Abstract
Roles for the non-coding small RNA RyhB in quorum-sensing and iron-dependent gene modulation in the human pathogen V. vulnificus were assessed in this study. Both the quorum sensing master regulator SmcR and the Fur-iron complex were observed to bind to the region upstream of the non-coding small RNA RyhB gene to repress expression, which suggests that RyhB is associated with both quorum-sensing and iron-dependent signaling in this pathogen. We found that expression of LuxS, which is responsible for the biosynthesis of autoinducer-2 (AI-2), was higher in wild type than in a ryhB-deletion isotype. RyhB binds directly to the 5′-UTR (untranslated region) of the luxS transcript to form a heteroduplex, which not only stabilizes luxS mRNA but also disrupts the secondary structure that normally obscures the translational start codon and thereby allows translation of LuxS to begin. The binding of RyhB to luxS mRNA requires the chaperone protein Hfq, which stabilizes RyhB. These results demonstrate that the small RNA RyhB is a key element associated with feedback control of AI-2 production, and that it inhibits quorum-sensing signaling in an iron-dependent manner. This study, taken together with previous studies, shows that iron availability and cell density signals are funneled to SmcR and RyhB, and that these regulators coordinate cognate signal pathways that result in the proper balance of protein expression in response to environmental conditions.
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Introduction
Bacterial pathogens are exposed to a variety of stressors either in the natural environment or in the host environment during the infection process, such as nutrient restrictions, temperature changes, osmotic stress, and oxidative stress1. These organisms have evolved sophisticated mechanisms to control gene expression in these diverse environments by sensing relevant environmental factors and rapidly adapting to improve both survival and pathogenicity. It is well known that iron plays an important role in regulating virulence factors in pathogenic bacteria2. Fur, ferric uptake regulator, is a well-studied regulatory factor that controls the expression of numerous genes in association with iron3,4. Similar examples are documented for V. vulnificus: Fur increases the expression of heme receptors in the presence of heme5, Fur represses genes encoding the biosynthesis and uptake of the siderophore vulnibactin6, and Fur suppresses quorum-sensing by modulating both the master regulator smcR and Qrrs, the quorum-sensing regulatory small RNAs7,8.
Bacterial cell density is another important factor influencing a wide range of cellular activities including the virulence of pathogenic bacteria. Regulation in response to cell density is achieved through a quorum-sensing pathway that monitors the accumulation of small diffusible signaling molecules produced by cognate cells in a closed space at high cell density, and this subsequently influences the expression of genes related to numerous functions such as survival, biofilm formation, and virulence9,10,11. The quorum-sensing pathway in V. vulnificus is similar to that of Vibrio harveyi and Vibrio cholerae. V. vulnificus harbors a homolog of V. harveyi luxS, which encodes an enzyme for the biosynthesis of the autoinducer-2 signaling molecule12,13. The genome sequence of the well-studied V. vulnificus strain MO6-24/O (GenBank accession number CP002469.1 for chromosome I and CP002470.1 for chromosome II)14 revealed that there is no biosynthetic gene for autoinducer-1 (AI-1; homoserine lactone molecule) or cholera autoinducer-1 (CAI-1). However, LuxPQ, the cognate receptor for autoinducer-2 (AI-2) and homologues of LuxU and LuxO involved in a phospho-relay in V. harveyi15,16,17,18,19 have been identified. The AI-2 signal converges to LuxO, a nitrogen regulatory protein (NtrC) homolog, and is transduced to the master regulator SmcR20,21,22. In addition, this pathogen harbors yet another quorum-sensing pathway that is mediated by cyclic dipeptide23,24,25.
RyhB, a non-translated small RNA, was first identified as important for iron metabolism in E. coli26. Since that time, genes homologous to RyhB have been found and characterized in the context of the iron regulon in many other bacteria such as V. cholerae, Salmonella typhimurium, Yersinia pestis, and Shigella dysenteriae27,28,29,30. Transcription is negatively regulated by Fur in the presence of iron. Under iron-limiting conditions, this repression is relieved and RyhB then represses genes encoding iron-containing proteins, such as superoxide dismutase and succinate dehydrogenase26,31. In addition, RyhB also activates the expression of shiA, a gene encoding shikimate permease, which is associated with siderophore synthesis, by directly pairing with the 5′-untranslated region of the shiA mRNA in E. coli32. RyhB also translationally down-regulates fur expression33. In V. cholerae, a ryhB mutant showed decreased mobility and poor biofilm formation compared to wild type27. RyhB represses SodB expression by pairing with the 5′-untranslated region of the sodB mRNA and causing the coupled degradation induced by RNaseE34,35. The sRNA chaperone Hfq is essential for this process as it binds and protects RyhB from RNase E degradation, thereby extending its half-life34,36,37,38.
Iron is essential for the growth of living organisms, but low solubility in the environment of a living cell makes this element a limiting factor for growth. High cell density leads to an even more severe iron stringency. Meanwhile, when intracellular iron is in excess, radicals that compromise the viability of cells are generated39. Therefore, it is expected that cell density and iron availability are intertwined and that these factors work together to control the expression of related genes. Here we report a new connection between quorum-sensing and the iron stress response mediated by the small non-coding RNA RyhB in V. vulnificus. The results of this study provide evidence for a close relationship between iron levels and cell density in terms of gene regulation and also add to the increasingly long list of roles for non-translated small RNA in pathogenic bacteria.
