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ijms International Journal of Molecular Sciences Int. J. Mol. Sci. 1422-0067 Molecular Diversity Preservation International (MDPI) 10.3390/ijms130911832 ijms-13-11832 Article Studies on the Interactions of Copper and Zinc Ions with β-Amyloid Peptides by a Surface Plasmon Resonance Biosensor YaoFujun1 ZhangRuiping2* TianHe2 LiXiangjun1* College of Chemistry and Chemical Engineering, Graduate University, Chinese Academy of Sciences, Beijing, 100049, China; E-Mail: yaofuj09b@mails.gucas.ac.cn Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China; E-Mail: tianhe@imm.ac.cn Authors to whom correspondence should be addressed; E-Mails: rpzhang@imm.ac.cn (R.Z.); lixiangj@gucas.ac.cn (X.L.); Tel.: +86-10-6316-5218 (R.Z.); +86-10-8825-6336 (X.L.); Fax: +86-10-6316-5218 (R.Z.); +86-10-8825-6093 (X.L.). 2012 19 09 2012 13 9 11832 11843 18 07 2012 07 09 2012 07 09 2012 © 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. 2012

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

The aggregation of β-amyloid peptide (Aβ) into fibrils plays an important role in the pathogenesis of Alzheimer’s disease (AD). Metal ions including copper and zinc are closely connected to the precipitation and toxicity of Aβ. In this study, a surface plasmon resonance (SPR) biosensor was constructed to investigate the interactions between Aβ and metal ions. Aβ peptide was immobilized on the SPR chip surface through a preformed alkanethiol self-assembled monolayer (SAM). Our observations indicate that the immobilized Aβ undergoes a conformational change upon exposure to the metal ions. A difference in metal binding affinity between Aβ1–28 and Aβ1–42 was also detected. The results suggest that SPR is an effective method to characterize the interactions between Aβ and metal ions.

Alzheimer’s disease β-amyloid peptide surface plasmon resonance metal ions
1. Introduction

Alzheimer’s disease (AD), which is characterized by irreversible and progressive neurodegeneration, is the dominant cause of dementia. The morphological hallmarks of AD are extracellular senile plaques and intracellular neurofibrillary tangles. The aggregated β-amyloid peptide (Aβ) containing 39–43 amino acid residues, which is generated from the amyloid precursor protein (APP) through the sequential cleavage by two enzymes, β-secretase and γ-secretase, is the principal component of the senile plaques. According to the amyloid cascade hypothesis, the aggregation of Aβ in AD leads to the formation of neurotoxic oligomers that are purportedly responsible for neuronal dysfunction and cell death [1]. Therefore, conditions that influence aggregation and the formation of oligomers are of great interest.

The aggregation of Aβ peptide is primarily affected by pH [2,3], peptide concentration [2], incubation time, membrane lipids [4] and temperature [5]. Moreover, a large body of evidence suggests that metal ions such as copper, zinc and iron may induce the aggregation of Aβ peptides, and these ligands may act as seeding factors in the formation of amyloid plaques [610]. In fact, elevated levels of zinc and copper have been found in amyloid plaques at concentrations reaching 1 mM and 400 μM, respectively [11]. These metal ions bind at the N-terminus (amino acids 1–16) and influence aggregation behavior. However, experimental evidence has shown that these metal ions in complex with Aβ peptides may have opposite functions with Zn accelerating aggregation, while Cu can reduce or accelerate aggregation [1214]. Furthermore, it has been proposed that copper and iron mediate the production of reactive oxygen species (ROS) and oxidative stress [15,16]. It has been reported that reactive oxygen species (ROS) such as H2O2 are produced during the association of Aβ with Cu2+ through the reduction of Cu2+ to Cu+, which mediates cell toxicity [17]. Therefore, intensive efforts have been made to study the primary interaction of Aβ peptide with metal ions.

Surface Plasmon resonance (SPR) spectroscopy is capable of detecting a mass or conformational change above a metal surface. At a specific angle, the absorption of incident light by a thin metal film causes a collective oscillation of electrons in the film that launches an evanescent wave into the dielectric layer adjacent to the metal film. The propagation of the evanescent wave decays exponentially away from the metal film and is thus significantly perturbed by the adsorption of a species on the metal film or changes in the adlayer structure. Due to the advantages of real-time, label-free and direct detection of molecules in various media, SPR has become a popular and powerful technique to study molecular interactions. In recent years, SPR has been successfully employed to study biomolecular interactions related to AD [18,19]. Various aspects of Aβ oligomerization, fibril formation and extension and Aβ interactions with biomolecules have been investigated by SPR [20,21]. A custom-built flow-injection (FI) SPR instrument equipped with a bicell detector was constructed in our lab and used to investigate the interactions between Aβ1–16 and metal ions [22]. Immobilization of monomeric Aβ was performed using a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) and amino coupling chemistry. To further test the feasibility of this biosensor, the interactions between metal ions and Aβ1–28 or the actual senile plaque component, Aβ1–42, were investigated.

2. Results and Discussion 2.1. Aβ Immobilization

The successful immobilization of the Aβ peptide on the sensor chip surface was tested by injecting a Zn2+ ion solution into the flow chamber and pumping it across the Aβ-immobilized sensing surface. In our experiment, the sample loop volume is 20 μL, the flowing rate is 10 μL/min, so that the Zn2+ ions start to flow onto the immobilized Aβ1–28 at about 80 s, the flowing of metal ions ends at about 200 s; the difference in the baseline SPR angles before and after the Zn2+ ion solution injection (termed as the SPR dip shift Δθ) is approximately 0.00246° (Figure 1a), considering the sophisticated interaction between Aβ peptide and metal ions, the detection of the SPR dip shift Δθ is selected at 550 s.

To confirm that the net change is induced by the metal ions, we conducted a control experiment in which Zn2+ ions flowed over an ethanolamine-blocked surface without the immobilization of Aβ peptide. Figure 1b shows that the SPR signal returned to its baseline within a short time. Additionally, the recovery of the original baseline in curve b suggests that the blocking process is efficient because it has been reported that alkanethiol SAM containing carboxylic acid can be used for SPR measurements of heavy metal ions [23]. Furthermore, the conversion of the negatively charged carboxyl groups on the MUA SAM to neutral amide groups during the peptide immobilization and blocking process could reduce the undesirable electrostatic attraction to metal ions in solution.

2.2. Real-Time Determination of Aβ1–28 Binding Zn2+ and Cu2+ by SPR

Figure 2 shows the sensorgrams of various Zn2+ concentrations flowing over the Aβ1–28 sensor chip. In these experiments, EDTA (10 mM) was used to regenerate the surface. Therefore, a single chip can be used repeatedly for multiple samples. As shown in Figure 2, increasing Zn2+ concentrations results in a greater SPR dip shift. When 600 μM Zn2+ flowed over the Aβ1–28-covered SPR sensor, the metal ions caused a net change of 0.0075°, which can be attributed to the binding of Zn2+ ions to Aβ1–28 resulting in conformational changes of the peptide molecules.

The SPR angle shift is directly correlated with the Zn2+ concentration ([Zn2+]). In Figure 3, the calibration curve generated from the sensor response to a series of different Zn2+ concentrations is shown. The calibration curve contains two regions; the first is from 50 to 300 μM, and the second from 600 to 800 μM. Linear regression analysis of both regions yielded the following equations:

Δ θ = - 3.07 × 10 - 4 + 1.39 × 10 - 5 [ Zn 2 + ] R 2 = 0.95 ( region  1 ) Δ θ = - 1.39 × 10 - 2 + 3.59 × 10 - 5 [ Zn 2 + ] R 2 = 0.99 ( region  2 )

The slope of the linear regression in the second region is higher than that in the first region, which reveals that the SPR angle shift elicited by Zn2+ ions is greater with increasing Zn2+ ion concentration. These results suggest that the Zn2+-induced Aβ1-28 conformation change is concentration dependent. This finding is in agreement with a previous report indicating that Aβ undergoes a conformational change from a random coil to a regular secondary structure in the presence of Zn2+ ions and forms stable 1:1 and 1:2 (peptide/zinc) complexes [8]. A possible explanation for this phenomenon is that the Aβ1–28 binds Zn2+ intramolecularly at low concentrations. When the Zn2+ ion concentration is sufficiently high, the excess Zn2+ ions may begin to bind with Aβ1–28 intermolecularly, and the Zn2+ ions behave like a bridge connecting two adjacent Aβ1–28 molecules. Our finding is supported by previous studies that have shown that Zn2+ can coordinate to Aβ in an intra- and inter-peptide mode [2426].

A similar trend was not found for the interactions between Cu2+ and Aβ1–28. The calibration curve depicted in Figure 4 only contains one linear region ranging from 100 to 600 μM. The linear regression equation with a correlation coefficient of R2 = 0.99 suggests a linear relationship between the SPR angle shift and the Cu2+ concentration.

Δ θ = - 4.67 × 10 - 5 + 1.49 × 10 - 6 [ Cu 2 + ] R 2 = 0.99

Compared with Zn2+-Aβ complexes having stoichiometry ranging from 1:1 to 3:1 [2729], most studies have demonstrated that the Aβ-peptide forms a 1:1 complex with Cu2+ [25,30,31]. NMR studies have demonstrated that Aβ1–28 forms a 1:1 complex with the Cu2+ ion via histidine residues [30]. We interpret these results to different coordination modes. Unlike Zn2+, which can bind to Aβ in an intra- and inter-peptide coordination mode, Cu2+ is primarily involved in intra-peptide binding in a fairly closed structure that protects the metal from further interactions. In addition, it has been reported that under physiological conditions, the coordination of the Cu2+ equivalent to the Aβ peptides leads to a mononuclear complex Cu1(Aβ)1 and is unlikely to form a Cu2(Aβ)1 complex [32].

