11 operon) were markedly reduced in sycrp2-disruptedmutants. However, Sycrp2 interacted with the cAMP receptor protein Sycrp1, which directlybinds to the upstream region of pilA9. These results indicate that Sycrp2 is involved in theregulation of the motility of Synechocystis 6803.

RESULTSDisruption of Sycrp2 results in the loss of motility of Synechocystis 6803. To

determine whether Sycrp2 of Synechocystis 6803 is involved in pilus biogenesis and cellmotility, we generated sycrp2::C.K2 mutants and the Sycrp2 complementary strain,sycrp2-com (Fig. 1). Sycrp2 was disrupted in sycrp2::C.K2 mutants (Fig. 1A) and wasrestored by the insertion of a modified Omega overexpression system (sycrp2 openreading frame with 1-kb upstream region of sycrp2) into the slr0168 neutral position ofthe sycrp2-com strain (Fig. 1B). Both strains were confirmed by PCR (Fig. 1C). The colonymorphological comparison is shown in Fig. 2. Cells of the wild-type strain formedirregularly shaped, flat, sheet-like colonies because of the motility of individual cells(Fig. 2A and B). However, sycrp2::C.K2 mutants showed a different colony morphology.They formed round-domed colonies, indicating the loss of cell motility (Fig. 2C and D).As shown in Fig. 2E and F, the sycrp2-com strain formed irregularly shaped, sheet-likecolonies similar to those of the wild-type strain, indicating the restoration of cellmotility in the sycrp2-com strain.

Phototactic movement was further monitored to confirm the different motilityabilities of the wild-type strain and the mutants. After 6 days under unilateral illumi-nation, the wild-type strain showed movement toward the light source—specifically,about 10 mm of movement with a moving trace tail (Fig. 2G). However, under the sameorientated illumination, sycrp2::C.K2 mutants did not move during the 6-day induction.The edge of the sycrp2::C.K2 mutant line was very sharp, confirming that mutants

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lacking Sycrp2 had lost motility. Within the 6-day induction with unilateral illumination,the sycrp2-com strain showed positive phototaxis and moved toward the light source,similarly to the wild-type strain (Fig. 2G). These results demonstrate that Sycrp2 inSynechocystis 6803 is critical for cell phototaxis.

Disruption of Sycrp2 results in the loss of piliation in Synechocystis 6803. Todetermine whether Sycrp2 of Synechocystis 6803 is involved in pilus biogenesis andmotility, the pilus appearance of the wild-type strain and mutants was examined usingtransmission electron microscopy at �10,000 magnification (Fig. 3). The sycrp2::C.K2mutant lacked pilus apparatus on the cell surface, although some “thin” pili remainedon the cell surface under our experimental conditions (Fig. 3B). The disruption of Sycrp2

FIG 1 Construction sketch map and PCR analysis of the sycrp2::C.K2 mutant and sycrp2-com strain. (A)Strategy for the disruption of sycrp2 in the Synechocystis 6803 genome. (B) Strategy for complementationof the sycrp2::C.K2 mutant. (C) PCR analysis of genomic DNA from the wild-type strain, sycrp2::C.K2mutant, and sycrp2-com strain. Lanes 1 through 4 used primers sll1924ko1 and sll1924ko2, and lanes 5to 6 used primers slr0168up and sll1924com-1. Lanes 1, 3, and 5, wild-type strain DNA; lane 2, sycrp2::C.K2mutant DNA; lanes 4 and 6, sycrp2-com strain DNA. The fragments are 1.2 kb in the wild-type strain and2.3 kb in the sycrp2::C.K2 mutant and sycrp2-com strain using primers sll1924ko1 and sll1924ko2. Thefragment is 1.7 kb in the sycrp2-com strain using primers slr0168up and sll1924com-1. This fragmentshould not be amplified in the wild-type strain. The bands in lane 5 are nonspecific.

FIG 2 Colony morphology and directional motility assay of the wild-type strain, sycrp2::C.K2 mutant, andsycrp2-com strain on 0.8% (wt/vol) solid BG-11 agar plates. For colony morphology assay, cells grew onplates for 5 days under illumination at 25 �mol photons · m�2 · s�1. For directional motility assays, cellsgrew on plates for 6 days under unidirectional illumination at 1 to 5 �mol photons · m�2 · s�1. (A andB) Wild-type strain. (C and D) sycrp2::C.K2 mutant. (E and F) sycrp2-com strain. (G) Motility state underunidirectional illumination (white light). The left lane is the wild-type strain, the middle lane is thesycrp2::C.K2 mutant, and the right lane is the sycrp2-com strain. The arrows indicate the direction of thelight source. Bars in panels A, C, and E, 1 mm; bars in panels B, D, and F, 0.5 mm.

