How does t4 adsorb to a cell

None of the Nik mutants could form plaques efficiently on BW Fig. As expected, Nik mutants did not adsorb to any other strains expressing OmpC, indicating that the Nik mutants lose the ability of adsorption dependent on OmpC. Characterization of T4 Nik mutants. A The solution containing the number of phage particles Nik1, left; Nik2, middle; Nik8, right panel indicated on the left was spotted on a lawn of the E.

Mutation sites of Niks, Nib, and Arl. A Amino acid sequence of the distal tips DT region in gp Arrowheads indicate substitutions of amino acids in Nik1, 2, 8, Nib, and Arl mutants. Schematic diagrams of the DT region viewed from the lateral surface B and the top surface C are shown. Lines indicate positions of Niks, Nib, and Arl mutations.

Although we isolated five Nib mutants, all of them had the same base substitution at CT of gene 37 , causing the amino acid substitution TI. Characterization of the T4 Nib mutant. A A solution containing the number of Nib phage particles indicated on the left was spotted on a lawn of the E. B Adsorption analyses with the E. As a result, three Arl mutants were isolated see Experimental Procedures.

Sequence analyses demonstrated that all three had the same two base substitutions at TC and AG of gene 37 , causing one amino acid substitution, YR Fig. Growth of the T4 Arl mutant. A solution containing the number of Arl phage particles indicated on the left was spotted on a lawn of the E. The requirements of LPS and OmpC for T4 adsorption were established decades ago and two different modes of adsorption, one of which depends on OmpC and the other is independent of OmpC, have been proposed Prehm et al.

In this study, we performed a comprehensive analysis of LPS structure in relation to each mode of T4 adsorption using various LPS mutants.

OmpC exists as a trimer in the outer membrane. The direct binding between OmpC and DT may be supported by our preliminary results; the amino acid substitution at a phenylalanine in the extracellular loop 4 of OmpC caused the inability of T4 adsorption, and this defect was recovered by the mutation of DT Unpublished data.

Also, Trojet et al. Similarly to OmpC, DT is also composed of a trimer of gp Additionally, Buehler et al. These observations suggest that the width of OmpC is compressed by the surrounding LPS in the outer membrane, rendering OmpC in a form favorable to interaction with DT.

The observations that shortening of the inner core, but not the outer core, drastically reduced adsorption Fig. Previous studies showed that T4 adsorbed to LPS which has only a glucose in outer core Yu and Mizushima and T4 adsorption to the B strain was inhibited by glucose Dawes All these strains commonly have a terminal glucose s in outer core.

Taken together, a terminal glucose s without a branch is an important factor for T4 adsorption independent of OmpC and should be a site of the DT interaction. Probably, one of them, the shorter, has a terminal glucose Glc II potentially eligible for an interaction with DT. When these two types of LPSs are randomly intermingled on a cell surface at a density of 0.

Also, Bartual et al. In these mutants, the adsorption dependent on OmpC is inactivated Fig. These results suggest that DT binds to the center of an OmpC trimer vertically and that the lateral surface of the DT head domain interacts at multiple sites with the extracellular loops of OmpC. We only obtained one Nib mutant with one amino acid substitution of TI Fig.

T is located at the top of the DT head domain and is flanked with glycines, making the side chain of threonine stand out. Valine has a side chain with a branch and is more bulky than glycine. Therefore, it would be reasonable to assume that a valine next to T hinders an interaction of T to LPS. Therefore, the top surface of the DT head domain containing T plays a key role in the recognition of a terminal glucose in the outer core.

In this connection, Bartual et al. Apparently, further study of these candidates in binding to a glucose or other sugars will give more insight into the interaction between DT and LPS. The Arl mutant conserves threonine at which is important for recognizing a terminal glucose in the outer core, and it is located at the top of the DT head domain. On the other hand, Y is located at the border between the lateral and top surfaces of the head domain Fig.

