CLONING AND SEQUENCING OF THE FUR GENE FROM MARINE PROBIOTIC ALTEROMO-NAS AURANTIA A 18 AND ITS POSSIBLE ROLE IN SIDEROPHORE PRODUCTION

The complete fur gene from the marine probiotic Alteromonas aurantia A18 was amplified by PCR and the cloned gene was sequenced. Results indicated that the similarity between the sequence of the fur gene from strain A18 and that from Alteromonas sp O-7 was 97%. Fur protein of strain A18 was 98% identical to that of Alteromonas sp. O-7 and 70% identical to that of E. coli. The putative fur open reading frame coded for 148 amino acids representing a protein of 16.637 kDa. The recombinant expression vector pQE30-fur was constructed to confirm function of the cloned fur gene in E. coli. We found that siderophore yields of the transformants bearing pQE30-fur (induced by IPTG) were much lower than those of the parental strain and the same transformants that were not induced by IPTG in the iron-rich medium. The results suggest that some enzymes responsible for biosynthesis and secretion of siderophores in E. coli were repressed by the Fur protein encoded by the fur gene from A. aurantia A18.


INTRODUCTION
In the past ten years, mariculture industry in China has been developing very rapidly.However, disease outbreaks are being increasingly recognized as a significant limitation on mariculture production in this country.Most of bacterial infections in marine animals were found to be caused by vibrios.So far, conventional approaches, such as the use of disinfectants and antimicrobial drugs had limited success in the prevention or cure of the bacterial diseases in marine animals.Furthermore, there is a growing concern about the abuse of antimicrobial drugs in mariculture.The massive use of antimicrobial drugs for bacterial disease control in marine animals increases the selective pressure exerted on the microbial population and encourages the natural emergence of bacterial resistance and the horizontal transfer of resistance genes in different organisms.The best alternative strategies to antimicrobial drugs in disease control would be the use of probiotic bacteria as vaccines or microbial control agents (Verschuere et al. 2000).
Iron is an essential nutrient for marine bacteria which is difficult to obtain due to its low solubility under the physiological conditions.Most of marine bacteria usually obtain iron by means of siderophore, which introduces iron into the bacterial cells by a specific cell membrane receptor (Li and Chi 2004).However, an excess of iron in the cells is toxic because of its ability to catalyze Fenton reactions and formation of active species of oxygen (Escolar et al. 1999).Therefore, iron uptake has to be exquisitely regulated to maintain the intracellular concentration of iron between desirable limits.It was found that Fur (ferric uptake regulate) protein encoded by fur gene in marine bacteria is involved in this regulation.The protein acts as a transcriptional repressor of iron-regulated promoters by virtue of its Fe 2+ -dependent DNA binding activity.There are two configurations of Fur protein in an equilibrium which is displaced by Fe 2+ towards the form competent for binding DNA and thus for repression of transcription.The lack of iron results in derepression of an entire collection of genes for biosynthesis and transport of siderophore (Escolar et al. 1999).
In the previous studies (Wang et al. 2002) we found that the marine probiotic A18 can be successfully applied to control the bacterial diseases in marine fish and improve the quality of rearing seawater.The probiotic strain A18 was identified and found to be Alteromonas aurantia (Li et al. 2001).In another study (Li et al. 2008) we found that a mutant of Alteromonas aurantia A18 can produce siderophores with hydroxamate group, which are related to the strong inhibition of the pathogenic bacteria.In order to elucidate the regulation mechanisms of the Fur protein in the probiotic strain A18, the fur gene from strain A18 was cloned, sequenced and expressed in E. coli in the present study.

Bacterial strains and plasmids
The bacterial strains and plasmids used in this study are listed in Table 1.

Growth media and cell cultivation
The marine Alteromonas aurantia A18 was cultivated at 27°C in marine broth 2216E and E. coli was grown at 37°C on LB medium with or without 50 mgml -1 of Ampicilin or/and Kanamycin.

Isolation of total genomic DNA and cloning the fur gene from Alteromonas aurantia A18 by PCR
The total genomic DNA was isolated from strain A18 according the methods described by Sambrook et al. (1989) and was used as the template for cloning of the fur gene by using PCR techniques.The primers of fur gene of A. aurantia A18 were designed by the homologous clone strategy according to the strain of Alteromonas sp.O-7 [8] for PCR were as follows: the forward primer was 5'-TGAAATG-ACAGACCACAACTTAGAGC-3' a n d t h e r e v e r s e p r i m e r w a s 5'-TGGCGAAGTGGAAGGCTAGTTAC-3.The conditions for PCR amplification were as follows: initial denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing temperature at 50 °C for 1 min, extension at 72°C for 1 min, final extension at 72°C for 10 min.The PCR was run for 30 cycles.0.25 ml of TaKaRa Ex Taq (5U•ml -1 ) was used as DNA polymerase.PCR cycler was GeneAmp Ò PCR System 2400 made by PERKIN ELMER.The size of PCR product was expected to be 0.5kb.The PCR product was electrophoresed on a 0.6% agarose gel.