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destroyed pilus biogenesis and resulted in the disappearance of pilus apparatus on thecell surface. The lack of a pilus structure in sycrp2::C.K2 mutants indicates that “thick” piliare essential for motility. The Sycrp2 complementary strain, sycrp2-com, showed re-stored pilus apparatus and similar cell morphology to that of the wild-type strain (Fig.3A and C), confirming the involvement of Sycrp2 in pilus biogenesis and cell motility inSynechocystis 6803.

The disruption of Sycrp1 resulted in nonmotile Synechocystis 6803 cells (21). Tocompare the functions of Sycrp1 and Sycrp2 in Synechocystis 6803, we constructed aSycrp1-disrupted mutant using the same methods as in described in the legend to Fig.1A. The sycrp1::C.K2 mutant had a similar phenotype to that of the sycrp2::C.K2 mutant(Fig. 3B and D). These results indicate that both Sycrp1 and Sycrp2 are critical for pilusbiogenesis and cell motility and that the deletion of either one results in similar cellmorphology, disappearance of pilus apparatus, and the loss of the phototactic capa-bility of cells.

Sycrp2 disruption markedly reduces expression of the pilA9-pilA11 cluster.More than 20 genes are required for the biogenesis of type IV pili or influence cellmotility in Synechocystis 6803 (8–10, 13–17). To understand the function of Sycrp2, thetranscriptional profiles of 23 genes essential for pilus assembly and retraction or cellmotility were compared between the wild-type strain and sycrp2::C.K2 mutants (Fig.4A). Genes encoding components for minor pilins (pilA9-pilA11) were downregulatedmore than 10-fold in sycrp2::C.K2 mutants (Fig. 4A), similarly to a previous report on theeffects of Sycrp1 disruption (27). Furthermore, the expression levels of sycrp2::C.K2mutants were recovered to the same level as those of the wild-type strain by thereintroduction of Sycrp2 (Fig. 4B). In addition, slr2018, pilJ (slr1044), and sycrp1 were

FIG 3 Electron micrographs of negatively stained cells from different strains. (A) Wild-type strain. (B)sycrp2::C.K2 mutant. (C) sycrp2-com strain. (D) sycrp1::C.K2 mutant. Bars, 1 �m.

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downregulated in sycrp2::C.K2 mutants compared with the wild-type strain (Fig. 4A).Yoshimura et al. suggested that the construction of the cell surface proteins CccS andCccP (encoded by slr1667 and slr1668) was involved directly in the biogenesis of thickpili (28), which were dramatically downregulated in the Sycrp1-disrupted mutant (27).However, Sycrp2 disruption had no influence on their expression; thus, the lack ofmotility of the Sycrp2-disrupted mutant was independent of the functions of CccS andCccP. Compared with those in the wild-type strain, the transcripts of most basic genesparticipating in the assembly and biogenesis of pili were unchanged in the Sycrp2-disrupted mutant, although pilM was upregulated 2-fold. The expression of other genesreported to be essential for the motility of Synechocystis 6803 was also investigated (17,29–36). However, the transcript levels of Sycrp2-disrupted mutants and the wild-typestrain were similar (see Fig. S1 in the supplemental material).

Sycrp1 and Sycrp2 interact with each other and themselves. CRP-like proteinsare widely distributed in cyanobacteria (see Table S1 in the supplemental material), andtwo copies of CRP-like proteins are found in many cyanobacterial species. We selected82 CRP-like proteins from 52 cyanobacterial species distributed in five orders toconduct phylogenetic analysis. CRP-like proteins mainly cluster into two branches (seeFig. S2 in the supplemental material). We defined proteins in the branch that containsSycrp1 and Sycrp2 as type I CRP-like proteins (CRP-I), while proteins in the other branchwere termed type II CRP-like proteins (CRP-II). The amino acid sequence alignment ofCRP-I and CRP-II revealed that although most CRP-like proteins retain only approxi-mately 40% identity, the cAMP binding motif is conserved in almost all CRP-likeproteins. The alignment of 16 CRP-like proteins from nine cyanobacterial speciesindicated that amino acids corresponding to the cAMP binding site are highly con-served, except in Sycrp2 from Synechocystis 6803 (see Fig. S3 in the supplementalmaterial). However, protein structural models demonstrated a high similarity betweenthe Sycrp1 and Sycrp2 proteins, although Sycrp2 does not bind to cAMP (see Fig. S4 inthe supplemental material).

