Keywords

Introduction

At any given moment, all parasites are engaged either in active parasitism of a host or in an extra-organismal transmission, i.e., a “search” that ends either with acquisition of a new host or instead in parasite death. For phages, all of the parasitizing stage is intracellular. In general terms, the process of phage acquisition of a host bacterium is described as adsorption as resulting in virion attachment to the host cell (chapter “Adsorption: Phage Acquisition of Bacteria”); the parasitizing stage is infection; and the initiation of the extracellular search stage is release. The life cycle of a phage thus consists of release ➔ movement ➔ attachment ➔ infection ➔ release. That is, virion release from a host cell, virion search for a new host cell (as largely though not entirely driven by virion diffusion), virion attachment to a host cell, phage infection of a host cell, and then the next round of release of virion progeny from phage-infected host cells. Phage infections that result in the release of phage progeny are said to be productive.

At an organismal level, these various phenomena typically can be assigned values that collectively are useful descriptors of phage life cycle properties, values we refer to here as growth parameters. Growth parameters include such things are phage virion adsorption rates (especially, the adsorption rate constant), phage infection durations (latent period), and the number of virions produced per infection (phage burst size). See Fig. 1 for summary. For the sake of phage use as antibacterial agents, i.e., for phage therapy, at a minimum some degree of phage attachment to and subsequent destruction of bacteria must occur. In addition, it often can be helpful in phage therapy for phage infections to produce new phage progeny, as described quantitatively by the phage burst size.

Fig. 1
figure 1

Adsorption, infection, and release are phage organismal-level properties that also can be described as life-history characteristics or organismal traits, and which are quantified for phages as growth parameters. These organismal aspects – including virion durability, adsorption rates (chapter “Adsorption: Phage Acquisition of Bacteria”), infection durations (latent period), and infection fecundities (burst sizes) – are all built upon numerous molecular aspects of phages, as well as molecular aspects of their bacterial hosts (“Biochemistry,” etc., in the figure). Those both molecular and organismal phenotypic aspects in turn are underlain by a phage’s genotype, as well as that of its host (“Genetics” in the figure). Lastly, to the right, phages interact with their environments as organisms, resulting in a phage’s ecology, which as an applied ecology we can also describe as phage therapy pharmacology (chapter “Bacteriophage Pharmacology and Immunology”). This figure is inspired by that of Abedon (2009)

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Infections can be successful or unsuccessful for the phage. Here we focus specifically on those infections that are successful, and especially on those that generate new, that is, progeny phage virions. Lysogenic cycles are covered elsewhere in this volume (chapter “Temperate Phages, Prophages, and Lysogeny”) as too are unsuccessful infections (chapter “Bacteria-Phage Antagonistic Coevolution and the Implications for Phage Therapy”). With lytic productive infections, the infection stage ends with phage-induced host-cell lysis, which results in virion release. With chronic productive infections, by contrast, virion release does not coincide with infection termination. The various steps of successful phage infection cycles are indicated in Fig. 2.

Fig. 2
figure 2

The phage life cycle, with emphasis on productive lytic cycles. This cycle can be differentiated into (1) periods of extracellular virion (or free phage) movement, encounter, and attachment (collectively, “Search” or extracellular search) and (2) a period of “Infection” that begins after virion attachment and which ends, at least for phage lytic cycles, with virion release from cells. “Infection” has been described also as a “Virocell” stage (chapter “Adsorption: Phage Acquisition of Bacteria”) and takes place entirely intracellularly, as contrasts especially with “Free virions,” as occur extracellularly. Note that “Post-eclipse” may be described also as period of virion accumulation but otherwise does not appear to have been formally named during the early development of phage biology nomenclature, versus, e.g., the phage latent period or the phage eclipse. Note also that “Free virion” and “Free phages” are used here synonymously. A phage’s generation time spans both the search and infection phase whereas a phage’s fecundity (burst size) is a product solely of the infection phase

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The mechanisms employed by different phages to effect these life cycle steps tend to vary considerably. Nonetheless, these steps also tend to possess certain commonalities. In this chapter, we consider these various steps of phage infection including in terms of mechanisms and variation. Mechanisms of phage-induced bacterial lysis from within are highlighted here (i.e., normal phage-induced bacterial lysis), while phage-induced bacterial lysis from without , particularly via the action of purified phage lysins used as antibacterial medicaments, is considered elsewhere in this volume (chapter “Enzybiotics: Endolysins and Bacteriocins”). Additionally, more ecologically oriented aspects of the material covered in this chapter as well as more evolutionary perspectives can be found in a subsequent chapter “Bacteriophage Ecology.”

Our aim in this chapter is to provide a broad overview of organismal properties associated with phage infections and this is rather than in-depth molecular descriptions. This emphasis is both for reasons of available space and because typically molecular details are less relevant to phage therapy success than simply appreciating a phage’s ability to display bactericidal, bacteriolytic, or virion production activities (chapter “Bacteriophage Pharmacology and Immunology”). Thus, numerous molecular mechanisms give rise to, for example, differences in phage adsorption rates, infection durations, and burst sizes, as well as virion durabilities – which also can vary under different conditions or while infecting different host strains – but distinctions between these mechanisms are not often relevant to phage therapy success. Definitions of terms associated with phage infections can be found in Table 1.

Table 1 Definitions of key terms
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Productive, Reductive, and Destructive Phage Infections

Phage infections, broadly speaking, can be differentiated into ones that are phage-productive versus phage-destructive versus phage-reductive (Abedon 2008, 2020; Abedon et al. 2009). See Fig. 3 for an overview (Fig. 2, by contrast, concentrates especially on lytic productive infections). In this section, we provide an introduction to these phenomena including how they fit into the concepts of phage growth parameters. In addition, we consider an important subset of productive infections, which include especially successful infections caused by strictly lytic phages.

Fig. 3
figure 3

Five possible outcomes of phage infection as falling under the categories of destructive infection, reductive infection, and productive infection. These differ in terms of whether the phage or the bacterium lives or dies, and whether new virions are either produced and released (productive infection) or neither produced nor released (reductive infection or destructive infection). Note that for phages, latent infections are caused by temperate phages and that such infections typically are described as lysogenic rather than using the term “latent.” Not listed in the figure under the heading of reductive infection is the concept of pseudolysogeny, which can involve phage-genome survival along with bacterial survival, but without phage replication (Abedon 2009c). Note further that the terms “Abortive infection” and “Restrictive infection” as used here are not employed necessarily as strictly by all authors. With meanings as used here, thus only abortive infections and lytic infections are bactericidal (“Bacterium dies”). For further discussion of phage infection types, see (Hobbs and Abedon 2016; Abedon 2020)

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Phage-Productive Infections

Productive phage infections are ones that result in both the production and release of phage progeny virions. Depending on the phages, this release can occur either with or without host-cell lysis. Those phages that release progeny virions without lysing their host cell can be described as displaying chronic infections or chronic release, and such infections generally are neither bacteriolytic nor bactericidal. For example are the filamentous phages of family Inoviridae (Russel and Model 2006) which release their virions chronically via a process of extrusion. Members of the seemingly much rarer phage family, Plasmaviridae (Maniloff et al. 1982; Maniloff and Dybvig 2006), instead release virions chronically via budding, which they are able to accomplish presumably because the bacteria they infect lack cell walls (Maniloff et al. 1982, Maniloff and Dybvig 2006). As this budding mechanism is associated with nonbactericidal phage infections, it is of little interest to phage therapy, at least thus far in its development, so are not covered in this chapter. The chapter instead emphasizes lytic cycles and especially those associated with the tailed phages, though all tailless phages other than members of phage families Inoviridae and Plasmaviridae also display lytic productive infections. Different phages types as considered from a perspective of virion structure are discussed in chapter “Structure and Function of Bacteriophages.”

