In situ burst size.
Burst size, i.e., the number of virus particles released upon host cell lysis, is an essential component in calculations that relate estimates of in situ viral production to the level of virus-mediated mortality of bacterioplankton. Intuitively, there is an inverse relationship of burst size and the level of host mortality due to viral infection. The greater the burst size, the smaller the number of host cells lysed to support a given level of virus production. Noting the dramatic effect of burst size estimates on modeling the impact of viral infection on host populations, many authors have chosen to use a range of burst sizes for these calculations (see Tables
2 through
6). However, it is likely that improvements in the precision of estimates of virus-mediated mortality can most easily be achieved through more accurate estimates of burst size under in situ conditions. To this end, several studies have used burst size estimates, based on in situ observations of bacterio- and phytoplankton, to calculate virus-mediated mortality. Burst size estimates are summarized in Table
2.
In a comprehensive review, Børsheim (
29) compiled a table of reported burst sizes for 26 marine bacteriophages. These estimates, based on one-step growth curve experiments, averaged 185 phages/lysed bacterium. This value is significantly higher than the estimates for in situ burst size reported in Table
2. It is not surprising that estimates of burst sizes, based on growth conditions in culture media, are larger than those in situ. In general, bacterial cells grown in the laboratory on bacteriological media are larger and support the production of greater numbers of phage particles (
29,
152,
200,
275,
347). Weinbauer et al. (
344,
347) found a significant correlation between in situ burst size and host cell volume. Increases in cell volume were paralleled by significant increases in burst size, especially for smaller phages (30 to <60 nm in capsid size). Stepwise multiple-regression analysis showed for Lake Plußsee water samples that cell size was the only parameter capable of explaining changes in burst size (
344). Furthermore, in situ observations of infected bacteria in the northern Adriatic Sea found that burst size estimates for bacterioplankton in eutrophic environments are significantly larger than those in mesotrophic environments (
342).
Nearly all of the estimates of in situ burst size (Table
2) are based on TEM observations of virus particles within intact (
176,
342) or thin-sectioned (
121) bacterioplankton cells. In most studies, in situ burst size estimates have been reported as averages from a number of single-cell observations; however, in a few instances, burst size has been reported simply as a range of individual observations (
33,
39,
44,
120,
121). While direct TEM estimates of in situ burst size are limited, there is surprisingly good agreement among the data. With the exception of the latter group of studies, the average burst size of bacterioplankton is 24 phage per cell across the range of environments examined (Table
2). This average estimate is lower than the burst size conversion factor of 50 used in other studies (
97,
120,
304,
318). For methods which utilize burst size estimates to calculate virus-mediated mortality of bacterioplankton, the use of the lower burst size estimate results in an increase in mortality estimates. Finally, TEM observations of intracellular viruses of the bloom-forming coccolithophorid
Emiliania huxleyi indicate that the in situ burst size for viruses of this important oceanic phytoplankter is ca. 400 to 500 viruses (
33,
44). With this burst size estimate, Bratbak et al. (
33) estimated that viral infection accounted for 25 to 100% of
E. huxleyi mortality during a bloom.
The most innovative method of estimating in situ burst size introduced thus far was reported by Heldal and Bratbak (
120). In their approach, bacteriophage within bacterioplankton cells are released by lysis of the cells from without through addition of streptomycin. Bratbak et al. (
39) show examples of this method in a series of TEM micrographs of streptomycin-lysed bacterioplankton cells. The authors regard estimates obtained by this method to be minimum, since all bacterioplankters may not respond to streptomycin treatment and since streptomycin, in some cases, prematurely terminates phage production; thus, the actual, full-term, burst size would be underestimated (
39,
120).
Another interesting approach to estimating in situ burst size is to calculate the burst size necessary to balance viral production with estimates of viral decay. Suttle and Chan (
312) used this approach for estimating in situ cyanophage burst sizes for near-coastal
Synechococcus populations. From estimations of
Synechococcus and cyanophage abundance, it was possible to calculate theoretical in situ contact rates between cyanophage and host. Assuming that all contacts result in infection, the burst size is simply the number of cyanophage produced per day, i.e., the cyanophage decay rate, divided by the abundance of cyanophage-infected
Synechococcus. Cyanophage burst sizes of between 92 and 324 were necessary to balance the range of cyanophage decay rates in Texas Gulf coastal water (Table
2) (
312). These in situ burst size estimates are the highest reported for any bacteriophage. It would be interesting to estimate in situ cyanophage burst size, both theoretically and directly, thereby allowing an assessment of the rigor of in situ burst size estimates and of the underlying assumptions required for the calculation.
The principal advantage of in situ burst size estimates is that these estimates account for the effect of environmental and biological variables on phage development. It has been demonstrated, using the in situ observation techniques discussed above, that burst size estimates can change significantly with temperature (
176), as well as with the trophic state of the aquatic environment (
342). It has also been observed that bacterioplankton morphological groups (rods, cocci, and spirilla) demonstrate significantly different burst sizes (
176,
342,
347), affecting the relative contributions of these groups to virioplankton production. Seasonal changes in burst size have also been noted (
121); however, these changes were only trends and were not statistically significant.
