3274–3293 Nucleic Acids Research, 2021, Vol. 49, No. 6 Published online 8 March 2021 doi: 10.1093/nar/gkab150 Binding site profiles and N-terminal minor groove interactions of the master quorum-sensing regulator LuxR enable flexible control of gene activation and repression Jun Zhang1,† , Bing Liu5,† , Dan Gu6,† , Yuan Hao1 , Mo Chen1 , Yue Ma1 , Xiaohui Zhou7 , David Reverter5,* , Yuanxing Zhang1,3,4 and Qiyao Wang 1,2,4,* 1 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China, 2 Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China, 3 Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), 519000 Zhuhai, China, 4 Shanghai Engineering Research Center of Maricultured Animal Vaccines, Shanghai, China, 5 Institut de Biotecnologia i Biomedicina, Dept. de Bioquı́mica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain, 6 Jiangsu Key Laboratory of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China and 7 Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT 06269, USA Received October 29, 2020; Revised February 19, 2021; Editorial Decision February 22, 2021; Accepted February 23, 2021 ABSTRACT derstanding of the high flexibility of mechanisms of LuxR control of gene activation and repression in LuxR is a TetR family master quorum sensing (QS) Vibrio QS, which may facilitate to design QS inhibit- regulator activating or repressing expression of hun- ing chemicals that interfere with LuxR regulation to dreds of genes that control collective behaviors in effectively control pathogens. Vibrios with underlying mechanism unknown. To illuminate how this regulator controls expression of various target genes, we applied ChIP-seq and INTRODUCTION DNase I-seq technologies. Vibrio alginolyticus LuxR Quorum sensing (QS) is a cell-to-cell communicating sys- controls expression of ∼280 genes that contain ei- tem widely employed by bacteria to regulate numerous ther symmetric palindrome (repDNA) or asymmet- genes’ expression in response to the accumulated extracel- ric (actDNA) binding motifs with different binding lular signaling molecules called autoinducers (1). In the profiles. The median number of LuxR binding sites model QS bacterium Vibrio harveyi, a critical-threshold for activated genes are nearly double for that of re- high concentration of autoinducers could trigger sophisti- pressed genes. Crystal structures of LuxR in com- cated transmembrane signaling that culminates to the con- plex with the respective repDNA and actDNA mo- trol of LuxR, the master QS regulator (MQSR) and LuxR- tifs revealed a new mode of LuxR DNA binding that mediated expression of genes required for group behaviors involves contacts of its N-terminal extension to the such as biofilm formation, motility, bioluminescence and pathogenesis (2,3). minor groove. The N-terminal contacts mediated by Most vibrios adopt V. harveyi-like QS cascades and Arginine-9 and Arginine-11 differ when LuxR binds to depend on conserved MQSR LuxR homologs, including repDNA vs actDNA, leading to higher binding affinity LuxR (V. harveyi), HapR (V. cholerae), SmcR (V. vulnifi- at repressed targets. Moreover, modification of LuxR cus), and OpaR (V. parahaemolyticus) for a wide range of binding sites, binding profiles, and N-terminal exten- QS outputs (4–6). LuxR homologs are unique TetR family sion have important consequences on QS-regulated transcription factors with a characteristic helix-turn-helix phenotypes. These results facilitate fundamental un- (HTH) DNA binding motif in the N-terminal domain (4,7). * To whom correspondence should be addressed. Tel: +86 21 64253306; Email: oaiwqiyao@ecust.edu.cn Correspondence may also be addressed to David Reverter. Tel: +34 93 5868955; Email: David.Reverter@uab.cat † The authors wish it to be known that, in their opinion, the first three authors should be regarded as Joint First Authors. C The Author(s) 2021. 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-NonCommercial 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 journals.permissions@oup.com Nucleic Acids Research, 2021, Vol. 49, No. 6 3275 In V. harveyi, the LuxR-binding motif in repressed promot- the gene repression and activation flexibility of Vibrio QS. ers has a dyad symmetry, whereas the motif in activated Collectively, the data from this study provides fundamen- promoters is an incomplete palindrome (8). Similarly, two tal molecular insights into the QS regulation mechanisms in distinct binding motifs have also been identified for HapR- vibrios. regulated genes in V. cholerae (9). The alignment of the se- quences for SmcR binding revealed a 22-bp consensus se- MATERIALS AND METHODS quence (10). The apo protein crystals of HapR and SmcR have been resolved (5,6). Despite the knowledge of LuxR Bacterial stains and culture conditions homologs in the regulation of QS genes, the molecular basis The strains and plasmids used in this study are listed in and mechanisms of LuxR protein action in simultaneously Supplementary Table S1. All strains were constructed as controlling the expression of hundreds of genes via their di- previously described using specific primers (Supplemen- rect activation or repression on regulated promoters remain tary Table S2) and isothermal assembly (28). Vibrio algi- unknown. nolyticus strains and derivatives were typically grown at Vibrio alginolyticus is a marine-born opportunistic 30◦ C in Luria-Bertani (LB) medium supplemented with 3% pathogenic bacterium, causing severe soft tissue infections, NaCl (LBS). Escherichia coli strains were grown at 37◦ C sepsis and ear infections in human and high-mortality in LB medium. When appropriate, the medium was sup- diseases in aquatic animals in summer (11). In V. algi- plemented with carbenicillin (100 ␮g/ml), chloramphenicol nolyticus, V. harveyi-like QS systems have been identified (25 ␮g/ml), kanamycin (50 ␮g/ml), IPTG (1 mmol/L) or L- that harbor three parallel systems and transmit signals arabinose (0.04%, w/v). via two-component system-mediated phosphorelay that ul- timately converge information at the regulatory protein LuxO (12,13). The AI-1 signal is synthesized by LuxM and Plasmids, strains and mutants recognized by LuxN (14,15). The LuxS/PQ system utilizes The full-length LuxR gene was cloned from the V. algi- the signal molecule AI-2 and the precursor of AI-2 is syn- nolyticus genome. Different constructs of LuxR were ampli- thesized by LuxS (12,16). The signal molecule CAI-1 is de- fied by PCR and cloned in to the BamHI/XhoI restriction pendent on CqsA for its synthesis and detected by CqsS enzyme sites of a pET28 vector using ligation-dependent (17). At low cell density (LCD), an insufficient concentra- cloning for expression with an N-terminal hexa-histidine tion of signal molecules drives the sensors to function as ki- (His6 ) tag for nickel affinity purification. Point mutations nases, and the phosphorylated LuxO leads to the lowest ex- were generated using a QuickChange site-directed mutage- pression of LuxR. However, at high cell density (HCD), the nesis Kit (Stratagene). high concentration of signal molecules triggers the transfer of phosphate groups, and the dephosphorylated LuxO pro- tein is inactivated to reduce the level of inhibiting sRNA Protein expression and purification qrrs, and eventually LuxR production is enhanced (Figure pET28 vectors harboring different constructs of LuxR were 1A) (12,18,19). transformed into E. coli BL21(DE3) cells, and the ex- The transcription of luxR itself is controlled by the pression was induced with 0.5 mM isopropyl ␤-D-1-thio- HCD- and LCD-induced MQSRs LuxR and AphA, re- galactopyranoside and grown overnight at 20◦ C. LuxR pro- spectively (18,19). These two factors, along with other tran- teins were purified by nickel affinity chromatography us- scriptional factors, i.e. RpoE and VqsA, bind to the luxR ing a buffer containing 300 mM NaCl, 20 mM Tris–HCl, promoter region and constitute a complicated regulatory pH 8.0, and 1 mM dithiothreitol (DTT) and eluted with network that enables the integration of AI signals and a similar buffer containing 300 mM imidazole. After His6 various physiological and environmental signals to con- removal using thrombin, LuxR proteins were further pu- trol luxR transcription and QS outputs, thus coordinating rified by gel filtration (Superdex 75 column, GE Health- the expression of virulence factors, including the exotoxin care) chromatography pre-equilibrated in 300 mM NaCl, Asp, hemolysin, extracellular products, lipopolysaccha- 20 mM Tris–HCl, pH 8.0, 1 mM DTT. The selenomethio- rides, siderophore, secretion systems and biofilm (20–27), nine (SeMet)-labeled LuxR was expressed in the E. coli but the molecular mechanisms by which LuxR controls the B834(DE3) strain in M9 medium. The purification steps for large regulon are still unclear in V. alginolyticus and other the SeMet-labeled LuxR were similar to those used for the vibrios. native protein. Here, we found that LuxR protein in V. alginolyticus is the core element for the QS at high cell density. High- Crystallization, data collection and structure determination resolution DNase I footprinting and sequencing (DNase I-seq) technology was used to determine the binding mo- All complementary single-stranded DNA oligonucleotides tifs and binding profiles of LuxR in the activated and re- of actDNA (5 -ATAATGACATTACTGTATATA-3 ) and pressed promoters, respectively. Furthermore, the crystal- repDNA (5 -TTATTGATAAAATTATCAATA-3 ) were lization of LuxR in complex with the DNA motifs revealed annealed to duplex strands in buffer containing 50 mM unique binding mechanisms of the N-terminal LuxR-arm NaCl, 10 mM Tris–HCl, pH 8.0, and 1 mM EDTA. LuxR domain with minor grooves, which determines the different and SeMet-labeled LuxR were concentrated to 876 ␮M binding affinities for repression and activation motifs. We and 771 ␮M for crystallization. LuxR-DNA complexes also showed that LuxR binding site sequence, location, and were prepared by mixing LuxR with duplex DNA oligonu- N-terminal extension interactions altogether contribute to cleotides at a 1:1.2 molar ratio. Diffraction quality crystals 3276 Nucleic Acids Research, 2021, Vol. 49, No. 6 Figure 1. LuxR is involved in QS regulation in V. alginolyticus. (A) QS systems in V. alginolyticus. At HCD, QS autoinducers AI-1, AI-2 and CAI-1 produced by LuxM, LuxS and CqsA, respectively, are sensed by corresponding histidine kinases to trigger signaling cascades and control the yields of MQSR AphA and LuxR which regulates exotoxin Asp and other various QS genes. The lines with arrows indicate positive regulation and bar-ended lines for negative regulation. (B) Growth of V. alginolyticus and luxR transcriptional levels. The transcriptional activity of luxR was determined by the PluxR - luxAB reporter with the luxR promoter fused to