To investigate the interactions between Sycrp1 and Sycrp2, the sycrp1 and sycrp2 genefragments were cloned into pGBKT7 and pGADT7 or pGADT7 and pGBKT7, respectively.The plasmids pGADT7-T-antigen and pGBKT7-p53 were used as positive controls, and theplasmids pGADT7-T-antigen and pGBKT7-lamin C were used as negative controls. As shownin Fig. 5A, Saccharomyces cerevisiae strain AH109 cultures harboring the plasmids pGADT7-T-antigen and pGBKT7-p53, pGADT7-sycrp1 and pGBKT7-sycrp2, pGADT7-sycrp2 andpGBKT7-sycrp1, pGADT7-sycrp1 and pGBKT7-sycrp1, pGADT7-sycrp2 and pGBKT7-sycrp2,

FIG 4 Comparison of transcript levels of essential genes involved in motility using qRT-PCR. (A) The transcript levels of essentialgenes involved in motility examined by qRT-PCR in the wild-type strain and sycrp2::C.K2 mutant. Transcript levels for thesycrp2::C.K2 mutant relative to those of the wild-type strain. (B) The transcript levels of pilA9 (slr2015), pilA10 (slr2016), andpilA11 (slr2017) were examined by qRT-PCR in the wild-type strain, sycrp2::C.K2 mutant, and sycrp2-com strain. CT values foreach gene were normalized to rnpB levels.

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or pGADT7-T-antigen and pGBKT7-lamin C grew on tryptophan and leucine dropoutsupplement (SD/-Trp-Leu) plates, indicating that all the plasmids were successfullycotransformed to S. cerevisiae AH109. Except for the negative control, all other strainsalso grew on the tryptophan, leucine, and histidine dropout supplement (SD/-Trp-Leu-His plates) (Fig. 5B), indicating that Sycrp1 and Sycrp2 interact with each other andthemselves. However, the S. cerevisiae AH109 cultures harboring plasmids pGADT7-sycrp2 and pGBKT7-sycrp2 did not grow on the tryptophan, leucine, histidine, and

FIG 5 Sycrp1 and Sycrp2 interaction analysis by yeast two-hybrid analysis (A to C) and pulldown assay (D to F). sycrp1 andsycrp2 gene fragments were cloned into pGBKT7 and pGADT7 or pGADT7 and pGBKT7, respectively. The resulting plasmidswere cotransformed into Saccharomyces cerevisiae AH109. S. cerevisiae AH109 was cotransformed with different plasmids andthen grown on yeast synthetic dropout medium with tryptophan and leucine dropout supplement (SD/-Trp-Leu) (A), withtryptophan, leucine, and histidine dropout supplement (SD/-Trp-Leu-His) (B), or with tryptophan, leucine, histidine, andadenine dropout supplement (SD/-Trp-Leu-His-Ade) (C) plates for 3 days. Then, 10 �l of yeast was dropped onto plates atoptical densities at 600 nm (OD600) of 0.1, 0.01, and 0.001. From top to bottom: S. cerevisiae AH109 harboring plasmidspGADT7-T-antigen and pGBKT7-p53 as positive controls; S. cerevisiae AH109 harboring plasmids pGADT7-sycrp1 and pGBKT7-sycrp2, pGADT7-sycrp2 and pGBKT7-sycrp1, pGADT7-sycrp1 and pGBKT7-sycrp1, pGADT7-sycrp2 and pGBKT7-sycrp2, orpGADT7-T-antigen and pGBKT7-lamin C as negative controls. Sycrp1-His, Sycrp1-GST, Sycrp2-His, and Sycrp2-GST fusionproteins were expressed in vitro. The fusion proteins Sycrp1-His and Sycrp1-GST, Sycrp1-His and GST-tag (D) or Sycrp2-GST andSycrp1-His, Sycrp1-His and GST-tag (E) or Sycrp2-GST and Sycrp2-His, and Sycrp2-His and GST-tag (F) were incubated withGST-Bind resin. The coeluted proteins from GST-Bind resin were confirmed by Western blotting analyses with GST- andHis-specific antibodies. The Western blotting signals are indicated by arrows. The sizes of Sycrp1-GST and Sycrp2-GST are about52 kDa, the size of the GST-tag is about 26 kDa, and the sizes of Sycrp1-His and Sycrp2-His are about 27 kDa.

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adenine dropout supplement (SD/-Trp-Leu-His-Ade) plates (Fig. 5C), nor did the neg-ative control, indicating that Sycrp2 only interacts weakly with itself.

Pulldown assays were used to confirm the interactions between Sycrp1 and Sycrp2.Sycrp1-His, Sycrp1– glutathione S-transferase (GST), Sycrp2-His, and Sycrp2-GST fusedproteins were expressed in vitro. Different combination groups were incubated withGST-Bind resin, and the coeluted proteins were identified by Western blotting analyseswith GST- and His-specific antibodies. As shown in Fig. 5D to F, the coeluted proteinsfrom the incubated pairs of Sycrp1-GST and Sycrp1-His, Sycrp2-GST and Sycrp1-His, andSycrp2-GST and Sycrp2-His were detected by GST- and His-specific antibodies. How-ever, the coeluted proteins from the incubated pairs of GST-tag and Sycrp1-His andGST-tag and Sycrp2-His, which were used as negative controls, could only be detectedby GST-specific antibodies. These results confirmed that Sycrp1 and Sycrp2 exist in cellsas heterodimers and homodimers by interacting with each other or themselves.

