Reciprocal Regulation between Fur and Two RyhB Homologs in Yersinia pestis, and Roles of RyhBs in Biofilm Formation＊

NI Bin, WU Hai Sheng, XIN You Quan, ZHANG Qing Wen,#, and ZHANG Yi Quan

1. School of Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, China; 2. Qinghai Institute for Endemic Disease Prevention and Control, Xining 811602, Qinghai, China; 3. Wuxi School of Medicine, Jiangnan University,Wuxi 214122, Jiangsu, China

Abstract Objective To investigate reciprocal regulation between Fur and two RyhB homologs in Yersinia pestis(Y. pestis), as well as the roles of RyhBs in biofilm formation.Methods Regulatory relationships were assessed by a combination of colony morphology assay,primer extension, electrophoretic mobility shift assay and DNase I footprinting.Results Fur bound to the promoter-proximal DNA regions of ryhB1 and ryhB2 to repress their transcription, while both RyhB1 and RyhB2 repressed the expression of Fur at the post-transcriptional level. In addition, both RyhB1 and RyhB2 positively regulated Y. pestis biofilm exopolysaccharide (EPS)production and the expression of hmsHFRS and hmsT.Conclusion Fur and the two RyhB homologs exert negative reciprocal regulation, and RyhB homologs have a positive regulatory effect on biofilm formation in Y. pestis.

Key words: Yersinia pestis; RyhB; Fur; Biofilm

INTRODUCTION

Yersinia pestis(Y. pestis) is the causative agent of the plague, which is a dangerous zoonotic disease that mainly circulates among reservoir animals and their fleas[1].Y. pestishas the ability to grow in the form of a biofilm in the flea's proventriculus, which blocks normal blood feeding and results in persistent starvation and feeding attempts, thereby promoting transmission of the plague among mammalian hosts[2].Y. pestisbiofilms are surfaced-associated bacterial communities enclosed by an extracellular matrix consisting primarily of a homopolymer of N-acetyl-Dglucosamine named exopolysaccharide (EPS)[3,4]. ThehmsHFRSoperon has been shown to be responsible for EPS production inY. pestis[5,6], and thehmsmutants failed to colonize the flea's proventriculus and to form biofilmsin vitro[7-9].

EPS production and biofilm formation are posttranscriptionally regulated by the intracellular concentration of c-di-GMP, a ubiquitous second messenger[10]. InY. pestis, c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP)by two diguanylate cyclases, HmsT and HmsD(encoded by y3730), and hydrolyzed to 5′-phosphoguanylyl-(3′-5′)-guanosine (pGpG) and/or guanosine monophosphate (GMP) by the phosphodiesterase HmsP[11-14]. Deletion ofhmsTsignificantly eliminatedin vitrobiofilm formation,but deletion ofhmsDhad a major negative effect onin vivobiofilm formation in fleas[12]. In contrast, thehmsPmutant formed hyperpigmented colonies and enhanced biofilm formation[12,14]. In addition,numerous transcriptional regulators have been identified that regulate biofilm formation inY. pestis,including major ones, such as the positive regulators CRP[15], CsrA[16], RovM[17], YfbA[18], and BfvR[19]and the negative regulators RcsB[20], Fur[21], and RovA[17].Overall, much has been learned about regulation ofY. pestisbiofilm formation, but its regulatory network still requires further investigation.

Small noncoding RNAs (sRNAs) control gene expression mostly by base pairing with their target mRNAs at the post-transcriptional level[22]. More than 100 sRNAs have been identified inY. pestisby using a cDNA cloning approach and RNA-seq technology, but their roles in gene regulation have not been well characterized[23-26]. RyhB, an Hfq-binding sRNA, plays a key role in bacterial iron homeostasis and is involved in regulating numerous cellular pathways, such as virulence, motility, and biofilm formation[27,28].Y. pestisencodes two RyhB homologs, named RyhB1 and RyhB2[29]. Stabilization of RyhB1 is mediated by Hfq,while RyhB2 does not require Hfq for stability[29].However, both RyhB1 and RyhB2 seem to be degraded by PNPase, because their expression levels increased significantly following PNPase inactivation[30]. Both RyhBs are upregulated in mouse lungs infected withY.pestis, suggesting that they are required for pathogen virulence[29]. However, the roles of RyhBs in other cellular pathways inY. pestisrequire further investigation. In addition, both RyhB homologs inY.pestishave been shown to be negatively regulated by the ferric uptake regulator (Fur)[29], but the detailed mechanisms also require further study. In the present study, we showed that Fur and RyhBs exerted reciprocal negative regulatory activity, and both RyhB homologs positively regulatedY. pestisbiofilm formation as well as the expression ofhmsHFRSandhmsT.