2.3. Real-Time Determination of Aβ1–42 Binding to Zn2+ and Cu2+ by SPR

1–40 and Aβ1–42 are the most prevalent in vivo Aβ forms. In particular, Aβ1–42 has a high propensity to self-assemble and deposit in senile plaques and is highly toxic to neurons [33]. While great progress has been achieved on the coordination chemistry of Zn2+ and Cu2+ with truncated Aβ1–16 and Aβ1–28, it is important to understand whether these peptides, which are missing a large part of the hydrophobic C-terminal residues, coordinate metal ions differently than the full-length peptides. Therefore, to better understand the mechanisms of AD, we continued to study the interactions of Zn2+ and Cu2+ with Aβ1–42 using the FI-SPR biosensor.

Figure 5 depicts SPR sensorgrams obtained from the sensor response to various Zn2+ concentrations. In contrast to the calibration curve of the Zn2+ interactions with Aβ1–28 that contains two regions, the calibration plot of the Zn interactions with Aβ1–42 only contains one region; the linear regression equation is as follows:

Δ θ = - 1.86 × 10 - 3 + 2.27 × 10 - 5 [ Zn 2 + ] R 2 = 0.96

1–42 peptides have 14 additional residues in the hydrophobic tail compared to Aβ1–28, which makes Aβ1–42 more prone to aggregation than the truncated Aβ1–28. A possible explanation for the decreased stoichiometric binding of Aβ1–42 compared to Aβ1–28 may be the interference of the hydrophobic tail in Aβ1–42 with the Zn2+ binding site. Our data are in agreement with a previous report indicating that Zn and Cu form a monomeric complex with Aβ1–42 [34].

Similar results were obtained for the Cu-Aβ1–42 interaction. As shown in Figure 6, the SPR angle shift increased with an increase of Cu2+ bound to the Aβ1–42-immobilized sensor chip. The calibration curve generated from the sensor exposed to different concentrations of Cu2+ is shown in the Figure 6 inset. A strong correlation coefficient (R2 = 0.99) was obtained for the linear regression equation calculated using Cu2+ concentrations ranging from 50 to 400 μM.

3. Experimental Section 3.1. Chemicals

Human amyloid-β peptide (1–28) (Aβ1–28) and human amyloid-β peptide (1–42) (Aβ1–42) were obtained from GL Biochem Ltd. (Shanghai, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), 11-mercaptoundecanoic acid (MUA), ARC-grade dimethyl sulfoxide (DMSO, 99%) and ethanolamine were purchased from J & K Chemical Ltd. K2HPO4·3H2O, KH2PO4, NaCl, ZnCl2 and CuCl2 were all of AR grade and purchased from Beijing Chemical Reagent Co. (Beijing, China). Water with a resistivity of 18.25 MΩ·cm−1 was collected from a Millipore Simplicity 185 system. EDC (0.4 M) and NHS (0.1 M) were prepared in a pH 7.4 PBS buffer (10.0 mM K2HPO4·3H2O and KH2PO4 prepared in 1 mM NaCl). MUA (4 mM) was prepared in pure ethanol. Stock solutions of 2.0 mM ZnCl2 and CuCl2 were prepared in water and then diluted to the desired concentrations.

3.2. Preparation of Fresh Aβ Solution

Uniform and nonaggregated monomeric Aβ1–28 and Aβ1–42 peptides were prepared as previously described [35]. Briefly, 0.1 mM of the peptide was dissolved in DMSO, which disrupts the β-sheet structure and renders Aβ monomeric [36]. The freshly dissolved monomeric Aβ was diluted with PBS buffer (pH 7.4) to a concentration of 10 μM.

3.3. Gold Film Preparation

BK7 glass cover slides (Fisher) were cleaned with a piranha solution of 70% concentrated H2SO4 and 30% H2O2 (7:3, v/v) at 80 °C for 30 min. Upon cooling to room temperature, the glass slides were rinsed thoroughly with deionized water. After drying with N2, each glass slide was coated with a 2 nm chromium layer and then covered with 50 nm of gold film using a sputter coater (Model 108, Kert J. Lester Inc., Clariton, PA, USA).

3.4. Aβ Peptide Immobilization

Aβ peptide immobilization was achieved using covalent bonding mediated by a chemical reaction. The N-terminus of the Aβ peptide was reacted with the functional group of the SAM of MUA on the surface of the Au film (as depicted in Figure 7). Briefly, the MUA gold chip was treated with a mixture of 0.4 M EDC and 0.1 M NHS (1:1) for 3 h to ensure that the carboxyl group of the SAM reacted fully with the EDC and NHS. Then, a freshly prepared 10 μM Aβ solution in PBS (pH 7.4) was reacted with the NHS-activated surface for 2 h. Finally, ethanolamine (1 M, pH 8.5) was used to block the remaining activated surface groups. The resulting film was either used immediately or stored at 4 °C for future use.

3.5. SPR Apparatus

SPR measurements were conducted with a custom-built flow injection-SPR equipped with a bicell detector as previously described [22]. The SPR instrument recorded the reflected light on two photodetectors (A and B). The differential (A − B) and sum (A + B) signals were then detected by a PCI-1371 interface card (Advantech, Taiwan) controlled by the Labview program. The resonance angles from the biosensor were measured by the division of the differential and sum signals, (A − B)/(A + B). The inlet of the flow cell was connected to a six-port valve. For each measurement, the sample solution was injected into a 20-μL loop with a microsyringe (Hamilton) and subsequently delivered to the flow cell at a flow rate of 10 μL/min, using a syringe pump (KDS100, KD Scientific Inc., Holliston, MA, USA).

4. Conclusions

In the present study, a SPR-based analytical method was used to investigate the interactions between Aβ peptides and metal ions. The coordination of metal ions with the truncated Aβ1–28 and the full-length Aβ1–42 were compared. The conformational transition of Aβ induced by metal ion binding can be readily detected by this highly sensitive FI-SPR sensor equipped with a bicell detector. At physiological pH, Zn2+ demonstrates different binding affinity for Aβ1–28 and Aβ1–42, while Cu2+ exhibits similar interactions with both Aβ peptides. The interactions between Zn2+ and the Aβ-peptides in an intra- and inter-molecular mode have been previously validated. These studies complement other analytical methods and should help elucidate the role of metal ions during Aβ aggregation.

This work was supported by the National Natural Science Foundation of China (21145006), Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry and Beijing Municipal Natural Science Foundation (2113046).

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The sensorgram of a 200 μM Zn2+ ion solution flowing over the surface plasmon resonance (SPR) sensor chip with (a) and without (b) immobilized β-amyloid peptides (Aβ1–28) peptides. Arrows indicate Zn2+ ions starting to flow onto immobilized Aβ1-28 and when the flowing of metal ions ends, respectively.

The sensorgrams for the interactions between Zn2+ ions and the Aβ1–28 peptides. Various concentrations (50, 100, 200, 300, 600, 700 and 800 μM, from top to bottom) of Zn2+ ions were injected onto the Aβ1–28 sensorchip. Arrows indicate Zn2+ ions starting to flow onto immobilized Aβ1–28 and when the flowing of metal ions ends, respectively.

Calibration curves for the interactions between various Zn2+ concentrations with Aβ1–28. Each value represents the mean ± standard deviation of three separate injections.

The calibration curve for the interactions between various Cu2+ concentrations with Aβ1–28. Each value represents the mean ± standard deviation of three separate injections.

The sensorgram for the interactions between Zn2+ ions and Aβ1–42. Various concentrations (50, 100, 300, 400, 500 and 800 μM, from top to bottom) of Zn2+ ions were injected onto the Aβ1–42 sensorchip. The inset shows the calibration curve for the interactions between various concentrations of Zn2+ with Aβ1–42. Each value represents the mean ± standard deviation of three separate injections. Arrows indicate Zn2+ ions starting to flow onto immobilized Aβ1–42 and when the flow of metal ions ends, respectively.

The sensorgram for the interactions between Cu2+ ions and Aβ1–42. Various concentrations (50, 100, 200, 300, and 400 μM, from top to bottom) of Cu2+ ions were injected onto the Aβ1–42 sensorchip. The inset shows the calibration curve for the interactions between various concentrations of Cu2+ with Aβ1–42. Each value represents the mean ± standard deviation of three separate injections. Arrows indicate Cu2+ ions starting to flow onto immobilized Aβ1–28 and when the flow of metal ions ends, respectively.

The procedure for the immobilization of Aβ. (a) The self-assembled monolayer (SAM) of MUA surface was activated with standard amine coupling chemistry using EDC/NHS; (b) The activated surface was covered with a fresh Aβ solution to form a bond between the amine group on the peptide and the carboxylic group on the MUA surface; (c) The remaining activated surface groups were blocked using ethanolamine.


