H-NS,Represses,Biofilm,Formation,and,c-di-GMP,Synthesis,in,Vibrio,parahaemolyticus＊

XUE Xing Fan ,ZHNAG Miao Miao ,SUN Jun Fang ,LI Xue ,WU Qi Min ,YIN Zhe ,YANG Wen Hui,NI Bin,HU Ling Fei,ZHOU Dong Sheng,#,LU Ren Fei,#,and ZHANG Yi Quan,#

1.School of Medicine,Jiangsu University,Zhenjiang 212013,Jiangsu,China;2.Department of Clinical Laboratory,Affiliated Nantong Hospital 3 of Nantong University,Nantong 226006,Jiangsu,China;3.State Key Laboratory of Pathogen and Biosecurity,Beijing Institute of Microbiology and Epidemiology,Beijing 100071,China

Abstract Objective This study aimed to investigate the regulation of histone-like nucleoid structuring protein(H-NS) on biofilm formation and cyclic diguanylate (c-di-GMP) synthesis in Vibrio parahaemolyticus RIMD2210633.Methods Regulatory mechanisms were analyzed by the combined utilization of crystal violet staining,quantification of c-di-GMP,quantitative real-time polymerase chain reaction,LacZ fusion,and electrophoretic-mobility shift assay.Results The deletion of hns enhanced the biofilm formation and intracellular c-di-GMP levels in V.parahaemolyticus RIMD2210633.H-NS can bind the upstream promoter-proximal DNA regions of scrA,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979 to repress their transcription.These genes encode a group of proteins with GGDEF and/or EAL domains associated with c-di-GMP metabolism.Conclusion One of the mechanisms by which H-NS represses the biofilm formation by V.parahaemolyticus RIMD2210633 may be via repression of the production of intracellular c-di-GMP.

Key words: Vibrio parahaemolyticus;Biofilm;H-NS;C-di-GMP

Vibrio parahaemolyticusis a Gram-negative halophilic bacterium that primarily causes seafood-associated gastrointestinal illness in humans[1].V.parahaemolyticusproduces multiple virulence determinants,such as two type-III secretion systems (T3SSs) and thermostable direct hemolysin (TDH) or TDH-related hemolysin,and forms robust biofilms on surfaces that support bacteria to survive in adverse conditions[1,2].V.parahaemolyticusemploys various specific structures,such as flagella,type-IV pili,and exopolysaccharide (EPS),to form mature biofilms[2].EPS is the main component of the biofilm matrix[2].InV.parahaemolyticus,thecpsA-Joperon is responsible for the synthesis of EPS[3].The deletion ofcpsA-Jled to smooth colonies on plates,whereas the wild-type (WT) strain formed wrinkled colonies[3].Previous studies showed that the variations between smooth and wrinkled colonies are strictly regulated by intracellular cyclic diguanylate (c-di-GMP) levels,CpsS-CpsR-CpsQ cascade,quorum sensing (QS),and histone-like nucleoid structuring protein (H-NS) inV.parahaemolyticus[4-10].

C-di-GMP is a ubiquitous secondary messenger involved in regulating multiple behaviors of bacteria,including biofilm formation,motility,and virulence[11].Increased intracellular c-di-GMP promotes biofilm formation and motility inhibition[12].The synthesis of c-di-GMP is catalyzed by diguanylate cyclase containing a GGDEF domain,whereas its degradation is associated with phosphodiesterases (PDE) possessing either an HDGYP or EAL domain[12].InV.parahaemolyticus,two proteins,namely,ScrG and ScrC,contain both GGDEF and EAL domains and act as PDEs to degrade c-di-GMP[4,6].In addition,the GGDEF-type proteins ScrO,GefA,ScrJ,and ScrL and the EAL type protein LafV are required for the regulation of motility and biofilm formation inV.parahaemolyticus[13-15].We recently demonstrated that a group of genes encoding the proteins with GGDEF and/or EAL domains,includingscrABCandscrG,were under the direct regulation of the master QS regulator OpaR,which acts as a repressor of biofilm formation and cdi-GMP production inV.parahaemolyticusRIMD2210633[9].