MAATERIALS AND METHODS

Bacterial Strains and Growth Conditions

Y. pestisbiovar microtus strain 201, an avirulent strain to humans, was used as the derivative (wild type,WT)[31]. NonpolarryhB1,ryhB2, andfursingle-gene deletion mutants derived from the WT strain, termedΔryhB1,ΔryhB2, andΔfur, respectively, were constructed previously using the λ-Red homologous recombination method[21,29,32]. Briefly, the entire coding regions offur,ryhB1, andryhB2were replaced with the kanamycin resistance cassette using the one-step inactivation method based on the lambda Red phage recombination system with the helper plasmid pKD46,which can express the highly efficient Red homologous recombination system. The polymerase chain reaction(PCR) fragment carrying the kanamycin resistance cassette flanked by regions homologous to thefur,ryhB1, orryhB2genes was introduced into the WT strain. The mutant strains were selected due to their kanamycin resistance and were verified by PCR and DNA sequencing.

For complementation of the mutants[21], a PCRgenerated DNA fragment comprising the corresponding coding region and transcriptional terminator was cloned into the pBAD33 vector,harboring a chloramphenicol resistance gene. Each recombinant plasmid was introduced into the corresponding mutant, yielding the complementation strain (termed C-ΔryhB1, C-ΔryhB2, and C-Δfur,respectively). All the primers used in this study are listed in Table 1.

Unless stated otherwise,Y. pestiswas cultivated in Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract, and 1% NaCl) or on LB agar plates[21]. Briefly,a single colony was inoculated on an LB agar plate an incubated for 1–2 d. The resultant bacterial cells were washed into LB broth to attain an OD620of approximately 1.5, and the resulting broth culture was stored in the presence of 30% glycerol at ?80 °C.Thereafter, 200 μL of bacterial glycerol stocks were inoculated into 18 mL of fresh LB broth and allowed to grow with shaking at 230 rpm to an OD620of approximately 0.4 prior to bacterial collection. When appropriate, the culture medium was supplemented with 34 μg/mL chloramphenicol.

Colony Morphology Assay

The colony morphology assay was performed as previously described[21]. Briefly, aliquots of 5 μL of bacterial glycerol stocks were spotted on an LB plate,followed by incubation for approximately 7 days.Thereafter, the surface morphology of each colony was recorded photographically.

Primer Extension Assay

The primer extension assay was performed essentially as previously described[21]. Briefly, total bacterial RNAs were extracted using TRIzol Reagent(Invitrogen), and then approximately 8 μg of total RNA was annealed with 1 pmol of 5′-32P-labeled reverse primer to generate cDNAs using the Primer Extension System (Promega) according to the manufacturer's instructions. The same labeled primer was used for sequencing with the AccuPower & Top DNA Sequencing Kit (Bioneer, Korea). The products of primer extension and sequencing were then analyzed by 6% polyacrylamide gel electrophoresis/8 M urea,and the results were detected by autoradiography using a Fuji Medical X-ray film.

Preparation of 6×His-tagged Fur (His-Fur) Protein

The entire coding region offurwas amplified,purified, and cloned into the pET28a vector(Novagen, USA), which was then verified by DNA sequencing.Escherichia coliBL21λDE3 were transformed with the recombinant plasmid encoding the His-Fur protein. Expression and purification of His-Fur protein was carried out as previously described[21]. The purified His-Fur protein was concentrated to a final concentration of approximately 0.2 mg/mL in storage buffer(phosphate-buffered saline, pH 7.5, 20% glycerol).The purity of the His-Fur protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified protein was stored at ?80 °C.