promoterless luxAB. The relative bioluminescence value was normalized by the CFU. (C, D) qRT-PCR (C) and western blot (D) analyses of the expression level of luxR at 2 h (LCD) and 10 h (HCD). The assays were performed in triplicate. *P < 0.05 (Student’s t-test). The numbers indicate the densitometric analysis of the bands. (E, F) HPA digestion assays (E) and western blot analysis (F) were used to determine the levels of extracellular Asp and cellular LuxR in the QS mutant strains. RNAP was used as the loading control. were obtained by hanging drop with vapor diffusion after and processed with XDS (30) and scaled, reduced and fur- 2–3 days using solutions containing 50 mM sodium for- ther analyzed using CCP4 (31). The heavy atom sites and mate, 50 mM Tris–HCl, pH 7.5, 22% PEG 2000 MME for initial density map were calculated using PHENIX SAD LuxR-actDNA; 50 mM bis–tris propane, pH 7.5, 20% PEG Autosol (32). After an initial automated build, the model 2000 MME for LuxR-repDNA, 50 mM sodium formate, 50 was manually built and corrected in Coot (33). This ini- mM bis–tris propane pH 7.5; and 16% PEG 2000 MME for tial model was used as a search model for molecular re- SeMet-labeled LuxR-repDNA. A single crystal was soaked placements in the native dataset with Phaser-Phenix (34). in a well solution supplemented with 25% ethylene glycol The final model for the native dataset was manually recon- (v/v) and flash-frozen in liquid nitrogen for data collection. structed using Coot (33) and further refined with PHENIX Native and SeMet-labeled datasets were collected at ALBA (32). More details on the data collection process and refine- synchrotron in Barcelona (BL13-XALOC beamline) (29), ment statistics are shown in Table 1. Nucleic Acids Research, 2021, Vol. 49, No. 6 3277 Table 1. Data collection and structure refinement statistics Data collection LuxR-actDNA (native) LuxR-repDNA (native) LuxR-repDNA (SeMet-Peak) Beamline ALBA-XALOC ALBA-XALOC ALBA-XALOC Space group P 41 P 41 P 41 Wave length (Å) 0.91918 0.97903 0.97903 Resolution (Å) 61.966–2.543 (2.552–2.543) 61.603–2.236 (2.244–2.236) 69.579–2.551 (2.560–2.551) a, b, c (Å) 68.91, 68.91, 123.93 69.34, 69.34, 123.21 69.58, 69.58, 125.76 ␣, ␤, ␥ (◦ ) ␣ = ␤ = ␥ = 90 ␣ = ␤ = ␥ = 90 ␣ = ␤ = ␥ = 90 Unique reflections 18 962 28 010 19 443 Data redundancy 13.7 (13.9) 5.0 (5.3) 13.7 (13.4) Rmerge 0.135 (1.910) 0.055 (1.350) 0.095 (2.990) CC (1/2) 0.984 (0.581) 0.999 (0.460) 0.998 (0.613) I/␴ 11.3 (1.5) 15.3 (1.4) 16.5 (1.0) Completeness (%) 99.7 (100.0) 96.4 (98.2) 99.8 (100.0) Refinement Resolution (Å) 60.223–2.600 49.030–2.300 Non-anomalous reflections 17 742 25 723 Rwork /Rfree 0.2216/0.2492 0.2115/0.2424 Number of all atoms 3850 3928 RMSD bond (Å)/angle (◦ ) 0.004/0.792 0.005/0.005 Ramachandran plot Favored (%) 94.99 96.71 Allowed (%) 5.01 3.29 Disallowed (%) 0.00 0.00 Total RNA extraction to each gene was determined using Picard tools (http:// ◦ broadinstitute.github.io/picard/faq.html) and then normal- Bacteria were incubated at 30 C and harvested after 2 h ized to the reads per kilobase of genic region per million (LCD) and 10 h (HCD). Total RNA was isolated using an mapped reads (RPKM) to obtain the relative level of ex- RNA extraction kit (Tiangen, Beijing, China). The RNA pression. ANOVA was performed to analyze the average samples were digested with DNase I (Promega, Madison, expression levels of the three biological replicates to deter- WI, USA) to eliminate genomic DNA contamination. mine the one which showed differential expression between any two conditions that were tested (FDR-value < 0.25 and Quantitative real-time reverse transcription PCR (qRT- 2-fold change). PCR) Equal amounts of RNA (1 ␮g) were used to generate com- Electrophoretic mobility shift assays (EMSAs) plementary DNA (Toyobo, Tsuruga, Japan) using random primers. Three independent qRT-PCR experiments were For the electrophoretic mobility shift assays (EMSAs), pu- performed, and each was run in triplicate on an Applied rified His6 -tagged LuxR was incubated with different Cy5- Biosystems 7500 Real Time System (Applied Biosystems). labeled DNA probes (Supplementary Table S2) in 20 ␮l of The specific primer pairs used are shown in Supplementary loading buffer (300 mM NaCl, 20 mM Tris–HCl (pH 8.0), Table S2. Transcript levels were normalized to 16 sRNA in 1 mM DTT, and 5%(V/V) glycerol). After the mixture was each sample using the CT method. incubated at 25◦ C for 30 min, the samples were resolved using 6% polyacrylamide gel electrophoresis in 0.5× TBE (Tris/boric acid/EDTA) buffer on ice at 100 V for 90 min. RNA-seq transcriptome generation and data analysis Then, the gels were scanned using a Typhoon FLA 9500 rRNA was removed from 1–5 ␮g of total RNA using a Ri- (GE healthcare, Uppsala, Sweden). boZero kit for Gram-negative bacteria (Epicentre, Chicago, IL, USA). First-strand cDNA synthesis from rRNA- DNase I footprinting assays depleted samples was performed using a TruSeq RNA sam- ple Prep kit (Illumina, San Diego, CA, USA). cDNA was Dye primer-based DNase I footprinting assays were per- purified using an RNA Clean and Concentrator-25 kit formed as previously described (36). Briefly, the promoter (Zymo Research, Irvine, CA, USA). After second-strand regions of RS20495, RS11155, RS21790 (ppkA2) and synthesis, the reactions products were cleaned up using RS21705 (hcp2) were amplified by PCR using pfu DNA AMPure XP beads according to the manufacturer’s instruc- polymerase (Supplementary Table S2) and 6-FAM labeled tions. The VAHTS Stranded mRNA-seq Library Prep Kit at the 5 end. For each assay, 400 ng of the probes were for Illumina (Vazyme, Nanjing, China) was used in the incubated with different amounts of LuxR in a total vol- construction of strand-specific RNA-seq libraries. The li- ume of 40 ␮l. After the mixture was incubated for 30 min braries were sequenced on a HiSeq 2000 platform (Illu- at 25◦ C, a 10-␮l aliquot of a solution containing approxi- mina, San Diego, CA, USA), yielding 100-base paired- mately 0.015 U DNase I (Promega) and 100 nmol of freshly end reads. Then, the BWA (35) program was used to align prepared CaCl2 was added. The mixture was then incubated the remaining reads to the reference sequences of V. algi- for 1 min at 25◦ C. The reaction was stopped by adding 140 nolyticus 12G01. The number of reads that was mapped ␮l of DNase I stop solution (36). The samples were first ex- 3278 Nucleic Acids Research, 2021, Vol. 49, No. 6 tracted using phenol/chloroform and then precipitated us- Briefly, after the DNA was fragmented to 100–500 bp, 12 ing ethanol, and the pellets were dissolved in 10 ␮l of MiniQ ␮l of DNase I (0.3 U/␮l) was added to 120 ␮l of frag- water. Approximately 2 ␮l of digested DNA was added to mented DNA solution and incubated at 25◦ C for 10 min, 7.9 ␮l of HiDi formamide (Applied Biosystems) and 0.1 ␮l then 330 ␮l of EDTA (50 mM) was added to the mix and of GeneScan-500 LIZ size standards (Applied Biosystems). incubated at 55◦ C for 1 min. The subsequent steps and anal- The samples were analyzed using a 3730 DNA Analyzer ysis were the same to those used for ChIP-seq. We iden- with a G5 dye set that was run with an altered default geno- tified the genes overlapped in the DNase I-seq and RNA- typing module that increased the injection time to 30 s and seq analyses, and divided them into activated genes and re- the injection voltage to 3 kV. The results were analyzed us- pressed genes. Then we used each promoter sequence of ing GeneMapper 4.0 (Applied Biosystems). the activated and repressed genes Blast against the trimmed reads of the DNase I-seq to identify the number of bind- ing sites of each promoter. The transcriptional start sites Chromatin immunoprecipitation (ChIP), quantitative PCR (TSS) was predicted by the Softberry-BPROM algorithm (ChIP-qPCR), and ChIP-seq (http://www.softberry.com) and manually checked with the ChIP was performed as previously described (27). luxR strand-specific RNA-seq determined TSS. cells harboring pBAD33::LuxR-flag and pBAD33::flag were harvested and treated in 1% formaldehyde at room Bioluminescence and fluorescence assays temperature for 10 min incubation. The bacteria were then washed twice with cold PBS and resuspended in 5 ml of SDS The bioluminescence assay was performed as previously de- lysis buffer (27). Then, the bacteria were sonicated for DNA scribed (26). In brief, the overnight culture of reporter strain fragmentation to 100–500 bp at 200 W. The supernatant was PluxR -luxAB/WT was diluted to 5×106 CFU/ml in fresh used as the input sample. Both the input and the IP samples LBS medium and then incubated at 30◦ C with shaking at were washed with 50 ␮l of protein G beads. The IP sam- 200 rpm. A 200-␮l aliquot was sampled every 2 h for 12 ples were then incubated overnight with 50 ␮l Flag-beads h, and after the addition of 1% decanal, the biolumines- (Sigma-Aldrich, St. Louis, MO, USA). The beads were se- cence was measured in an Orion II bioluminescence reader quentially washed twice using low-salt wash buffer, high- (Berthold Detection Systems, Pforzheim, Germany). In ad- salt wash buffer, LiCl wash buffer, and TE buffer (27). The dition, the cultures were sampled, and the cells on the plates beads were then resuspended in 200 ␮l of elution buffer (27), were counted to normalize the bioluminescence values of incubated at 65◦ C for 2 h. The supernatants containing the each time point sample. immunoprecipitated DNA were collected, and 8 ␮l of 5 M For the fluorescence assays, cultures were incubated NaCl was added to all of the IP and input tubes followed by overnight and then diluted 1:100 in fresh LBS medium. Af- incubation at 65◦ C overnight to reverse the DNA-protein ter 9 h of growth with shaking at 30◦ C, the bacterial cells crosslinking. After treatment with RNase A and proteinase were washed twice with PBS. Then, EGFP fluorescence K, ChIP DNA was purified using phenol-chloroform. was measured using a florescence plate reader (BioTek, ChIP-qPCR was performed with the purified DNA and Winooski, VT, USA). specific primers (Supplementary Table S2). The enrich- ment of DNA targets was calculated using the following Extracellular protease activity assay (HPA) CT method. For each DNA target, CT of the in- put fraction and IP fraction was calculated for both the ECP activity was determined using hide powder azure pBAD33::flag/wt and pBAD33::LuxR-flag/luxR sam- (HPA) (Sigma-Aldrich) digestion as previously described ples. Each value was then divided by the corresponding (12). Briefly, the strains were grown in LBS medium at 30◦ C CT that was obtained for the nonspecific gyrB intragenic for 9 h. The cell density was measured at 600 nm (OD600 ). region in the strains. Then, the enrichment ratio was calcu- The bacterial cultures were centrifuged, and the super- lated from