Effect of (p)ppGpp on the Expression of the Vibrioferrin-Mediated Iron Acquisition System in Vibrio parahaemolyticus

Abstract

Bacteria have a stringent response system mediated by guanosine pentaphosphate and tetraphosphate ((p)ppGpp), which suppresses the expression of genes involved in cell growth and promotes the expression of genes involved in nutrient uptake and metabolism under nutrient-limited stress. In environments with limited availability of iron, an essential trace element, bacteria generally produce and secrete siderophores to efficiently utilize water-insoluble ferric iron (Fe³⁺) in the environment. In Vibrio parahaemolyticus, Fur (iron-responsive repressor) and RyhB (Fur-regulated small RNA) regulate the expression of genes involved in the utilization of vibrioferrin (VF), a siderophore produced by this bacterium. In this study, we examined whether (p)ppGpp is also involved in regulating the expression of genes related to the VF utilization system. Results of the chrome azurol S plate assay revealed that the strain in which 3 (p)ppGpp synthetases were deleted (∆relA∆spoT∆relV) produced less VF than the parental strain. Growth test results showed that the growth rate of ∆relA∆spoT∆relV in an iron-limited medium was suppressed compared with that of the parental strain but was restored with the addition of VF. Furthermore, RT-quantitative (q)PCR results showed that the expression levels of pvsA (VF biosynthesis gene) and pvuA2 (ferric VF receptor gene) in ∆relA∆spoT∆relV under iron limitation were significantly reduced compared with those in the parental strain. Western blot results demonstrated that the expression level of PvuA2 in ∆relA∆spoT∆relV was lower than that in the parental strain. These results suggest that (p)ppGpp promotes the expression of genes related to VF biosynthesis and the ferric VF uptake system under iron limitation.

INTRODUCTION

Iron is an essential trace element for many organisms, including bacteria, because it is involved in biological processes such as electron transport, the tricarboxylic acid cycle, redox reactions, and DNA metabolism.¹⁾ However, although iron is one of the most abundant elements on Earth, it is rapidly oxidized from ferrous iron (Fe²⁺) to ferric iron (Fe³⁺) under oxygen-rich conditions, such as in aquatic environments, resulting in the formation of insoluble ferric hydroxide complexes, which have low bioavailability.²⁾ Furthermore, within mammals, iron is mainly bound to host-derived proteins (e.g., transferrin and ferritin), making it largely inaccessible to invading bacteria.³⁾ To overcome these iron-limited conditions, many bacteria have evolved iron acquisition systems mediated by siderophores, low-molecular-weight iron chelate molecules with a high affinity for Fe³⁺.⁴⁾ In response to iron-limiting stress, bacteria synthesize siderophores intracellularly and secrete them into the environment to chelate Fe³⁺ from insoluble iron hydroxide complexes and host iron-binding proteins. The formed Fe³⁺–siderophore complex is transported into the bacterial cell via a specific transporter, such as a TonB-dependent specific uptake system consisting of an outer membrane receptor protein and an ATP-binding cassette transporter in Gram-negative bacteria.⁵⁾ Furthermore, the expression of these iron acquisition systems in bacteria is negatively controlled at the transcriptional level by a global iron-binding repressor protein called Fur (ferric uptake repressor), which functions by forming a complex with Fe²⁺ as a corepressor when intracellular iron levels are high.⁵⁾ Such bacterial siderophore-mediated iron acquisition systems are also considered a virulence factor because they contribute to host colonization by the pathogen and to increased severity of the pathogenic disease.¹⁾