The side chain of Y protrudes toward the top surface Bartual et al. In addition, the side chain of arginine is longer than that of tyrosine. Antibiotics are now losing potency because of increasing antibiotic resistance and the lack of new types of antibiotics.

This situation spotlights a resurgence of phage therapy. However, in contrast to antibiotics that are effective against a wide range of bacterial species, phages infect only a limited range. Because of this strict host specificity, when a new pathogenic bacterium appears, a naturally isolated phage that can infect it is necessary and its safety should be established before use for therapy.

This approach takes much time, labor and cost, and isolation of useful phage is not guaranteed. This problem might be solved if we could control the host specificity of a known phage to infect a given strain.

Isolation of Arl mutants implies that it is not difficult to change host range by manipulating the structure of the DT head domain, especially the top surface, to recognize various types of LPSs.

Since current technology can replace any amino acid, even all, with designated one s , T4 might be largely expanded to become a tool for phage therapy. Table S1. Changes in T4 host range by mutations located in genes 37 and 38 in Nik and Nib mutants. Figure S1. Analysis of LPS purified from E. We cordially thank Dr. John W. Drake at the U. National Institute of Environmental Health Sciences for invaluable help with the manuscript.

National Center for Biotechnology Information , U. Journal List Microbiologyopen v. Published online Jun 6. Author information Article notes Copyright and License information Disclaimer. Yuichi Otsuka, Email: pj. Corresponding author.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. This article has been cited by other articles in PMC.

Abstract Bacteriophages have strict host specificity and the step of adsorption is one of key factors for determining host specificity. Introduction Bacteriophages Phages are viruses that infect and kill bacteria, but the host range of phages is generally narrow. Experimental Procedures E. Table 1 E. Open in a separate window.

Construction of a plasmid To clone E. Bacteriophage adsorption assay E. Purification of lipopolysaccharides LPS from E. Figure 1. Figure 2. Figure 4. Figure 3. Figure 5. Figure 6. Figure 9. Figure 7. Figure 8. Discussion The requirements of LPS and OmpC for T4 adsorption were established decades ago and two different modes of adsorption, one of which depends on OmpC and the other is independent of OmpC, have been proposed Prehm et al. Conflict of Interest The authors have no conflict of interest to declare.

Supporting information Table S1. Click here for additional data file. Acknowledgments We cordially thank Dr. References Amor, K. Distribution of core oligosaccharide types in lipopolysaccharides from Escherichia coli. Crystal structure of osmoporin OmpC from E.

Plasticity of Escherichia coli porin channels. Dependence of their conductance on strain and lipid environment. The function of tail fibers in triggering baseplate expansion of bacteriophage T4.

FEMS Immunol. Characterisation of the bacteriophage T4 receptor site. Nature — Adsorption curves of of R -type and M -type phage stocks isolated from individual plaques of the R 8 and M 8 populations are compared: R 8 open diamonds , R 8 P 1 open triangles , R 8 P 2 open circles , M 8 closed diamonds , M 8 P 1 closed triangles , M 8 P 2 closed circles , and the plaque isolated residuals of M 8 P 1 open squares.

Error bars represent the standard deviation of three replicates for R 8 and M 8. For the plaque-isolated cultures, error bars present the standard deviation of at least three individual plaque-isolated cultures. DNA sequencing revealed genetic differences between the R -type and M -type phage stocks in gene 37, which codes for the protein constituting the distal half of the phage T4 long tail-fiber [23] Fig. The phage T4 long tail-fiber is the organelle of attachment and recognition [24].

Gene 38 was also sequenced since proper trimeric assembly of gp37 is assured with the assistance of gp38 [25]. Genes 34, 35, 36, and 37 code for the major structural proteins of the phage T4 long tail-fiber diagram of the long tail-fiber structure is adapted from Fig. The residual stocks contained two point mutations resulting in changes to the amino acid sequence of domains D5 and D7 of the long tail-fiber.