Results
Identification of the transcription start site of RyhB and evidence for Hfq-dependent stabilization of the RNA
Our previous study showing the repression of smcR by the Fur-iron complex in V. vulnificus7 led us to further investigate the relationship between quorum-sensing and iron in this pathogen. In both E. coli and V. cholerae, the Fur-iron complex represses ryhB, a gene encoding a small RNA26,27,28. Therefore we speculated that if any small RNAs affect quorum-sensing in an iron-dependent manner in V. vulnificus, the most likely candidate would be RyhB. A search of the genome sequence of V. vulnificus revealed a 224-bp gene with 56% similarity to the ryhB of V. cholerae. This gene mapped between the genes encoding DNA polymerase I (GenBank accession number VVMO6_02895) and porphobilinogen (VVMO6_02896). The transcription start site was identified through primer extension experiments and typical − 10 and − 35 consensus sequences also were observed to be present (Supplementary Fig. S1). The chaperone Hfq is required to stabilize RyhB in E. coli35. Therefore, we looked for a similar role in V. vulnificus and compared levels of the RyhB transcript in wild type and a Δhfq mutant at several time points after rifampicin was added to pause transcription. As shown in Fig. 1, RyhB from wild type is still detected up to 30 min after rifampicin treatment with a half-life of about 30.7 min. On the other hand, in the hfq mutant RyhB was degraded quickly with a half-life of about 7.5 min, and was barely detectable at 15 min following the rifampicin treatment (Fig. 1).
Hfq stabilizes the transcript of ryhB. V. vulnificus MO6 and hfq mutant (Δhfq) were cultured in LB broth for 3 h, at which point 200 μM of 2, 2′-dipyridyl was added and incubation continued for another hour. Samples were then treated with 250 g/ml of rifampicin and collected at time intervals for RNA quantification. Intensities of bands were quantitatively measured using a densitometer and the results are shown as a graph in the lower panel.
Fur represses RyhB expression in the presence of iron
We assessed the expression levels of ryhB under different iron conditions. RNA was extracted from V. vulnificus grown in either iron rich medium (LB broth with or without 25 μM of FeSO4) or under iron limiting conditions (LB broth with 200 μM of 2, 2′-dipyridyl) (Fig. 2a). In wild type MO6-24/O cells, the ryhB transcript is barely detectable in cells grown in LB broth with or without 25 μM of FeSO4, while an abundance of transcript was detected when the iron chelator 2, 2′-dipyridyl was added. In contrast, a fur-deletion mutant (Δfur) had high amounts of the RyhB transcript regardless of iron concentrations. These results suggest that iron strongly represses the transcription of ryhB and that Fur is necessary for this repression.
Expression of RyhB is dependent on Fur and iron concentrations. (a) Primer extensions were carried out to assess the level of RyhB expression with and without Fur and iron. Wild type MO6 and a fur mutant were cultured in LB supplemented with either 25 μM of FeSO4 or 200 μM 2, 2′-dipyridyl. After RNA extraction, the RyhB transcript was quantified by primer extension using primer RyhB-PE (Supplementary Table S2). Sequencing ladders generated by the same primer are included. (b) Gel shift assay of a 32P-labeled DNA fragment of the region upstream of ryhB with increasing amounts of Fur in the presence of the divalent cation MnSO4. Lanes 1 through 5 are Fur concentrations of 0, 100, 200, 400, and 600 nM, respectively. Lanes 6 and 7 include 600 nM of Fur incubated with probe in the presence of either 260 ng or 720 ng of non-labeled probe, respectively. (c) Gel shift assay of 32P-labeled ryhB probe with increasing amounts of Fur in the presence of 10 mM EDTA. Lanes 1 through 5 are Fur concentration of 0, 100, 200, 400, and 600 nM, respectively. Lane 6 includes 600 nM of Fur without EDTA, and lane 7 includes 600 nM of Fur and MnSO4. In figs. 2b and c, the upper bands are to be labeled with ‘Fur-probe complex’ and the lower bands with ‘Free probe’ just like in Fig. 4a.
Fur regulates gene expression in response to environmental iron conditions by binding to a specific site called the ‘Fur box’ in the promoter region of target genes6,40,41. To verify that Fur regulates ryhB expression in a similar manner, a gel-mobility shift assay was performed using a DNA fragment containing the region upstream to ryhB and purified Fur protein in the presence of the divalent ion Mn2+ (Fig. 2b). Fur specifically bound to this DNA sequence in the presence MnSO4 and binding was abolished if EDTA was added to sequester the divalent ion (Fig. 2c). The binding site for Fur within the ryhB promoter region was defined through a DNase I footprinting assay in the presence of 100 μM MnSO4 (Supplementary Fig. S2). The result showed that Fur protected a region from − 64 to + 6 with respect to the transcription start site, covering the consensus − 10 and − 35 promoter sequences (Supplementary Fig. S3).
SmcR represses transcription of ryhB by binding to the promoter region
Our previous study showed that regulation of vvsAB, which encodes a biosynthetic enzyme for the siderophore vulnibactin, is coordinately regulated by both iron and quorum-sensing6. Furthermore, expression of the qrr genes, encoding small RNAs associated with quorum-sensing modulation9, also are regulated by iron8. These results point to a connection between RyhB and quorum-sensing regulation and that these are somehow associated with iron levels. In support of this prediction, analysis of the nucleotide sequences upstream of ryhB revealed similarities to the consensus binding motif for the quorum-sensing master regulator SmcR42 (Supplementary Fig. S3).