the CT value of the wt strain divided by that natants were then filtered through 0.22-␮m filters (Milli- of the luxR strain. pore, Bedford, MA, USA), and 1 ml of the filtered super- ChIP-seq was performed with the purified ChIP DNA natant was mixed with 1 ml of phosphate-buffered saline fragment. Sequencing libraries were constructed with the (PBS, pH 7.2) and 10 mg of HPA. This mixture was incu- VAHTATM Turbo DNA library Prep Kit (Vazyme) and bated with shaking at 37◦ C for 2 h. After the reaction was subjected to sequencing with a MiSeq sequencer (Illu- terminated by adding 10% trichloroacetic acid, total pro- mina). The following analysis was performed as previously tease activity was measured at 600 nm. ECP activity was described with default parameters (18). Briefly, ChIP-seq normalized for each strain by dividing the total activity by reads were mapped to the V. alginolyticus 12G01 genome OD600 value. (18). Binding peaks were called using MACS software (37), followed by MEME analysis (http://meme-suite.org) to gen- Western blot assay erate a representative LuxR-binding logo with ‘strand only’, ‘palindrome only’ search mode or default parameters. For the immunoblotting assay, supernatants and bacterial cell pellets were harvested to the same OD600 . Then, 15 ␮l of each sample was loaded onto a 12% denaturing poly- DNase I-seq acrylamide gel; the proteins were resolved by electrophore- DNase I-seq was performed in a manner similar to ChIP- sis and finally transferred to a PVDF membrane (Milli- seq with DNase I added to remove the unprotected DNA. pore, Bedford, MA, USA). The membranes were blocked Nucleic Acids Research, 2021, Vol. 49, No. 6 3279 with a 10% skim milk powder solution and incubated with assays with LuxR-specific antibody also showed low luxR a Asp-, LuxR- (GL Peptide Ltd., Shanghai, China), Flag- transcription and expression levels at LCD and high levels or Myc-specific antiserum at a 1:2000 dilution (Sigma- at HCD (Figure 1C-D, and Supplementary Figure S1), in- Aldrich, St. Louis, MO, USA) and then incubated with dicating that LuxR expression is cell density-dependent. horseradish peroxidase-conjugated goat anti-rabbit or anti- We were intrigued how three parallel QS systems con- mouse IgG at a 1:2000 dilution (Santa Cruz Biotechnol- tribute to LuxR production and its downstream gene ex- ogy, Santa Cruz, CA, USA). Finally, the blots were visu- pression in V. alginolyticus. Single or combined deletion alized with an ECL reagent (Thermo Fisher Scientific Inc., mutants for each of the autoinducer synthetases, i.e. luxM Waltham, MA USA). for AI-1, luxS for AI-2, and cqsA for CAI-1, were con- structed (Supplementary Tables S1–2) and the expression Bacterial killing analysis of LuxR and Asp, a LuxR-controlled exotoxin (Supplemen- tary Figure S1) (20), was determined. As previously estab- The bacterial killing analysis was performed as previously lished (24), the luxM strains and cqsA showed minimal described (38). In brief, after the bacteria were cultured Asp production while luxS displayed slightly decreased overnight, the density was adjusted to OD600 = 0.5, and Asp activity compared to the wt strain (Figure 1E-F). In then, the predator (V. alginolyticus strains) and prey cells line with the Asp production, LuxR production was unde- (E. coli) were mixed at a ratio of 4:1. Then, 25 ␮l of this tectable in cqsA and the luxM strains and was slightly mixture were spotted on LBS agar plates and cocultured at decreased in luxS. The luxM, cqsA and luxS double and 30◦ C for 4 h. CFU of the strains in the mixtures spotted triple deletion mutants were abolished in LuxR production on LBS agar plates at t = 0 were determined by plating 10- at all (Figure 1F). Taken together, these results indicated fold serial dilutions on appropriate plates. After 4 h, bacte- that the LuxM/N and CqsA/S systems are essential for rial spots were collected from the LBS agar plates, and the LuxR and QS gene expression, while the LuxS/LuxPQ sys- CFUs of the surviving predator and prey were determined. tem may be partially involved in the expression of LuxR and An empty pBAD33 or pRK415 plasmid was used to render genes downstream from LuxR. E. coli and V. alginolyticus resistant to chloramphenicol and tetracycline, respectively. Genome-wide identification of the LuxR regulon and binding sites Infection of fish Transcriptomics analysis of the wt and luxR strains indi- All of the overnight cultured strains were harvested and cated that a total of 168 genes were upregulated and 112 then serially diluted with PBS. Zebrafish weighing approx- genes were downregulated in luxR compared to the wt imately 0.25 g were infected with the strains at a dose of strain (|log2 FC| ≥ 1-fold, Padj < 0.05) (Figure 2A, and Sup- 105 CFU/fish via intramuscular (i.m.) injection according plementary Figure S2, Supplementary Tables S3–S4), in- to a previous description (27). Zebrafish were anesthetized dicating that LuxR serves as both a repressor and activa- with tricaine methanesulfonate (MS-222) (Sigma-Aldrich) tor in V. alginolyticus. We performed chromatin immuno- at a concentration of 80 mg/l. Thirty fish were infected precipitation and nucleotide sequencing (ChIP-seq) to de- with each dilution, and three parallel experiments were per- termine the LuxR regulon (Supplementary Table S5). Be- formed. Then, the fish death that was caused by vibriosis cause the sizes of the DNA fragments analyzed by ChIP- was recorded. seq assays (∼200 bp) are much larger than the LuxR ho- molog binding consensus sequences (16–28 bp) (8–10), the RESULTS respective ChIP samples were subjected to DNase I-seq, i.e. DNA fragmentation, DNase I digestion, and nucleotide se- LuxR is a master QS regulator in V. alginolyticus quencing of the LuxR protected fragments (∼50 bp), thus Three parallel autoinducer receptor systems are deployed allowing mapping with higher resolution of LuxR bind- in a V. harveyi-like QS system and culminate in the con- ing sites (Supplementary Table S6). We used the luxR trol of the production of LuxR, the TetR family transcrip- strain harboring vector expressing Flag-tagged LuxR in tion factor known as a MQSR (Figure 1A). In HCD, QS ChIP assays with a strain expressing Flag alone as a nega- signal transduction induces LuxR production and thereby tive control. Flag-LuxR behaved similarly to the wt LuxR, regulates the expression of virulence-associated genes. To as Asp activity in luxR was fully restored by introducing characterize the cell density-dependent LuxR yield in V. al- the vector expressing Flag-LuxR (Supplementary Figure ginolyticus, we introduced a PluxR -luxAB reporter with the S3A and B), thus validating the following ChIP- associated luxR promoter fused to luxAB gene into the wild-type (wt) analysis. strain. The luxR promoter activity was low at low cell den- The independent ChIP-seq assays and subsequent DNase sity stage (∼6E+8 CFU/ml, 2 h or LCD) as a result of I-seq analysis enabled mapping of the LuxR binding se- AphA repression (18,27). Although a slight decrease in bi- quences to the V. alginolyticus genome. We identified 297 oluminescence between 4 h to 8 h with an unknown mech- enriched loci harboring LuxR-binding peaks (enriched > 2- anism, high luxR promoter activity was observed at high fold compared with the control sample) by ChIP-seq (Sup- cell density (∼6E+10 CFU/ml, 10 h or HCD) (Figure 1B). plementary Table S5) and 101 enriched loci by DNase I-seq Then the luxR promoter activity was decreased after 10 h, (enriched > 2-fold) (Supplementary Table S6), respectively. probably due to the inhibition of LuxR to its own transcrip- Moreover, 76 enriched loci revealed by DNase I-seq were tion (27). Similarly, analysis via qRT-PCR and Western blot overlapped with those identified by ChIP-seq (Figure 2A). 3280 Nucleic Acids Research, 2021, Vol. 49, No. 6 Figure 2. Genome-wide analysis of LuxR-binding sites in genes activated or repressed by LuxR. (A) Venn diagrams indicate the number of genes regulated by LuxR through RNA-seq, ChIP-seq, and DNase I-seq. (B, C) Pie chart of LuxR binding peak locations in various genes as revealed by ChIP-seq (B) and DNase I-seq (C). (D–G) LuxR binding motifs derived from MEME analysis of the ChIP-seq and DNase I-seq peaks under ‘strand only’ search mode. The binding motifs of all peaks from the ChIP-seq (D) and DNase I-seq (E) analyses, enriched peaks in the repressed promoters (F) and the activated promoters (G) are shown. Arrowed lines denote the complete (solid) or incomplete (dashed) inverted repeat relative to the left half part. (H) The number of binding sites in the LuxR repressed and activated genes, respectively. ***P < 0.001 (Student’s t-test). (I, J) The normalized DNase I-seq reads of LuxR-binding sites in the subregions in activated (I) or repressed (J) promoters controlled by LuxR. The dashed boxes indicate the subregions containing a high density of LuxR binding sites. These loci were located across the genome, including in in- Analysis with the MEME (multiple EM for motif elic- tergenic regions and coding regions (Figure 2B and C, Sup- itation) algorithm under the ‘strand only’ search mode re- plementary Figure S4A–C). Based on the DNase I-seq data, vealed a 22-bp binding motif (5 -TTATTGATAAATTTAT peaks covering ∼30–50 bp sequences were identified in the CAATAA-3 ) showing sequence palindrome in the LuxR- strain expressing LuxR-Flag but not in the strain producing binding sequences investigated by ChIP-seq (Figure 2D) Flag alone. Validating the DNase I-seq analysis, the bind- and a 22-bp highly consistent sequence (5 -TTATTGATAN ing sites of LuxR protein at luxR promoter were consistent TTTTATCAATAA-3 ) detected by DNase I-seq (Figure with those identified with the previous DNase I footprinting 2E). These analyses showed an overall 9–4-9 LuxR bind- analysis (27). ing motif with 9-mer consensus palindrome sequence and Nucleic Acids Research, 2021, Vol. 49, No. 6 3281 4-mer spacer sequence of low conservativeness. Highly sim- purified LuxR protein. As expected, the results showed ilar logos were also generated with the same datasets under that LuxR protein protected 5 -TACGGTAATGACAAA default or ‘palindrome only’ search mode (Supplementary TCGATCAGTAA-3 (the underlined nucleotides indicate Figure S4D–G). highly conserved sequences in the repDNA logo identified Further analysis by RNA-seq and DNase I-seq showed with DNase I-seq) in the promoter region of RS20535, and that 37 enriched peaks were associated with LuxR- 5 -CCATAAATATGTGATTTATCAGTAA-3 in the pro- regulated genes (Figure 2A, Supplementary Table S6). We moter region of RS11155 (Figure 3D, H). Similarly, LuxR then separately analyzed the LuxR-enriched peaks in acti- fully or partially protected two distinct AT-rich sequences vated promoters (n = 9) and repressed