Viral inactivation and particle destruction.
Since the earliest studies of bacteriophage physiology (
79), researchers have routinely reported data on the stability of purified viral isolates. Initially, data on viral inactivation were of practical use only in research; however, public health concerns about the safety of recreational, drinking, and shellfish-producing waters soon added significance to investigations on the survival of viruses in aquatic environments. Thus, most studies on the survival and fate of viruses in natural waters has focused directly on enteroviruses or on coliphages used as indicators of enteroviral pollution. The latest stimulus for investigating virus decay and destruction in natural waters has come from interest in the impact of viral infection on bacterial productivity. If, as enumeration studies seem to indicate, virioplankton abundance is relatively stable over seasonal scales, rates of viral production and loss should be equal. Therefore, estimations of viral loss and inactivation in aquatic environments should indicate the virus production rate needed for steady-state conditions. In turn, estimates of viral loss rates can lead to estimates of the level of virus-mediated mortality of bacterioplankton hosts. For excellent, comprehensive reviews of the early literature on viral inactivation, see references
5 and
141.
The difficulty in detecting viral inactivation factors in natural waters arises from the complex interplay of physical, chemical, and biological variables which influence the survival of an infective virus. Nevertheless, from the wealth of data on this topic, it is possible to draw conclusions about the viricidal qualities of various physical, chemical, and biological factors. Among studies of environmental virus inactivation, the most prevalent finding is that the presence of a natural bacterial community and particulate organic matter (POM) greatly increases the rate of viral inactivation. As shown in Table
3, in nearly every case the removal of bacterioplankton and POM (>0.22 μm) from water by filtration results in a lower decay rate than in untreated natural water. The rare exception to this general conclusion was the observation that water filtration slightly increased the decay of coliphage φX174 (
190,
191) or did not change the decay of poliovirus (
6).
Further support for the involvement of bacteria, as well as heat-labile substances, in viral inactivation comes from the general observation that either heating or autoclaving natural water samples prior to viral inoculation improves virus survival. The positive effect of heating on virus survival has been observed in viral inactivation experiments with enteroviruses (
168,
178,
217,
291) coliphage (
21,
46,
190), and marine bacteriophage (
215). It is likely that observations of increased viral inactivation at higher incubation temperatures (4 to 25°C) are related to temperature-mediated enhancement of bacterial and enzymatic activity in natural waters (
101,
107,
166,
176,
213,
377). The exact nature of the virucidal heat-labile substances within natural waters is not known; however, it is suspected that these substances exhibit both protease and nuclease activity. Attempts to re-create the virus-inactivating capability of activated sludge, with RNase and trypsin (
340), or of seawater, with a bacterial cell lysate (
178), were unsuccessful. However, Noble and Fuhrman (
215) were able to reconstitute 20 to 25% of the virucidal activity of natural seawater by addition of high-molecular-weight DOM (colloids and dissolved particles in seawater, 0.2 μm and 30,000 Da size range) to heat-treated or filtered seawater. Finally, the implication of nonliving biological or colloidal material in viral decay is supported by the observation that a low level of viral inactivation occurs in cyanide-treated seawater (
318).
While some biological and chemical components of natural waters have aggressive virucidal properties, others serve to promote virus survival. In particular, the inactivating effects of bacterioplankton and heat-labile dissolved compounds can be offset by the protective effect of viral adsorption to sediment and particulates suspended in the water column. Indeed, the general sorptive properties of viruses to suspended particulates in water (see the review by Bitton [
26]) have been exploited in several methods for concentrating viruses from natural water samples (
7,
81,
82,
252,
287,
288,
336). In controlled experiments, the addition of clay or sediment to water, prior to the addition of virus, lowered the decay rate of both enteroviruses (
158,
297) and coliphages (
27,
105). In addition to inorganic particulates, heat- or UV-killed bacterial cells can protect viruses through adsorption (
191). The degree to which sorption onto particulates counteracts viral inactivation is affected by the ionic environment and whether adsorption is reversible. It is possible that the differential and sometimes contradictory effects of salinity on viral inactivation in natural waters (
106,
166,
178,
193,
378) are related to the influence of ionic environment on the adsorption of a particular virus strain to water column particulates.
From the wealth of scientific literature gathered prior to 1990, concerning viral inactivation in natural waters, it appeared that most of the important environmental factors involved in decay had been identified. However, the role of sunlight as a dominant factor in the decay of marine bacteriophages brought a new perspective to the issue (
318). The most recent review, to 1992, by Kapuscinski and Mitchell (
141) on the topic of viral inactivation in seawater, concluded that the levels of solar radiation penetrating surface waters is not important in the degradation of enteric viruses discharged into natural waters. In studies using coliphages that were conducted prior to 1992, sunlight was reported to have only a mildly enhancing effect on viral inactivation (
21,
213). More recent studies, utilizing various marine bacteriophages (
215,
312,
318,
357,
369), cyanophage (
101,
312), and a virus of
Micromonas pusilla, a cosmopolitan marine phytoplankter (
62), have shown that unattenuated sunlight is a dominant factor in controlling the decay of viral infectivity in surface waters. In most cases, exposure of experimental treatments to sunlight results in a doubling of viral inactivation rates (Table
3).
