THE GROWTH OF BACTERIOPHAGE AND LYSIS OF THE HOST

1. A new strain of B. coli and of phage active against it is described, and the relation between phage growth and lysis has been studied. It has been found that the phage can lyse these bacteria in two distinct ways, which have been designated lysis from within and lysis from without. 2. Lysis from within is caused by infection of a bacterium by a single phage particle and multiplication of this particle up to a threshold value. The cell contents are then liberated into solution without deformation of the cell wall. 3. Lysis from without is caused by adsorption of phage above a threshold value. The cell contents are liberated by a distension and destruction of the cell wall. The adsorbed phage is not retrieved upon lysis. No new phage is formed. 4. The maximum yield of phage in a lysis from within is equal to the adsorption capacity. 5. Liberation of phage from a culture in which the bacteria have been singly infected proceeds at a constant rate, after the lapse of a minimum latent period, until all the infected bacteria are lysed. 6. If the bacteria are originally not highly in excess, this liberation is soon counterbalanced by multiple adsorption of the liberated phage to bacteria that are already infected. This leads to a reduction of the final yield.     

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants.

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage lambda forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage lambda development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage lambda to the dnaA seqA mutant cells is decreased at 0 degrees C , but not at 30 degrees C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for beta-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.     

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 lambda-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 lambda-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.     

Metabolic characterization of the viable, residually dividing and nondividing cell classes of recombination-deficient strains of Escherichia coli.

The abilities of rec+, recB- recC-, recA-, and recA- recB- rec C- strains to support growth of bacteriophage T4, to take up oxygen, and to maintain cell integrity have been measured. (i) With respect to bacteriophage T4 growth, T4 phage is produced with identical lysis time in single -step growth curves with all strains tested. rec- strains show reduced phage production (fewer infected centers), but the average burst size per infected center is the same for all strains tested. Some rec- cells are unable to produce any phage, whereas the remainder of the rec-cells produce phage as rapidly and as efficiently as rec+ cells. (ii) With respect to oxygen consumption, rec- strains are deficient relative to the rec+ control to the same extent as the deficiency in phage production by theculture. The reduction in oxygen consumption is coordinate with reduction in rate of mass increase of the rec- culture. (iii) With respect to cell integrity, rec- cultures show increased lysis as measured by release of beta-galactosidase into the medium. The kinetics of release suggest that rec- nondividing cells all eventually lyse. These results are most consistent with the idea that rec- residually dividing cells and viable cells are metabolically normal according to the parameters we have measured, whereas nondividing cells are metabolically inactive.     

Coliphage N4 N-acetylmuramidase defines a new family of murein hydrolases

Escherichia coli phage N4 infection leads to delayed host cell lysis, 3000 particles per infected bacterium and a small plaque phenotype. We show that bacteriophage N4 encodes a murein hydrolase (gp61) that is essential for N4 plaque-forming ability. gp61 has a high level of sequence similarity to hypothetical proteobacterial proteins, and Vibrio harveyi phage VHML ORF 19. Nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS) analysis of muropeptides from purified gp61 digestion of E. coli peptidoglycan indicates that gp61 is an N-acetylmuramidase. The EGGY motif present near the N terminus of gp61 and its homologs contains the glutamic acid residue essential for enzymatic activity. These results provide evidence that N4 gp61 and its homologs define a new family of N-acetylmuramidases (pfam05838.4, DUF847, COG3926). In contrast to its homologs, gp61 contains an N-terminal signal sequence. When expressed at levels present during phage infection, gp61 localizes primarily to the cell inner membrane; in contrast, over-expression of recombinant N4 gp61 is sufficient for rapid cell lysis. Overproduction of the recombinant Salmonella typhimurium (STM0016) homolog is sufficient for cell lysis only when fused to the gp61 N-terminal signal sequence. The results of subcellular localization and of mutagenesis of the gp61 N-terminal signal sequence indicate that gp61 must be released from the inner membrane to be catalytically active.     

Fixation probability for lytic viruses: the attachment-lysis model

The fixation probability of a beneficial mutation is extremely sensitive to assumptions regarding the organism's life history. In this article we compute the fixation probability using a life-history model for lytic viruses, a key model organism in experimental studies of adaptation. The model assumes that attachment times are exponentially distributed, but that the lysis time, the time between attachment and host cell lysis, is constant. We assume that the growth of the wild-type viral population is controlled by periodic sampling (population bottlenecks) and also include the possibility that clearance may occur at a constant rate, for example, through washout in a chemostat. We then compute the fixation probability for mutations that increase the attachment rate, decrease the lysis time, increase the burst size, or reduce the probability of clearance. The fixation probability of these four types of beneficial mutations can be vastly different and depends critically on the time between population bottlenecks. We also explore mutations that affect lysis time, assuming that the burst size is constrained by the lysis time, for experimental protocols that sample either free phage or free phage and artificially lysed infected cells. In all cases we predict that the fixation probability of beneficial alleles is remarkably sensitive to the time between population bottlenecks.     

