Microbial Ecology
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Microbial Ecology Isolation and Genetic Analysis of Haloalkaliphilic Bacteriophages in a North American Soda Lake Shereen Sabet, Weiping Chu and Sunny C. Jiang Department of Environmental Health, Science, & Policy, University of California, 1367 SE II, Irvine, CA 92697-7070, USA Received: 19 January 2006 / Accepted: 19 January 2006 / Online Publication: 6 May 2006 Abstract bacteria [7] and some of the highest recorded viral abundance of any natural aquatic system [11], yet little is Mono Lake is a meromictic, hypersaline, soda lake that known about the viral community that resides in this harbors a diverse and abundant microbial community. A extreme environment. previous report documented the high viral abundance in Bacteriophages, along with their hosts, make up the Mono Lake, and pulsed-field gel electrophoresis analysis largest biomass on earth, residing mostly in aquatic of viral DNA from lake water samples showed a diverse habitats. Viruses are believed to play a critical role in the population based on a broad range of viral genome sizes. mortality of aquatic bacteria [6], thereby affecting the To better understand the ecology of bacteriophages and microbial food web and biogeochemical processes, as their hosts in this unique environment, water samples well as affecting bacterial diversity by restructuring the were collected between February 2001 and July 2004 for microbial community [15, 30]. Bacteriophages can also isolation of bacteriophages by using four indigenous affect their hosts via lysogeny and transduction and by bacterial hosts. Plaque assay results showed a differential mutually providing genes that enhance host survival [6, seasonal expression of cultured bacteriophages. To reveal 13, 25]. the diversity of uncultured bacteriophages, viral DNA Viruses and viruslike particles have been detected from lake water samples was used to construct clone and isolated in hypersaline environments such as the libraries. Sequence analysis of viral clones revealed Dead Sea, a marine solar saltern in Australia, the Great homology to viral as well as bacterial proteins. Further- Salt Lake in Utah, and the Little Salt Pond in Yallahs, more, dot blot DNA hybridization analyses showed that Jamaica [16, 20, 23, 31]; however, the literature is sparse the uncultured viruses are more prevalent during most regarding the ecology of these extremophilic aquatic seasons, whereas the viral isolates (A7 and 72) were less bacteriophages. As a first step toward better understand- prevalent, confirming the belief that uncultured viruses ing these viruses, especially in an extreme environment, represent the dominant members of the community, this study aims to track the seasonal presence of Mono whereas cultured isolates represent the minority species. Lake’s representative bacteriophage community as well as uncultured phages. Fragments of viral genomes from lake waters were cloned and analyzed, and their sequences Introduction provide insight as to the nature of the viruses that reside in a hypersaline, alkaline lake. Mono Lake is a closed basin, hypersaline, alkaline soda lake located in central California, USA, presently charac- terized by a salt concentration of 8% (80 g/L) and a pH Methods of 10. Mono Lake is a meromictic lake that undergoes Mono Lake Water Samples. Small-volume water holomixis approximately once every 10 years [19], with samples (50 mL) were collected monthly by researchers the most recent event recorded in October of 2003 (R. at the Sierra Nevada Aquatic Research Laboratory, Jellison, pers. comm.). The lake has a very productive University of California, from Mono Lake stations 2, 6, microbial community [9] with a high diversity of 7, and 11 (Fig. 1)[21] between March 2003 and July 2004. A single, upper 9-m integrated water column Correspondence to: Sunny C. Jiang; E-mail: [email protected] sample was taken from all of the stations, whereas a DOI: 10.1007/s00248-006-9069-1 & Volume 51, 543–554 (2006) & * Springer Science+Business Media, Inc. 2006 543 544 S. SABET ET AL.: SEASONAL PRESENCE OF HYPERSALINE PHAGES Figure 1. Map of Mono Lake showing locations of sampling stations. All stations were used for water sample collection for the duration of this study, except for stations 4, 5, and 9. detailed vertical profile at 2, 8, 10, 12, 16, 20, 24, 28, and sterilized by boiling. Plates were incubated for 24–72 h at 35 m was sampled at station 6 using a Niskin bottle room temperature and observed for colony growth. onboard a small boat. Integrated water samples were Individual colonies were picked and cultivated further in collected by lowering a 1-in.