2725346
Effects of bacteriophage traits on plaque formation
BackgroundThe appearance of plaques on a bacterial lawn is one of the enduring imageries in modern day biology. The seeming simplicity of a plaque has invited many hypotheses and models in trying to describe and explain the details of its formation. However, until now, there has been no systematic experimental exploration on how different bacteriophage (phage) traits may influence the formation of a plaque. In this study, we constructed a series of isogenic λ phages that differ in their adsorption rate, lysis timing, or morphology so that we can determine the effects if these changes on three plaque properties: size, progeny productivity, and phage concentration within plaques.ResultsWe found that the adsorption rate has a diminishing, but negative impact on all three plaque measurements. Interestingly, there exists a concave relationship between the lysis time and plaque size, resulting in an apparent optimal lysis time that maximizes the plaque size. Although suggestive in appearance, we did not detect a significant effect of lysis time on plaque productivity. Nonetheless, the combined effects of plaque size and productivity resulted in an apparent convex relationship between the lysis time and phage concentration within plaques. Lastly, we found that virion morphology also affected plaque size. We compared our results to the available models on plaque size and productivity. For the models in their current forms, a few of them can capture the qualitative aspects of our results, but not consistently in both plaque properties.ConclusionsBy using a collection of isogenic phage strains, we were able to investigate the effects of individual phage traits on plaque size, plaque productivity, and average phage concentration in a plaque while holding all other traits constant. The controlled nature of our study allowed us to test several model predictions on plaque size and plaque productivity. It seems that a more realistic theoretical approach to plaque formation is needed in order to capture the complex interaction between phage and its bacterium host in a spatially restricted environment.
9309392
A quantifiable phenotype of viral propagation.
A system has been identified where a virus, replicating continuously on its host, displays a distinct and quantifiable phenotype, and thereby continuously reports on the state of the virus-host relationship. When bacteriophage T7 is plated out with its host, Escherichia coli, it establishes a constant-velocity infection wave, which is driven by an autocatalytic reaction-diffusion mechanism. The velocity--which is easily measured--continuously reflects the infection environment. The simplicity of the system extends to the investigator an unprecedented ability to monitor and control a viral infection process.
30238723
Evolution of bacteriophage T7 in a growing plaque
The emergence of mutants during the 10(9)-fold amplification of a bacteriophage was spatially resolved in a growing plaque. When wild-type phage T7 was grown on an Escherichia coli host which expressed an essential early enzyme of the phage infection cycle, the T7 RNA polymerase, mutant phage relying on this enzyme appeared in 10(8) phage replications and outgrew the wild type. Spatial resolution of the selection process was achieved by analyzing stab samples taken along a plaque radius. Different mutants were selected at different rates along different radii of the plaque, based on host range and restriction patterns of the isolates. The mutants deleted up to 11% of their genomes, including the gene for their own RNA polymerase. They gained an advantage over the wild type by replicating more efficiently, as determined by one-step growth cultures.
10882052
Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal Virus.
Research on the growth characteristics and genetic properties of animal viruses has stood greatly in need of improved quantitative techniques, such as those used in the related field of bacteriophage studies.
43166401
The ability of single phage particles to form plaques and to multiply in liquid cultures.
SUMMARY: The results of testing a bacteriophage to a strain of clover nodule bacteria using young (1 day) and old (5 days) bacterial cultures both fit to the hypothesis that phage multiplication can be initiated by single phage particles. As the same phage preparations gave more plaques on solid media and higher proportions of liquid cultures in which phage multiplication could be detected, with young than with old bacterial cultures, the fit to the hypothesis is not an evidence that every single phage particle will multiply. It may be so when young bacterial cultures are used, although there is no positive evidence for it. With older bacterial cultures definitely only a proportion of viable phage particles succeed in starting phage multiplication, the proportion decreasing with the increasing age of bacterial cultures used for testing.
83948981
Co-adaptation of Escherichia coli and coliphage λvir in continuous culture
Populations of the bacterium Escherichia coli and of its phage λvir appeared to equilibrate in continuous cultures. The bacterial end-populations were heterogeneous in respect of their resistance to λvir and their ability to utilize maltose. The most competitive of the selected bacteria were mutants which had a reduced rate of synthesis of λ-receptor so as to become highly, but not totally, resistant to the phage. The coexisting phage had an increased affinity for the receptor and an altered antigenic specificity, suggesting adaptation of its adsorption site in response to the evolution of resistance in the bacteria.
1894386
Evolvability of an RNA virus is determined by its mutational neighbourhood
The ubiquity of mechanisms that generate genetic variation has spurred arguments that evolvability, the ability to generate adaptive variation, has itself evolved in response to natural selection. The high mutation rate of RNA viruses is postulated to be an adaptation for evolvability, but the paradox is that whereas some RNA viruses evolve at high rates, others are highly stable. Here we show that evolvability in the RNA bacteriophage φ6 is also determined by the accessibility of advantageous genotypes within the mutational neighbourhood (the set of mutants one or a few mutational steps away). We found that two φ6 populations that were derived from a single ancestral phage repeatedly evolved at different rates and toward different fitness maxima. Fitness measurements of individual phages showed that the fitness distribution of mutants differed between the two populations. Whereas population A, which evolved toward a higher maximum, had a distribution that contained many advantageous mutants, population B, which evolved toward a lower maximum, had a distribution that contained only deleterious mutants. We interpret these distributions to measure the fitness effects of genotypes that are mutationally available to the two populations. Thus, the /evolvability of φ6 is constrained by the distribution of its mutational neighbours, despite the fact that this phage has the characteristic high mutation rate of RNA viruses.