INCREASE IN BACTERIOPHAGE AND GELATINASE CONCENTRATION IN CULTURES OF BACILLUS MEGATHERIUM

1. The increase in bacteria, phage concentration, and gelatinase concentration in cultures of B. megatherium has been determined. 2. With lysogenic cultures the phage concentration, gelatinase concentration, and bacteria concentration increase logarithmically at first. The phage and gelatinase concentration then decrease while the bacteria concentration increases to a maximum. 3. The results are the same with sensitive cultures if the ratio of phage to bacteria is small. If the ratio of phage to bacteria is large phage, gelatinase, and bacteria concentration all increase at first and then decrease. The maximum rate of increase coincides approximately with the maximum rate of oxygen consumption of the culture. 4. 60–90 per cent of the phage is free from the cells. 5. The amount of phage produced is determined by the combined phage and not by the total phage. 6. Phage is produced during growth of the cells and not during lysis. 7. In a very narrow range of pH near 5.55 no increase in bacteria occurs but large increases in phage may be obtained.     

THE GROWTH OF BACTERIOPHAGE

1. An anti-Escherichia coli phage has been isolated and its behavior studied. 2. A plaque counting method for this phage is described, and shown to give a number of plaques which is proportional to the phage concentration. The number of plaques is shown to be independent of agar concentration, temperature of plate incubation, and concentration of the suspension of plating bacteria. 3. The efficiency of plating, i.e. the probability of plaque formation by a phage particle, depends somewhat on the culture of bacteria used for plating, and averages around 0.4. 4. Methods are described to avoid the inactivation of phage by substances in the fresh lysates. 5. The growth of phage can be divided into three periods: adsorption of the phage on the bacterium, growth upon or within the bacterium (latent period), and the release of the phage (burst). 6. The rate of adsorption of phage was found to be proportional to the concentration of phage and to the concentration of bacteria. The rate constant k_a is 1.2 x 10^–9 cm.⁸/min. at 15°C. and 1.9 x 10^–9 cm.⁸/min. at 25°. 7. The average latent period varies with the temperature in the same way as the division period of the bacteria. 8. The latent period before a burst of individual infected bacteria varies under constant conditions between a minimal value and about twice this value. 9. The average latent period and the average burst size are neither increased nor decreased by a fourfold infection of the bacteria with phage. 10. The average burst size is independent of the temperature, and is about 60 phage particles per bacterium. 11. The individual bursts vary in size from a few particles to about 200. The same variability is found when the early bursts are measured separately, and when all the bursts are measured at a late time.     

多重耐药肺炎克雷伯菌噬菌体KP002的生物学特性及基因组学研究

以上海某些医院临床分离到的多重耐药肺炎克雷伯菌为宿主菌,从不同环境的污水中分离获得1株肺炎克雷伯菌噬菌体KP002。电子显微镜显示其为有尾噬菌体,头部直径约70nm,尾长约80nm,尾宽约20nm。对其生物学特性进行研究,结果显示此株噬菌体在pH 3~9及4~50℃的环境中具有较高活性;6min吸附率达95%以上;潜伏期为10min,爆发期为50min;裂解量为172pfu/cell。结果表明,该噬菌体对pH值和温度适应范围较宽。对其全基因组进行测序分析,结果显示其基因组为环状双链DNA,全长47 173bp,GC含量为48%。本研究筛选获得1株对pH值和温度适应范围较宽的耐药肺炎克雷伯菌烈性噬菌体KP002,为建立耐药肺炎克雷伯菌的噬菌体库以用于治疗临床多重耐药菌感染提供了新的思路。     

Mutations in the new gene stIII of bacteriophage T4B suppressing the lysis defect of gene stII and a gene e mutant.