The effect of sunlight on bacteriophage viability in seawater is directly proportional to the amount of sunlight (
101,
318). From data correlating decay rates of five marine bacteriophages with irradiance, Suttle and Chan (
312) developed a predictive model for estimating the phage inactivation rate from measurements of incident quantum irradiance. By combining surface irradiance measurements with sunlight attenuation coefficients for water samples of different turbidities, they found that sunlight-induced inactivation was significant to a depth of 200 m in clear oceanic waters (
318). Even in turbid estuarine and near-coastal waters, a modest effect of sunlight on viral decay can occur at a depth of 2.5 m (
62). Predictably, light wavelengths of <320 nm (UV-B) had the greatest virucidal effect on marine bacteriophages (
207,
318,
357,
369), accounting for up to two-thirds of the total decay (
215). Exclusion of UV-B or reduction of incident sunlight to 20% of surface levels still resulted in significant decay, compared to dark controls (
318). Recent experiments with natural cyanophage populations in the Gulf of Mexico found UV-A (320 to 400 nm) to have the greatest impact on the inactivation of this virioplankton group (
101). However, involvement of wavelengths longer than 400 nm in marine phage inactivation has not been supported (
215).
Conclusions presented to date on virucidal factors in natural water come from experiments assessing the loss of viral infectivity under various environmental conditions. A connection between a loss of viral infectivity (inactivation) and destruction of virus particles has a special relevance for our understanding of in situ virioplankton production. If the mechanism of viral inactivation is directly related to, or coincident with, destruction of the virus particle, then VDC should be an accurate estimate of the abundance of infective viruses. In a few studies (Table
3), the loss of viral particles has been examined using natural seawater (
97,
101,
120,
176,
357). The rate of viral particle loss found in these studies ranged from 1% h
−1, which compares well with viral inactivation rates, up to an astounding 54% h
−1 (
120). The broad range of these estimates does not support or nullify a connection between particle destruction (loss) and inactivation. Similarly, experiments examining the destruction of enterovirus in natural water have not supported a direct connection between particle loss and inactivation. Studies of the destruction of poliovirus and coxsackievirus in experimental microcosms concluded that inactivation of enteroviruses coincides with cleavage or damage of viral RNA (
217,
340). However, loss of infectivity precedes destruction of viral capsids (
124,
325).
Wommack et al. (
369) determined rates of inactivation and loss of VDC in artificial seawater microcosms seeded with bacteriophage strains CB 7Φ and CB 38Φ isolated from Chesapeake Bay (K. E. Wommack, R. T. Hill, and R. R. Colwell, Abstr. 93rd Gen. Meet. Am. Soc. Microbiol. 1993, abstr. Q-288, p. 399, 1993). The results are shown in Fig.
2 and
3. As expected, for both phages the infectivity declined rapidly under surface sunlight conditions, at a rate twice that of low-light, control, and dark treatments. CB 7Φ, however, was more resistant to the inactivating effects of sunlight than was CB 38Φ. Others have also noted marked differences in the UV tolerance of bacteriophage strains (
133). Under surface sunlight conditions, the abundance of virus particles significantly exceeded the number of infectious viruses, whereas, under low-light, control, and dark treatments, the titers were in close agreement. Unlike inactivation rates, the rate of loss for CB 7Φ and CB 38Φ phage particles was identical, regardless of experimental treatment.
Altogether, the findings of Wommack et al. (
369) indicate that destruction of phage particles is a process separate from loss of infectivity, especially for sunlit waters. The marked difference between inactivation and particle destruction was also noted for phages LB1VL (
317) and PWH3a-P1 (
357) in Texas coastal waters. In the absence of sunlight, similar processes may be responsible for the loss of infectivity and the destruction of virus particles. Finally, while CB 7Φ and CB 38Φ had substantially different tolerances to the inactivating effects of sunlight, they showed similar rates of particle loss, regardless of treatment, indicating that in seawater, all viral capsids may have a characteristic degradation rate. The notion of an “expiration date” for viral capsids in aquatic environments is supported by the observation that in lake water, capsid destruction rates were similar for coxsackievirus and poliovirus but inactivation rates were not (
124). Conversely, however, data from studies documenting total virioplankton loss rates indicate that there is a range of capsid degradation rates. Two studies in which virioplankton loss was calculated from changes in VDC reported that the <60-nm capsid size class exhibited the most rapid decrease whereas larger viruses comprised the more refractory portion of the virioplankton (
120,
176). Overall, the important implication of these findings for understanding viral abundance is that VDC most probably overestimate the actual number of infectious virioplankton in sunlit surface waters.
The extreme sunlight sensitivity of viruses leads to a prediction of very low or nonexistent titers of infective viruses in surface waters. However, as noted previously, titers of infective cyanophages can be as high as 10
4 to 10
5 ml
−1 in surface waters (
312,
313,
341). As pointed out by Suttle et al. (
317), this suggests a paradox, especially for viruses such as cyanophages and algal viruses, which infect photoautotrophic hosts. A plausible explanation of this paradox is that the infectivity of sunlight-inactivated bacteriophages is restored by either host- or phage-mediated DNA repair. The phenomenon of photoreactivation of UV-inactivated bacteriophage is well known. Indeed, this phenomenon alerted researchers to the existence of specific DNA repair machinery in bacteria (
74,
75,
231). Subsequently, a great deal of the basic enzymatic and genetic mechanisms involved in DNA repair have been elucidated using coliphages T4 and λ (
20). However, the importance of photoreactivation or other repair mechanisms for the survival of viruses in natural waters has only recently been investigated.