Phage-Reductive Infections

Reductive infections are characterized by a combination of phage genome survival and a temporary lack of phage virion production (Abedon 2008, 2020; Abedon et al. 2009). The term “reductive” comes from Lwoff (1953), p. 272 (emphasis his), “When the infection is going to be reductive, when the bacterium is going to be lysogenized , the genetic material of the infecting phage or germ is “reduced” into prophage.” With respect to phage latent states, the term lysogeny refers specifically to latent states where the bacteriophage genome exists as a replicating prophage. Lysogeny is considered in greater depth in a separate chapter “Temperate Phages, Prophages, and Lysogeny.” Alternatively, phage genomes may persist intracellularly without integration into the host genome. These latent states also include pseudolysogeny (Miller and Day 2008; Abedon 2009c; Los and Wegrzyn 2012). Reductive infections can transition into productive infections, which, for lysogenic cycles, is described as prophage induction. Reductive infections generally are considered to be problematic in effecting phage therapy since, with reductive infections, phage-mediated bacterial death (bactericidal activity) typically is avoided, or at least delayed.

Phage-Destructive Infections

Infections where neither the phage genome survives nor are phage progeny produced may be described as destructive. That is, during such infections, phages are subjected to processes that result in destruction of the infecting phage. This destruction, though, should not be confused with destruction of the bacterial host as seen upon phage-induced bacterial lysis. Such losses of phage viability in the course of infection can be the result of bacterial restriction-modification systems, abortive infection systems, CRISPR-Cas systems, lysogen-expressed superinfection immunity, superinfection exclusion, or simply can occur as a consequence of biochemical incompatibilities between infecting phages and infected hosts (Hyman and Abedon 2010; Labrie et al. 2010; Stern and Sorek 2011; Samson et al. 2013; Chaturongakul and Ounjai 2014) (chapter “Bacteria-Phage Antagonistic Coevolution and the Implications for Phage Therapy”).

As noted, phage destruction does not necessarily coincide with bacterial destruction, i.e., bactericidal or bacteriolytic phage infection activities do not necessarily coincide with failures of phages to successfully infect. Indeed, of the various phenomena listed in Fig. 3, only those resulting in either lytic infections or abortive infections will, by definition, result in bacterial death. Thus, phage destructive infections generally are not always useful for phage therapy. It is important, however, to distinguish among destructive infection types in terms of impacts on bacteria, particularly abortive infections versus restrictive infections, as restrictive infections by definition (as used here) are not also destructive of bacteria (Table 1; Fig. 3). Indeed, even abortive infections will be more limited in their usefulness in phage therapy than lytic productive infections.

Phage Growth Parameters

The above descriptors, productive, reductive, and destructive, are qualitative. Alternatively, it is possible to consider phage life cycles from quantitative perspectives, i.e., in terms of growth parameter values. Four such measures are commonly considered, and these are the length of time required for virion adsorption to occur (adsorption rate), the length of the phage infection period (latent period), the number virion progeny produced per phage-infected bacterium (burst size), and the durability of the virion particles (as measured quantitatively in terms of virion inactivation rates). The middle two of these are inextricably linked to the topics covered in this chapter, and thus are of primary interest here. Adsorption instead is covered in chapter “Adsorption: Phage Acquisition of Bacteria.” Also, we can define productive infections, reductive infections, and destructive infections in terms of associated growth parameter values, which is the emphasis of this subsection.

Latent Periods

Phage-productive infections and phage-reductive infections can be distinguished in terms of lengths of latent period. A productive infection has a latent period that often is well-defined in duration and which otherwise is finite in length. A reductive infection, by contrast, can last indefinitely, coming to a close only with loss of the infecting phage (e.g., curing) or transition to productive infection (induction). Thus, productive infections are associated with relatively short latent periods (e.g., much less than many days long) whereas reductive infections are associated with relatively long latent periods (e.g., often much more than many days long).

Burst Sizes

Productive infections, reductive infections, and destructive infections in turn can be distinguished in terms of burst sizes. A productive infection has a burst size of greater than zero whereas both reductive and destructive infections have burst sizes of zero. Reductive infections, unlike destructive infections, however, retain a potential to display infections in which burst sizes are greater than zero, i.e., as following prophage induction. Indeed, with lysogeny, an infection’s lineage has the potential to display multiple bursts, i.e., multiple new productive infections. This potential is a consequence of lysogen binary fission in combination with multiple prophage induction events, while an obligately productive infection typically will display only one burst per initial phage adsorption (at least for lytic, productive infections), and a phage-destructive infection by definition will display no burst at all.

Utility for Phage Therapy

From a perspective of phage impact on target bacteria, e.g., as during phage therapy, generally higher adsorption rates are preferable to lower adsorption rates, and thus greater adsorption rate constants are preferable, particularly constants associated with that adsorption as achieved in situ (chapter “Adsorption: Phage Acquisition of Bacteria”). There can be limits to the utility of possessing ever higher adsorption rates, however, due to diminishing returns. These issues are considered in greater detail in the ecology chapter “Bacteriophage Ecology.”

For lytically infecting phages, a burst size of greater than zero is always associated with bacterial death and always implies that a fully phage-destructive infection has not occurred. Such bactericidal phage infections are crucial for successful phage therapy (chapter “Bacteriophage Pharmacology and Immunology”). In addition, for phage therapy larger burst sizes often are more desirable than smaller burst sizes since release of new virions has the effect of increasing the in situ dosing of phage particles, as typically is considered to be a good thing (chapter “Bacteriophage Pharmacology and Immunology”). Lastly, generally reductive infections, or destructive infections in which the phage dies but the bacterium does not (i.e., restrictive infections), are considered to lack utility in phage therapy, and can even be antagonistic to phage therapy success as they have the explicit effect of reducing the bactericidal as well as bacteriolytic impacts of phages.

Strictly Lytic Phages for Phage Therapy

For phage therapy, at minimum and as noted, phages must be bactericidal. In addition, it is helpful for phage infections to be productive, since this can lead to increases in phage numbers (and associated titers) within treated individuals, especially in direct, spatial association with phage impact on target bacteria (Fig. 4). Reductive infections, and to a certain degree, destructive infections as well, interfere with phage bactericidal activity, and both reductive infections and destructive infections interfere with the production of new phage virions in situ. Indeed, phage-reductive infections can be worse than phage-destructive infections because not only are bacteria by definition not killed, but the resulting bacterial lysogens are now phage-killing entities, i.e., due to the expression by bacterial lysogens of what is known as superinfection immunity (Blasdel and Abedon 2012), as gives rise to restrictive infections.

