Molecular characterization of Vibrio harveyi ... - Wiley Online Library

Environmental Microbiology (2007) 9(2), 322–331

doi:10.1111/j.1462-2920.2006.01140.x

Molecular characterization of Vibrio harveyi bacteriophages isolated from aquaculture environments along the coast of India Mockshanath M. Shivu,1 Bettada C. Rajeeva,1 Shivani K. Girisha,1 Indrani Karunasagar,1 Georg Krohne2 and Iddya Karunasagar1* 1 Department of Fishery Microbiology, Karnataka Veterinary, Animal and Fisheries Sciences University, College of Fisheries, Mangalore – 575 002, India. 2 Division of Electron Microscopy, University of Wuerzburg, Biozentrum, Am Hubland 97074, Wuerzburg, Germany. Summary Seven bacteriophages specific to Vibrio harveyi, the causative agent of luminous vibriosis in shrimp, were isolated from coastal aquaculture systems like shrimp farms, hatcheries and tidal creeks along the east and west coast of India. All the seven phages were found to have the typical head and tail morphology with double-stranded DNA as genetic material. Morphologically, six phages were grouped under family Siphoviridae and one under Myoviridae. These phages were further characterized with respect to host range, morphology and structural proteins. Genomic fingerprinting was carried out using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD). Major capsid proteins of all the phages detected by SDSPAGE were distinct from one another. All the phages were found to be highly lytic for V. harveyi and had different lytic spectrum for the large number of isolates tested. Six of the seven phages isolated had a broad lytic spectrum and could be potential candidates for biocontrol of V. harveyi in aquaculture systems.

Introduction Luminous Vibrio harveyi, the causative agent of luminous vibriosis, is recognized as a primary pathogen of penaeid shrimp throughout Asia and Latin America (Sunaryanto Received 29 July, 2006; accepted 10 August, 2006. *For correspondence. E-mail [email protected]; karuna8sagar@ yahoo.com; Tel. (+91) 824 2246384; 2243180; Fax (+91) 824 2246384; 2243180.

and Mariam, 1986; Jiravanichpaisal et al., 1994; Pizzutto and Hirst, 1995; Liu et al., 1996; Alvarez et al., 1998; Vandenberghe et al., 1998) where shrimp aquaculture is a major industry. Vibrio harveyi has also been reported to cause diseases in finfish (Kraxenberger-Beatty et al., 1990; Saeed, 1995; Hispano et al., 1997; Ishimaru and Muroga, 1997; Alvarez et al., 1998). Until recently various antibiotics were used extensively as prophylactics and therapeutics in hatcheries and farms, resulting in emergence and spread of antibiotic resistant bacteria in the environment (Karunasagar et al., 1994). A recent dimension to the problem has been the ban imposed by seafood-importing countries on shrimp containing even traces of antibiotic residues. This has virtually resulted in total ban on the use of antibiotics in shrimp culture. Hence the search for alternative ecofriendly methods to control bacterial diseases has gained considerable importance. Historically, phages have been employed as biological control agents and have been suggested as alternative for antibiotic therapy in both human and veterinary (Barrow and Soothill, 1997; Alisky et al., 1998; Barrow et al., 1998; Sulakvelidze et al., 2001; Summers, 2001; Cerveny et al., 2002) medicine and in aquaculture (Wu et al., 1981; Nakai et al., 1999; Park et al., 2000; Nakai and Park, 2002). The study of marine bacteriophages is of immense ecological significance. It is now widely accepted that viruses are the most abundant biological entities in the sea and that most marine viruses are bacteriophages (Bergh et al., 1989; Fuhrman, 1999). Presently, a major role has been assigned to bacteriophages not only in maintaining the population densities and diversity of bacterial species, but also in significantly influencing the biogeochemical and ecological processes including nutrient cycling, carbon flow and genetic transfer (Gill et al., 2003). Vibrios are common microflora of the coastal marine environments and phages infecting many Vibrio spp., including V. cholerae and V. parahaemolyticus have been recorded in the coastal environments (Nakanishi et al., 1966; Koga and Kawata, 1981). The association of bacteriophage with V. harveyi was first reported by Ruangpan and colleagues (1999), who demonstrated the toxicity of V. harveyi to Penaeus monodon when associated with a bacteriophage specific to V. harveyi. Oakey and Owens (2000) isolated a new

© 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd

Molecular characterization of Vibrio harveyi bacteriophages 323 Table 1. Phage isolates with respective host bacteria, source, plaque size and genome size. Phage isolates

