Nucleic acid economy in bacteria infected with bacteriophage T2. I. Purine and pyrimidine composition

1. The phosphorus content per infective particle of isolated bacteriophage T2 has been redetermined. It does not exceed 1.8 to 2.2 x 10^–11 µg. The equivalent amount of DNA has been defined in terms of several analytical methods and taken as a unit of measurement of intrabacterial DNA. 2. The DNA of E. coli contains guanine, adenine, cytosine, and thymine in approximately equal amounts, but no hydroxymethylcytosine. One bacterial cell contains 40 to 150 units of DNA, depending on the conditions of growth and the method of measurement. 3. The DNA of phage T2 (one unit per particle by definition) contains guanine, 5-hydroxymethylcytosine, and relatively large amounts of adenine and thymine, but no cytosine. 4. Infected bacteria contain DNA of a composition that varies systematically during the course of viral growth. At all times it resembles a mixture of bacterial and viral DNA. 5. The characteristic bacterial DNA is decomposed after infection, measuring about one-third its initial amount at 20 minutes. The characteristic viral DNA increases in amount, after a short delay, reaching a level of 100 to 400 units per bacterium 30 minutes after infection. At 10 minutes after infection, the two kinds of DNA are approximately equal in amount. 6. The characteristic viral DNA present in infected cells exists in two forms, one consisting of infective particles and one not. The portion not contained in infective particles builds up to 40 to 80 units per cell during the first 10 minutes after infection and afterwards remains roughly constant in amount. Infective particles begin to appear at 10 minutes and account for all or most of the increase thereafter.     

THE PHOTODYNAMIC INACTIVATION OF PHAGE PRECURSOR BY METHYLENE BLUE

Methylene blue added to suspensions of activated staphylococci in amounts sufficient to furnish 1 x 10⁵ molecules of dye/bacterium inactivates the phage precursor content of the cells without causing cell death when the mixtures are exposed to strong light of 4000–8000 Å. There is a lag phase of approximately 15 minutes in the photodynamic inactivation of phage precursor by methylene blue. This delay seems to be due to a primary reaction between the cell and methylene blue after the completion of which exposure to light brings about the inactivation of precursor quite promptly.     

Reduction in Exopolysaccharide Viscosity as an Aid to Bacteriophage Penetration through Pseudomonas aeruginosa Biofilms

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 × 10⁸ purified phage per ml for 24 h at 37°C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10¹⁰ and 10¹² phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.     

Influence of environmental variables in the efficiency of phage therapy in aquaculture

Aquaculture facilities worldwide continue to experience significant economic losses because of disease caused by pathogenic bacteria, including multidrug-resistant strains. This scenario drives the search for alternative methods to inactivate pathogenic bacteria. Phage therapy is currently considered as a viable alternative to antibiotics for inactivation of bacterial pathogens in aquaculture systems. While phage therapy appears to represent a useful and flexible tool for microbiological decontamination of aquaculture effluents, the effect of physical and chemical properties of culture waters on the efficiency of this technology has never been reported. The present study aimed to evaluate the effect of physical and chemical properties of aquaculture waters (e.g. pH, temperature, salinity and organic matter content) on the efficiency of phage therapy under controlled experimental conditions in order to provide a basis for the selection of the most suitable protocol for subsequent experiments. A bioluminescent genetically transformed Escherichia coli was selected as a model microorganism to monitor real-time phage therapy kinetics through the measurement of bioluminescence, thus avoiding the laborious and time-consuming conventional method of counting colony-forming units (CFU). For all experiments, a bacterial concentration of ≈ 10⁵ CFU ml⁻¹ and a phage concentration of ≈ 10^6–8 plaque forming unit ml⁻¹ were used. Phage survival was not significantly affected by the natural variability of pH (6.5–7.4), temperature (10–25°C), salinity (0–30 g NaCl l⁻¹) and organic matter concentration of aquaculture waters in a temperate climate. Nonetheless, the efficiency of phage therapy was mostly affected by the variation of salinity and organic matter content. As the effectiveness of phage therapy increases with water salt content, this approach appears to be a suitable choice for marine aquaculture systems. The success of phage therapy may also be enhanced in non-marine systems through the addition of salt, whenever this option is feasible and does not affect the survival of aquatic species being cultured.     