23620800
Bacteriophage plaques: theory and analysis.
Laboratory characterization of bacteriophage growth traditionally is done either in broth cultures or in semisolid agar media. These two environments may be distinguished in terms of their spatial structure, i.e., the degree to which they limit diffusion, motility, and environmental mixing. Well-mixed broth, for example, represents the microbiological ideal of a non-spatially structured environment. Agar, by contrast, imposes significant limitations on phage and bacterial movement and therefore gives rise to spatial structure. The study of phage growth within spatially structured environments, such as that seen during phage plaque formation, is important for three reasons. First, a large fraction of environmental bacteria live within spatially structured environments such as within biofilms, within soil, or when growing in or on the tissues of plants and animals. Second, phage growth as plaques is a central technique to phage studies, yet appears to be under appreciated by phage workers in terms of its complexity. Third, selective pressures acting on phage during plaque growth differ from those seen during broth growth. In this chapter we will discuss just what a plaque is, how one forms, and what can affect plaque size. We will describe methods, both experimental and theoretical, that have been employed to study plaque growth. As caveats we will discuss why plaque formation failure is not necessarily equivalent to virion inviability (Note 1). We also will consider problems with inferring phage broth growth fitness as a function of plaque characteristics (Note 2).
23387241
Different Biological Characteristics of Wild-Type Porcine Reproductive and Respiratory Syndrome Viruses and Vaccine Viruses and Identification of the Corresponding Genetic Determinants
ABSTRACT Two attenuated vaccines, Ingelvac PRRS MLV and Ingelvac PRRS ATP, derived from VR2332 and JA142, respectively, have been used to control porcine reproductive and respiratory syndrome (PRRS) virus. However, there have been several field reports concerning the reversion of the vaccine virus to virulence. Furthermore, viruses genetically indistinguishable from the vaccines and wild-type parental viruses have been detected in clinical PRRS cases, raising the need for a better differential tool. As the vaccine viruses replicated better and produced bigger plaques in MARC-145 cells than did the wild-type parental strains, the following study was conducted to determine if the growth difference in MARC-145 cells can be utilized to differentiate a vaccine-like virus (VLV) from a wild-type virus and to identify genetic markers corresponding to such phenotype of the vaccine viruses. The relatedness of 83 field isolates collected between 1996 and 2005 to VR2332 and JA142 was classified genetically and antigenically. Thirteen of 25 VR2332-related viruses and 9 of 10 JA142-related viruses were determined as VLVs, since those viruses produced plaques similar to those by the vaccine viruses. Four unique amino acids each were identified throughout structural genes for MLV and ATP. Among those, F10 in open reading frame 2 (ORF2) of MLV and E85 and Y165 in ORF3 of ATP were stable during pig passages. When the sequences unique for MLV were incorporated into an infectious clone constructed based on VR2332, the virus growth and resultant plaque size in MARC-145 cells were increased, suggesting that these sequences can be used as genetic markers for VLVs.
8267654
Evolution of a persistent aphthovirus in cytolytic infections: partial reversion of phenotypic traits accompanied by genetic diversification
Foot-and-mouth disease virus (FMDV) shows a dual potential to be cytolytic or to establish persistent infections in cell culture. FMDV R100, a virus rescued after 100 passages of carrier BHK-21 cells persistently infected with FMDV clone C-S8c1, showed multiple genetic and phenotypic alterations relative to the parental clone C-S8c1. Several FMDV R100 populations have been subjected to 100 serial cytolytic infections in BHK-21 cells, and the reversion of phenotypic and genetic alterations has been analyzed. An extreme temperature sensitivity of R100 reverted totally or partially in some passage series but not in others. The small-plaque morphology reverted to normal size in all cases. The hypervirulence for BHK-21 cells did not revert, and even showed an increase, upon cytolytic passage. Most of the mutations that had been fixed in the R100 genome during persistence did not revert in the course of cytolytic passages, but the extended polyribocytidylate tract of R100 (about 460 residues, versus 290 in C-S8c1) decreased dramatically in length, to the range of 220 to 260 residues in all passage series examined. In passages involving very large viral populations, a variant with two amino acid substitutions (L-144-->V and A-145-->P) next to the highly conserved Arg-Gly-Asp (RGD motif; positions 141 to 143) within the G-H loop of capsid protein VP1 became dominant. A clonal analysis allowed isolation of a mutant with the single replacement A-145-->P. Viral production and growth competition experiments showed the two variants to have a fitness very close to that of the parental virus. The results provide evidence that the repertoire of variants that could potentially become dominant in viral quasispecies may be influenced by the population size of the evolving virus. The net results of a series of persistent-infection passages followed by a series of cytolytic passages was progressive genomic diversification despite reversion or stasis of phenotypic traits. Implications for the evolution of RNA viruses are discussed.