promoters (n = 22). resembling the repDNA and actDNA binding motifs in MEME analysis with various parameters, i.e. ‘strand only’, the promoter of ppkA2 and hcp2 that were identified with ‘palindrome only’ or default search mode, showed that the DNase I footprinting analysis (Figure 3L, P), further un- 20- or 21-bp LuxR-binding motifs in the repressed promot- derpinning lower DNA-binding affinity at these regions in ers had nearly palindromic sequence (Figure 2F and Sup- the LuxR-activated genes as compared to those repressed plementary Figure S4H and I), whereas the 18-bp LuxR- genes revealed by DNase I-seq (Figure 2I and J) and EM- enriched motifs in the activated promoters had an incom- SAs (Figure 3C, G, K, O). Taken together, these results con- plete inverted 9-mer repeat ‘TTATTGATA’ (Figure 2G and firmed the DNase I-seq and ChIP-seq data and the derived Supplementary Figure S4J). Notably, MEME analysis with binding logos. ‘palindrome only’ parameter could not yield a significant We further illuminated the genome-wide regulon of (E-value < 0.05) logo from the enriched promoters of acti- LuxR in V. alginolyticus (Supplementary Figure S5, Supple- vated genes. The logos from these repressed and activated mentary Table S3). Several functional categories of genes, promoters were highly consistent with the motifs found in including type II secretion system (T2SS), type VI se- the luxR promoter region as revealed by the DNase I foot- cretion system 2 (T6SS2), cell motility and chemotaxis, printing assay (27). two-component systems, pathogenesis-associated genes, Moreover, the median number of LuxR binding sites for metabolism pathways related to phosphotransferase sys- activated genes was 3 while that for repressed genes was 1.6 tems, and the TCA cycle, were regulated by LuxR. In partic- (Figure 2H), respectively, indicating that the binding site ular, previous investigations indicated that AphA, the LCD distribution patterns associated with the LuxR-activated MQSR, and LuxR were essential for exotoxin Asp produc- and LuxR-repressed genes were significantly different (P tion and the bacterial virulence towards hosts (18,27). The < 0.001). The normalized reads for the LuxR-bound re- promoter region of asp and aphA also carries a putative pressed promoters were higher than those for the activated LuxR-binding motif. EMSA verified that LuxR binds the promoter sequences (Figure 2I and J), suggesting a LuxR promoter region of asp and aphA (Supplementary Figure binding model with stronger binding affinity for repressed S6A and B). The increased level of LuxR led to a signifi- promoter sequence than for activated sequence. In addition, cant increase in Asp and a decrease in AphA (Supplemen- the peaks for activated genes were located at approximately tary Figure S6C and D), thus confirming that asp and aphA –35 to –100 bp and 0 to +25 (Figure 2I), while those for are included in the LuxR regulon. RNA-seq showed that all the inhibited genes were located at 0 to –50 bp (Figure 2J). genes in the T6SS2 cluster were all downregulated in luxR Taken together, more precise LuxR-binding sites were un- compared to their expression in the wt strain (Supplemen- raveled by ChIP-seq and DNase I-seq assays, which demon- tary Figure S7A, Supplementary Table S3), and the pro- strated totally distinct LuxR binding patterns for activated moters of the T6SS2 genes were enriched by LuxR in ChIP- and repressed genes. seq and DNase I-seq assays (Supplementary Table S4). In addition, Western blot assays demonstrated that there was a strong reduction in hcp2 expression in luxR compared Validation of LuxR-mediated gene regulation to the wt or luxO strains (Supplementary Figure S7B). To validate the ChIP-seq and DNase I-seq data, four pro- Moreover, interbacterial competition assays showed that moter regions of LuxR-targeted genes (RS20535, RS11155, luxR lost the ability to kill E. coli (Supplementary Fig- ppkA2 and hcp2) (Figure 3A, E, I, M) were selected for ure S7C), together with the finding that LuxR directly binds qRT-PCR analysis and electrophoretic mobility shift assays to the promoter regions of T6SS2 (Figure 3I-P, and Sup- (EMSA). In a strain lacking luxR (luxR), the transcripts plementary Figure S7A), further demonstrating that LuxR of RS20535 and RS11155 were significantly upregulated, positively regulates the T6SS2 mediated bacterial killing while those of ppkA2 and hcp2 were significantly down- function in V. alginolyticus. Taken together, the data show regulated, and the reintroduction of luxR into the strain that LuxR is globally involved in the control of genes re- restored the respective gene transcriptional levels to those lated to metabolism and virulence by binding to repDNA of the wt strain, confirming the roles of LuxR in control and actDNA to exert transcriptional regulation. of these genes (Figure 3B, F, J, N). In addition, retarda- tion was also observed in the EMSAs with LuxR and the Overall structure of the LuxR-DNA complex DNA probes harboring the respective promoter sequences in a concentration-dependent manner, indicating that LuxR To understand the structural basis for DNA recognition by protein can bind to all four promoters (Figure 3C, G, K, LuxR, we crystallized recombinant full-length LuxR in the O). To further confirm the binding motif in the promoter presence of activation or repression double-stranded DNA. of LuxR-repressed or LuxR-activated genes, we performed According to our in vivo DNase I-seq results, LuxR rec- DNase I footprinting for each of the four promoters with ognizes 22 DNA bases, but crystallization with a 22-mer 3282 Nucleic Acids Research, 2021, Vol. 49, No. 6 Figure 3. Verification of the LuxR-activated and LuxR-repressed genes by EMSA, qRT-PCR and DNase I footprinting analyses. (A–P) Verification of the ChIP-seq identification of LuxR-binding target genes RS20535 (A), RS11155 (E), ppkA2 (I) and hcp2 (M) by qRT-PCR (B, F, J, N), EMSA (C, G, K, O) and DNase I footprinting (D, H, L, P) analyses, respectively. The transcript levels of target genes in the WT, luxR and luxR+ strains were determined by qRT-PCR. *P < 0.05, ***P < 0.001 (Student’s t-test). An increasing amount of purified LuxR protein with the promoter regions was used for the EMSA and DNase I footprinting assay. The poly(dI:dC) was used as a non-specific competitor DNA, and the unlabeled DNA was used to compete the Cy5-labeled DNA. B, bound DNA; U, unbound DNA. failed due to low diffraction quality. We resort to use an tal lattice of the LuxR-repDNA structure, Arg11 of subunit activation 21-mer DNA containing the LuxR-binding site A binds the T-A base pair of the adjacent DNA duplex in I (LBSI or RBS3) from the luxR promoter (actDNA: 5 -AT the pseudofilament (Supplementary Figure S9). AATGACATTACTGTATATA-3 ), established by DNase Similar to the TetR-DNA complex, which was the first I footprinting analysis (27) and DNase I-seq (Supplemen- well-characterized member of the TetR-family transcrip- tary Figure S8C), and a 21-mer repression DNA contain- tional regulators (Figure 4A), both crystal structures of ing the whole logo sequence (repDNA: 5 -TTATTGATAA LuxR in complex with actDNA or repDNA, display one AATTATCAATA-3 ) (note that A4 and C8 were modified homodimer in the asymmetric unit, arranged as subunits A to T4 and T8, respectively, as they show the same frequency and B, which interacts with a double-stranded DNA via two bits) (Supplementary Figure S4H) for the LuxR-DNA crys- conserved N-terminal DNA-binding domain (DBD). Each tallization and structural analysis. We resolved the crys- subunit of the LuxR homodimer displays two alpha heli- tal structures of LuxR in complex with the actDNA and cal domains with a similar fold to that of SmcR and HapR repDNA at 2.6 and 2.3 Å respectively (Table 1). In the crys- structures, which are the LuxR orthologs in V. vulnificus and Nucleic Acids Research, 2021, Vol. 49, No. 6 3283 Figure 4. Structure of the LuxR-DNA complex. (A) Sequence alignment of TetR with LuxR family members in V. alginolyticus (LuxR Va), V. harveyi (LuxR Vh), V. vulnificus (SmcR), V. cholerae (HapR), V. fischeri (LitR), V. parahaemolyticus (OpaR) and V. anguillarum (VanT), labeling with the N- terminal extension (LuxR-arm), HTH motif and core DNA-binding domain (DBD). Residues involved in the interactions with DNA bases are marked. (B, C) Structures of the LuxR homodimer bound to an actDNA 21-mer (B) and repDNA 21-mer (C) are displayed. (D) Close-up view of the LuxR- arm interaction with the DNA minor groove in the LuxR-repDNA structure. Arg9 and Arg11 are shown with a 2Fo – Fc electron density map. (E, F) Structural superposition between LuxR-repDNA (magenta) with LuxR-actDNA (blue), and LuxR-repDNA (red) with SmcR (green). (G) Mixed surface and ribbon representation of LuxR-repDNA shows the highly positively charged DNA-binding surface. (H) Schematic representations of LuxR-actDNA and LuxR-repDNA contacts. Only interactions with <4 Å separating the amino acid residues from the bases of the cognate DNA are indicated by arrows for simplicity. The 22th T-A base pair from the adjacent DNA duplex in the pseudo-filament is marked. (I) Ribbon display of the superpositions of repDNA (magenta) and actDNA (blue) and of repDNA (magenta) and B-DNA (orange). The distances of the major grooves are indicated with dashed lines. 