Table 1. Oligonucleotide primers used in this study

Electrophoretic Mobility Shift Assay (EMSA)

For the EMSA[21,33], the 5′-ends of the regulatory DNA regions of each target gene were labeled with[γ-32P]-ATP. EMSA was performed in a 10 μL reaction volume containing binding buffer [100 mmol/L MnCl2, 1 mmol/L MgCl2, 0.5 mmol/L DTT, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 7.5), 0.05 mg/mL sheared salmon sperm DNA, 0.05 mg/mL BSA, and 4% glycerol], labeled DNA probe (1,000–2,000 CPM/μL), and increasing quantities of His-Fur.Three controls were included: (1) cold probe as a specific DNA competitor (corresponding regulatory DNA region unlabeled), (2) negative probe as a nonspecific DNA competitor (the unlabeled coding region of 16S rRNA), and (3) nonspecific protein competitor (rabbit anti-F1-protein polyclonal antibodies). The binding products were analyzed in a native 4% (w/v) polyacrylamide gel, and the results were detected by autoradiography after exposure to Fuji Medical X-ray film.

DNase I Footprinting Assay

For the DNase I footprinting assay[21,33], the promoter-proximal DNA regions of each target gene with a single32P-labeled end were generated by PCR and purified using QiaQuick columns (Qiagen,Germany). DNA binding was performed in a 10 μL reaction volume containing the same binding buffer as for EMSA, labeled DNA fragment (2–5 pmol), and increasing quantities of His-Fur and incubated at room temperature for 30 min. Prior to digestion,10 μL of Ca2+/Mg2+solution (5 mmol/L CaCl2and 10 mmol/L MgCl2) was added to each reaction and incubated for 1 min at room temperature. Then, the optimized RQ1 RNase-Free DNase I (Promega) was added to each reaction mixture and then incubated at room temperature for 30–90 s. The cleavage reaction was quenched by adding 9 μL of stop solution(200 mmol/L NaCl, 30 mmol/L EDTA, and 1% SDS),followed by incubation for 1 min at room temperature. The partially digested DNA samples were extracted with phenol/chloroform, precipitated with ethanol, and analyzed on a 6% polyacrylamide/8 mol/L urea gel. Protected regions were identified by comparison with DNA sequencing size markers. The results were detected by autoradiography after exposure to Fuji Medical X-ray film.

RESULTS

Fur Directly Represses the Transcription of ryhB1 and ryhB2

The primer extension assay was employed to detect the transcriptional start sites of each target gene and to compare the yields of primer extension products in WT andΔfurAs shown in Figure 1A, the primer extension assay detected a single transcription start site for eachryhBhomologous gene, which were considered as the 5’-ends of RyhB1 and RyhB2[21], respectively. In addition, the yields of the primer extension products ofryhB1andryhB2significantly increased inΔfurrelative to those in WT, indicating that the transcription of bothryhB1andryhB2was repressed by Fur inY. pestis. The promoter-proximal DNA regions ofryhB1andryhB2were amplified, purified, radioactively labeled, and then subjected to EMSA with the purified His-Fur protein. As shown in Figure 1B, His-Fur was able to specifically bind to these DNA fragments in a dosedependent mannerin vitro. The DNase I footprinting assay further revealed that His-Fur protected a single DNA region within each of the promoter-proximal DNA regions ofryhB1andryhB2, located from ?37 to+6 and ?41 to +2 against DNase I digestion(Figure 1C). In short, Fur directly represses the transcription ofryhB1andryhB2inY. pestis.

The Two RyhB Homologs Repress the Expression of fur

The results of the primer extension assay showed that the mRNA levels offursignificantly increased in bothΔryhB1andΔryhB2relative to those in WT(Figure 2A), indicating that the expression offurwas repressed by the two RyhB homologs. The RyhB homologs regulate gene expression by base pairing with their target mRNAs[22,27]. Thus, the online IntaRNA program was applied to predict potential base pairing of the nucleotides of RyhB1 and RyhB2 with those offurmRNA. As shown in Figure 2B and 2C, both RyhB1 and RyhB2 might act on thefurmRNA with base pairing occurring at the deep coding sequences, and the region of RyhB1 predicted to hybridize to the target mRNA overlaps entirely with that of RyhB2. Therefore, the two RyhB homologs inhibitedfurexpression possibly by acceleratingfurmRNA cleavage catalyzed by RNase E[22,34].