Host bacteriaa

Source

Plaque size (diameter, mm)

Plaque

Genome sizeb (kb)

Viha1 Viha2 Viha3 Viha4 Viha5 Viha6 Viha7

VH VH VH VH VH VH VH

Hatchery water Hatchery water Hatchery water Creek water Hatchery water Hatchery water Hatchery water

3–5 3–5 4–6 1–3 0.5–1 1–2 5–6

Clear Clear Turbid Clear Clear Clear Clear

94 94 70 85 83 60 44

017 020 025 042 102 036 039

a. Bacterial isolates from our own culture collection. b. Approximate size of genome estimated by RFLP pattern using Kodak 1D software.

temperate bacteriophage of V. harveyi, which they termed VHML (Vibrio harveyi Myovirus-Like). Munro and colleagues (2003) and Austin and colleagues (2003) demonstrated that VHML conferred virulence to V. harveyi isolates by infecting avirulent V. harveyi with the bacteriophage and converting them to virulent state. Karunasagar and colleagues (2005) and Vinod and colleagues (2006) isolated a bacteriophage, which had lytic activity against several V. harveyi cultures isolated from shrimp culture environments. Studies carried out in laboratory microcosms and consequent trials in shrimp hatcheries suggested that the phage could be used to control V. harveyi populations during a disease outbreak in a hatchery (Vinod et al., 2006). The bacteriophages were also found to be useful in the control of biofilm formed by V. harveyi (Karunasagar et al., 2006). Pasharawipas and colleagues (2005) isolated and characterized a bacteriophage termed Vibrio harveyi siphoviridae-like phage (VHS1) from black tiger shrimp rearing pond in Thailand. Narrow host range and phage-induced virulence limits the use of phages as biocontrol agents. Vibrio harveyi is the most abundant aerobic normal flora in shrimp hatchery systems and is responsible for producing what is perceived as the widest spectrum of disease of any bacterial pathogen in aquaculture. Therefore, phages that are capable of infecting a wide range of V. harveyi strains would be potentially valuable as therapeutic and diagnostic agents for control of luminous vibriosis. In this context, for better understanding of phages as biocontrol agent

A

B

C

D

against V. harveyi, we attempted isolation of several phages from the aquaculture environment, studied their host range, protein profile and genetic similarity. Results Isolation of bacteriophages Out of hundred samples of water analysed, 42 V. harveyi (data not shown) as well as seven bacteriophages for V. harveyi were isolated in this study. The samples that were positive for the phage did not yield any V. harveyi isolates. Six phages were isolated from west coast samples – five from shrimp hatchery water (Viha1, Viha2, Viha5, Viha6 and Viha7) and one from creek water (Viha4). The seventh phage Viha3 was isolated from east coast hatchery water. Host bacteria used for the isolation of each phage, source, plaque size, plaque turbidity and approximate genome size is summarized in Table 1. All phages except Viha3 produced clear plaques and on repeated subcultures, there was no lysogenization as indicated by lack of appearance of any colonies within the clear plaques. Morphology of phages The seven bacteriophages isolated were examined by electron microscope in order to study their morphology. All the phages had an isometric head with a tail (Fig. 1A–G), thus belonging to the order Caudovirales. Phages Viha1,

E

F

G

Fig. 1. Electron micrographs of phages isolated from aquaculture environments. Panels A to G, phages Viha1, Viha2, Viha3, Viha4, Viha5, Viha6 and Viha7 respectively.

© 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 322–331

324 M. M. Shivu et al. Table 2. Morphological features of V. harveyi phagesa. Additional features Phage

Family

Head diameter (nm)

Viha1 Viha2 Viha3 Viha4 Viha5 Viha6 Viha7

Siphoviridae Siphoviridae Siphoviridae Myoviridae Siphoviridae Siphoviridae Siphoviridae

56 ⫾ 5 53 ⫾ 3 56 ⫾ 5 114 ⫾ 9 92 ⫾ 6 Elongatedb 58 ⫾ 3

Tail length (nm)

Tail diameter (nm)

Collar

Base plate

Tail pins

Terminal bulb

176 ⫾ 9 200 ⫾ 18 211 ⫾ 22 192 ⫾ 22 175 ⫾ 19 126 ⫾ 12 194 ⫾ 16

9⫾1 8⫾1 9⫾1 24 ⫾ 3 19 ⫾ 2 11 ⫾ 1 9.5 ⫾ 1

– – – + – – –

– – – + – – –

– – – + – – –

+ + + – + + +

a. The values are the means of nine independent measurements (mean ⫾ standard deviation) for different phage particles. b. Head length 112 ⫾ 6 nm and breadth 48 ⫾ 3 nm.