Weinbauer et al. (
351) demonstrated that infectivity could be restored and virus production could be increased by incubation of sunlight-damaged viruses with a host population treated with photoreactivating wavelengths of light. Generalized light-dependent repair of the virioplankton was demonstrated by the observation that incubation of sunlight-damaged virioplankton with bacterioplankton under photoreactivating conditions always resulted in a greater production of viruses than did incubation of sunlight-damaged virioplankton nonactivated bacterioplankton (
351). The degree to which host cell light-dependent repair was capable of restoring viral infectivity varied according to the aquatic environment. In oligotrophic ocean samples, VDC of sunlight-damaged virioplankton incubated with activated bacterioplankton recovered to only 25% of control titers, whereas for coastal-water samples, VDC recovery was nearly 50% of control titers. The authors proposed that due to water clarity, oceanic virioplankton suffered a greater degree of nonrepairable damage (
351). The repair of virioplankton infectivity has also been estimated from the difference between loss rates of infectivity and virioplankton particles in mesocosm incubations. Wilhelm et al. (
357) argued that to maintain the steady state (i.e., a stable virioplankton population size) … “infectivity cannot be lost faster than virus particles,” assuming that the pool of infectious viruses is derived from the production of new viruses. Based on these assumptions, it was calculated that for Gulf of Mexico waters, between 54 and 78% and between 39 and 67% of viral infectivity was restored daily at oligotrophic and mesotrophic stations, respectively.
The principal mechanism of UV-mediated viral inactivation is the formation of DNA photoproducts such as cyclobutane pyrimidine dimers (CPDs). Immunochemical detection and quantification of CPDs within viral DNA was recently utilized to determine the relationship between DNA damage and loss of infectivity in marine virus strains (
358). As with experiments in sunlight, viral strains showed marked differences in susceptibility to UV inactivation; however, in each of the four phage-host systems examined, host-mediated photorepair systems were responsible for significant recovery of inactivated viruses. Subsequently, the relationship of CPD abundance to loss of infectivity for one virus strain, PWH3a-P1, was used to estimate inactivation in natural viral communities. From measurements of CPD abundance within sunlight-exposed virioplankton communities, it was determined that under photoreactivating conditions, more than 50% of the viruses remained active, even at the height of daily CPD formation (
358).
The findings reviewed above indicate that host-dependent, light-mediated repair may be the primary mechanism of phage recovery; however, phage-encoded light-independent repair mechanisms exist, the best studied of which is mediated by T4 endonuclease V, encoded by
denV (
20). Recently, a genetic and phenotypic homolog of
denV (A50L) was discovered in
Chlorella virus PBCV-1 (
100). DNA sequences homologous to the PBCV-1 A50L gene were found in several other
Chlorella virus strains. The possibility that other, unique, phage-encoded repair systems remain to be discovered was recently demonstrated with a
P. aeruginosa phage, UNL-1, which is capable of
recA-independent repair in UV-A-irradiated hosts (
290). The existence of both host-mediated photoreactivation repair and phage-encoded endonuclease repair may explain how high titers of viruses infecting photoautotrophic hosts are maintained in nature (
333,
379). Furthermore, the discovery of similar dark repair enzymes in unrelated viruses is exciting, since it suggests that such mechanisms may be widespread among viruses (
100).
Differences in sunlight inactivation rates of CB 7Φ and CB 38Φ (Figs.
2 and
3) demonstrate that environmental persistence is a trait particular to each phage strain. As is evident from the data on decay rates presented in Table
3, viruses exhibit a range of susceptibilities to the virucidal effects of the aquatic environment, related in part to phenotypic traits, such as phage-encoded DNA repair. Under in situ conditions, autochthonous bacteriophages appear to survive better than their allochthonous counterparts (
107,
215,
220). The difference between sunlight tolerance of native (Southern California Bight) and nonnative (North Sea) bacteriophages was especially pronounced when both were exposed to southern California sunlight conditions (
215), suggesting that virucidal factors provide a selective pressure influencing the composition of virioplankton communities. Support for this hypothesis includes the recent observation that there is a strong seasonal component to the susceptibility of natural cyanophage populations to light-dependent decay (
101). In environments where the amounts of particulates and UV-adsorbing compounds are small and the level of incident sunlight is high, a large proportion of the virioplankton may be nonviable or inactivated. However, it is probable that in more turbid waters, a large proportion of viruses remain infective. Through mathematical modeling, it has been demonstrated that the degree to which virioplankton populations are affected by sunlight is also strongly influenced by the degree of stratification and mixing that occurs within the water column (
207).
Host cell concentration and viral replication.