H-NS is an abundant nucleoid-associated DNAbinding protein involved in chromosome folding and gene regulation in Gram-negative bacteria[16].H-NS prefers to bind AT-rich DNA sequences and has been generally described as a negative regulator of gene expression[16].InV.parahaemolyticus,H-NS is a negative regulator of the major virulence determinants,including T3SS1,T3SS2,TDH,T6SS1,and T6SS2[17,18].The deletion ofhnsled to enhanced swarming motility andlafexpression inV.parahaemolyticus[19].In addition,previous studies showed that H-NS acts as a positive regulator of biofilm formation byV.parahaemolyticusRIMD2210633 because thehnsmutant formed smooth colonies and manifested lower expression ofcpsA-J[8,20].However,the regulatory mechanisms remain not fully understood.The data on crystal violet (CV) staining presented here showed that HNS acted as a repressor of biofilm formation byV.parahaemolyticusRIMD2210633.H-NS was a negative regulator of intracellular c-di-GMP synthesis and transcription ofscrA,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979,encoding a set of proteins containing GGDEF and/or EAL domains.

Construction and Complementation of the hns Mutant

V.parahaemolyticusstrain RIMD2210633 was used as the WT[21].Non-polarhnsdeletion mutant(Δhns) and complementaryΔhns(C-Δhns) were constructed in our previous study[17].Control strains were also constructed by transferring the empty pBAD33 into the WT andΔhnsstrains to counteract the effects of arabinose and chloramphenicol on the results[17].Table 1lists all the primers used in the current study.

Table 1.Oligonucleotide primers used in this study

Growth Conditions

TheV.parahaemolyticusstrains were similarly cultured as described previously[9].Briefly,V.parahaemolyticuswas cultivated overnight (about 12 h) in 2.5% (w/v) Bacto heart infusion (HI) broth (BD Biosciences,USA) at 37 °C with shaking at 200 rpm.The resultant cultures were diluted 40-fold into the phosphate-buffered saline buffer (PBS,pH 7.2).Then,150 μL of the diluted cultures were spread onto the HI plate with a diameter of 5 cm.After incubation at 37 °C for 6 h,the bacterial cells were harvested by adding 2 mL pre-cold PBS.When necessary,the media were supplemented with 100 μg/mL gentamicin,5 μg/mL chloramphenicol,or 0.1% arabinose.

CV Staining

The CV staining was carried out as previously described[9,10].Briefly,overnight cell cultures were diluted 50-fold into 5 mL fresh HI broth and cultured at 37 °C with shaking at 200 rpm and OD600of approximately 1.4.Thereafter,the resultant cultures were 50-fold diluted into 2 mL fresh Difco marine(M) broth 2216 (BD Biosciences,USA) containing 0.1% arabinose and 5 μg/mL chloramphenicol in glass tubes and cultured at 30 °C with shaking at 150 rpm for 48 h.Media with planktonic cells were collected for the measurement of OD600values.The surface-attached cells were stained with 0.1% CV.The bound CV was dissolved with 20% ethanol,and the OD570values were measured.The relative biofilm formation was calculated with the formula:100 × OD570/OD600.

Quantification of c-di-GMP

The intracellular c-di-GMP levels were measured similarly as previously described[22].Briefly,V.parahaemolyticuswas grown on HI plates at 37 °C for 6 h,and the bacterial cells were harvested by adding 2 mL pre-chilled PBS.Two OD600bacterial suspensions were centrifuged at 10,000×gfor 3 min at 4 °C.The cell pellet was washed twice with icecold PBS,resuspended in 2 mL ice-cold PBS,and then sonicated for 30 min (power 100%,frequency 37 kHz) in an ice-water bath.After centrifugation at 10,000×gfor 10 min at 4 °C,the supernatant containing extracted c-di-GMP was collected to determine the intracellular c-di-GMP concentration with a c-di-GMP enzyme-linked immunosorbent assay kit (Mskbio,Beijing,China).Total proteins in the cell extracts were also determined by a Micro BCA Protein Assay Kit (ThermoFisher Scientific,USA),in accordance with the manufacturer’s instructions.The intracellular c-di-GMP concentration was expressed as pmol/mg of protein.