Sycrp1 specifically binds to the upstream region of pilA9, but Sycrp2 does not.Purified His tag-fused Sycrp2 and Sycrp1 were used for gel mobility shift assays toinvestigate whether they directly bind to the upstream region of pilA9 (se Fig. S5 in thesupplemental material). As shown in Fig. 6A, Sycrp2 binds to the probes and formsretarded DNA bands with an additional mass amount of Sycrp2. However, retardedDNA bands were dispersed and free probes accumulated in the presence of unlabeledDNA strands or poly(deoxyinosinic-deoxycytidylic) acid [poly(dI-dC)]. This indicated thatSycrp2 does not specifically bind to the upstream region of pilA9. In contrast, Sycrp1binds to the probes and formed retarded DNA bands with an additional mass of Sycrp1,and the retarded DNA band was markedly weakened by the addition of unlabeled DNA

FIG 6 Gel mobility shift assay of Sycrp2 and Sycrp1 with the upstream region of pilA9. The reactioncomponents are listed above. Retarded DNA and free DNA are indicated by arrows. Purified Sycrp2 (A)or Sycrp1 (B) was added at the amounts indicated above each row and incubated with the labeled DNA.The unlabeled DNA or poly(dI-dC) was added to the last two lanes to exclude nonspecific binding.

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strands. Furthermore, the retarded DNA band was still present when poly(dI-dC) wasadded (Fig. 6B). Different concentrations of poly(dI-dC) were also added to test thebinding affinities of Sycrp1 and Sycrp2 to the upstream region of pilA9 (see Fig. S6 inthe supplemental material). Sycrp1 binds to the probes and formed retarded DNAbands when the mass of poly(dI-dC) added was between 0 and 2,000 ng. The retardedDNA bands were not weakened by increasing the mass of poly(dI-dC) addition (Fig. S6,lanes 2 to 5). The retarded DNA bands formed by Sycrp2 binding to the probes weremarkedly weakened when the mass of poly(dI-dC) added was between 0 ng and 500ng (Fig. S6, lanes 6 to 9). The negative control indicated that Sycrp2 also binds toprobes without a CRP consensus binding motif, and it formed retarded DNA bandswhen poly(dI-dC) was not added (Fig. S6, lane 10). Taken together, these resultsindicate that Sycrp1 specifically binds to the upstream region of pilA9, while Sycrp2does not specifically bind to the upstream region of pilA9. These results suggest thatSycrp1 regulates the expression levels of the pilA9-pilA11 cluster with cooperation fromSycrp2.

DISCUSSION

CRP-like proteins are widely distributed in various prokaryotes, including cyanobac-teria. These proteins bind to cAMP in a dimeric form and then bind to the promoter ofgenes to activate or repress the expression of related genes (37–39). CRPs are involvedin regulating a host of cellular processes in Gram-negative bacteria, including catabo-lism, fatty acid utilization, and flagellum synthesis (23, 40). In Synechocystis 6803, Sycrp1binds to the secondary messenger cAMP and directly regulates the expression of genesinvolved in motility (27). Here, we report that the disruption of Sycrp2 also results in theloss of cell motility of Synechocystis 6803. This phenotype is similar to that of Sycrp1-disrupted mutants reported in a previous study (21). The observation of type IV pilusappearance shows that piliation loss is probably the reason for the loss of cell motilityin sycrp2::C.K2 mutants.

The CRP/cAMP dimer binds to the 22-bp conserved DNA sequence 5=-AAATGTGANTCACATTT-3=6, which is a CRP-binding sequence (CBS) (41). The underlined sequencesare the most important for CRP/cAMP-DNA interactions (41). According to the CBSposition and transcription-activated mechanism, CRP-dependent promoters can beclassified into three classes (42, 43). Class I and class II promoters contain CBS, and theactivation of transcription requires interactions between CRP and the carboxy-terminaldomain of RNA polymerase (RNA-P) �-subunit (�-CTD), as in the lac and galP1 promot-ers (44). In class III promoters (for example, the acsP2 promoter), CRP interacts with DNAat multiple sites in combination with additional proteins (45). In Synechocystis 6803,Sycrp1 binds in vitro to proposed CRP target sites upstream of slr1351 (murF), sll1874(chlAII), sll1708 (narL), slr0442, and sll1268 (46), and the genes slr1351, sll1874, andslr0442 were shown to be targets regulated by Sycrp1 (47). The transcriptional profilesof cccS and cccP appeared to be contradictory between Sycrp1- and Sycrp2-disruptedmutants. The operon regions of the cccS and cccP genes contain CBS in the samelocation as the cAMP receptor protein (Sycrp1) binding site. Compared with those inthe wild-type strain, the transcript levels of cccS and cccP were significantly reduced inSycrp1-disrupted mutants (27). Yoshimura et al. reported that the disruption of CccS orCccP resulted in a lack of thick pili and a nonmotile phenotype, demonstrating theessential roles of these two genes in motility (28). The mechanism of transcriptionactivation of these genes is similar to that for the CRP regulation of class I and class IIpromoters reported in a previous study (44). Sycrp2 disruption had no influence on theexpressions of cccS and cccP (Fig. 4), suggesting that the underlying mechanisms of thenonmotile morphology resulting from the deletion of Sycrp1 and Sycrp2 were notidentical.