The Two RyhB Homologs Affect the Colony Morphology of Y. pestis

The ability ofY. pestisstrains to synthesize biofilm exopolysaccharide was detected by the rugose colony morphology on the LB plate[21]. As shown in Figure 3, bothΔryhB1andΔryhB2developed much smoother colony morphology than that of WT, C-ΔryhB1, and C-ΔryhB2, while WT,C-ΔryhB1, and C-ΔryhB2produced similar colony morphology results. Moreover, the double gene mutantΔryhB1ΔryhB2produced with similar rugose morphology relative to that ofΔryhB1orΔryhB2,suggesting that RyhB1 and RyhB2 may have no cooperative activities when they were involved in regulating the biofilm formation. In short, both RyhB1 and RyhB2 acted as positive regulators of biofilm formation in Y. pestis.

Figure 1. Regulation of ryhB1 and ryhB2 by Fur. Bacterial cells were harvested at an OD600 value of approximately 0.4 to investigate Fur-mediated ryhB1 and ryhB2 transcription. The negative and positive numbers represent the nucleotide positions upstream and downstream of each target gene. (A) Primer extension. An oligonucleotide primer was designed to be complementary to the RNA transcript of each target gene. The primer extension products were analyzed using an 8 mol/L urea-6% acrylamide sequencing gel. The underlined bases were transcription start sites. (B) EMSA. The radioactively labeled promoter-proximal DNA fragments of each target gene were incubated with increasing amounts of His-Fur and then subjected to 4% (w/v) polyacrylamide gel electrophoresis. The EMSA design is shown below.(C) DNase I footprinting. Lanes G, A, T, and C represent the Sanger sequencing reactions. Labeled coding or noncoding DNA probes were incubated with increasing amounts of purified His-Fur (Lanes 1, 2, 3, and 4 contained 0, 5, 10, and 15 pmol, respectively), and were subjected to DNase I footprinting. The protected regions are indicated with vertical bars with the corresponding sequence positions.(D) Structural organization of the RyhBs promoters. Transcription start sites are marked with bent arrows.The ?10 and ?35 boxes are enclosed in boxes. The Fur sites are underlined with solid lines.

Figure 2. Regulation of fur by RyhB1 and RyhB2. (A) Primer extension was carried out as described in Figure 1. The transcription start site is indicated with arrows, with the corresponding nucleotide and sequence positions. (B and C) RyhB1 and RyhB2 exhibited complementarity with the coding region of fur mRNA. Complementary nucleotides are marked with vertical lines. The numbers flanking the fur or ryhB mRNA sequence refer to the nucleotide positions relative to the translation or transcription start sites(+1).

Figure 3. Bacterial colony morphology. Bacterial glycerol stocks were spotted on the LB plate and incubated at 26 °C for approximately 7 days.

The Two RyhB Homologs Have a Positive Regulatory Effect on hmsHFRS

The rugose colony morphology on the agar plate is due to the synthesis of abundant EPS[21]. The products of thehmsHFRSgene have been shown to be required for production of EPS inY. pestis[6]. Thus,regulation ofhmsHFRSby RyhB1 and RyhB2 was investigated in the present study. The primer extension assay detected two transcription start sites forhmsHFRS, which were located at 322 bp and 228 bp upstream of the translation start site,respectively, and their transcribed activities were under the positive control of RyhB1 and RyhB2(Figure 4A). In addition, we predicted, using the IntaRNA program, two independent base pairings occurring at the deep coding sequence of thehmsHFRSmRNA with the nucleotides of each of RyhB1 and RyhB2 (Figures 4B and 4C, respectively). A noncoding RNA that base pairs with its target mRNA at the deep coding sequence generally acts as a post-transcriptional repressor[22]. It is still unknown to us what mechanisms RyhBs adopt to activate the expression ofhmsHFRSinY. pestis.

The Two RyhB Homologs Have a Positive Regulatory Effect on hmsT

ThehmsTgene encodes a protein with the GGDEF domain and thus contributes to the c-di-GMP pool and biofilm formation inY. pestis[6,35]. The primer extension assay detected a single transcription start site forhmsT, located at 128 bp upstream of the translation start site, and its transcriptional activity was also under the positive control of RyhB1 and RyhB2 (Figure 5A). In addition,both RyhB1 and RyhB2 also might act on thehmsTmRNA, with base pairing occurring at the deep coding sequence (Figures 5B and 5C).

