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NI Bin, WU Hai Sheng, XIN You Quan, ZHANG Qing Wen, ZHANG Yi Quan. Reciprocal Regulation between Fur and Two RyhB Homologs in Yersinia pestis, and Roles of RyhBs in Biofilm Formation[J]. Biomedical and Environmental Sciences, 2021, 34(4): 299-308. doi: 10.3967/bes2021.039
Citation: NI Bin, WU Hai Sheng, XIN You Quan, ZHANG Qing Wen, ZHANG Yi Quan. Reciprocal Regulation between Fur and Two RyhB Homologs in Yersinia pestis, and Roles of RyhBs in Biofilm Formation[J]. Biomedical and Environmental Sciences, 2021, 34(4): 299-308. doi: 10.3967/bes2021.039

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

doi: 10.3967/bes2021.039
Funds:  This work was supported by the Basic Application Research Project of Science and Technology Department of Qinghai province [2020-ZJ-788]; the Key Laboratory of National Health Commission on Plague Control and Prevention [2019PT310004]; the Key Laboratory for Plague Prevention and Control of Qinghai Province [p2020-ZJ-Y23]; and the Qinghai Province High-end Innovative Talents Thousand Talents Program (2019)
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  • Author Bio:

    NI Bin, male, born in 1978, Doctor, majoring in gene regulation of pathogenic bacteria

    WU Hai Sheng, male, born in 1982, Assistant Researcher, majoring in plague and other infectious disease prevention and control

  • Corresponding author: ZHANG Yi Quan, E-mail: zhangyiquanq@163.com; ZHANG Qing Wen, E-mail: 13519783993@126.com
  • &These authors contributed equally to this work.
  • Received Date: 2020-08-07
  • Accepted Date: 2020-12-04
  •   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.
  • &These authors contributed equally to this work.
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Reciprocal Regulation between Fur and Two RyhB Homologs in Yersinia pestis, and Roles of RyhBs in Biofilm Formation

doi: 10.3967/bes2021.039
Funds:  This work was supported by the Basic Application Research Project of Science and Technology Department of Qinghai province [2020-ZJ-788]; the Key Laboratory of National Health Commission on Plague Control and Prevention [2019PT310004]; the Key Laboratory for Plague Prevention and Control of Qinghai Province [p2020-ZJ-Y23]; and the Qinghai Province High-end Innovative Talents Thousand Talents Program (2019)
  • Author Bio:

  • Corresponding author: ZHANG Yi Quan, E-mail: zhangyiquanq@163.com ZHANG Qing Wen, E-mail: 13519783993@126.com
  • &These authors contributed equally to this work.

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.

&These authors contributed equally to this work.
NI Bin, WU Hai Sheng, XIN You Quan, ZHANG Qing Wen, ZHANG Yi Quan. Reciprocal Regulation between Fur and Two RyhB Homologs in Yersinia pestis, and Roles of RyhBs in Biofilm Formation[J]. Biomedical and Environmental Sciences, 2021, 34(4): 299-308. doi: 10.3967/bes2021.039
Citation: NI Bin, WU Hai Sheng, XIN You Quan, ZHANG Qing Wen, ZHANG Yi Quan. Reciprocal Regulation between Fur and Two RyhB Homologs in Yersinia pestis, and Roles of RyhBs in Biofilm Formation[J]. Biomedical and Environmental Sciences, 2021, 34(4): 299-308. doi: 10.3967/bes2021.039
    • 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. pestis has 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. pestis biofilms are surfaced-associated bacterial communities enclosed by an extracellular matrix consisting primarily of a homopolymer of N-acetyl-D-glucosamine named exopolysaccharide (EPS)[3, 4]. The hmsHFRS operon has been shown to be responsible for EPS production in Y. pestis[5, 6], and the hms mutants failed to colonize the flea's proventriculus and to form biofilms in vitro[7-9].

      EPS production and biofilm formation are post-transcriptionally regulated by the intracellular concentration of c-di-GMP, a ubiquitous second messenger[10]. In Y. 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 of hmsT significantly eliminated in vitro biofilm formation, but deletion of hmsD had a major negative effect on in vivo biofilm formation in fleas[12]. In contrast, the hmsP mutant formed hyperpigmented colonies and enhanced biofilm formation[12, 14]. In addition, numerous transcriptional regulators have been identified that regulate biofilm formation in Y. 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 of Y. pestis biofilm 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 in Y. pestis by 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. pestis encodes 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 with Y. pestis, suggesting that they are required for pathogen virulence[29]. However, the roles of RyhBs in other cellular pathways in Y. pestis require further investigation. In addition, both RyhB homologs in Y. pestis have 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 regulated Y. pestis biofilm formation as well as the expression of hmsHFRS and hmsT.