Quantitative Real-time Polymerase Chain Reaction(qPCR)

The qPCR assay was performed as previously described[23].Briefly,the total RNAs were extracted from the WT andΔhnsstrains,and the cDNAs were generated from 1 μg total RNAs using the FastKing First-Strand cDNA Synthesis Kit (Tiangen Biotech,Beijing,China) following the manufacturer’s instructions.The relative mRNA levels of each target gene were determined using the classic 2-ΔΔCtmethod.

LacZ Fusion and β-galactosidase Assay

The promoter-proximal DNA region of each target gene was amplified and cloned into thepHRP309 vector containing a promoterlesslacZgene and a gentamicin resistance gene[24].After verification by DNA sequencing,the recombinant pHRP309 was transferred to the WT andΔhnsstrains,respectively.Thereafter,the transformants were cultured and lysed to measure the βgalactosidase activity in the cell extracts using the β-Galactosidase Enzyme Assay System (Promega,USA),in accordance with the manufacturer’s instructions.

The two-plasmid reporter assay inE.coliwas performed as previously described[25].Briefly,theE.coli100 λpir (Epicenter) bearing the indicatedhnsexpression plasmid pBAD33-hnsor the empty pBAD33 vector and a recombinantlacZplasmid was cultivated overnight in Luria-Bertani (LB) broth at 37 °C with shaking at 200 rpm.The overnight cultures were diluted 1:100 into 5 mL fresh LB broth containing 0.1%arabinose and 20 μg/mL chloramphenicol and incubated at 37 °C with shaking at 200 rpm to reach an OD600value of about 1.2.TheE.colicells were harvested and lysed to measure the β-galactosidase activity in the cell extracts.

Electrophoretic Mobility Shift Assay (EMSA)

The coding region ofhnswas amplified and cloned into the pET28a vector (Novagen,USA) and then transferred toE.coliBL21λDE3 to express 6 × Histagged H-NS (His-H-NS).The expression and purification of His-H-NS were performed as previously described[17].The dialyzed His-H-NS was concentrated to a final concentration of 1.4 mg/mL.EMSA was performed exactly as previously described[26].The results of EMSA were detected by ethidium bromide(EB) staining,and the gel images were displayed with an ultraviolet (UV) transilluminator.

Statistical Methods

The EMSA was performed at least twice with similar results.The CV staining,c-di-GMP quantification,LacZ fusion,and qPCR were performed at least thrice with three replications each,with values expressed as the means ± standard deviation (SD).Analysis of variance with the Tukey-Kramer test was applied to calculate statistical significance,withP＜0.01 considered significant.

H-NS Repressed Biofilm Formation by V.parahaemolyticus

We previously reported that H-NS activated the EPS-dependent bacterial colony morphology and the transcriptioncpsA-JinV.parahaemolyticusRIMD2210633[8].In this work,we investigated whether H-NS regulated biofilm formation byV.parahaemolyticusRIMD2210633 using the CV staining assay.As shown inFigure 1,the normalized CV staining byΔhns/pBAD33 significantly increased relative to that by WT/pBAD33,and C-Δhnsproduced a restored CV staining.This result indicated that H-NS repressed biofilm formation byV.parahaemolyticusRIMD2210633,as assessed by CV staining.

Mutation of hns Increased the Intracellular c-di-GMP Level

An increased c-di-GMP level leads to enhanced biofilm formation in bacteria[2].Thus,the increased CV staining byΔhnsprompted us to investigate whether H-NS affects the production of c-di-GMP inV.parahaemolyticusRIMD2210633.As shown inFigure 2,the intracellular c-di-GMP level was significantly enhanced inΔhnsrelative to that in WT,indicating that H-NS inhibited the synthesis of c-di-GMP inV.parahaemolyticusRIMD2210633.

Figure 1.H-NS repressed biofilm formation by V.parahaemolyticus.Biofilms of V.parahaemolyticus were measured by CV staining.*P ＜ 0.01.