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viruses Viruses Viruses Viruses 1999-4915 MDPI 10.3390/v4091410 viruses-04-01410 Review Structural Aspects of the Interaction of Dairy Phages with Their Host Bacteria Mahony Jennifer 1 van Sinderen Douwe 1 2 * Department of Microbiology, University College Cork, Western Road, Cork, Ireland; Email: Alimentary Pharmabiotic Centre, Biosciences Institute, University College Cork, Western Road, Cork, Ireland Author to whom correspondence should be addressed: Email:; Tel.: +353-21-4901365; Fax: +353-21-4903101. 31 08 2012 09 2012 4 9 1410 1424 13 07 2012 22 08 2012 23 08 2012 © 2012 by the authors; licensee MDPI, Basel, Switzerland. 2012

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (

Knowledge of phage-host interactions at a fundamental level is central to the design of rational strategies for the development of phage-resistant strains that may be applied in industrial settings. Phages infecting lactic acid bacteria, in particular Lactococcus lactis and Streptococcus thermophilus, negatively impact on dairy fermentation processes with serious economic implications. In recent years a wealth of information on structural protein assembly and topology has become available relating to phages infecting Escherichia coli, Bacillus subtilis and Lactococcus lactis, which act as models for structural analyses of dairy phages. In this review, we explore the role of model tailed phages, such as T4 and SPP1, in advancing our knowledge regarding interactions between dairy phages and their hosts. Furthermore, the potential of currently investigated dairy phages to in turn serve as model systems for this particular group of phages is discussed.

bacteriophage milk fermentation receptor
1. Introduction

Phages are the most abundant biological entities on Earth [1] and are responsible, at least in part, for driving the evolution of their bacterial hosts [2]. The selective pressure imposed by phages on their hosts also requires an equivalent genomic plasticity and adaptability by the phages themselves in order to successfully produce progeny in a host that tries to escape its parasites. All characterized phages known to infect lactic acid bacteria (LAB) possess a tail and therefore this review will solely consider tailed phages.

One of the first physical phage-host interactions is that between the receptor on the host bacterium and the distal end of the tail of the phage. Many isolated phages have been characterised morphologically by electron microscopy analysis [3] and the information derived from such analyses may be useful to gain an insight into the position and nature of host receptor (see below). In recent years structural information has emerged related to phage proteins that are involved in binding to these receptors, usually referred to as anti-receptors, receptor binding proteins (RBPs) or adhesins. Depending on the macromolecular nature of the receptor, i.e., being a protein or carbohydrate, the phage tail tip of Siphoviridae phages, which contains the receptor-binding protein, appears to display a particular morphology. For example, phages that interact with protein moieties generally possess either a pointed or “stubby” end, while those that interact with carbohydrate moieties more often display a larger, more obvious structure, which is called the baseplate. While there are exceptions to this generalisation, including many undefined phage-host interactions, this may perhaps be the primary indicator of the type of moiety required by a phage to interact with its host. Here we will explore the role of electron microscopy and structural analysis in defining the interactions between phages and their hosts, and examine the various types of receptor-binding proteins encoded by phages and their corresponding host-encoded receptor. The interactions and wealth of structural data for phage RBPs that now exists will be examined with a view to understand the interactions between dairy phages and their hosts. Strains of Lactococcus lactis and Streptococcus thermophilus are the most widely utilised LAB in the dairy industry, with certain species of Lactobacillus also being used intensively. Consequently, phages infecting these species dominate LAB phage research. Since the most frequently isolated, and hence the most problematic phages of dairy lactic acid bacteria (LAB) are those belonging to the Siphoviridae (isolated for Lactococcus, Streptococcus and Lactobacillus spp., among others) and, to a lesser extent, Myoviridae families (isolated for Lactobacillus spp.), we will focus on the former for purposes of clarity.

2. Phages that Recognize a Proteinaceous Receptor

Phages recognizing a proteinaceous receptor can achieve a strong phage-bacterium interaction through protein-protein interactions and therefore only a single phage protein or small complex is required due to the high affinity, avidity and specificity of this interaction [4]. In electron micrographic images such phages possess a pointed or stubby tail tip, which is where the RBP is located (See Table 1 for examples). A very well established example of this is the Bacillus subtilis-infectingsiphophage SPP1, which interacts irreversibly with its host-encoded receptor protein YueB via its pointed tail tip following an initial and reversible attraction to a carbohydrate moiety on the cell surface [4]. Additionally, the lactococcal c2-type siphophages interact with the phage infection protein (PIP) through a RBP that is presumed to be located at the blunt-ended tail tip [5]. It is noteworthy that both YueB and PIP are members of the Type VII secretion system and are closely related proteins and therefore functional analogies are perhaps not surprising. The tail adsorption protein of c2 is proposed to be encoded by orf l10, a 75 kDa protein identified through mass spectrophotometry and immune‑gold electron microscopy [6]. The bacterial receptors of the Siphoviridae coliphages λ and T5 (both of these phages possess pointed tail tips) have been defined and isolated (LamB and FhuA, respectively), which has permitted the development of in vitro DNA ejection assays to positively identify these receptor proteins [7,8]. The coliphages belonging to the Myoviridae T4 superfamily possess six long tail fibers that interact reversibly with lipopolysaccharide (LPS) or a porin protein, such as OmpC [9,10]. Subsequently, the baseplate is lowered toward the cell surface and the six short tail fibers are released to allow reversible binding to occur, thereby allowing the pointed puncturing device to initiate DNA injection into the host after the baseplate is switched to the star conformation [11]. Of the above-mentioned phages, T4 is one of the best studied model phages with respect to the structural analysis of the tail fibers and injection devices. The 3D structure of the long tail fiber receptor-binding domain of gp37 has been resolved, which revealed that the aromatic and positive residues on the surface of the fiber tips are most likely receptor-binding determinants with the receptor, although the authors do not specify whether the receptor material is LPS or the OmpC porin protein [9]. The proteins constituting the T4 baseplate complex have been studied by means of cryo‑electron microscopy and X-ray crystallography [11,12,13]. Through these analyses, at least twelve proteins have been identified as baseplate components: gp5, gp6, gp7, gp8, gp9, gp10, gp11, gp12, gp25, gp27, gp53, gp54. The six gp10 trimers are a crucial component of the baseplate and structural analysis of this protein has revealed its role in extension of the short tail fibers encoded by gp12 by means of a partner shift: the pre-infection state sees an interaction between the C-terminal end of gp10 and gp12, while infection requires an interaction between the baseplate protein gp9 and gp10 so as to release the short tail fibers, thus permitting interaction with the host receptor [13]. This fascinating and complex phage serves as an excellent model for the physical interactions between a phage and its host. However, this model is much more sophisticated than many of the phages of the Siphoviridae family and for this purpose phage SPP1 may be a better representative for this family of phages.

viruses-04-01410-t001_Table 1

Proven and putative phage receptors.

Host Phage Receptor type Host receptor Reference
Bacillus subtilis SPP1 Protein YueB [14]
Escherichia coli T4-like Protein OmpC/LPS [10]
T5 Protein FhuA [8]
Λ Protein LamB [7]
Lactococcus lactis c2 Protein PIP [5]
p2 Saccharide Unknown [15]
bIL170 Saccharide Unknown [16]
TP901–1 Saccharide Unknown [17]
Tuc2009 Saccharide Unknown
Lactobacillus LL-H Lipoteichoic acid Poly-Glycerophosphate LTAs [18]
Streptococcus thermophilus OBJ Saccharide Glucosamine/Ribose [19]
CYM Saccharide Glucosamine/Rhamnose [19]
3. SPP1 as a Model for Protein-Interacting Siphophages of Gram-Positive Bacteria

SPP1 possesses a long non-contractile tail, which is the trademark property of the Siphoviridae phages. At the base of the tail is a narrow device, the tail tip, which interacts with a membrane-anchored protein (YueB) on the host cell surface [14]. Significant structural data has emerged in recent years relating to the capsid, tail, head-tail connector and the tail tip of SPP1, thus rendering it a well understood model for other siphophages infecting Gram-positive bacteria, including those that target LAB [20,21,22,23,24,25,26,27].

One of the first structural studies of phage SPP1 focused on the tail at pre- and post-infection stages, and detailed the loss of the tail tip following DNA ejection [14]. The tail of SPP1 is composed of an estimated 40 stacked rings (gp17.1 and gp17.1*), inside which is a channel containing the tail tape measure protein (gp18) and through which the phage genome is passed upon release from the capsid [14]. The outside of the tail rings exhibit a six-fold symmetry (gp17.1 and gp17.1*, see below), while the inner tube has a continuous appearance that is composed of several gp18 subunits [14]. The tail tip, which has no channel to allow DNA passage, interacts with YueB, which prompts its release from the tail cap, thereby triggering a sequence of conformational changes that result in the ejection of the viral genome from the capsid. Following DNA ejection, the internal channel diameter appears to undergo a size reduction from 56 Å to 42 Å due to rearrangements in the major tail proteins gp17.1 and gp17.1* and not negating the fact that the tail tape measure protein fills the channel prior to DNA ejection [14]. These gp17.1 and gp17.1* proteins are present in a 3:1 ratio and protein microsequencing has demonstrated that the product of gp17.1* contains an additional C-terminal 10 kDa region, which is thought to be exposed on the outer surface of the tail [28]. This feature also seems to be encoded by prophages of Bacillus licheniformis and Bacillus halodurans, while such extensions also appear to be present in the phage receptor-binding proteins and major tail protein‑associated sequences of other Siphoviridae phages such as the lactococcal phage Q54 and Listeria phage A118 [28].