Viha2, Viha3, Viha5, Viha6 and Viha7 were found to have long flexible non-contractile tails and hence placed in family Siphoviridae while phage Viha4 had a rigid contractile tail and was classified as a member of the family Myoviridae. The size of head and tail for each of the seven phages is given in Table 2. The capsid size of phage Viha4 was twice that of the other phages except for Viha5 which had a comparatively large capsid among Siphoviridae members. All were double-stranded DNA phages. Burst size and latent period The one-step growth curve of Viha1 was determined. The latent period was about 90 min and the average burst size was around 105 pfu per cell, calculated as the ratio of the final count of the liberated phage particles to the initial count of infected bacterial cells during the latent period. Host range of phage isolates The lytic spectrum of all the phages was tested on 183 V. harveyi cultures isolated from India and a collection from different parts of the world. Viha1, Viha2, Viha3, Viha4, Viha5, Viha6 and Viha7 each lysed 127 (69.40%), 101 (55.19%), 120 (65.57%), 74 (40.44%), 27 (14.75%), 71 (40.80%) and 124 (67.76%) isolates respectively. Table 3 represents the host range of each phage (only results of few isolates are shown). All phages showed broad lytic spectrum, lysing more than 40% of host culture except for Viha5, which had a narrow lytic spectrum of 15%. None of the phages in this study lysed the other Vibrio species like V. parahaemolyticus, V. cholerae, V. vulnificus, V. alginolyticus that are commonly encountered in coastal environments and also other genera of bacteria such as Escherichia coli (Table 3). Characterization of phage nucleic acid Digestion with RNase and S1 nuclease did not affect the phage nucleic acids thus confirming that all the seven

phages in this study are double-stranded DNA phages. DNA of all the phages was subjected to digestion by using 14 different restriction enzymes. Of these, only HincII and HpaII were able to digest DNA of all the phages and yielded discriminatory patterns (Fig. 2A–D). Phages identified as belonging to Siphoviridae exhibited different but closely related digestion patterns, except for Viha1 and Viha2 that were unique in their digestion pattern for the entire enzyme range tested. In contrast, the Myoviridae phage, Viha4 DNA exhibited a unique band pattern with the enzyme XbaI (data not shown). DNA of all the phages in this study were found to be highly refractory to digestion by several commonly used enzymes including BamHI, HaeIII, BclI and PstI. The phage genome sizes estimated from restriction fragment length polymorphism (RFLP) pattern were approximately 94 kb for Viha1 and Viha2, and for Viha3, Viha4, Viha5, Viha6 and Viha7 it was 70 kb, 85 kb, 83 kb, 60 kb and 44 kb respectively. Thus, the difference in RFLP pattern gives evidence that the phages isolated in this study were distinct from one another except for Viha1 and Viha2. Phage structural protein profiles To compare the relatedness of the phage isolates, which are distinguishable by RFLP, the structural protein profile of these phages was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 3). All the phages showed two or three main protein bands as well as several minor bands. The phage Viha1 (lane 7, Fig. 3) showed a major band at 68 kDa and another at 35 kDa. The phage Viha3 (lane 6, Fig. 3) showed similar protein pattern. The phage Viha2 (lane 5) also showed similar pattern except that the 68 kDa band was very faint. The lone representative of Myoviridae, Viha4, showed a different pattern with a double band at about 70 kDa and at about 35 kDa (lane 4). The phage Viha7 had major bands at 68 and 40 kDa. The phage Viha5 had a prominent band at 32 kDa and at 90 kDa (lane 2). On the other hand, Viha6 had prominent bands at 60 and 65 kDa (lane 1).


Molecular characterization of Vibrio harveyi bacteriophages 325 Table 3. Lytic activity of different phages on some representative bacterial isolates. Lysis by bacteriophagesa Bacterial reference strains b

V. harveyi (VH 017) V. harveyi (VH 020)b V. harveyi (VH 025)b V. harveyi (VH 036)b V. harveyi (VH 039)b V. harveyi (VH 042)b V. harveyi (VH 102)b V. harveyi (BB120)b V. harveyi (BB886)b V. harveyi (LMG10946)c V. harveyi (LMG11224)c V. harveyi (ACMM 20)c V. harveyi (VIB 585)c V. harveyi (LMG16863)c V. harveyi (STD3-986)c V. harveyi (STD3-1024)c V. harveyi (LMG11755)c V. harveyi (LMG13949)c V. harveyi (LMG19714)c V. harveyi (LMG10947)c V. harveyi (LMG11659)c V. harveyi (LMG7890)c V. harveyi (LMG4044)c V. harveyi (LMG11660)c V. parahaemolyticus VP014b V. alginolyticus (VA 012)b V. cholerae (ATCC 39315)b V. vulnificus (ATCC 27562)b E. coli (ATCC 25922)b