To maintain a steady-state concentration of a virulent bacteriophage population requires that a single progeny phage from each burst event survive to infect and replicate (
89,
208). Intuitively, as the host cell concentration declines, the theoretical time to successful host-virus contact increases. At a critical threshold of host cell density, viral inactivation and destruction rates exceed the contact rate and therefore the number of virulent phage declines. Viewed in these terms, virus-host contact has been described mathematically as diffusive transport (
208).
Considering only the physical properties of the water column, (e.g., size, abundance, and motility of a virus and its single cell host) Murray and Jackson (
208) devised a predictive model for contact rates between viruses and planktonic hosts of various sizes. From simulations, close agreement was observed between theoretical and empirically derived attachment rates for a number of virus-host systems. Important conclusions derived from the diffusive-transport model were that the enhancing effect of host motility on viral adsorption is inversely related to host size and that the process of contact and adsorption to nonhost particles may be an important factor in virus mortality. Despite a significantly greater contact rate for viruses and large host cells, it is more probable that a virus and bacterium will connect because of the sheer abundance of bacterioplankton within aquatic ecosystems.
The diffusive-transport model, while useful for estimating an ideal host-virus adsorption rate, has not been widely applied to predicting the concentration of host cells required for the maintenance of a population of virulent viruses. This is principally because important model parameters, e.g., in situ concentration, burst size, and production rate of a specific virus, are not known. An elegant example of the application of diffusive transport theory to predicting the concentration of hosts necessary for viral propagation was a study of the dynamics of a lytic virus,
MpV, infecting the ubiquitous marine picoflagellate
Micromonas pusilla (
62). Because in situ titers of
MpV could be easily obtained, Cottrell and Suttle (
62) were able to estimate the concentration of 1.1 × 10
3 to 5.4 × 10
3M. pusilla ml
−1 required to maintain the
MpV production rate in Texas Gulf Coast water samples. The fivefold difference in threshold density of
M. pusilla arose from differences between theoretical and measured adsorption rates. The lower adsorption rate measured for the
MpV clone from the Texas water samples illustrated that the rate can vary among virus clones and that theoretical rates are likely to be maximal estimates (
62).
Other studies carried out to examine changes in viral production relative to host cell concentration have relied on in vitro approaches. In experiments using three bacteriophage-host systems, 80α (
Staphylococcus aureus), T4 (
Escherichia coli), and SPβ
cI (
Bacillus subtilus), it was found that bacteriophage production did not occur until the host cell concentrations reached approximately 10
4 cells ml
−1 (
355). Because similar threshold concentrations were found in phylogenetically dissimilar hosts, the authors suggested that this trait may be universal for virulent phage-host systems. This idea was challenged, however, when a series of one-step growth curve experiments, employing three
Pseudomonas aeruginosa bacteriophages, demonstrated both successful phage attachment and production at low host concentrations (10
2cell ml
−1), with little or no change in burst size or latent period (
156).
The discrepancy between these two studies most probably arises from differences in experimental design. Kokjohn et al. (
156) separated the effect of host cell density on the two independent processes of infection and replication by using the classical one-step growth curve experimental design (
79), whereas Wiggins and Alexander (
355) examined coincident phage production in a batch culture of growing host cells. Of the two studies, the former, employing
Pseudomonas aeruginosa phage-host system (PHS) models, lends stronger support for survival of a virulent phage population in an aquatic environment. Even if a higher concentration, such as that suggested by Wiggins and Alexander (
355), is necessary, virulent-phage survival would theoretically still be possible. Near-coastal and estuarine waters, where bacterioplankton concentrations are typically 10
6cells ml
−1, may contain up to 100 different bacterial hosts, each at a concentration of 10
4 ml
−1.
In addition to pure-culture studies, observations of natural virioplankton dynamics indicate the existence of a minimum host cell concentration necessary for viral replication. Through linear regression analysis, it was found that a minimum bacterioplankton concentration of between 4.7 × 10
5 and 7.2 × 10
5 bacteria ml
−1 is necessary for measurable levels of virioplankton production in Southern California Bight water samples (
306). A similar statistical analysis of bacterioplankton and virioplankton abundance data for Southern California Bight and Gulf of Bothnia water samples did not yield a minimum concentration of bacterioplankton before a measurable number of viruses could be obtained (
56). However, in the latter study it is possible that the actual threshold concentration of host cells was below the detection limit for both viral and bacterial direct counts (10
4 ml
−1). The former approach of comparing the viral production rate with bacterial abundance is more likely to detect an in situ threshold host cell concentration, because, as shown in Table
1, it appears that no natural aquatic environment is devoid of viruses as free virus particles. Finally, FVIC data from northern Adriatic Sea water samples indicated that when the numbers of rod-shaped bacteria was below 2 × 10
5ml
−1, no intracellular viruses were observed in this dominant morphological group (
347).
Observations of the in situ dynamics of marine
Synechococcusphage indicate that a minimum number of host cells is necessary for replication of these phage. In an analysis of changes in the natural abundance of cyanophages infecting specific strains of marine
Synechococcus and total
Synechococcus abundance, it was demonstrated that synechophage numbers increased dramatically when the total
Synechococcus abundance reached ca. 10
3 ml
−1 (
312,
313). Long-term, seasonal observations of marine
Synechococcus and cyanophages in Woods Hole Harbor indicated that if a minimum number of
Synechococcus cells, i.e., a threshold density, exists, it is likely to be very small, since titers of specific synechophage were detected when the number of
Synechococcus cells was as small as 10
2 ml
−1 (
341).
