1. Introduction
Iron plays a significant role in many biological processes [1] [2] [3] , and is an essential nutrient that Streptococcus iniae (S. iniae) needs to survive. Despite its abundance in the natural environment, iron has low solubility in physiological condition which made iron capture is an important act in bacteria. Our previous study indicated that the iron-transporter mtsABC of S. iniae HD-1 was involved in hemeutilization [4] [5] , but very little was known about the mechanisms involved in regulating and maintaining iron balance in S. iniae. The iron acquisition processes in bacteria are tightly regulated, and the homeostasis of iron was typically controlled by iron-dependent transcription regulators belonging to the DtxR or the Fur family [6] . These two families regulated the production of iron-transport systems, and in pathogens, they often controlled the expression of virulence factors as well.
S. iniae is one of the most important fish pathogens that causes serious infections in kinds of fish [7] [8] [9] [10] , and has been reported to cause opportunistic infection in humans [11] . In present study, BLAST-mediated sequences similarity search of S. iniae genome sequences resulted in the identification of gene mtsR that shared amino acid sequence homologies with iron-dependent transcription regulators of other Streptococcal pathogens. Therefore, the role of iron-dependent transcriptional regulator MtsR in S. iniae has been characterized, and the results demonstrated that MtsR regulated the expression of iron-transporter mtsABC in response to iron availability intracellular. This might provide information about the role of MtsR in iron homeostasis in S. iniae HD-1.
2. Materials and Methods
2.1. Iron Is Essential for S. iniae
To detect the iron requirement of S. iniae, HD-1 cells were cultured. HD-1 was isolated from Plectorhynchus cinctus in China, and had been described previously and characterized thoroughly by Zhou et al. [12] . HD-1 cells were grown in complete medium, brain heart infusion (BHI) at 28˚C, iron-restricted medium was prepared by adding 0.1285 g nitrilotriacetic acid trisodium salt (NTA), 3.6 g brain heart infusion in 100 ml dd H2O, and supplementing it with 0.0043 g MnCl2, 0.0038 g ZnCl2, 0.0031 g CaCl2, and 0.0033 g MgCl2 [13] .
2.2. Cloning and Sequence Analysis of mtsR
Genomic DNA was extracted from the S. iniae HD-1 using the Wizard genomic DNA purification kit, and the products were quantified by measuring the absorbance at 260 nm. PCR was carried out with 1 μg of genomic DNA using the primers 5'-TTTTGTGACATATAGTTGGCGGGCA-3' and 5'-ATGACGCCTAACAAAGAAGATT-3' as described by Zou [4] , and the PCR products were sequenced at Invitrogen corporation to confirm their specificity. Briefly, the cycling conditions used for mtsR ORF were as follows: 1 cycle of 94˚C for 2 min, 35 cycles of 94˚C for 45 sec, 35 cycles of 61˚C for 45 sec, 35 cycles of 72˚C for 1 min, 1 cycle of 72˚C for 7 min. Nucleotide and deduced amino acid homology analysis of MtsR were carried out by NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The Conserved Domains of mtsR were detected by NCBI CD Search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).
2.3. qPCR Analysis
Duplicate cultures of S. iniae HD-1 were harvested at early logarithmic phase (OD600 nm = 0.35), mid-logarithmic phase (OD600 nm = 0.65), and final-logarithmic phase (OD600 nm = 0.80) in BHI orin iron-restricted medium. The total RNA was extracted and was reverse transcribed to cDNA. Real-time fluorescence PCR (qPCR) analysis was performed with a LightCycler480 system. The primers of mtsABC, mtsR and gyrA used in qPCRas listed in Table 1 [4] , and the cycling protocol were an initial denaturation at 94˚C for 2 min followed by 40 cycles of 94˚C for 15 s, 56˚C for 50 s, and 79˚C for 10 s. Finally, a melting curve was performed to ensure that there was no contamination. qPCR of the genes of interest, mtsA, mtsB, mtsC, and mtsR and a normalizer gene, gyrA, were performed in triplicate for each sample, and included a no-template control to rule out contamination and primer-dimer formation. Gene gyrA was chosen as a normalizing gene because its expression in other streptococci is stable under different test conditions [14] . The expression fold change of gene was calculated base on the comparison with the normalized gyrA. All statistical analyses were performed using the SPSS 16.0 software (SPSS Inc., USA).
3. Results
3.1. Iron Supports S. iniae HD-1 Growth
To detect the importance of iron for S. iniae, the growth curves of HD-1 in BHI and iron-restricted medium were characterized. The results showed that the growth of S. iniae HD-1 was inhibited by adding NTA to BHI medium supplemented with additional cations, and this inhibition was the result of iron limitation. In iron-restricted medium, the HD-1 cells needed 9.5 h to reach the stationary phase, which was 3 h more than that of in BHI medium, and the growth level was dropped 10% (Figure 1). The gram stain was used to observe the morphological features of S. iniae HD-1 cells at the logarithmic phase, and the results showed that the cells in iron-restricted medium had long chain length (Figure 2(b)) than that of in BHI medium (Figure 2(a)), which indicated that
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Table 1. Primer pairs used in qPCR analysis of mtsABC and mtsR.
*Gene correspond to mtsABC gene designations in S. iniae HD-1. F, forward, R, reverse.
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Figure 1. Iron influences on the growth of S. iniae HD-1. (BHI: Positive control; 5 mM NTA: Add 5 mM NTA to BHI medium).