Sycrp2 lacks several amino acids (Glu-73, Arg-83, and Ser-84) corresponding to thecAMP binding motif of CRP in E. coli and does not bind to cAMP (26), even though ithas a high structural similarity to Sycrp1 (Fig. S4). Functional CRPs are formed of dimersthat bind to specific DNA sequences when activated by binding to cAMP (25, 26),

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suggesting that a dimeric form of Sycrp2 may be involved in the signal transductionpathway. The evidence for interactions between Sycrp1 and Sycrp2 and the loss of cellmotility by Sycrp1 or Sycrp2 disruption suggests that Sycrp1 and Sycrp2 might coop-erate in the process of cell motility in Synechocystis 6803. Genes encoding the minorpilin components PilA9, PilA10, and PilA11 were significantly downregulated in Sycrp2-disrupted (Fig. 4A) and Sycrp1-disrupted mutants (27). It is known that minor pilins playa vital role in type IV pilus assembly in Pseudomonas aeruginosa (48, 49) and Syn-echocystis 6803, because deletion of pilA9 or pilA11 resulted in complete nonmotility(50). No significant differences in the levels of transcripts for the main components forpilus assembly were observed among the wild-type strain and Sycrp1- and Sycrp2-disrupted mutants, implying that the loss of cell motility by deletion of either Sycrpgene might be related to a significant decrease in minor pilin accumulation (down-regulated expressions of pilA9, pilA10, and pilA11). In wild-type Synechocystis, minorpilins form a major population as secreted “mini” pilins, which may aid the main pilusassembly and biogenesis (50).

Gel mobility shift assays demonstrated that Sycrp1 specifically binds to the up-stream region of pilA9 (Fig. 6B). This conclusion contradicts the study of Yoshimura etal. (27). This might be explained by the different upstream region of pilA9 used in theprevious study. In the genome of Synechocystis 6803, sycrp2 was also located upstreamof pilA9, in the opposite transcription direction. Therefore, the upstream region of pilA9is also upstream of sycrp2. To test whether Sycrp1 regulates the transcription of sycrp2,the relative expression levels of sycrp2 was compared in the wild-type strain andsycrp1::C.K2 mutants using reverse transcription-quantitative PCR (qRT-PCR). As shownin Fig. S7 in the supplemental material, similar levels of sycrp2 transcripts were observedbetween the wild-type strain and sycrp1::C.K2 mutants. Thus, Sycrp1 mainly regulatestranscription of the pilA9 operon. It was reported that another transcription regulator,lexA (sll1626), also binds to the upstream region of pilA9 (34). Compared with thewild-type strain, the transcriptional level of sycrp2 was downregulated in LexA-disrupted mutants (34). This implies that LexA may bind to the upstream region ofsycrp2 to regulate its expression. Because no typical CBS motif exists in the regionupstream of pilA9, its transcription-activated mechanism might be similar to thoseof genes containing the CRP-regulated class III promoter. It was reported that CRPand MalT (the maltose regulon activator) synergistically activate transcription fromthe E. coli malKp promoter (51). In the presence of CRP, MalT binds to thelower-affinity set of sites and triggers transcription initiation. The synergistic actionof MalT and CRP therefore relies on MalT repositioning via the formation of anucleoprotein structure involving the entire regulatory region. However, CRP orMalT alone did not efficiently activate malKp transcription (51). The crystal structureof the cAMP receptor protein from Mycobacterium tuberculosis, in complex withcAMP and its DNA binding element (PDB accession number 3mzh), was used as atemplate to predict the structure of the Sycrp1/Sycrp2 heterodimer and its DNAbinding element (see Fig. S8 in the supplemental material). The binding affinitybetween the Sycrp1/Sycrp2 heterodimer and its DNA binding element might not be astight as the cAMP receptor protein homodimer. This may be useful for the transcriptioninitiation of pilA9. Based on results of the present and previous studies, the two CRP-likeproteins (Sycrp2 and Sycrp1) are essential for twitching motility, and the disruption ofeither of them influenced pilus formation and resulted in the loss of cell motility inSynechocystis 6803. We suggest that the Sycrp2/Sycrp1 heterodimer is involved in theregulated pathway for cell motility in Synechocystis 6803, as shown in Fig. 7. TheSycrp2/Sycrp1 heterodimer binds to cAMP to activate transcription of the pilA9-pilA11cluster. However, the Sycrp1 homodimer and Sycrp2 homodimer did not efficientlyactivate their expression. This functional model is different from the expression ofanother gene cluster, slr1667-slr1668, whose transcription was positively regulated bythe Sycrp1 homodimer.