Fig. 4
figure 4

Depiction of phage infection effectiveness (here, “performance”) as toward phage therapy utility. Passive treatment requires only that phage virions adsorb (attach) and then kill bacteria (bactericidal activity). Active treatment, by contrast, requires that phages adsorb, attach, and kill bacteria as well as produce new phage virions (productive infection). Bacteriolytic activity, as considered distinctly, may have utility especially toward bacterial biofilm eradication. These aspects of phage therapy pharmacology are considered further in the chapter “Bacteriophage Pharmacology and Immunology.” Particularly, it is strictly lytic phages which display the greater phage therapy infection performance. At least arguably, greater phage performance and therefore antibacterial activity is seen with greater virion durability, faster virion adsorption, shorter phage latent periods, and larger phage burst sizes

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Phage-destructive infections can be avoided through informed phage choice, as mechanisms of resistance to phages is a determinant of phage host range (Hyman and Abedon 2010; Ross et al. 2016; Hyman 2019). Thus, if a phage is able to kill a bacterium, then that bacterium by definition will be within that phage’s bactericidal host range. Similarly, if a phage is able to produce new virion particles upon infection (virion production), such as leading in the laboratory to plaque formation, then by definition that bacterium is part of a phage’s productive host range.

Lysogenic cycles may be fully avoided only by not employing temperate phages for phage therapy. We describe phages that cannot display lysogenic cycles variously as obligately productive or strictly productive, or instead as virulent (the latter, i.e., strictly lytic/obligately lytic). As chronically infecting phages generally are not bactericidal, then they should be avoided for phage therapy as well. Thus, ideally for phage therapy one employs strictly lytic, aka., obligately lytic phages. Furthermore, for reasons such as avoidance of phages that can transduce bacterial DNA (chapter “Bacteriophage-Mediated Horizontal Gene Transfer: Transduction”), it is best also to avoid phages for phage therapy that have recently descended from temperate phages. Such strictly lytic phages that also are not closely related to temperate phages may be described as professionally lytic (Hobbs and Abedon 2016). Here emphasis is primarily on productive infections that end with phage-induced host lysis, though examples especially from phage λ, a temperate phage that has been subject to substantial characterization in this area, nevertheless are provided.

Infection

Bacteriophage infections by definition occur within host bacteria, turning bacteria into what can be described instead as virocells (Forterre 2012). The virocell stage of phage life cycles spans the period from virion acquisition of a bacterial host, that is, given phage genome uptake into the bacterial cytoplasm, at least to the point of release of new virion particles, and indeed beyond this point given chronic rather than lytic release (Fig. 2). As noted above, once infections have been initiated, they can be either phage-reductive or phage-destructive, but also phage-productive (Fig. 3), with our emphasis here especially on the latter. Particularly for lytic productive infections, the duration of infections is traditionally described as a latent period (Fig. 5). In-between steps include an eclipse and then a post-eclipse period of virion accumulation (Fig. 2). A third step, as seen with single-step growth experiments (Hyman and Abedon 2009), is known as the rise. This technically is a period of termination of the latent period that coincides with phage-induced bacterial lysis. We discuss the rise in this section rather than as a step that occurs after infection because of its association with the mentioned single-step growth experiments (Fig. 5), but do so also to make sure that readers are unambiguously aware that the rise is not equivalent to the intracellular, post-eclipse , period of virion accumulation. Rather, the rise is a period of phage-progeny extracellular virion accumulation.

Fig. 5
figure 5

Depiction of a single-step growth experiment, including with determination of intracellular period of virion accumulation (PVA). Adsorption is synchronized to initiate infections, and this is followed by dilution of resulting infective centers into warm broth to allow for unimpeded phage metabolism throughout the experiment. Traditionally, initial phage multiplicities are somewhat less than 1, e.g., 0.1, and resulting “infective centers” are diluted prior to the end of latent period (and therefore prior to lysis) to limit virion adsorption to new bacteria once those virions have been released from phage-infected bacteria. The end of the “eclipse” is defined as the point at which titers of accumulated intracellular virions, obtained via bacterial artificial lysis (dashed blue curve), equal the number of phage-infected bacteria (the dashed horizontal line started at 100). As noted, PVA stands for period of virion accumulation, i.e., as occurring after the eclipse. The latent period is followed by a rise in extracellular phage titers, as distinct from the rise in extracellular titers instead seen with artificial lysis. The number of phages indicated below the label, “Lysis from Within,” is designated as “mostly” extracellular because some phage-infected bacteria are assumed to persist until the end of the rise. A phage’s burst size is equal to the number of phages present at the end of the rise divided by the number of phage-infected bacteria that are present prior to the start of the rise. This figure was adapted from the experiment presented by Doermann (1952). For more on single- or one-step growth experiments, see Hyman and Abedon (2009) as well as Kropinski (2018)

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The Eclipse

Productive phage infections generally can be reduced to two consecutive steps, that which occurs prior to the intracellular production of the first progeny virion and that which happens after. This dividing line reflects observations from the 1950s (Doermann 1952) that there is a period following phage attachment when no phage virions can be recovered as plaque-forming units even given artificial lysis of otherwise still-intact phage-infected bacteria. This initial period is then followed by a period during which phage virions instead can be recovered upon this artificial lysis of phage infections (Fig. 5). The first period was termed the “eclipse,” as perhaps can be viewed as an allusion to the occasional disappearance of celestial objects due to intervening bodies, in this case it being the phage virion stage that is “eclipsed” by the early phage-infected bacterium stage (Fig. 2). It is important to realize, however, that nearly all steps associated with phage infections, save for the period of virion accumulation and subsequent virion release, occur during the eclipse. That is, nearly all aspects of gene expression, host takeover, and complete intracellular virion morphogenesis are initiated prior to the later, post-eclipse stage of phage infection. This makes sense as all aspects of phage infection necessary to produce phage virions must occur prior to the end of the eclipse.

The length of the eclipse, i.e., the eclipse period, represents another phage growth parameter, or at least a sub-parameter, one that affects the number of virion progeny produced by a phage infection. Though to a degree the eclipse as a concept is physiologically almost arbitrary, its duration nonetheless is both real and has real consequences regarding the kinetics of phage progeny production during infections. We can ask, therefore, why does a given phage, under a given set of circumstances, display the eclipse duration that it does? The answer to this question presumably has to do with a balancing of the length of the eclipse period – where all else held constant shorter presumably is better (thereby giving rise to overall shorter phage latent periods and therefore shorter phage generation times), – with the complexity of the phage infections (where more complex infections presumably would require more time, but also presumably supply some sort of as yet not well understood advantage to phages). Given that all phage infections must display some maturation steps along with gene expression, eclipse periods cannot be of length zero (i.e., with the period of virion accumulation hypothetically beginning immediately after phage genome uptake). So too, however, there presumably are costs, particularly in terms of overall phage generations times, of having excessively long eclipse periods. In addition, a phage may be able to more effectively resist destruction upon infection (destructive infection) at the expense of eclipse period lengthening, such as one sees with phage T7, which delays the full initiation of infections so as to first block, via the expression of an antirestriction protein, the action of host restriction endonucleases (Molineux 2006).