3284 Nucleic Acids Research, 2021, Vol. 49, No. 6 V. cholerae (5,6), containing the canonical helix-turn-helix important loop rearrangements and the creation of novel motif (HTH), followed by a large ␣-helix C-terminal do- interface contacts (Supplementary Figure S10B–D). It is main responsible for dimerization (Figure 4B and C). The particularly interesting to observe residue rearrangements overall structure of each subunit in the LuxR homodimer around the hinge region between the two DBDs, where the is very similar, with root mean square deviation (rmsd) val- Arg32, Arg36 (from the ␣1–␣2 loop) and Arg60 interaction ues of 0.23 and 0.38 Å for the A and B subunits of LuxR- with the phosphate backbone in the central minor groove repDNA and LuxR-actDNA, respectively. Additionally, induces important changes in the adjacent residues at the the structural comparison of the actDNA and repDNA dimer interface (Supplementary Figure S10A-B). For ex- LuxR homodimers with a rmsd value of 0.256Å (Supple- ample, DNA binding triggers the formation of an electro- mentary Figure S10A), revealed that the bound DNA frag- static interaction among Arg60–Glu124–Arg122–Glu116, ments consist of a distorted B-DNA double helix (rmsd of which is not present in the apo form, with Arg122 playing 0.744 Å), indicating a similar mode of interaction for both the central role in this novel interface contact. Addition- LuxR complexes (Figure 4E, I). Alignment with an ideal ally, it is quite remarkable that, upon displacement by ∼5 Å, B-DNA revealed notable alterations in the regular double Glu124 interacts with Arg60 and that the novel hydropho- helix geometry, specifically in the two major grooves that bic pocket around Trp128 (Supplementary Figure S10C-D) interact with LuxR. Notably, the size differed from 17.9 Å is completely different from that of the apo LuxR structure. for a standard B-DNA to 21.8 Å for the B-DNA in actDNA and repDNA bound to LuxR (Figure 4I). Overall DNA recognition by LuxR LuxR contains a 12-residue N-terminal extension that precedes the core DBD, herein termed LuxR-arm, which is In both LuxR-actDNA and LuxR-repDNA structures, the quite conserved in all LuxR homologs but is absent in TetR subunit A is bound to the left half-site of the palindrome, and many of other TetR family proteins (Figure 4A). In the whereas the subunit B binds to the right half-site of the crystal structures of apo SmcR and HapR, the conserved palindrome (Figure 5A and B). The DBD of LuxR binds LuxR-arm is partially displayed and disordered. Although the major and minor DNA grooves through the HTH mo- LuxR and TetR DBDs display a similar mode of binding tif and LuxR-arm, respectively, forming extensive interac- to the DNA major groove through the HTH motif (rmsd tions with phosphates and bases in actDNA and repDNA of 0.440 Å), the LuxR-arm extension establishes an un- (Figure 4G and H). The DNA-binding surface of LuxR is expected interaction with the DNA minor groove through highly positively charged, thus displaying extensive contacts Arg9 (R9) and Arg11 (R11) (Figure 4B-D). Arg9 and Arg11 with the negative backbone phosphates, particularly in the are fully conserved in LuxR-Vh, SmcR, HapR and LitR, three minor grooves formed by the DNA structure (Fig- and probably play a similar role in these LuxR-type tran- ure 4G). The most important difference between the LuxR- scription factors (Figure 4A). This N-terminal extension actDNA and LuxR-repDNA structures corresponds to the is not found in other TetR transcription factor members, unique interaction of LuxR-arm with the two external mi- namely, DesT, TetR, CgmR, RcsB and QacR, for which nor grooves of the repDNA structure (Figure 4B and C). protein-DNA structures are available (39–43). LuxR shares low sequence identify with TetR, but ap- LuxR-DNA backbone interactions proximately 73% and 92% identity with HapR and SmcR, respectively (44). Since the apo structure of LuxR is not In both structures, the DNA phosphate backbones of the available, this high sequence similarity allows us to com- actDNA and repDNA sequences establish extensive po- pare the structures of apo SmcR with that of the LuxR- lar interactions (including hydrogen bonds) in their ma- DNA complex to analyze the structural changes upon jor and minor grooves with Arg17, Arg32, Arg36, Ser49, binding to DNA. Superimposition of one subunit of the Thr52, Tyr56, Pro58, Thr59 and Arg60 (Figure 5A and B). apo-SmcR to holo-LuxR displayed rmsd values of 1.759, Among these interactions, the interactions between the 1.402 and 0.643 Å for the whole molecule, the C-terminal guanidinium-positive groups of Arg32 and Arg36 with the dimerization/regulatory domain (residues 60–199), and the phosphate DNA groups of the nucleotides at positions 13 N-terminal DBD domains (residues 9–59), respectively, in- and 14 (and the equivalent positions, 9 and 10 , in the com- dicating a similar fold (Figure 4F and Supplementary Fig- plementary DNA strand) are particularly relevant. These ure S10B). contacts are produced after a notable rearrangement of the ␣1–␣2 loop that allows the interaction with the minor cen- tral groove and corresponds to a spacer in the middle of LuxR dimer interface rearrangement the palindromic DNA sequence, as observed by compari- Alignment of one subunit in the homodimer of LuxR-DNA son with the apo LuxR (SmcR structure) (Supplementary and SmcR (apo LuxR) causes a notable rigid-body dis- Figure S10B). placement in other subunits, including important confor- Other backbone contacts include the interaction of the mational changes in the homodimer interface (Figure 4E side chains of Arg17 and Tyr56 with the backbone phos- and F). DNA binding triggers a shift of ∼1.5 Å and a tor- phates of DNA nucleotides at positions 4 and 19 in each sion of 10.2◦ between the two HTH motifs of the DBD do- complex, respectively. The main and side chains of Pro58, mains (using the C␣ of Ala51 as a reference point) to enable Thr59 and Arg60, located in the loop between helices ␣3 the protein to fit in the DNA duplex (Figure 4F). This fitting and ␣4, interact with the backbone phosphates at positions of the two N-terminal DBD domains into the DNA grooves 14 and 15 (8 and 9 in the complementary DNA strand). produces a displacement at the dimer interface, resulting in The side chains of Ser49 and Thr55, located in the ␣3 of Nucleic Acids Research, 2021, Vol. 49, No. 6 3285 Figure 5. Interactions between the HTH motif and the DNA major groove. (A–D) Interactions between amino acid residues with phosphate backbone or DNA bases in actDNA (A and C) and repDNA (B and D) are represented by short lines and ribbon display. The colors of the interaction lines indicate the corresponding subunits. The detailed interactions between major groove bases and the HTH motif are shown in C-i and C-ii for actDNA, and in D-i and D-ii for repDNA. (E) Structural superposition shows that the repDNA (red) has the overall closer contact distances with LuxR than the actDNA (blue) has with LuxR. 3286 Nucleic Acids Research, 2021, Vol. 49, No. 6 the HTH motif of the major groove, are in close proxim- nucleobases and sugar phosphate backbones of T2, A3 and ity to the phosphate backbone at position 5 (or 18 in the A1 (or T1 for actDNA) (Figure 6B, E). Since the repDNA other strand). Although LuxR establishes basically simi- sequence is palindromic, the LuxR-arm extension of the lar contacts through the backbone phosphates with both subunit B establishes equivalent contacts with nucleobases the repression and activation DNA sequences, the LuxR– and sugar phosphate backbones, specifically with T20, A21, repDNA complex displays globally closer contact distances T18 and T19 for Arg9 and with T22, A20 and T21 for than the LuxR-actDNA complex, probably reflecting a Arg11 (Figure 6G and H). Notably, position 22 is occupied strong binding affinity (Figure 5A and B). by a thymine, not an adenine, as would be the case for a palindrome, because it is from the symmetric crystal neigh- LuxR-DNA nucleobase interactions at the major groove bor; however, it displays a similar H-bond pattern as ade- nine. Finally, in contrast to the repression complex, in the Normally, an analysis of specific contacts between the LuxR-actDNA structure only the side chain of Arg11 in the DNA nucleobases and LuxR would be essential to establish subunit A forms H-bond interactions with the O2 atoms in the structural basis for the different transcription activities the nucleobases of T2 and T1 , as well as H-bond interac- between the actDNA and repDNA sequences. Nucleobase tions with the O4 atoms in the sugar phosphate backbone contacts in the major groove are realized by side chains of of T2 and A3 (Figure 6B and C). the residues emanating from ␣3 of the HTH motif in the The major difference between the activation and repres- DBD domain, namely Ser49, Ala51, Phe54 and Asn55 (Fig- sion DNA sequences in the LuxR-arm interaction is re- ure 5C and D). Ser49 only contacts T17 in the B subunit vealed at positions 1 and 4, which may be either a thymine of the activation complex, due to the unique presence of a or adenine. Whereas both nucleobases in position 1 can thymine. Ala51 establishes close contacts with T5, G6 and establish equivalent H-bond interactions with Arg11, as C6 (G17 , T18 and C17 in the complementary strand) in shown in our structures (Figure 6A, D), it is possible that the repression complex ((Figure 5C, i and ii). However, in the presence of adenine in position 4 might play a role in the activation complex, due to the semipalindromic DNA the decrease in the affinity of the sequence for LuxR in the sequence, some of these contacts are not established (Fig- activation complex. ure 5C ii), partly because of the adenine at position 18 . Phe54 establishes an extensive van der Waals and hy- drophobic contacts with T14, A15 and T16 nucleobases In vitro validation of DNA recognition by the LuxR-arm do- (T7 , A8 and T9 in the complementary DNA) in the re- main pression complex (Figure 5D, i, ii). However, in the activa- Our structures revealed the DNA recognition by the LuxR- tion complex, only contacts with G14 and T15 (G8 and T9 arm domain. To determine how the interaction affects in complementary DNA) are maintained (Figure 5C, i, ii). DNA-binding affinity, we performed EMSA experiments Finally, the side chain of Ans55 is within van der Waals dis- with wt or variant LuxR proteins and various DNA tance of atom C7 in the methyl group of T4 (T19 ) in the probes. First, we investigated the contribution of the full- repression complex, which differs from the activation com- length LuxR-arm and the LuxR-arm with mutated R9/R11 plex, in which this interaction is maintained only in subunit residues to LuxR-arm binding with actDNA (Figure 7A B (T19 ), since position 4 in subunit A is occupied by ade- and Supplementary Figure S11) and repDNA (Figure 7B nine. Similar to the phosphate backbone interactions, the and Supplementary Figure S11). Compared to wt LuxR distances of the nucleobases to LuxR residues in the major (Kd = 449 nM for actDNA) and the LuxR variant with groove are shorter in the repression complex, displaying an a residue substitution K16A not related to DNA binding average distance difference of 0.5 Å in comparison to the ac- (Kd = 329 nM), the R11A and R9/11A mutants (LuxRR11A tivation complex (Figure 5E), further suggesting a stronger and LuxRR9/11A ) as well as a variant with the LuxR-arm binding affinity of LuxR for the repDNA palindromic se- domain deleted in LuxR (LuxRLuxR-arm ) were unable to quence. bind with actDNA, except for LuxRR9A which showed sig- nificantly decreased binding affinity for actDNA (Kd = 698 LuxR-arm interaction with the minor groove nM), which is consistent with the observation that R11 of The major difference between LuxR-actDNA and LuxR- LuxR is the essential residue for the minor groove interac- repDNA structures is revealed by the extensive contact in- tion in the LuxR–actDNA structure (Figure 6A). Although terface displayed between the N-terminal extension LuxR- the involvement of R9 in the LuxR–actDNA interaction arm domain, mainly composed of Arg9 and Arg11, and was not observed, the residue is assumed to be involved in the nucleobases of the two minor grooves in the