H-NS Repressed the Transcription of a Set of Genes Associated with c-di-GMP Metabolism

The transcription of a group of genes encoding proteins with GGDEF and/or EAL domains,namely,scrABC,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979,were directly regulated by the QS master regulator OpaR[9].H-NS is also a transcriptional repressor ofscrABC[8],but whether H-NS can directly bindscrABCpromoters is still unknown.In this study,we investigated the regulatory mechanisms of H-NS on the transcription of these seven genes.The results of qPCR demonstrated that the mRNA levels of all of the seven genes significantly increased inΔhnsrelative to those in WT (Figure 3A),suggesting that their transcription was repressed by H-NS.The upstream DNA region of each of the seven genes was cloned into the pHRP309 plasmid containing a gentamicin resistance gene and a promoterlesslacZgene and then transferred intoΔhnsand WT to investigate the β-galactosidase activities in the cell extracts.As shown inFigure 3B,the promoter activities of all the seven genes were considerably higher inΔhnsthan those in WT,suggesting that the expressions of these genes were under the negative control of H-NS.Altogether,H-NS acted as a repressor ofscrABC,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979 inV.parahaemolyticusRIMD2210633.

H-NS Repressed the Expression of Target Genes ina Heterologous Host

To test whether H-NS can control the expressions of the seven target genes in a heterologous host,we expressed H-NS from the pBAD33 vector inE.coli100 λpir,which also contained alacZfusion reporter plasmid[25].As shown inFigure 4,after induction by the addition of 0.2% arabinose in the culture media,the promoter activities for all of the seven target genes in theE.coli100λpir containing the recombinant pBAD33-hnswere significantly lower than those inE.coli100λpir containing the empty pBAD33 vector.These results suggested that H-NS can bind the promoter DNA region of each of the seven target genes to repress their expression.

Figure 2.Intracellular c-di-GMP levels in V.parahaemolyticus.The data are expressed as the mean ± SD of at least three independent experiments.WT: wild-type.

Binding Activities of His-H-NS to the Promoter DNA Regions of Target Genes

The EMSA was employed to detect whether His-H-NS had binding activities to the regulatory DNA regions of the seven target genesin vitro.As shown inFigure 5,His-H-NS can bind the regulatory DNA regions of all of the seven target genes in dosedependent manners,but it cannot bind the DNA fragment of 16S rDNA,which was used as the negative control.These results further suggested that H-NS directly inhibited the transcription ofscrABC,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979 inV.parahaemolyticusRIMD2210633.

H-NS has been shown to be a positive regulator of biofilm formation byV.parahaemolyticusBB22 because the deletion ofhnsforms defective submerged biofilms relative to the WT strain[20].Thehnsmutant ofV.parahaemolyticusRIMD2210633 formed smooth colonies and expressed lowcpsA-J,indicating that H-NS may also act as a positive regulator of biofilm formation byV.parahaemolyticusRIMD2210633[8].However,the data on CV staining presented here showed that HNS repressed biofilm formation byV.parahaemolyticusRIMD2210633 (Figure 1).This result is not only contrary to the previous observations onV.parahaemolyticusBB22 but alsoV.parahaemolyticusRIMD2210633[8,20].The genetic information contained in the genomes of BB22 and RIMD2210633 are different,and RIMD2210633 contains many unique genes relative to BB22[27].Thus,the discrepancy between the data presented here and the previous conclusion regarding BB22 may be explained by these genetic differences.However,why the results of CV staining and colony morphology are contrary to each other in RIMD2210633 is difficult to understand.Colony morphology is mainly attributed to the EPS content,which is associated with the expression ofcpsA-J[3].However,a previous study showed that the biofilm formation by theV.parahaemolyticus trhpositive strain ATCC17802 was strongly correlated with extracellular DNA and protein content in the extracellular polymeric substances but not with the EPS content[28].Thus,similar observations may be obtained withV.parahaemolyticusRIMD2210633.Such a condition may be also due to the global influence of H-NS[16].Specifically,studies showed that H-NS inducedopaRexpression and polar flagellum production but repressed the transcription of lateral flagellar genes inV.parahaemolyticusRIMD2210633[19,29,30].Flagella-mediated motilities promote not only the initial adhesion of biofilm formation but also the active dispersal of biofilms[31];OpaR is an inhibitor of biofilm formation byV.parahaemolyticusTIMD2210633[9].OpaR may have some effects on the H-NS-mediated inhibition of biofilm formation inV.parahaemolyticusRIMD2210633.Although regulatory mechanisms may be different,the negative regulation of H-NS on biofilm formation has been confirmed inV.cholerae[32,33].

