Over the last two decades, there had been growth in aquaculture due to the increased in population growth and urbanisation. The aquatic organisms such as fish provide high-protein animal food and are digestible with high biological value as well. Globally, aquatic organisms such as finfish and shellfish had been cultured and provide about two-thirds of the total fish supply (FAO, 2003). Aquaculture with its resource-efficient method of producing aquatic organisms provide one-third of aquatic animals to the total supply of fish (Shruti and Soumya, 2012). Aquaculture faces some setbacks mainly disease outbreaks where Vibrio harveyi and Vibrio anguillarum are contributing candidates. These Vibrio spp causes serious economic losses globally that hinder the development of the aquaculture sector. The World Bank in 1997 reported that, US $3 Billion per year is global estimation of disease losses (Shruti and Soumya, 2012).
According to the Harikrisknan et al. (2011), Vibrio harveyi is a major gram-negative bacterium pathogen that cause severe Vibriosis, that lead to immense mortalities and economic loss to some animals but found everywhere around gut of some marine animals, water column and marine environments (Mahmoud and Manal, 2013). V. harveyi affects a varied range of marine vertebrates, invertebrates and marine teleosts including penaeids, sea horse, bivalves and cephalopods (Pass et al., 1987; Abraham et al., 1997; Ramesh et al., 1999; Thompson et al., 2002; Tendencia, 2004; Pang et al., 2016). According to the Kim et al. (2010), LuxR is a homologous protein that control quorum-sensing responses in all Vibrios, including HapR (Vibrio cholerae), SmcR (Vibrio vulnificus) and LitR (Vibrio fischeri). Julia et al. (2013) reported that LuxR proteins belong to bacteria regulatory protein of TetR family and located in the N-terminal domains which are part of helix-turn-helix (HTH) DNA binding.
V. harveyi, LuxR gene makes it unlike other characterized TetR-type proteins is that it clearly represses expression of some genes. LuxR gene functions as an activator, repressor, directly and indirectly controls the 625 gene expression (Van Kessel et al., 2013). LuxR with its homologs, positively and negatively control large regulons of genes (Lee et al., 2008). However, the virulence factors of V. harveyi are not completely understood (Kumaran and Citarasu, 2016).
In the present study, LuxR gene of V. harveyi HY99 was cloned, sequenced and bioinformatics analysed to provide a theoretical basis for further study of the function of LuxR protein.
1.1 Strain and vectors
Strains and vectors of V. harveyi HY99, which is a highly virulent strain, was isolated from a diseased fish in Zhanjiang watershed in Guangdong Province. The cloning vector pMD18-T was purchased from TaKaRa Biotechnology (Dalian) Company Ltd. Escherichia coli DH5 were preserved in our laboratory.
Ampicillin stock solution (100 mg/ml) was prepared with sterile water and stored at -20°C. The working concentration was 100 μl/100 ml in medium. Chloramphenicol stock solution was prepared with anhydrous ethanol, and the working concentration was 25 μg/ml. Calcium chloride solution (0.1 mol/L) used to prepared competent cells was prepared by dissolving 1.47 g of CaCl2·2H2O in 100 ml of sterile water, filtered through 0.22 μm filter and stored in a refrigerator at 4°C.
UNIQ-10 Column Bacterial Genomic DNA Extraction Kit was purchased from Sangon Biotech (Shanghai) Company Ltd. EasyPure Plasmid MiniPrep Kit and EasyPureTM Quick Gel Extraction Kit were purchased from Beijing Transgen Biotech Co., Ltd. TIANamp Bacteria DNA Kit was purchased from Tiangen Biotech (Beijiing) Co., Ltd. TaKaRa Ex Taq DNA Polymerase and T4 DNA ligase were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. Easy Pfu DNA polymerase was purchased from Tiangen Biotech (Beijiing) Company Ltd. Primer synthesis and sequence analysis were done by Sangon Biotech (Shanghai) Company Ltd.
The media used in this experiment include TSA medium (pH 7.3±0.2), TSB medium (pH 7.3±0.2), LB liquid mediumand LB solid medium (containing 1.5% agar).
2.1 DNA extraction
A single colony of V. harveyi HY99 was cultured on TSB medium, and genomic DNA was extracted using UNIQ-10 Column Bacterial Genomic DNA Extraction Kit following the manufacture’s instruction. Cloning of LuxR gene from V. harveyi HY99. The primers were designed using Primer version 5.0, according to the whole LuxR sequence. The forward primer was 5’-TAGTGATGTTCACGGTTGTAGATGCACA-3’ and the reverse primer was 5’-ATGGCAAGGAAAATGGATATGGACT-3’.
1 ml of bacterial solution 5,000 r/min centrifugation 5 min, PBS washing twice resuspended in 200 µl of double distilled water (ddw), boiled for 5 min at 100°C and centrifuged for 5 min, the supernatant was in sterile EP tube, centrifugation at 12,000 r/min for 5 min and the clear supernatant were the bacterial genomic DNA template and kept at -20°C until use.