Other elements of the distal end of the tail of SPP1 have been characterised including the distal tail protein (Dit, gp19.1) and the N-terminal region of the tail spike protein (gp21) [23,25]. The SPP1 Dit appeared as a dimer of hexamers in a back-to-back orientation which is structurally similar to a component of the lactococcal phage p2 baseplate (ORF 15), however it is now clear that this apparent dimer is a crystallographic artifact in the case of SPP1 [23]. Two trimers of the SPP1 truncated tail spike protein have been shown to interact with Dit, where one trimer is presumed to be in a “closed” conformation while the other trimer is in the “open” conformation (Figure 1) [25], a system which is consistent with observations made for the lactococcal phage p2 baseplate [25,29]. The Dit-tail spike complex appears to constitute the tail cap of SPP1 and the structural conservation of these features in lactococcal phages may indicate a common lineage of such systems [25]. Calcium ions stabilise the “open” conformation of tail cap, which is also consistent with the lactococcal phage p2 [25,29]. The Dit-tail complex acts as a hub upon which the baseplate (in the case of phages that recognize a saccharidic receptor) or the tail tip/spike (for phages that bind to a proteinaceous receptor) is hinged, at the distal end of the tail. Furthermore, (multimers of) the major tail protein (MTP) and tail tape measure protein (TMP) attach to this structure to form the tail tube. It is suggested that the interaction of the RBP with the host-encoded receptor induces tail base to change to the open conformation triggering the movement of the first ring of MTP which is then sequentially continued up through the MTP rings with ensuing release of DNA from the phage capsid [14,25]. The structural conservation of the Dit-tail cap complex and conformational arrangements in SPP1 and lactococcal phages endorses SPP1 as a model for Siphoviridae phages, and as such is a basis for understanding the interactions between dairy phages and their hosts.

Schematic representation of a top view of the re-organisation of the p2 (top) and SPP1 (bottom) baseplate regions before (right) and after (left) binding to the host cell. The p2 baseplate, composed of a hexamer of ORF15 (orange), a trimer of ORF16 (green) and six trimers of ORF18 (blue) which represents the RBP. In parallel, the SPP1 distal tail region is composed of two back-to-back hexamers of gp19.1 (orange) and a trimer of gp21 (green) bound to each of the Dit (gp19.1) hexamers, one trimer in the open conformation and the other in the closed conformation. A representation of the genomic regions that encode the tail structural components are presented in the lowermost section, including the genes encoding the proposed major tail protein (pink), the tail tape measure protein (purple), the distal tail protein (Dit)/hub protein (orange), the tail spike (SPP1) or tail tip (p2) (green) and the RBP (blue, p2). Those in grey encode non-structural proteins or have not been functionally assigned.

4. Structural Approaches to Understand Dairy Phage Interactions and Carbohydrate-Recognizing Phages

Lactococcal phages have, among the dairy phages, received the most extensive scientific scrutiny with regards to the structural analysis of protein/protein complexes [17,29,30,31,32]. Isolated lactococcal phages are grouped into ten distinguishable species based on DNA homology and particle morphology [33]. Of these, the 936, c2 and P335 species are most frequently encountered in dairy facilities world-wide. Members of the 936 and P335 species are believed to bind a saccharide component that is present on the surface of the cell envelope, while the c2 phages are known to bind the so-called phage infection protein (PIP) [5]. Members of the 936 and P335 species have been studied at a structural level and constitute the backbone of emerging structural data relating to dairy phages.

4.1. Lactococcal P335 and 936 Phage-Host Interactions

Over the past decade, significant efforts have been made to determine the molecular nature and mechanism of the interactions between lactococcal phages and their hosts, and members of the P335 and 936 species have been central to such studies due to their industrial relevance. It has long been suggested that these phages recognize a carbohydrate moiety on their host, a notion that is consistent with carbohydrate inhibition assays [34]. Through mutational analyses several adjacent genes within an operon, which encodes the biosynthetic machinery for a cell envelope-associated polysaccharide (also called the pellicle), have been shown to be required for adsorption of 936-type phages. The involvement of this pellicle biosynthesis operon in phage infection has been demonstrated in two separate studies for three 936-type phages, namely bIL170, 645 and sk1, although the precise role of the mutated genes as a phage receptor has not yet been established [35,36]. The 20–25 kb pellicle operon contains elements that are conserved among all sequenced lactococcal strains, while it also contains strain-specific regions that are expected to lead to the production of pellicles with a distinct saccharidic structure and composition. The so-called “adsorption genes”, or receptor-encoding genes, involved in 936-type phage recognition and adsorption encode either a glycosyltransferase or a membrane-associated protein [35]. The involvement of these gene-products may imply two possibilities of the mode of interaction and DNA injection pathway of these phages. Firstly, that the glycosyltransferase inactivation may prevent the transfer and decoration of the required pellicle monosaccharide component to the surface thus negating the primary contact between the phage RBP and its saccharide receptor; or secondly, if primary contact is established and a membrane component is required for the translocation of the DNA across the membrane, the membrane-associated protein inactivation prevents internalisation of the phage DNA. Chemical mutagenesis of the lactococcal host strain 3107, which is sensitive to the P335 phages LC3 and TP901–1, resulted in the isolation of five genetically and phenotypically distinct mutants resistant to one or both phages [37]. This analysis revealed that the injection pathways employed by these phages of the same species are distinct. It is through studies such as these that more attention has been diverted towards the identification of phage RBPs on lactococcal phages in order to determine the relationship between a phage and its host(s).

The 936 phage RBPs has been identified through the isolation of a chimeric sk1/bIL170, which also was shown to exhibit a “host-range swapping” phenotype [38]. The identification of the RBPs of these phages presented the opportunity to define the structure of the corresponding proteins and their associated complexes. This study applied an approach previously used by Duplessis and Moineau (2001) in which the variable C-terminal VR2 region of ORF18 of DT1 was replaced by the corresponding fragment of MD4. The resultant chimeric DT1 infected the host for MD4, thereby elucidating the host specificity function of this protein [39]. Similarly, a chimeric derivative of phage TP901–1 (designated TP901–1C), harbouring the lower baseplate protein of phage Tuc2009 instead of its original TP901–1 equivalent, was shown to have acquired the host range of Tuc2009, thereby identifying the lower baseplate protein as the RBP of both of these P335 phages [40]. Such discoveries have been very useful in the assignment of RBPs of other P335 phages through homology and genomic positioning of the encoding gene [41]. Also noteworthy is the observation of the altered baseplate morphology in the case of the chimeric phage TP901–1C as compared to the parent phage [36]. TP901–1 has a clear double-disc baseplate while the chimeric phage TP901–1C was described to possess “hanging droplets” rather than a distinct lower baseplate and furthermore the lower disc appeared narrower than that of the parent phage (23 nm versus 28 nm in the parent phage) and intermediate between that of the wild-type TP901–1 and Tuc2009. The genetic plasticity of the lactococcal phages has permitted the development of recombinant phages of both the 936 and P335 species and may present a mechanism by which these phages alter their host specificity or enlarge their host spectrum as their environment dictates.

4.2. 936 Phage Baseplates

The first lactococcal phage RBP to be structurally defined was that of the 936 phage p2 [42]. The p2 RBP is a homotrimeric protein comprising three domains: the head, neck and shoulder. The actual receptor binding site was identified within the “head” domain through the application of the heavy chain neutralising llama antibody fragment VHH5, which had previously been determined to prevent infection of the lactococcal host by phage p2 [15,42,43]. The shoulder domain possesses eight ß‑strands interspersed with an α-helix between ß-strands one and two, and a coil between ß-strands two and three [27]. The neck is a ß-prism of three segments that yields a rigid structure to which analogy has been drawn to the gp12 short tail fiber structure of the coliphage T4. Finally, the head domain is a ß-barrel of seven anti-parallel ß-strands [42]. Seven charged or polar and five non-polar residues were identified in the RBP head domain interacting with the VHH5.

The receptor binding site was further characterised through the isolation of escape mutants that could bypass the llama neutralising antibody fragments [15]. The majority of the mutations that enable the mutant phages to overcome the llama antibodies are within the interacting site. Just twelve out of fifty assessed lactococcal 936 phages were neutralised by VHH5 antibodies, highlighting the apparent variability of RBPs found in members of this closely related phage species. Mutants overcoming the VHH5 were readily isolated for eleven of the twelve phages neutralised and in all cases a single point mutation in the corresponding RBP-encoding gene was shown to be sufficient to prevent antibody recognition. This study also served to improve the resolution of the RBP structure of p2 from 2.3 to 1.7 Å and identified a glycerol molecule bound to the structure originating from the cryoprotectant. The binding of the glycerol molecule was observed to be tighter in the head domain than the shoulder domain where three hydrogen bonds are formed between head domain residues and the glycerol O-1 and O-2 atoms (versus one in the shoulder domain). Other saccharides were assessed for their ability to bind the RBP in solution and it was suggested that phosphoglycerol, which is a component of lipoteichoic acid and teichoic acid, represented a potential receptor. This was the first direct proof of carbohydrate binding of a lactococcal 936-type phage RBP, confirming biological analyses that suggested the role of specific carbohydrates as receptors for 936-type phages [15,34].