Perhaps the strongest evidence that maintenance of a stable virus population requires a specific number of host cells was provided by the observations of Wilcox and Fuhrman (
356). In mesocosm experiments, natural virioplankton and bacterioplankton densities were altered and changes in VDC and BDC were monitored for 6 days. For all experimental treatments, an initial titer of >10
6 viruses ml
−1 was necessary for VDC to rise to in situ levels. Interpreting these results in light of the diffusional transport theory (
208), the authors concluded that the product of bacterioplankton and virioplankton concentrations had to be >10
12 in order for lytic virus production to occur (
356). Therefore, in aquatic environments, such as estuarine and near-coastal waters, where the product of the number of bacteria and virus is typically on the order of 10
13(10
6 bacteria ml
−1 × 10
7viruses ml
−1), lytic viral production would be high.
While these in situ observations indicate the population densities of bacterioplankton and virioplankton at which virus production seems to occur, they cannot predict the threshold density of a single bacterial host species necessary for replication of a particular virulent phage strain. Estimates of threshold density under in situ conditions require data on the temporal dynamics of the several naturally occurring PHS. With current technology, direct observation of PHS dynamics is difficult. However, based on theoretical calculations of phage-host encounters (
303), production of a lytic virus appears to occur under natural conditions. In an examination of bacterial populations in a meromictic lake, Tuomi et al. (
329) postulated that lytic-virus production could explain in situ fluctuations in the densities of individual bacterial strains. In this system, VBR was a relatively constant high value of ca. 50; therefore, if individual bacterial populations were on the order of 10
4 ml
−1 and each virus strain was, on average, 50-fold more abundant than its host, then ca. 750 virus contacts h
−1 occurred. The authors concluded that in this system, lytic-virus infection could account for dramatic changes in the abundance of bacterioplankton populations.
Host metabolic state.
It is widely understood that the physiological state of bacteria under laboratory culture conditions is dramatically different from that under natural conditions. Even in the most productive aquatic environments, bacteria are hypothesized to exist in a condition of “feast-or-famine” (
157), in which periods of nongrowth are punctuated by short periods of rapid growth. Therefore, the normal state of aquatic bacteria is generally believed to be approximated by the stationary phase of a typical bacterial growth curve (
157). Besides obvious metabolic slowdown, the transition from exponential growth to the stationary state results in dramatic changes in cell morphology, rates of macromolecular synthesis and degradation, and constitution and surface characteristics of the cell wall (
146,
294). The genetic mechanisms behind these adaptations to starvation survival in nonsporulating bacteria are only beginning to be revealed (see the review by Matin et al. [
177]). After some time in a nongrowth state, cells may enter the viable-but-nonculturable state characterized by loss of culturability on standard media but with retention of substrate responsiveness (viability) (
61,
277).
The bias toward the study of exponentially growing bacteria is based on the requirement for balanced growth in physiological experiments. Similarly, this bias has extended to the study of the biology of phage-host interactions, since nearly all of the thousands of described bacteriophages infect and replicate apparently only in rapidly growing hosts (
272). The belief that most aquatic bacteria exist in a slow-growth or starvation state is at odds with our current understanding of the nature of phage-host interactions and calls into question whether bacteriophage infection and replication occur in aquatic environments.
Detailed studies of bacteriophage infection of senescent host cells are few. The one detailed examination of phage infection and growth in stationary-phase cells utilized
Achromobacter bacteriophage α3a (
375). Compared with growth on logarithmic-phase, wild-type
Achromobacter, the growth of α3a on strain 14 (an
Achromobacter strain supporting α3a replication only during stationary-phase growth) showed dramatic increases during the latent period (100 min versus 6 to 9 h) and in burst size (153 versus 700). Significant production of α3a by stationary-phase strain 14 cells required microaerophilic conditions, was observed in stationary (nonshaken) cultures for up to 19 days, and was not due to infection of a small subset of growing cells (
273,
375). Increases in the latent period and changes in the burst size of bacteriophages infecting starved or stationary-phase host cells were recently documented for several bacteriophages specific for either
P. aeruginosa or
E. coli hosts. Astoundingly, two of the
P. aeruginosa phages, ACQ and UT1, were able to infect and replicate in host cells which had existed under starvation conditions for 5 years (
286). Delayed plaque formation or continuous growth in plaque size on an established lawn of host cells have been speculated to be characteristic of a stationary-phase bacteriophage (
272). When isolating several PHS from Chesapeake Bay and Bahamian waters, we have observed two instances of bacteriophages demonstrating growing plaques. We concluded these bacteriophages are likely to infect host cells both during rapid growth and during host cell senescence (Wommack et al., Abstr. 93rd Gen. Meet. Am. Soc. Microbiol. 1993).
