The bacterial strains, mutants, plasmids, and primers used in this study are listed in Tables 1, 2. Escherichia coli (E. coli) strains were grown at 37°C in Luria-Bertani (LB) medium (Sambrook and Russel, 2001). M. gryphiswaldense strains were grown at 30°C in sodium lactate/ ammonium chloride/yeast extract (L AY) medium as described previously (Liu et al., 2008). Heat-killed cells of V. parahaemolyticus strain 09vp109 were from the Beijing Entry-Exit Inspection and Quarantine Bureau (Beijing, China).

Bicinchoninic acid (BCA) kits were from Pierce Biotechnology (Rockford, Illinois, USA). Abs were prepared by Kirkegaard and Perry Laboratories (KPL) Biotechnology (Gaithersburg, Maryland, USA). Other chemical reagents were from Beijing Chemical Reagents Co., China.

Primers and probes were designed using ABI 7000 Primer Express software (http://www.lifetechnologies.com/global/en/home/technical-resources/software-downloads/abi-prism-7000-sequence-detection-system.html). The probe used for real-time fluorescence quantitative PCR (FQ-PCR) was labeled with 6-carboxyfluorescein reporter dye (FAM) at the 5′-end and 6-carboxy-tetramethylrhodamine quencher dye (TAMRA) at the 3′-end.

Mutant strain M. gryphiswaldense ΔF was constructed by replacing mamF with the gentamicin resistance gene (aminoglycoside acetyltransferase gene, aac). Plasmid pUC-GM was digested by KpnI to generate an aac gene fragment. The upstream and downstream fragments of mamF gene were amplified by the corresponding primers (Table 2) from genomic DNA of MSR-1, and referred to as U and D, respectively. Fragments U and D were digested by XbaI/KpnI and KpnI/PstI and then connected with the aac gene fragment by T4 DNA ligase to generate a U-aac-D fragment. The U-aac-D fragment was cloned into a suicide plasmid pUX19. The recombinant plasmid was transformed into E. coli S17-1 and then transferred into MSR-1 by biparental conjugation. Mutant bacterial strains were screened as described previously (Liu et al., 2008).

mamC, mamF, and spa genes were amplified from genomic DNA of MSR-1 or S. aureus ATCC 6538 by the corresponding primers (Table 2). The start codon of spa and the stop codons of mamC and mamF were removed during amplification. The three above fragments were recovered to generate mamC-spa and mamF-spa fragments by fusion PCR (Komeili et al., 2004). These two fragments were respectively cloned into pMD18-T simple cloning vector and transformed into E. coli DH5α. After overnight culture of the recombinant strains, two plasmids were extracted and digested with EcoRI and BamHI. The recovered fragments were then cloned into the broad host plasmid vector pBBR1MCS-2, resulting in plasmids pBBR-mamC-spa and pBBR-mamF-spa, respectively (Figure 1). These plasmids contained a linker consisting of 15 amino acids between the spa and mam genes, and recognition sites of restriction endonucleases BamHI and EcoRI flanking the sides of the mam-spa fusion genes.

Plasmids pBBR-mamC-spa and pBBR-mamF-spa were introduced into E. coli S17-1 by transformation (Sambrook and Russel, 2001) and then transferred into MSR-1 or M. gryphiswaldense ΔF by conjugation as described previously (Liu et al., 2008).

Magnetosomes or recombinant magnetosomes (BMP-As) of M. gryphiswaldense strains were isolated and purified as described previously (Li et al., 2010). The membrane proteins of BMPs or BMP-As were extracted as described by Grünberg et al. (2004) and identified by Northern blotting. The proteins of each sample (generally from 0.25 mg magnetosomes) were separated by SDS-PAGE, and the bands were transferred onto a nitrocellulose membrane by electroblotting and blocked overnight at 4°C. A dilute solution of primary Ab (mouse mAb, 0.5–5.0 mg/mL) was incubated with the membrane under gentle agitation for 1 h at room temperature. The membrane was rinsed to remove unbound primary Ab and then placed in a solution of secondary Ab (goat anti-mouse IgG) for 1 h. The secondary Ab was linked to alkaline phosphatase, which was then used to catalyze 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) into a blue precipitate in proportion to the amount of protein.

