Microb Ecol (2003) 45:399–410 DOI: 10.1007/s00248-002-1059-3 2003 Springer-Verlag New York Inc.
The Vertical Distribution and Diversity of Marine Bacteriophage at a Station off Southern California S. Jiang,1 W. Fu,1 W. Chu,1 J.A. Fuhrman2 1 2
Environmental Analysis and Design, University of California, Irvine, CA 92697-7070, USA Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-0371, USA
Received: 19 August 2002; Accepted: 20 January 2003; Online publication: 22 April 2003
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B S T R A C T
Sixty-two bacteriophages were isolated on eight indigenous bacteria from a Pacific Ocean station spanning 887-m vertical depth, on two occasions between 1999 and 2000. On the basis of 16S rRNA sequences, six hosts were tentatively identified to be in the genus Vibrio and the other two were closely related to Altermonas macleodii (W9a) and Pseudoalteromonas spp. (W13a). Restriction fragment length polymorphism (RFLP) analysis of phage genomes using AccI and HapI showed that 16 phages infecting host C4a (Vibrio) displayed 14 unique RFLP patterns. However, identical phages infecting host C4b, C6a, and C6b (all Vibrio) were obtained from both the surface layer and the hypoxic zone at 850 m. Most phage isolates from the second year had a different RFLP pattern but shared genetic similarity to the phages infecting the same host from the previous year based on a hybridization study using phage genome probes. Cluster analysis of RFLP patterns and hybridization results also indicated that phages infecting the same or genetically related hosts, in general, shared higher degrees of homology in spite of the diverse RFLP patterns. Pulsed field gel electrophoresis (PFGE) analysis of native viral genomes indicated a range in genome size from less than 40 to 200 kb, and the dominant band shifted up by about 5– 10 kb in the deep samples compared to the shallow ones. Hybridization of phage genome probes with total viral community DNA from various depths suggests these isolates, or at least some of their genes, represent a detectable portion of the natural viral community and were distributed throughout the water column. Thus, the results of this study demonstrated that the genetic diversity of bacteriophage in the ocean is far greater than that of their bacterial hosts. However, host range may have contributed to the evolution of the diverse phage population in the marine environment.
Introduction
Correspondence to: S. Jiang; E-mail:
[email protected]
Viruses are numerically the most abundant microorganisms in the marine environment [32]. They play an
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important role in controlling their host population density and contributing to bacterial gene exchange [13]. Direct microscopic observations indicate that most of the marine viruses are tailed bacteriophage resembling those of double stranded DNA coliphages [4]. In addition, nearly 50% of marine bacterial isolates have been shown to contain inducible prophage, suggesting lysogens are an important portion of marine prokaryotes [12, 14]. However, little is known about the characteristics of those marine phages, how they interact with their bacterial hosts, or their genetic diversity and evolutionary origins. The diversity of marine viral isolates infecting Vibrio parahaemolyticus, members of the genus Pseudoalteromonas, and the microalga Micromonas pusilla has been investigated using both viral genome hybridization and restriction fragment length polymorphism (RFLP) analysis [7, 17, 31]. Since many environmental bacteria (and thus their viruses) can be difficult to cultivate, many investigators rely on culture-independent molecular approaches to investigate genetic diversity in natural viral assemblages. Viral diversity studies using direct PCR amplification combined with sequence analysis or denaturing gradient gel electrophoresis (DGGE) have shown great promise. Recent studies have focused on eukaryotic algal viruses [5, 6, 27, 28] and myo-cyanophages infecting Synechococcus using group specific primers [9, 36]. These molecular approaches, however, are typically limited to characterizing only a small portion of the viral assemblage at a time because of a lack of shared genetic determinants (genes) between virus strains. Application of pulsed field gel electrophoresis (PFGE) in field studies has revealed that viruses with different genomic sizes are present in any given viral community [29, 33]. This method is capable of detecting 105 to 106 viruses of either the same species or a group of viruses with a similar genome size. Therefore only the dominating viral groups in the community can be observed by PFGE. Analysis of PFGE viral fingerprints from the Chesapeake Bay indicated that changes in virioplankton community structure varied temporally and spatially, and with the extent of water column stratification [33, 34]. The study presented here investigated the genetic diversity of marine bacteriophage isolated from a Pacific Ocean station over a 2-year period to elucidate their vertical distribution and relationship with their resident host population.
S. Jiang et al.
Materials and Methods Sample Collection Water samples were collected from a single station in the Pacific Ocean (33 33¢ N; 118 24¢ W) off the coast of Southern California during two cruises on the R/V Point Sur between August 11 and 13, 1999, and again between August 10 and 17, 2000. Samples were collected using 20-liter Niskin bottles mounted on a rosette fitted with a conductivity–temperatures–depth recorder (SeaBird Electronics model 911+). Dissolved oxygen was determined by Winkler titration [22]. Chlorophyll a concentrations were determined fluorometrically [11, 19, 35].
Host Bacteria Isolation Water samples of 20 to 100 L from 20 m, 40 m, or 850 m were immediately concentrated using a Vortex Flow Filtration system (VFF) using a 100-kD, molecular weight cutoff filter cartridge to 40 to 60 mL as previously described [15]. The efficiency of viral recovery with this system averages 72% [23]. One hundred microliters of each of the concentrates was spread onto Marine Agar (Zobell 2216, Difco) to isolate bacterial hosts. Bacterial colonies with diverse morphology were picked after 48 h incubation at room temperature (25C) and used for isolation of bacteriophages. Susceptible hosts were further subcultured to ensure the purity of the isolates. The rest of the concentrates were stored at 4C until being used for bacteriophage isolation.
Isolation of Bacteriophages Concentrates used for phage isolation were either the same as used for host isolation or from a different depth. The top agar overlay technique of Adams [3] was used for isolation of bacteriophage. In brief, 1 mL of mid-log culture of each bacterial isolate and 1 mL of seawater concentrate was mixed with 3 mL of melted soft agar and overlayed on a marine agar plate. The plates were incubated at 25C and monitored for the development of plaques. Plaques with diverse morphology were picked, and each individual plaque was propagated on the original host three times before being considered a single phage isolate. Single phage isolates were grown as above and eluted from petri dishes of confluent infection using 0.5 M Tris buffer (pH 8.0), then centrifuged to remove residual host bacteria and cell debris, and cellfree lysates were stored at 4C. All phage isolates formed clear plaques on the host bacterial lawn, suggesting they are lytic to the susceptible host used for isolation.
Direct Counts of Bacteria and Viruses The epifluorescence procedure of Noble and Fuhrman [21] was used. In brief, samples were preserved with 1% 0.02 lm filtered formalin; 2 mL from the euphotic zone and 5–10 mL from the deeper waters were filtered onto 0.02-lm pore size 25 mm diam.
Marine Bacteriophage Diversity Anodisc filters (Whatman). The filters were stained with SYBR Green I (Molecular Probes) for 15 min, mounted in antifade solution (0.1% ethylenediamine in glycerol:PBS 1:1), and counted under blue excitation on an Olympus BX60 microscope. Both bacteria and viruses were counted from the same slide, and at least 200 particles of each kind per filter were counted. Some of the counts in this study were based on duplicate slides, but for the detailed depth profile, most were from single slides. The average variation between duplicates is generally