    • Y. pestis biovar microtus strain 201, an avirulent strain to humans, was used as the derivative (wild type, WT)[31]. Nonpolar ryhB1, ryhB2, and fur single-gene deletion mutants derived from the WT strain, termed ΔryhB1, ΔryhB2, and Δfur, respectively, were constructed previously using the λ-Red homologous recombination method[21, 29, 32]. Briefly, the entire coding regions of fur, ryhB1, and ryhB2 were 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 the fur, ryhB1, or ryhB2 genes 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 PCR-generated 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.

      TargetPrimers (forward/reverse, 5’-3’)
      Construction of mutants
       furCAGCCTTAATTTGAATCGATTGTAACAGGACTGAATCCGCTGTAACGCACTGAGAAGC/
      GTGCTTAAAATCTTTATAAGAGTAATGCGATAAAACGATAAGATTGCAGCATTACACG
       ryhB1CATATTCCCCCTGAGTCAAAT/CGTGTAATGCTGCAATCTGGCAATGATAATCATTATCAC
      GCTTCTCAGTGCGTTACATTTGCCTTTTTCTCACCCCGTTC/GGTAAATCAACTTAATCCGAGAG
       ryhB2GGCGTAAACCAGTCGGTAGTCT/CGTGTAATGCTGCAATCTAAAATGATAATACTTATCAATAT
      GCTTCTCAGTGCGTTACAGTGCCCAGAAAACCCCCAGC/TTCCGGTGAGTGAGTACAGC
      Complementation of the mutants
       ryhB1CACGAATTCTGCTTTCAGATGAGCGCATCAAAAGTTTAGGTG/TTGAAGCTTAAAAAAGCCAGCACCCGGCTGGCTAAGTAAAC
       ryhB2CACGAATLTCTGCGATTCAGAACAAGGCAGGCAGTCTTTGG/TTGAAGCTTAAAAAAGCCAGCACCCGAGCTGGCTTAAAATAC
      Protein expression
       furGCGGGATCCATGACTGACAACAACAAAG/GCGAAGCTTTTATCTTTTACTGTGTGCAGA
      Primer extension
       fur/CCAAATGAAAACGGTGGTTG
       ryhB1/CCGGCTGGCTAAGTAAACAC
       ryhB2/GCTTTACTGAACCCCCAGCC
       hmsT/GGTATTTATTCCGACATCACGAC
       hmsH/TATTGTTGCAAAGTCATTATAGGAT
      EMSA
       ryhB1ATCCCAGGACAGGTTCTCTC/CCGGCTGGCTAAGTAAACAC
       ryhB2GCACCGCCTGATTATTCATCG/CACCCGAGCTGGCTTAAAATAC
       furCTGAGTATTTCTGTGATGCGATG/CTGACGTGGTGACACGCAGG
      DNase I footprinting
       ryhB1ATCCCAGGACAGGTTCTCTC/CCGGCTGGCTAAGTAAACAC
       ryhB2GCACCGCCTGATTATTCATCG/CACCCGAGCTGGCTTAAAATAC

      Table 1.  Oligonucleotide primers used in this study

      Unless stated otherwise, Y. pestis was 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 OD620 of 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 OD620 of approximately 0.4 prior to bacterial collection. When appropriate, the culture medium was supplemented with 34 μg/mL chloramphenicol.

    • 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.

    • 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.

    • The entire coding region of fur was amplified, purified, and cloned into the pET28a vector (Novagen, USA), which was then verified by DNA sequencing. Escherichia coli BL21λDE3 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.

    • 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.

    • For the DNase I footprinting assay[21, 33], the promoter-proximal DNA regions of each target gene with a single 32P-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 CaCl2 and 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.

    • 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 and ΔfurAs shown in Figure 1A, the primer extension assay detected a single transcription start site for each ryhB homologous gene, which were considered as the 5’-ends of RyhB1 and RyhB2[21], respectively. In addition, the yields of the primer extension products of ryhB1 and ryhB2 significantly increased in Δfur relative to those in WT, indicating that the transcription of both ryhB1 and ryhB2 was repressed by Fur in Y. pestis. The promoter-proximal DNA regions of ryhB1 and ryhB2 were 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 dose-dependent manner in vitro. The DNase I footprinting assay further revealed that His-Fur protected a single DNA region within each of the promoter-proximal DNA regions of ryhB1 and ryhB2, located from −37 to +6 and −41 to +2 against DNase I digestion (Figure 1C). In short, Fur directly represses the transcription of ryhB1 and ryhB2 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.

