Microsoft Word - 1 BIOTROPIA VOL. 13 NO. 1, 2006 : 1 - 10 TRANSPOSITION AND EXPRESSION OF GFP GENE IN THE GENOME OF Vibrio harveyi TO MONITOR ITS ADHERENCE IN SHRIMP LARVAE WlDANARNI0, ANTONIUS SUWANTO^SUKENDA1', AND BlBIANA WlDIATI LAY3) '' Faculty of Fisheries and Marine Science, Bogor Agricultural University, Bogor 16680, Indonesia '' Faculty of Science and Mathematics, Bogor Agricultural University, Bogor 16680. Indonesia ''Faculty of Veterinary Science, Bogor Agricultural University, Bogor 16680, Indonesia ABSTRACT Expression of green fluorescent protein encoded by gfp gene in Vibrio harveyi was investigated to understand the ability of the gene as a molecular marker for adherence of this pathogenic Vibrio in shrimp larvae. The gfp gene was inserted into pl/ClSNot and pUTmini-Tn.') to generate a recombinant plasmid pWG02 and p\VG03, respectively; which was transferred into the three isolates of V. han'eyi employing diparental mating. Recombinant E. coli carrying p\VG02 and pWG03 resulted in green-fluorescent colonies and cells due to the production of GFP. However, all of mini-Tn.J, including mini-Tn.5-gfp were not successfully transferred to V. harveyi. Therefore, we used mini-Tn/fl (pLOFKm-gfp) for inserting of gfp gene into V. han'eyi genome. Although we could obtain relatively high (10's) transconjugans employing Tn/rt, only one of TnJO derived isolate of V. harveyi G3 (G3-Tn/flgfp) showed gfp expression and was further employed for adherence assay. Fluorescent G3-Tn70gfp cells could be observed inside the digestive tract of shrimp larvae and could be distinguished from Vibrio that naturally exist in shrimp larvae. Key words: gfp gene, Vibrio han'eyi, gene expression, shrimp larvae, molecular marker INTRODUCTION Vibrio harveyi was identified as a causative agent of mass mortalities of shrimp larvae and were frequently associated with luminous Vibrio (Lavilla- Pitogo et al. \ 990; Karunasagar et al. 1994; Ruangpan 1998, Suwanto et al. 1998). Luminescent vibriosis in shrimp larvae is characterized by lethargy, anorexia, muscle opacity, bacterial masses in the hemocoel, and luminosity of the larvae (Lavilla-Pitogo et al. 1990). Electron microscopy observation has revealed that bacteria colonize the feeding apparatus, forming bacterial plaques in heavily infected larvae, therefore, it is highly probable that the mouth is the main entrance for colonization of inner tissues (Lavilla-Pitogo et al. 1990). Pathogenicity assays based on Koch's Postulates (Madigan et al. 2003) were practically difficult to be conducted in shrimp larvae due to their relatively small size and lack of availability of Vibrio-free larvae (Widanarni and Suwanto 2000). Investigations into adherence and pathogenicity processes of this disease might ' Corresponding author: E-mail: asuvvanto@indo.net.id BIOTROPIA VOL. 13 NO. 1, 2006 greatly facilitate if a visible marker could be introduced into the bacterial cells. Pathogenic V. harveyi have observed their attachment to crustacean larvae by epifluorescence microscopy using 5-(4,6-dichorotriazin-2-yl) aminofluorescein (5-DTAF, D-16) as a marker (Soto-Rodriguez et al. 2003). One visible molecular marker which has extensively been used for studying bacterial activity in the environment is gfp, i.e. a gene encoding green fluorescent protein (GFP) from a jellyfish (Aequorea victoria) (Manning 1997). As a molecular gene marker, GFP has some advantages, such as no requirement for exogenous substrate or energy source for their visualization, sensitivity of detection, high stability, lack of toxicity, and no disturbance in cell function and growth (Josenhans et al. 1998; Ling et al. 2000). GFP as a molecular marker has been used to demonstrate the mechanism of Edwardsiella tarda infection on epithelial cells of giant gouramy (Ling et al. 2000); and Pseudomonas plecoglosicida infection in ayu (Plecoglossus altivelis) (Sukenda and Wakabayashi 2001). GFP was also successfully used as a marker in lactic acid bacteria (Lactobacillus plantarum and L. lactis) to study the possibility of using the bacteria as live vaccine carriers (Geoffroy et al. 2000). A broad host-range plasmid expressing gfp gene (pWGOl) was constructed and has been successful to tag K harveyi (Widanarni et al. 2005). However, under non selective long- term experiments without antibiotic pressure, pWGOl was highly unstable. Therefore, transposon insertion may