Characterization of a cold-active bacteriophage on two psychrophilic marine hosts

Abstract

A cold-active bacteriophage designated 9A was isolated against Colwellia psychrerythraea Strain 34H at near in situ temperature (-1°C) by enrichment of seawater from an Arctic nepheloid layer, using a newly developed isothermal overlay technique. Phage 9A is classified as a Siphoviridae with a genome size of 80 to 90 kb. In addition to 34H, 9A infects C. demingiae ACAM 459 T ; no other hosts (of 22 tested) were identified. In replete media, 9A formed plaques on 34H from -6 to 4°C and on C. demingiae from -6 to 8°C; the temperature range of plaque formation on 34H could be extended to 8°C by prior host starvation. An indirect plating method and microscopic evaluation also determined phage production at temperatures between -13 and -10°C. At -1°C, 34H had a broader salinity range of plaque formation than C. demingiae: 20 to 50 (but not 65) psu vs. 27 to 34 (but not 50) psu. As monitored by epifluorescence microscopy, phage production by 34H was observed at 1, 10, 100 and 200 atm (all at -1°C), but not at 400 or 600 atm. The 9A-34H system commonly had a low efficiency of plating (EOP; typically ~1%) which varied with culture age. Despite repeated attempts, no meaningful adsorption rate could be determined at -1 or 8°C. This result, the low EOP, and the effect of starvation on plaque formation suggest that fluctuating host phenotypes may play an important role in the dynamics of this system. One-step growth curves (using 34H as host) revealed a longer latent period (4 to 5 vs. 2.5 to 3 h) and greater burst size (55 vs. 5) at -1 than 8°C; at temperatures between -10 and -12°C, the estimated latent period was 5-10 d and the burst size 5. At both -1 and 8°C, rise times were comparable to latent periods. Although the cycle from infection to burst at -1°C required only 10 to 20% of the generation time of 34H at this temperature, the amount of viral DNA synthesized was comparable to the size of the host genome, suggesting very efficient and cold-active virus-encoded enzymes.KEY WORDS: Virus · Phage 9A · Colwellia psychrerythraea 34H · Colwellia demingiae · Arctic · Adsorption · One-step growth curve · Pressure · Salinity Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 45: [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] 2006 'psychrophilic ' (e.g. Stamatin 1963, Olsen 1967, Ackermann & DuBow 1987 or 'psychrotrophic' (Greer 1982, 1983, Patel & Jackman 1986, the terms are ambiguous or misleading, since they imply growth characteristics of the host, not features necessarily specific to the virus (e.g. the temperature range of infection by a given virus may change with different hosts). For the sake of clarity, we define as 'cold-active' those viruses capable of infection and production at ≤ 4°C. This upper limit accords with the sparse literature about phage infection at low temperature and encompasses the temperatures of the perennially cold regions of the ocean. By this definition, several phage or phage-host systems (PHS) termed 'psychrophilic' or 'p...

Effects of temperature, salinity and clay particles on inactivation and decay of cold-active marine Bacteriophage 9A

“…A high titer phage lysate was prepared from silica overlay plates as described by Wells & Deming (2006c). The phage were stored at 4°C in phage buffer: 0.1 M NaCl, 50 mM Tris-HCl pH 8.0, 8 mM MgSO 4· 7H 2 0, 2 mM CaCl 2· 2H 2 0, 5% glycerol.…”

“…Following mixing and periodically thereafter, 50 µl samples were removed to sterile 0.5 ml microcentrifuge tubes on ice. When all samples had been collected (within 15 to 60 min, depending on the temperature), a dilution series of phage was plated using the silica-gel overlay technique (described by Wells & Deming 2006c) and Colwellia psychrerythraea strain 34H (henceforth 34H) first harvested in mid-exponential growth phase (A600 of 0.2 to 0.3) and concentrated 2 to 4-fold by centrifugation (6000 × g for 20 min at 2°C). Plates were incubated at 2°C; plaques took approximately 1 wk to form.…”

“…As discussed in Wells & Deming (2006c) and despite efforts to standardize 34H growth and infection conditions, the efficiency of plating (EOP) of Phage 9A on 34H was highly variable in a way that appears to reflect changing bacterial phenotypes. The exact cause of variability remains unknown.…”

“…High viral concentrations in the deep ocean (e.g. Hara et al 1996, Weinbauer et al 2003, Ortmann & Suttle 2005) also imply viral activity at low temperatures, but in situ or laboratory research pertinent to viral processes there has been rare (Wiebe & Liston 1968, Weinbauer et al 2003, Wells & Deming 2006c.…”

“…Isolated from an Arctic nepheloid layer, a distinct particle-rich region of the water column often associated with the bottom, and characterized by unprecedented cold activity to at least -10°C (Wells & Deming 2006c), Phage 9A is an obvious candidate for studies that explore whether, as in enzymes, cold activity is associated with greater lability in viruses and, if so, whether particles offset that lability. For this work, we focused on clay particles, which (along with silts) dominate the particle load of the Mackenzie River (Carson et al 1998), the major sediment source for the Mackenzie Shelf area (Macdonald et al 1998), whence 9A was isolated.…”