    • The results of the primer extension assay showed that the mRNA levels of fur significantly increased in both ΔryhB1 and ΔryhB2 relative to those in WT (Figure 2A), indicating that the expression of fur was 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 of fur mRNA. As shown in Figure 2B and 2C, both RyhB1 and RyhB2 might act on the fur mRNA 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 inhibited fur expression possibly by accelerating fur mRNA cleavage catalyzed by RNase E[22, 34].

      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).

    • The ability of Y. pestis strains to synthesize biofilm exopolysaccharide was detected by the rugose colony morphology on the LB plate[21]. As shown in Figure 3, both ΔryhB1 and ΔryhB2 developed much smoother colony morphology than that of WT, C-ΔryhB1, and C-ΔryhB2, while WT, C-ΔryhB1, and C-ΔryhB2 produced similar colony morphology results. Moreover, the double gene mutant ΔryhB1ΔryhB2 produced with similar rugose morphology relative to that of ΔryhB1 or ΔryhB2, 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 3.  Bacterial colony morphology. Bacterial glycerol stocks were spotted on the LB plate and incubated at 26 °C for approximately 7 days.

    • The rugose colony morphology on the agar plate is due to the synthesis of abundant EPS[21]. The products of the hmsHFRS gene have been shown to be required for production of EPS in Y. pestis[6]. Thus, regulation of hmsHFRS by RyhB1 and RyhB2 was investigated in the present study. The primer extension assay detected two transcription start sites for hmsHFRS, 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 the hmsHFRS mRNA 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 of hmsHFRS in Y. pestis.

      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).

    • The hmsT gene encodes a protein with the GGDEF domain and thus contributes to the c-di-GMP pool and biofilm formation in Y. pestis[6, 35]. The primer extension assay detected a single transcription start site for hmsT, 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 the hmsT mRNA, with base pairing occurring at the deep coding sequence (Figures 5B and 5C).

      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).

    • Fur-dependent transcription of RyhB has been demonstrated in some bacterial species, such as Klebsiella pneumoniae and E. coli, in which Fur was shown to be a direct repressor of RyhB[27, 36, 37]. In Y. pestis, transcription of ryhB1 and ryhB2 is 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 of ryhB1 and ryhB2 to repress their transcription. The Fur binding site for each ryhB1 and ryhB2 promoter overlaps the core −10 and −35 elements and the transcription start site (Figure 1D), and thus, Fur-mediated ryhB1 and ryhB2 transcriptional repression would be via blocking the entry of the RNA polymerase.

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

      Fur has been shown to be a repressor of Y. pestis biofilm formation and c-di-GMP production[21]. Therefore, the RyhB1- and RyhB2-mediated repression of fur expression indicated that the two RyhB homologs might also be involved in regulating biofilm formation in Y. pestis. Indeed, the data presented here showed that single or double gene mutants of ryhB1 and ryhB2 produced smooth colony morphology, while the WT and complementary mutants produced wrinkled colony morphology (Figure 3). Moreover, the RyhB homologs stimulated expression of hmsHFRS (Figure 4) and hmsT (Figure 5), both of which promote biofilm formation in Y. pestis[5, 6, 12, 13].

      Y. pestis ryhB RNAs were estimated to be approximately 110 nt long, which is slightly longer than E. coli ryhB RNA (90 nt) but shorter than V. cholerae ryhB RNA (> 200 nt)[29, 36, 39]. V. cholerae RyhB has been shown to be involved in regulating the expression of many genes that are not regulated by RyhB in E. coli, including genes involved in biofilm formation, flagellar biosynthesis, and chemotaxis[39]. The V. cholerae ryhB mutant exhibited reduced chemotactic motility and biofilm formation in low-iron 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 the ryhB mutants 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 in Yersinia.

      In summary, the data presented here show that Fur and RyhBs exhibit reciprocal negative regulation of expression, and the RyhB homologs promote Y. pestis biofilm formation, probably via activation of the expression of hmsT and hmsHFRS. However, whether the RyhB homologs activate Y. pestis biofilm formation via regulation of genes other than hmsT and hmsHFRS requires further characterization.

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