2.2 Cloning of LuxR gene from the V. harveyi HY99
The PCR reaction system contained 1 µl of each primer, 1 µl of extracted DNA sample from V. harveyi HY99, 12.5 µl r-taq buffer, 9.5 µl of double distilled water (ddw). The PCR reaction was started with a pre denaturation step at 96°C for 5 min followed by 33 thermal cycles of denaturation step at 96°C for 30 s, annealing/renaturation at 55°C for 45 s and extension at 72°C for 1 min, terminated with a final extension of 10 min at 72°C. Finally, the PCR products were electrophoresed on a 1.0% agarosed gel.
2.3 Ligation, transformation and sequencing of target gene
The PCR product was incubated with the vector pMD-18T at 16°C overnight. The ligation product was sequenced Sangon Biotech (Shanghai) Company Ltd. LuxR gene contain 635 bp open reading frame (ORF) and encode 211 amino acids.
2.4 Bioinformatics analysis
The physical and chemical properties of protein, protein signal peptide prediction, N-glycosylation sites prediction and protein structure domain were analysed respectively (Leehouts et al., 1990; Maldonado et al., 2003; Rojo-Bezares et al., 2007; and de Jong et al., 2010). The N-terminal signal peptide sequence of LuxR protein was predicted using SignalP 4.1 Server software. Transmembrane domains were predicted using TMHMM Server 2.0 software. Subcellular localization was performed using PSORT. The potential phosphorylation sites and glycosylation sites were predicted using Soft Berry-Psite software. The amino acid sequences of other Vibrio strains similar to the studied protein were searched in NCBI using the provided BLAST online searching software. These amino acid sequences were analyzed using MEGA7.0 software by neighbour-joining method (NJ). Multiple sequence analysis of amino acids was performed using ClustalX2 software, so as to deduce the evolutionary relationship of this protein with different strains. Homologous modelling of V. harveyi LuxR protein was performed using SWISS-MODEL software, to predict its 3-D structure.
3.1 PCR amplification of LuxR gene
Strain LuxR gene was amplified by PCR, and then amplification products were analysed by agarose gel electrophoresis. The sequencing results showed that LuxR gene amplified to 1,400 bp encoded 211 amino acid residues. The resulting gene sequence was submitted to GenBank under accession number MH193370. The result of PCR amplification of LuxR gene was shown in Figure 1.
Figure 1 Cloning of LuxR
Note: M: DNA marker DL2000; 1-5: LuxR PCR product
3.2 Physiochemical properties and sequence analysis
The physicochemical properties of V. harveyi LuxR protein (Table 1) was analyzed using ExPASy (http://web.expasy.org/protparam/) software. The molecular structural formula of LuxR was C108H1696N310O316S1 with a total atom number of 3,414. LuxR is a stable hydrophilic protein with a theoretical pI value of 5.98.
Table 1 Physicochemical properties of LuxR protein
The LuxR protein was subjected to signal peptide prediction using SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) online software, and the results show that the protein contains no significant signal peptide cleavage sites. PORST (https://psort.hgc.jp/) subcellular localization prediction showed that LuxR exists in cytoplasm. The prediction using TMHMM Server 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) online software shows that LuxR contains no transmembrane domain. The prediction using SoftBerry-Psite software (http://linux1.softberry.com/berry.phtml?topic=psite&group=programs&subgroup=proloc) online software shows that the amino acid sequence of LuxR potentially contains five casein kinase II phosphorylation site, two protein kinase C phosphorylation, three N-glycosylation site, two N-myristoylation site and three microbodies C-terminal targeting signal as shown in Figure 2.
Figure 2 LuxR gene sequence and amino acid sequence
Note: N-glycosylation site (aa 101-104, 139-142,184-187); Protein kinase C phosphorylation site (aa 126-128, 138-140); Casein kinase II phosphorylation site (aa 65-68, 126-129, 127-130, 186-189, 188-191); N-myristoylation site (aa 39-44, 174-179); Microbodies C-terminal targeting signal (aa 16-18, 44-46, 75-77): * as terminator
3.3 Homology analysis with similar protein sequences of other Vibrio strains
The LuxR protein sequence of V. harveyi HY99 was subjected to homology comparison with Vibrios strains including V. campbellii and V. jasicida through NCBI Blast (http://blast.ncbi.nlm.Gov/Blast.cgi). The results show that LuxR protein is relatively stable in Vibrio (Figure 3). A phylogenetic tree was built with LuxR protein sequence of V. harveyi HY99 and similar protein sequences of other Vibrio strains using MEGA7.0 system software, and the results showed that V. campbellii and V. jasicida were clustered together, indicating that the genetic relationship between the two species was closest (Figure 4).