THE RELATIONSHIP BETWEEN BACTERIAL GROWTH AND PHAGE PRODUCTION

1. The effects of temperature and H-ion concentration on the reaction between antistaphylococcus phage and a susceptible staphylococcus have been studied. 2. The temperature optimum for phage production is in the neighborhood of 35°C. and that for bacterial growth is approximately 40°C. 3. With increasing H-ion concentrations there occur: (a) an increase in the lag phase of bacterial growth without any corresponding increase in the lag phase of phage production; (b) a diminution in the total bacterial population accumulating in the medium without any corresponding drop in the total amount of phage formed. 4. With increasing alkalinity there is no pronounced change in the curves of bacterial growth and phage formation. At pH 8.5 the lytic threshold is increased to about 1000 phage units per bacterium instead of 100–140 as is usually the case and the time of lysis is delayed. 5. By adjusting the medium to pH 6 and 28°C. bacterial growth can be completely inhibited while phage production continues at a rapid rate. 6. Apparently, the previously stressed importance of bacterial growth as the prime conditioning factor for phage formation does not hold, for under certain experimental conditions the two mechanisms can be dissociated.     

Life-Style and Genome Structure of Marine Pseudoalteromonas Siphovirus B8b Isolated from the Northwestern Mediterranean Sea

Marine viruses (phages) alter bacterial diversity and evolution with impacts on marine biogeochemical cycles, and yet few well-developed model systems limit opportunities for hypothesis testing. Here we isolate phage B8b from the Mediterranean Sea using Pseudoalteromonas sp. QC-44 as a host and characterize it using myriad techniques. Morphologically, phage B8b was classified as a member of the Siphoviridae family. One-step growth analyses showed that this siphovirus had a latent period of 70 min and released 172 new viral particles per cell. Host range analysis against 89 bacterial host strains revealed that phage B8b infected 3 Pseudoalteromonas strains (52 tested, >99.9% 16S rRNA gene nucleotide identity) and 1 non-Pseudoaltermonas strain belonging to Alteromonas sp. (37 strains from 6 genera tested), which helps bound the phylogenetic distance possible in a phage-mediated horizontal gene transfer event. The Pseudoalteromonas phage B8b genome size was 42.7 kb, with clear structural and replication modules where the former were delineated leveraging identification of 16 structural genes by virion structural proteomics, only 4 of which had any similarity to known structural proteins. In nature, this phage was common in coastal marine environments in both photic and aphotic layers (found in 26.5% of available viral metagenomes), but not abundant in any sample (average per sample abundance was 0.65% of the reads). Together these data improve our understanding of siphoviruses in nature, and provide foundational information for a new ‘rare virosphere’ phage–host model system.     

Distribution of Viruses and Dissolved DNA along a Coastal Trophic Gradient in the Northern Adriatic Sea

The distribution of viral and other microbial abundances as well as the concentrations of dissolved DNA (D-DNA) along a trophic gradient in the northern Adriatic Sea were determined. Virus abundances, covering a range of 1.2 × 10⁹ to 8.7 × 10¹⁰ liter^-1 were on average 2.5-fold higher in eutrophic than in mesotrophic stations. A 2.5-fold enrichment was also measured for chlorophyll a concentrations, whereas the densities of bacteria and heterotrophic nanoflagellates were only approximately 1.5-fold higher. The frequency of bacteria containing mature phage increased linearly with bacterial abundance. Assuming that mature phage is only visible during the last 14 to 27% of the latent period (L. M. Proctor, A. Okubo, and J. A. Fuhrman, Microb. Ecol. 25:161-182, 1993), we estimated that between 3.5 and 7.3% of the bacterial population was infected at mesotrophic stations versus between 7.0 and 19.5% at eutrophic stations, indicating that the bacterial mortality due to viral lysis might increase with the degree of eutrophication. The frequency of bacteria with mature phage and the burst size varied significantly with the bacterial morphotype; rod-shape cells, the most abundant morphotype, showed low infection rates but a high burst size. Concentrations of D-DNA varied significantly with season but not with trophic conditions. The estimated percentage of viral DNA on total D-DNA concentrations averaged 17.1% (range, 0.7 to 88.3%). Some kind of interaction between heterotrophic nanoflagellates and viruses is proposed. We conclude (i) that the significance of viruses varies with changing trophic conditions and (ii) that viral activity may play a significant role in food web structure under changing trophic conditions.     