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MATERIALS AND METHODSStrains and culture conditions. A glucose-tolerant motile strain of Synechocystis sp. PCC 6803 was

cultured in BG-11 medium (52) under continuous illumination of 25 �mol photons · m�2 · s�1 at 30°C.Kanamycin was added at 30 �g · ml�1 when mutants were screened and maintained. For the comple-mentary mutants, 30 �g · ml�1 kanamycin and 25 �g · ml�1 spectinomycin were added to BG-11medium.

Construction of mutant and complementary strains of Synechocystis 6803. The sycrp2::C.K2 orsycrp1::C.K2 mutants were generated by introducing a C.K2 (kanamycin resistance) cassette into the BglIIor Eco52I site of the open reading frame of sycrp2 or sycrp1 by homologous recombination in Syn-echocystis 6803 (53). DNA fragments containing the sycrp2 gene (1,227 bp) or the sycrp1 gene (1,772 bp)were amplified from the genomic DNA of Synechocystis 6803 using the primers sll1924ko1/sll1924ko2 orsll1371ko1/sll1371ko2, respectively (see Table S2 in the supplemental material). The sequences werecloned into pMD18-T (TaKaRa, Japan) and confirmed by sequencing. The C.K2 cassette was inserted intothe BglII site of the plasmid pMD18-T::sycrp2, resulting in plasmid pS1531, or into the Eco52I site of theplasmid pMD18-T::sycrp1, resulting in plasmid pHS1532, for the inactivation of sycrp2 or sycrp1 mutants(Fig. 1A). The method to obtain the complementary strain of sycrp2 was performed as described by Jianget al. (54). The sycrp2 open reading frame with a 1-kb upstream region of sycrp2 fragment was amplifiedfrom the genomic DNA of Synechocystis 6803 using the primers sll1924com-1/sll1924com-2 (Table S2)and then cloned into the NdeI and HpaI sites of the Omega overexpression system (pHS298) (Fig. 1B).

Motility assays. Cells were grown in liquid BG11 medium to late log phase (optical density at 730nm [OD730], 0.8 to 1.0), spread on solid BG-11 medium containing 0.8% (wt/vol) agar, 8 mMN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES; pH 8.0), 5 mM glucose, and 0.3% (wt/vol)sodium thiosulfate, and then cultivated under continuous illumination at a light intensity of 25 �molphotons · m�2 · s�1 at 30°C for 6 days. Colony morphology was monitored using an MZ16 F stereomi-croscope equipped with a DFC480 digital camera and ICM50 software (Leica, Solms, Germany).

Phototactic movement was examined as described by Wilde et al. (55), with some modifications.Strains streaked on solid BG-11 medium plates (0.8% agar, wt/vol) were placed into opaque boxes withan open slit (3 cm width) at one side and then cultivated under continuous illumination at a lightintensity of 1 to 5 �mol photons · m�2 · s�1 at 30°C for 6 days. The movements were monitored byphotography at day 6.

Electron microscopy. Cells of each strain growing on 0.8% (wt/vol) agar plates were suspended inBG-11 medium without physical shaking or stirring. The suspended cells were stained with 0.8% (wt/vol)phosphotungstic acid (pH 7.0) for 5 min and examined by transmission electron microscopy (H-7000FA,Hitachi, Japan) according to Yoshihara et al. (14).

RNA isolation and transcriptional analysis. Total RNA was isolated using the TRIzol reagent(CWbio, China), following the manufacturer’s instructions. The Perfect real-time kit (TaKaRa, Japan) wasused to remove DNA contamination and for the reverse transcription of cDNA. Synthesized cDNAs wereused as a template for real-time PCR amplification. The RNase P subunit B gene (rnpB) was used as aninternal control for normalizing target gene transcriptional abundance. PCR amplifications were per-formed in 10-�l reaction mixtures using the SYBR green real-time PCR master mix (CWbio, China) and 40cycles. Primers used in these experiments are shown in Table S2 in the supplemental material.

Yeast two-hybrid assay. The putative protein-protein interactions of Sycrp2 and Sycrp1 were detectedby the Matchmaker GAL4 two-hybrid system 3 (Clontech, Palo Alto, CA). Saccharomyces cerevisiae AH109 is a

FIG 7 Hypothesis model of Sycrp1 and Sycrp2 heterodimers on the transcription initiation of the pilA9operon. The Sycrp1 and Sycrp2 heterodimer binds to the upstream region of pilA9 (blue region) and activatestranscription efficiently. The homodimer of Sycrp1 does not activate the transcription of pilA9 operonefficiently, and the homodimer of Sycrp2 does not specifically bind to the upstream region of pilA9.