Origin

Viha1

Viha2

Viha3

Viha4

Viha5

Viha6

Viha7

India India India India India India India India India India India Australia Australia Spain Ecuador PRC Bahamas Thailand Japan Red sea USA USA USA USA India India Bangladesh Florida FDA

+ + + + + + – – – + + – – (+) + + – + – + + + + + – – – – –

+ (+) – + + – – – + + – – – + (+) – – + + + + – + – – – – – –

+ + + – + + + – – (+) + – + + + + – + – + (+) (+) (+) + – – – – –

+ + – + + + + – + – + – – – + – + (+) – + + – + – – – – – –

+ + – – + – – (+) – + – + (+) + – – – – – – + – – – – – – – –

+ + – – + + + – – – – – + – + + + + + – + – + + – – – – –

+ + + + + + – + + + + + + + + + + + + + + + + + – – – – –

a. Lysis patterns of bacteriophages, + plaques; – no plaques; (+) turbid plaques. b. Our culture collection. c. Culture collection of microorganisms from ARC, University of Ghent, Belgium.

Genomic fingerprinting by randomly amplified polymorphic DNA analysis Two primers P-2 and P-1 were used for randomly amplified polymorphic DNA (RAPD) fingerprinting of seven bacteriophage DNA samples. All the Siphoviridae phages were found to yield different pattern except for Viha1 and Viha2 which showed a nearly similar band pattern when primer P-2 was used (Fig. 4B). On the other hand, when primer P-1 was used, four Siphoviridae phages designated Viha1, Viha2, Viha3 and Viha7 showed fairly similar band patterns (Fig. 4A). In contrast to Siphoviridae, the Myoviridae phage was found to be yielding distinct pattern by both primers P-2 and P-1.

Discussion Bacteriophages are ubiquitous in nature and are generally isolated from the ecosystems that are habitats for the host bacterial strains. Though there are many reports of isolation of Vibrio phages from coastal and marine environment (Nakanishi et al., 1966; Barros et al., 1978; Koga and Kawata, 1981; Ackermann et al., 1984; Muramatsu

and Matsumoto, 1991; Matsuzaki et al., 1992; DePaola et al., 1998; Miller et al., 2003), there are very few published reports on isolation of V. harveyi phages, including lysogenic ones (Ruangpan et al., 1999; Oakey et al., 2002) and lytic phages (Vinod, 2002; Karunasagar et al., 2005; 2006; Pasharawipas et al., 2005; Vinod et al., 2006). But there is no information on the lytic spectrum of naturally occurring phages against host bacteria isolated from different geographical regions. From 100 samples analysed in this study, seven lytic phages were isolated. In parallel to this, 42 V. harveyi isolates were also isolated from these samples, which provides evidence that phage and host bacteria coexist in aquatic environment. However, samples that were positive for bacteriophages did not yield V. harveyi isolates, suggesting that the lytic phage population was dominating at the time of sampling. Six of the seven phages isolated were from hatchery water. This gives a clear indication that shrimp hatchery environment, being the habitat for enrichment of V. harveyi, would be a good source for isolation of phages. The phages isolated in this study were classified into two families Siphoviridae and Myoviridae based on ICTV rules of nomenclature (Van Regenmortel et al.,


326 M. M. Shivu et al. 1

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7

8

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3

4

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8

M

21,226

21,226 5,148 3,530

5,148 3,530

2,027 1,375

2,027

831 564

831 564

B

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1

21,226

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21, 226

5,148

5,148 3,530 2,027

2,027 1,584

831

Fig. 2. A. Agarose gel (0.8%) showing electrophoretic patterns of HincII restriction digestion of phage DNA. Lanes 1, 3, 5 and 7, HincII digests of Viha1, Viha3, Viha2 and Viha4 phage DNA. Lanes 2, 4, 6 and 8, respective undigested phage DNA. Lane M, l/EcoRI/HindIII digest. B. Lanes 2, 4 and 6 HincII digests of Viha6, Viha5 and Viha7 phage DNA. Lanes 1, 3 and 5, respective undigested phage DNA. Lane M, l/EcoRI/HindIII digest. C. Lanes 1, 3, 5 and 7 – HpaII digests of DNA of Phage Viha1, Viha3, Viha2, Viha4. Lanes 2, 4, 6 and 8 – undigested DNA of phage Viha1, Viha3, Viha2, Viha4. Lane M – l/EcoRI/HindIII digest. D. Lane 1 – l/EcoRI/HindIII digest. Lanes 2, 4 and 6 – HpaII digests of DNA of phage Viha6, Viha5, Viha7. Lanes 3, 5 and 7 – undigested DNA Viha6, Viha5, Viha7.