13075305
Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus.
An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 degrees C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed.
22460796
Bacteriophage evolution given spatial constraint.
Spatial structure can impede mixing, diffusion, and motility. In microbiology laboratories, spatial structure is commonly achieved via formation of agar gels, within which bacteriophage (phage) replication results in localized clearings called plaques. Developing a better understanding of phage plaque formation is relevant because of the ubiquity of phage plaquing in the laboratory; because plaque size has been employed as a measure of phage fitness; because many bacteria exist within environments that display significant spatial structure (e.g., biofilms, soils, sediments, and in or on plant or animal tissues); and because spatial structure could impede phage exploitation of bacterial communities. There is, however, a relative dearth of experimentation and analysis considering phage plaque formation from the perspective of selection acting on individual phage growth parameters-latent period, burst size, and adsorption rate. Here we consider the impact of these parameters on rates of plaque wavefront velocity (rates of radial plaque enlargement), especially as functions of existing phage and environmental properties. We do so based on analyses of published equations which predict plaque enlargement rates. These indicate that greater wavefront velocities should be associated with (i) latent period reductions, (ii) larger burst sizes, or (iii) faster virion binding to bacteria. We suggest, however, that deviations could occur, respectively, (i) if virion adsorption is "slow" or if burst sizes are large, (ii) if burst sizes are already large, or (iii) if virion binding rates are already fast, bacterial densities are especially high, or burst sizes are large. Higher initial lawn bacterial densities could also contribute to faster plaque expansion, but only if adsorption is otherwise slow or burst sizes are large. By contrast, faster virion diffusion is always expected to result in greater plaque wavefront velocities. Overall, we provide a snapshot of how phage populations may respond evolutionarily to selection for more-rapid propagation during spatially constrained growth.
6721553
High adsorption rate is detrimental to bacteriophage fitness in a biofilm-like environment
BackgroundBacterial biofilm is ubiquitous in nature. However, it is not clear how this crowded habitat would impact the evolution of bacteriophage (phage) life history traits. In this study, we constructed isogenic λ phage strains that only differed in their adsorption rates, because of the presence/absence of extra side tail fibers or improved tail fiber J, and maker states. The high cell density and viscosity of the biofilm environment was approximated by the standard double-layer agar plate. The phage infection cycle in the biofilm environment was decomposed into three stages: settlement on to the biofilm surface, production of phage progeny inside the biofilm, and emigration of phage progeny out of the current focus of infection.ResultsWe found that in all cases high adsorption rate is beneficial for phage settlement, but detrimental to phage production (in terms of plaque size and productivity) and emigration out of the current plaque. Overall, the advantage of high adsorption accrued during settlement is more than offset by the disadvantages experienced during the production and emigration stages. The advantage of low adsorption rate was further demonstrated by the rapid emergence of low-adsorption mutant from a high-adsorption phage strain with the side tail fibers. DNA sequencing showed that 19 out of the 21 independent mutant clones have mutations in the stf gene, with the majority of them being single-nucleotide insertion/deletion mutations occurring in regions with homonucleotide runs.ConclusionWe conclude that high mutation rate of the stf gene would ensure the existence of side tail fiber polymorphism, thus contributing to rapid adaptation of the phage population between diametrically different habitats of benthic biofilm and planktonic liquid culture. Such adaptability would also help to explain the maintenance of the stf gene in phage λ's genome.
23733781
Replication of viruses in a growing plaque: a reaction-diffusion model.
An understanding of the viral replication process commonly referred to as "plaque growth" is developed in the context of a reaction-diffusion model. The interactions among three components: the virus, the healthy host, and the infected host are represented using rates of viral adsorption and desorption to the cell surface, replication and release by host lysis, and diffusion. The solution to the full model reveals a maximum in the dependence of the velocity of viral propagation on its equilibrium adsorption constant, suggesting that conditions can be chosen where viruses which adsorb poorly to their hosts will replicate faster in plaques than those which adsorb well. Analytic expressions for the propagation velocity as a function of the kinetic and diffusion parameters are presented for the limiting cases of equilibrated adsorption, slow adsorption, fast adsorption, and large virus yields. Hindered diffusion at high host concentrations must be included for quantitative agreement with experimental data.
19301239
Optimizing bacteriophage plaque fecundity.