Mechanisms responsible for stationary-phase-specific infection and replication of α3a on strain 14 were suggested to be related to template specificity of the host RNA polymerase (
274) and not to selective attachment of α3a only to stationary-phase cells (
273). Similarly, the RNA coliphage MS2 demonstrated the curious ability to attach to, infect, and replicate in both stationary- and logarithmic-phase host cells; however, MS2 production in stationary-phase hosts was not followed by immediate lysis. Delay of cell lysis appeared to be linked to cell division, since infected stationary-phase cells released progeny virus only after resumption of cell growth (
259,
260). In an ecological sense, the behavior of coliphage MS2 is a possible explanation for the persistence of a virulent phage within a population of nongrowing hosts. By delaying lysis but not production until cell division, progeny phage are released during periods of rapid growth when conditions are more favorable for further rounds of phage infection and replication.
The aforementioned studies demonstrate that phage attachment and production can occur in physiologically stationary cells under ideal culture conditions; however, the stationary state of bacterial host cells under natural conditions has not been examined. The effect of the natural starvation state on phage infection was examined in experiments with two bacteriophages, UT1 and Φ116L, and starved
P. aeruginosa cells placed in autoclaved river water (
156). Both the virulent UT1 and the temperate Φ116L phages were capable of infecting and replicating in starved (
156) cells under riverine conditions, albeit with a lengthened latent period and greatly reduced burst size, compared to logarithmic-phase infection. Significant attachment (≥44%) of UT1 and Φ116L to starved
P. aeruginosa cells was observed as long as 40 days after the onset of starvation conditions.
At present, the number of bacteriophage species capable of infecting nongrowing hosts appears to be limited; however, due to the wealth of evidence for active viral infection in aquatic microbial communities, it is intuitive that stationary-phase infection should be common within the virioplankton. Recent studies by Moebus (
199,
200) offer a first step toward wider demonstration of this phenomenon. Four of six marine PHS that were assayed showed some degree of stationary-phase infection after host propagation on low-nutrient media. Dramatic differences were observed in the ability of senescent cells to propagate phage. For example, PHS H40-H40/1 showed no decrease in the ability to produce phage throughout the 30-day assay period, whereas, bacteriophage H2/1 could not infect and grow on stationary-phase H2 host cells that were more than 3 days old. All PHS showed decreases in the rate and extent of phage production during the logarithmic-phase-to-stationary-phase transition (
199). This observation may indicate that cellular changes occurring during prolonged nongrowth are necessary for phage infection. It is interesting that, like phage α3a, production of phage H3/1 on stationary-phase H3 cells was significantly faster under microaerophilic conditions. It is exciting to speculate that physiological changes occurring with the adjustment to microaerophilic conditions may coincide with those necessary for phage infection and production in nongrowing cells.
From the few studies presented, it is not possible to estimate the fraction of marine bacteriophage within the virioplankton which are capable of infection and replication on nongrowing hosts. The interaction between phage and bacterial host cells in a natural state of physiological stasis, such as the viable-but-nonculturable state, also remains an open question. Finally, it has been suggested that study of stationary-phase bacteriophage infection may provide insight into this enigmatic physiological state (
273).
Host range.
It is possible to view the bacterioplankton host community as comprising nongrowing cells within a diverse assemblage of species, each present at small numbers. Under these conditions, propagation of highly strain-specific viruses seems unlikely; however, a bacteriophage capable of infecting a broad range of bacterial hosts would, conceivably, have better potential for replication under in situ conditions. In general, phage do not “trespass generic boundaries” (
2). Therefore, among the ca. 4,000 known bacteriophages, host specificity is the rule. Similarly, most aquatic viruses (
102,
264) and bacteriophages (
13,
22,
64,
117,
151,
321) examined to date have demonstrated either species or strain specificity. Nonetheless, broad-host-range (polyvalent) bacteriophage have been demonstrated on several occasions.
Most notable of these are the lipid-containing phages of the virus family
Tectiviridae (see reference
10 for a review of the
Tectiviridae type phage PRD-1). Several phages within the
Tectiviridae have demonstrated the ability to infect multiple genera within either gram-positive or gram-negative domains (
10). This unusually broad host range is explained by the fact that the phage receptor is encoded on conjugative plasmids (
32,
374). Marine bacteriophages described to date have generally shown species specificity with commonly observed resistant strains (see reference
29 for a review). One exception is the vibriophage KVP40, which was shown to infect eight
Vibrio species and the closely related bacterium
Photobacterium leiognathi (
180). The basis of the broad host range of KVP40 is the OmpK receptor, which, from sequence information and immunoblotting, is unique to
Vibrio and
Photobacterium strains (
130,
131). The central role of the receptor in determining host specificity is also evident from the observation that the phage host range is often extended by mutations in the phage genome, which allow the use of an additional receptor molecule for attachment (
203). Investigation of bacterial strains showing a reduced efficiency of plating for KVP40 revealed a restriction-modification system which may be common to several species of the
Vibrionaceae (
179). The existence of a common restriction-modification system is noteworthy, since R-M systems are generally species or strain specific (
31). It is interesting to speculate that the widespread R-M phenotype found in the
Vibrio strains is a result of the selective pressure of the broad host range of KVP40.