Figure 3 Homology comparison of amino acid sequence of LuxR protein
Figure 4 Phylogenetic tree of HY99 LuxR constructed by neighbour-joining method
3.4 Structure domain analysis of LuxR
The amino acid sequence of LuxR protein was committed to NCBI online to perform protein Blast to achieve conserved domain database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to be analysed as shown in Figure 5. The result of LuxR shows TetR_N superfamily. The SOPMA secondary structure prediction method was performed (https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl). The LuxR secondary structure was composed of 61.14% alpha helix (Hh), 5.21% extension strand (Ee), 27.49% random coil (Cc) and 6.16% beta turn (Tt). The protein transmembrane helical was no signal. The result indicated that the protein was not secreted proteins, and there was no transport across the membrane as shown in Figure 6.
Figure 5 Analysis of Functional Domain of LuxR protein
Figure 6 Secondary structure of LuxR protein in Vibrio harveyi
3.5 Prediction of tertiary structure
The homologous modelling of V. harveyi HY99 LuxR protein was performed using SWISS-MODEL online software (http://swissmodel.expasy.org/). 3-D structure model of LuxR protein subunit was obtained (Figure 7). The result described that LuxR gene belongs to LuxR family transcriptional regulator.
Figure 7 Three-dimensional structure of LuxR protein in Vibrio harveyi HY99
The fundamental of LuxR extracts protein that can bind acyl-homoserine lactone (AHL) but bound to acyl-homoserine lactone (AHL) to transcription from the right hand lux promoter (pLuxR) (http://parts.igem.org/Lux). Quorum sensing in Vibrios, LuxR-type transcription factors are good regulators (Kim et al., 2010). LuxR proteins activate and repress large regulons of genes but are rare to TetR superfamily of transcription factors.
Compared with other secreted proteins, LuxR protein had a small number of amino acids, theoretical molecular mass of 24.4 kDa and has an acidic isoelectric point. Additionally, the GRAVY value of the protein was-0.238 and instability index was 37.75 which suggested the LuxR protein can be activated in vivo. LuxR protein was hydrophilic protein, presence of no transmembrane region and had three N-glycosylation sites. LuxR peptide chain was distributed with multiple functional domains and binding sites such as three N-glycosylation site, two protein kinase C phosphorylation site, five casein kinase II phosphorylation site, three microbodies C-terminal targeting signal and two N-myristoylation site. These sites enable the protein to enter into the host cells and anchor in intracellular structures. The finding of researches of Kim et al. (2004) on prediction of phosphorylation sites in SVMS, Tian et al. (2013) on phosphoproteomic analysis of protein phosphorylation in Tetrahymena thermophila are in line with the present study showing that protein phosphorylation is a post-translation modification of protein.
After translation of LuxR protein of V. harveyi, contains no Golgi apparatus and endoplasmic reticulum which could not further process the proteins obtained, and could not perform modification of amidation or glycosylation. However, phosphorylation is not affected (Hunter, 2000) but in further studies, the construction of mutant strains with the deletion of phosphorylation sites to further perform in-depth function analysis.
The aligned protein sequences of various Vibrio strains were found that, the LuxR protein in V. harveyi was highly similar, indicating that the protein is relatively stable in the evolutionary process of Vibrio. A built of the phylogenetic tree on V. harveyi HY99 was clustered together with V. alginolyticus 40B indicating that the genetic relationship between the two species were closest, satisfying the results of morphological and biochemical classification.
The analysis on the structure of LuxR protein show that the protein contains a TetR_N superfamily conserved domain. LuxR protein belongs to HTH-type transcriptional regulator and the domain is correlated with transcriptional regulator. It has been demonstrated that the LuxR protein causes an auto inducer-dependent activation of transcription of operonL (Shadel and Baldwin, 1990). The 3-D structure model of LuxR protein subunit was obtained using SWISS-MODEL software and found that LuxR gene belongs to LuxR family transcriptional regulator. Tertiary structure of LuxR is a homodimer. The structure and configuration of homodimers vary under different environmental conditions.
In conclusion, the LuxR protein of V. harveyi HY99 was subjected to bioinformatics analysis, which the secondary structure, 3D structure and conserved domains were predicted and analyzed, with an attempt to lay a foundation for the profound understanding of the action mechanism of transcriptional regulator of LuxR protein. Also, LuxR function as an activator, repressor and can control gene expression directly and indirectly. This study laid a foundation for further research and development of vaccine.
MES carried out the cloning, molecular studies, bioinformatics analysis and drafted the manuscript. FKAK participated in the molecular studies and also assisted in the bioinformatics analysis. HP and JC conceived the study, and participated in its design, JW participated in its coordination. All authors read and approved the final manuscript.
I express gratitude to Stephen Ayiku, Beatrice Kyei-Amankwah, Gyan Ray Watson and Linda Adzigbli for their immense support during laboratory work. I express profound gratitude to Emmanuel Delwin Abarike and Adele Aglago for inspiring and motivated during the cloning of the gene. This paper was supported by Natural Science Foundation of Guangdong Province (2017A030313174); Natural Science Foundation of Guangdong Ocean University (C17379); Undergraduate Innovative and Entrepreneurial Team Project (CCTD201802); Science and Technology Program of Guangdong Province (2015A020209163).
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