Phage Gene Expression

During the eclipse, phage genes are expressed, the phage genome is replicated, and the processes of virion morphogenesis are initiated. Often phage genes are differentiated into those whose expression begins early in the phage infection and therefore early during the eclipse (early genes) versus those genes whose expression begins late in the phage infection and therefore late in the eclipse (late genes) (Yang et al. 2014). With some phages, e.g., T4, there are also genes that are expressed at intermediate times, or middle genes (Hinton 2010). Early genes are generally involved in host takeover or are simply expressed over long periods. Late genes, by contrast, tend to be relevant to the generation of progeny virions and lysis of the host. Early genes and late genes often occupy separate regions on phage genomes and are associated with characteristic gene expression promoter sequences. In the transition from early to late gene expression, there tend to be modifications in the specificity of RNA polymerases. This often occurs, for example, through the phage expression of RNA polymerase specificity-changing, gene promoter-binding proteins known as sigma factors, e.g., Mosig and Eiserling (2006).

Phage Genomes and Replication

Phage genome replication provides not only more templates for transcription but also new phage DNA (or RNA for RNA-genomed phages) for packaging into newly generated phage procapsids (chapter “Structure and Function of Bacteriophages”). Different phage types display a number of variations on the standard semiconservative DNA replication scheme. In terms of tailed phages, which possess dsDNA genomes, phage λ provides an illustrative example. Early after infection, the circularized λ genome, i.e., now closed-circular rather than a linear chromosome, is replicated bidirectionally from its origin of replication, in a process called θ replication (i.e., theta replication; or circle-to-circle rather than circle-to-concatemer replication). This is equivalent to how bacteria with their circular chromosomes replicate. In the first 15 min following genome uptake into the bacterial cytoplasm, about 5–7 consecutive rounds of θ replication take place, resulting in the production of 50–100 λ circular chromosomes, and all of these copies can serve as templates for RNA transcription (Wegrzyn and Wegrzyn 2005; Narajczyk et al. 2007). At some point, via an unknown mechanism, θ replication gives way to unidirectional σ replication (i.e., sigma replication, which is a form of rolling-circle replication; Fig. 6). The latter produces long, linear DNA molecules containing many copies (concatemers) of the λ genome. These concatemeric molecules are cut by the enzyme, terminase, into appropriately sized λ genomes, which are then packaged into newly produced procapsids (Wegrzyn and Wegrzyn 2005, Narajczyk et al. 2007).

Fig. 6
figure 6

Four variations on phage DNA and its replication. These are separate examples rather than a sequence of steps, and most of the DNA replication involved originates at specific DNA sequences known as origins of replication. 1. In rolling circle replication (upper, left), generation of multiple copies of a genome can be initiated from only a single origin of replication, generating a linear concatemer of DNA. This DNA can either remain single-stranded or instead can be converted through further DNA replication to double-stranded DNA, depending on the phage. 2. Alternatively (lower, right), replication can originate by nonstandard means, such as due to strand invasion by other DNAs (recombination initiation), which is particularly possible given phage genome circular permutation. 3. Circular permutation (lower, left) is not equivalent to circular chromosomal DNA but genomes instead are slightly longer than necessary to contain all of a phage’s genes, resulting in each phage genome containing two copies of a small proportion of genes, as found at the ends of these linear chromosomes. How many genes are found in excess varies between individual mature virions. The amount of DNA packaged into virions with circularly permuted genomes is determined by capsid size, hence headful packaging. 4. Alternatively, rather than being circularly permutated (middle, right), DNA packaging into capsids can be facilitated by DNA sequences known as Pac sites, which are locations on DNA where cutting is targeted to result in conversion of DNA concatemers into single rather than single-plus genome lengths

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For phage T4, also a tailed phage, DNA replication too involves origins of replication. In addition, however, it involves multiple rounds of DNA replication-priming intragenomic recombination steps, producing what are described as large, multibranched concatemers of T4 DNA. These concatemers are then cut into slightly longer than single-genome length units (as resulting in circular permutation) as they are inserted into phage procapsids in a process known as headful packaging. To a degree, phage T4 DNA replication and subsequent packaging are temporally coordinated since in phage T4 transcription from late-gene promotors, and therefore of virion structural genes, does not begin until DNA replication begins (Mosig and Eiserling 2006).

Rolling-circle replication is used by a number of phages as well as other viruses and even plasmids. The process is efficient as it requires only a single priming step, though is limited to replicating closed-circular DNAs. Key is the use of one parental strand as a template and then ongoing displacement of the complementary strand, i.e., as it is replaced by newly synthesized complementary strand. Indeed, the newly synthesized DNA strand itself can be displaced by even more recently synthesized DNA. In this manner, a long, linear concatemer of DNA can be produced off of a single template strand, which will then require further DNA synthesis to generate a complementary strand for dsDNA genomes. Packaging then involves cutting either genome-sized (at Pac sites) or headful-sized DNA lengths (headful packaging). Alternatively, the displaced DNA can be cut and ligated to generate closed-circular DNA. See Fig. 6 for illustration of various DNA-replication-associated concepts.

The Latent Period Continues…

The stage of phage infection that follows the eclipse does not, to the best of our knowledge, possess a consistently employed formal name. It has been referred to (Hyman and Abedon 2009) as a post-eclipse , intracellular phage growth period, reproductive period, adult period, period of phage-progeny accumulation, or a period of virion maturation. The latter is inaccurate, however, as the process of intracellular virion maturation begins prior to the end of the eclipse. For the sake of convenience, we designate this post-eclipse stage here also as a “period of virion accumulation,” which we will abbreviate as PVA (and which more accurately for lytic phages is a period of virion intracellular accumulation). Note that the phage PVA should not be described in terms of a “rise” in phage numbers within infected bacteria since a different aspect of phage infection is already described as the phage rise (see below).

The PVA , by definition, begins following the phage eclipse, and continues until phage-induced bacterial lysis for lytic phages, or the loss of infection viability or otherwise cessation of phage production for chronically infecting phages. Indeed, for phages that chronically release, the PVA is not an intracellular period of virion accumulation, but instead one of ongoing extracellular virion accumulation. The overall PVA duration appears to vary between infections by the same phage type even given otherwise identical infection conditions (Singh and Dennehy 2014), and certainly varies among phage types, as well as with certain types of phage mutations, the latter as considered below in terms of mechanisms of phage-induced bacterial lysis. Over the course of the PVA, phage virions appear to accumulate intracellularly in a linear, that is, constant rate (Wang et al. 1996), or at least linear accumulation is a common assumption. Such kinetics of intracellular virion accumulation makes logical sense, though, assuming that some aspect of virion morphogenesis must serve as a virion-morphogenesis rate-limiting step and so long as the rate of this step does not change over the course of an infection. For example, this might include the number of ribosomes available or the infected bacterium’s rate of ATP production.

