Mathematical Analysis of Growth and Interaction Dynamics of Streptomycetes and a Bacteriophage in Soil

We observed the infection cycle of the temperate actinophage KC301 in relation to the growth of its host Streptomyces lividans TK24 in sterile soil microcosms. Despite a large increase in phage population following germination of host spores, there was no observable impact on host population numbers as measured by direct plate counts. The only change in the host population following infection was the establishment of a small subpopulation of KC301 lysogens. The interaction of S. lividans and KC301 in soil was analyzed with a population-dynamic mathematical model to determine the underlying mechanisms of this low susceptibility to phage attack relative to aquatic environments. This analysis suggests that the soil environment is a highly significant component of the phage-host interaction, an idea consistent with earlier observations on the importance of the environment in determining host growth and phage-host dynamics. Our results demonstrate that the accepted phage-host interaction and host life cycle, as determined from agar plate studies and liquid culture, is sufficient for quantitative agreement with observations in soil, using soil-determined rates. There are four significant effects of the soil environment: (i) newly germinated spores are more susceptible to phage lysis than are hyphae of developed mycelia, (ii) substrate mycelia in mature colonies adsorb about 98% of the total phage protecting susceptible young hyphae from infection, (iii) the burst size of KC301 is large in soil (>150, 90% confidence) relative to that observed in liquid culture (120, standard error of the mean [SEM], 6), and (iv) there is no measurable impact on the host in terms of reduced growth by the phage. We hypothesize that spatial heterogeneity is the principal cause of these effects and is the primary determinant in bacterial escape of phage lysis in soil.     

Survival of the temperate actinophage φC31 and Streptomyces lividans in soil and the effects of competition and selection on lysogens

Abstract Lysogenic infections were demonstrated in microcosms of sterile soil inoculated with Streptomyces lividans and the φC31 derivative, KC301, in free state or via lysogenized hosts. Intermittent soil mixing caused liberation of KC301 due to lysis of germinating lysogenized and uninfected spores. The presence of lysogenized host ensured that KC301 was maintained at a constant density. The lysogen S. lividans TK24 (KC301) achieved a population density lower than that of its non-lysogenized counterpart. Thiostrepton in the soil did not select for the thiostrepton resistance gene KC301. The long-term survival in soil of a temperate actinophage was demonstrated.     

驯化噬菌体提高噬菌体对碳青霉烯类耐药肺炎克雷伯菌的杀菌能力

【目的】本研究旨在通过驯化提高噬菌体的裂解能力并降低其宿主菌耐受性产生的速度,从而提高对重要病原菌-碳青霉烯类耐药肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumoniae, CRKp)的杀菌效果。【方法】以临床CRKp菌株Kp2092为宿主菌,利用双层琼脂平板法从污水中分离噬菌体并分析其裂解谱;对其中的广谱强裂解性噬菌体通过透射电镜观察其形态特征并进行全基因组测序;通过噬菌体-宿主连续培养进行噬菌体驯化,并比较驯化前后噬菌体生物学特性的差异。【结果】分离得到的9株肺炎克雷伯菌噬菌体中,噬菌体P55anc裂解能力强且裂解谱广,透射电镜观察发现其为短尾噬菌体。P55anc基因组全长40 301 bp,包含51个编码序列,其中27个具有已知功能,主要涉及核酸代谢、噬菌体结构蛋白、DNA包装和细胞裂解等。噬菌体P55anc经9 d的驯化后,得到3株驯化噬菌体。驯化后噬菌体杀菌能力增强,主要表现为细菌生长曲线显著下降、噬菌体暴发量增多、裂解谱扩大,且宿主菌对其产生抗性的概率显著降低。与此同时,驯化后的噬菌体在热处理、紫外暴露以及血清等环境下保持较好的稳定性。【结论】利用噬菌体-宿主连续培养的方法可对噬菌体进行驯化和筛选,驯化后的噬菌体杀菌效果更强,且在不同压力处理下的稳定性良好,而细菌产生噬菌体抗性的概率也降低。     