Bacteriophages (phages), the viruses of bacteria, form visible lesions within bacterial lawns (called plaques), which are employed ubiquitously in phage isolation and characterization. Plaques also can serve as models for phage population growth within environments that display significant spatial structure, e.g. soils, sediments, animal mucosal tissue, etc. Furthermore, phages growing within plaques, in experimental evolution studies, may become adapted to novel conditions, may be selected for faster expansion, or may evolve toward producing more virions per plaque. Here, we examine the evolution of the latter, greater plaque fecundity, considering especially tradeoffs between phage latent period and phage burst size. This evolution is interesting because genetically lengthening latent periods, as seen with phage lysis-timing mutants, should increase phage burst sizes, as more time is available for phage-progeny maturation during infection. Genetically shortening latent periods, however, is a means toward producing larger phage plaques since phage virions then can spend more time diffusing rather than infecting. With these larger plaques more bacteria become phage infected, resulting in more phage bursts. Given this conflict between latent period's impact on per-plaque burst number versus per-infection burst size, and based on analysis of existing models of plaque expansion, we provide two assertions. First, latent periods that optimize plaque fecundity are longer (e.g. at least two-fold longer) than latent periods that optimize plaque size (or that optimize phage population growth within broth). Second, if increases in burst size can contribute to plaque size (i.e. larger plaques with larger bursts), then latent-period optima that maximize plaque fecundity should be longer still. As a part of our analysis, we provide a means for predicting latent-period optima-for maximizing either plaque size or plaque fecundity-which is based on knowledge of only phage eclipse period and the relative contribution of phage burst size versus latent period toward plaque size.
44888493
Approximate solution to the speed of spreading viruses.
Recently, it has been shown that the speed of virus infections can be explained by time-delayed reaction-diffusion [J. Fort and V. Méndez, Phys. Rev. Lett. 89, 178101 (2002)], but no analytical solutions were found. Here we derive formulas for the front speed, valid in appropriate limits. We also integrate numerically the evolution equations of the system. There is good agreement with both numerical and experimental speeds.
23694999
Adsorption of bacteriophage lambda on the LamB protein of Escherichia coli K-12: point mutations in gene J of lambda responsible for extended host range
LamB is the cell surface receptor for bacteriophage lambda. LamB missense mutations yielding resistance to lambda group in two classes. Class I mutants block the growth of lambda with the wild-type host range (lambda h+) but support the growth of one-step host range mutants (lambda h). Class II mutants block lambda h but support the growth of two-step host range mutant (lambda hh*) phages. To identify amino acid residues in the J protein (the tail fiber of phage lambda) responsible for the extended host range phenotype of mutants of phage (lambda h+), we selected a series of one-step (lambda h) and two-step (lambda hh*) host range mutants and analyzed their corresponding J genes. Three different class I LamB missense mutants (mutations at sites 247, 245, and 148) were used to select 11 independent, new, one-step host range mutants (lambda h phages). DNA sequence analysis revealed a single-amino-acid change in each case. The 11 alterations affected only three residues in the distal part of J, corresponding to a Val-->Ala change at site 1077 in five cases, a Thr-->Met change at site 1040 in three cases, and a Leu-->Pro change at site 1127 in three cases. Recombination experiments confirmed that in the cases tested, the mutations identified were indeed responsible for the extended host range phenotype. The class II LamB mutant (Gly-->Asp at site 151) was used to select two-step extended host range mutants (lambda hh* phages) from three new lambda h phages, corresponding to different amino acid modifications in the J protein (at sites 1040, 1077, and 1127). The new lambda hh* phages analyzed corresponded to either double or triple point mutations located at the distal end of the J protein. In all, seven residues involved in the extended host range properties of lambda mutants were identified in the distal part of the J protein, suggesting that the last C-terminal portion of the J protein participates directly in the adsorption of the phage onto LamB. In agreement with the fact that the lambda h mutants (and the lambda hh* mutants) could grow on all of the lamB class I mutations tested, we found tha the nature of the J mutations did not depend on the LamB class I mutant used to select them. This is interpreted as meaning that the mutated residues in the J protein and in the LamB mutants are not involved in allele-specific protein-protein interactions. Rather, the LamB mutations would block a step in phage adsorption, and this block would be overcome by the mutations in the J protein.
1884524
Lysis Timing and Bacteriophage Fitness
The effect of lysis timing on bacteriophage (phage) fitness has received little theoretical or experimental attention. Previously, the impact of lysis timing on phage fitness was studied using a theoretical model based on the marginal value theorem from the optimal foraging theory. An implicit conclusion of the model is that, for any combination of host quantity and quality, an optimal time to lyse the host would exist so that the phage fitness would be the highest. To test the prediction, an array of isogenic λ-phages that differ only in their lysis times was constructed. For each phage strain, the lysis time, burst size, and fitness (growth rate) were determined. The result showed that there is a positive linear relationship between lysis time and burst size. Moreover, the strain with an intermediate lysis time has the highest fitness, indicating the existence of an optimal lysis time. A mathematical model is also constructed to describe the population dynamics of phage infection. Computer simulations using parameter values derived from phage λ-infection also showed an optimal lysis time. However, both the optimum and the fitness are different from the experimental result. The evolution of phage lysis timing is discussed from the perspectives of multiple infection and life-history trait evolution.