Bacteria are often exposed to various stressors, including iron availability, pH, temperature, and nutrient availability, in their natural environment and within their hosts. Therefore, to survive and proliferate in unfavorable environments, bacteria have evolutionarily acquired gene expression networks to sense and respond to various stresses.⁶⁾ The stringent response is a conserved bacterial stress response network that allows bacteria to respond to various nutritional stresses and is accompanied by increased production of alarmones, guanosine-5′-triphosphate 3′-diphosphate, and guanosine-3′,5′-bis(diphosphate) (ppGpp), typically referred to as (p)pGpp.^7,8) Intracellular concentrations of (p)ppGpp are regulated by RelA/SpoT homolog family proteins, such as RelA (synthesis enzyme of (p)ppGpp) and SpoT (synthesis and hydrolysis enzyme of (p)ppGpp) in γ-proteobacteria, such as Escherichia coli.⁹⁾ RelA is a ribosome-associated (p)ppGpp synthetase that is activated upon binding to ribosomes during amino acid depletion, specifically when uncharged tRNAs are present in the ribosome A site.¹⁰⁾ SpoT is a bifunctional enzyme that synthesizes and hydrolyzes (p)ppGpp and balances intracellular (p)ppGpp concentrations in response to various nutrient stresses, such as carbon,¹¹⁾ fatty acid,¹²⁾ and iron limitation.¹³⁾ Unlike other γ-proteobacteria, Vibrio cholerae possesses a third (p)ppGpp synthetase, RelV, in addition to RelA and SpoT, and the relV gene is highly conserved among different Vibrio species, such as Vibrio parahaemolyticus and Vibrio vulnificus.¹⁴⁾ Furthermore, in V. cholerae, the relA, spoT, and relV triple gene mutants cannot produce (p)ppGpp even under amino acid or carbon limitation, and no accumulation of (p)ppGpp occurs within the cells.¹⁴⁾

Vibrio parahaemolyticus is a halophilic Gram-negative bacterium found in warm brackish water and rivers that causes watery diarrhea when transmitted by eating raw or uncooked contaminated seafood. We previously demonstrated that V. parahaemolyticus has an iron acquisition system that utilizes the siderophore vibrioferrin (VF) produced by itself. We also identified 2 divergent operons involved in VF biosynthesis and ferric VF transport in V. parahaemolyticus (i.e., pvsABCDE and pvuA1A2BCDE, respectively)^15–17) and revealed that the transcription of these operons is released from repression by the Fur-Fe²⁺ complex under iron-limited conditions.^15,17) Furthermore, we identified the Fur-regulated small RNA RyhB, which can translationally down-regulate genes encoding many iron-containing and iron storage proteins¹⁸⁾; intriguingly, it positively regulates a polycistronic mRNA from pvsABCDE as a direct target.¹⁹⁾ In the present study, we report that the stress-responsive expression of the 2 divergent operons involved in VF utilization under iron-limited conditions in V. parahaemolyticus can be affected not only by Fur and RyhB but also by intracellular levels of (p)ppGpp.

MATERIALS AND METHODS

Bacterial Strains and Growth Conditions

The V. parahaemolyticus strains used in this study are listed in Table 1. The strains were routinely cultivated in Luria–Bertani (LB) medium containing 3% NaCl. E. coli β2155,²¹⁾ a diaminopimelate (DAP) auxotroph conjugal donor, was cultured in an LB medium containing 0.5% NaCl with 0.5 mM DAP. LB media with and without the ferric iron-specific chelator ethylenediamine-di(o-hydroxyphenylacetic acid) (Sigma, St. Louis, MO, U.S.A.) at 25 μM were used for growth under iron-limited (−Fe) and iron-replete (+Fe) conditions, respectively.¹⁶⁾ When appropriate, the following antibiotics were added: 10 μg/mL chloramphenicol and 10 μg/mL tetracycline. The Tris-buffered succinate (TS) medium²²⁾ containing 2% NaCl and 1 μM FeCl₃¹⁹⁾ was used as an amino acid-deficient medium, and all 20 amino acids (each 20 μg/mL) were added to the medium as necessary.

Table 1. Vibrio parahaemolyticus Strains Used in the Present Study

Vibrio parahaemolyticus strain	Description	Reference or source
RIMD2210633	Clinical isolate of serotype O3:K6, parental strain for all deletion mutants	20)
∆pvsB (formerly known as VPD5)	pvsB deletion mutant from RIMD2210633 (vibrioferrin nonproducer mutant)	16)
∆relA	relA deletion mutant from RIMD2210633	Present study
∆relA∆spoT	relA and spoT double deletion mutant from RIMD2210633	Present study
∆relA∆relV	relA and relV double deletion mutant from RIMD2210633	Present study
∆relA∆spoT∆relV	relA, spoT, and relV triple deletion mutant from RIMD2210633	Present study
∆relA∆spoT∆relV-pRK415	∆relA∆spoT∆relV harboring pRK415	Present study
∆relA∆spoT∆relV-pSpoT	∆relA∆spoT∆relV harboring pSpoT	Present study