Specific Lysogenicity in Streptomyces azureus

Slope (or plate) cultures of thiostrepton-producing Streptomyces azureus (ATCC 14921) often showed spontaneously developing plaques. Plaques increased in number during serial subcultures. The production of aerial mycelia and sporulating aerial hyphae was interrupted by the overlapping plaques, whereas the growth of substrate mycelia continued in the plaques. These abnormal (eroded) cultures were easily restored to their normal conditions once they were passed through liquid cultures under shaking conditions. A few phage particles were found in the plaques, together with some headless tails and numerous tail tips which formed a hexagonal crystal or a large crystal mass when viewed in an electron microscope. No lytic phenomenon and no phage production were found in the liquid cultures, although all mycelia and spores harbored phage-producing abilities. It was also found that the propagation of phages was successful in solid culture, but not in liquid culture. The whole phage was named SAt2, which belongs to group B of Bradley's morphological classification. From these results, it is considered that S. azureus is lysogenic with temperate phage SAt2, of which virulent mutants are able to infect the aerial mycelia and sporulating hyphae of their lysogenic host.     

Epifluorescence and atomic force microscopy: Two innovative applications for studying phage-host interactions in Lactobacillus helveticus

Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥ 6 log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5 h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions.     

Large variabilities in host strain susceptibility and phage host range govern interactions between lytic marine phages and their Flavobacterium hosts

Phages are a main mortality factor for marine bacterioplankton and are thought to regulate bacterial community composition through host-specific infection and lysis. In the present study we demonstrate for a marine phage-host assemblage that interactions are complex and that specificity and efficiency of infection and lysis are highly variable among phages infectious to strains of the same bacterial species. Twenty-three Bacteroidetes strains and 46 phages from Swedish and Danish coastal waters were analyzed. Based on genotypic and phenotypic analyses, 21 of the isolates could be considered strains of Cellulophaga baltica (Flavobacteriaceae). Nevertheless, all bacterial strains showed unique phage susceptibility patterns and differed by up to 6 orders of magnitude in sensitivity to the same titer of phage. The isolated phages showed pronounced variations in genome size (8 to >242 kb) and host range (infecting 1 to 20 bacterial strains). Our data indicate that marine bacterioplankton are susceptible to multiple co-occurring phages and that sensitivity towards phage infection is strain specific and exists as a continuum between highly sensitive and resistant, implying an extremely complex web of phage-host interactions. Hence, effects of phages on bacterioplankton community composition and dynamics may go undetected in studies where strain identity is not resolvable, i.e., in studies based on the phylogenetic resolution provided by 16S rRNA gene or internal transcribed spacer sequences.     

Characterization of Streptococcus thermophilus host range phage mutants

To investigate phage-host interactions in Streptococcus thermophilus, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn917 library of phage-sensitive strain S. thermophilus SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10(-6). Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (orf15, orf17, and orf18) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in S. thermophilus.     

Dynamics of Mycobacteriophage-Mycobacterial Host Interaction: Evidence for Secondary Mechanisms for Host Lethality

Mycobacteriophages infect mycobacteria, resulting in their death. Therefore, the possibility of using them as therapeutic agents against the deadly mycobacterial disease tuberculosis (TB) is of great interest. To obtain better insight into the dynamics of mycobacterial inactivation by mycobacteriophages, this study was initiated using mycobacteriophage D29 and Mycobacterium smegmatis as the phage-host system. Here, we implemented a goal-oriented iterative cycle of experiments on one hand and mathematical modeling combined with Monte Carlo simulations on the other. This integrative approach lends valuable insight into the detailed kinetics of bacterium-phage interactions. We measured time-dependent changes in host viability during the growth of phage D29 in M. smegmatis at different multiplicities of infection (MOI). The predictions emerging out of theoretical analyses were further examined using biochemical and cell biological assays. In a phage-host interaction system where multiple rounds of infection are allowed to take place, cell counts drop more rapidly than expected if cell lysis is considered the only mechanism for cell death. The phenomenon could be explained by considering a secondary factor for cell death in addition to lysis. Further investigations reveal that phage infection leads to the increased production of superoxide radicals, which appears to be the secondary factor. Therefore, mycobacteriophage D29 can function as an effective antimycobacterial agent, the killing potential of which may be amplified through secondary mechanisms.     

