While bacteriophages have been the subjects of intense scientific study for decades, knowledge of their replication potential in situ is limited. If optimal environmental conditions for host cell growth are prevalent only on an infrequent basis, the production of bacteriophages may be severely limited and sporadic. Models of the dynamics of bacteriophages created by the simple extrapolation of laboratory observations of systems maintained under artificially optimal conditions may not faithfully represent situations in natural ecosystems. At the present time, because of the highly limited nature of the knowledge base, it is impossible to make accurate predictions of the magnitude of the effect of bacteriophages on bacterial populations in marine ecosystems.

In order to more accurately model bacteriophage dynamics in marine ecosystems, we have undertaken a study of the effects of environmental stresses on marine and freshwater bacteria. The effects of solar ultraviolet (UV) irradiation and starvation on the replication of bacteriophages and host cell survival have been quantified. These experiments have examined both the kinetics of progeny phage production as well as burst sizes subsequent to application of stress. Prophage induction due to DNA damage has been quantified in lysogens exposed to solar UV or starvation stress. In addition, the effects of broad-band solar UV irradiation on the survival of free virions have been quantified.

Key Words: Bacteriophage, dynamics, lysogeny, Weigle reactivation, starvation

Studies of both marine and freshwater ecosystems have revealed the presence of very substantial numbers of virus (bacteriophage) particles (Torrella and Morita, 1979; Bergh et al., 1989; Proctor and Fuhrman, 1990; Paul et al., 1991; Wommack et al., 1992). The exact origin(s), ecological importance, and functions of these bacteriophage particles are uncertain at the present time, but those studies that have been done (Proctor and Fuhrman, 1990; Suttle and Chen, 1992) coupled with the sheer magnitude of the abundance of these particles suggest significant ecological impact.

Bacteriophages have the potential to significantly affect microbial ecology in several distinct ways important for bioremediation and risk assessment. Infection of host bacteria results in the death of susceptible cells and production of progeny virions. Under laboratory (optimal) conditions, the production of bacteriophage at the expense of host cells can be so efficient that dense, turbid cultures are rapidly cleared due to the lysis of bacterial cells. Thus, under some conditions, bacteriophages may act to destroy sensitive bacterial cell populations. This destructive process is almost never total, however. The destruction by infection of host populations is a powerful selection for cells that do not support bacteriophage adsorption (loss of receptor is common) or replication. This effectively results in a rapid evolution of the host bacterial population to bacteriophage resistance. While it may then seem a simple matter to develop bacterial populations with the property of bacteriophage resistance, other properties of the host may also change, rendering such strains less useful or not useful for the intended process.

In addition to the potential for dynamic control of bacterial populations and resultant genetic changes exerted due to selection processes, bacteriophages are likely to be important mediators of genetic exchange processes in bacterial populations. The genetic exchange mediated by the direct transport of host genetic material encapsidated in virion particles (transduction) or possible stimulation of cell transformation as a result of cell lysis is both a potential accelerator and confounder of engineered bioprocesses. Evolution stimulated by genetic exchange may aid the adaptation of remediative communities to stress and metabolism of xenobiotic compounds. However, the risk assessement of such processes is complicated by the tendency of bacteriophages to disseminate genetic materials, both chromosomal and plasmid.

In order to understand and effectively model the effects of bacteriophages in situ, it is necessary to address questions concerning their origins, rates of production, and mechanisms of decay. While there is an excellent scientific understanding of the basic mechanisms of replication of bacterial viruses, primarily models are based on a (necessarily) limited number of defined host species growing under optimal or very favorable conditions. Estimates of potentials to produce bacteriophages subsequent to infection of microbial populations in situ, populations often subsisting under radically different physicochemical conditions with grossly different growth rates, may not faithfully represent true in situ situations. Moreover, bacterial hosts in natural ecosystems have a high probability of being subject to stresses, potentially concurrent, that usually have not been factored into predictions of in situ situations. Concurrent stress effects are most problematic since it is not trivial to predict whether interactions would be antagonistic, synergistic, or even detectable.

To refine models of phage dynamics in situ, we have initiated a study of the effects of stress agents known to impact populations of microbes at our freshwater and marine sampling sites on the replication of bacteriophages.

Bacterial and bacteriophage strains. Bacterial strains used were Pseudomonas aeruginosa PAO303 for experiments in freshwater environments and Vibrio alginolyticus ATCC 17749 for marine studies. Bacteriophages infecting P. aeruginosa were D3 and F116 (Kokjohn et al., 1991), Antelope Creek 1994 (T), a newly isolated bacteriophage obtained at our field site on the University of Nebraska-Lincoln, and Phage S, a clear-plaque forming bacteriophage isolated in 1994 during studies of ultraviolet radiation resistance of P. aeruginosa. Bacteriophage M-24, a phage infecting V. alginolyticus ATCC 17749 was isolated by our group from water samples obtained from Key West, FL.

Bacteriophage replication. One-step growth curves were conducted using modifications of the procedures of Ellis and Delbruck (1939). Starved cell cultures were produced by growing host cells in rich medium, Luria broth for P. aeruginosa, MBL/2 for marine hosts, to log phase, harvesting by centrifugation, and suspension of cells in 0.85% saline (P. aeruginosa) or MBL/2 salts (marine strains).