The overall length of a phage infection, particularly as associated with lytic infections, is called the phage latent period. If one considers only a single phage-infected bacterium, then the end of the latent period coincides with phage-induced bacterial lysis. Alternatively, for an adsorption-synchronized phage population, the latent period can be considered to end with the release of phage virions from the first lysing bacterium. The ensuing period, during which the other infected bacteria in the population are lysed, is termed the phage rise, as considered below.

…And Continues?

To the extent that new virions are produced at a more or less constant rate by a single lytic-phage infected bacterium, then the length of the PVA multiplied by the rate of intracellular virion accumulation should approximate the resulting infection burst size. Burst size, that is, is the total number of phage progeny virions produced per host–cell infection, and which serves as another key phage growth parameter. Longer latent periods, so long as the increase in length is associated with the PVA, as well as faster virion accumulation rates, thus will give rise to larger phage burst sizes. Postponing host lysis nonetheless directly delays the initiation of subsequent infections by newly produced phages since lytic phages retain their virion progeny intracellularly prior to bacterial lysis. This trade-off between producing more phage progeny intracellularly and allowing those progeny to initiate their own progeny-producing infections has given rise to a series of models devoted to calculating the optimal lysis timing for a lytic phage (Abedon 1989; Wang et al. 1996; Abedon et al. 2001; Bull et al. 2004; Bull 2006; Wang 2006; Bonachela and Levin 2014).

The general findings of these models relate latent period length optima – that is, as allowing maximal rates of phage population growth – to numbers of permissive hosts available for infection if the current cell is lysed. Specifically, the more host bacteria that are present, then the faster progeny numbers can be increased by their infecting new hosts, versus increasing numbers solely by continuing intracellular maturation and virion accumulation within a still unlysed host. Thus, more opportunities for creation of new phage infections should provide a benefit to shorter phage latent periods. If the eclipse period is fixed in length, however, then this means that such latent period shortening can only come at the expense of the length of the PVA, and therefore directly at the expense of the phage burst size. The length of a given phage’s PVA thus presumably represents some compromise between a phage displaying shorter generation times (via a shorter PVA) and displaying larger burst sizes (via a longer PVA). It is important to recognize, however, that the PVA can be shortened and burst size increased simultaneously due to improved host physiological conditions (Hadas et al. 1997). Thus, one should not overly generalize the idea that shorter phage latent periods automatically result in smaller phage burst sizes. Rather, the key perspective is that PVAs that have been shortened due to phage mutation will tend to result in smaller burst sizes.

Burst size dependence on the PVA helps to explain advantages associated with the lysis inhibition phenotype that is seen in some phages. Here phage adsorption to already phage-infected bacteria results in an induced PVA extension and associated increased infection burst size (Abedon 1990, 2009a, 2019). The attachment of phages to a phage-infected bacterium, i.e., secondary adsorption, signals to the infecting phage, that is, the virocell, that there may be strong competition for hosts in the surrounding environment. In this situation, the phage is better-off staying put within the bacterium it is already infecting, and this is rather than exit the host but then fail to find an uninfected bacterium to infect. With temperate phages such as phage λ, rather than multiple adsorptions inducing an extension of PVA, instead multiple adsorptions can result in an increased tendency for infections to display lysogenic rather than lytic cycles (Abedon 2017a). That is, an extension of a period preceding the PVA (though for lysogenic cycles that period is not typically described as an eclipse, nor the resulting lysogenic cycle described as a latent period). With chronically released phages there is no equivalent inherent conflict between phage production and infection duration, though faster rates of phage production nonetheless may result in reduced infection durations (Breitbart et al. 2005). For phage therapy, lysis inhibition would be the most important of these phenomena as, unlike the others, it is seen with obligately lytic phages, but it nevertheless is uncertain the extent of prevalence of this phenotype among therapeutic phages.

The Rise

The transition from infection to free virions occurs over the course of what is traditionally described as the phage rise. The phage rise is defined as the period during single-step growth experiments over which phage titers increase, that is, rise in number (Fig. 5). Specifically, these are phage populations in which the start of infections has in some manner been synchronized, such as in terms of adsorption. In these experiments, variance in the timing of lysis results in a more gradual rather than instantaneous increase in phage numbers, up to some maximum number of new virions as determined by a phage’s burst size. The rise begins when the first phage-infected cell lyses, which during single-step growth experiments is when the number of infective centers increases above the number of phage-infected bacteria. This also, for many authors, serves to define the end of the phage latent period. The rise in phage numbers then continues until all phage-infected bacteria in the culture have lysed.

Note that it is essential to make sure that released phages during the phage rise, during single-step growth experiments (Hyman and Abedon 2009), are unable to adsorb bacteria. This serves three purposes. First, any phages which happen to adsorb bacteria that are already phage infected (secondary adsorptions) will no longer be available as plaque-forming units, i.e., they will instead experience destructive infections (Fig. 3). This point is true even if the phage in question is unable to display superinfection exclusion (Abedon 1994, 2017b). Thus, one will expect an underestimation of burst sizes if such adsorptions to already phage-infected bacteria area allowed to occur. Second, for phages that are able to display lysis inhibition, these secondary adsorptions of already phage-infected will extend the adsorbed phage infection’s latent periods and also substantially multiply the resulting burst size. Lastly, if released phages adsorb to phage-uninfected bacteria, then these new infections can give rise to new phage bursts, also artificially inflating the phage burst size. Nonetheless, it is not entirely uncommon to see in the literature so-called single-step growth experiments which appear, in reality, to be “multiple”-step growth experiments, which at best are illustrations of phage exponential growth under a given set of laboratory conditions rather than measures of a phage’s rise or burst size. We return to this concern in a subsequent section, since arguably there is no more valuable single phage-characterization assay concerning phage infection and determination of associated growth parameters than single-step growth experiments, properly performed.

Host Physiology Considerations

For most phages, the robustness of productive infections will vary as a function of not only host genetics but also of host physiology including as can vary with environmental conditions (Hadas et al. 1997). At an extreme, phage-productive infections will tend to stall when bacterial hosts enter stationary phase (Miller and Day 2008; Bryan et al. 2016). This outcome can be seen during batch culture phage population growth when bacteria grow to high densities, thereby resulting in failures of phages to lyse these cultures, but also during phage plaque growth (chapter “Detection of Bacteriophages: Phage Plaques”). Phage plaques for many phages, that is, are limited in size, and these limitations largely are thought to be consequence of bacterial lawn entrance into stationary phase. For certain phages, however, bacterial lawn entrance into stationary phase does not block a continuation of plaque growth, as can result in extremely large plaques such as seen with coliphage T7 (Yin 1991).