The crystal structure of the RBP head domain of another 936 phage, bIL170, was solved and represents one of a number of phages that were not neutralised by the VHH5 antibodies due to its variable C-terminal sequence [16]. The phylogeny of the RBPs of 936 phages has demonstrated clear links to host range and VHH5 antibody-neutralisation, and it is well established that while the N‑terminal region is well-conserved, it is the C-terminal (head) domain that determines the specific host interaction [15,45,46,47]. Interestingly, while there is little sequence conservation between the head domains of p2 and bIL170, their structure is strikingly similar [16]. Furthermore, it bears structural similarity to the head domain of the lactococcal P335 phage TP901–1 [16]. Akin to the RBP of p2, the head domain of bIL170 is proposed to be a homotrimer that binds saccharides near a tyrosine residue (Tyr226) whose side chain forms the upper wall of the crevice between the trimer components and is accessible to solvents [16].

Expression and purification of ORFs 15, 16, 17 and 18 of p2 by affinity chromatography revealed that the baseplate of p2 is composed of three proteins, ORFs 15, 16 and 18. The structural data generated from crystals, obtained from the in vitro expressed recombinant baseplate complex, was mapped onto the baseplate structure, generated from electron microscopy images of the virion, provided comparative data to verify the accuracy of the expressed baseplate complex structure [29]. ORF15 and 16 readily mapped onto the virion structural data while ORF18 displayed some variation. This variation was proven to be the result of conformational swapping of the RBP head domains either upward, facing the capsid in an inactive form when calcium is absent, or in the “activated” form in the presence of calcium (or strontium) in which case the RBP head domains rotate 200° downward. Calcium is required for infection by many phages and therefore it is not surprising that it may be involved in the initial stages of the phage-host interaction, however, the conformational swapping of the RBP head domains is the first report of any such baseplate activation for Siphoviridae phages [29]. Such studies highlight the importance of electron microscopy analysis to define the native virion baseplate structures because, while the use of camelid antibodies provided data on the baseplate/RBP in the activated state, the state of the free phage baseplate revealed a previously unknown mechanism. While calcium is known to be required for the activation of the baseplate, it is unclear as yet if there is also as role for calcium in the physiology and organization of the bacterial envelope, thereby endorsing phage infection or if the effect is limited to the activation of the phage baseplate.

The application of these “block cloning” strategies to the expression and purification of baseplate complexes of the lactococcal phage p2 and TP901–1 is an important tool to facilitate the study of lactococcal base plates [47,48]. This block cloning also provides discerning information that may define which proteins of an operon form part of the complex and this approach may be applied to any operon encoding structural components of heteromeric organelles. A model for the construction and assembly of lactococcal P335 phage tails has been proposed in which the tail tape measure (TMP), distal tail protein (Dit) and tail-associated lysin (Tal) converge to act as the “initiation complex” upon which the baseplate is assembled [40,41]. Mass spectrometry has recently been used as an alternative method to define the assembly pathway of the baseplates of p2 and TP901–1 [49]. Using this approach, the p2 baseplate is proposed to assemble through the interaction of a hexamer of ORF15 with a preassembled ORF16 trimer followed by the capture of six ORF18 trimers [49]. This approach was also applied to the P335 phage TP901–1 and an assembly pathway was proposed which is outlined below.

4.3. P335 Phage Baseplates

The possible assembly pathway for TP901–1 that was proposed involves the interaction of homotrimers of ORF48 (upper baseplate protein) and ORF49 (lower baseplate protein) [49]. These then form tripods comprised of 3×ORF48 and 9×ORF49, and after interaction between ORF48 with ORF46 (distal tail protein or Dit), oligomerisation of ORF46 may proceed with concomitant baseplate formation with a proposed final composition of 6×ORF46, 18×ORF48 and 54×ORF49 [49]. Furthermore, through electron microscopic analysis of the TP901–1 baseplate together with that of TP901–1 mutants lacking either BppU (upper baseplate) or BppL (lower baseplate), the structure and composition of the TP901–1 baseplate have been determined [31]. Structural analysis of the RBP of TP901–1 has revealed the similarity of the modularity of the RBP of TP901–1 and the previously studied p2, pointing to common ancestral origins of such structures (Figure 2) [17]. This is consistent with the development of a chimeric RBP with the N-terminal portion of TP901–1 and the C-terminal head domain of p2 [50]. The resulting structure presented domains almost indistinguishable from the parental structures highlighting the modular conservation of these lactococcal phages of distinct species [49]. The head domain of TP901–1 RBP was also observed to bind glycerol and fluorescence quenching further consolidated the carbohydrate affinity of this phage protein, identifying affinity for glycerol and muramyl-dipeptide [17].

Unlike the 936-type phage p2, the TP901–1 baseplate is maintained in a so-called “infection-ready conformation” [31,51]. This observation highlights the bipartite baseplate conformations either maintained in an infection-ready state or in a state requiring activation. It is suggested that the signalling to induce DNA ejection after adsorption of TP901–1 may require a more subtle conformational change than that of p2 and the T4 Myoviruses [51]. In this study, two potential infection pathway scenarios were proposed: first is the possibility that the tail-associated lysin (Tal) may interact with the cell wall peptidoglycan resulting in triggering of DNA release through a so‑called cascade effect channelled up through the tail tube in a similar manner proposed for SPP1 [14,51]. The second proposed injection pathway involves the binding of the RBP tripod complexes to the carbohydrate moiety on the cell surface with mechanical alterations leading to signalling to the lowermost ring of the major tail protein with ensuing “un-screwing” of the tail to allow DNA ejection to occur [51]. Furthermore, comparisons have been drawn between the activation systems of p2 and the Myophage T4 upon interaction with a carbohydrate moiety, which is an interesting analogy and further corroborates theories of common ancestral elements and conservation of systems and modular structures in the case of RBPs [46].

Tuc2009 is a temperate P335 phage that is closely related to TP901–1 [52], although its baseplate‑encoding operon contains an additional gene designated bppA (accessory baseplate protein). This appears to be a non-essential structural element of the Tuc2009 baseplate that is possibly involved in the determination or extension of host range. A low resolution model of the Tuc2009 baseplate has been determined in which the baseplate was proposed to consist of six tripods, each comprising 3×BppU, 3×BppA and 3×BppL, although this model is currently being refined that might align Tuc2009 more closely to TP901–1 in terms of its baseplate structure [30]. Structural analysis of this baseplate may uncover a potential role for BppA, for which a functional assignment remains to be provided. The presence of homologues of BppA in other lactococcal P335 phages (ul36 ORF303, accession no. NP_663688.1; P335 ORF45, accession no. ABI54248.1), indicates that there may be a biological significance and ecological advantage to phages possessing the protein in their baseplate. Furthermore, the recent finding that Tuc2009 is one of the few sequenced members of the P335 species that requires calcium to produce plaques may highlight the subtleties that distinguish individual phages within a species [51]. Although significant data is emerging relating to the structure of P335 phage baseplates and individual components thereof, there remains a large gap between our knowledge of these phages and those of the model phages T4 and SPP1. While analogies may be drawn between these models and dairy phages it is imperative to also define relevant structural models within the dairy phage group.

A schematic representation of the distal tail region of Tuc2009, TP901–1 and p2. The stacks of tail rings composed of the major tail protein (MTP) (blue) with the distal tail (Dit) protein (orange) beneath, upon which the baseplate components are hinged (green and purple). In the case of Tuc2009 the green area represents the upper baseplate disc (BppU and BppA) while the TP901–1 upper disc is composed of BppU only. The lower baseplate (BppL) is presented in purple with a disc representation for TP901–1 and a so‑called “petticoat” representation for Tuc2009. The protrusion from the baseplates of TP901–1 and Tuc2009 represent the tail associated lysin (Tal) (red). The schematic of the baseplate region of p2 highlights ORF15 (orange) at the base of the tail with ORF16 beneath (red) and the RBP in the active downward orientation (purple).

5. Conclusions and Future Perspectives

Structural studies relating to T4 in the 1990’s and early 2000’s paved the way for the structural analyses of many phage receptor binding proteins and their associated complexes and have permitted the detailed analysis of several phage-host interactions [9,10,11,12]. This, together with the isolation of the receptor for T5 and lambda in the 1990’s and the 1970’s prompted some of the early phage-host interaction studies for lactococcal phages [7,8,34]. Since then, numerous studies relating to the interactions of dairy phages and their hosts have been reported and have led to a surge of interest in structural analyses of these systems. The lactococcal phages p2, TP901–1 and Tuc2009 are emerging as model phages for the dairy Siphoviridae phages in terms of structural analyses. These data present a fresh perspective on studies attempting to define the host molecules with which dairy phages interact. The type of baseplate as defined by electron microscopy may be the initial indicator of the material that acts as receptor for the individual phage although this is merely a first glimpse. Structural analysis and/or detailed electron microscopic analysis may provide detailed information on the baseplate organisation and structure, which may be applied to phage-host interaction studies. The analysis of such structures is paramount to understanding how dairy phages bind to and subsequently infect their hosts, and an improved knowledge of phage-host interactions will aid in developing novel and effective anti-phage strategies such as those already explored through the use of camelid antibodies and designed ankyrin repeat proteins (DARPins) to inactivate phages [15,32].

Since structural data relating to these model dairy phages continue to accrue, it is inevitable that these will become the dominant model for the analysis of other dairy phages including those of S. thermophilus and Lb. delbrueckii, typical cultures used in dairy fermentations. It has been suggested that the receptor material for the Lb. delbrueckii phage LL-H is polyglycerophosphate type lipoteichoic acids [18], which is reminiscent of affinity of the 936 phage p2, further demonstrating the usefulness of the lactococcal phage data as a suitable model for other dairy phages. However, while structural analyses are essential to furthering our knowledge of phage-host interactions, it is equally important to apply biological and functional characterisation to verify conclusions drawn from structural analyses. The marriage of molecular microbiology with structural biology represents one of the most powerful modern tools to understand complex interactions and pathways involved in host recognition and infection, and may be the key to developing next generation anti-phage systems with potential application in the dairy industry.