To verify this, we measured the expression of ryhB using a luxAB transcriptional fusion as a reporter in the following strains: wild type MO6-24/O; a mutant with a deletion in luxO (ΔluxO) such that it lacks the cytoplasmic signal transducer that degrades SmcR at low cell density via Qrr and Hfq8; an smcR-deletion mutant (ΔsmcR); and a luxOsmcR double mutant (ΔluxOΔsmcR). In both ΔsmcR and ΔluxOΔsmcR, transcription of ryhB was significantly higher than that of wild type at early stationary phase (A600 ≅ 1.0) (Fig. 3). Conversely, in a ΔluxO mutant, transcription was just half that of wild type. These results indicated that SmcR represses ryhB expression at the transcriptional level. A DNA fragment containing the upstream region of ryhB was incubated with increasing amounts of purified SmcR and analyzed in a gel shift assay (Fig. 4a). As expected, SmcR bound directly to the promoter region of ryhB. DNase I footprinting identified the binding region as − 27 to + 1 with respect to the transcriptional start site, including a putative − 10 promoter sequence (Fig. 4b and Supplementary Fig. S3). These results indicate that SmcR represses ryhB transcription by directly binding to a cis-element upstream of ryhB to prevent the binding of RNA polymerase.
The quorum sensing master regulator SmcR represses the expression of ryhB. Quantitative analysis of ryhB transcription levels using luxAB as reporter genes. Luminescence activity representing the level of ryhB transcription was compared at early stationary phase of growth (A600 ≅ 1.0) for V. vulnificus MO6-24/O, ΔluxO, ΔsmcR, and ΔluxOΔsmcR harboring pHK-ryhB. Relative light units (RLU) were normalized to cell density (luminescence/A600). Values are averages from three independent experiments, and error bars denote standard deviations. The p-values for comparison with MO6-24/O are indicated (Student’s t-test; *, 0.005 ≤ P < 0.05).
SmcR binds directly to the upstream region of ryhB. (a) Gel mobility shift assay of purified SmcR binding to a DNA fragment of the upstream region of ryhB. Lanes 1 to 6 represent 20 ng of 32P-labeled ryhB probe incubated with 0, 50, 100, 150, 200, and 300 nM of SmcR, respectively. Lanes 5 and 6 represent 20 ng of the labeled ryhB probe incubated with 250 nM of SmcR with the addition of either 260 ng or 780 ng of unlabeled probe as a competitor, respectively. (b) DNase I footprinting to identify the SmcR binding site in the region upstream of ryhB. 32P-labeled ryhB probe (100 ng) with SmcR at 0, 62.5, 125, 250, 500 and 1000 nM is included in lanes 1 to 5, respectively. Sequencing ladders were included for comparison.
RyhB promotes the production of AI-2 in V. vulnificus
The results described above suggested that RyhB is involved in the iron-dependent regulation of the quorum-sensing pathway in V. vulnificus. To examine this further, we compared the transcription of genes related to the quorum-sensing pathway in wild-type and a ryhB-deletion mutant using transcriptome analysis. Expression levels of luxPQUO and SmcR were not significantly different in the two isotypes. However, the expression of luxS, encoding an AI-2 biosynthetic enzyme, was approximately 40% lower in a ΔryhB mutant than in wild type (Supplementary Table S3). To verify this result, we compared both AI-2 production and LuxS expression in wild type and ΔryhB mutant backgrounds. Supernatants from each of the two V. vulnificus strains cultured in AB minimal medium with a low iron concentration were assessed for the production of AI-2 using the bio-indicator V. harveyi BB17043 and measuring luminescence. The luminescence induced by the wild type culture supernatant was significantly higher than that of ΔryhB, and introduction of an exogenous ryhB-expressing clone into ΔryhB in trans restored luminescence to wild type levels, whereas introduction of vector alone did not (Fig. 5a). These results indicate that RyhB promotes expression of LuxS under iron-limiting conditions. We then assessed the effects of RyhB on the expression of a gene normally regulated by the AI-2 quorum-sensing system. We quantitatively measured the expression of vvpE, which encodes elastase, a virulence factor that is positively regulated by quorum sensing7,44. Iron repressed vvpE expression regardless of RyhB. However, in the absence of iron, expression in the ΔryhB mutant was significantly lower than in wild type (Fig. 5b). This result supports the idea that enhanced production of AI-2 by RyhB promotes quorum-sensing signaling.
RyhB promotes AI-2 production and vvpE expression. (a) Effects of RyhB on AI-2 production were measured using the AI-2 indicator V. harveyi strain BB170. V. vulnificus strains wild type MO6-24/O, ΔryhB, ΔryhB with a ryhB clone (ΔryhB pBBR12-ryhB), Δhfq, Δhfq with a hfq clone (Δhfq pBBR12-hfq), and ΔluxS were cultured in AB minimal medium and supernatants were collected to measure AI-2 production. (b) Effects of RyhB on the expression of vvpE. vvpE expression was measured using a transcriptional fusion (pHK-vvpE) in either V. vulnificus wild type or the ΔryhB mutant. V. vulnificus was cultured in LBS medium supplemented with 100 μM of 2, 2′-dipyridyl to A600 of about 0.1. Samples were collected at late stationary phase (A600 ≅ 2.5) and both cell density and luminescence were measured. Relative light units (RLU) were normalized to cell density (luminescence/A600). Values are an average of three independent experiments and error bars denote the standard deviations. The p-values for comparison with MO6-24/O (without iron) are indicated (Student’s t-test; *, 0.005 ≤ P < 0.05; **, P < 0.005).
In summary, these results indicate that RyhB increases AI-2 mediated quorum-sensing signaling by enhancing the production of AI-2 itself, and that SmcR represses RyhB as a mechanism for feedback inhibition to repress AI-2 production. The presence of iron, as part of the Fur-iron complex, down-regulates overall quorum-sensing signaling by inhibiting the transcription of both SmcR and RyhB.