The parent T4 stock is a wild-type strain that aligned nearly completely in genes 37 and 38 with wild-type phage T4T except for two nucleotides positions 1, and 1, of gene The main fraction stocks M 4 and M 8 contained the exact nucleotide sequence of the parent T4 stock S in both genes 37 and 38; the residual fraction stocks, R 4 and R 8 , contained two important differences.

While gene 38 of R 4 and R 8 aligned completely with gene 38 of S , gene 37 contained two distinct point mutations which resulted in new amino acid residues on gp37 Fig. The substitution of adenine A with cytosine C at nucleotide 1, replaced a negatively charged aspartic acid Asp at residue of the wild-type T4 with a nonpolar alanine Ala.

At nucleotide 1, of R 4 and R 8 , guanine G was substituted by adenine A , resulting in a positively charged lysine Lys supplanting the negatively charged glutamic acid Glu at residue of the wild-type strain. Residues and are found in domains D5 and D7, respectively, of the distal half of the T4 long-tail fiber [23] Fig. The authors suggested that the existence of the residual fraction may have evolved as a form of diversified bet-hedging [21]. According to this theory, the slow adsorbing members of the population would be the result of phenotypic variations of the same genotype, evolved to guarantee some phages remain unadsorbed during periods of poor growth conditions that inhibit productive infection of the host cell while some phages such as T7 can replicate in a stationary phase culture, most cannot.

This can be thought of as an alternative strategy to lysogeny or pseudolysogeny, which are common tactics employed by bacterial viruses to survive long periods of nutrient limitation [26] — [28]. Another proposed explanation by the authors for the existence of a residual fraction is errors in protein processing [8] , which can be as high as 0. Transcription and translation errors may even comprise an evolutionary advantage in the development of complex mutations [29] , [30].

At least two lactococcal phages have been shown to exhibit heterogeneity in their adsorption characteristics due to proteolytic processing of tail fiber proteins [31]. Regardless of the explanation, the hypothesis that the heterogeneity of phage populations provides an evolutionary advantage to the virus is quite plausible and can explain residual fractions in many phage-host systems. Adsorption data reported for a wide range of tailed phage families all suggest the existence of a residual fraction [1] — [11] , [19] , [20].

Therefore, it is possible that the heterogeneity of a phage stock is a universal phenomenon common to most or even all phages. While the work of Gallet et al. Should the adsorption trait be due to phenotypic variations of a specific T4 genotype, one would expect that the poorly adsorbing phage trait would be superseded by the efficiently adsorbing phages during the amplification process.

Similarly, if the adsorption traits were brought about by errors in protein processing, isolated phages from the residual fraction should produce progeny with adsorption behavior equivalent to that of the original stock S. In actuality, this did not occur, even when isolating individual plaques Fig. Instead, the poor adsorption efficiency of the residual fraction was passed on to progeny phages during amplification Figs. The heritability of the adsorption characteristics of R 1 , which was isolated from the original stock S , thus suggests at least some modifications to the phage genome are at work and passed on to further generations in a phage population.

Importantly, the heterogeneity of the phage population is not merely the result of R -mutants already present in the phage population. All plaques isolated in this way produced phage populations with observable residual fractions M 8 P 1 and M 8 P 2 , Fig. If these phages were adsorption delinquent merely due to random transcriptional or translational errors, they would have produced progeny with the same adsorption traits as the parent strain.

The results of Fig. The adsorption characteristics of R -type and M -type phages were not the only inherited traits observed in the study. The poor adsorption efficiency of the R -type phages in liquid culture translated to slow plaque development on agar plates Fig. While the residual fraction consists of phages that are unable to adsorb to a host cell over the course of an adsorption experiment, clearly they are not completely deficient as they have the ability to form plaques on agar lawns.

Stocks amplified from individual plaques of R -type phages yielded plaques of similar morphology to those shown in Fig. The reduction in phage productivity observed for R -type phages during stock amplification Fig. It is a well understood phenomenon in phage ecology that lowering the initial MOI in an amplification experiment results in a longer growth period for the host cells, usually resulting in higher final titers.