UV radiation resistance. Survival of cells and bacteriophages after exposure to ultraviolet (UV) radiation was determined by modifications of the methods of Simonson et al., 1990. Weigle reactivation of UV-irradiated bacteriophages was as described by Simonson et al., (1990.) Sources of UV radiation were a GE germicidal lamp (UV-C) or an Oriel Corp (Stratford, CT) Model 66002 Solar simulator for UV-B and UV-A radiation.

Bacteriophage inactivation by solar ultraviolet radiation. Using solar ultraviolet radiation delivered from our simulator, we have quantified the survival of free virions exposed to damaging effects of this portion of sunlight. Our findings are that all phages tested to date will show inactivation, although exact rates differ for varieties of virus.

One-step growth curves. For our typical bacteriophage-host system when growing under optimal conditions, the infected cells will have a latent period of approximately 60-90 minutes followed by a burst of, on average, 50-100 progeny bacteriophage particles.

Starvation of the host cells radically changes the kinetics of progeny phage production, although the precise magnitude of change differs with different host-bacteriophage systems. Starvation of host P. aeruginosa will result in an extension of the latent period of infecting D3 and F116 viruses from approximately 90 minutes to somewhere around 24 hours post infection if a burst of progeny phage occurs at all. Thus, starvation of the host in this case dramatically lengthens the latent period and in some cases may disallow bacteriophage replication. Replication of vibriophage M-24 is less affected by starvation of the host. While burst size is decreased, the kinetics of production of progeny bacteriophage is nearly the same as for fed infected cultures.

If host cells were exposed to environmental stimuli that seem to enhance production of bacteriophage would this counteract the starvation-induced decrease in replication potential? Exposure of starved host cells to heat shock, a treatment shown previously to potentiate bacteriophage replication (Wiberg et al., 1988), does seem to counteract the effects of starvation and allow phage production in starved host cells.

Reactivation of UV-damaged bacteriophages. Bacteriophage lysates damaged by exposure to UV-C radiation were infected into lightly-irradiated host cells to quantify Weigle (damage-inducible) reactivation. Solar UV radiation is an ineffective inducer of Weigle reactivation for some, but not all, bacteriophages. Phage S is reactivated when infected into a host cell irradiated with UV-A radiation (Figure 1).

Effects of lysogeny on host cell survival. Bacterial hosts were lysogenized with phage F116 by cross-streaking cultures against high-titer phage lysates. Survivors were streaked for isolation and tested for spontaneous phage release, a general property of lysogens. Confirmed lysogens were exposed to solar UV and percentage survival quantified. P. aeruginosa lysogens of phage F116 are more resistant to solar UV radiation than the parental (nonlysogenic) strain (Figure 2).

What is the potential for bacteriophage replication in natural aquatic ecosystems in which host cells will frequently be starved and subjected to stress such as solar UV radiation? Both starvation and solar UV radiation have deleterious effects on bacteriophage replication. Clearly, the clock is ticking once bacteriophage virions are released into the environment and their half-lives are limited. Starvation definitely affects the replication potential of bacteriophages and causes a slow-down in the rate of production. Indeed, our first experiments revealed that for some systems, reproduction may be disallowed in starved host cells. Moreover, other investigators have demonstrated that starvation of lysogens eliminates prophage induction (Lwoff, 1953).

Is the actual situation then one in which the replication of bacteriophage is limited in nature? First, such conclusions are difficult to reconcile with the observations of very high abundance of bacteriophage particles observed in natural ecosystems (Torrella and Morita, 1979; Bergh et al., 1989; Proctor and Fuhrman, 1990; Paul et al., 1991; Wommack et al., 1992) unless the additional assumption of slow turnover rates of these particles is made. Our experiments do reveal that some environmental stresses will act to override the negative effects of starvation on bacteriophage replication. Heat shock will allow replication to proceed in infected starved hosts, although heat shock apparently does not enable prophage induction to proceed in starved lysogens. In addition, our first experiments following infected starved host cells for long periods have revealed the apparent ability of such cells to ultimately produce a burst of progeny bacteriophage, although with greatly extended latent periods. Thus, the situation in reality will be complicated.

Studies of the ability of bacteriophages to reverse DNA damage due to solar UV irradiation have revealed that some apparently may be reactivated by host cells exposed to solar UV radiation (Weigle reactivation). While the actual ability of potential hosts in situ to reactivate damaged bacteriophages is unknown at present, these findings do suggest that some bacteriophages may have the capacity to resist UV-mediated destruction by repair of DNA lesions.

One of the more intriguing observations concerning the ecology of bacteriophages is that establishment of lysogeny may sometimes offer an apparent selective advantage to the host cell. Bacteriophage F116 and others present as prophage result in cells more resistant to solar UV radiation than the parental (nonlysogenized) strains. Thus, in solar UV irradiated environments, the condition of lysogeny may enable the host cells to be more competitive or survive for longer periods. For certain bacteriophages in some environments there may be a positive selection for lysogeny.

This work was supported by the U.S. Environmental Protection Agency through Cooperative Agreement CR-822163-01-0 and the University of Nebraska-Lincoln Water Center.

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Kokjohn Figure 1

Kokjohn Figure 2