Virion Release

In this section, we consider phage virion release from infected bacteria. Emphasis is on phage-induced bacterial lysis along with major variations in lysis mechanisms. More formally, this has been described as a lysis from within, or LI (Kao and McClain 1980; Young 1992), in order to distinguish it from a pair of somewhat unrelated phenomena that are described instead as lysis from without, or LO (Abedon 2011). One form of LO is described elsewhere in this volume as induced by purified phage lysins (chapter “Enzybiotics: Endolysins and Bacteriocins”), but historically LO denotes lysis induced by high multiplicities of phage adsorption to bacteria (Delbrück 1940). Generally, phage-induced bacterial lysis from within possesses two defining components, which are the timing of initiation of lysis, on one hand, and the mechanism of bacteria cell-wall destruction on the other. Associated with one or both of these phenomena is a combination of the cessation of infection metabolism and release of phage progeny virions that up to this point are trapped intracellularly. The physiological purpose of LI is this phage progeny release. Ecologically, however, phage-induced bacterial lysis is important as well to nutrient cycling within ecosystems, that is, as lysis represents the first step of bacterial decomposition as stems from the action within environments of phages (chapter “Bacteriophage Ecology”).

In simpler systems, i.e., as associated with single-stranded lytic phages, only a single protein is required to effect LI. For double-stranded phages, however, two or more proteins can be involved. Typically these will include both a holin and an endolysin, the latter, a.k.a. a lysin (chapter “Enzybiotics: Endolysins and Bacteriocins”), though various lysis timing regulating proteins including antiholins may be involved as well. The holins themselves can vary fundamentally in their actions, though in all cases they perform two basic functions: (1) metabolically poisoning and thereby terminating phage infections and (2) allowing endolysins to enzymatically degrade cells walls. More recently described are proteins known as spanins which are involved in breaching the outer membrane of Gram-negative bacteria during the lysis process. Numerous general reviews of phage lysis exist, published particularly by Young and colleagues (Young 1992, 2005, 2013, 2014; Young et al. 2000; Bernhardt et al. 2002; Young and Wang 2006; Chamakura and Young 2018; Cahill and Young 2019).

Holin-Mediated Lysis from Within

The majority of phage lysis systems involve, minimally, a combination of two molecules: a holin and an endolysin. The holins are responsible for controlling the timing of LI while the endolysins are responsible for enzymatically degrading the cell wall of the infected bacterium. The enzymatic nature of endolysins, or lysins as they are described for short, is covered elsewhere in this volume (chapter “Enzybiotics: Endolysins and Bacteriocins”). Here we focus instead on phage holins as well as additional protein factors involved in controlling or effecting the lysis process.

Lysis-Mediating Phage Proteins

Holins, as their name partially implies, are responsible for producing holes in the bacterial plasma membrane. So far as is understood, all double-stranded phages employ holins to effect LI. The holes can be large enough to allow endolysins to pass through the plasma membrane to gain access to the bacterial peptidoglycan layer (Wang et al. 2003), or alternatively the holes, known as pinholes because of their roughly nanometer rather than roughly micrometer size, can release endolysins that are tethered to the plasma membrane in the periplasm (Young 2014). In either case, the formation of these holes results in cessation of host metabolism as the proton motive force that fuels ATP synthesis is disrupted . The disruption of the inner membrane by holins and the subsequent enzymatic digestion of cell walls by endolysin are followed, in Gram-negative phages, by the disruption of the outer membrane by spanin proteins (Berry et al. 2012; Kongari et al. 2018). Once the last cellular barrier is subverted, the cell’s turgor pressure ensures the explosive expulsion of the cytoplasmic contents, including progeny phages, into the surrounding medium.

Holin-mediated timing of cell lysis is controlled in part by holin-gene transcription and then translation rates in combination with the threshold concentrations of holin protein required to form holes. The holin concentrations required to initiate hole formation depend on the holin protein sequence, modifications of which can produce early or late lysing phage phenotypes (Dennehy and Wang 2011; Kannoly et al. 2020). As general transcription and translation rates are functions in part of environmental conditions, growth in low nutrient media can extend the duration of the latent period (Hadas et al. 1997; Dennehy and Wang 2011). Furthermore, modification of the holin-gene promoter and Shine–Dalgarno sequences, as these also can impact gene transcription and translation rates, each can speed up or delay holin accumulation and eventual lysis. Notwithstanding these various mechanisms that can control the timing of hole formation, once a hole small hole forms in the membrane, the cell rapidly lyses in seconds (Wang et al. 2000; Gründling et al. 2001). The mechanism for the rapid, or saltatory progression of lysis is believed to be the accumulation of previously dispersed membrane-associated holin molecules at the site of the lesion, and also the transformation of antiholin, the holin inhibitor, into functional holin because of the removal of the proton motive force. Thus, after the collapse of the proton motive force, holin, and antiholin jointly will contribute, for many phages, to an approximately micron-sized hole in the membrane sufficient to allow large numbers of endolysin molecules to access their substrate, the peptidoglycan cell wall. See Fig. 7 for a model of hole formation.

Fig. 7
figure 7

Model of holin action. The holin as encoded by phage λ is a plasma membrane protein with three transmembrane domains. The inactive holin is depicted with two hydrophilic faces on two of the transmembrane domains oriented toward each other (inactive monomer, upper left; note the green faces oriented toward the middle as found at the top of the holin depiction). These monomers can oligomerize while retaining this inactive orientation. Once holin protein has accumulated within the plasma membrane in sufficient quantities, forming into so-called death rafts of holin proteins, the inactive form partially or fully switches to an active form (see model, lower left), which then spontaneously forms holes with the hydrophilic faces now oriented toward the lumen of the holes. This hole formation poisons the plasma membrane, which prompts further holin switching from inactive to active form. Endolysin (not shown) is now able to digest the cell wall of the phage-infected bacterium and lysis ensues. Also not shown here is the role of antiholin proteins. This figure is derived from that presented by Cahill and Young (2019)

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Antiholins

As noted, some phages, such as bacteriophage λ, produce a lysis inhibitor known as an antiholin. The phage λ antiholin is expressed from the same open reading frame as its holin, and only differs from the holin by virtue of an extra two amino acids at the n-terminus (Young 2014). The net positive charge exhibited by the extra amino acids prevents the antiholin from accessing the holin’s three transmembrane-domain configuration (Gründling et al. 2000). Consequently, the antiholin cannot participate in initiation of hole formation, but nonetheless can dimerize with the holin prior to hole formation. This dimerization with antiholin inhibits holin from participating in hole formation and thus represents the molecular basis of the antiholin activity. The inhibition lasts until sufficient sufficient numbers of holin dimers have accumulated, resulting in hole formation despite the antiholin’s presence and thereby abolishing the bacterium’s proton motive force. The resulting membrane depolarization enables antiholin to access the three transmembrane-domain configuration characteristics of the holin, thereby allowing the antiholin to become functionally equivalent to a holin. See Box 1 for further discussion of the possible utility of antiholin expression by phage λ.

Box 1 Why Have an Antiholin?

In wild type phage λ, holin and antiholin are expressed in a 2:1 ratio (Chang et al. 1995). Since each antiholin inhibits one holin (antiholin’s dimerization with itself is negligible), two-thirds of the output of the phage λ S gene, which encodes both proteins, is initially functionally inactive. Given that proteins are energetically expensive to produce, the existence of this system thus constitutes an evolutionary puzzle. We must assume that nature is not profligate; nonetheless, no fitness-enhancing function for antiholin has been experimentally demonstrated. It is conceivable, however, that antiholin serves as many as three roles.