Large Variabilities in Host Strain Susceptibility and Phage Host Range Govern Interactions between Lytic Marine Phages and Their Flavobacterium Hosts

Phages are a main mortality factor for marine bacterioplankton and are thought to regulate bacterial community composition through host-specific infection and lysis. In the present study we demonstrate for a marine phage-host assemblage that interactions are complex and that specificity and efficiency of infection and lysis are highly variable among phages infectious to strains of the same bacterial species. Twenty-three Bacteroidetes strains and 46 phages from Swedish and Danish coastal waters were analyzed. Based on genotypic and phenotypic analyses, 21 of the isolates could be considered strains of Cellulophaga baltica (Flavobacteriaceae). Nevertheless, all bacterial strains showed unique phage susceptibility patterns and differed by up to 6 orders of magnitude in sensitivity to the same titer of phage. The isolated phages showed pronounced variations in genome size (8 to >242 kb) and host range (infecting 1 to 20 bacterial strains). Our data indicate that marine bacterioplankton are susceptible to multiple co-occurring phages and that sensitivity towards phage infection is strain specific and exists as a continuum between highly sensitive and resistant, implying an extremely complex web of phage-host interactions. Hence, effects of phages on bacterioplankton community composition and dynamics may go undetected in studies where strain identity is not resolvable, i.e., in studies based on the phylogenetic resolution provided by 16S rRNA gene or internal transcribed spacer sequences.     

The effect of Streptomyces species on the survival of Salmonella in soil

Abstract A modified Chelex 100 ion-exchange extraction method was used to monitor streptomycete spores, streptomycete mycelia and Salmonella in soil. Salmonella dusseldorf in soil was inhibited by the bactericidal effect of streptomycin and by the growth of Streptomyces bikiniensis . The soil used in the experiments exerted an antimicrobial effect on S. dusseldorf .
Competition between S. dusseldorf, Streptomyces lividans TK24 and Stm. bikiniensis ATCC 11062 was monitored in soil. In sterile amended soil Stm. lividans increased the survival of S. dusseldorf , whereas survival was reduced in the presence of the known streptomycin producer, Stm. bikiniensis . In the presence of S. dusseldorf the production of spores and mycelia by Stm. bikiniensis was reduced, and Stm. lividans sporulation was reduced but mycelia production increased. Evidence was seen for a beneficial effect between S. dusseldorf and Stm. lividans mycelia.
In non-sterile unamended soil the survival of S. dusseldorf was increased in the presence of Stm. lividans , whereas Stm. bikiniensis had no effect. Stm. lividans and Stm. bikiniensis reduced the survival of S. dusseldorf in non-sterile amended soil, with the most dramatic reduction caused by Stm. bikiniensis . No such changes in the survival of S. dusseldorf were observed with non-sterile amended soil that had been treated with sludge. The presence of sludge in unamended soil increased the rate of Salmonella die-off. In unamended soil containing sludge the presence of Stm. lividans increased the survival of S. dusseldorf , whereas survival was reduced in the presence of Stm. bikiniensis . The data provided evidence of antibiosis in soil, relating to the possible production of streptomycin by Stm. bikiniensis .     

Elevated Lytic Phage Production as a Consequence of Particle Colonization by a Marine <Emphasis Type="Italic">Flavobacterium</Emphasis> (<Emphasis Type="Italic">Cellulophaga</Emphasis> sp.)