Construction of Deletion Mutants of relA, spoT, and relV

The deletion of relA, spoT, and relV in V. parahaemolyticus is accompanied by allelic exchange using the suicide vector pXAC623,²³⁾ as previously described.¹⁶⁾ Each deletion fragment was prepared by PCR-driven overlap extension, as described previously.^24,25) Deletion fragments were constructed using the following primers: for relA deletion, relA-#1 (5′-CAGCCATCTAGAATCGATGGCACTGTTTG-3′), relA-#2 (5′-gagctgttcactgctagcttGATGACAACG-3′), relA-#3 (5′-aagctagcagtgaacagctcGAAGAACTGC-3′), and relA-#4 (5′-GACCAATCTAGATTCTGAATCGCATCCACC-3′); for spoT deletion, spoT-#1 (5′-AGAAGTTCTAGACGCTCGTGAACGTCAAG-3′), spoT-#2 (5′-cggtcgacagccagtttgatCAGGATAACG-3′), spoT-#3 (5′-atcaaactggctgtcgaccgAAGAACGTGA-3′), and spoT-#4 (5′-AACATTTCTAGACTGCACCATTCCAATCATC-3′); and relV-#1 (5′-CCCTTCTCTAGAATCATGTTTCGTTGCTAACC-3′), relV-#2 (5′-aagcttttgataccaaaagcATCAATGCTG-3′), relV-#3 (5′-gcttttggtatcaaaagcttGAACGTCAAG-3′), and relV-#4 (5′-ATATGTTCTAGAATTGGTAGGGTAAAACTGC-3′) (the underlined sequences are XbaI sites, and the lowercase letter sequences within primers #2 and #3 are each complementary to the corresponding gene sequences). The PCR fragments were ligated into the XbaI site of pXAC623, and the resulting hybrid plasmids were then transformed into E. coli β2155 to generate donor strains for mating with V. parahaemolyticus strains.

Construction of a Trans-Complementation Strain

A broad-host-range vector pRK415²⁶⁾ was used to construct a trans-complementation strain of spoT. The full-length spoT fragment was amplified by PCR using primers spoT-#1 and spoT-#4 and ligated into the XbaI site of pRK415 to obtain pSpoT harboring the spoT gene controlled by its own promoter. pSpoT and pRK415 were mobilized into ∆relA∆spoT∆relV to construct ∆relA∆spoT∆relV-pSpoT (trans-complementation strain) and ∆relA∆spoT∆relV-pRK415 (vector control), respectively, as described previously.²⁵⁾

Measurement of Siderophore-Producing Activity Using Chrome Azurol S (CAS) Plates

A CAS plate assay was carried out to assess the VF-producing activities of various V. parahaemolyticus strains.²⁷⁾ In brief, a single colony of each V. parahaemolyticus strain was picked up using an autoclaved 10 μL pipette tip, spotted onto a CAS plate, and incubated for 48 h at 25°C. Subsequently, the diameters of the colonies and the orange halo around the colonies, which indicate the production of siderophores, were measured and then calculated as the ratio of halo/colony diameters (n = 5 for each V. parahaemolyticus strain).

Growth Assay

V. parahaemolyticus strains cultured overnight were inoculated into the −Fe medium at an optimal density of 600 nm (OD₆₀₀) of 0.005. When appropriate, the −Fe medium was supplemented with VF²⁸⁾ at a final concentration of 20 μM (−Fe + VF medium). The cultures were then shaken at 37°C at 70 rpm and measured at OD₆₀₀ using a biophotorecorder TVS062CA (Advantec, Tokyo, Japan) every hour for 20 h.

RT-Quantitative PCR (RT-qPCR)

V. parahaemolyticus cells were diluted with the +Fe or −Fe medium from the overnight culture to an OD₆₀₀ of 0.005. The cultures were shaken at 37°C until the OD₆₀₀ reached 0.4–0.6 and then treated with RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. The treated cell pellets were stored at −80°C until RNA extraction. Total RNA extraction, deoxyribonuclease (DNase) treatment, and cDNA synthesis were performed as previously described.¹⁹⁾ qPCR analysis was performed using cDNA generated from 0.5 μg of DNase-treated RNA, the primer pairs VppvsA-qF/VppvsA-qR¹⁹⁾ and VppvuA2-qF (5′-AGGAGTATTACTTCCGTGGGATT-3′)/VppvuA2-qR (5′-GTCCCGGAGCTGGGTATC-3′), specific for pvsA and pvuA2, respectively, and the Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) in a Thermal Cycler Dice Real-Time System III (TaKaRa, Shiga, Japan). Relative mRNA expression levels were determined using the comparative threshold cycle method, with recA expression as an internal control.²⁹⁾