repres- DNA binding in support of R11 inserting into the minor sion complex (Figure 6A, D). In the activation complex, groove to interact with the specific nucleobases. only Arg11 in the subunit A is observed to be in contact An overall higher affinity was observed for the LuxR- with one end of the semipalindromic DNA sequence (Fig- repDNA interaction as compared to the LuxR-actDNA ure 6B and C). However, in the LuxR-repDNA complex, interaction (Figure 7B and Supplementary Figure S11). both of the subunits establish extensive contacts with the Compared to wt LuxR (Kd = 157 nM for repDNA), the minor groove through Arg9 and Arg11 (Figure 6E–H). In LuxRR9A and LuxRR11A variants exhibited severely de- the LuxR-arm extension of the subunit A, Arg9 forms H- creased binding affinity for repDNA, with Kd values of 434 bond interactions with the nucleobases of T4 and T3 and and 1057 nM, respectively (Figure 7B). Although a sig- with the sugar phosphate backbone of T4, T5 and A2 (Fig- nificant reduction was observed for repDNA affinity for ure 6E), and Arg11 also forms H-bond interactions with the LuxRR9A , the R11A mutant was more close to LuxR-arm Nucleic Acids Research, 2021, Vol. 49, No. 6 3287 Figure 6. Interactions between the LuxR-arm and the DNA minor groove. (A, D) The interactions between amino acid residues and nucleotides in the minor grooves in actDNA (A) and repDNA (D) are shown by short lines. (B, C, E–H) The detailed interactions in the minor grooves are presented by ribbons and sticks in (B) and (C) for subunit A of LuxR-actDNA, and (E), (F) for subunit A, (G), (H) for subunit B of LuxR-repDNA. deletion and R9/11A-double mutant, resulting in drasti- for AM7) for LuxR (Figure 7C). Nevertheless, the mutant cally decreased DNA binding (Figure 7B). These data indi- containing the substitution of A4T (AM6) resulted in the cated that both R9 and R11 residues of LuxR are essential increased affinity for LuxR, which might be due to the cre- for the LuxR-repDNA recognition, although R11 mediates ation of interactions of R9 in subunit A, as shown in the a stronger interaction than R9 (Figure 7B). Intriguingly, the LuxR-repDNA structure (Figure 6A). Similarly, the mul- LuxRLuxR-arm showed residual but extremely low (i.e. the tiplex substitutions, i.e. A1T, A4T and A22T in actDNA Kd could not be calculated (ND)) affinity to repDNA as (AM5), also resulted in the increase in affinity for LuxR compared to the mutant with R9/R11A double substitu- (Figure 7C). The other single or multiplex mutant contain- tions, suggesting that the presence of a flexible N-terminal ing T18A (AM2–4) in actDNA did not seem to significantly extension that cannot interact with the minor groove at all affect the binding affinity, which might be due to the gener- (R9/R11A) might perturb or hinder the major groove bind- ation of possible interactions for R9 or R11 and disruption ing by the DBD domain in the LuxRR9/11A variant. of the HTH mediated DNA-protein interactions (Figures We also carried out in vitro assays to validate the contri- 6A and 7C). As expected, the T22G (RM1), A21G (RM2), bution of key nucleobases mediating actDNA or repDNA T20G (RM3), T2G (RM4) and A18T (RM5 and RM6) mu- recognition by LuxR (Figure 7C, D and Supplementary tations that eliminated the interactions of the mutants at Figure S11A). In comparison to the wt actDNA (Kd = 449 R9 and R11 residues showed a significant reduction in the nM), only the substitutions at T2A (AM1) and A3C (AM7), binding affinity of LuxR-repDNA and verified that these which abolish the observed R11 interaction, caused reduced nucleobases mediate key interactions during repDNA bind- binding affinities (Kd = 1686 nM for AM1and Kd = 636 nM ing with LuxR. The multiplexed mutations of T1A, T4A 3288 Nucleic Acids Research, 2021, Vol. 49, No. 6 Figure 7. The residues Arg9 (R9) and Arg11 (R11) in the LuxR-arm domain are essential for LuxR-DNA interactions and QS regulation. (A, B) DNA binding of wt and variants of LuxR. DNA binding curves from the quantitative analysis of bound bands in EMSAs (see Supplementary Figure S11 for a representative example) are shown for LuxRR9A , LuxRR11A , LuxRR9/11A , LuxRLuxR-arm , LuxRK16A and LuxR with actDNA (A) and repDNA (B). (C, D) LuxR protein binding to the respective mutant actDNA (C) and repDNA (D) fragments (Supplementary Figure S11). The densitometric intensities of the bound DNA fragments were determined and plotted against the protein concentrations. Triplicate assays were performed. Dissociation constants (Kd , nM) are shown in brackets. ND, not detectable. (E, F) Scatter plots of gene expression in luxR and the luxR strains chromosomally expressing LuxRR9A (E) and LuxRR11A (F) relative to wt. (G) Survival analysis for zebrafish challenged with wt, luxR, or the luxR strains chromosomally expressing LuxRR9A or LuxRR11A . The P values were calculated using a Kaplan–Meier survival analysis with a log-rank test. *P < 0.05, **P < 0.01. (H) Activities of PluxR and its variants fused to egfp. PluxR harbors two LuxR-binding sites, LBSI (actDNA, green) and LBSII (close to repDNA, light blue). The variant PluxR was constructed with the substitutions of repDNA (blue) or 22-bp nonrelated control DNA (yellow). All promoter activities were assayed in the context of the luxR or the luxR strains chromosomally expressing wt LuxR, LuxRR9A or LuxRR11 cultured for 9 h and the results were normalized by the activity of the wt strain. The results are presented as the means ± S.D. (n = 3). *P < 0.05, **P < 0.001, ***P < 0.0001 (Student’s t test). Nucleic Acids Research, 2021, Vol. 49, No. 6 3289 and T22A in RM7, which abrogates the R9 interaction and actDNA motif (LBSI, –91 to –70 bp) and a motif sequence somewhat transforms the DNA residues into actDNA, also resembling repDNA (LBSII, +3 to +24 bp), respectively, in results in significantly reduced binding affinity of LuxR- the PluxR region (Figure 7H). This finding represents a com- repDNA (Figure 7D). Collectively, these data validated that mon profile of LuxR-binding sites in the promoter region of R9 and R11 in LuxR-arm interactions with specific nucle- the LuxR-repressed or activated genes (Supplementary Fig- obases play important roles in LuxR–DNA binding. ure S8). We substituted chromosomal LBSI or LBSII with the palindromic sequence (repDNA) used for the crystal structure determination and a nonrelated GC-rich 22-bp se- In vivo analysis of DNA recognition and QS gene regulation quence that is not bound by LuxR as a control (Supplemen- by the LuxR-arm domain tary Figure S11B), to test their effect on PluxR activities in We characterized the QS regulation behavior of the LuxR the context of V. alginolyticus expressing wt or variant LuxR variants in vibrios. The luxR mutant stains chromosoma- (LuxRR9A , and LuxRR11A ). Except in the PluxR strains with lly expressing wt or mutant forms of LuxR, LuxR-arm, LBSI or LBSII sequences replaced with control DNA that LuxRR9A , LuxRR11A , LuxRR9/11A , LuxRR9E , LuxRR11E , appeared to totally destroy the PluxR activity (Figure 7H, were constructed and showed various levels of Asp pro- d and f), all the strains with luxR deleted (luxR) showed duction (Supplementary Figure S12). The significantly de- high level of GFP fluorescence, indicating the overall repres- creased Asp yields in the strain expressing R9 and R11 sion of LuxR on PluxR activity, consistent with the previous and the LuxR-arm-related LuxR variants further demon- finding showing that LuxR acts as a repressor of luxR ex- strated their essential roles in QS regulation (Supplemen- pression (27). Compared to the luxR strain, the LuxR- tary Figure S12). Notably, the LuxRR9E strain exhibited a producing strain reduced the wt PluxR activity by 4.6-fold much lower level of Asp production than LuxRR9A , prob- (Figure 7H, a). The repression effect of LuxR on the PluxR ably due to the electric repulsion of the DNA interaction variants (with LBSI or LBSII replaced by repDNA) (b, c, by the Glu substitution, demonstrating the important role and e) was significantly increased, by 8.6-,12.9-, and 4.0- of R9 in regulation of asp expression (Supplementary Fig- fold, respectively. Similarly, LuxR repression to PluxR vari- ure S12). Comparative transcriptomic assays and ChIP- ants in which LBSII was replaced with actDNA (g) de- seq analysis facilitated the identification of the regulon by creased to 3.6-fold, indicating the expected derepression ac- these LuxR proteins. The low level of consistency between tivity mediated by actDNA. These analyses indicated that LuxRR9A -controlled genes (R2 = 0.07) and genes affected the LuxR-binding sequences and their relative positions in by luxR deletion, compared with LuxRR11A and that of PluxR together determine the extent of LuxR repression or luxR deletion-regulated genes (R2 = 1) (Figure 7E, F, Sup- activation at specific promoters. Moreover, LuxRR9A and plementary Figure S2), demonstrated that R11 is more im- LuxRR11A mutant proteins exhibited various degrees of at- portant than R9 for LuxR function in QS regulation. More- tenuated repression, compared to that of wt LuxR in PluxR , over, the LuxRR11A regulated considerably fewer of genes further demonstrating that the R9 and R11 in the LuxR- than LuxRR9A or wt LuxR as revealed by RNA-seq (Sup- arm domain play key roles in regulating QS gene expression. plementary Figure S2E) and ChIP-seq (Supplementary Fig- We also detected the overall much lower activities of PluxR ure S13), confirming that R9 and R11 in the LuxR-arm play and its variants at LCD (2 h) (Supplementary Figure S14). pivotal roles in DNA binding and transcriptional regula- The substitution of +3 to +24 bp repDNA sequence with tion. the LBSI DNA in a strain producing LuxR also activated Quorum sensing plays an important role in the pathogen- the luxR transcription as it did at HCD (Figure 7H and esis of V. alginolyticus (27). We used zebrafish as a model Supplementary Figure S14, a and g). In contrast, the re- system to test the effects of R9 and R11 in the LuxR-arm placement of LBSI sequence at –91–70 bp with repDNA on the virulence of V. alginolyticus (Figure 7G). Groups of significantly impaired the promoter activities in the LuxR zebrafish (n = 30) were acclimated for at least 4 weeks before generating strain at both LCD and HCD stages (Figure 7H the infection experiments were performed with a dose of 105 and Supplementary Figure S14, a and c), demonstrating CFU/fish. A significant difference (P < 0.05) in the survival the potential activation function of LBSI in luxR transcrip- rates was observed between the groups of fish treated with tion. Collectively, these analyses demonstrated that the fea- wt and luxR strains, consistent with a previous observa- tures of LuxR-binding sequences, the profiles of the binding tion (27). The survival of the fish in the groups treated with site position in the controlled promoters, and the R9/R11 strains producing LuxRR11A or LuxRR9A was also signifi- recognition mode of the LuxR-arm domain enable the flex- cantly higher (P < 0.001) than that of those treated with ible control of gene expression and coordinate QS output in wt, indicating that R9A and R11A mutants significantly at- vibrios. tenuated the virulence of V. alginolyticus in fish. Taken to- gether, vibrios seem to adopt a regulatory mechanism by using the N-terminal LuxR-arm domain for versatile gene DISCUSSION modulation in V. harveyi-like QS cascades. LuxR-mediated gene regulation is conserved among Vibrios V. harveyi-type QS is common to most vibrios and repre- LuxR binding profiles and LuxR-arm domain coordinate QS sents the major model for QS studies for which TetR fam- output ily LuxR homologs is known as MQSR to control the QS Our DNase I-seq analysis and a previous investigation output (2). LuxR is unique as it cannot bind ligands and is revealed two distinct LuxR-binding sites harboring the distinct from TetR, as it acts as a global regulator with high 3290 Nucleic Acids Research, 2021, Vol. 49, No. 6 DNA binding flexibility to control the expression of hun- this case, stronger binding affinity leads to greater repres- dreds of genes by both activation and repression activity, sion (Figure 7H). The activation role of LuxR is accom- with the underlying mechanisms remaining unknown (4,8). plished by the recruitment of RNAP, which requires LuxR Many tiers of factors are assumed to govern the LuxR- to bind to the upstream site of RNAP (LBSI, actDNA). centered QS output for various collective phenotypes, In this case, the strong binding affinity of LuxR for LBSI e.g. the upstream autoinducer signaling cascades of luxR positions would lead to repression since the strong binding expression, the DNA binding sites and their distribution affinity of LuxR for DNA arrests RNAP to prevent its re- in genes, and the manner by which LuxR interacts with lease. Thus, our results support a mechanism for the mas- specific DNA (45–48). Although further analysis of the ter regulator LuxR with different binding affinities (strong signaling dynamics of the QS input is warranted, con- or weak) that account either for repression, the RNA poly- served QS components, such as LuxO, qrrs, and AphA, have merase cannot displace LuxR from the binding site and been identified, demonstrating that V. alginolyticus harbors transcription is repressed, or for activation, the RNA poly- QS systems resembling that of V. harveyi (12,18,49). Here merase machinery goes through and transcribes the LuxR- we also validated that LuxR behaves similarly in V. algi- dependent gene. nolyticus and V. harveyi because our RNA-seq and ChIP- We thus proposed a model in which LuxR binds to the seq analysis revealed a large group of overlapping genes repDNA with high affinity at –50 bp to +1 bp for occlu- (∼67) controlled by LuxR, including genes associated with sion of RNAP binding at the promoter region and tran- T1/T3/T6SS, c-di-GMP signaling, flagellin, the ABC trans- scription initiation (Figure 8, bottom). In contrast, most porter system and QS genes, such as luxR (Supplementary of the LuxR-binding sites at an activated promoter are lo- Figure S5) (50). The gene encoding the V. alginolyticus- cated from –30 bp to –100 bp relative to the transcriptional specific exotoxin Asp was revealed to be directly con- start site, indicating that LuxR can be classified as type I trolled by LuxR (Supplementary Figure S6), demonstrat- transcriptional factor (52) and facilitates the recruitment of ing the presence of a species-specific QS regulon in vibrios. RNAP as it interacts with the C-terminus of the ␣-subunit Taken together, these analyses indicate that LuxR regulon (51,53). The nearly doubled LuxR binding site number in is largely shared by V. harveyi-like QS systems, but the tem- the activated genes, the larger range of distribution in up- poral variance in the regulons controlled by LuxR and QS stream or downstream of the promoter region and the lower remains to be elucidated. affinity of LuxR for actDNA, as discussed below, may facil- Moreover, our ChIP-seq analysis coupled with DNase I- itate LuxR alterations of the chromosome conformation to seq assays allowed us to dissect at a high-resolution LuxR- recruit RNAP and trigger transcription initiation complex binding motifs i.e. actDNA and repDNA. In particular, formation for gene expression (Figure 8, bottom). the actDNA binding sequences generated logos with an asymmetric half right of the motif (Figure 2G). Both the Structural bases of unique DNA binding by LuxR actDNA and repDNA sequences are highly similar to that established for LuxR homologs in V. harveyi with ChIP-seq Crystal structures of the LuxR-DNA complex revealed a analysis (8), suggesting conserved regulatory mechanisms new mode of DNA binding by a TetR-family transcrip- adopted by LuxR homologs in vibrios. tional regulator that involves a N-terminal extension bind- ing to the minor groove. Compared to the LuxR–actDNA interaction, stronger binding affinity for the palindromic re- Distinct features of LuxR binding sites dictate gene activation pression sequence is basically caused by both-end contacts and repression involving the N-terminal LuxR-arm extension (Figure 8, DNase I-seq facilitated determination of the binding-site up). In particular, Arg9 and Arg11 from both dimer sub- distribution profiles in the LuxR-targeted promoters. Mul- units establish 18 H-bond contacts with nucleobases in the tiple binding sites in a single LuxR-controlled promoter two minor grooves of the DNA double helix in the LuxR– normally correlates with activation, but it is not always repDNA complex. DNA recognition by the binding of Arg9 the case, as some exceptional LuxR-controlled promoters and Arg11 to nucleobases in the minor grooves is unique containing a mixture of both repDNA or actDNA bind- to the LuxR proteins in the TetR family. Other TetR fam- ing sites in the otherwise activated or repressed promoters, ily members also have a disordered N-terminal extension e.g. the previous established PluxR (Figure 7H and Supple- that mediates its binding in the DNA minor groove, such as mentary Figure S8). This indicated that other levels of com- SimR, in which Arg18 interacts with thymine in the minor plexity, such as the presence of other DNA-binding proteins groove, and Arg19 and Arg22 interact with phosphate back- that modulate the binding complex (51), can affect LuxR- bones (54). However, the N-terminal extensions of LuxR dependent genes’ expression. However, our structural anal- and SimR are not conserved, and their modes of interac- ysis and cellular assays using a single binding site appear tion with DNA minor groove are distinct. Moreover, LuxR to support a model in which the LuxR binding affinity is can flexibly bind to the actDNA and repDNA with one end sufficient to determine activation or repression (Figure 7H, or both LuxR-arm structures, while SimR only binds to e). DNA using both of its N-terminal extensions, which ren- The different binding affinities for the DNA sequence ders the roles of LuxR and SimR in modulating DNA bind- might determine the fate of polymerase activity, i.e., tran- ing highly divergent. Notably, an adjacent C-terminal arm scription or inhibition. Taking PluxR as an example, LuxR of TZAP 11th C2H2 zinc finger also recognizes the mi- represses transcription by binding to the positions of LBSII nor groove of telomeric DNA through the arginine-thymine (close to repDNA) where RNAP binds or passes over. In pattern in eukaryotes (55). Nucleic Acids Research, 2021, Vol. 49, No. 6 3291 Figure 8. The working model of LuxR interaction with actDNA and repDNA and their binding patterns to control QS gene expression. The numbers indicate the location relative to transcription start site; arrows indicate transcription activation and bar-ended short lines show transcription repression (lower panel). Finally, fundamental understanding of the LuxR me- SUPPLEMENTARY DATA diated QS regulation in vibrios may facilitate to develop Supplementary Data are available at NAR Online. strategies, such as designing QS inhibiting chemicals that interfere with LuxR regulation to effectively control the pathogen threatening health to both humans and the ACKNOWLEDGEMENTS aquatic animals. The knowledge of regulation machinery of Author contributions: Conceptualization: W.Q.; Data cura- the MQSR LuxR in vibrios also help to design promising tion: W.Q., R.D., Z.J., L.B., GD. Funding acquisition: W.Q., genetic parts or circuits useful for the synthetic biology. M.Y., R.D.; Investigation: Z.J., L.B., G.D., H.Y., C.M., M.Y., Z.X. Project administration: W.Q.. Supervision: W.Q. ETHICS STATEMENT Validation: R.D., W.Q.. Visualization: W.Q., R.D. Writing All animal protocols used in this study were approved by – original draft: W.Q., R.D., Z.X., Z.J., L.B., G.D. Writing the Animal Care Committee of the East China University – review & editing: W.Q. of Science and Technology (2006272). The Experimental Animal Care and Use Guidelines from Ministry of Science FUNDING and Technology of China (MOST-2011-02) were strictly fol- National Natural Science Foundation of China [31772891 lowed. to Q.W., 31772893 to Y.M.]; Ministry of Agriculture of China [CARS-47-G17]; Science and Technology Com- DATA DEPOSITION mission of Shanghai Municipality [17391902000]; Fun- The ChIP-seq, DNase I-seq and RNAS-seq sequence reads damental Research Funds for the Central Universities were deposited in the NCBI SRA database (PRJNA660595 [22221062017019]; Ministerio de Ciencia, Innovación y and PRJNA673251). The accession numbers for LuxR- Universidades [PGC2018-098423-B-I00 to D.R.]. Funding repDNA and LuxR-actDNA structures are 7AMN and for open access charge: National Natural Science Founda- 7AMT, respectively. tion of China. 3292 Nucleic Acids Research, 2021, Vol. 49, No. 6 Conflict of interest statement. None declared. 20. Wang,Q.Y., Liu,Q., Ma,Y., Rui,H.P. and Zhang,Y.X. (2007) LuxO controls extracellular protease, haemolytic activities and siderophore production in fish pathogen Vibrio alginolyticus. J. Appl. Microbiol., REFERENCES 103, 1525–1534. 1. Papenfort,K. and Bassler,B.L. (2016) Quorum sensing 21. Rui,H.P., Liu,Q., Ma,Y., Wang,Q.Y. and Zhang,Y.X. (2008) Roles of signal-response systems in Gram-negative bacteria. Nat. Rev. LuxR in regulating extracellular alkaline serine protease A, Microbiol., 14, 576–588. extracellular polysaccharide and mobility of Vibrio alginolyticus. 