The hydrated radii and zeta potentials of magnetosomes were analyzed by a Zeta Potential Analyzer (Brookhaven Instruments Corp., Long Island, State of New York USA). Samples were prepared as described by Takahashi et al. (2010).

Chemical modification of magnetosomes by bis(sulfosuccinimidyl) suberate (BS³) was performed as described previously (Li et al., 2010). The procedure for self-assembly of recombinant magnetosome was as follows. BMP-A or Ab (each 1 mg) was resuspended in 1 mL of 1 mM phosphate-buffered saline (PBS; pH 7.4). The magnetosome and Ab suspensions were mixed, placed in a mild ultrasonic bath (30 W) for 2 min, and incubated on a rotary shaker at 150 × g for 60–90 min. The resulting BMP-A-Ab complexes were isolated by a NdFeB magnet, washed three times with 1 mL of 10 mM PBS (pH 7.4), and resuspended in 500 μL of 1 mg/mL Ab solution. After repeated dispersion, incubation, collection, and washing as above, the complexes were blocked with sterile 0.5% BSA solution overnight at 4°C and stored.

Ab concentrations before and after reaction with magnetosomes were determined using a BCA kit, and linkage rates were calculated by the equation:

Linkage rate (μg Ab/mg magnetosomes) = (C1–C2) × V/M

where C1 = Ab concentration before reaction with magnetosomes; C2 = Ab concentration after reaction with magnetosomes; V = volume of Ab solution reacting with magnetosomes; M = weight of magnetosomes conjugated with Ab. C1 and C2 were calculated by the equation γ = kx, where γ = OD₅₆₂ detected by the BCA kit; x = Ab concentration; k = slope.

Serially 10-fold diluted Vibrio suspensions (10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶; each 1 mL) were mixed with 1 mg BMP-A-Ab complexes. The mixtures were dispersed by sonication for 2 min and incubated on a rotary shaker (150 × g) for 1 h at room temperature. BMP-A-Ab-Vibrio complexes were isolated using a magnet and washed three times with 1 mL of 10 mM PBS buffer to remove free bacterial cells. Control mixtures of Vibrio and BMP-As (without Ab) were incubated in PBS under the same conditions. The resulting precipitate was resuspended in 100 μL PBS. The concentrated cell suspension was incubated for 10 min at 98°C and centrifuged at 10,000 × g for 3 min at room temperature. The supernatants were used as templates. FQ-PCR was performed as described previously (Li et al., 2010).

V. parahaemolyticus strains produce species-specific TLHs, and the tlh gene has been used as a probe to confirm the identity of Vibrio species (McCarthy et al., 1999). We measured the fluorescence intensity of tlh FQ-PCR products to estimate the number of Vibrio cells trapped by BMP-A-Ab complexes. Standard curves were constructed using a 10⁻¹–10⁻⁶ serial 10-fold dilution of Vibrio genomic DNA, and the threshold cycle (Ct) value was plotted against the log of DNA weight (ng) by linear regression. The average weight of a pair of DNA bases is 1 × 10⁻²¹ g, and the whole genome of V. parahaemolyticus (5165770 bp; Makino et al., 2003) was estimated to weigh 5.2 × 10⁻¹⁵ g. The number of Vibrio was calculated as the amount of DNA divided by the weight of genome.

SPA is an immunoglobulin G-binding protein. Magnetosome-specific expression of SPA could facilitate efficient localization and appropriate orientation of various Abs on the surface of magnetosomes. In the present study, SPA was expressed on magnetosomes by fusing its gene (spa; 1503 bp) with the abundant MM protein genes mamC (378 bp) or mamF (336 bp) from M. gryphiswaldense. The fusion genes mamC-spa and mamF-spa were generated by fusion PCR from mamC, mamF, or spa PCR products. These genes were cloned into the broad host plasmid vector pBBR1MCS-2 (5144 bp), resulting in plasmids pBBR-mamC-spa and pBBR-mamF-spa, which were then introduced into MSR-1 by conjugation. The recombinant strains harboring plasmid pBBR-mamC-spa and pBBR-mamF-spa were termed MSR-CA and MSR-FA, respectively.