DNA ligation and transformation
The amplified DNA fragment with 0.5 kb by PCR was excised and purified from the gel by using PCR Fragment Recovery Kit (Shanghai Bioasia Biotechnology CO.LTD) according to the procedures offered by the manufacturer.The DNA fragment was ligated into pUCm-T Vector by using T 4 DNA ligase at 16°C for 16 h and was transformed into the competent cells of E. coli JM109.The transformants with pUCm-T carrying the fur gene was selected on LB plate with Ampicilin.

Identification of the fur gene on the plasmids
The transformants were cultivated in liquid LB medium with Ampicilin at 37°C for 12-16 h.The plasmids were isolated from the cultivated transformant cells according to the methods described by Sambrook et al. (1989).The fur gene was amplified by PCR using the isolated plasmid as the template.The plasmid with the fur gene was named pUC-fur.

Sequencing of the fur gene
Sequencing of the fur gene on the pUC-fur was carried out by the Shanghai Bioasia Biotechnology CO.LTD.

Expression of the fur gene in E. coli QC1732
The fur gene was amplified by PCR using pUC-fur as template.The primers for PCR were as follows: the forward primer was 5'-CGGGATCC(BamH I) ATGACAGA CCACAAC-3' and the reverse primer was 5'-CCCAAGCTT(Hind III) CTACTCA CCATTTTC-3'.The conditions for PCR amplification were the same as those described above.The PCR products were ligated into pGEM T-easy by overnight and the fur gene on pQE30 in E. coli QC1732 (pQE30-fur) was confirmed by using the same methods described above.The plasmid obtained from the positive transformants was named pQE30-fur.Expression of the fur gene in the transformants was induced in liquid LB medium with 1mmol•l -1 of IPTG and 50 mg•ml -1 of Kanamycin for 10h.

Determination of siderophore
The non-specific assay of siderophore in the supernatants of the culture was carried out using the modified CAS method described by Schwyn et al. 1987.The amount of siderophore in the solution was calculated according to the following equation: A is OD 630nm value of the reaction mixture of the supernatant of the culture and the same volume of CAS solution.The OD 630nm value of the liquid LB medium was used as a blank.Ar is OD 630nm value of the reaction mixture of the liquid LB medium and the same volume of CAS solution.

Statistical methods
Results of the amount of siderophore production between induced and uninduced fur expressions were compared by repeated measures ANOVA.
Results are expressed as means ± SD.Differences with a value of P < 0.05 were considered significant Carlos H et al. 2002).

Amplification of the fur gene by PCR
When the total genomic DNA from strain A18 and the primers were used for PCR amplification, only one single band with about 500 bp was obtained in the PCR products (Figure 1).

Sequencing of the PCR products
After purification of the PCR products from the gel, the PCR products were ligated into plasmid pUCm-T and transformed into the competent cells of E. coli JM109.The positive transformants on LB plate including Amp, X-gal and IPTG were selected and the plasmids in the transformants were isolated.
After the fur gene on the plasmid was confirmed by PCR (Figure 2) the fur gene was sequenced.The whole nucleotide sequence of the fur gene is shown in Figure 3.

Analysis of the Fur proteins
Amino acid sequence of the Fur protein deduced from the sequence of the fur gene in strain A18 is shown in Figure 4.This results showed that the putative fur open reading frame encoded 148 amino acids with molecular mass of 16.637kDa.The homology analysis proves that strain A18 was closely related to Alteromonas sp 0-7.

Involvement of the fur gene in siderophore biosynthesis in E. coli QC1732
Figur 5 shows the analysis of the PCR products from the expression vector containing the fur gene (PQ 30-FUR).The positive clones were used for the experiments to determine the amount of siderophores.
The amount of siderophores in the cultures used in this experiment are given in table 2. Results have shown that the QC1732 (PQE30fur) when induced to express Fur protein in iron rich medium has resulted in significantly low amount of siderophores.Tsujibo et al. (2000) observed that a serine protease-encoding gene of marine Alteromonas sp.O-7 is regulated by the Fur protein.However, it is still unknown whether the Fur protein is related with siderophore biosynthesis and secretion in the marine Alteromonas sp.O-7.