26701896
Bacteriophage Adsorption Rate and Optimal Lysis Time
The first step of bacteriophage (phage) infection is the attachment of the phage virion onto a susceptible host cell. This adsorption process is usually described by mass-action kinetics, which implicitly assume an equal influence of host density and adsorption rate on the adsorption process. Therefore, an environment with high host density can be considered as equivalent to a phage endowed with a high adsorption rate, and vice versa. On the basis of this assumption, the effect of adsorption rate on the evolution of phage optimal lysis time can be reinterpreted from previous optimality models on the evolution of optimal lysis time. That is, phage strains with a higher adsorption rate would have a shorter optimal lysis time and vice versa. Isogenic phage λ-strains with different combinations of six different lysis times (ranging from 29.3 to 68 min), two adsorption rates (9.9 × 10−9 and 1.3 × 10−9 phage−1 cell−1 ml−1 min−1), and two markers (resulting in “blue” or “white” plaques) were constructed. Various pairwise competitions among these strains were conducted to test the model prediction. As predicted by the reinterpreted model, the results showed that the optimal lysis time is shorter for phage strains with a high adsorption rate and vice versa. Competition between high- and low-adsorption strains also showed that, under current conditions and phenotype configurations, the adsorption rate has a much larger impact on phage relative fitness than the lysis time.
8145168
Enumeration of bacteriophage particles
Bacteriophages are increasingly being utilized and considered for various practical applications, ranging from decontaminating foods and inanimate surfaces to human therapy; therefore, it is important to determine their concentrations quickly and reliably. Traditional plaque assay (PA) is the current “gold standard” for quantitating phage titers. However, it requires at least 18 h before results are obtained, and they may be significantly influenced by various factors. Therefore, two alternative assays based on the quantitative real-time polymerase chain reaction (QPCR) and NanoSight Limited (NS) technologies were recently proposed for enumerating phage particles. The present study compared the three approaches’ abilities to quantitate Listeria monocytogenes-, Escherichia coli O157:H7-, and Yersinia pestis-specific lytic phages quickly and reproducibly. The average coefficient of variation (CVS) of the PA method including all three phages was 0.15. The reproducibility of the PA method decreased dramatically when multiple investigators performed the assays, and mean differences of as much as 0.33 log were observed. The QPCR method required costly equipment and the synthesis of phage-specific oligonucleotide primers, but it determined phage concentrations faster (within about 4 h) and more precisely than did PA (CVS = 0.13). NS technology required costly equipment, was less precise (CVS = 0.28) than the PA and QPCR methods, and only worked when the phages were suspended in clear medium. However, it provided results within 5 min. After the overall correlation is established with the PA method, either of the two assays may be useful for quickly and reproducibly determining phage concentrations.
24223605
Quantification of Shiga toxin 2‐encoding bacteriophages, by real‐time PCR and correlation with phage infectivity
Aims:  To evaluate a qPCR‐based protocol for the enumeration of Shiga toxin (Stx) 2 phages and to compare the results of qPCR with the number of infective Stx phage particles.
8937352
Bacteriophage infection and multiplication occur in Pseudomonas aeruginosa starved for 5 years.
Bacteriophages specific for Pseudomonas aeruginosa and Escherichia coli were examined for their ability to multiply in stationary phase hosts. Four out of five bacteriophages tested, including E. coli bacteriophage T7M, were able to multiply in stationary phase hosts. The bacteriophage ACQ had a mean burst size of approximately 1000 in exponential phase P. aeruginosa hosts and 102 in starved hosts, with corresponding latent periods that increased from 65 to 210 min. The bacteriophage UT1 had a mean burst size of approximately 211 in exponential phase P. aeruginosa hosts and 11 in starved hosts, with latent periods that increased from a mean of 90 min in exponential phase hosts to 165 min in starved hosts. Bacteriophage multiplication occurred whether or not the hosts had entered stationary phase, either because the cultures had been incubated for 24 h or were starved. Significantly, bacteriophage multiplication occurred in P. aeruginosa, which had been starved for periods of 24 h, several weeks, or 5 years. Only one P. aeruginosa virus, BLB, was found to be incapable of multiplication in stationary phase hosts. These results reveal that starvation does not offer bacterial hosts refuge from bacteriophage infection and suggest that bacteriophages will be responsible for significant bacterial mortality in most natural ecosystems.
21111752
HOST DENSITY IMPACTS RELATIVE FITNESS OF BACTERIOPHAGE Φ6 GENOTYPES IN STRUCTURED HABITATS
Abstract Spatially structured environments may impact evolution by restricting population sizes, limiting opportunities for genetic mixis, or weakening selection against deleterious genotypes. When habitat structure impedes dispersal, low-productivity (less virulent) infectious parasites may benefit from their prudent exploitation of local hosts. Here we explored the combined ability for habitat structure and host density to dictate the relative reproductive success of differentially productive parasites. To do so, we allowed two RNA bacteriophage Φ6 genotypes to compete in structured and unstructured (semi-solid versus liquid) habitats while manipulating the density of Pseudomonas hosts. In the unstructured habitats, the more-productive phage strain experienced a relatively constant fitness advantage regardless of starting host density. By contrast, in structured habitats, restricted phage dispersal may have magnified the importance of local productivity, thus allowing the relative fitness of the less-productive virus to improve as host density increased. Further data suggested that latent period (duration of cellular infection) and especially burst size (viral progeny produced per cell) were the phage “life-history” traits most responsible for our results. We discuss the relevance of our findings for selection occurring in natural phage populations and for the general evolutionary epidemiology of infectious parasites.