provide an alternative method to insert gfp gene directly into the genomic DNA of V. harveyi in order to yield stable recombinants. In this report, we describe the construction in Tn vector and expression of V. harveyi carrying gfp gene in the genomic DNAs to monitor its adherence in shrimp larvae. MATERIALS AND METHODS Bacterial strains and plasmids Bacterial strains and plasmids used in this study and their relevant characteristics are described in Table 1. Escherichia coli and V. harveyi were grown in Luria Bertani (LB) medium at 37°C and Seawater Complete (SWC) medium at 28°C, respectively. LB medium was made as previously described (Sambrook et al. 1989) and SWC medium contained 5 g bactopeptone, 1 g yeast extract, 3 ml glycerol, 15 g agar, 750 ml seawater, and 250 ml distilled water. Plasmid construction and molecular techniques Recombinant plasmid pWG02 which has the lac promoter was constructed and used to drive the expression of gfp. The promoter and gfp gene were isolated from pSKLOl using £coRI sites and ligated into pUClSNot linearized with £coRI. The recombinant plasmid vector (pWG02) (Figure 1) was digested with Notl and then promoter and gfp gene were ligated into pUTmini-TnJ linearized with Noil resulting Transposition and expression of GFP gene - Widanami et al. in recombinant plasmid (pWGOS) (Figure 2). The recombinant plasmid vector was transformed into E. colt DH5a using a standard heat shock transformation (Sambrook et al. 1989) and the colonies carrying pWG02 and pWG03 were examined for green fluorescence under UV-trans- illuminator at 260 nm (Biometra Ti 1, Gottingen). Plasmid extraction, restriction enzyme digestions, agarose gel electrophoresis, gel isolated DNA fragment purification, and ligation were carried out using standard methods (Sambrook et al. 1989), and following the manufacturer's instructions. Restriction endonucleases and other enzymes were obtained from New England Biolabs Inc (Beverly, MA, USA). Bacterial mating To transfer recombinant plasmid pWG03 harboring mini-Tn5gfp into V. harveyi we used diparental mating (Suwanto and Kaplan 1992). Escherichia coli DH5a (pWGOS) donors were grown overnight in LB medium supplemented with spectinomycin and streptomycin (Sp/Sm) 50 ugmT1 at 37°C; whereas V. harveyi recipients were grown in SWC medium at 28°C. Each 1.5 ml of the donor and recipient were pelleted in a micro-centrifuge at maximum speed for 1 min, and then the cells were washed with 1.0 ml 0.85% NaCl, re-centrifuged, and suspended in 40 ul of LB medium before being spotted onto a filter (1 cm diameter; pore size 0.45 Urn; Millipore) on LB medium agar. The bacteria were allowed to conjugate at 28°C for 16 to 18 hours. At the end of the mating period, the filter containing the bacterial Transposition and expression of GFP gene — Widanarni et al. mixture was transferred into 1.5 ml microfuge tube containing 0.8 ml of 0.85% NaCl. The bacterial cells were suspended thoroughly by agitation on a vortex mixer. The transconjugants were selected on Thiosulphate Citrate Bile Salt (TCBS, Oxoid) medium supplemented with Sp/Sm (50 jugm!"1). The selective medium TCBS was used to inhibit the growth of E. coli, while allowing V. harveyi transconjugants harboring TnJgfp (resistant to spectinomycin and streptomycin) to grow. The same method was conducted to transfer recombinant plasmid pLOFKm-gfp (resistant to kanamycin) into V. harveyi and the transconjugants were selected on TCBS medium supplemented with kanamycin (100 (agml"1). Some of V. harveyi transconjugants were analyzed by Pulsed-Field Gel Electrophoresis (PFGE) with Notl restriction enzyme (Suwanto and Kaplan 1992; Widanarni and Suwanto 2000) to show the place of TnlOgfp inserted in the genomic of V. harveyi. GFP stability and pathogenicity assay Vibrio harveyi strains harboring g/p both in the plasmid pWGOl (Widanarni et al. 2005) and in the genomic's DNA were grown overnight in SWC broth supplemented with kanamycin. Sequential propagation under non selective conditions were performed by inoculating with 1:100 (v/v) to assess gfp existence by comparing duplicate colony counts on selective and non selective plates. For pathogenicity assay, two groups with three duplicates of shrimp post-larvae (PL4) were immersed for 30 min in 106 CFUml"1 of gfp recombinants and wild type of V. harveyi (final concentration), respectively, and then placed in a 2 L shrimp rearing