Bacteria growing on marine particles generally have higher densities and cell-specific activities than free-living bacteria. Since rapidity of phage adsorption is dependent on host density, while infection productivity is a function of host physiological status, we hypothesized that marine particles are sites of elevated phage production. In the present study, organic-matter-rich agarose beads and a marine phage-host pair (Cellulophaga sp., PhiS(M)) were used as a model system to examine whether bacterial colonization of particles increases phage production. While no production of phages was observed in plain seawater, the presence of beads enhanced attachment and growth of bacteria, as well as phage production. This was observed because of extensive lysis of bacteria in the presence of beads and a subsequent increase in phage abundance both on beads and in the surrounding water. After 12 h, extensive phage lysis reduced the density of attached bacteria; however, after 32 h, bacterial abundance increased again. Reexposure to phages and analyses of bacterial isolates suggested that this regrowth on particles was by phage-resistant clones. The present demonstration of elevated lytic phage production associated with model particles illustrates not only that a marine phage has the ability to successfully infect and lyse surface-attached bacteria but also that acquisition of resistance may affect temporal phage-host dynamics on particles. These findings from a model system may have relevance to the distribution of phage production in environments rich in particulate matter (e.g., in coastal areas or during phytoplankton blooms) where a significant part of phage production may be directly linked to these nutrient-rich "hot spots."     

Mycoparasitism of Acremonium strictum BCP on Botrytis cinerea, the gray mold pathogen

A fungal strain BCP, which parasitizes Botrytis cinerea gray mold pathogen, was isolated and identified as Acremonium strictum. BCP strain overgrew the colonies of B. cinerea and caused severe lysis of the host hyphae. Frequent penetration and hyphal growth of A. strictum BCP inside the mycelia of B. cinerea were observed under light microscopy. In addition, some morphological abnormalities such as granulation and vacuolation of the cytoplasm were observed in mycelia and spores of B. cinerea. In dual culture test, A. strictum BCP strongly inhibited the mycelial growth of several plant pathogenic fungi as well as B. cinerea. To our knowledge, this is the first report on mycoparasitism of Acremonium species on B. cinerea.     

Characterization of Streptococcus thermophilus Host Range Phage Mutants

To investigate phage-host interactions in Streptococcus thermophilus, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn917 library of phage-sensitive strain S. thermophilus SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10⁻⁶. Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (orf15, orf17, and orf18) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in S. thermophilus.     

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.     

铜绿假单胞菌噬菌体K5中DNA聚合酶等基因在宿主中的表达

本研究分析了铜绿假单胞菌噬菌体K5基因在宿主中的表达及其影响因素. 通过测定融合报告基因dnaP-lacZ、capP-lacZ、bapP-lacZ和rdr-lacZ编码的β 半乳糖苷酶活力,分析了噬菌体K5相关基因的表达水平,发现噬菌体K5的不同基因在宿主细胞内表达水平存在较大差异,其中噬菌体K5的DNA聚合酶基因dnaP的表达水平最高,而主要衣壳蛋白基因capP的表达水平最低. 加入噬菌体后,除二磷酸核糖核苷酸还原酶基因rnr外,其它基因的表达水平均有明显提高,说明噬菌体自身因子能够调控噬菌体部分基因在宿主细胞中的表达. 进一步分析显示,噬菌体基因在对数生长前期细胞中的表达水平显著高于平衡期. 同时,噬菌体感染对数生长前期的宿主菌,其释放量为12.8 PFU/感染中心,是平衡期释放量的9.2倍. 噬菌体以对数生长期宿主为指示菌时噬菌体的滴度为4.7×108 PFU/mL,而以平衡期宿主菌为指示菌噬菌体K5滴度仅能达到2.5×104 PFU/mL,噬菌体K5的裂解能力显著降低. 这些结果对研究噬菌体与宿主细胞的相互作用机制具有重要作用.     

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 lambda-strains with different combinations of six different lysis times (ranging from 29.3 to 68 min), two adsorption rates (9.9 x 10(-9) and 1.3 x 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.     