First, antiholin may prevent premature lysis by dampening stochastic gene expression from the λ late gene promoter. Our reasoning for this hypothesis stems from the fact that gene expression systems are well-known to generate unpredictable protein numbers in identical cells in the same environment (Raj and van Oudenaarden 2008). Consider that phages will be encountering cells with variable numbers of RNA polymerases and ribosomes. Moreover, due to the inherent probabilistic nature of biochemical reactions, these complexes are engaged in a stochastic manner. Given the holin’s relatively low threshold for nucleation, ~1000 molecules (Chang et al. 1995), it is plausible that this threshold could be reached during the eclipse period. This outcome would be tantamount to phage suicide – or more specifically, to an abortive infection – because progeny virions would not have yet been produced.

Second, if the cell should die before holin-induced lysis occurs, then any replicated progeny would be trapped within the dead cell. Antiholin may prevent this circumstance because it is converted to holin following the termination of the proton motive force. While insufficient holin may be present to lyse the cell, the combined amount of holin and antiholin may be enough to form holes and lyse the cell even though the cell is otherwise physiologically dead. In other words, the phage infection in effect produces emergency but normally nonfunctional holin in reserve, thereby making it easier to balance the lysis-timing and lysis-effecting roles of holins since no longer must sufficient quantities of holin necessary to start lysis also be sufficient quantities of holin to finish lysis.

Third, antiholin may reduce the impact of noise in gene expression on the timing of lysis via an incoherent feed-forward regulatory system (Ghusinga et al. 2017). Incoherent feed-forward regulatory systems are control system variants (others include, for example, positive and negative feedback) where an effector and its inhibitor are expressed from the same promoter (Singh and Dennehy 2014, Ghusinga et al. 2017). For stable proteins, such as holin, it has been shown that incoherent feed-forward control reduces gene expression noise more effectively than feedback control (Chang et al. 1995). Other features of the λ holin expression system, including the high transcription rate, the low translational “burst size” (i.e., the average number of proteins produced per mRNA molecule), and the optimal holin lysis-triggering threshold concentration, also ensure that lysis occurs at precisely scheduled times (Kannoly et al. 2020).

Less well studied but no less interesting are the antiholins of phage T4. T4 antiholins are the products of the phage rI and rIII genes. The resulting RIII antiholin is unusual in that it acts on the cytoplasmic rather than periplasmic-side the phage T4 holin, which is encoded by the phage T4 gene t (Chen and Young 2016). The RI antiholin is even more unusual than RIII in that it serves as an extrinsically activatable antiholin, that is, activated by something that comes from outside of the phage-infected bacterium rather than inside, i.e., rather than by cell metabolic poisoning. Specifically, the secondary adsorption signal that stimulates the expression of T4 lysis inhibition is in some manner received and acted upon by the RI antiholin, which then serves to interfere with T-holin lysis-timing activity (Tran et al. 2005).

Inhibition of Peptidoglycan Production

Toward phage-induced lysis from within, there exists an alternative strategy to that of destroying intact cell-wall peptidoglycan via phage endolysin-mediated digestion following holin-based hole formation. That other approach is instead to prevent cell wall formation and remodeling during the process of bacterial growth. The primary advantage to this latter approach toward lysis from within is that interfering with the completion of cell wall formation is simpler in terms of the gene products involved, and it may also more directly link lysis timing with resulting burst size. For example with phage MS2, an RNA phage, translation of its lysis protein and capsid protein are linked, suggesting that sooner lysis would be associated with larger phage burst sizes and vice versa (Bernhardt et al. 2002).

Notwithstanding the exact utility for their doing so, it appears to be particularly the simpler of phages which employ such single-gene, nonholin-endolysin -based lysis systems. These are the single-stranded, nontailed, lytic phages, members of phage families Leviviridae (ssRNA phages) and Microviridae (ssDNA phages, both with icosahedral capsids (chapter “Structure and Function of Bacteriophages”)). These nonholin and nonendolysin phage lytic proteins have been described as “protein antibiotics” (Bernhardt et al. 2002), that is, phage-encoded proteins that interfere with peptidoglycan production (Chamakura and Young 2018) as analogous to the interference with peptidoglycan production seen with the nonprotein antibiotic, penicillin. More prominent as potential cell-wall inhibiting protein antibiotics, however, are the endolysins of tailed phages, as have been described instead as “Enzybiotics” (chapter “Enzybiotics: Endolysins and Bacteriocins”).

Chronic Release

The primary, though certainly not only challenge for phages, in terms of their release to the extracellular environment, is breaching the bacterial cell wall. Most phages accomplish this by degrading the cell wall to a point that it fails, physiologically terminating both the phage infection and the phage-infected bacterium. The resulting bacterial lysis has the effect of creating relatively large holes in the cell envelope of host bacteria through which phage virions can pass, as well as to a degree solubilizing the bacterium.

Alternatively, certain bacteria exist which lack cell walls and at least one phage is known that passes through a cell-wall-less cell envelope via budding, i.e., as is commonly seen also with numerous enveloped eukaryotic viruses. This phages is Acholeplasma phage L2 and it is the sole isolated member of the phage family, Plasmaviridae (Maniloff and Dybvig 2006).

A third approach is seen with members of phage family Inoviridae, the filamentous phages. These phages extrude their virions across intact cell-wall-containing cell envelopes and do so in a likely much more common form of phage virion chronic release than virion budding. From a phage therapy perspective, extrusion as a means of virion release is not highly relevant since while such phages may be engineered in various ways to kill bacteria (Hagens et al. 2004; Yacoby and Benhar 2008; Moradpour et al. 2009; Ngo-Duc et al. 2020; Peng et al. 2020), actual phage release, should it even occur, is not involved in the antibacterial process. Thus, unlike lysis, chronic release is not mechanistically considered in much detail here. Nevertheless, as follows is a short overview of the process.

The DNA of the filamentous inoviruses is single-stranded and does not accumulate in the infected bacterium cytoplasm. Instead that DNA complexes with phage protein (pV) and this complex is then recognized by a second phage protein (pI) that is host plasma-membrane associated. Protein pI is found at regions in the cell envelope where the inner and outer membranes interact, due to associations of pI and pXI proteins in the inner membrane and pIV protein in the outer membrane (all of which are phage proteins). In the process of extrusion, the pV protein is stripped from the phage DNA while various other phage proteins are added (pIII, pVI, pVII, pVIII, pIX). The now mature phage is released through a channel formed by the pIV protein and thereby from the bacterium. Unlike lytic phages which exit phage-infected bacteria through large holes, filamentous phages, presumably due in large part to the narrowness of their virions (about 6.5 nm), are able to pass through small holes in the bacterial cell envelope, holes which are not highly disruptive to either ongoing cell or infection metabolism (Russel and Model 2006).