D van Sinderen is a recipient of a Science Foundation Ireland (SFI) Principal Investigatorship award (Ref. No. 08/IN.1/B1909).

Conflict of Interest

The authors declare no conflict of interest.

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Rossmann M.G. Three-dimensional structure of bacteriophage T4 baseplate Nat. Struct. Biol. 2003 10 688 693 10.1038/nsb970 Leiman P.G. Shneider M.M. Kostyuchenko V.A. Chipman P.R. Mesyanzhinov V.V. Rossmann M.G. Structure and location of gene product 8 in the bacteriophage T4 baseplate J. Mol. Biol. 2003 328 821 833 10.1016/S0022-2836(03)00366-8 Leiman P.G. Shneider M.M. Mesyanzhinov V.V. Rossmann M.G. Evolution of bacteriophage tails: Structure of T4 gene product 10 J. Mol. Biol. 2006 358 912 921 10.1016/j.jmb.2006.02.058 Plisson C. White H.E. Auzat I. Zafarani A. Sao-Jose C. Lhuillier S. Tavares P. Orlova E.V. Structure of bacteriophage SPP1 tail reveals trigger for DNA ejection EMBO J. 2007 26 3720 3728 10.1038/sj.emboj.7601786 Tremblay D.M. Tegoni M. Spinelli S. Campanacci V. Blangy S. Huyghe C. Desmyter A. Labrie S. Moineau S. Cambillau C. Receptor-binding protein of Lactococcus lactis phages: Identification and characterization of the saccharide receptor-binding site J. Bacteriol. 2006 188 2400 2410 10.1128/JB.188.7.2400-2410.200616547026 Ricagno S. Campanacci V. Blangy S. Spinelli S. Tremblay D. Moineau S. Tegoni M. Cambillau C. Crystal structure of the receptor-binding protein head domain from Lactococcus lactis phage bil170 J. Virol. 2006 80 9331 9335 10.1128/JVI.01160-0616940545 Spinelli S. Campanacci V. Blangy S. Moineau S. Tegoni M. Cambillau C. Modular structure of the receptor binding proteins of Lactococcus lactis phages. The RBP structure of the temperate phage TP901-1 J. Biol. Chem. 2006 281 14256 14262 10.1074/jbc.M60066620016549427 Raisanen L. Schubert K. Jaakonsaari T. Alatossava T. Characterization of lipoteichoic acids as Lactobacillus delbrueckii phage receptor components J. Bacteriol. 2004 186 5529 5532 10.1128/JB.186.16.5529-5532.2004 Quiberoni A. Stiefel J.I. Reinheimer J.A. Characterization of phage receptors in Streptococcus thermophilus using purified cell walls obtained by a simple protocol J. Appl. Microbiol. 2000 89 1059 1065 10.1046/j.1365-2672.2000.01214.x11123479 Vinga I. Baptista C. Auzat I. Petipas I. Lurz R. Tavares P. Santos M.A. Sao-Jose C. Role of bacteriophage SPP1 tail spike protein gp21 on host cell receptor binding and trigger of phage DNA ejection Mol. Microbiol. 2012 83 289 303 10.1111/j.1365-2958.2011.07931.x Lhuillier S. Gallopin M. Gilquin B. Brasiles S. Lancelot N. Letellier G. Gilles M. Dethan G. Orlova E.V. Couprie J. et al. Structure of bacteriophage SPP1 head-to-tail connection reveals mechanism for viral DNA gating Proc. Natl. Acad. Sci. U. S. A. 2009 106 8507 8512 10.1073/pnas.081240710619433794 Veesler D. Blangy S. Lichiere J. Ortiz-Lombardia M. Tavares P. Campanacci V. Cambillau C. Crystal structure of Bacillus subtilis SPP1 phage gp23.1, a putative chaperone Protein Sci. 2010 19 1812 1816 10.1002/pro.46420665904 Veesler D. Robin G. Lichiere J. Auzat I. Tavares P. Bron P. Campanacci V. Cambillau C. Crystal structure of bacteriophage SPP1 distal tail protein (gp19.1): A baseplate hub paradigm in gram-positive infecting phages J. Biol. Chem. 2010 285 36666 36673 10.1074/jbc.M110.15752920843802 Veesler D. Blangy S. Spinelli S. Tavares P. Campanacci V. Cambillau C. Crystal structure of Bacillus subtilis SPP1 phage gp22 shares fold similarity with a domain of lactococcal phage p2 RBP Protein Sci. 2010 19 1439 1443 10.1002/pro.416 Goulet A. Lai-Kee-Him J. Veesler D. Auzat I. Robin G. Shepherd D.A. Ashcroft A.E. Richard E. Lichiere J. Tavares P. et al. The opening of the SPP1 bacteriophage tail, a prevalent mechanism in Gram-positive-infecting siphophages J. Biol. Chem. 2011 286 25397 25405 10.1074/jbc.M111.24336021622577 Chagot B. Auzat I. Gallopin M. Petitpas I. Gilquin B. Tavares P. Zinn-Justin S. Solution structure of gp17 from the Siphoviridae bacteriophage SPP1: Insights into its role in virion assembly Proteins 2012 80 319 326 10.1002/prot.23191 White H.E. Sherman M.B. Brasiles S. Jacquet E. Seavers P. Tavares P. Orlova E.V. Capsid structure and its stability at the late stages of bacteriophage SPP1 assembly J. Virol. 2012 86 6768 6777 10.1128/JVI.00412-12 Auzat I. Droge A. Weise F. Lurz R. Tavares P. Origin and function of the two major tail proteins of bacteriophage SPP1 Mol. Microbiol. 2008 70 557 569 10.1111/j.1365-2958.2008.06435.x18786146 Sciara G. Bebeacua C. Bron P. Tremblay D. Ortiz-Lombardia M. Lichiere J. van Heel M. Campanacci V. Moineau S. Cambillau C. Structure of lactococcal phage p2 baseplate and its mechanism of activation Proc. Natl. Acad. Sci. U. S. A. 2010 107 6852 6857 10.1073/pnas.100023210720351260 Sciara G. Blangy S. Siponen M. Mc Grath S. van Sinderen D. Tegoni M. Cambillau C. Campanacci V. A topological model of the baseplate of lactococcal phage Tuc2009 J. Biol. Chem. 2008 283 2716 2723 18045876 Bebeacua C. Bron P. Lai L. Vegge C.S. Brondsted L. Spinelli S. Campanacci V. Veesler D. van Heel M. Cambillau C. Structure and molecular assignment of lactococcal phage TP901-1 baseplate J. Biol. Chem. 2010 285 39079 39086 10.1074/jbc.M110.17564620937834 Veesler D. Dreier B. Blangy S. Lichiere J. Tremblay D. Moineau S. Spinelli S. Tegoni M. Pluckthun A. Campanacci V. et al. Crystal structure and function of a darpin neutralizing inhibitor of lactococcal phage TP901-1: Comparison of DARPin and camelid VHH binding mode J. Biol. Chem. 2009 284 30718 30726 10.1074/jbc.M109.03781219740746 Deveau H. Labrie S.J. Chopin M.C. Moineau S. Biodiversity and classification of lactococcal phages Appl. Environ. Microbiol. 2006 72 4338 4346 10.1128/AEM.02517-0516751549 Geller B.L. Ngo H.T. Mooney D.T. Su P. Dunn N. Lactococcal 936-species phage attachment to surface of Lactococcus lactis J. Dairy Sci. 2005 88 900 907 10.3168/jds.S0022-0302(05)72756-9 Dupont K. Janzen T. Vogensen F.K. Josephsen J. Stuer-Lauridsen B. Identification of Lactococcus lactis genes required for bacteriophage adsorption Appl. Environ. Microbiol. 2004 70 5825 5832 10.1128/AEM.70.10.5825-5832.2004 Chapot-Chartier M.P. Vinogradov E. Sadovskaya I. Andre G. Mistou M.Y. Trieu-Cuot P. Furlan S. Bidnenko E. Courtin P. Pechoux C. Cell surface of Lactococcus lactis is covered by a protective polysaccharide pellicle J. Biol. Chem. 2010 285 10464 10471 10.1074/jbc.M109.08295820106971 Ostergaard-Breum S. Neve H. Heller K.J. Vogensen F.K. Temperate phages TP901-1 and phiLC3, belonging to the P335 species, apparently use different pathways for DNA injection in Lactococcus lactis subsp. cremoris 3107 FEMS Microbiol. Lett. 2007 276 156 164 10.1111/j.1574-6968.2007.00928.x17956421 Dupont K. Vogensen F.K. Neve H. Bresciani J. Josephsen J. Identification of the receptor-binding protein in 936-species lactococcal bacteriophages Appl. Environ. Microbiol. 2004 70 5818 5824 10.1128/AEM.70.10.5818-5824.2004 Duplessis M. Moineau S. Identification of a genetic determinant responsible for host specificity in Streptococcus thermophilus bacteriophages Mol. Microbiol. 2001 41 325 336 10.1046/j.1365-2958.2001.02521.x Vegge C.S. Vogensen F.K. Mc Grath S. Neve H. van Sinderen D. Brondsted L. Identification of the lower baseplate protein as the antireceptor of the temperate lactococcal bacteriophages TP901-1 and Tuc2009 J. Bacteriol. 2006 188 55 63 10.1128/JB.188.1.55-63.200616352821 Mc Grath S. Neve H. Seegers J.F. Eijlander R. Vegge C.S. Brondsted L. Heller K.J. Fitzgerald G.F. Vogensen F.K. van Sinderen D. Anatomy of a lactococcal phage tail J. Bacteriol. 2006 188 3972 3982 10.1128/JB.00024-0616707689 Spinelli S. Desmyter A. Verrips C.T. de Haard H.J. Moineau S. Cambillau C. Lactococcal bacteriophage p2 receptor-binding protein structure suggests a common ancestor gene with bacterial and mammalian viruses Nat. Struct. Mol. Biol. 2006 13 85 89 10.1038/nsmb102916327804 Ledeboer A.M. Bezemer S. de Hiaard J.J. Schaffers I.M. Verrips C.T. van Vliet C. Dusterhoft E.M. Zoon P. Moineau S. Frenken L.G. Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama J. Dairy Sci. 2002 85 1376 1382 10.3168/jds.S0022-0302(02)74204-512146467 De Haard H.J. Bezemer S. Ledeboer A.M. Muller W.H. Boender P.J. Moineau S. Coppelmans M.C. Verkleij A.J. Frenken L.G. Verrips C.T. Llama antibodies against a lactococcal protein located at the tip of the phage tail prevent phage infection J. Bacteriol. 2005 187 4531 4541 10.1128/JB.187.13.4531-4541.2005 Mahony J. Deveau H. Mc Grath S. Ventura M. Canchaya C. Moineau S. Fitzgerald G.F. van Sinderen D. Sequence and comparative genomic analysis of lactococcal bacteriophages jj50, 712 and p008: Evolutionary insights into the 936 phage species FEMS Microbiol. Lett. 2006 261 253 261 10.1111/j.1574-6968.2006.00372.x Dupont K. Vogensen F.K. Josephsen J. Detection of lactococcal 936-species bacteriophages in whey by magnetic capture hybridization pcr targeting a variable region of receptor-binding protein genes J. Appl. Microbiol. 2005 98 1001 1009 10.1111/j.1365-2672.2005.02548.x15752347 Castro-Nallar E. Chen H. Gladman S. Moore S.C. Seemann T. Powell I.B. Hillier A. Crandall K.A. Chandry P.S. Population genomics and phylogeography of an Australian dairy factory derived lytic bacteriophage Genome Biol. Evol. 2012 4 382 393 10.1093/gbe/evs017 Campanacci V. Veesler D. Lichiere J. Blangy S. Sciara G. Moineau S. van Sinderen D. Bron P. Cambillau C. Solution and electron microscopy characterization of lactococcal phage baseplates expressed in Escherichia coli J. Struct. Biol. 2010 172 75 84 10.1016/j.jsb.2010.02.00720153432 Shepherd D.A. Veesler D. Lichiere J. Ashcroft A.E. Cambillau C. Unraveling lactococcal phage baseplate assembly by mass spectrometry Mol. Cell Proteom. 2011 10 M111 009787 Siponen M. Sciara G. Villion M. Spinelli S. Lichiere J. Cambillau C. Moineau S. Campanacci V. Crystal structure of orf12 from Lactococcus lactis phage p2 identifies a tape measure protein chaperone J. Bacteriol. 2009 191 728 734 10.1128/JB.01363-08 Veesler D. Spinelli S. Mahony J. Lichiere J. Blangy S. Bricogne G. Legrand P. Ortiz-Lombardia M. Campanacci V. van Sinderen D. Structure of the phage TP901-1 1.8 MDa baseplate suggests an alternative host adhesion mechanism Proc. Natl. Acad. Sci. U. S. A. 2012 109 8954 8958 10.1073/pnas.120096610922611190 Seegers J.F. Mc Grath S. O'Connell-Motherway M. Arendt E.K. van de Guchte M. Creaven M. Fitzgerald G.F. van Sinderen D. Molecular and transcriptional analysis of the temperate lactococcal bacteriophage Tuc2009 Virology 2004 329 40 52 10.1016/j.virol.2004.07.00315476873

