Genome sequences characterizing five mutations in RNA polymerase and major capsid of phages ϕA318 and ϕAs51 of Vibrio alginolyticus with different burst efficiencies

Abstract

Background: The burst size of a phage is important prior to phage therapy and probiotic usage. The efficiency for a phage to burst its host bacterium can result from molecular domino effects of the phage gene expressions which dominate to control host machinery after infection. We found two Podoviridae phages, ϕA318 and ϕAs51, burst a common host V. alginolyticus with different efficiencies of 72 and 10 PFU/bacterium, respectively. Presumably, the genome sequences can be compared to explain their differences in burst sizes.

Results: Among genes in 42.5 kb genomes with a GC content of 43.5%, 16 out of 47 open-reading frames (ORFs) were annotated to known functions, including RNA polymerase (RNAP) and phage structure proteins. 11 strong phage promoters and three terminators were found. The consensus sequence for the new vibriophage promoters is AATAAAGTTGCCCTATA, where the AGTTG bases of -8 through -12 are important for the vibriophage specificity, especially a consensus T at -9 position eliminating RNAP of K1E, T7 and SP6 phages to transcribe the genes. ϕA318 and ϕAs51 RNAP shared their own specific promoters. In comparing ϕAs51 with ϕA318 genomes, only two nucleotides were deleted in the RNAP gene and three mutating nucleotides were found in the major capsid genes.

Conclusion: Subtle analyses on the residue alterations uncovered the effects of five nucleotide mutations on the functions of the RNAP and capsid proteins, which account for the host-bursting efficiency. The deletion of two nucleotides in RNAP gene truncates the primary translation due to early stop codon, while a second translational peptide starting from GTG just at deletion point can remediate the polymerase activity. Out of three nucleotide mutations in major capsid gene, H53N mutation weakens the subunit assembly between capsomeres for the phage head; E313K reduces the fold binding between β-sheet and Spine Helix inside the peptide.

Figure 1

Different characteristics between phages ϕA318 and ϕAs51. (A) The plaque sizes within 16 h after phage infection at 25°C. (B) Transmission electron micrographs of phage particles. Virions were negatively stained with uranyl acetate. The bars represent a length of 100 nm. (C) One-step growth curves of phages in V. alginolyticus at 25°C for ϕA318 and 37°C for ϕAs51. The burst sizes were calculated by the amplifying ratio of Pt/P0 (phage titer at the plateau phase is divided by the inoculating phage titer). (D) Thermal stability of phages at various temperatures one hour. Samples were taken to determine the titer of the surviving particles and to calculate the percentage of infectious phage. In the stationary phase, standard errors (SE) for each phage were consistent at 16-20% (SE/mean), while the standard errors (SE) were approximate 16-72% (SE/mean) during the exponential phase.

Figure 2

Genome alignment and annotation for phages ϕA318 and ϕAs51 with the genomes from phages SP6 and K1E. (A) Genome alignment using query of ϕA318 against to phages ϕAs51, SP6 and K1E, (B) Alignment using query of SP6 against phages K1E, ϕA318, and ϕAs51. ϕAs51 map is highly similar to ϕA318 with 5 single nucleotide mutations (marked by m) and annotation as shown in Table 1 and GenBank access no. KF322026 and KF800937. This draw was done by BLAST [9] and Ring Image Generator [32]. Purple line represents skew(-); green for skew(+); black for GC%; grey for K1E; pink for SP6; blur red for ϕA318; cyan for ϕAs51; red for specific genes. (C) Mauve was used to efficiently construct multiple genome alignments, which provides a basis for research into comparative genomics among phages ϕA318 to its related phage K1E.

Figure 3

The sixteen promoters in phage ϕA318 genome were predicted and aligned with those of T7, SP6, K11, and T3 phages. The position of each promoter in ϕA318 genome was recorded as the number following the name of phage. The names of predicted promoters are given using their positions in ϕA318 genome, while the positions are shifted by two nucleotides in ϕAs51 genome.

Figure 4

Location of nucleotide deletion in ϕs51 and proposed products of subunits. (A) Two nucleotides were deleted found after 1545 nt in ϕAs RNAP. The proposed new ribosomal binding site was underlined. (B) Two fragments of peptides were predicted by PHYRE² for 3D structures. Green represents the N-term of full RNAP, while the cyan is for C-term. The predicted structures were superimposed together onto T7 RNAP skeleton.

Figure 5

Point mutations found in ϕAs51 and losses of interaction in the predicted structures of subunits. Dot lines between atoms represent the distances. βJ is located in P-domain. The pair of His-53 is in the F-loops of two capsomere subunits or between the subunit G and neighboring hexamer.