Mutations of bacteriophage T4B were found which suppress the lysis defect of both gene stII mutants and gene e mutants. The suppressor mutations belong to a new gene, stIII, of phage T4B. Gene stIII is located on the genetic map of T4B between genes stI and e. stIII mutants sometimes form star plaques on Escherichia coli B. The latent period on E. coli 594, but not E. coli B, is shorter with stIII mutants than that with wild-type phage. The premature lysis of E. coli 594 infected with stIII phage does not depend on the expression of both stII+ and e+ function. StIII allele is dominant over the stIII+ with respect to both the ability to suppress the stII defect and the early lysis of infected E. coli 594 cultures.     

Marine transducing bacteriophage attacking a luminous bacterium

The isolation and partial characterization of a marine bacteriophage attacking a strain of luminous bacteria is described, including some physical, biological, and genetic properties. It is a DNA phage of density of 1.52 with a long flexible tail and an apparently icosohedral head. With respect to stability in suspension, it has a rather specific requirement for the sodium ion in high concentration; it is further stabilized by the addition of calcium and magnesium ions. These same ions are likewise all required for both good plating efficiency and plaque uniformity. Although it goes through a typical lytic growth cycle (about 45 min), with a burst size of 100, and no stable lysogens have been isolated, it is nevertheless a transducing phage specifically for the tryptophan region, transducing several, but not all, independently isolated Trp⁻ auxotrophs to protrophy. No other auxotrophs of a variety of amino acids were transduced by this phage to prototrophy. Phage infection does not change the normal expression of the luminescent system, and light remains at near normal levels until cell lysis occurs.     

Genomic Investigation of Lysogen Formation and Host Lysis Systems of the Salmonella Temperate Bacteriophage SPN9CC

To understand phage infection and host cell lysis mechanisms in pathogenic Salmonella, a novel Salmonella enterica serovar Typhimurium-targeting bacteriophage, SPN9CC, belonging to the Podoviridae family was isolated and characterized. The phage infects S. Typhimurium via the O antigen of lipopolysaccharide (LPS) and forms clear plaques with cloudy centers due to lysogen formation. Phylogenetic analysis of phage major capsid proteins revealed that this phage is a member of the lysogen-forming P22-like phage group. However, comparative genomic analysis of SPN9CC with P22-like phages indicated that their lysogeny control regions and host cell lysis gene clusters show very low levels of identity, suggesting that lysogen formation and host cell lysis mechanisms may be diverse among phages in this group. Analysis of the expression of SPN9CC host cell lysis genes encoding holin, endolysin, and Rz/Rz1-like proteins individually or in combinations in S. Typhimurium and Escherichia coli hosts revealed that collaboration of these lysis proteins is important for the lysis of both hosts and that holin is a key protein. To further investigate the role of the lysogeny control region in phage SPN9CC, a ΔcI mutant (SPN9CCM) of phage SPN9CC was constructed. The mutant does not produce a cloudy center in the plaques, suggesting that this mutant phage is virulent and no longer temperate. Subsequent comparative one-step growth analysis and challenge assays revealed that SPN9CCM has shorter eclipse/latency periods and a larger burst size, as well as higher host cell lysis activity, than SPN9CC. The present work indicates the possibility of engineering temperate phages as promising biocontrol agents similar to virulent phages.     

Bacteriophage lysins as effective antibacterials

Lysins are highly evolved enzymes produced by bacteriophage (phage for short) to digest the bacterial cell wall for phage progeny release. In Gram-positive bacteria, small quantities of purified recombinant lysin added externally results in immediate lysis causing log-fold death of the target bacterium. Lysins have been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and infected tissues. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Thus, lysins may be a much needed anti-infective in an age of mounting antibiotic resistance.     

Inhibition of bacterial cell wall mucopeptide synthesis: A new function of RNA bacteriophage Qβ

Studies of the morphology of Escherichia coli showed that these bacilli when infected with RNA phage Qβ in broth containing hypertonic sucrose and Mg²⁺ formed osmotically labile spherical cells or spheroplasts. Phage-induced spheroplasts readily released their burst of phage when diluted into ordinary culture broth without sucrose. Investigation of the mechanism of host cell lysis revealed that incorporation of [³H]diaminopimelic acid (DAP) into the mucopeptide layer of the cell wall was markedly inhibited starting at about the midpoint of the phage replication cycle. The major site of inhibition is the DAP-containing mucopeptide layer since the synthesis of the lipoprotein-lipopolysaccharide layer, making up the bulk of the cell wall of E. coli, was not affected. A model for Qβ-mediated cell lysis is discussed which is analogous to penicillin-induced cell rupture.     

Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage

Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E. coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid. To inactivate phage lytic activity, we used the T4e(-) phage, which does not produce the lysozyme responsible for host cell lysis. Infection of E. coli K12 cells with the GFP-labeled T4e(-) phage (T4e(-)/GFP) enabled the visualization and distinction of E. coli K12 cells from T4 phage-insensitive cells, Pseudomonas aeruginosa. Prolonged incubation of E. coli K12 cells with the T4e(-)/GFP phage did not lead to cell lysis. Propagation of T4e(-)/GFP in host cells increased the intensity of green fluorescence, making the distinction of E. coli cells from other cells simple and effective. This method enables the rapid, conclusive quantitation of E. coli cells within an hour.     

Within-host competition determines reproductive success of temperate bacteriophages

Within-host competition between parasites is frequently invoked as a major force for parasite evolution, yet quantitative studies on its extent in an organismal group are lacking. Temperate bacteriophages are diverse and abundant parasites of bacteria, distinguished by their ability to enter a facultative dormant state in their host. Bacteria can accumulate multiple phages that may eventually abandon dormancy in response to host stress. Host resources are then converted into phage particles, whose release requires cell death. To study within-host competition between phages, I used the bacterium Escherichia coli and 11 lambdoid phages to construct single and double lysogens. Lysogenic bacterial cultures were then induced and time to host cell lysis and productivity of phages was measured. In double lysogens, this revealed strong competitive interactions as in all cases productivity of at least one phage declined. The outcome of within-host competition was often asymmetrical, and phages were found to vary hierarchically in within-host competitive ability. In double infections, the phage with the shorter lysis time determined the timing of cell lysis, which was associated with a competitive advantage when time differences were large. The results emphasize that within-host competition greatly affects phage fitness and that multiple infections should be considered an integral part of bacteriophage ecology.     

噬菌体裂解细菌过程中冷光实时监测活菌方法的建立

产毒素的霍乱弧菌Vibrio cholerae可导致严重腹泻,已引起7次全球大流行.对于烈性噬菌体清除霍乱弧菌的效果评价上,一般使用传统的活细胞培养计数及噬菌斑进行观察分析,但操作费时耗力,尤其不能实时获得菌株被裂解及残存细胞的数量变化.进一步探索简便、能够实时监测噬菌体裂解霍乱弧菌的方法是非常必要的.利用荧光报告质粒...     

Phospholipid metabolism in Pseudomonas BAL-31 infected with lipid-containing bacteriophage PM2.

Infection of Pseudomonas BAL-31 with the lipid-containing bacteriophage PM2 resulted in no detectable change in the rate of phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) biosynthesis. An increase in the PG content of infected cultures was not seen until the cultures began to lyse, and this increase was in fact only a relative increase resulting from the extensive turnover of PE at the onset of culture lysis. Turnover studies revealed that the glycerol, phosphorus fatty acid, and ethanolamine moieties of PE turned over simultaneously at the time of lysis, and therefore made it unlikely that there was a PE to PG conversion during the latent period of the phage. The lipid found in the bacteriophage did not reflect a preferential selection for lipid synthesized before or after infection, but in fact reflected the composition of the host membrane at the time the phage were assembled. The use of a modified medium that allowed the cultivation of Pseudomonas BAL-31 as a prototroph and resulted in reliable lysis times of infected cultures led us to the conclusion that PM2 infection effects little change in host phospholipid metabolism, and that there is sufficient PG in the host cytoplasmic membrane to account for a full burst of phage. As a result of the reliable lysis times that we have achieved, we concluded that certain metabolic events, i.e., PE turnover, are lytic phenomena and must not be confused with events relevant to the biosynthesis and maturation of the phage.     

Characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages and the physicochemical analysis of phage adsorption

AIMS: Three indigenous Lactobacillus delbrueckii subsp. bulgaricus bacteriophages and their adsorption process were characterized. METHODS AND RESULTS: Phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed low burst size and short latent periods. A remarkably high sensitivity to pH was also demonstrated. Indigenous phage genomes were linear and double-stranded DNA molecules of approx. 31-34 kbp, with distinctive restriction patterns. Only one phage genome appeared to contain cohesive ends. Calcium ions did not influence phage adsorption, but it was necessary to accelerate cell lysis and improve plaque formation. The adsorption kinetics were similar on viable and nonviable cells, and the adsorption rates were high between 0 and 50 degrees C. SDS and proteinase K treatments did not influence the phage adsorption but mutanolysin and TCA reduced it appreciably. No significant inhibitory effect on phage adsorption was observed for the saccharides tested. This study also revealed the irreversibility of phage adsorption to their hosts. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on phages of thermophilic lactic acid bacteria.