Vibriophages Differentially Influence Biofilm Formation by Vibrio anguillarum Strains

Vibrio anguillarum is an important pathogen in marine aquaculture, responsible for vibriosis. Bacteriophages can potentially be used to control bacterial pathogens; however, successful application of phages requires a detailed understanding of phage-host interactions under both free-living and surface-associated growth conditions. In this study, we explored in vitro phage-host interactions in two different strains of V. anguillarum (BA35 and PF430-3) during growth in microcolonies, biofilms, and free-living cells. Two vibriophages, ΦH20 (Siphoviridae) and KVP40 (Myoviridae), had completely different effects on the biofilm development. Addition of phage ΦH20 to strain BA35 showed efficient control of biofilm formation and density of free-living cells. The interactions between BA35 and ΦH20 were thus characterized by a strong phage control of the phage-sensitive population and subsequent selection for phage-resistant mutants. Addition of phage KVP40 to strain PF430-3 resulted in increased biofilm development, especially during the early stage. Subsequent experiments in liquid cultures showed that addition of phage KVP40 stimulated the aggregation of host cells, which protected the cells against phage infection. By the formation of biofilms, strain PF430-3 created spatial refuges that protected the host from phage infection and allowed coexistence between phage-sensitive cells and lytic phage KVP40. Together, the results demonstrate highly variable phage protection mechanisms in two closely related V. anguillarum strains, thus emphasizing the challenges of using phages to control vibriosis in aquaculture and adding to the complex roles of phages as drivers of prokaryotic diversity and population dynamics.     

Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis.

During the first 6 hr of sporulation, infection of Bacillus subtilis by by phi105 wild type or the clear-plaque mutant phi105 c30 was nonproductive, but phage DNA was trapped inside developing spores. After infection with either wild-type or mutant phage at early times of sporulation (T1-T3), phage DNA entered the developing spores in a heat-stable form, which may represent integration of the phage DNA into the host chromosome. Phage DNA in carrier spores produced by infection at later times (T4-T6) was much more heat sensitive. Spore preparations containing either phi105 wild type or phi105 c30 carrier spores gave rise to a spontaneous burst of phage during outgrowth, although the fraction of carried wild-type phage that chose lysis over lysogeny at germination has not been determined. Heat induction of the thermoinducible lysogen 3610 (phi105 cts23) was also abortive during sporulation. Furthermore, induction neither prevented eventual spore formation nor resulted in the conversion of prophage DNA to the carrier state; during outgrowth, the previously induced lysogenic spores remained stable lysogens. However, if the sporulating lysogenic cells were plated immediately after induction, they did not form colonies at high efficiency, as though transfer to fresh medium allowed sufficient phage expression to kill the host.     

Characterization of bacteriophage phi C69 of Saccharopolyspora erythraea and demonstration of heterologous actinophage propagation by transfection of Streptomyces and Saccharopolyspora

A bacteriophage, designated phi C69, isolated from a culture of Saccharopolyspora erythraea was characterized. The phage propagates on Sac. erythraea NRRL 2338 but does not infect 10 Streptomyces or 3 Micromonospora species tested. It infects Sac. erythraea NRRL 2359 but does not produce infectious phage particles in this host. phi C69 is approximately 40 kb in length and contains cohesive ends. A cos fragment containing ligated phage DNA ends was cloned in Escherichia coli. Restriction maps of the phage DNA and the cos fragment for several enzymes are shown. Transfection of both Sac. erythraea and Streptomyces lividans with phi C69 resulted in approximately equal titres of infectious phage particles produced from approximately the same number of regenerating cells. Transfection of Sac. erythraea with DNA from Streptomyces phages SH10 and KC404 also resulted in the production of infectious phage particles. The basis for differences among hosts in susceptibility to infection by various actinophages is discussed.     

松口蘑人工菌塘诱导形成的初步研究

本研究通过土壤筛选找到适合松口蘑菌丝体生长的土壤,并在其土壤中添加适当浓度的橄榄油(0.5%-2.0%),有效地促进了菌丝体在土壤中的生长,菌丝生长状况酷似野外的松口蘑菌塘,且随着添加浓度的增加,菌丝体生物量呈明显的正相关。当土壤中橄榄油浓度为0.5%和1.0%时,松口蘑共生植物赤松苗的生长不但没有受到抑制,而且还能与菌丝体在这种“菌塘”中形成菌根。本研究成功地建立了中国松口蘑人工菌塘诱导体系,同时也为其人工子实体的诱导研究提供了必要的前提条件。