tank. A control group was immersed in sterile scawater. Daily survival rate of shrimp larvae for 5 days were recorded and compared with the control group. Adherence assay Samples of shrimp larvae from control and treatment groups were directly observed under a fluorescence microscope. Samples from dead shrimp larvae were also inoculated onto SWC plates containing kanamycin (100 figrnl"1) to show that the dead shrimp larvae were infected by recombinant V. harveyi. RESULTS AND DISCUSSION GFP-containing plasmid construction The GFP-plasmid vector was constructed for molecular marker in V. harveyi. Two GFP vectors i.e. pWG02 and pWGOS, were constructed with lac promoter to drive the expression of gfp. Recombinant E. coli carrying pWG02 or pWG03 (mini-TnJgfp) resulted in green-fluorescent colonies and cells due to the production of GFP (Figure 3). However, all of mini-Tn5, including mini-TnJ-gfp was not successfully transferred to V. harveyi. The same results were observed by Stretton e? BIOTROPIA VOL. 13 NO. I, 2006 a b Figure 3. Fluorescence micrograph of (a) E. coli DH5a (pWG02) and (b) E. coli DHSct (pWG03) al. (1998). Therefore, we used mini-Tn70 (pLOFKm-gfp) that has been constructed by Stretton et al. (1998) for inserting ofgfp gene into V. harveyi genome. Construction of V. harveyi gfp+ employing mini-Tn/0 (pLOFKm-gfp) We could obtain relatively high transconjugants employing TnlO (Table 2). Pulsed-Field Gel Electrophoresis (PFGE) analysis of some V. harveyi transconjugants demonstrated that Tn70gfp was randomly inserted in the gcnomic V. harveyi G3 (Figure 4). However, only one of TnlO derived isolate of V. harveyi G3 (G3-Tn70gfp) resulted in green-fluorescent colonies and cells due to the expression of GFP and that fluorescence levels qualitatively was almost the same with G3 (pWGOl) (Figure 5). This result occurred due to the fact that gfp in pLOFKmg/p was constructed promoterless, so its expression depended on promoter strength in the insertion site within V. harveyi genome. GFP stability and pathogenicity assay Colonies of V, harveyi G3 (G3-Tn70gfp) that were grown on media with or without antibiotic exhibited uniform fluorescence appearance. This was not the case Transposition and expression of GFP gene - Widanarni et al. M Figure 4. PFGE profiles of genomic DNA of V. harveyi G3 (Lane M: Axel-digested genotnic DNA of R. sphaeroides 2.4.1 as a molecular size marker (Suwanto and Kaplan 19 89). Lane 1 and 8: G3 wild type, Lane 2: G3 - Tn/flgfp (KmR and showed gfp expression), Lane 3 -7: mutants 03 (KmK) Figure 5. Fluorescence micrograph of (a) V. harveyi G3 (pWGOi) and (b) G3-Tn/Ogfp for V. harveyi G3 (pWGOl). Their colonies grown on antibiotic-containing media exhibited uniform fluorescence appearance, whereas those grown on media without antibiotic showed mixture of fluorescent and non fluorescent colonies which might indicate plasmid loss. The stability of the GFP on V. harveyi G3 both in plasmid and in the genomic's DNA were investigated during sequential propagation in the absence of antibiotic selection for five successive days. Under non- selective long-term experiments without antibiotic pressure, pWGOl was highly unstable but Tn/0gfp was stably maintained in V. harveyi G3 strain (Figure 6). Insertion of Tn70gfp also did not show alteration in G3 pathogenicity to shrimp larvae (Figure 7), so that it was further employed for adherence assay. Adherence assay Sample from dead shrimp larvae showed that the dead larvae were infected by V. harveyi. Vibrio harveyi G3-Tn/0gfp could be isolated from dead shrimp larvae placed on TCBS+Km media and fluorescent G3-Tn70gfp could also be observed directly in the carcasses of dead larvae. Fluorescent G3-TnJOgfp cells were observed in the oral region at 15-30 min after inoculation and could be observed inside the digestive tract at 2-3 h (Figure 8V The concentration of V. harveyi G3-TnlOgfp used in this study was 10 CFUml". Soto-Rodriquez et al. (2003) reported that 105 CFUml"1 V. harveyi labeled with 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (5-DTAF, D-16) could be observed in the oral region of Litopenaeus vannamei mysis at 0 and 2 h after ingestion. After 4 h inoculation, individual cells could already be seen inside the middle intestine, and at 18 or 24h, the fluorescent V. harveyi were observed throughout the intestinal tract. 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