Regulation of the intracellular concentration of T4 induced tRNA

Summary We have studied the biosynthesis of T4 induced tRNA's upon infection of E. coli B ^E cells in low phosphate (l.p.) medium (10^-4 M PO ₄ ^--- ). Under our experimental conditions the onset of phage DNA synthesis occurs at about 15 min after infection, while the first intracellular phage appears one hour later. Amounts of newly synthesized DNA and phage burst size are equivalent to the values obtained in standard (M9) medium (10^-1 M PO ₄ ^--- ). We present evidence that the synthesis of mature tRNA's and of at least one dimeric precursor drastically declines 20 min after infection. In addition we show that T4 induced tRNA molecules are stable and that the triphosphate nucleoside precursor pool does not change significantly during infection. Therefore we conclude that T4 induced tRNA molecules behave similarly to other early gene products.     

Ultrasound-mediated DNA transfer for bacteria

In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell–pilus–cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5α and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl₂, temperature of 22°C, plasmid concentration of 0.8 ng/µl, P. putida UWC1 cell concentration of 2.5 × 10⁹ CFU (colony forming unit)/ml and reaction volume of 500 µl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 ± 2.3 × 10⁻⁶ transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5α and P. fluorescens SBW25 with efficiencies of 1.16 ± 0.13 × 10⁻⁶ and 4.33 ± 0.78 × 10⁻⁶ transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.     

Chemical mapping of DNA and counter-ion content inside phage by energy-filtered TEM

Double-stranded DNA in many bacterial viruses (phage) is strongly confined, which results in internal genome pressures of tens of atmospheres. This pressure is strongly dependent on local ion concentration and distribution within the viral capsid. Here, we have used electron energy loss spectroscopy (EELS), energy-filtered TEM (EFTEM) and X-ray energy dispersive spectroscopy to provide such chemical information from the capsid and the phage tail through which DNA is injected into the cell. To achieve this, we have developed a method to prepare thin monolayers of self-supporting virus/buffer films, suitable for EELS and EFTEM analysis. The method is based on entrapment of virus particles at air–liquid interfaces; thus, the commonly used method of staining by heavy metal salts can be avoided, eliminating the risk for chemical artifacts. We found that Mg^2 + concentration was approximately 2–4 times higher in the DNA-filled capsid than in the surrounding TM buffer (containing 10 mM Mg^2 +). Furthermore, we also analyzed the DNA content inside the phage tail by mapping phosphorus and magnesium.     

Divided genomes and intrinsic noise

Summary Segmental genomes (i.e., genomes in which the genetic information is dispersed between two or more discrete molecules) are abundant in RNA viruses, but virtually absent in DNA viruses. It has been suggested that the division of information in RNA viruses expands the pool of variation available to natural selection by providing for the reassortment of modular RNAs from different genetic sources. This explanation is based on the apparent inability of related RNA molecules to undergo the kinds of physical recombination that generate variation among related DNA molecules. In this paper we propose a radically different hypothesis. Self-replicating RNA genomes have an error rate of about 10^–3–10^–4 substitutions per base per generation, whereas for DNA genomes the corresponding figure is 10^–9–10^–11. Thus the level of noise in the RNA copier process is five to eight orders of magnitude higher than that in the DNA process. Since a small module of information has a higher chance of passing undamaged through a noisy channel than does a large one, the division of RNA viral information among separate small units increases its overall chances of survival. The selective advantage of genome segmentation is most easily modelled for modular RNAs wrapped up in separate viral coats. If modular RNAs are brought together in a common viral coat, segmentation is advantageous only when interactions among the modular RNAs are selective enought to provide some degree of discrimination against miscopied sequences. This requirement is most clearly met by the reoviruses.     