phages (Viha1, Viha3 and Viha7) lysed over 65% of the strain tested while Viha2, Viha4 and Viha6 lysed over 40% of the host strains. Only Viha5 had a narrow spectrum of 14% lytic activity of host strain. This is the first report on the lytic spectrum of naturally occurring phages against a large collection of host bacteria. The study is also unique

2000). Six of the seven phages isolated belonged to the family Siphoviridae suggesting that members of this family are more prevalent in aquaculture environment compared with members of Myoviridae. This study illustrates the diversity and host range of V. harveyi phages in aquaculture systems. Three of the 1

2

3

M

4

5

M

6

7 97,400 68,000 43,000

Fig. 3. SDS-polyacrylamide gel (12%) electrophoretic profiles of phage structural proteins. Lane M, protein molecular weight marker (in Daltons), Lanes 1–7, Viha6, Viha5, Viha7, Viha4, Viha2, Viha3 and Viha1 respectively.

29,000

20,000

14,300


Molecular characterization of Vibrio harveyi bacteriophages 327

A

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3,000

2,000 1,500 1,200 1,031 900 800 700 600 500

B

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3,000 2,000 1,500 1,200 1,031 900 700 600

Fig. 4. A. RAPD pattern of bacteriophage DNA with primer P-1. B. RAPD pattern of bacteriophage DNA with primer P-2. Lane 1, Viha6; lane 2, Viha5; lane 3, Viha7; lane 4, Viha4; lane 5, Viha3; lane 6, Viha2; lane 7, Viha1.

in that isolates from different parts of the world were used to examine the lytic spectrum. The wide spectrum of activity of these phages suggests that these have a potential for application as agents for biocontrol of luminous V. harveyi in aquaculture environments. Vinod and colleagues (2006) have recently published evidence to show that lytic bacteriophages of V. harveyi belonging to family Siphoviridae could be used for biocontrol of this pathogen in experimental hatchery system. In this study, Siphoviridae family had phage members with both broad and narrow host range (Table 1). Phage Viha5 with narrow host range also showed a smaller plaque size and the plaque size generally correlated with the host range (Table 1). The susceptibility of bacterial strains to phage lysis varies and this may be due to variation of receptor molecules, restriction modification system in the host, or phage resistant system (Duckworth et al., 1981). The bacteriophages isolated in this study showed divergence in size and structure (Table 2). The members of V. harveyi phage belonging to Siphoviridae family described so far by Pasharawipas and colleagues (2005) and Vinod and colleagues (2006) had features similar to the members of the family isolated here. The other

reported VHML phage reported by Oakey and Owens (2000) had a head size much smaller (40–50 nm diameter) compared with the member of Myoviridae reported here (114 ⫾ 9). The size of the Myoviridae member reported here is in the range reported for this family of phages by Ackermann (1999). The genome size of the phages described here ranged from 44 to 94 kb. The genome size of siphophages has been reported to be ranging from 40 to 134 kb and the genome size of the only V. harveyi siphophage reported so far, VHS1 was 83 kb (Pasharawipas et al., 2005). The member of Myoviridae reported here had a genome size of 85 kb and members of this family have been reported to have a larger genome size of 200–300 kb (Hill et al., 1989). However, the myovirus-like phage reported by Oakey and colleagues (2002) had a genome size of 43 kb. The larger genome size of the myovirus-like phage reported here is consistent with the larger head size reported here. To further characterize the seven phages, the structural protein composition was analysed by SDS-PAGE. Two to three major structural proteins and several minor proteins were detected in all the phages. The banding pattern of the phages revealed differences among them. However, the phages Viha1, Viha2 and Viha3, had almost similar protein profile. Although six phages belong to one family, there were differences in the structural protein. It has been observed previously that the major structural proteins of phages classified in the same DNA homology group are conserved (Prevots et al., 1990). It may be speculated that some difference in the minor proteins found in this study may be responsible for the diversity of host range, exhibited by these phages, as the same case was reported for T-even phages of E. coli (Hantke, 1978). Hence it may be suggested that, though the phages belong to same family, the structural proteins are unique to some of the phages. This study shows that all the phages isolated had double-stranded DNA genome. Restriction digestion pattern of genomic DNA indicated a high degree of similarity between Viha1 and Viha2 while rest of the phages appeared to be distinct from one another. Though the phages Viha1 and Viha2 showed similar protein and RFLP pattern, they differed in their host range. Similar results have been reported in Erwinia amylovora (Gill et al., 2003). An important observation made in this study was that most of Siphoviridae phages had a restriction site for HpaI, but Viha6 which showed uniqueness in its genome was refractory to this enzyme. Viha6 also showed uniqueness in its morphology in that it had a much elongated head with short tail which none of the other Siphoviridae phages had in this study. Another important observation made was the insensitivity of phages to some of commonly used restriction endonucleases like BamHI, SmaI and PstI. This is in contrast to