-diameter Tygon tubing their respective media. Frozen stocks were made and weighted at one end into the water down to the 9-m stored at _80-C. M12-2c and M12-chla hosts were depth and then lifting the weighted end out of the water identified by 16S rRNA sequence analysis in a previous and transferring the water to a bucket. Water samples study (Hollibaugh, pers. comm.) and are listed under were filtered through 0.22-mm pore-size syringe filters GenBank accession numbers AY730244 and AY730242, (Millex GP PES membrane; Millipore, Carrightwohill, respectively. M12-2c is 99% similar to Vibrio metschniko- Co. Cork, Ireland) to remove bacteria and phytoplankton, vii and M12-chla only has a close match with a bac- and viral fractions were shipped cold overnight to the terium previously isolated from Mono Lake sediment in University of California Irvine laboratory. the database. In this study, the MN12-2a and MN1-12.5a Large-volume water samples (5–10 L) were collected bacterial isolates were also characterized by 16S rRNA at sampling stations 1, 3, 6, 8, 10, 11, and 12 from both sequence analysis. Those sequences are listed under the oxygenic (2–10 m) and anoxic (35 m) layers during the accession numbers AY856383 and AY856384, field trips in February and August 2001, in August 2002, respectively. and in February and October 2003 for isolation of To isolate bacteriophages, 5–10 L of water samples bacterial hosts and viruses. All samples were processed were prefiltered through glass fiber and 0.45-mm filters on site within 4 h of collection. (Millipore) to remove large plankton when plankton concentrations were high. The samples were then Bacterial Host and Viral Isolation. Four Mono sequentially filtered via TFF, first by using a 0.22-mm Lake bacterial hosts were isolated in February (designated pore-size filtration cartridge followed by a 30-kDa M12-2c, M12-chla) and in August (designated MN1- molecular weight cutoff cartridge. The first cartridge 12.5a, MN12-2a) of 2001 by first concentrating 10 L of removes bacteria, whereas the second cartridge concen- water using a tangential flow filtration (TFF) system with a trates viruses in the retentate. The final retentate volume 30-kDa molecular weight cutoff filtration cartridge (PALL is approximately 100 mL. Corp., Hauppauge, NY, USA). One hundred microliters Plaque assays were carried out by using the top agar of TFF retentate was then spread onto either marine agar overlay method [1]. Briefly, 1 mL of bacterial host plates (Difco, Becton, Dickinson and Co., Sparks, MD, (described above) in 3 mL of top agar was inoculated USA) supplemented with 1% NaCl (M12-2c and M12- with 1 mL of TFF-concentrated viral sample, then mixed chla), or onto agar plates made with Mono Lake water and poured over a bottom nutrient plate. Both top and supplemented with 0.5% peptone, which was then bottom agar contain the same ingredients as the bacterial S. SABET ET AL.: SEASONAL PRESENCE OF HYPERSALINE PHAGES 545 host isolation and growth media. Plates were incubated cut from the gel and stained with SYBR Gold (Molecular at room temperature for 48 h before the number of viral Probes) to visualize the DNA bands. The rest of the gel plaques was counted. Selective individual plaques were was not stained in order to protect the viral DNA from picked and suspended in 300 mL of either 0.5 M Tris (pH UV damage and to increase cloning efficiency. The 8.0) or MSM buffer (450 mM NaCl/50 mM MgSO4/50 stained lane was then used as a guide to cut out gel mM Tris, pH 8.0). Each isolate was purified by slices containing the 35- to 55-kb desired band from the reisolation of individual plaques four more times before remainder of the unstained gel. DNA was then purified it was considered to be a pure isolate. Viral lysates were from the gel by the GELase product (Epicentre Technol- harvested by plate elution using MSM buffer. Lysates ogies, Madison, WI, USA) according to the manufactur- were then filtered through 0.22-mm pore-size syringe fil- er’s protocol. The DNA and digested agarose were then ters to remove any remaining bacteria. Viral filtrates were transferred to Centricon 100-kDa filter tubes (Millipore) either frozen at _80-C in a 1:1 ratio of MSM buffer and and washed two times with 1Â TE (1000 Â g, 15 min). 50% sterile glycerol or stored at 4-C for further analyses. After gel extraction, DNA concentration was again measured via the fluorometer as described above. Bacterial Artificial Chromosome Cloning. To Viral DNA was ligated into the pCC1 copy control understand the diversity and dynamics of uncultured BAC vector and cells were transformed according to the bacteriophages in Mono Lake, a bacterial artificial manufacturer’s protocol (Epicentre). Electrocompetent chromosome (BAC) clone library was constructed. First, transformations were carried out with the Eppendorf viruses were concentrated from 240 mL of 0.22-mm- Electroporator model 2510. Clones were screened for filtered lake water, collected from station 3 at 2 m in size and similarity by using BglII, HindIII, and NotI February 2003, to a final volume of 2 mL via the restriction enzymes (Promega).