RyhB delays the decay of luxS mRNA by binding directly to the 5′-UTR of LuxS
The next question was how RyhB enhances the production of AI-2 at the molecular level. The non-coding sRNA RyhB post-transcriptionally regulates gene expression by affecting the turnover of mRNAs in E. coli26. We assumed that RyhB would affect the luxS mRNA in the same manner. To test this, levels of the luxS transcript were measured over time after cells were treated with rifampicin. As shown in Fig. 6, luxS mRNA degraded faster in ΔryhB than in wild type. Previous studies reported that Hfq is required for the functioning of RyhB. To test for this possibility in V. vulnificus, we also compared the levels of luxS mRNA following rifampicin treatment in wild type, ΔryhB, Δhfq, and a ΔryhBΔhfq double mutant. As shown in Fig. 6, in the absence of Hfq, luxS mRNA levels decreased significantly, independent of RyhB, indicating that even though RyhB appears to stabilize luxS mRNA, this function requires Hfq.
Effects of RyhB on the stability of luxS mRNA. Effects of RyhB and Hfq on the stability of luxS mRNA. The relative levels of luxS mRNA following rifampicin treatment were measured using qRT-PCR. V. vulnificus MO6, ryhB mutant (ΔryhB), hfq mutant (Δhfq) or ryhB, hfq double mutant (ΔryhBΔhfq) were cultured in AB broth for 5 h until reaching an A600 of 0.3. Samples were then collected at 0, 2, 4, 6, and 8 min after the addition of rifampicin (500 μg/ml), and RNA was quantified by qRT-PCR.
Generally, non-coding RNAs modulate gene expression by base pairing with target mRNAs at the 5′-UTR45, and the sRNA chaperone Hfq either stabilizes the sRNA or facilitates binding of the sRNA to the target46. We predicted that RyhB delays the decay of luxS mRNA by binding directly to the 5′-UTR. A 32P-labeled 102-bp luxS RNA fragment including bases − 62 to + 40 with respect to the luxS translation start site was expressed by in vitro transcription using T7 RNA polymerase. Full-length RyhB was also transcribed using in vitro transcription, and increasing amounts of this transcript were incubated with the labeled luxS mRNA probe in the presence or absence of Hfq. In the presence of Hfq, RyhB binds to the 5′-UTR of luxS mRNA in a concentration-dependent manner (Fig. 7a), whereas no binding was observed in the absence of Hfq. We also assessed the effect of Hfq on the half-life of RyhB and observed that stability was significantly reduced in Δhfq compared to wild type (Fig. 7b). Together these indicate that RyhB binds directly to the 5′-UTR of luxS mRNA and stabilizes it with the assistance of Hfq.
Effects of Hfq on interactions between RyhB and luxS mRNA. (a) Gel shift assay comparing the binding of RyhB to luxS mRNA with and without Hfq. The LuxS 5′-UTR was transcribed and labeled with 32P-UTP using T7 RNA polymerase. Increasing concentrations of purified RyhB were incubated with LuxS 5′-UTR with or without the addition of 1 μM of Hfq. Lanes 1 to 4 include RyhB concentrations of 0, 100, 200, and 400 nM without Hfq, respectively. Lanes 5 to 8 include RyhB concentrations of 0, 100, 200, and 400 nM with Hfq, respectively. The probes bound by RyhB are indicated with arrows. (b) Effects of Hfq on the stability of RyhB mRNA. Relative RyhB mRNA levels following rifampicin treatment were measured using qRT-PCR. V. vulnificus MO6 or the hfq mutant (Δhfq) were cultured in AB broth for 5 h until reaching an A600 of 0.3. Samples were then collected at 0, 2, 4, 6, and 8 min after treatment with 500 μg/ml rifampicin for RNA purification and qRT-PCR analysis.
Confirmation of base pairing between RyhB and the 5′-UTR of luxS mRNA
The mFold software47 was used to predict secondary structures that may form in the 5′-UTR of luxS mRNA (Fig. 8a), one of which is a stem-and-loop structure (SL2) that would obscure the start codon. Hybridization between RyhB and the 5′-UTR of luxS mRNA was predicted to include base pairing in three regions labeled HR (Hybridized Region) 1, 2, and 3 (Fig. 8b). If hybridization occurs at these sites, loops SL1 and SL2 would be resolved and the start codon of luxS mRNA would be exposed (Fig. 8b).
Predicted secondary structure of the 5′ end of luxS mRNA and potential hybridization sites between RyhB and luxS 5′-UTR mRNA. (a) The secondary structure of the 5′ end of luxS mRNA was predicted using mFold software. The nucleotides that potentially form stem structures are shaded in blue (SL1) or red (SL2) and the start codon is noted. (b) Possible regions where RyhB and the luxS 5′-UTR may hybridize. Three possible regions of hybridization (HR1, HR2, and HR3) are indicated and the potential stem structures from (a) are noted with the corresponding colored dots and arrows.
To confirm these predictions, we performed a primer extension in the presence or absence of the 5′-UTR of luxS mRNA by reverse transcription using RyhB RNA as a template and primer RyhB-PE (Supplementary Table S3, Supplementary Fig. S3) which is complementary to the 3′-end of RyhB RNA. We expected that binding of RyhB to the luxS mRNA in the presence of Hfq would produce shorter immature cDNA products due to the formation of secondary structures between two RNA molecules that partially block the primer extension. In fact, we observed partially extended cDNA molecules that terminated at cytosine at position 108 and uracil at position 84 relative to the first base of the ryhB transcript (Fig. 9a,b). These two sites correspond to the 3′-end of the HR2 and HR3 regions of RyhB, respectively. cDNA terminated at adenine at position 97 also was observed. This residue is the first base at the 3′-end of Loop 2 (Fig. 9b). These results suggested that hybridization to luxS mRNA does indeed occur at HR2 and HR3. We speculate that there may be weak hybridization at HR1 such that termination of cDNA at this region was barely detectable. To confirm the involvement of the HR2 and 3 regions of RyhB in regulation of the luxS expression, we constructed derivatives of RyhB with mutations in either or both of HR2 or 3 (named HR2m, HR3m, and HR2&3m) by site-directed mutagenesis. A mutation called HR0m at a site outside of the hybridized region (HR0) was generated as a positive control (Fig. 9b and Supplementary Fig. S3).