This is due to the fact that the reduction in the initial number of virus particles allows the cell culture to grow to a higher density before the phage overtakes the population and population-wide lysis is observed. The authors have previously shown that reducing the adsorption efficiency of a phage stock through the manipulation of environmental parameters has a similar effect to lowering the initial MOI in an amplification experiment [9].

Comparison of the amplification dynamics of the M 1 and R 1 phage stocks shown in Fig. The dynamics of infection were greatly modified by simply using a phage stock with inherently low adsorption efficiency — a phenomenon that normally requires environmental manipulation — providing another validation of the assumption that the poor adsorption efficiency of the R -type phages is a conserved trait passed on to progeny.

There are many possible genetic causes for the poorly adsorbing behavior observed in the residual fraction. For example, mutations to any number of genes coding for phage proteins could lead to adverse structural or morphological changes to the mature phage particle.

In this study, DNA sequencing focused on genes coding for what were considered likely candidate proteins influencing adsorption capability: the long tail fibers. The phage T4 long tail-fiber, as shown in Fig.

The distal half, which interacts directly with the host cell, is composed of a trimer of gp37 [23]. Proper trimeric assembly of gp37 is assured by the product of gene 38 [25]. Consequently, mutations to genes 37 and 38 are most likely to directly affect the adsorption behavior of the virus. The results of DNA sequencing highlighted two mutations in gene 37 of the R 4 and R 8 stocks that offer the most comprehensive explanation for the differences in adsorption behavior among the phage stocks tested Fig.

Located in domains D5 and D7 of the distal half of the T4 long tail-fiber, these mutations introduce amino acid residues with significant differences in their side chain structure and charge. The distribution of polar and nonpolar side chains is an important factor governing the folding of a protein. Therefore, disrupting the charge balance on these important structural globules could potentially lead to a weakened or dysfunctional tail fiber.

For example, in D5, a hydrophobic, nonpolar molecule Ala replaces a negatively charged hydrophilic molecule Asp. The fact that both the R 4 and R 8 stocks possessed the same genetic defects provides conclusive evidence that point mutations are conserved from one generation to the next Fig. It should be noted that samples for DNA sequencing were taken from amplified stocks, rather than from single plaques. This may seem, at first, an unconventional practice.

It does not enable the identification of the full genotypic variations present in the stock. It will only allow the determination of the sequence of the predominant genotype present, as opposed to the genotype of a specific phage particle. However, considering the implications of the results presented here and that even a single plaque is the product of multiple rounds of progeny, samples taken from any stock solution or any plaque will contain a variety of genotypes.

This is supported by the results of Fig. In fact, a T4 plaque likely consists of millions of phage particles propagated over multiple generations. Therefore, the DNA sequence obtained from any sample will be representative of the predominant genotype within that plaque or stock. The sampling method for DNA sequencing in no way diminishes the fact that the residual fraction phages isolated did, in fact, contain two conserved point mutations to gene Is the existence of two point mutations in the long tail-fiber of a phage T4 mutant enough to explain the drastic changes in adsorption behavior among the R -type and M -type populations?

The results of similar studies suggest that it is. For example, one study on phage T4 found that it could switch hosts from E. Finally, a comprehensive analysis of the T2-like phage Ox2 found that it contained specific, hypervariable regions in gene 38 which enabled the virus to expand its host receptor specificity to include various outer membrane proteins [34] , [35].

In fact, some mutants of Ox2 were able to switch from a protein receptor to a carbohydrate [34]. Most studies have suggested that fast mutation rates within phage species can lead to broader host ranges or more efficient adsorption.

Some have even speculated that the high rates of mutation observed are a direct result of the Darwinian struggle between predator and prey [34]. A common method to shield a cell from virus invasion is to prevent the very first step in the process: attachment to the host [36] , [37]. Changes in the receptors used for the reversible or irreversible step in virus adsorption can lead to resistant bacterial strains. Consequently, evolutionary driving forces may have placed pressure on the regions within some phage genomes that code for the phage adsorption machinery to be more prone to mutations.