Figure 4. Regulation of hmsHFRS by RyhB1 and RyhB2. (A) Primer extension was carried out as described in Figure 1. The transcription start site is indicated with arrows, with the corresponding nucleotide and sequence position. (B and C) RyhB1 and RyhB2 exhibited complementarity with the coding region of hmsH mRNA. Complementary nucleotides are marked with vertical lines. The numbers flanking the hmsH or ryhB mRNA sequence refer to the nucleotide positions relative to the translation or transcription start sites (+1).

DISCUSSION

Fur-dependent transcription of RyhB has been demonstrated in some bacterial species, such asKlebsiella pneumoniaeandE. coli, in which Fur was shown to be a direct repressor of RyhB[27,36,37]. InY.pestis, transcription ofryhB1andryhB2is negatively regulated by Fur, as demonstrated by northern blot analysis, but the detailed mechanisms are lacking[29].In the present study, the data showed that Fur binds to the promoter-proximal DNA regions ofryhB1andryhB2to repress their transcription. The Fur binding site for eachryhB1andryhB2promoter overlaps the core ?10 and ?35 elements and the transcription start site (Figure 1D), and thus, Fur-mediatedryhB1andryhB2transcriptional repression would be via blocking the entry of the RNA polymerase.

Figure 5. Regulation of hmsT by RyhB1 and RyhB2. (A) Primer extension was carried out as described in Figure 1. The transcription start site is indicated with arrows, with the corresponding nucleotide and sequence position. (B and C) RyhB1 and RyhB2 exhibited complementarity with the coding region of hmsT mRNA. Complementary nucleotides are marked with vertical lines. The numbers flanking the hmsT or ryhB mRNA sequence refer to the nucleotide positions relative to the translation or transcription start sites (+1).

InE. coli, thefurtranscript comprises an open reading frame consisting of 28 codons, which is located immediately upstream of and overlaps with the 5’-coding region offur[38]. RyhB interacts with that open reading frame to post-transcriptionally inhibit the translation offur[38]. The data presented here showed that both RyhB1 and RyhB2 can repress the expression of Fur inY. pestis(Figure 2A).However, the interaction sequences of RyhB1 and RyhB2 with thefurmRNA is most likely to occur at the deep coding sequence of thefurgene (Figure 2B and 2C). Thus, diverse regulatory mechanisms may be adopted by RyhB homologs to posttranscriptionally inhibitfurexpression in different bacterial species.

Fur has been shown to be a repressor ofY. pestisbiofilm formation and c-di-GMP production[21].Therefore, the RyhB1- and RyhB2-mediated repression offurexpression indicated that the two RyhB homologs might also be involved in regulating biofilm formation inY. pestis. Indeed, the data presented here showed that single or double gene mutants ofryhB1andryhB2produced smooth colony morphology, while the WT and complementary mutants produced wrinkled colony morphology (Figure 3). Moreover, the RyhB homologs stimulated expression ofhmsHFRS(Figure 4) andhmsT(Figure 5), both of which promote biofilm formation inY. pestis[5,6,12,13].

Y. pestis ryhBRNAs were estimated to be approximately 110 nt long, which is slightly longer thanE. coli ryhBRNA (90 nt) but shorter thanV.cholerae ryhBRNA (> 200 nt)[29,36,39].V. choleraeRyhB has been shown to be involved in regulating the expression of many genes that are not regulated by RyhB inE. coli, including genes involved in biofilm formation, flagellar biosynthesis, and chemotaxis[39].TheV. cholerae ryhBmutant exhibited reduced chemotactic motility and biofilm formation in lowiron medium, but the capacity for biofilm formation was restored by growing the mutant in the presence of excess iron or succinate[39]. Similarly, the presented data showed that theryhBmutants exhibited reduced production of EPS and lower expression of genes involved in biofilm formation. LB is likely already iron-replete; nevertheless, this is the first report of the sRNA RyhB regulating biofilm formation inYersinia.

In summary, the data presented here show that Fur and RyhBs exhibit reciprocal negative regulation of expression, and the RyhB homologs promoteY.pestisbiofilm formation, probably via activation of the expression ofhmsTandhmsHFRS. However,whether the RyhB homologs activateY. pestisbiofilm formationviaregulation of genes other thanhmsTandhmsHFRSrequires further characterization.

Received: August 7, 2020;

Accepted: December 4, 2020