Identification of regions of RyhB RNA that hybridize with luxS mRNA. (a) Primer extension assays to determine the hybridization sites between RyhB and luxS RNA. Lanes 1 to 4 include Hfq at 0, 125, 250, and 500 nM, respectively. Lanes 5 to 9 include luxS RNA at 100, 200, 400, 600, and 800 nM incubated with 1 μM Hfq, respectively. The three distinct bands that represent higher intensities in the presence of Hfq are indicated. Base positions are numbered relative to the transcription start site of RyhB. (b) A model of base pairings between LuxS 5′-UTR and RyhB. Hybridization regions (HR) 1, 2, and 3 are highlighted in yellow. Loops 1 and 2 of RyhB, which form during the hybridization also are indicated. The region labeled HR0 is the site that was mutagenized to generate HR0m as a positive control.
Wild type RyhB and each of the RyhB mutations (HR0m, 2m, 3m, and 2&3m) were introduced into a ryhB-null mutant strain (ΔryhB) and assessed for both AI-2 production using the V. harveyi indicator strain BB170 (Fig. 10a) and levels of LuxS by western hybridization using antibody against purified LuxS (Fig. 10b). RyhB with HR0m did not differ significantly from wild type. However, the presence of either HR2m or 3m led to significant decreases in both AI-2 production and LuxS expression, suggesting that HR2 and 3 are critical for LuxS regulation. However, the results of luxS qRT-PCR for wild type and the ryhB mutants showed that these mutations did not affect transcription levels of luxS under experimental conditions (Fig. 10c), suggesting that the regulation of the LuxS expression by RyhB is exerted at the translational level.
Effects of the hybridization between RhyB and luxS mRNA on expression of LuxS. (a) Influence of hybridization regions HR2 and HR3 on the production of AI-2 by LuxS as measured using the V. harveyi indicator strain BB170. The following V. vulnificus strains were cultured in AB minimal medium and sampled for the presence of AI-2 in the supernatant: 1, wild type (MO6 pBBR12); 2, ryhB deletion mutant (ΔryhB pBBR12); 3, ΔryhB with a wild type ryhB clone (ΔryhB pBBR12-ryhB); 4, ΔryhB with a mutated region HR2 clone (ΔryhB pBBR12-ryhB HR2m); 5, ΔryhB with mutated region HR3 clone (ΔryhB pBBR12-ryhB HR3m); 6, ΔryhB carrying a clone with both HR2 and HR3 mutated (ΔryhB pBBR12-ryhB HR2&3m); 7, ΔryhB with a clone carrying an unrelated mutated region as a control (ΔryhB pBBR12-ryhB HR0m). The presence of AI-2 was measured in RLU, relative light units (luminescence/A600). Values are averages from three independent experiments and error bars denote the standard deviations. The p-values as compared with MO6-24/O pBBR12 are indicated (Student’s t-test; *, 0.005 ≤ P < 0.05; ns, no significant). (b) Effects of HR2 and HR3 on the production of LuxS as measured by western hybridization. Western blot analysis of LuxS levels in whole cell lysates was measured. As a control, insulinase enzyme (SidC) (Kim et al., 2015. J. Biol. Chem. 290:18708), which is not regulated by quorum-sensing, was included. (c) The relative mRNA levels of luxS were measured using qRT-PCR for each of the strains listed in 10A. Cells were cultured in AB broth for 5 h until they reached an A600 of 0.3, at which point samples were collected for RNA purification and qRT-PCR analysis. The p-values for comparison with MO6-24/O pBBR12 are indicated (Student’s t-test; *, 0.005 ≤ P < 0.05; ns, no significant).
Discussion
It is well known that an adequate concentration of iron is required for the survival of bacteria2,48. Nevertheless, iron is not readily available in natural environments due to its low solubility at neutral pH49. Furthermore, it is very difficult for pathogenic bacteria to compete with host cells for iron50. Therefore, pathogenic bacteria are equipped with various mechanisms to more effectively scavenge iron in the host environment. Our previous studies showed that iron levels affect quorum-sensing pathways6,8. This study more specifically defines the relationship between iron levels and quorum-sensing by showing that iron inhibits the production of AI-2, an initiation signal for quorum-sensing. Our observations lead us to propose that one of the most important roles for quorum-sensing is to control the timing of expression of virulence factors in pathogens as they prepare to attack host cells to acquire nutrients such as iron.
Fur is the most common regulator of iron-dependent target genes. We previously showed that cell density signals and iron converge on the AI-2 quorum-sensing pathway in V. vulnificus, and that the regulation is also mediated by Fur7,8. In the presence of iron, Fur represses the expression of major components in this pathway such as luxO, five qrr genes, and smcR. However, a deletion in fur did not fully abolish this repression8. This observation led us to search for additional factors known to be involved with iron-dependent regulation. We examined these factors and found that RyhB affects the expression of luxS, which encodes the biosynthesis of AI-2. Neither genetic analysis using a reporter fusion to luxS nor gel-shift assays using purified Fur-iron along with the upstream region of luxS provided any evidence that Fur directly controls luxS expression in V. vulnificus (our unpublished data). Interestingly, unlike other components of the quorum-sensing pathway, luxS expression appears to be controlled by RyhB at the translational level.
