Alternative stable states in host–phage dynamics

Bacteriophage are ubiquitous in nature, yet many central aspects of host–phage biology have not been integrated into mathematical models. We propose a novel model of host–phage population dynamics that accounts for the decreased ability of phages to lyse hosts as hosts approach their carrying capacity. In contrast to existing predator–prey-like models, we find a parameter regime in which phages cannot invade a host-only system but, nonetheless, can stably coexist with hosts at lower densities. The finding of alternative stable states suggests clear linkages with observed life history strategies of phages. In addition, we solve a limiting case of the proposed model and show that conservative predator-prey like systems do not inevitably exhibit population cycles. Finally, we discuss possible extensions of the present model and scenarios for experimental testing.     

Regulation of oxidative DNA damage repair: The adenine:8-oxo-guanine problem

Reactive oxygen species (ROS) constantly attack DNA. One of the best-characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). Many human diseases, such as cancer and neurodegenerative disorders, have been correlated with oxidative DNA damage. In the last few years, DNA polymerase (Pol) λ, one of the 15 cellular Pols, has been identified to play an important role in performing accurate translesion synthesis over 8-oxo-G. This is eminently important, since normally faithful replicative Pols α, δ and ε, with their tight active center, often wrongly incorporate adenine (A) opposite the 8-oxo-G lesion. A:8- oxo-G mispairs are accurately repaired by the pathway identified in our laboratory involving MutY DNA glycosylase homolog (MutYH) and Pol λ. Until now, very little was known about the spatial and temporal regulation of Pol λ and MutYH in active repair complexes. We now showed in our latest publication that the E3 ligase Mule can ubiquitinate and degrade Pol λ, and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. In contrast, phosphorylation of Pol λ by Cdk2/cyclinA counteracts this degradation by recruiting it to MutYH on chromatin to form active 8-oxo-G repair complexes.     

Rapid label-free detection of E. coli using a novel SPR biosensor containing a fragment of tail protein from phage lambda

Abstract

In efforts to speed up the assessment of microorganisms, researchers have sought to use bacteriophages as a biosensing tool, due to their host-specificity, wide abundance, and safety. However, the lytic cycle of the phage has limited its efficacy as a biosensor. Here, we cloned a fragment of tail protein J from phage lambda and characterized its binding with the host, E. coli K-12, and other microorganism. The N-terminus of J was fused with a His-tag (6HN-J), overexpressed, purified, and characterized using anti-His monoclonal antibodies. The purified protein demonstrated a size of ～38?kDa upon SDS-PAGE and bound with the anti-His monoclonal antibodies. ELISA, dot blot, and TEM data revealed that it specifically bound to E. coli K-12, but not to Pseudomonas aeruginosa. The observed protein binding occurred over a concentration range of 0.01–5?μg/ml and was found to inhibit the in vivo adsorption of phage to host cells. This specific binding was exploited by surface plasmon resonance (SPR) to generate a novel 6HN-J-functionalized SPR biosensor. This biosensor showed rapid label-free detection of E. coli K-12 in the range of 2?×?10⁴ ?2?×?10⁹ CFU/ml, and exhibited a lower detection limit of 2?×?10⁴ CFU/ml.     

噬菌体Qβ病毒样颗粒的制备、纯化及鉴定

目的: 利用大肠杆菌原核表达系统制备噬菌体Qβ VLPs,验证其自组装能力,纯化后免疫动物以确定其免疫原性,同时制备兔多克隆抗体验证其在哺乳动物细胞中内化能力。方法: 合成pET-28-Qβ-CP质粒,利用大肠杆菌原核表达系统制备Qβ VLPs,通过蔗糖密度梯度离心纯化VLPs,将经凝胶层析柱Sephacryl S-400纯化的Qβ VLPs用透射电镜观察颗粒形态;纯化后的Qβ VLPs加入或不加入佐剂分别免疫新西兰兔,获得血清后用Protein G纯化得到兔多克隆抗体,通过Western blot确定制备的兔多克隆抗体特异性,并利用间接免疫荧光法对Qβ VLPs的细胞内化能力进行鉴定。结果: 制备并获得纯度较高的Qβ VLPs,通过透射电镜观察到大量直径约为28 nm的颗粒;Western blot结果表明制备的兔多克隆抗体能特异性识别Qβ VLPs,且在免疫实验中加入佐剂与不加入佐剂分别免疫动物,对抗体水平的影响不显著。间接免疫荧光法结果表明Qβ VLPs在哺乳动物细胞中具有内化能力。结论: 成功制备Qβ VLPs为后续研发以噬菌体Qβ VLPs为载体的相关疫苗奠定基础。     