Figure 3.H-NS repressed the transcription of the seven genes associated with c-di-GMP metabolism in V.parahaemolyticus.The negative and positive numbers indicate the nucleotide positions upstream and downstream of indicated genes,respectively.(A) qPCR.After incubation at 37 °C for 6 h on HI plates,the bacterial cells were harvested,and total RNA was extracted using TRIzol Reagent.qPCR assay was used to determine the relative mRNA levels for target genes in Δhns and WT using the classical 2-ΔΔCt method.(B)LacZ fusion.The promoter DNA region of each target gene was cloned into the pHRP309 and then transferred into Δhns and WT to test the promoter activity in cellular extracts.

Figure 4.Two-plasmid reporter assay in E.coli.The E.coli 100λpir containing the pBAD33 plasmid or recombinant pBAD33-hns plasmid and a LacZ fusion reporter plasmid were grown in LB broth containing 0.2% arabinose until the mid-log phase (OD600=1.2).The bacterial cells were collected and assayed for lacZ expression using the β-galactosidase assay.The negative and positive numbers indicate the nucleotide positions upstream and downstream of indicated genes,respectively.

Figure 5.Binding of His-H-NS to the target DNA fragments.The regulatory DNA region of each target gene was incubated with increased amounts of His-H-NS and then subjected to 6% (w/v) polyacrylamide gel electrophoresis.The results were detected by EB staining and a UV transilluminator.A schematic representation of the EMSA design is shown below.The negative and positive numbers denote the nucleotide positions upstream and downstream of indicated genes,respectively.

The increased c-di-GMP content results in enhanced biofilm formation and motility inhibition[2].The data presented here showed that the deletion ofhnssignificantly increased the intracellular c-di-GMP level relative to that in the WT strain (Figure 2).The genome ofV.parahaemolyticusRIMD2210633 contains more than 50 genes encoding proteins that may be associated with the c-di-GMP metabolism[21],among which the transcriptions ofscrABC,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979 are directly regulated by the master QS regulator OpaR[9].Here,we showed that H-NS can bind the promoter-proximal DNA regions ofscrA,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979 to repress their transcription (Figures 3,4,and 5).Notably,OpaR showed the reverse regulation ofscrGand VP2979 compared with those of H-NS regulation[9];OpaR and H-NS exerted the same effects on c-di-GMP production.However,such findings should be easy to understand because the effect degrees of dozens of putative enzymes on intracellular c-di-GMP metabolism should be inconsistent under various genetic backgrounds (or growth conditions).Except forscrAandscrG,the functions of the other 5 target genes are completely unknown,and the data presented here showed that H-NS repression of the intracellular c-di-GMP levels inV.parahaemolyticusmay occurviathe direct regulation of the transcription of enzyme genes involved in the synthesis of c-di-GMP.The H-NS binding sites are usually long and overlap with the core -10 and -35 elements[17,19].Thus,H-NS is usually thought to repress gene transcription by directly interfering with the action of RNA polymerase.

Altogether,this work reported that H-NS was a negative regulator of the biofilm formation byV.parahaemolyticusRIMD2210633.H-NS directly repressed the transcription ofscrA,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979,which encode a group of GGDEF and/or EAL type proteins and thus decrease the intracellular c-di-GMP level.Therefore,H-NS repression of the biofilm formation byV.parahaemolyticusRIMD2210633 may occur via the repression of the production of intracellular c-di-GMP.However,the regulation mechanisms of H-NS on the biofilm formation byV.parahaemolyticusstill need to be further studied.

Received:December 24,2021;

Accepted:March 29,2022