Category:TeamBlue

Figure 4: A comparison of DNA affinity for two versions of the proposed exonuclease using EMSA (elecrophoretic mobility shift assay). When starved of ATP, TmMRE loses affinity for DNA binding. MRE requires dimerization followed by ATP hydrolysis to perform exonuclease activity.

Figure 5: Schematic of DNA binding mechanism via the Antibody DNA-binding Assay.

These results allowed the researchers to conclude with confidence taht Q9X1X0 is a DNA-binding and exonuclease protein.

The portal complex can work against large internal forces. Internal forces and the rate of DNA packaging were measured. In addition to demonstrating the phi29 motor complex produces force when DNA is being packaged into the capsid.

Usage of microspheres were utilized by attaching it to partly prepackaged complexes and to the tethered end of the DNA that was captured in the optical trap. Optical tweezers measured the forces acting upon the packaging on DNA while the other microsphere was held by a micropipette (Fig 1a). The force increases when the portal motor pulls in the tethered DNA and less force when the DNA is not being pulled in (Fig. 2). Stalling of DNA packaging occurs at higher forces (Fig. 3). Build up of internal force is created when more DNA is packaged in which it exerts opposing forces causing the motor to slow. Internal forces are also created in order to initiate the injection of phage genomic DNA into a bacterium.

Troll's tail sheath genome obtained good BLASTP hits towards Listeria Bacteriophage A511's tail sheath protein, tsh. With a 98% query cover, E-value of 9e-156 and 45% identity, it suggests that A511's tsh gene shows an significant homology towards Troll's tail sheath protein gene.

An HHPred job of S5YPI2 provides evidence that G4FG24 and S5YPI2 have sequence alignment.

BLASTP: BLAST YP_008430900.1 (S5YPI2) YP_007977993 (Q9X1X0) is a result with E-value of 2e-10, score of 70.9, query coverage of 91%, 26% identity.

HHPred: The protein sequence of S5YPI2 returns the first result 3THO which is part of Q9X1X0 (Score: 296.59; E-value: 2e-38; Similarity: 0.301; % Identity: 23)

Protein may be dual exonuclease/metallophosphatase with DNA-binding activity.

Peptidoglycan of B. cereus was incubated with 5 micrograms LysBPS13 for 30 min to determine the amidase activity of LysBPS13 by the amount of free N-acteylmuramic acid liberated. The N-acteylmuramic acid product was first degraded to lactic acid, and then acetaldehyde to be identified colorimetrically with p-hydroxydiphenyl (PHD). The results showed an increase in free N-acteylmuramic acid from cleavage of the bond between N-acetylmuramic acid and L-alanine (Figure 2), demonstrating LysBPS13 has N-acetylmuramyl-L-alanine amidase activity. A glycosidase assay ruled-out LysBPS13 as a glucosaminidase or a muramidase by showing free reducing sugars were not generated by the peptidoglycan after LysBPS13 treatment. Additionally, when 5 micrograms/milliliter of purified, recombinant LysBPS13 was added to B. cereus cells, almost all the B. cereus cells were effectively lysed within 10 min (Figure 1.c), further supporting N-acetylmuramoyl-L-alanine amidase activity of LysBPS13.

The author's exposed Listeria Bacteriophage A511 baseplate to a-Gp proteins, purified antibodies for cross-linking (with Staphylococcus ISP), and immunogold labeling. The results were found by use of transmission electron micrograph.

In Figure 7, TEM images depict the antibody cross-linking of A511 and ISP. Immunogold labelling is seen as well by black dots on the image. A contracted tail fiber was identified in the A511 tail through the identification of Gp108.

In Figure 8, an immunogold label TEM shows the contracted tail of A511. In addition to antibody cross-linking and in silico prediction, a model of the A511 tail was generated and compared to the TEM image. The presumed location of gene products including tsh (tail sheath protein) are seen in the model.

Targeted BlastP of the Enterobacteriophage T4 td gene (T4p237) against myoviridae shows strong homology with conservation of thymidylate synthase function among hits. Bacillus phage troll gene TROLL_50 (ThyX) displays 99% query cover (Identity 38%) with an e-value of 8e-60, which is within threshold limits. Therefore, it reasonable to assume that the ThyX gene product has a similar thymidylate synthase function to the annotated Enterobacteria phage T4 cousin.

Experiments were performed on wild type T4 td and T4D td6 (a variant containing a missense mutation in the relevant region).

A concentration of ~10^13/ml phage ghost particles was collected and thymidylate synthetase activity measured using a radioactive assay of [3H dUMP] for two hours.

Table 1 demonstrates thymidylate synthase activity is significantly lower in the td6 mutant (< 0.01 mol/min) than the wild type (0.16 mol/min), indicating that the td gene in T4 phage is involved with thymidylate synthase activity.

Researchers generated rabbit-based polyclonal antibodies against purified J proteins along with secondary antibodies of biotinylated anti-rabbit IgG conjugated to horseradish peroxidase (Amersham) with immunogold labeling. The results were observed using electron microscopy.

Figure 7 shows the presence of gold-conjugated antibodies at the side of the phage baseplate, which causes phage tails to clump together in 58 of the 78 total phage particles observed. The remaining samples were visualized with conjugated gold around the baseplate area. This allowed the researches to conclude with confidence that P2 protein J is a component of the baseplate.