Western Blot Analysis of Whole-Cell Lysates from V. parahaemolyticus

V. parahaemolyticus cells were diluted with the +Fe or −Fe medium from the overnight culture to an OD₆₀₀ of 0.005. The cultures were shaken at 37°C until the OD₆₀₀ reached 0.4–0.6. V. parahaemolyticus cells were harvested, washed once with PBS, and then resuspended in a 1/5th volume of 2 × Laemmli buffer. Each cell lysate (5 μL per OD₆₀₀ of 0.5) was separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and subjected to Western blot analysis using a rabbit antiserum against PvuA2, as previously described.¹⁷⁾ The band intensity of Western blots of PvuA2 was quantified using ImageJ software (version 1.54g, https://imagej.net/ij/).

RESULTS AND DISCUSSION

Effect of Mutations in (p)ppGpp Synthetase Genes on VF Production in V. parahaemolyticus

The (p)ppGpp-mediated stringent response is one of the bacterial stress response mechanisms, and (p)ppGpp synthesis is enhanced under amino acid starvation^30,31) and deficiencies in carbon¹¹⁾ and iron.¹³⁾ γ-Proteobacteria have RelA and SpoT as regulators of (p)ppGpp concentration.³²⁾ In addition, many Vibrio species, including V. cholerae, have a third (p)ppGpp synthetase, RelV. V. parahaemolyticus also has RelA,³³⁾ SpoT,³³⁾ and RelV¹⁴⁾ homologs (VP2564, VP0159, and VP1295, respectively) that show 87, 91, and 70% identities with V. cholerae RelA, SpoT, and RelV, respectively, by BLAST search (https://www.genome.jp/tools/blast/), but the role of these (p)ppGpp synthetases in V. parahaemolyticus has not been well studied. Therefore, we first constructed deletion mutants of the relA, spoT, and relV genes in V. parahaemolyticus and examined the growth phenotypes in the amino acid-deficient medium. The RIMD2210633, ∆relA, ∆relA∆spoT, and ∆relA∆relV strains were able to grow on TS medium agar plates, whereas the triple gene-deletion mutant ∆relA∆spoT∆relV barely grew on it (Fig. 1A), just as a (p)ppGpp nonproducer mutant of V. cholerae lacking the 3 corresponding genes cannot grow on the amino acid-deficient medium.¹⁴⁾ In addition, the spoT complementation strain ∆relA∆spoT∆relV-pSpoT, but not the vector control strain ∆relA∆spoT∆relV-pRK415, was restored to the ability to grow on TS medium agar plates (Fig. 1A). Furthermore, both ∆relA∆spoT∆relV and ∆relA∆spoT∆relV-pSpoT showed growth on TS medium agar plate containing amino acids (Fig. 1B). Meanwhile, the ∆spoT and ∆spoT∆relV deletion strains (i.e., relA⁺spoT ⁻ strains), and relA and relV complemented strains of ∆relA∆spoT∆relV could not be created like V. cholerae^14,34) because these deletions and complemented strains displayed a lethal or severely slow growth phenotype even in LB medium, probably due to the accumulation of excessive levels of (p)ppGpp resulting from the absence of (p)ppGpp hydrolase activity of SpoT.^14,34,35) Taken together, these results strongly suggested that V. parahaemolyticus RelA, SpoT, and RelV were functionally similar to those of V. cholerae.

Fig. 1. Growth on TS Medium Agar Plates without (A) and with (B) Amino Acids, VF Production Activity on CAS Agar Plates (C, D), and Halo/Colony Diameter Ratio in CAS Agar Plates (E, F)

(A, B) Each strain of V. parahaemolyticus was grown overnight in LB medium, washed twice, resuspended with TS medium, and streaked on TS medium agar plates without (A) and with (B) all 20 amino acids, followed by incubation for 16 h at 37°C. (C, D) VF production abilities of various deletion mutants of (p)ppGpp synthetase genes (C) and the spoT-complemented strain (D) were tested using CAS agar plates. The distinctive orange halos observed in RIMD2210633, ∆relA, ∆relA∆spoT, ∆relA∆relV, and ∆relA∆spoT∆relV-pSpoT are indicative of VF production. The ∆pvsB strain (i.e., the VF nonproducer mutant) exhibited no orange halo. Faint orange halos in ∆relA∆spoT∆relV and ∆relA∆spoT∆relV-pSpoT represent poor VF production. (E, F) Halo/colony diameter ratios of each V. parahaemolyticus strain on CAS agar plates were scored after incubation for 48 h at 25°C. Each bar depicts the mean ± standard deviation (S.D.) (n = 5) and dots represent individual values. p-Values were estimated using the Kruskal–Wallis test, followed by Dunn’s post hoc test for multiple comparisons. p-Values < 0.05 were considered statistically significant.