This mode of regulation may allow for a continuous basal level of AI-2 expression even in the presence of iron rather than the tight suppression that would occur if Fur were involved. If cells do not produce any AI-2 at all, the quorum-sensing pathway cannot quickly resume once it is shut down by the Fur-iron complex. This scenario may also explain the presence of the strong promoter sequence of ryhB, which is very similar to the canonical consensus promoter sequence in this organism. In this way, expression of ryhB is initiated swiftly once the repression exerted by Fur-iron is relieved. Employing RyhB may also provide a particular advantage in that this small RNA functions at the translational level to alter expression quickly and, due to the short half-life of the molecule, repression is rapidly relieved if the SmcR or Fur-iron signals disappear. The chaperone Hfq is absolutely required for RyhB activity. Furthermore, levels of luxS mRNA in a hfq-deletion mutant were even lower than in a ryhB-deletion isotype (Fig. 6), suggesting that yet other factor may exist, possibly an additional small RNA specific to luxS. A search for non-coding small RNAs modulated by RyhB may provide more insight into the iron-dependent modulation of genes.
Quorum-sensing signaling pathways are a valuable way for pathogenic bacteria to adapt to host environmental conditions but they are energetically expensive due to the numerous factors and variety of regulatory mechanisms that are employed. Therefore, it is necessary for cells to be equipped with the ability to control this signaling when it is not needed. For example, once a quorum-sensing signaling molecule accumulates within a closed environment, it is possible that the cell would continue to sense the signal even when expression of target genes are no longer required. The best way to avoid such a waste of energy is to lower the concentration of signaling molecules. Two possible mechanisms are either to degrade the signal molecule or to cease synthesis of the signal molecule. It is known that bacteria harbor enzymes to degrade the homoserine lactone signal molecule51,52. However, whether the AI-2 molecule is degraded in a similar way remains to be elucidated. To the best of our knowledge, ours is the first study to show that feedback inhibition of quorum signaling occurs through the inhibition of AI-2 synthesis. Other small RNAs called Qrrs are also known to be involved in the feedback control of quorum-sensing8,53,54. However, these appear to be involved in elaborate control of the signal to properly adapt to changes in cell density and not in feedback control of the quorum-sensing pathway as a whole.
AI-2 is a signal that plays a very important role in bacterial community structure, affecting inter-species communication as well as intra-species communication9. AI-2 signaling from pathogenic bacteria can be transmitted to other recognizable bacteria, and a decrease in AI-2 levels in response to the presence of iron can certainly affect other microbiota present in the infected regions of a host. A comparison of the gut microbiota in mice infected with wild type V. vulnificus and mice infected with a luxS-deleted isotype are significantly discernable (our unpublished data). This suggests that the presence of iron alone can indeed affect the community structure of the microbiota, in addition to influencing levels of the quorum-sensing signal molecules and related target functions. Studies on the effect of AI-2 on host microbiota in the context of iron concentrations may provide interesting insight into host-microbe interactions.
Small RNAs have been widely studied especially in the context of virulence regulation in pathogenic bacteria31,37,38,55. In Vibrionacea, small RNAs has been intensively studied in V. cholerae56. However, in other species, the role of small RNAs awaits further elucidation. The nucleotide sequences of RyhB and luxS mRNAs and possible hybridization between these two molecules in three related Vibrio spp. (V. cholerae, V. parahaemolyticus, and V. harveyi) demonstrated that, for all three, the start codon for luxS is hidden within stem-loop structures, unless binding with RyhB resolves this secondary structure (Supplementary Fig. S4), implying that this type of regulation is common among Vibrionaceae. Considering that major virulence factors are controlled by quorum sensing within these species, appreciation of the role of RyhB in mechanisms related to pathogenicity will provide useful information about the disease process.
Iron, or the molecules with which it interacts, may be good targets for the development of agents to control pathogenic bacteria. This study demonstrated that iron inhibits the quorum sensing signaling pathway associated with expression of virulence factors, and any tactics to enhance iron solubility may be of value to control pathogenic bacteria. Development of molecules such as anti-sense RNAs that interfere with the action of RyhB or possibly direct inhibitors of Hfq may be useful clinical approaches.
Materials and methods
Strains and culture condition
Strains and plasmids used in this study are listed in Supplementary Table S1. Escherichia coli cells were cultured in Luria–Bertani (LB) medium at 37 °C. V. vulnificus strains were grown at 30 °C in LB medium supplemented with 2.0% (w/v) NaCl (LBS) or in AB minimal medium (0.3 M NaCl, 0.05 M MgSO4, 0.2% casamino acid, 1 mM KPO4, 1 mM l-arginine, pH 7.5)57. All media components were purchased from Difco (Detroit, USA), and antibiotics were purchased from Sigma (St. Louis, USA).
Determination of the transcription start site of ryhB
RNA was isolated from V. vulnificus using the RNase Easy Mini Kit (Qiagen, Valencia, CA), and RNA concentration was determined using a Biophotometer (Eppendorf, Hamburg, Germany). A 500 ng sample of RNA extracted from V. vulnificus was incubated with 5′-labeled primer RyhB-PE at 65 °C and chilled on ice. Reverse transcription was performed using a PrimeScript RT reagent kit (Takara, Tokyo, Japan). The resulting product and sequencing ladder were resolved on a 6% polyacrylamide sequencing gel to identify the transcription start site.