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Figure 2. Morphological features of S. iniae HD-1 in BHI and iron-restricted medium. (Gram stain and microscopic examine of S. iniae HD-1. A: HD-1 in BHI medium at 5 h; B: HD-1 in iron-restricted medium at 5 h).
the reproduction of S. iniae HD-1 cells had been affected without the support of iron. These results indicated that iron was the essential nutrient that S. iniae needed to survive.
3.2. Cloning and Sequence Analysis of mtsR
Iron acted as a regulatory factor having influence on proteins production, but the mechanisms processes in S. iniae for iron homeostasis have not been characterized. Screening of S. iniae genome sequences resulted in the identification of gene mtsR (GeneBank No: JN177478) that shared amino acid sequence homologies with DtxR family which were the metal-dependent transcription regulators. The closest homologs for MtsR are the iron-dependent repressors from Streptococcus pyogenes MGAS9429 (Table 2, http://blast.ncbi.nlm.nih.gov/Blast.cgi). mtsR has 624 bp which located at 5' proximal of the iron-transporter mtsABC, and was transcribed in the opposite direction (Figure 3(a)). The localization of mtsR, and its similarity to other iron-dependent transcriptional regulators suggested that MtsR may function as the mtsABC repressor, which can regulate
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Table 2. The proteins to which MtsR have close identity and similarity.
the transcription of mtsABC in response to iron availability intracellular.
The TBLASTN analysis showed that the predicted amino acid sequence of MtsR was highly conserved (Table 2), and the NCBI CD Search predicted that MtsR has three conserved domains: WHTH_GntR, Fe_dep_repr_C, and FeoA (Figure 3(c)). WHTH_GntR, and Fe_dep_repr_Cdomains are highly conserved in MtsR, which found in other DtxR homologues thatare responsive to iron, manganese, or both (Figure 1(b)). FeoA domain was found at the C-terminus of a variety of metal-dependent transcriptional regulators, which in most cases likely to be either iron or manganese [15] . Based on these observations, we concluded that mtsR is a member of the metal-dependent transcriptional regulators.
3.3. Determination of MtsR as the Iron-Dependent Transcriptional Regulator
Total RNA was isolated from HD-1 cells in BHI and in iron-restricted medium, and qPCR analysis was performed to determine whether MtsR regulated the expression of iron-transporter mtsABC at the transcriptional level. The housekeeping gene gyrA was used as an internal control in qPCR, and similar levels of
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Figure 3. The streptococcal iron transport repressor (MtsR).
amplification confirmed that the RNA quantities used as the templates in all qPCR reactions were equal. When use the RNA isolated from the HD-1 grown in BHI medium as templates, the expression of mtsA, mtsB, and mtsC were significantly up-regulated and reached the peak at the early logarithmic, de-regulated at the mid-logarithmic, and back to housekeeping gene level at the final-logarithmic phase (p < 0.05, Figure 4). This representation owed to the high-efficiency of procaryotic cells. The cells needed iron for growth, they stored iron through up-regulate iron-transporter, and once they captured enough iron, the expression of iron-transporter was up-regulated [16] . The expression tendency of the mtsABC in iron-restricted medium was consistent with that of in BHI medium, but the expression levels of mtsA, mtsB, and mtsC were showed 1.83, 13.3, and 2.11 times higher than that of in BHI medium, respectively (Figure 5). In contrast, both in Figure 4 and Figure 5 the results showed that the high transcription of the mtsABC is observed when the mtsR remain inactivate, suggesting that MtsR up-regulate mtsABC expression was very likely to lead to an increase in iron uptake by S. iniae. Meanwhile, activation of MtsR resulted in de-regulation of the mtsABC transcription, which demonstrated that mtsR
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Figure 4. The relative gene expression levels of mtsABC and gyrA in BHI medium.
up-regulated the expression of mtsABC in response to iron availability intracellular to control the iron homeostasis in S. iniae HD-1.
4. Discussion
Iron is an important nutrient for various pathogens, which can often use low environmental iron levels as a signal for the induction of virulence genes [2] . Bacteria face the problem that in acquiring sufficient iron from their surroundings is particularly acute for pathogens. For bacterial pathogen, scavenging iron from the environment is less effortless than synthesizing it de novo. Our study showed that S. iniae HD-1 in the iron-restricted medium caused by addition of NTA in the final concentration 5 mM postpones the log phase of bacterial growth 3 h. When the morphological features were examined by the Gram stain, HD-1 cells showed a long chain appearance in iron-restricted medium than that of in BHI medium. These results indicated that iron was required for the growth of S. iniae HD-1.
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Figure 5. The relative gene expression levels of mtsABC and gyrA in BHI-NTA medium.
Iron withholding by the human host is a challenge for pathogen, as the bacterium requires iron for optimal growth. At the same time, maintaining iron homeostasis is important for the bacterial physiology as well. Therefore, like other bacterial pathogens, S. iniae needs to modify iron uptake in response to changes of iron availability in the environment. To address the conundrum of iron homeostasis in S. iniae, the gene of putative iron-dependent transcriptional regulators, mtsR, was cloned fromHD-1. MtsR may have both negative and positive roles in mtsABC expression, depending on the iron availability in the cells. Using qPCR analysis, we have demonstrated that high transcription of the mtsABC genes is observed when the mtsR maintained inactivate, suggesting that MtsR repressed mtsABC expression in cells growing in BHI medium. MtsR de-regulated the expression of mtsABC in cells grown in restricted-medium, and this de-regulation as a result of MtsR activation is very likely to lead to an increase in iron uptake by S. iniae. This has demonstrated that MtsR is a DtxR homologue with an important role in iron homeostasis.
Acknowledgements
Project support was provided by grants from The Hubei Office of Education Foundation (grant nos.B2016022 to J.W.).