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Method Of Exam

Author: A. Chandrasekhar, MD Consultant: M. Klamut, MD

Have the patient stand and learn forward, resting his hands on the examining table. Place a small amount of lubricant on your gloved index finger.

An alternate method: The rectal exam can also be done in the left lateral decubitus.

Rectal exam is unpleasant.


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Blog ››› ››› SERGIO MUNOZ

Fox News is continuing to baselessly claim that Senate Majority Leader Harry Reid's proposal to eliminate the ability of the GOP minority to filibuster executive branch nominations is unwarranted.

On the July 12 edition of America Live, Fox News guest host Alisyn Camerota brought on Fox contributors Joe Trippi and Ed Rollins to discuss Reid's announcement that his caucus will enact limited filibuster reform, perhaps as early as next week.

The proposal currently being floated would change Senate rules so a president's picks to fill leadership positions in his cabinet and the executive branch automatically receive up-or-down votes, as opposed to being held hostage to GOP filibusters. Although this proposal wouldn't affect the unjustified filibusters of judicial nominations, this limited reform would finally allow simple majority votes on the nominees for labor secretary, Environmental Protection Agency administrator, Consumer Financial Protection Bureau director, and the bipartisan slate for the National Labor Relations Board.

Camerota and her guests, however, adopted Senate Minority Leader Mitch McConnell's argument that because many of President Obama's nominees were eventually confirmed, not only is there no problem, but disallowing subsequent filibusters on these cabinet and agency selections will result in the death of the institution.

Trippi and Rollins' supposed concern for the partisan harm that filibuster reform might cause to the "decorum" of the Senate was too much for even Camerota, who laughed off the concern as "quaint."

The Republican minority - cheered on by right-wing media - has relentlessly engaged in an all-out blockade of the president's agenda, just as McConnell unashamedly admitted he would do in hopes of limiting the administration to one term. Part of this nihilistic approach of blanket obstructionism has included delaying as long as possible the confirmation of the personnel the president needs to carry out his initiatives. Taking into account this significant delay, the GOP talking points that Camerota recycled are true only in that the president's executive nominees, for the most part, were eventually seated.

But what neither Camerota nor McConnell mention is the fact that the Republican minority has required Reid to break filibusters on these nominees in unprecedented fashion. That is, by denying these executive nominees timely up-or-down votes and instead requiring Reid to regularly break filibusters on them (by filing or voting for "cloture"), GOP senators have successfully stalled parts of the executive branch from carrying out the initiatives of a twice-elected president. And this obstructionism is indeed unprecedented, as Marge Baker of People For The America Way points out:

From 1949 (when Senate rules were changed to provide for cloture on nominations) through 2008, cloture votes were forced on only 20 executive branch nominations. However, in just the first four and a half years of the Obama administration, there have already been 16 such cloture votes...That's an increase of more than 1,000% in the rate of forced cloture votes on nominations - 20 nominations on which cloture votes were forced in sixty years compared to 16 in just four and a half.  Republicans are on pace to force 28 cloture votes by the end of the Obama administration - more than under all previous presidents combined.

An examination of President George W. Bush's term reveals that the Senate subjected only seven executive nominations to cloture votes in the entire eight years of his presidency--compared to 16 in just the first four and a half of the Obama administration.

Furthermore, Camerota is absolutely right when she considered her guests' slippery slope argument - that the Senate's traditions will be lamentably shattered - laughable. Ezra Klein of The Washington Post went farther and called such claims "utter nonsense." From the Post:

[H]ere's where the claims of tradition break down into utter nonsense. The Senate never used to work like this. The minority could have an unconstrained right to obstruct because they rarely invoked it. The majority could govern more easily because the two parties hadn't polarized into disciplined war machines. To airily talk about the Senate of the past is almost an obscenity: It was killed long before today, and there's no resolution to this crisis that will bring it back.

The question should not be who is doing a better job protecting the Senate of the past. It's which set of rules will build a stronger Senate for the future.

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Fox News Channel
Alisyn Camerota, Ed Rollins
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Media Plan: KSL, Media UGG Australia, The Invisible Game

KSLMediaThe success of UGG’s classic sheepskin boot eclipsed the brand’s other offerings. In order to highlight the men’s brand, UGG Australia enlisted KSL Media to craft “The Invisble Game” campaign. KSL engaged men through a multichannel approach that integrated video, digital, rich media, print, out of home, social and mobile to court the affluent 25-44 year old male. Footballer Tom Brady was the brand ambassador and played a large role in creating awareness. As a result, the brand saw a 354 percent lift in visits to the men’s landing page.

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Video Tour   FAQ
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Chronic Obstructive Pulmonary Disease is often called by its abbreviated name which is COPD. Actually, COPD occurs due to inhalation of noxious particles which are the consequence of tobacco smoking. COPD can be diagnosed by performing certain tests on liver. The important management strategies for COPD are smoking cessation, vaccination and appropriate dosages of drug therapy. The chronic obstructive pulmonary disease PowerPoint templates which we offer are highly professional and have the potential to lead to the preparation of attractive and exclusive chronic obstructive pulmonary disease PowerPoint presentations so that the audience can effectively understand the topic. These PPT layouts have exotic chronic obstructive pulmonary disease PowerPoint backgrounds which have the potential to add a thrill to the monotonous presentation and make it lively. Also, these chronic obstructive pulmonary disease PPT slides are available with a total number of 30 slides at an unbelievable but highly affordable price.
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Extra Help: Assistance paying for the Medicare drug benefit

Section IX.e. The Extra Help Program
Question 1 of 6 (use "Last" or "Next" buttons to see more)

If your monthly income is up to $1,471 for singles ($1,991 for couples) and your assets are below specified limits (see chart below for details), you may be eligible for Extra Help, a federal program that helps you pay for some or most of the costs of Medicare prescription drug coverage.