16272814
The use of antibiotics to improve phage detection and enumeration by the double-layer agar technique
BackgroundThe Double-Layer Agar (DLA) technique is extensively used in phage research to enumerate and identify phages and to isolate mutants and new phages. Many phages form large and well-defined plaques that are easily observed so that they can be enumerated when plated by the DLA technique. However, some give rise to small and turbid plaques that are very difficult to detect and count. To overcome these problems, some authors have suggested the use of dyes to improve the contrast between the plaques and the turbid host lawns. It has been reported that some antibiotics stimulate bacteria to produce phages, resulting in an increase in final titer. Thus, antibiotics might contribute to increasing plaque size in solid media.ResultsAntibiotics with different mechanisms of action were tested for their ability to enhance plaque morphology without suppressing phage development. Some antibiotics increased the phage plaque surface by up to 50-fold.ConclusionThis work presents a modification of the DLA technique that can be used routinely in the laboratory, leading to a more accurate enumeration of phages that would be difficult or even impossible otherwise.
13152773
Mutations of Bacterial Viruses Affecting Their Host Range.
In this follow up to his 1943 article with Delbruck on phage-resistant bacteria, Luria demonstrated that not only do bacteria
 mutate into forms resistant to certain phages, but that phages can mutate to compensate for resistant mutations in their host.
20344889
Allelic and Nonallelic Genes Controlling Host Specificity in a Bacteriophage.
Preliminary crosses ( HERSHEY and ROTMAN 1948, and unpublished) between pairs of independently arising host-range mutants of the same virus, however, have failed to yield recombinants, even when the two mutants were clearly different. LURIA (personal communication) has had the same experience with host-range mutants of the related phage T2L. If the results with the two kinds of mutant are to be given a consistent interpretation, it has to be assumed that different host-range mutations, unlike the r mutations, tend to occur at a single genetic locus. This assumption is not implausible, inasmuch as the host-range character can be readily combined with the r character in the appropriate genetic crosses (HERSHEY and ROTMAN 1949). Nevertheless, we felt that the genetic interpretation could be greatly strengthened by establishing one authentic example of multiple allelism in the virus. This we have done, by three independent means. The demonstration is all the more convincing because one mutant was found that proved to be carrying a mutation in a second locus controlling host specificity. M ETH ODs The technique of crossing viral mutants has been modified slightly since last described (HERSHEY and ROTMAN 1949). The bacteria are now grown in nutrient broth, suspended in buffered saline, admixed with virus, and diluted in broth after allowing five minutes for adsorption to occur. Adsorption in the absence of nutrient is physiologically equivalent to simultaneous adsorption of the added virus particles ( BENZER et al. 1950), and the effects we observe are understandable on the assumption that this causes a more
7405036
S gene expression and the timing of lysis by bacteriophage lambda
The S gene of bacteriophage lambda encodes the holin required for release of the R endolysin at the onset of phage-induced host lysis. S is the promoter-proximal gene on the single lambda late transcript and spans 107 codons. S has a novel translational initiation region with dual start codons, resulting in the production of two protein products, S105 and S107. Although differing only by the Met-1-Lys-2... N-terminal extension present on S107, the two proteins are thought to have opposing functions, with the shorter polypeptide acting as the lysis effector and the longer one acting as an inhibitor. The expression of wild-type and mutant alleles of the holin gene has been assessed quantitatively with respect to the scheduling of lysis. S mRNA accumulates during the late gene expression period to a final level of about 170 molecules per cell and is maintained at that level for at least the last 15 min before lysis. Total S protein synthesis, partitioned at about 2:1 in favor of the S105 protein compared with the other product, S107, accumulates to a final level of approximately 4,600 molecules per cell. The kinetics of accumulation of S is consistent with a constant translational rate of less than one S protein per mRNA per minute. Mutant alleles with alterations in the translational initiation region were studied to determine how the translational initiation region of S achieves the proper partition of initiation events at the two S start codons and how the synthesis of S105 and S107 relates to lysis timing. The results are discussed in terms of a model for the pathway by which the 30S ribosome-fMet-tRNA complex binds to the translational initiation region of S. In addition, analysis of the relationship between lysis timing and the levels of the two S gene products suggests that S107 inhibits S105, the lethal lysis effector, by a stoichiometric titration.
33743946
Local Interactions Select for Lower Pathogen Infectivity
Theory suggests that the current rapid increase in connectivity and consequential changes in the structure of human, agricultural, and wildlife populations may select for parasite strains with higher infectivity. We carried out a test of this spatial theory by experimentally altering individual host movement rates in a model host/pathogen system by altering the viscosity of their environment. In our microevolutionary selection experiments, the infectivity of the virus was, as predicted by the theory, reduced in the most viscous populations. We therefore provide empirical support for the theory that population structure affects the evolution of infectious organisms.