THE GROWTH OF BACTERIOPHAGE

1. An anti-Escherichia coli phage has been isolated and its behavior studied. 2. A plaque counting method for this phage is described, and shown to give a number of plaques which is proportional to the phage concentration. The number of plaques is shown to be independent of agar concentration, temperature of plate incubation, and concentration of the suspension of plating bacteria. 3. The efficiency of plating, i.e. the probability of plaque formation by a phage particle, depends somewhat on the culture of bacteria used for plating, and averages around 0.4. 4. Methods are described to avoid the inactivation of phage by substances in the fresh lysates. 5. The growth of phage can be divided into three periods: adsorption of the phage on the bacterium, growth upon or within the bacterium (latent period), and the release of the phage (burst). 6. The rate of adsorption of phage was found to be proportional to the concentration of phage and to the concentration of bacteria. The rate constant k_a is 1.2 x 10^–9 cm.⁸/min. at 15°C. and 1.9 x 10^–9 cm.⁸/min. at 25°. 7. The average latent period varies with the temperature in the same way as the division period of the bacteria. 8. The latent period before a burst of individual infected bacteria varies under constant conditions between a minimal value and about twice this value. 9. The average latent period and the average burst size are neither increased nor decreased by a fourfold infection of the bacteria with phage. 10. The average burst size is independent of the temperature, and is about 60 phage particles per bacterium. 11. The individual bursts vary in size from a few particles to about 200. The same variability is found when the early bursts are measured separately, and when all the bursts are measured at a late time.     

Diel,seasonal, and depth-related variability of viruses and dissolved DNA in the northern Adriatic Sea

The depth-dependent, seasonal, and diel variability of virus numbers, dissolved DNA (D-DNA), and other microbial parameters was investigated in the northern Adriatic Sea. During periods of water stratification, we found higher virus abundances and virus/bacterium ratios (VBRs) as well as a larger variability of D-DNA concentrations at the thermocline, probably as a result of higher microbial biomass. At the two investigated stations, virus densities were highest in summer and autumn (up to 9.5 × 10¹⁰ 1^–1) and lowest in winter (< 10⁹ 1^–1); D-DNA concentrations were highest in summer and lowest in winter. The VBR as well as an estimated proportion of viral DNA on total D-DNA showed a strong seasonal variability. VBR averaged 15.0 (range, 0.9–89.1), and the percentage of viral DNA in total D-DNA averaged 18.3% (range, 0.1–96.1%). An estimation of the percentage of bacteria lysed by viruses, based on 2-h sample intervals in situ, ranged from 39.6 to 212.2% d^–1 in 5 m and from 19.9 to 157.2% d^–1 in 22 m. The estimated contribution of virus-mediated bacterial DNA release to the D-DNA pool ranged from 32.9 to 161% d^–1 in 5 m and from 10.3 to 74.2% d^–1 in 22 m. Multiple regression analysis and the diel dynamics of microbial parameters indicate that viral lysis occasionally could be more important in regulating bacterial abundances than grazing by heterotrophic nanoflagellates. Correspondence to: M.G. Weinbauer     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Elevated Abundance of Bacteriophage Infecting Bacteria in Soil

Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 × 10⁷ g⁻¹), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 × 10⁸ g⁻¹, which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.     

Significant contribution of the 3′→5′ exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ?

Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3′→5′ exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 10⁶–10⁸ incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3′→5′ exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10⁻⁴–10⁻⁷) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3′→5′ exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 10² to 1.2 × 10⁴-fold. The resulting overall fidelity of hPolϵ (10⁻⁶–10⁻¹¹) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1–1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation.     