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defective yeast strain that does not grow on yeast synthetic dropout medium with tryptophan dropoutsupplement (SD/-Trp), leucine dropout supplement (SD/-Leu), histidine dropout supplement (SD/-His) oradenine dropout supplement (SD/-Ade). It contains two reporter genes, Gal1-His3 and Gal2-Ade2, which areonly activated by Gal4 protein (56), which contains a DNA binding domain (BD) and DNA activation domain(AD). The plasmid pGADT7 harbors a leucine-producing gene and a Gal4-DNA AD-encoding region withmultiple cloning sites to fuse with other genes. The plasmid pGBKT7 harbors a tryptophan-producing geneand a Gal4-DNA BD-encoding region with multiple cloning sites to fuse with other genes. When the twoplasmids pGADT7 and pGBKT7 were cotransformed to S. cerevisiae AH109, the yeast strain grew on yeastsynthetic dropout medium with tryptophan and leucine dropout supplement (SD/-Trp-Leu). Then one genewas fused to the Gal4-DNA AD-encoding region, and the other gene was fused to the Gal4-DNA BD-encodingregion. The two resulting plasmids were cotransformed to S. cerevisiae AH109, and the two fused proteinswere expressed in this strain. If the two fused proteins can interact with each other, the reporter genesGal1-His3 and Gal2-Ade2 should be activated, and the yeast strain will grow on yeast synthetic dropoutmedium with tryptophan, leucine, and histidine dropout supplement (SD/-Trp-Leu-His) or yeast syntheticdropout medium with tryptophan, leucine, histidine, and adenine dropout supplement (SD/-Trp-Leu-His-Ade).Fragments of sycrp2 and sycrp1 genes were cloned into either pGBKT7 and pGADT7 or pGADT7 and pGBKT7,respectively. The resulting plasmids were cotransformed into Saccharomyces cerevisiae AH109 and selected onyeast synthetic dropout medium with tryptophan and leucine dropout supplement (SD/-Trp-Leu) agar plates.The transformants were then grown on yeast synthetic dropout medium with tryptophan, leucine, andhistidine dropout supplement (SD/-Trp-Leu-His) or with tryptophan, leucine, histidine, and adenine dropoutsupplement (SD/-Trp-Leu-His-Ade) agar plates for 3 days to detect the potential protein-protein interactions.

Pulldown assay. For in vitro pulldown assay, fragments of sycrp2 and sycrp1 were generated by PCRamplification, using genomic DNA as a template and using the primers shown in Table S2. The PCRproducts of sycrp2 and sycrp1 fragments were digested with NdeI and XhoI or EcoRI and XhoI, and theresulting fragments were cloned into the pET28a plasmid, to generate the plasmids pHS1538 andpHS1539, or into the pGEX-4T-1 plasmid, to generate the plasmids pHS1540 and pHS1541, respectively.Plasmids were transformed into E. coli strain BL21 to obtain four protein-overexpressing E. coli strains.The two protein-overexpressing E. coli strains containing pHS1538 and pHS1539 were grown in LBmedium and treated with 1 mmol · liter�1 isopropyl-�-D-thiogalactopyranoside (IPTG) for 4 h at 37°C. Thetwo protein-overexpressing E. coli strains containing pHS1540 and pHS1541 were grown in LB mediumand treated with 0.4 mmol · liter�1 IPTG for 4 h at 28°C. Then, cells were collected by centrifugation andresuspended with phosphate-buffered saline (PBS) buffer (10 mmol · liter�1 Na2HPO4, 1.8 mmol · liter�1

KH2PO4, 140 mmol · liter�1 NaCl, and 2.7 mmol · liter�1 KCl; pH 7.3). Subsequently, cells were disrupted5 times in a French pressure cell at 1 � 108 Pa, then centrifuged to obtain the supernatants. Thesupernatants from the E. coli strains overexpressing two proteins were mixed and incubated on ice for1 h, and then purified on a GST binding resin matrix. The coeluted proteins were confirmed by Westernblotting analyses with GST- and His-specific antibodies. Protein samples were separated by 12%SDS-PAGE, transferred to nitrocellulose filters (Millipore), and detected with anti-GST (10000-0-AP;Proteintech) and anti-His (66005-1-Ig; Proteintech). All samples were visualized with goat anti-rabbitalkaline phosphatase antibody (Invitrogen) with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Amresco, USA) as the substrates. GST protein was used as a negative control toexclude the influence of tag and nonspecific binding to resins.

Gel mobility shift assays. The procedures for purifying recombinant His-tagged Sycrp1 and Sycrp2proteins from E. coli cells were as follows. E. coli strain BL21 cultures containing pHS1538 and pHS1539were grown in LB medium and treated with 1 mmol · liter�1 IPTG for 4 h at 37°C. Then, cells werecollected by centrifugation and resuspended with binding buffer (20 mmol · liter�1 Tris-HCl, 5 mmol ·liter�1 imidazole, and 500 mmol · liter�1 NaCl as the substrate; pH 7.9). Subsequently, cells weredisrupted 5 times in a French pressure cell at 1 � 108 Pa and centrifuged to obtain the supernatant. Then,1 ml of His-Bind resin was added to a 10-ml column and washed with 3 ml deionized water, 5 ml chargingbuffer (50 mM NiSO4), and 3 ml binding buffer. The supernatant was incubated with the His-Bind resin,and this was then washed with 10 ml binding buffer, 6 ml washing buffer (20 mmol · liter�1 Tris-HCl,60 mmol · liter�1 imidazole, and 500 mmol · liter�1 NaCl; pH 7.9) and eluted by 6 ml elution buffer (20mmol · liter�1 Tris-HCl, 1 mol · liter�1 imidazole, and 500 mmol · liter�1 NaCl; pH 7.9).