328 M. M. Shivu et al. the observation of Pasharawipas and colleagues (2005) that VHS1 was susceptible to these enzymes. Even within the family Siphoviridae, different phages had difference in sensitivity for the various enzymes used. Similar results have also been reported for the Campylobacter phages (Atterbury et al., 2003). Bacteriophages refractory to restriction enzyme are not uncommon and have been reported previously (Prevots et al., 1990; Johansson et al., 1995; Ishimaru and Muroga, 1997). Several explanations have been proposed to explain phage DNA resistance to restriction enzyme, usually referred to as ‘antirestriction mechanism’ (Moineau et al., 1993). Foremost among these explanations is that, phage genomes lose restriction sites naturally during evolution (Moineau et al., 1993) and another suggestion for the refractory nature of some phages is the integration of unusual bases in the viral DNA (Jensen et al., 1998). Alternatively, the phages may encode methyltransferase that modify specific nucleotides within the recognition site (Sails et al., 1998). Whatever the mechanism, this present study suggests low sensitivity of V. harveyi phages to commonly used restriction enzymes and data suggest that though all the seven phages were similar to each other, they were distinct by RFLP analysis. This study further shows that RAPD is a useful technique to study phage genome diversity. Most of the RAPD patterns with V. harveyi phages were distinct with variation in number of bands, fragment size and intensity. Phages Viha1 and Viha2 showed similarity as seen in the protein profile, RFLP and RAPD pattern. In conclusion, this study demonstrates that genetically diverse bacteriophages with wide host range are prevalent in shrimp hatchery environments. Experimental procedures Bacterial strains and media One hundred and eighty-three isolates of V. harveyi from various geographical locations and aquaculture environments were used in this study. Twenty cultures of V. harveyi from our own department collection were used as hosts for phage isolation. One hundred and eighty-three V. harveyi isolates drawn from different parts of the world, other Vibrio species and non-vibrios such as E. coli ATCC 25922 isolate were also used to test the host range and cross reactivity of phages. The V. harveyi isolates used in this study were from our culture collection, identified as described earlier (Karunasagar et al., 1994) and from Artemia Reference Center (ARC), University of Ghent, Belgium. The cultures were maintained in glycerol broth (15%) at -80°C (Sanyo Corporation, Japan). Vibrio harveyi cultures and all other Vibrio isolates maintained at -80°C used in the study were propagated overnight in tryptone soya broth (Himedia, Mumbai) supplemented with 0.5% (wt/vol) sodium chloride (TSBS) prior to experiments. All bacterial strains from TSBS were subcultured on tryptone

soya agar (Himedia, Mumbai) supplemented with 0.5% (wt/ vol) sodium chloride (TSAS) and incubated at ambient temperature (28°C ⫾ 1°C) aerobically.