2. Henke,J.M. and Bassler,B.L. (2004) Three parallel quorum-sensing FEMS Microbiol. Lett., 285, 155–162. systems regulate gene expression in Vibrio harveyi. J. Bacteriol., 186, 22. Cao,X., Wang,Q., Liu,Q., Liu,H., He,H. and Zhang,Y. (2010) Vibrio 6902–6914. alginolyticus MviN is aluxO-regulated protein and affects cytotoxicity 3. Plener,L., Lorenz,N., Reiger,M., Ramalho,T., Gerland,U. and towards epithelioma papulosum cyprini (EPC) cells. J. Microbiol. Jung,K. (2015) The phosphorylation flow of the Vibrio harveyi Biotechnol., 20, 271–280. quorum-sensing cascade determines levels of phenotypic 23. Cao,X., Wang,Q., Liu,Q., Rui,H., Liu,H. and Zhang,Y. (2011) heterogeneity in the population. J. Bacteriol., 197, 1747–1756. Identification of a luxO-regulated extracellular protein Pep and its 4. Ball,A.S., Chaparian,R.R. and van Kessel,J.C. (2017) Quorum roles in motility inVibrio alginolyticus. Microb. Pathog., 50, 123–131. sensing gene regulation by LuxR/HapR master regulators in vibrios. 24. Liu,H., Gu,D., Cao,X.D., Liu,Q., Wang,Q.Y. and Zhang,Y.X. (2012) J. Bacteriol., 199, e00105-17. Characterization of a new quorum sensing regulator luxT and its 5. De Silva,R.S., Kovacikova,G., Lin,W., Taylor,R.K., Skorupski,K. roles in the extracellular protease production, motility, and virulence and Kull,F.J. (2007) Crystal structure of the Vibrio cholerae in fish pathogen Vibrio alginolyticus. Arch. Microbiol., 194, 439–452. quorum-sensing regulatory protein HapR. J. Bacteriol., 189, 25. Sheng,L.L., Lv,Y.Z., Liu,Q., Wang,Q.Y. and Zhang,Y.X. (2013) 5683–5691. Connecting type VI secretion, quorum sensing, and c-di-GMP 6. Kim,Y., Kim,B.S., Park,Y.J., Choi,W.C., Hwang,J., Kang,B.S., production in fish pathogen Vibrio alginolyticus through phosphatase Oh,T.K., Choi,S.H. and Kim,M.H. (2010) Crystal structure of SmcR, PppA. Vet. Microbiol., 162, 652–662. a quorum-sensing master regulator of Vibrio vulnificus, provides 26. Gao,X., Wang,X., Mao,Q., Xu,R., Zhou,X., Ma,Y., Liu,Q., Zhang,Y. insight into its regulation of transcription. J. Biol. Chem., 285, and Wang,Q. (2019) VqsA, a novel LysR-type transcriptional 14020–14030. regulator, coordinates quorum sensing (QS) and is controlled by QS 7. Ramos,J.L., Martı́nez-Bueno,M., Molina-Henares,A.J., Terán,W., to regulate virulence in the pathogen Vibrio alginolyticus. Appl. Watanabe,K., Zhang,X., Gallegos,M.T., Brennan,R. and Tobes,R. Environ. Microbiol., 84, e00444-18. (2005) The TetR family of transcriptional repressors. Microbiol. Mol. 27. Gu,D., Guo,M., Yang,M., Zhang,Y., Zhou,X. and Wang,Q. (2016) A Biol. Rev., 69, 326–356. ␴ E -mediated temperature gauge controls a switch from 8. Kessel,J.C., Ulrich,L.E., Zhulin,L.B. and Bassler,B.L. (2013) Analysis LuxR-mediated virulence gene expression to thermal stress of activator and repressor functions reveals the requirements for adaptation in Vibrio alginolyticus. PLoS Pathog., 12, e1005645. transcriptional control by LuxR, the master regulator of quorum 28. Gibson,D.G., Young,L., Chuang,R.Y., Venter,J.C., Hutchison,C.A. sensing in Vibrio harveyi. mBio, 4, e00378-13. 3rd and Smith,H.O. (2009) Enzymatic assembly of DNA molecules 9. Tsou,A.M., Cai,T., Liu,Z., Zhu,J. and Kulkarni,R.V. (2009) up to several hundred kilobases. Nat. Methods., 6, 343–345. Regulatory targets of quorum sensing in Vibrio cholerae: evidence for 29. Juanhuix,J., Gil-Ortiz,F., Cunı́,G., Colldelram,C., Nicolás,J., two distinct HapR-binding motifs. Nucleic Acids Res., 37, 2747–2756. Lidón,J., Boter,E., Ruget,C., Ferrer,S. and Benach,J. (2014) 10. Lee,D.H., Jeong,H.S., Jeong,H.G., Kim,K.M., Kim,H. and Developments in optics and performance at BL13-XALOC, the Choi,S.H. (2008) A consensus sequence for binding of SmcR, a Vibrio macromolecular crystallography beamline at the ALBA synchrotron. vulnificus LuxR homologue, and genome-wide identification of the J. Synchrotron Radiat., 21, 679–689. SmcR regulon. J. Biol. Chem., 283, 23610–23618. 30. Kabsch,W. (2010) XDS. Acta Crystallogr. D, 66, 125–132. 11. Jacobs Slifka,K.M., Newton,A.E. and Mahon,B.E. (2017) Vibrio 31. Winn,M.D., Ballard,C.C., Cowtan,K.D., Dodson,E.J., Emsley,P., alginolyticus infections in the USA, 1988–2012. Epidemiol. Infect., Evans,P.R., Keegan,R.M., Krissinel,E.B., Leslie,A.G., McCoy,A. 145, 1491–1499. et al. (2011) Overview of the CCP4 suite and current developments. 12. Rui,H., Liu,Q., Wang,Q., Ma,Y., Liu,H., Shi,C. and Zhang,Y. (2009) Acta Crystallogr. D, 67, 235–242. Role of alkaline serine protease,asp, in Vibrio alginolyticus virulence 32. Adams,P.D., Afonine,P.V., Bunkóczi,G., Chen,V.B., Davis,I.W., and regulation of its expression by LuxO-LuxR regulatory system. J. Echols,N., Headd,J.J., Hung,L.W., Kapral,G.J., Microbiol. Biotechnol., 19, 431–438. Grosse-Kunstleve,R.W. et al. (2010) PHENIX: a comprehensive 13. Tian,Y., Wang,Q., Liu,Q., Ma,Y., Cao,X., Guan,L. and Zhang,Y. Python-based system for macromolecular structure solution. Acta (2008) Involvement of LuxS in the regulation of motility and flagella Crystallogr. D, 66, 213–221. biogenesis in Vibrio alginolyticus. Biosci. Biotechnol. Biochem., 72, 33. Emsley,P., Lohkamp,B., Scott,W.G. and Cowtan,K. (2010) Features 1063–1071. and development of Coot. Acta Crystallogr., D, 66, 486–501. 14. Swem,L.R., Swem,D.L., Wingreen,N.S. and Bassler,B.L. (2008) 34. McCoy,A.J., Grosse-Kunstleve,R.W., Adams,P.D., Winn,M.D., Deducing receptor signaling parameters fromin vivo analysis: Storoni,L.C. and Read,R.J. (2007) Phaser crystallographic software. LuxN/AI-1 quorum sensing in Vibrio harveyi. Cell, 134, 461–473. J. Appl. Crystallogr., 40, 658–674. 15. Timmen,M., Bassler,B.L. and Jung,K. (2006) AI-1 influences the 35. Li,H. and Durbin,R. (2009) Fast and accurate short read alignment kinase activity but not the phosphatase activity of LuxN of Vibrio with burrows-Wheeler transform. Bioinformatics, 25, 1754–1760. harveyi. J. Biol. Chem., 281, 24398–24404. 36. Liang,H., Deng,X., Li,X., Ye,Y. and Wu,M. (2014) Molecular 16. Ye,J., Ma,Y., Liu,Q., Zhao,D.L., Wang,Q.Y. and Zhang,Y.X. (2008) mechanisms of master regulator VqsM mediating quorum-sensing Regulation ofVibrio alginolyticus virulence by the LuxS and antibiotic resistance in Pseudomonas aeruginosa. Nucleic Acids quorum-sensing system. J. Fish Dis., 31, 161–169. Res., 42, 10307–10320. 17. Kelly,R.C., Bolitho,M.E., Higgins,D.A., Lu,W., Ng,W.L., 37. Feng,J.X., Liu,T., Qin,B., Zhang,Y. and Liu,X.L. (2012) Identifying Jeffrey,P.D., Rabinowitz,J.D., Semmelhack,M.F., Hughson,F.M. and ChIP-seq enrichment using MACS. Nat. Protoc., 7, 1728–1740. Bassler,B.L. (2009) The Vibrio cholerae quorum-sensing autoinducer 38. Yang,Z., Zhou,X., Ma,Y., Zhou,M., Waldor,M.K., Zhang,Y. and CAI-1: analysis of the biosynthetic enzyme CqsA. Nat. Chem. Biol., Wang,Q. (2018) Serine/threonine kinase PpkA coordinates the 5, 891–895. interplay between T6SS2 activation and quorum sensing in the marine 18. Gu,D., Liu,H., Yang,Z., Zhang,Y.X. and Wang,Q.Y. (2016) pathogen Vibrio alginolyticus. Environ. Microbiol., 20, 903–919. Chromatin immunoprecipitation sequencing technology reveals 39. Miller,D.J., Zhang,Y.M., Subramanian,C., Rock,C.O. and White,S.W. global regulatory roles of low-cell-density quorum-sensing regulator (2010) Structural basis for the transcriptional regulation of AphA in the pathogen Vibrio alginolyticus. J. Bacteriol., 198, membrane lipid homeostasis. Nat. Struct. Mol. Biol., 17, 971–975. 2985–2999. 40. Orth,P., Schnappinger,D., Hillen,W., Saenger,W. and Hinrichs,W. 19. Rutherford,S.T., van Kessel,J.C., Shao,Y. and Bassler,B.L. (2011) (2000) Structural basis of gene regulation by the tetracycline inducible AphA and LuxR/HapR reciprocally control quorum sensing in Tet repressor–operator system. Nat. Struct. Biol., 7, 215–219. vibrios. Genes Dev., 25, 397–408. Nucleic Acids Research, 2021, Vol. 49, No. 6 3293 41. Itou,H., Watanabe,N., Yao,M., Shirakihara,Y. and Tanaka,I. (2010) 49. Liu,H., Liu,W., He,X., Chen,X., Yang,J., Wang,Y., Li,Y., Ren,J., Crystal structures of the multidrug binding repressor Xu,W. and Zhao,Y. (2020) Characterization of a cell Corynebacterium glutamicum CgmR in complex with inducers and density-dependent sRNA, Qrr, and its roles in the regulation of the with an operator. J. Mol. Biol., 403, 174–184. quorum sensing and metabolism inVibrio alginolyticus. Appl. 42. Filippova,E.V., Wawrzak,Z., Ruan,J., Pshenychnyi,S., Schultz,R.M., Microbiol. Biotechnol., 104, 1707–1720. Wolfe,A.J. and Anderson,W.F. (2016) Crystal structure of 50. Bagert,J.D., van Kessel,J.C., Sweredoski,M.J., Feng,L., Hess,S., nonphosphorylated receiver domain of the stress response regulator Bassler,B.L. and Tirrell,D.A. (2016) Time-resolved proteomic RcsB from Escherichia coli. Protein Sci., 25, 2216–2224. analysis of quorum sensing in Vibrio harveyi. Chem. Sci., 7, 43. Schumacher,M.A., Miller,M.C., Grkovic,S., Brown,M.H., 1797–1806. Skurray,R.A. and Brennan,R.G. (2002) Structural basis for 51. Chaparian,R.R., Tran,M.L.N., Conrad,L.C.M., Rusch,D.B. and van cooperative DNA binding by two dimers of the multidrug-binding Kessel,J.C. (2020) Global H-NS counter-silencing by LuxR activates protein QacR. EMBO J., 21, 1210–1218. quorum sensing gene expression. Nucleic Acids Res., 48, 171–183. 44. Campanella,J.J., Bitincka,L. and Smalley,J. (2003) MatGAT: an 52. Browning,D.F. and Busby,S.J. (2016) Local and global regulation of application that generates similarity/identity matrices using protein transcription initiation in bacteria. Nat. Rev. Microbiol., 14, 638–650. or DNA sequences. BMC Bioinformatics, 4, 29. 53. Ball,A.S. and van Kessel,J.C. (2019) The master quorum-sensing 45. Coquant,G., Grill,J.P. and Seksik,P. (2020) Impact of regulators LuxR/HapR directly interact with the alpha subunit of N-acyl-homoserine lactones, quorum sensing molecules, on gut RNA polymerase to drive transcription activation in Vibrio harveyi immunity. Front. Immunol., 11, 1827. and Vibrio cholerae. Mol. Microbiol., 111, 1317–1334. 46. Kim,B.S., Jang,S.Y., Bang,Y.J., Hwang,J., Koo,Y., Jang,K.K., Lim,D., 54. Le,T.B., Schumacher,M.A., Lawson,D.M., Brennan,R.G. and Kim,M.H. and Choi,S.H. (2018) QStatin, a selective inhibitor of Buttner,M.J. (2011) The crystal structure of the TetR family quorum sensing in Vibrio species. mBio, 9, e02262-17. transcriptional repressor SimR bound to DNA and the role of a 47. Lee,K.J., Jung,Y.C., Park,S.J. and Lee,K.H. (2018) Role of heat shock flexible N-terminal extension in minor groove binding. Nucleic Acids proteases in quorum-sensing-mediated regulation of biofilm Res., 39, 9433–9447. formation by Vibrio species. mBio, 9, e02086-17. 55. Zhao,Y., Zhang,G., He,C., Mei,Y., Shi,Y. and Li,F. (2018) The 11th 48. Brameyer,S., Plener,L., Müller,A., Klingl,A., Wanner,G. and Jung,K. C2 H2 zinc finger and an adjacent C-terminal arm are responsible for (2018) Outer membrane vesicles facilitate trafficking of the TZAP recognition of telomeric DNA. Cell Res., 28, 130–134. hydrophobic signaling molecule CAI-1 between Vibrio harveyi cells. J. Bacteriol., 200, e00740-17.