Determination of Phage Growth Parameter Values

Phage organismal characteristics have long been subject to experimental determination. Experimental approaches include though are not limited to virion survival experiments under various conditions, virion adsorption rate determinations, variations on single-step growth curves, and also determinations of phage population growth rates, chemostat dynamics, and the study of plaque formation. In this section, we provide brief overviews of these approaches. Many of these methods involve determinations of phage titers, or numbers of phage-infected bacteria, which usually involves plaque-based enumeration (chapter “Detection of Bacteriophages: Statistical Aspects of Plaque Assay”).

Virion Durability Determination

Virion survival curves are commonly generated to observe the impact of environmental extremes, such as in pH or temperature. Survival curves also are used to determine the effects of virion processing, or instead the impact of virion storage or exposure to disinfecting agents. Other aspects involve issues of virion interaction with animal immune systems (chapter “Bacteriophage Pharmacology and Immunology”) as well as phage survival following spraying on crops (chapter “Crop Use of Bacteriophages”). The standard protocol for determination of phage survival characteristics is straightforward, involving repeated enumeration of a virion population that has been subjected to some constant, often challenging condition over some span of time. Inactivation is typically exponential (exponential decline). In some cases, such as following exposure to ultraviolet light, the inactivation also can be conditional depending on the circumstances associated with or taking place prior to enumeration (Weinbauer et al. 1997).

Eclipse, Latent Period, Burst Size, and Rise

Single- or one-step growth curves involve, ideally, a synchronized start to phage infections. This can be accomplished via phage adsorption to a population of metabolically inhibited target bacteria, usually at relatively low phage multiplicities, or instead in the course of lysogen induction. These now productively infected bacteria are then enumerated at regular intervals until phage numbers increase, corresponding to phage release, and then stabilize in number at a level determined by a phage’s burst size. When it is that virion release first occurs can serve to define the end of the phage latent period as well as the start of the phage rise. It is possible, however, to skip many of the middle time points involved in latent period and rise determination so as to determine burst size only. In retaining these middle time points, enumeration following artificial lysis of cells allows characterization of the phage eclipse, which ends at the point of the intracellular maturation of the first virion progeny (Fig. 5). Further discussion can be found in Delbrück (1946), Benzer et al. (1950), Hyman and Abedon (2009), and Kropinski (2018). Doermann provides explorations of means of artificially lysing phage-infected bacteria to determine eclipse periods in association with single-step growth determinations (Doermann 1951, 1952; Anderson and Doermann 1952).

Phage Population Growth Rates

Phage population growth rates can be determined either for a single phage type or instead for mixtures of different phage types, the latter, e.g., such as a phage mutant versus its wild-type parent. With single phage types, for a given set of conditions, host type, and host densities, a phage exponential growth rate, called a Malthusian parameter, can be determined or instead, equivalently, a phage population doubling time may be assessed. The rate of this growth will be a function of phage generation times and burst sizes. Phage durability need not play a substantial role, though antiphage antagonists, such as relatively low titers of antiphage serum, can be incorporated into growth-rate determinations if that is desired.

Population growth rates with mixtures of phages typically will be determined for two competing phages with the intention of determining what can be described as a relative fitness. That is, the rate at which one phage population increases in number relative to the other. This approach is employed to determine the degree to which distinct populations of the same phage isolate differ in terms of their evolutionary fitness, such as in competing a viable phage mutant with a wild type phage. Both separate and competing phage population growth curves are presented in Abedon et al. (2001, 2003), and see Dennehy et al. (2007) and Shao and Wang (2008) for relative fitness calculations.

Continuous Culture

Continuous culture growth, as occurs in devices known as chemostats, can be somewhat more complex than simple batch phage population growth. This occurs because at least four distinct phage population states can exist with continuous culture: (1) phage population growth, (2) phage population decline, (3) phage population extinction, and (4) phage- as well as bacterium-population-size steady states. Extinction, furthermore, can occur either because phages cannot replicate faster than they are lost from growth vessels, such as due to too-low densities of susceptible host bacteria, or instead, at an extreme, because phages have driven the population of host bacteria to extinction. By including the possibility of phage-resistant hosts as well as phage host-range mutations, yet additional scenarios are possible (chapter “Bacteria-Phage Antagonistic Coevolution and the Implications for Phage Therapy”). Nevertheless, basic phage organismal characteristics still determine which of these scenarios will be present in a continuous culture, particularly as determined by phage burst size, but also virion adsorption rates and latent periods. An important article documenting phage chemostat growth is that of Bohannan and Lenski (1997), as has been deconstructed numerically in terms of growth parameters (Abedon 2009b).

Plaques

Plaque formation involves not just phage population growth but also variations in both phage location and the physiological states of host bacteria, all at the same time (chapter “Detection of Bacteriophages: Phage Plaques”). As with phage population growth within more fluid environments, it is those phages with optimal combinations of shorter latent periods and larger bursts sizes that are expected to give rise to faster increases in plaque size. Perhaps particularly latent period length impacts these rates (Abedon and Culler 2007a), though burst size of course should be highly relevant as well in terms of increases in numbers of phages present per plaque (Abedon and Culler 2007b). The impact of virion adsorption rates (chapter “Adsorption: Phage Acquisition of Bacteria”), by contrast, is less straightforward, with particularly high affinity of virions for target bacteria potentially slowing down phage movement into the periphery of plaques and therefore slowing down rates of plaque-size increase. In any case, for most phages the entrance of their bacterial hosts into stationary phase blocks further phage population growth and limits phage-induced bacterial lysis. Stationary phase, therefore, will impose limits on the size of phage plaques. For study of the theoretical impact of different phage growth parameters of rates of plaque-size increase, see Abedon and Culler (2007a) and see Gallet et al. (2011) for experimental exploration.

Conclusion

Since especially the 1960s, the study of phage biology has been substantially a molecular endeavor. Phages, however, exist as collections of different molecules rather than as the more isolated molecules that are required for precise molecular study. These phage-molecule collections we can describe as either free virions or virocells, both of which are representative of individual phages as organisms. Indeed, predating the intensive study of phage molecules was the study instead of phages primarily as whole organisms and in terms of whole-organism traits (Fig. 8), e.g., such as in terms of single-step growth experiments or abilities to form plaques (chapter “Detection of Bacteriophages: Phage Plaques”). Many of the phage organismal-level phenotypes can be described quantitatively as phage-growth parameters, e.g., latent period, burst size, or adsorption rate constant, and in this chapter we have concentrated on those phenotypes associated especially with virocells. Together with the organismal phenotypes associated with phage virions (chapter “Adsorption: Phage Acquisition of Bacteria”), these phage organismal phenotypes largely define a phage’s interaction with its environments, whether these are the natural environments outside of the body (chapter “Bacteriophage Ecology”) or instead as seen during phage treatment of bacterial infections within bodies (chapter “Bacteriophage Pharmacology and Immunology”).

Fig. 8
figure 8

Summary of phage organismal properties and underlying factors. For discussion of especially adsorption rates (see chapter “Adsorption: Phage Acquisition of Bacteria”)

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Cross-References