To increase the amount of SPA on the recombinant magnetosomes (BMP-As), a mamF mutant strain of M. gryphiswaldense was constructed by allelic gene replacement. The native mamF gene in the MSR-1 genome was replaced by a gentamicin resistance gene (aminoglycoside acetyltransferase gene; aac), resulting in strain ΔF. The mutant strain harboring plasmid pBBR-mamF-spa was termed ΔF-FA. Recombinant magnetosomes from strains MSR-1, MSR-CA, MSR-FA, and ΔF-FA were termed BMP-WT, BMP-CA, BMP-FA, and ΔF-BMP-FA, respectively.

The expression of SPA on magnetosomes was confirmed by western blotting. The membrane proteins of purified magnetosomes were separated by SDS-PAGE and transferred onto a nitrocellulose membrane by electroblotting. The protein bands were colored by sequential treatment with primary Ab, secondary Ab, and BCIP-NBT. SPA was found in all the recombinant magnetosomes (Figure 2).

Strains as above were collected by centrifugation after 3 days of culture, and their magnetosomes were isolated and purified. Transmission electronic microscopy (Figure 3) showed generally similar morphology of WT magnetosomes (diameter range 35–84 nm) and recombinant magnetosomes. The magnetosomes of ΔF-FA were slightly smaller (24–48 nm) than those of the other strains.

The hydrated radii and zeta potentials of magnetosomes were analyzed by a Zeta Potential Analyzer. All zeta potentials were lower than −30 mV, indicating that the aqueous colloids were moderately stable. The expression of foreign proteins can affect the amount of electrical charge on interfaces between magnetosomes and aqueous solutions. The large hydrated radii of BMP-WTs indicated that several BMP-WT gathered into a larger particle in suspension. The expression of foreign proteins on magnetosomes in the case of BMP-CA and BMP-FA was associated with smaller values of hydrated radii and polydispersity, indicating improved dispersity and uniformity. ΔF-BMP-FA had the smallest hydrated radii and best dispersity because they had the largest amount of foreign proteins.

Polyclonal Abs were linked with SPA on the surface of magnetosomes by self-assembly to form functional magnetic carriers (BMP-Ab) for capturing Vibrio. In an attempt to improve linkage rate, various values of pH (6.0, 7.2, 7.9), incubation time (30, 60, 90, 120 min), and ratio of Ab to magnetosomes (250 μg, 500 μg, 1.0 mg, and 1.5 mg Ab per mg magnetosomes) were used for the linkage of BMP-CA to Ab (Figure 4). The maximal linkage rate was achieved with pH 7.2, incubation time 120 min, and ratio 1:1.

Linkage rates of different magnetosome-Ab linking methods were compared under these optimal conditions (Figure 5). BMP-WTs were linked to Ab by nonspecific adsorption or BS³, and BMP-CA and ΔF-BMP-FA were linked to Ab by self-assembly.

The percentage of Ab linked to BMP-WT by nonspecific adsorption was almost 20% and could not be ignored. However, the percentages of Ab coupled to magnetosomes by covalent linkage or self-assembly were significantly higher. The linkage rate of ΔF-BMP-FA magnetosomes to Ab was much higher than that of other magnetosomes, indicating that ΔF-BMP-FA is the best candidate for magnetic carrier carriers for IMS (Table 3).

Anti-Vibrio rabbit polyclonal Ab was linked to recombinant magnetosomes (BMP-CA, BMP-FA, and ΔF-BMP-FA) by self-assembly. WT magnetosomes (BMP-WT) were linked to Ab by BS³. Magnetosome-Ab complexes (BMP-A-Ab) were used to capture V. parahaemolyticus. FQ-PCR was performed with the species-specific gene tlh as template. A standard curve was constructed by plotting the threshold cycle (Ct) value against the log of Vibrio DNA weight (ng) by linear regression. The number of Vibrio cells was estimated based on the amount of DNA attached to the magnetosomes (Table 4).

Each type of magnetosome (1 mg) was mixed with 1 mL of diluted V. parahaemolyticus suspension (10⁻⁴; approximately 2 × 10⁸ cfu). WT magnetosomes bound a much lower number of Vibrio than did the other magnetosomes, indicating that many Ab molecules failed to work. ΔF-BMP-FA captured the highest number of Vibrio, indicating that ΔF-BMP-FA is the best candidate for a magnetic carrier of IMS for detection of pathogenic Vibrio.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.