Next, to determine the effect of (p)ppGpp synthetases on the expression of the siderophore utilization system during iron-limiting stress in V. parahaemolyticus, deletion mutants of relA, spoT, and relV were examined for their ability to produce VF using CAS plates. On CAS plates, V. parahaemolyticus strain RIMD2210633 exhibited an orange halo indicating VF production, but VF production was lost in the VF biosynthesis gene deletion strain ∆pvsB (Figs. 1C, 1D). The ∆relA, ∆relA∆spoT, and ∆relA∆relV strains formed halos and showed similar halo/colony diameter ratios to the RIMD2210633 strain, but the ∆relA∆spoT∆relV strain, in which the 3 (p)ppGpp synthetase genes were deleted, showed a lower halo/colony diameter ratio than the RIMD2210633 strain (Figs. 1C, 1E). Moreover, genetic complementation of the ∆relA∆spoT∆relV strain with pSpoT in trans resulted in a larger halo/colony diameter ratio than that of the vector control strain and was restored to the same level (p = 0.957) as that of the RIMD2210633 strain (Figs. 1D, 1F). These results suggested that VF production was lower in the ∆relA∆spoT∆relV strain than in the RIMD2210633 strain.

Growth Assay of Deletion Mutants of (p)ppGpp Synthetase Genes

To determine whether (p)ppGpp affects growth under iron limitation in V. parahaemolyticus, we performed growth assays using the ∆relA∆spoT∆relV strain, in which 3 (p)ppGpp synthetase genes were deleted. Both RIMD2210633 and ∆relA∆spoT∆relV strains showed growth in the −Fe medium, where the growth of ∆pvsB strains was suppressed, but the change from lag phase to logarithmic phase was slower in the ∆relA∆spoT∆relV strain than in the RIMD2210633 strain (Fig. 2A). By contrast, in the −Fe + VF medium, the growth of the ∆pvsB strain was restored, and the ∆pvsB and ∆relA∆spoT∆relV strains showed a change from the lag phase to the logarithmic phase 1 h after the start of growth (Fig. 2A). Moreover, the ∆relA∆spoT∆relV-pSpoT-complemented strain grew as well as the RIMD2210633 strain in the −Fe medium, whereas the ∆relA∆spoT∆relV-pRK415 vector control strain did not grow well unless VF was present (Fig. 2B). These results suggested that (p)ppGpp synthetases enabled efficient growth under iron-limited conditions by increasing VF production and transport by V. parahaemolyticus.

Fig. 2. VF-Mediated Growth of Various Deletion Mutants of (p)ppGpp Synthetase Genes (A) and the spoT-Complemented Strain (B)

The growth rates of V. parahaemolyticus RIMD2210633 (circle), ∆pvsB (square), ∆relA∆spoT∆relV (triangle), ∆relA∆spoT∆relV-pRK415 (inverted triangle), and ∆relA∆spoT∆relV-pSpoT (diamond) were assessed in the −Fe (closed symbols) or −Fe + VF medium (open symbols). OD₆₀₀ was measured every hour for 20 h. Representative results from three independent experiments are shown.

RT-qPCR Analysis of the Transcription Levels of VF Biosynthesis and Transport Genes in the ∆relA∆spoT∆relV Strain

The cluster of genes involved in VF biosynthesis and transport consists of 2 divergent operons, pvsABCDE and pvuA1A2BCDE.^15–17) Based on the results of the CAS and growth assays, transcriptional analysis of the pvsA and pvuA2 genes was performed by RT-qPCR to determine whether (p)ppGpp is involved in regulating the expression of these 2 operons. Both pvsA and pvuA2 were induced under iron-limited conditions in all strains, but the expression of these genes was lower in the ∆relA∆spoT∆relV strain than in the RIMD2210633 strain (Fig. 3). Additionally, in the gene complementation test for spoT, the expression of pvsA and pvuA2 was significantly higher in the ∆relA∆spoT∆relV-pSpoT-complemented strain than in the ∆relA∆spoT∆relV-pRK415 vector control strain (Fig. 3).