A side-effect of this variability is the incidental production of phage particles with poor adsorption capabilities to the specific host being studied, leading to the residual fraction observed in most phage T4 adsorption experiments. It may thus be tempting to consider the residual fraction of phage T4 a by-product of evolution; engineering mishaps in the race for improved phage fitness; however, the more complete view sees the potential benefits of poorly adsorbing members of the phage population.

As noted by Gallet et al. In addition, the adsorption efficiency of a population has been shown to strongly correlate with the conditions of infection [9] , [10] , suggesting the residual fraction contains phages able to adsorb well in a different environment. Residual fraction phages consistently demonstrated poor adsorption efficiencies, small plaque morphology, and lower amplification productivities.

Numerous rounds of amplification could not yield phage populations characteristic of the parent strain. Even single plaques of M -type phages produced small numbers of R -type phages whose adsorption traits were both distinct from the parent strain and passed on to progeny.

The best explanation for the differences between the residual fraction phages and the main fraction phages observed in this study is the presence of two point mutations in gene 37, which are likely to impact the structure of the long tail-fiber used in host cell attachment and recognition. The results suggest further experimentation would lead to additional mutations that could either hinder or improve phage adsorption.

These small defects, while generally detrimental to phage fitness in the experiments performed, might represent an overall, long-term evolutionary benefit by assuring some phage particles remain free in solution during adverse growth conditions. These mutations can also explain the residual fraction observed in phage T4.

Adsorption of R 1 Mi and M 1 Ri phage stocks. The adsorption dynamics of M 1 closed diamonds , M 2 closed circles , and M 3 closed triangles are shown as a reference. The adsorption dynamics of R 1 closed diamonds , R 2 closed circles , and R 3 closed triangles are shown as a reference.

Adsorption data is plotted as the concentration of free phages remaining in solution normalized to the initial titer. The curves indicate trends and are not the result of a modeling equation. Conceived and designed the experiments: ZS DS. Performed the experiments: ZS.

Analyzed the data: ZS DS. Wrote the paper: ZS DS. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Many bacteriophage populations display heterogeneity in their adsorption characteristics; a portion of the phage population remains free in solution throughout adsorption experiments residual fraction.

Introduction Heterogeneity in the adsorption characteristics of a bacteriophage population has been widely reported since the first half of the 20 th century. Materials and Methods Organisms and media Bacterial cultures of E. Adsorption experiments Adsorption experiments were carried out at room temperature in 1.

Isolation of two distinct subgroups The heterogeneity of the phage population was studied by dividing the original stock solution into two subgroups: the main fraction and the residual fraction. Phage amplification Amplification dynamics of some phage stocks were studied in detail. Download: PPT. Results Nomenclature and experimental design The following nomenclature is introduced to describe different sets of phage T4 subgroups presented in this study refer to Fig.

Figure 1. Schematic of adsorption experiments and nomenclature. Adsorption characteristics of the isolated phage stocks The heterogeneity of the T4 population is prominently displayed in the adsorption data presented in Fig. Plaque morphology While the residual fraction consisted of phages with a very poor aptitude for adsorption in liquid culture, they were still able to form plaques on an agar plate. Dynamics of infection differ between main fraction and residual fraction phages While the adsorption curves of Fig.

Characterization of the heterogeneity of phage T4 populations The nature of the heterogeneity of the T4 population was studied through a series of isolation and amplification experiments. Figure 5. Residual fractions of the M -type and R -type phage stocks. Figure 6.

Adsorption of populations originating from single plaques. Genetic analysis DNA sequencing revealed genetic differences between the R -type and M -type phage stocks in gene 37, which codes for the protein constituting the distal half of the phage T4 long tail-fiber [23] Fig.

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