45124462
‘Small worlds’ and the evolution of virulence: infection occurs locally and at a distance
Why are some diseases more virulent than others? Vector–borne diseases such as malaria and water–borne diseases such as cholera are generally more virulent than diseases spread by direct contagion. One factor that characterizes both vector– and water–borne diseases is their ability to spread over long distances, thus causing infection of susceptible individuals distant from the infected individual. Here we show that this ability of the pathogen to infect distant individuals in a spatially structured host population leads to the evolution of a more virulent pathogen. We use a lattice model in which reproduction is local but infection can vary between completely local to completely global. With completely global infection the evolutionarily stable strategy (ESS) is the same as in mean–field models while a lower virulence is predicted as infection becomes more local. There is characteristically a period of relatively moderate increase in virulence followed by a more rapid rise with increasing proportions of global infection as we move beyond a ‘critical connectivity’. In the light of recent work emphasizing the existence of ‘small world’ networks in human populations, our results suggests that if the world is getting ‘smaller’ (as populations become more connected) diseases may evolve higher virulence.
23136957
Dynamical types of bacteria and bacteriophages interaction: shielding by debris.
The dynamics of the bacteria and bacteriophages interaction process was explored in depth, and laid on a firm basis through simulation and analysis. A modified Campbell model of phage-bacteria interaction was used to simulate three interacting species: bacteria, phages and bacterial debris, and their time behavior in terms of three parameters, in selected range values: the phages burst size (beta), the dissolution rate of the bacterial debris (q), and the lysing time delay (tau). Six types of dynamical behavior were identified, occupying various zones in the two-dimensional (2D) plane (q, beta) for several values of tau, allowing the determination of the region where shielding by bacterial debris is in effect. The problem of the occurrence of stability transitions between stable and unstable dynamical regimes was also addressed analytically, and compared with simulation results.
38725904
Bacterial debris-an ecological mechanism for coexistence of bacteria and their viruses.
A model of bacteria and phage survival is developed based on the idea of shielding by bacterial debris in the system. This model is mathematically formulated by a set of four nonlinear difference equations for susceptible bacteria, contaminated bacteria, bacterial debris and phages. Simulation results show the possibility of survival, and domains of existence of stable and unstable solutions
3103192
The lysis mechanism of phage T4: mutants affecting lysis.
Abstract A selection technique is used to isolate a new class of lysis-defective mutants of phage T4, the T4 amber t mutants. Under normal conditions, T4-infected bacteria lyse at a characteristic time after infection, each infected cell releasing a few hundred progeny phage. However, Su − bacteria infected with amber t mutant phage fail to lyse and continue to manufacture phage beyond the normal latent period even though phage lysozyme is present. Genetic crosses and complementation tests show that t mutant phage define a new T4 gene, gene t. Functional t gene product is required for the cessation of phage-infected cell metabolism at the characteristic lysis time. The T4 lysis mechanism is shown to be a sequential process of at least two steps: function of phage t gene product followed by function of phage e gene product. The e gene product, phage lysozyme, is known to degrade the host cell wall. It is proposed that the t gene product may be involved in degradation of the host cytoplasmic membrane.
27205068
Lysis defective mutants of bacteriophage lambda: genetics and physiology of S cistron mutants.
Abstract Five lysis-deficient isolates of the S type, (including sus, def , and one ts mutant), were characterized genetically and physiologically. All the isolates were found to share two distinctive phenotypic properties; susceptibility of host cells to chloroform-induced lysis, and overproduction of phage relative to wild-type or R-mutant yields. Complementation tests demonstrated that the sus and def isolates belong to the S cistron. Two- and three-factor genetic crosses showed that all five markers are closely linked, and located the S cistron between cistrons Q and R. Comparison of the kinetics of production of phage and phage components (serum-blocking material and DNA) by S and R mutants and wild-type phage, showed that overproduction by S mutants is achieved by prolongation of component synthesis and maturation. The failure of R mutants to overproduce even though the host cells do not lyse shows that expression of the S function is responsible for the cessation of phage synthesis at the end of the normal latent period.
80970290
The process of infection with bacteriophage FX174X. Mutations in a FX lysis gene
The ability of conditional lethal mutants of phage ΦX174 to induce host cell lysis during infection under restrictive conditions has been studied. We have found amber (am) and temperature-sensitive (ts) mutants which present a variety of alterations in the normal lytic process. In particular, there is a class of am mutants which do not produce cell lysis but otherwise replicate normally in the restrictive host. These mutants constitute a single complementation group. The existence of these mutants implicates a phage-coded protein in the lytic process. This protein is not an essential structural component of the phage, since normal phage particles are produced in the absence of lysis.