DISCUSSION
One single band with about 500bp obtained from PCR amplification reactions (Figure 1) indicated

Ar A Ar
The amount of siderophore(%)= ×100% that the primers designed in this study could specifically bind to the total genomic DNA of strain A18 and the expected 500 bp fragments were amplified during PCR.
When the primers for amplification of the fur gene by PCR were designed, the start codon (ATG) and the stop codon (TAG) were inserted into the primers.Therefore, the amplified nucleotide sequence of the fur gene contained the whole Open Reading Frame (ORF).The analysis of the whole nucleotide sequence shows that ORF of the fur gene was composed of 447 bp.(Tsujibo et al. 2000).reported that the ORF of the fur gene from marine Alteromonas sp.O-7 has ATG start codon at 497.
The sequences of the fur genes from some negative bacteria available from GenBank and the sequence of the fur gene obtained in this study were aligned and the phylogenetic tree was constructed by using the software DNAstar.The phylogenetic tree shows that the nucleotide sequences reveal significant similarity with the fur genes from the same genus.For example, the similarity between the sequence of the fur gene from strain A18 used in this study and that from Alteromonas sp O-7 was 97%.This means that strain A18 was closely related to Alteromonas sp O-7 and the fur gene was highly conserved.
It can be observed from Figure 4 that the putative fur open reading frame coded for 148 amino acids representing a protein of 16.637 kDa.It has been reported the fur gene from Alteromonas sp.O-7 encodes a protein of 148 amino acids with a calculated molecular mass of 16.682 kDa (Tsujibo et al. 2000).The Fur protein of E. coli is a cytoplasmic 17 kDa polypeptide which binds iron as corepressor and consequently binds to the Fur-box, repressing gene transcription under iron-rich conditions (Escolar et al., 1998).Then, the amino acid sequences of the Fur protein from some negative bacteria available from GenBank and the sequence of the Fur protein from strain A18 were aligned and the phylogenetic tree was constructed by using the software DNAstar.The results of the phylogenetic analysis (data not shown) show that the similarity between the amino acid sequence of Fur protein from A. aurantia A18 and that from Alteromonas sp O-7 was 98.6% whereas the similarity between the aminci acid sequence of Fur protein from A. aurantia A18 and that from E. coli, S. oneidensis MR-1, Y. pestis was 70.3%, 72.7% and 68.2%, respectively.The results of the phylogenetic analysis (data not shown) also show that the similarity between the amino acid sequence of Fur protein from A. aurantia A18 and that from three species of Vibrio was over 69%, whereas the similarity between the amino acid sequences of Fur proteins from different species of Vibrio was over 91%.This again proves that strain A18 was closely related to Alteromonas sp O-7 and the Fur proteins from different Gram negative bacteria were highly conserved.E. coli QC1732 is a fur null mutant strain and thus, under iron-rich conditions, there is no repression of the Fur-regulated genes including those for siderophore biosynthesis and secretion.After the recombinant expression vector pQE30-fur constructed was transformed into the competent cells of E. coli QC1732, the fur gene in E. coli QC1732 (pQE30-fur) was confirmed by PCR (Figure 5).
When the single colony of E. coli QC1732 (pQE30-fur) was cultivated in liquid LB medium plus 50 mg•ml -1 of Ampicilin and Kanamycin at 37°C and OD 600nm value of the culture reached to 0.5, expression of the fur gene in E. coli QC1732 (pQE30-fur) was induced by adding 1mmol•l -1 of IPTG for 10 h.Then the amount of siderophore in the supernatant of the culture was determined (Table 2).The culture from a single colony of E. coli QC1732 without pQE30-fur was used as the control (Table 2).In addition in order to reduce the amount of Fe 2+ in the culture medium, 2,2-dipyridyl (the final concentration of it was 150 mm) was added to the medium to chelate iron Aso H et al.

2002),
The results in Table 2 indicate that when iron was scare, the amount of siderophore in the 2,2dipyridyl-adding cultures of E. coli QC1732, E. coli QC1732 (pQE30-fur) and QC1732 (pQE30-fur) induced by IPTG, was almost the same.This means that siderophore biosynthesis in E. coli was derepressed in the iron-deficient medium, resulting in increase in the amount of siderophores in the medium plus 2, 2-dipyridyl.However, the amount of siderophore was greatly reduced in the cultures grown in the iron-rich media, suggesting that siderophore biosynthesis in E. coli grown in the iron-rich medium was significantly repressed.

CONCLUSION
The amplified nucleotide sequence of the fur gene is contained 447bp.The homology analysis proved that the strain A18 is closely related to Alteromonas sp O-7.Statistically significant reduction of siderophore production by E coli strain with fur gene expression vector was observed when induced with IPTG.

ACKNOWLEDGEMENT
This study was supported by the National Natural Science Foundation of China (Grant No is 30328021).

Figure 5 .
Figure 5. Analysis of PCR products by agarose gel electrophoresis for positive clone of pQE30-fur

Table 1 . Bacterial strains and plasmids JUNFENG
LI ET AL: CLONING AND SEQUENCING OF THE FUR GENE using T 4 DNA ligase at 16°C for 16 h and transformed into the competent cells of E. coli JM109.