Perhaps the most careful analysis of host specificity among a group of aquatic bacteriophages has been done for cyanophages. In a collection of 14 cyanophage isolates infecting marine
Synechococcus strains, only 2 phage were specific to a single host strain. All other phage infected at least three different strains of
Synechococcus marine cluster A (
341). Among seven cyanophages isolated from Texas Gulf coast waters, most were specific to a single host strain (five out of seven), while two, S-PWM3 and S-PWM4, were polyvalent (
313). In both studies, host range assays identified marine
Synechococcus strains which were particularly susceptible to cyanophage attack and thus were useful as indicator strains. Among freshwater cyanophages, a similar variability in host range has been reported, ranging from a virus strain which infects several filamentous species across three genera (
279) to more host-specific strains infecting only two or three strains of heterocystous cyanobacteria (
295) or unicellular forms (
278,
280) (see reference
229 for a review).
It appears that polyvalence may be more common among cyanophages. However, none of the studies gave results that correlated host phylogeny with cyanophage susceptibility. Both studies involving marine
Synechococcus assayed strains containing either phycocyanin or phycoerythrin. Based on this criterion, only three cyanophages were capable of infecting representatives of both pigment groups (
313,
341). As pointed out by Ackerman and DuBow (
3), coliphages often appear to be polyvalent because the family
Enterobacteriaceae is such a closely related bacterial group. At present, there is not enough information on the host range of aquatic bacteriophages to determine the degree of polyvalence among virioplankters. A study of activated-sludge PHS showed that polyvalence could be as high as 10% in that natural bacteriophage consortia (
114). A method, recently developed by Hennes et al. (
123), which involves labeling of bacteriophage particles with fluorescent dyes, could be a promising means of rapidly determining whether a new phage isolate is potentially polyvalent. With this approach, they demonstrated that phage PWH3a specifically attached only to its host and not to other cells within a natural bacterioplankton consortia. Conversely, the known polyvalent cyanophage S-PWM3 attached to ca. 3% of
Synechococcus cells in water samples.
The varied susceptibility of bacteria to phage infection and the generally restricted host range of bacteriophage have been exploited to construct highly selective schemes for typing bacterial strains (
250). In an application of phage typing, Moebus and coworkers (
201,
202) utilized a large collection of marine PHS to examine the geographic distribution of bacterial populations. From the large set of phage-typing data, it was evident that bacterioplankton populations west of the Azores were phenotypically distinct from those east of the Azores. Furthermore, eastern North Atlantic bacteria were often susceptible to infection by “western” phages, whereas the converse was only rarely seen (
202). These susceptibility patterns, the authors argued, reflect the general circulation of surface waters in the North Atlantic from west to east. Interestingly, out of the 217 Atlantic series phages positive for infection, only 41 (18.9%) were specific solely to the original host strain. Because the taxonomy of the bacterial hosts tested was unknown, the authors made no claim that phages infecting several strains were actually polyvalent.
More recently, the phage-mapping approach was applied to German coastal waters. Unlike the Atlantic series, the Helgoland series of bacteriophages appeared to have a higher incidence of host specificity. Among the 194 phages of this typing set, 88 were propagated solely on their original host (
194). However, the Helgoland collection also contained a few polyvalent members, since a recent study extensively characterizing 85 of these phages showed that 27% had moderate to broad host ranges (
353). In general, these phage-typing experiments found a greater degree of polyvalence among phages collected from low-nutrient, pelagic ocean waters than among those collected from productive coastal waters. This observation raises the intriguing possibility that bacteriophages occurring in oligotrophic oceanic waters with a low bacterioplankton abundance have a tendency toward polyvalence whereas phage found in nutrient- and host-rich coastal waters are generally host specific. This hypothesis is tenable because, in a low-host-density environment, broad-host-range phages would intuitively have a greater chance for successful host encounter. Conversely, high-host-density environments, where host-phage encounter is more common, would presumably select for hosts with broad-range resistance to abundant cooccurring viruses.
Finally, recent work suggests that the dogma of extreme host specificity among bacteriophages may be a methodological artifact (
132). Tests of two bacteriophage collections revealed that 90% of the phages were polyvalent. The first group, phages infecting
Sphaerotilus natans, were capable of infecting
Pseudomonas aeruginosa with the same efficiency as the original host due to a general resistance of these phages to type I and II restriction endonucleases. The second group was more typical of polyvalent phages, since these phages plated much less efficiently on the alternate host. The latter group was isolated using a novel two-host enrichment protocol instead of the traditional single-host enrichment. The authors argued that the two-host enrichment prevented selection for narrow-host-range phages and thus demonstrates that polyvalent phages can be readily isolated from environmental samples (
132). The implications of these findings are best illustrated in the observation that two of the nine polyvalent phages mediated generalized transduction. Therefore, if polyvalent viruses are common in the virioplankton, it is possible that indiscriminate transfer of genetic material between bacterioplankton species occurs frequently.
There are two open questions concerning the relationship of phage-host range and virioplankton ecology. First, in most aquatic environments, it seems that a broad-host-range phenotype would be favored; however, most aquatic phages are thought to have highly specific host ranges. Therefore, what is the selective advantage of a narrow host range for a bacteriophage? The second question relates to the hypothesis discussed above. What is the relationship between the frequency of polyvalent phenotypes and environmental conditions?