12869257
From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli
In Escherichia coli, levels of the two major outer membrane porin proteins, OmpF and OmpC, are regulated in response to a variety of environmental parameters, and numerous factors have been shown to influence porin synthesis. EnvZ and OmpR control porin‐gene transcription in response to osmolarity, and the anti‐sense RNA, MicF, influences ompF translation. In contrast to these characterized factors, some of the components reported to influence porin expression have only modest effects and/or act indirectly. For others, potential regulatory roles, although intriguing, remain elusive. Here we review many of the components that have been reported to influence porin expression, address the potential regulatory nature of these components, and discuss how they may contribute to a regulatory network controlling porin synthesis.
45204300
Regulation of the Escherichia coliσE‐dependent envelope stress response
The Escherichia coliσE‐dependent stress response pathway controls the expression of genes encoding periplasmic folding catalysts, proteases, biosynthesis enzymes for lipid A (a component of lipopolysaccharide or LPS) and other proteins known or predicted to function in or produce components of the envelope. When E. coli is subjected to heat or other stresses that generate unfolded envelope proteins, σE activity is induced. Four key players in this signal transduction pathway have been identified: RseA, an inner membrane σE antisigma factor; RseB, a periplasmic protein that binds to the periplasmic face of RseA; and the DegS and YaeL proteases. The major point of regulation, the interaction between σE and RseA, is primarily controlled by the stability of RseA. Envelope stress promotes RseA degradation, which occurs by a proteolytic cascade initiated by DegS. There is evidence that one σE‐inducing stress (OmpC overexpression) directly activates DegS to cleave RseA. Secondarily, envelope stress may relieve RseB‐mediated enhancement of RseA activity. Additional levels of control upon σE activity may become evident upon further study of this stress response pathway.
26328062
A component of the side tail fiber of Escherichia coli bacteriophage lambda can functionally replace the receptor-recognizing part of a long tail fiber protein of the unrelated bacteriophage T4
The distal part of the long tail fiber of Escherichia coli bacteriophage T4 consists of a dimer of protein 37. Dimerization requires the catalytic action of protein 38, which is encoded by T4 and is not present in the virion. It had previously been shown that gene tfa of the otherwise entirely unrelated phage lambda can functionally replace gene 38. Open reading frame (ORF) 314, which encodes a protein that exhibits homology to a COOH-terminal area of protein 37, is located immediately upstream of tfa. The gene was cloned and expressed in E. coli. An antiserum against the corresponding polypeptide showed that it was present in phage lambda. The serum also reacted with the long tail fibers of phage T4 near their free ends. An area of the gene encoding a COOH-terminal region of ORF 314 was recombined, together with tfa, into the genome of T4, thus replacing gene 38 and a part of gene 37 that codes for a COOH-terminal part of protein 37. Such T4-lambda hybrids, unlike T4, required the presence of outer membrane protein OmpC for infection of E. coli B. An ompC missense mutant of E. coli K-12, which was still sensitive to T4, was resistant to these hybrids. We conclude that the ORF 314 protein represents a subunit of the side tail fibers of phage lambda which probably recognize the OmpC protein. ORF 314 was designated stf (side tail fiber). The results also offer an explanation for the very unusual fact that, despite identical genomic organizations, T4 and T2 produce totally different proteins 38. An ancestor of T4 from the T2 lineage may have picked up tfa and stf from a lambdoid phase, thus possibly demonstrating horizontal gene transfer between unrelated phage species.
40317301
On kinetics of phage adsorption.
Adsorption of lambda-phage on sensitive bacteria Escherichia coli is a classical problem but not all issues have been resolved. One of the outstanding problems is the rate of adsorption, which in some cases appears to exceed the theoretical limit imposed by the law of random diffusion. We revisit this problem by conducting experiments along with new theoretical analyses. Our measurements show that upon incubating lambda-phage with bacteria Ymel, the population of unbound phage in a salt buffer decreases with time and in general obeys a double-exponential function characterized by a fast (tau(1)) and a slow (tau(2)) decay time. We found that both the fast and the slow processes are specific to interactions between lambda-phage and its receptor LamB. Such specificity motivates a kinetic model that describes the interaction between the phage and the receptor as an on-and-off process followed by an irreversible binding. The latter may be a signature of the initiation of DNA translocation. The kinetic model successfully predicts the double exponential behavior seen in the experiment and allows the corresponding rate constants to be extracted from single measurements. The weak temperature dependence of the reversible and the irreversible binding rate suggests that phage retention by the receptor is entropic in nature and that a molecular key-lock interaction may be an appropriate description of the interaction between the phage tail and the receptor.
8220963
Appendix: a model of plaque formation.
Equations describing plaque formation in soft agar have been based on certain simplifying assumptions, for which data are presented. The derived equations permit one to calculate (i) average plaque size as a function of the initial density of indicator cells (Do), (ii) the number of cells lysed per plaque as a function of Do, and (iii) the cumulative number of cells lysed at various stages of plaque development. The calculated values agree well with those determined experimentally.
20970395
Isolation of the Bacteriophage Lambda Receptor from Escherichia coli
A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.
29149629
Bacteriophage lysis: mechanism and regulation.
Bacteriophage lysis involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm. The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains. The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis. The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself. In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized. Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope. These lysis proteins function by activation of cellular autolysins. A host locus is required for the lytic function of the phi X174 lysis gene E.