Construction of ryhB deletions in V. vulnificus
A DNA fragment comprising the upstream region of ryhB was amplified by primers RyhB-KO-upF and RyhB-KO-upR, and ligated into a pGEM-T easy vector, generating plasmid pGEM-T-RyhBup. The downstream region of ryhB was amplified by primers RyhB-KO-downF and RyhB-KO-downR and ligated into a pGEM-T easy vector, generating plasmid pGEM-T-RyhBdown. Plasmid pGEM-T-RyhBup was digested with restriction enzymes PstI and BamHI, and the resulting DNA fragment containing the ryhB upstream region was ligated into plasmid pGEM-T-RyhBdown to construct pGEM-T-RyhBKO. The construction was digested with XhoI and XbaI, and subsequently ligated into pDM4 to obtain pDM4-RyhBKO which was subsequently introduced into E. coli S17-1 λpir to be mobilized into V. vulnificus by conjugation. Double crossover selection was performed on a 10% sucrose plate as described previously58. The ryhB deletion mutant, ΔryhB, was confirmed by PCR and DNA sequencing.
Gel shift assay
A 336-bp DNA fragment including the ryhB promoter region (− 213 to + 123 with respect to the transcriptional start site) was PCR-amplified using the 32P-labeled primers RyhB-F1 and RyhB-R1 (Supplementary Table S2). For the gel shift assay, 10 ng of labeled DNA fragment was incubated with increasing amounts of purified SmcR (0 to 1 μM) or Fur (0 to 1 μM) in a 20 μl reaction for 30 min at 37 °C. The SmcR binding reaction buffer contained 10 mM HEPES, 100 mM KCl, 200 μM EDTA, and 10% glycerol at pH 7.5. The Fur binding reaction contained 10 mM HEPES, 100 mM KCl, and 10% glycerol at pH 7.5 and was supplemented with either 100 μM MnSO4 or 1 mM EDTA. The binding reaction was terminated by the addition of 3 μl sucrose dye solution (0.25% bromophenol blue, 0.25% xylene cyanol, 40% sucrose) and the samples were resolved on a 6% neutral polyacrylamide gel. For gel shift assays of RyhB and LuxS 5′-UTR, 10 ng of 32P-labeled LuxS 5′-UTR and increasing amounts of RyhB were incubated at 70 °C for 10 min and then chilled on ice for at least 1 min. Then 2 μl of 10 × structure buffer (100 mM Tris–Cl, 50 mM magnesium acetate, 1 M ammonium chloride, 5 mM DTT, pH 7.5) and Hfq were added and incubated at 30 °C for 10 min. The binding reaction was terminated by the addition of 4 μl of sucrose dye and resolved on a 6% neutral polyacrylamide gel.
Construction of ryhB::luxAB and vvpE::luxAB transcriptional fusions
Primers PryhB-F and PryhB-R were used for PCR amplification of the ryhB promoter region, which is − 251 to + 123 relative to the transcription start site. Primers PvvpE-F and PvvpE-R were used for PCR amplification of the vvpE promoter region. The resulting products were digested with KpnI and XbaI (Takara, Ohtsu, Japan) and cloned into vector pHK0011 to construct pHryhB and pHvvpE, respectively, and each was conjugated into V. vulnificus MO6-24/O wild type, single mutants of luxO, smcR, and the luxO/smcR double mutant.
Bioluminescence assay
Overnight cultures of V. vulnificus strains were washed with either LBS or AB medium and inoculated into fresh AB medium containing the appropriate antibiotic. At various growth stages, 0.006% (v/v) n-decylaldehyde was added and luminescence was measured using a luminometer (Mithras LB 940, Berthold Technologies, Bad Wildbad, Germany). Transcription levels were measured as light units normalized to cell density (relative light units: RLU) as described previously6.
DNase I footprinting assay for binding of SmcR and Fur to the upstream region of ryhB
An end-labeled 340-bp DNA fragment consisting of bases − 217 to + 123 relative to the transcription start site of ryhB was generated by PCR amplification using primers 32P-labeled RyhB-F1 and RyhB-R1 (Supplementary Table S2). To determine the binding site of SmcR, 200 ng of the amplified DNA fragments was incubated with increasing amounts of purified SmcR or Fur for 30 min at 37 °C in 50 μl buffer (10 mM HEPES, 100 mM KCl, 200 μM EDTA, 10% glycerol, pH 7.5). To identify the Fur binding region, 200 ng of the resulting DNA fragments was incubated for 30 min with increasing amount of purified Fur in 50 μl of binding solution (10 mM HEPES, 100 mM KCl, 10% glycerol, 100 μM MnSO4, pH 7.5). After supplementing 50 μl of CaCl2-MgCl2 solution (5 mM CaCl2, 10 mM MgCl2) to the binding reaction, 0.25 unit of DNase I (Promega, Madison, USA) was added and allowed to react for 1 min at 37 °C before terminating with 90 μl stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS). To precipitate the DNA, 500 μl ethanol was added and samples were incubated on ice for 30 min, after which pellets were collected, washed with 70% ethanol, and suspended in 10 μl loading buffer (98% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue). The resulting product and sequencing ladder generated with the 32P-labled RyhB-F1 primer (Supplementary Table S2) were resolved on a 6% polyacrylamide sequencing gel.
Detection of AI-2 production
The AI-2 assay was performed using the V. harveyi reporter strain BB170 as previously described43. Briefly, BB170 was prepared by culturing overnight in LB broth at 30 °C, then washing twice and diluting 1:3000 in fresh AB minimal medium. Test cultures of V. vulnificus were grown overnight in LBS broth and then washed once before diluting 1:250 in fresh AB broth. Each hour, A600 was measured and 10 μl of cell-free supernatant was collected and mixed with 90 μl of diluted BB170, prepared as described above, in 96 well plates at 30 °C. The luminescence was measured using a Mithras LB 940 multimode microplate reader (Berthold, Germany).