Effects of Hydration on Steric and Electric Charge-Induced Interstitial Volume Exclusion—a Model

The presence of collagen and charged macromolecules like glycosaminoglycans (GAGs) in the interstitial space limits the space available for plasma proteins and other macromolecules. This phenomenon, known as interstitial exclusion, is of importance for interstitial fluid volume regulation. Physical/mathematical models are presented for calculating the exclusion of electrically charged and neutral macromolecules that equilibrate in the interstitium under various degrees of hydration. Here, a central hypothesis is that the swelling of highly electrically charged GAGs with increased hydration shields parts of the neutral collagen of the interstitial matrix from interacting with electrically charged macromolecules, such that exclusion of charged macromolecules exhibits change due to steric and charge effects. GAGs are also thought to allow relatively small neutral, but also charged macromolecules neutralized by a very high ionic strength, diffuse into the interior of GAGs, whereas larger macromolecules may not. Thus, in the model, relatively small electrically charged macromolecules, such as human serum albumin, and larger neutral macromolecules such as IgG, will have quite similar total volume exclusion properties in the interstitium. Our results are in agreement with ex vivo and in vivo experiments, and suggest that the charge of GAGs or macromolecular drugs may be targeted to increase the tissue uptake of macromolecular therapeutic agents.     

FLUCTUATING TEMPERATURE LEADS TO EVOLUTION OF THERMAL GENERALISM AND PREADAPTATION TO NOVEL ENVIRONMENTS

Environmental fluctuations can select for generalism, which is also hypothesized to increase organisms’ ability to invade novel environments. Here, we show that across a range of temperatures, opportunistic bacterial pathogen Serratia marcescens that evolved in fluctuating temperature (daily variation between 24°C and 38°C, mean 31°C) outperforms the strains that evolved in constant temperature (31°C). The growth advantage was also evident in novel environments in the presence of parasitic viruses and predatory protozoans, but less clear in the presence of stressful chemicals. Adaptation to fluctuating temperature also led to reduced virulence in Drosophila melanogaster host, which suggests that generalism can still be costly in terms of reduced fitness in other ecological contexts. While supporting the hypothesis that evolution of generalism is coupled with tolerance to several novel environments, our results also suggest that thermal fluctuations driven by the climate change could affect both species’ invasiveness and virulence.     

Identification of essential loops and residues of glucosyltransferase V (GtrV) of Shigella flexneri

Lipopolysaccharide (LPS), particularly the O-antigen component, is one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen modification is mediated by glucosyltransferase (gtr) genes encoded by temperate serotype-converting bacteriophages. The gtrV and gtrX genes encode the GtrV and GtrX glucosyltransferases, respectively. These are integral membrane proteins, which catalyze the transfer of a glucosyl residue via an α1,3 linkage to rhamnose II and rhamnose I of the O-antigen unit. This mediates conversion of S. flexneri serotype Y to serotype 5a and X, respectively. Essential regions in the topology of GtrV protein were identified by in vivo recombination and a PCR-mediated approach. A series of GtrX-GtrV and GtrV-GtrX chimeric proteins were constructed based on the fact that GtrV and GtrX share sequence similarity. Analysis of their respective serotype conversion abilities led to the identification of two important periplasmic loops: loops No 2 and No 10 located in the N- and C-termini, respectively. Within these two loops, three conserved motifs were identified; two in loop No 2 and one in loop No 10. These conserved motifs contain acidic residues which were shown to be critical for GtrV function.     

A reliable method for storage of tailed phages

A universal and effective method for long-term storage of bacteriophages has not yet been described. We show that randomly selected tailed phages could be stored inside the infected cells at −80 °C without a major loss of phage and host viability. Our results suggest the suitability of this method as a standard for phage preservation.