Isolation of bacteriophages Water samples were collected during January 2002– February 2004 from shrimp hatcheries, grow-out ponds, estuaries and creeks from the west and east coast of India. Enrichment technique (Crosse and Hingorani, 1958) was followed for the isolation of bacteriophages. To avoid selection bias due to use of few hosts, 20 different V. harveyi isolates from various locations in India were used for phage isolation. One millilitre of each water sample was added to 5 ml of 20 different host V. harveyi broth cultures for enrichment and incubated at 28°C ⫾ 1°C for 6–8 h. The enrichment was subjected to high speed centrifugation at 10 000 g in a cooling centrifuge (Biofuge, Germany) and the supernatant filtered through 0.22 mm pore size syringe filter (Millipore Corporation, USA). Fifteen microlitres of the filtrate was spotted on the V. harveyi lawn culture on TSAS plates and the plates incubated at 28°C ⫾ 1°C for up to 16–18 h to observe for any clearing. The filtrates were also tested for the presence of bacteriophages by diluting and plating onto lawns of V. harveyi by the soft agar overlay method (Adams, 1959). One millilitre of host bacterial cell suspension and 100 ml of the filtrate were mixed with 5 ml of molten agarose held at 40°C and poured over nutrient agar plates. The plates were incubated at 28°C ⫾ 1°C for up to 18–24 h and observed for formation of plaques. Individual plaques were picked using sterile loop, transferred to microcentrifuge tubes containing 100 ml SM buffer (50 mM Tris HCl, pH 7.5, 100 mM NaCl, 10 mM MgSO4, 0.01% gelatin) and stored at 4°C. Further dilutions were made from this suspension and plated again for confirmation and purification by serial passage.

Propagation of phages This was done according to the method described by Su and colleagues (1998). Phage lysates were first prepared by flooding a confluently lysed plate with 5 ml of SM buffer and placed on a shaker incubator maintained at 28°C for 2 h. The suspension was transferred to a tube and the bacterial debris was cleared by centrifugation at 4000 g for 10 min (Biofuge, Heraeus, Germany) and the entire supernatant was used to infect a 50 ml host bacterial culture in midexponential growth phase (A600 = 0.5). The host–phage mixture was incubated in a shaker incubator at 28°C for 3 h and the degree of lysis was observed by reduction in A600 to about 0.2. The bacterial debris was pelleted by high speed centrifugation at 10 000 g for 30 min at 4°C, the supernatant containing phage filtered through 0.22 mm membrane filter (Millipore Corporation, USA) and the filtrate stored at -80°C for further studies.

One-step growth One-step growth experiment was performed as described by Pajunen and colleagues (2000) with some modifications.


Molecular characterization of Vibrio harveyi bacteriophages 329 Vibrio harveyi cells were centrifuged and resuspended in fresh TSB (108 cfu ml-1). Phage Viha1 was added at a moi of 0.005 and allowed to adsorb for 5 min at room temperature. The mixture was centrifuged at 10 000 g for 10 min and the pellet containing infected cells was suspended in 25 ml TSB and incubated at 30°C. Samples were drawn at 30 min intervals and the phage titrated by soft agar overlay method (Adams, 1959).

digest 10 mg of the nucleic acid at 37°C for 1 h, and the product was electrophoresed through 0.7% agarose gel. To determine whether the phage DNA was single/doublestranded, 10 mg of DNA was incubated with 20 U of S1 nuclease (MBI Fermentas) at 37°C for 1 h as detailed by Sambrook and colleagues (1989), and the resulting product was electrophoresed through 0.7% agarose gel.

Restriction digestion of phage DNA Determination of host range of phages The phages obtained were tested for their ability to lyse different strains of V. harveyi, other species of genus Vibrio and E. coli. Bacteriophage lysis assay was based on the modified method of the traditional double-layer plaque technique (Gill et al., 2003). Lysis assay was conducted in 10 cm diameter Petri plates (Hi media, India). The top agar layer consisted of 1% tryptone, 0.5% yeast extract, 1% NaCl, 0.1% MgCl2, 1 ml of 1 M CaCl2, 0.4% agar. For each strain tested, 3 ml of top agar was steamed for approximately 10 min and allowed to cool to 47°C. One hundred microlitres of overnight culture of bacterium to be tested was added to the top layer and the mixture was vortexed and poured on to the bottom agar (TSAS). The top agar was allowed to solidify at room temperature and 20 ml phage suspension (1010 pfu ml-1) was spotted onto the top layer. The plate was incubated at 30°C overnight and then examined for the presence of zone of lysis. Positive spot test was confirmed by titration assay using diluted phage preparation.

Isolation of phage nucleic acid Phage nucleic acid was extracted by the method described by Su and colleagues (1998). To the phage lysates obtained by confluent plate lysis, DNase I was added to a final concentration of 10 mg ml-1 and incubated at 37°C for 30 min to lyse any bacterial DNA. Filter sterilized 2 M ZnCl2 was added to the phage lysate at a ratio of 1:50 (v/v) and incubated at 30°C for 5 min. Then, the phage particles were pelleted by centrifugation at 4000 g for 5 min at room temperature (28°C ⫾ 1°C). The supernatant was discarded and the pellet dissolved in 700 ml TENS buffer (50 mM Tris-HCl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 0.3% Sodium dodecyl sulfate) for each ml of phage lysate and proteinase K was added to a final concentration of 100 mg ml-1 and incubated at 65°C for 10 min. The mixture was further deproteinated by extracting the solution with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1 v/v) twice. The aqueous phase was collected in a new tube and an equal volume of isopropanol was added and gently mixed. The aggregated DNA was pelleted at 4000 g for 10 min. Finally the DNA pellet was washed with 70% ethanol and resuspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