RNA synthesis by in vitro transcription
Template DNA of ryhB and luxS was prepared for in vitro transcription by PCR using primers containing the T7 promoter sequences as shown in Supplementary Table S2. RNA was synthesized by in vitro transcription using T7 RNA polymerase (Takara, Tokyo, Japan) using the prepared template DNA at 37 °C following the protocol provided and was further purified using a Monarch RNA Cleanup Kit (NEB, Cambridge, USA).
Determination of the regions of RyhB RNA and luxS mRNA that hybridize with each other using primer extension with reverse transcriptase
RyhB (50 ng) was incubated with LuxS 5′-UTR and 32P-labeled primer RyhB-PE, which is complementary to RyhB from residues 167 and 189 relative to the transcription start site of ryhB, at 65 °C for 10 min and then chilled on ice. Hfq and 10 × structure buffer (100 mM Tris–Cl, 50 mM magnesium acetate, 1 M ammonium chloride, 5 mM DTT, pH 7.5) were added and the mixture was incubated at 30 °C for 10 min. Lastly, dNTPs and 1 unit of SuperScript III reverse transcriptase (Invitrogen, Carsbad, USA) were added and incubated at 30 °C for 1 h. Reactions were terminated at 85 °C for 5 min, and samples were resolved on a 6% polyacrylamide sequencing gel alongside a sequencing ladder generated by the same primer.
Purification of LuxS and Hfq proteins
A DNA fragment covering the 519-bp luxS open reading frame (ORF) encoding the 172-amino acids of LuxS was PCR-amplified using primers STREP-LUXSF and STREP-LUXSR (Supplementary Table S2). The amplified fragment was sub-cloned into pASK-IBA-7 (IBA, Göttingen, Germany) which generates a fusion between the Strep-tag and the N-terminus of the expression protein. The resulting Strep-tagged luxS construct was transformed into E. coli BL21 (DE3) (Novagen, Madison, USA), and over-expressed by inducing with 10 μg/ml anhydrotetracycline. The bacterial pellets were suspended in W buffer (100 mM Tris–Cl, 150 mM NaCl, pH 8.0), sonicated, and then centrifuged at 13,000 rpm for 15 min. The resulting supernatant was applied to 1 ml of Strep-Tactin-Sepharose resin (IBA, Göttingen, Germany) and bound proteins were eluted with E buffer (100 mM Tris–Cl, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin, pH 8.0). The eluted proteins were assessed for purity using 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). To purify the Hfq protein, DNA spanning the 261-bp Hfq ORF encoding 86 amino acids of Hfq was PCR-amplified using primers STREP-HFQF and STREP-HFQR (Supplementary Table S2). The amplified fragment was cloned into pASK-IBA-7 to generate pASK-IBA-Hfq (Supplementary Table S1), and Hfq was purified as described above.
Construction of a ryhB-deletion and series of site-directed mutations in ryhB
A DNA fragment containing the complete sequence of ryhB was PCR-amplified using primers CryhB-F and CryhB-R (Supplementary Table S2). The resulting product was ligated into pGEM-T easy vector (Promega). After confirmation by sequencing, the DNA fragment containing the ryhB sequence was cut with KpnI and XbaI and cloned into pRK415, generating pRK-RyhB. Nucleotides that potentially bind to LuxS 5′-UTR were mutated using the EZchange Site-directed Mutagenesis Kit (Enzynomics, Deajeon, Korea). Primers ryhB_SDM2_F and ryhB_SDM2_R were used to construct pRK-RyhB2m; primers ryhB_SDM3_F and ryhB_SDM3_R were used to construct pRK-RyhB3m; all four primers (ryhB_SDM2_F&R, ryhB_SDM3_F&R) were used to construct pRK-RyhB2&3m; and primers ryhB_SDM_CON_F and ryhB_SDM_CON_R were used to construct the control pRK-RyhB0m (Supplementary Table S2). The resulting constructs were mobilized from S17-1 to mutant strain ΔryhB. Conjugants were selected in thiosulfate-citrate-bile salts-sucrose agar (TCBS) plate medium supplemented with 1 μg/ml tetracycline.
Preparation of polyclonal rabbit antibody against purified LuxS and western hybridization
Purified LuxS was used to produce polyclonal rabbit antibodies (Ab Frontier, Seoul, South Korea). For LuxS expression western blot analysis, V. vulnificus MO6-24/O wild type or mutants were cultured in LBS broth for 6 h, then diluted 1:100 in fresh LB broth. After 3 h of growth, cells were collected and washed twice with PBS, after which 20 μg of each lysate was resolved by SDS-PAGE and transferred to Hybond P membrane (Amersham, Arlington Heights, USA). The membrane was incubated first with polyclonal rabbit antibodies against LuxS (1:2000) and then with goat anti-rabbit IgG-AP (1:5000) (Santa Cruz Biotechnology, CA, USA). LuxS expression was visualized using Western blotting luminal reagent (Santa Cruz Biotechnology, CA, USA).
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This work was supported by grant from the National Research Foundation, Republic of Korea (NRF-2019R1A2C2084282).
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K.L., Y.W., and N.P. performed the experiments. K.L., Y.W., and K.K. were involved in the conception and design of the study. K.L. and K.K. were involved in drafting the manuscript.
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Lee, KW., Wen, Y., Park, NY. et al. Quorum sensing and iron-dependent coordinated control of autoinducer-2 production via small RNA RyhB in Vibrio vulnificus. Sci Rep 12, 831 (2022). https://doi.org/10.1038/s41598-021-04757-9
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DOI: https://doi.org/10.1038/s41598-021-04757-9
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