Fig. 3. RT-qPCR Analysis of pvsA and pvuA2 in the Relevant V. parahaemolyticus Strains

The mRNA levels of pvsA and pvuA2 were assessed by RT-qPCR using the total RNA samples extracted from the V. parahaemolyticus RIMD2210633, ∆relA∆spoT∆relV, ∆relA∆spoT∆relV-pRK415, and ∆relA∆spoT∆relV-pSpoT strains grown in the +Fe or −Fe medium. The expression was normalized to that of recA. Each bar depicts the mean ± S.D. (n = 4), and dots represent individual values. p-Values were estimated using the Kruskal–Wallis test, followed by Dunn’s post hoc test for multiple comparisons. p-Values < 0.05 were considered statistically significant.

Analysis of PvuA2 Expression Level in Various V. parahaemolyticus Strains by Western Blot

The results thus far obtained suggested that (p)ppGpp increased VF production and utilization by upregulating the expression of the pvsABCDE and pvuA1A2BCDE operons. Western blot was performed using whole-cell lysates prepared from various V. parahaemolyticus strains to confirm whether the expression of PvuA2, a 78 kDa ferric VF receptor,^16,17) is decreased in the ∆relA∆spoT∆relV mutant strain under iron-limited conditions. In Western blot, the protein amount of each lysate sample was confirmed to be approximately the same as that in Coomassie-stained SDS-PAGE (Fig. 4A). In all V. parahaemolyticus strains, PvuA2 levels were induced when cultured in the −Fe medium, but the expression of PvsA2 in the ∆relA∆spoT∆relV and ∆relA∆spoT∆relV-pRK415 strains was reduced to approx. 40% of that in the RIMD2210633 strain (Fig. 4B). By contrast, in the spoT complementation strain ∆relA∆spoT∆relV-pSpoT, the expression of PvuA2 was restored to approximately 80% of that in the RIMD2210633 strain (Fig. 4B). These results suggested that the presence of (p)ppGpp synthetases led to transcriptional induction of the pvuA1A2BCDE operon involved in VF utilization and thus increased the expression of its encoded proteins, such as PvuA2.

Fig. 4. SDS-PAGE (A) and Western Blot (B) of Whole-Cell Lysates Prepared from the Relevant V. parahaemolyticus Strains

Whole-cell lysates of V. parahaemolyticus RIMD2210633, ∆relA∆spoT∆relV, ∆relA∆spoT∆relV-pRK415, and ∆relA∆spoT∆relV-pSpoT strains were prepared from cells grown at 37°C in the +Fe or −Fe medium until the OD₆₀₀ reached 0.4–0.6. Whole-cell lysates equivalent to 25 μL of culture medium with an OD₆₀₀ of 0.5 were electrophoresed on a 12.5% SDS-PAGE gel at 20 mA/gel for 80 min in duplicate; one was stained with Coomassie blue (A), and the other was transferred to a PVDF membrane and subjected to Western blot with antiserum against the PvuA2 protein (B). M, Molecular weight marker (Protein Ladder One Plus, triple-color for SDS-PAGE; Nacalai Tesque, Kyoto, Japan); arrow, PvuA2 protein (78 kDa); NS, non-specific bands.

In conclusion, VF production and expression of the ferric VF uptake system were significantly decreased in the ∆relA∆spoT∆relV strain of V. parahaemolyticus, in which (p)ppGpp synthesis was probably absent or markedly reduced. Similar to the results of this study, the results obtained by Vinella et al.¹³⁾ demonstrated that E. coli induces the SpoT-dependent accumulation of (p)ppGpp under iron-limiting stress and that the induced (p)ppGpp promotes the expression of the utilization system for enterochelin, a siderophore produced by E. coli. Although the mechanisms by which (p)ppGpp regulates the expression of the VF biosynthesis operon pvsABCDE and the ferric VF transport operon pvuA1A2BCDE remain unclear, (p)ppGpp appears to control the homeostasis of intracellular iron concentration by regulating the expression of the iron acquisition system in γ-proteobacteria. In many Gram-negative bacteria, (p)ppGpp is involved in the expression of virulence factors other than the iron acquisition system, such as biofilm formation, adhesion, and motility.^36–38) Further investigation into the regulation of the expression of various virulence factors of V. parahaemolyticus, including the VF-mediated iron utilization system, by (p)ppGpp, is warranted to understand the pathogenicity of this bacterium.

Acknowledgments

This work was supported by JSPS KAKENHI (Grant Number: JP18K07130).

Conflict of Interest

The authors declare no conflict of interest.

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