Even if your income or assets are above the limit you may still qualify for Extra Help because certain types of income and assets may not be counted. For example, part of your earned income and your house will not be counted.

If you are enrolled in Medicaid, Supplemental Security Income (SSI) or a Medicare Savings Program (MSP), you automatically qualify for Extra Help. You do not have to apply for this extra assistance.

If you are not enrolled in one of the programs that automatically qualify you for Extra Help, you will have to fill out an application. You can do that through the Social Security Administration (SSA), using either the agency's print or online application, or at your local Medicaid office.

If you become eligible, you will get a Special Enrollment Period to enroll in a Medicare private drug plan. You will not have to pay a Part D premium penalty, even if you enroll in a Medicare private drug plan after you were first eligible.

Depending on your income and assets, you may qualify for either “full” or “partial” Extra Help. With either, you will never have to pay the full cost of your drugs as long as you take medications that are on your plan’s list of covered drugs (formulary) and you buy them at a pharmacy in your plan’s network.

If you use mail order to get your prescriptions, you may be able to get a 90-day supply of your prescription for the same amount of money that you would normally pay for a one-month supply. Your costs will be even lower after your out of pocket drug costs reach $4,700 (catastrophic coverage).

The chart below outlines how you qualify for and what you will pay under the different levels of Extra Help.

Even if you qualify for Extra Help, you must choose a private plan offering Medicare drug coverage in your area in order to get the Medicare prescription drug benefit. For Extra Help to fully cover your premium, you must choose a plan that offers basic coverage and has a premium at or below the Extra Help premium amount for your state. If you do not choose a plan, you will be automatically enrolled in one in most cases (in some cases, if you have Medicaid and certain kinds of employer coverage, you may not be enrolled or can ask not to be).*

You should check with 800-MEDICARE (800-633-4227) to make sure the plan you choose offers basic coverage.

Note: If you have Extra Help, you will be able to change Medicare private drug plans once a month.

For a list of Extra Help premium amounts by state, click on the MI Extra.

2015 Extra Help Premium Amounts by State

For the 2015 Income and Asset Limits Please click on the flier below.

Extra Help Program Income and Asset Limits 2015

If you have prescription drug insurance through a retiree plan, check with your former employer or union to see if you can get a Part D plan and still keep your current health coverage. If you cannot have both, you will want to think carefully about whether you should get a Part D plan, especially if your retiree health plan also covers your spouse or dependents who are not yet eligible for Medicare coverage.

*If you have Medicaid and have certain kinds of employer, retiree or union prescription drug coverage, you will not be automatically enrolled in a Part D plan. If you will lose your retiree or union health coverage by enrolling in a Medicare private drug plan, you may not want to take Medicare drug coverage. You will need to contact your state Medicaid office to find out what steps you should take to decline Part D coverage and still keep your Medicaid.

  • If you are not automatically enrolled into a Part D plan, but later want one, you can choose to enroll in a Part D plan at any time without penalty (as long as your are still enrolled in Medicaid or Extra Help).

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 Bulging Eyes and Dogs on MedicineNet

My Dog's Eyes Are Bulging

If you are the proud owner of one of the many brachycephalic breeds, such as the pug, Pekingese, Boston terrier, or shih tzu, you are quite familiar with bulging eyes! These dogs are distinguished by their protruding exposed eyes, extremely short pushed-in noses, and small nostrils. You are also likely to be all too familiar with what a nuisance it can be to treat the problems that go along with those exposed eyeballs.

When your dog's eyes appear to be bulging more than you are accustomed to, it is always a reason to be concerned. Take some time to examine both eyes carefully and pay special attention to how willing your dog is to tolerate the examination process.

What to Look For

Put on a pair of rubber gloves. With your dog in a comfortable position in a well-lit room, cradle his head in your hands and look him straight in the eyes. Pay strict attention to the symmetry of his eyes, looking for differences in shape, position, color, sensitivity, and response to light. Look at all of the structures associated with the eyes, including the upper and lower lids, the conjunctiva, or soft pink tissue immediately surrounding the eyeball, the sclera, or white of the eye, iris, pupil, and the third eyelid. The last of these will be difficult to view in a bulging-eyed dog. To get a look at it, place your thumb on the upper lid and, by placing gentle pressure on that lid, force the globe of the eye back into its socket. In the process, the third eyelid should rise passively from the lower corner of the eye closest to the muzzle. Use a bright flashlight to check your dog's pupils' response to light.

In addition, check the anterior chambers of your dog's eyes by shining the flashlight beam from the side, checking for any cloudiness in the forward-most portion (anterior chamber) of each globe. Finally, evaluate your dog's ability to see out of each eye. This test is to check his menace reflex. With the palm of your hand facing your dog, move your hand swiftly from a position about eighteen inches from your dog's eye to within about three inches of your dog's eye. A sighted dog will exhibit a menace reflex by blinking. The absence of that response is a strong indication of reduced or absent vision. Repeat the maneuver a few times to check your result, since some blind dogs may actually blink in response to the movement of air created by your hand. Be cautious, however, as most dogs do not find this procedure easy to tolerate.


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 Testicular Cancer Symptoms, Causes, Treatment - What is testicular cancer? - MedicineNet

Testicular Cancer (cont.)

What is testicular cancer?

Testicular cancer is a disease in which cells become malignant (cancerous) in one or both testicles.

The testicles (also called testes or gonads) are a pair of male sex glands. They produce and store sperm and are the main source of testosterone (the major male hormone) in men. These hormones control the development of the reproductive organs and other male physical characteristics. The testicles are located under the penis in a sac-like pouch called the scrotum.

Based on the characteristics of the cells in the tumor, testicular cancers are classified as seminomas or nonseminomas. Other types of cancer that arise in the testicles are rare and are not described here. Seminomas may be one of three types: classic, anaplastic, or spermatocytic. Types of nonseminomas include choriocarcinoma, embryonal carcinoma, teratoma, and yolk sac tumors. Testicular tumors may contain both seminoma and nonseminoma cells.

Testicular cancer accounts for only 1 percent of all cancers in men in the United States. About 8,000 men are diagnosed with testicular cancer, and about 390 men die of this disease each year. Testicular cancer occurs most often in men between the ages of 20 and 39, and is the most common form of cancer in men between the ages of 15 and 34. It is most common in white men, especially those of Scandinavian descent. The testicular cancer rate has more than doubled among white men in the past 40 years, but has only recently begun to increase among black men. The reason for the racial differences in incidence is not known.

What are the risk factors for testicular cancer?

The exact causes of testicular cancer are not known. However, studies have shown that several factors increase a man's chance of developing this disease.

  • Undescended testicle (cryptorchidism): Normally, the testicles descend from inside the abdomen into the scrotum before birth. The risk of testicular cancer is increased in males with a testicle that does not move down into the scrotum. This risk does not change even after surgery to move the testicle into the scrotum. The increased risk applies to both testicles.
  • Congenital abnormalities: Men born with abnormalities of the testicles, penis, or kidneys, as well as those with inguinal hernia (hernia in the groin area, where the thigh meets the abdomen), may be at increased risk.
  • History of testicular cancer: Men who have had testicular cancer are at increased risk of developing cancer in the other testicle.
  • Family history of testicular cancer: The risk for testicular cancer is greater in men whose brother or father has had the disease.
Male Illustration - Testicular Cancer
Male Illustration - Testicular Cancer
Medically Reviewed by a Doctor on 3/28/2014

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Featured: tobramycin ophthalmic solution (Tobrex) Main Article

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Return to the Poitiers index page
Panel 06 - Moses tells Aaron to take his censer and make atonement (Num 16:46)
Following the failed rebellion of Korah and the other priests against the authority of Moses, God unleashes a plague on the congregation. Moses instructs Aaron to light his censer and hurry to make atonement for the rebels to lessen God's wrath. Something of the urgency in the words of Num 16:46-47 is conveyed by Moses' twisting posture as he hands his brother the censer and points him towards the action.


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The 10 Most Expensive Health Insurance Markets

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Texas Psychiatrist

Is the Colorado mountain resort area issue a matter of healthy people engaging in athletic injuries? If so, it would seem that although the hospital would have higher costs, it would be spread amongst insurers from all areas when their insureds are traveling. Or, are insurance companies spreading the risk from us Texas folk trying to ski on to the local population?.

Stephen Sorsby, MD, MHA, FAAFP

One of the basic flaws in the idea of setting up marketplaces is that competition between insurers will drive down the cost of care. That's akin to believing that having more mortgage companies will drive down the cost of housing. Yes, there are some costs to be wrung out of the insurance system, on the administrative side. But, by law, 85% of premiums go toward actual medical costs. What is needed is competition on price between health care providers, either between physicians or between hospitals. Where that happens, premiums are lower. Where it doesn't, an insurance company can either pay the demanded price (and raise its premium), leave the market, or go out of business..

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