Halobacterium halobium phage øH

Phage øH, a novel virus of the archaebacterium Halobacterium halobium, resembles in size and morphology two other Halobacterium phages. One-step growth curves show a 5.5 h eclipse, a latent period of 7 h, and an apparent burst size of 170. Phage øH contains linear, double-stranded DNA which has a molecular weight of 39 x 10⁶ and a GC content of 65%. A packaging model accounting for the partial circular permutation and terminal redundancy of øH DNA is suggested. Partial homology of øH DNA with the DNA of H. halobium, predominantly with the AT-rich satellite DNA, was observed. The presence of minor restriction fragments of øH DNA which could be removed by purification of phage from single plaques suggests the existence of phage variants with rearranged DNA. A strain of H. halobium containing øH DNA was isolated which is resistant to infection by phage øH.     

Simian Immunodeficiency Virus Replicates to High Levels in Sooty Mangabeys without Inducing Disease

A serologic survey of primates living in a French zoo allowed identification of three cases of infection with simian immunodeficiency virus in sooty mangabeys (Cercocebus atys) (SIVsm). Viral isolates, which were designated SIVsmFr66, SIVsmFr74, and SIVsmFr85, were obtained after short-term culture of mangabey lymphoid cells. Phylogenetic analysis of gag and env sequences amplified directly from mangabey tissues showed that the three SIVsmFr were genetically close and that they constituted a new subtype within the diverse SIVsm–SIVmac–human immunodeficiency virus type 2 (HIV-2) group. We could reconstruct the transmission events that likely occurred in 1986 between the three animals and evaluate the divergence of SIVsmFr sequences since transmission. The estimated rate of mutation fixation was 6 × 10⁻³ substitutions per site per year, which was as high as the rate found for SIVmac infection in macaques. These data indicated that SIVsmFr replicated at a high rate in mangabeys, despite the nonpathogenic character of infection in this host. The viral load evaluated by competitive PCR reached 20,000 viral DNA copies per 10⁶ lymph node cells. In addition, productively infected cells were readily detected in mangabey lymphoid tissues by in situ hybridization. The amounts of viral RNA in plasma ranged from 10⁵ to 10⁷ copies per ml. The cell-associated and plasma viral loads were as high as those seen in susceptible hosts (humans or macaques) during the asymptomatic stage of HIV or SIVmac infections. Thus, the lack of pathogenicity of SIVsm for its natural host cannot be explained by limited viral replication or by tight containment of viral production.     

Virus dynamics in a large epishelf lake (Beaver Lake,Antarctica)

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Abundance,Distribution, and Diversity of Viruses in Alkaline,Hypersaline Mono Lake,California

Mono Lake is a large (180 km²), alkaline (pH ~10), moderately hypersaline (70–85 g kg^–1) lake lying at the western edge of the Great Basin. An episode of persistent chemical stratification (meromixis) was initiated in 1995 and has resulted in depletion of oxygen and accumulation of ammonia and sulfide beneath the chemocline. Although previous studies have documented high bacterial abundances and marked seasonal changes in phytoplankton abundance and community composition, there have been no previous reports on the occurrence of viruses in this unique lake. Based on the high concentrations and diversity of microbial life in this lake, we hypothesized that planktonic viruses are also abundant and diverse. To examine the abundance and distribution of viruses and bacteria, water samples were collected from four stations along 5 to 15 vertical depths at each station. Viral abundance ranged from 1 × 10⁸ to 1 × 10⁹ mL^–1, among the highest observed in any natural aquatic system examined so far. Increases (p < 0.1) in viral densities were observed in the anoxic bottom water at multiple stations. However, regression analysis indicated that viral abundance could not be predicted by any single environmental parameter. Pulsed field gel electrophoresis revealed a diverse viral community in Mono Lake with genome sizes ranging from ~14 to >400 kb with most of the DNA in the 30 to 60 kb size range. Cluster analysis grouped the anoxic bottom-water viral community into a unique cluster differentiating it from surface and mid-water viral communities. A hybridization study using an indigenous viral isolate as a probe revealed an episodic pattern of temporal phage distribution with strong niche stratification between oxic and anoxic waters.