Nature of the nucleic acid The nucleic acid extracted above was digested with DNasefree RNase (Gibco BRL) to determine whether the nucleic acid was DNA or RNA. 20 U of the enzyme was used to

To determine the diversity of phage isolates, and to determine the approximate genome sizes from the fragments generated, restriction digestion analyses were carried out with 14 restriction enzymes. The restriction enzymes AluI, BamHI, EcoRI, EcoRV, HaeIII, HincII, HindIII, HpaI, HpaII, KpnI, PstI, SmaI, TaqI and XbaI were used according to manufacturer’s (MBI Fermentas) instructions. The restriction digests were separated on 0.7% agarose gel containing 0.5 mg ml-1 ethidium bromide at a constant voltage of 80 V in TAE buffer. The approximate phage genome sizes were estimated from the RFLP pattern generated. The fragments of phage DNA digested by restriction endonucleases were calculated from relative mobility to standard size markers using Kodak 1D Software. Finally individual fragment sizes were added up for each enzyme to calculate the approximate genome size.

Genomic fingerprinting by RAPD analysis Random amplification of polymorphic DNA for phage was done by modification of method described by Johansson and colleagues (1995) by using decamer primer. Two primers were used in this study: P-1 (5′-CCG CAG CCA A-3′) and P-2 (5′-AAC GGG CAG A-3′). Each 30 ml reaction mixture for RAPD PCR analysis of bacteriophage DNA contained 19.8 ml of distilled water, 3 ml of Taq DNA polymerase 10¥ PCR buffer, 2.4 ml of deoxynucleoside triphosphate mixture, 30 pmol of primer, 0.3 mg DNA template and 0.3 ml of Taq DNA polymerase. DNA amplification was performed with a gradient thermocycler (PTC 200 MJ, Research, USA). Themocycler programme was as follows: for P-2: Initial delay 94°C for 3 min; 35 cycles of 94°C for 5 s, 36°C for 45 s, 72°C for 1.3 min and 5 min at 72°C; for P-1: Initial delay 94°C for 5 min; 35 cycles of, 94°C for 20 s, 45°C for 30 s, 72°C for 1 min and a final delay of 7 min at 72°C. The DNA banding patterns were examined in 1.5% agarose gel electrophoresis and 100 bp ladder (MBI Fermentas) was used to provide size standards.

SDS-PAGE of proteins The profiles of phage structural proteins were analysed by SDS-PAGE as described by Laemmeli (1970). To 100 ml of purified phage suspension obtained by CsCl gradient centrifugation (Sambrook et al., 1989), 5 ml of b-mercaptoethanol was added and samples were boiled for 5 min with equal volume of sample buffer (0.075 M Tris hydrochloride, 0.1% sulfuric acid, 15% glycerol, 2% SDS, 0.01% bromophenol blue [pH 6.8]). Electrophoresis was carried out in 15% discontinuous gel (15% total monomer, 0.5% cross-linking


330 M. M. Shivu et al. monomer) in the vertical slab gel electrophoresis system and stained with 0.1% Coomassie blue. Molecular weight marker (Bangalore Genei, India) was included in the run with samples to determine molecular weights of bands obtained.

Electron microscopy Phage-containing solutions were mixed with 1/10 volume of 2.5% buffered glutaraldehyde (50 mM cacodylate pH 7.2; 50 mM KCl, 2.5 mM MgCl2) for 5 min. Twenty-five microlitres of fixed phages were added to the surface of a pioloform coated grid and left for 2 min. Excess fluid was removed, grids washed for 2 s in distilled water and negatively stained with 0.5% uranyl acetate for 2 min. Excess stain was removed and the grids were air dried. Grids were then inspected with a Zeiss EM10 transmission electron microscope (Zeiss/LEO, Oberkochen, Germany).

Acknowledgements This research was supported by grants from the Department of Biotechnology, Govt. of India through their ‘Programme Support in Fisheries Biotechnology’. Thanks are due to the Laboratory of Aquaculture and Artemia Reference Center, University of Ghent, Belgium for kindly providing their collection of Vibrio harveyi cultures for the study.

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