A 474-bp fragment as a DNA probe was generated by PCR with the synthetic primers listed in TableS2. The 5= side of the upstream primer was labeled with biotin. This 474-bp fragment corresponds to the5= upstream region (�473 to �1) of pilA9. The biotin-labeled probe (50 ng) was incubated withSycrp1-His or Sycrp2-His in a total volume of 10 �l of the binding buffer (10 mM Tris-HCl, 50 mM KCl, 1mM dithiothreitol [DTT], 0.1% bovine serum albumin [BSA], and 5% glycerol; pH 7.5) with a finalconcentration of 20 �M cAMP for 90 min at 24°C. The poly(dI-dC) or unlabeled 474-bp DNA strands wereadded to exclude unspecific binding. Samples were loaded onto 5% polyacrylamide gels (acrylamide:N,N=-methylenebisacrylamide, 29:1). The electrophoresis buffer was 1� Tris-borate-EDTA (TBE) with 20�M cAMP. Electrophoresis was conducted at 60 V for 1 h and at 80 V for 1 h. After electrophoresis, thegels were soaked with 0.5� TBE and then transferred to nylon membranes (Millipore, USA) at 20 V for1 h using a Trans-Blot SD semidry transfer cell (Bio-Rad, USA). The nylon membrane was placed undera UV lamp for 10 min for UV cross-linking. Then, the nylon membrane was soaked with 5% dried skimmedmilk for 30 min and 1% dried skimmed milk containing horseradish peroxidase (HRP)-conjugatedstreptavidin for 30 min. After washing 3 times with Tween 20 –triethanolamine-buffered saline solution(0.05% Tween 20, 2.5 mM Tris Cl, and 0.5 M NaCl; pH 7.5), the samples were visualized with enhancedchemiluminescence (ECL) solution (Yeasen, China) using FluorChem M (ProteinSimple, USA).

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Sycrp protein structure modeling, sequence alignment and phylogenetic analysis. The entriesof GenBank accession numbers P73234 (sycrp2) and P74171 (sycrp1) were modeled by searching in a 3Dprotein structure database (UniProt; http://www.uniprot.org/uniprot/). We presented the predicted 3Dprotein structures of Sycrp2 (covering amino acids 25 to 238) and of Sycrp1 (covering amino acids 27 to231). The 3D structures were downloaded from the Protein Model Portal (http://www.proteinmodelportal.org/). The sequences of CRP-like proteins were downloaded from UniProt and aligned using Clustal X(http://www.ch.embnet.org/software/BOX_form.html).

The phylogenetic tree shows the relationship of 82 CRP-like proteins from 52 species in fiveorders of cyanobacteria. Sequences used for phylogenetic analysis were obtained using BLAST-Phttps://www.uniprot.org/blast/ with the Sycrp2 amino acid sequences as queries. Protein sequenceswere aligned in MEGA 6.06 using Clustal W, and a phylogenetic tree was constructed using theneighbor-joining method with the Jones-Taylor-Thornton (JTT) model. Bootstrap values were calculatedwith 1,000 replications.

The crystal structure of the Sycrp1/Sycrp2 heterodimer and its DNA binding element was predictedusing Swiss Model (https://www.swissmodel.expasy.org) and ClusPro (https://cluspro.bu.edu), using thecrystal structure of the cAMP receptor protein from Mycobacterium tuberculosis in complex with cAMPand its DNA binding element (3mzh) as a template. The presented images were produced using PyMolsoftware.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/JB.00436-18.

SUPPLEMENTAL FILE 1, PDF file, 0.8 MB.

ACKNOWLEDGMENTSWe thank Yi-Wen Yang and Guo-Wei Qiu for experimental assistance. We also thank

Hai-Bo Jiang for help with the experimental designs.This study was financially supported by the National Natural Science Foundation of

China (grant 31670332) and the Fundamental Research Funds for the Central Univer-sities (grant CCNU17QN0023).

B.-S.Q. and W.-Y.S. designed the study. W.-Y.S., S.-S.Z., and Z.-K.L. performed allexperiments. W.-Y.S. and K.L. did protein structure modeling, sequence alignment andphylogenetic analysis. W.-Y.S., G.-Z.D., M.C., and B.-S.Q. wrote the manuscript. Allauthors have read and approved the manuscript.

We declare that the research was conducted in the absence of any commercial orfinancial relationships that could be construed as a potential conflict of interest.

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