Physiological Properties and Genome Structure of the Hyperthermophilic Filamentous Phage φOH3 Which Infects Thermus thermophilus HB8

Abstract

A filamentous bacteriophage, φOH3, was isolated from hot spring sediment in Obama hot spring in Japan with the hyperthermophilic bacterium Thermus thermophilus HB8 as its host. Phage φOH3, which was classified into the Inoviridae family, consists of a flexible filamentous particle 830 nm long and 8 nm wide. φOH3 was stable at temperatures ranging from 70 to 90°C and at pHs ranging from 6 to 9. A one-step growth curve of the phage showed a 60-min latent period beginning immediately postinfection, followed by intracellular virus particle production during the subsequent 40 min. The released virion number of φOH3 was 109. During the latent period, both single stranded DNA (ssDNA) and the replicative form (RF) of phage DNA were multiplied from min 40 onward. During the release period, the copy numbers of both ssDNA and RF DNA increased sharply. The size of the φOH3 genome is 5688 bp, and eight putative open reading frames (ORFs) were annotated. These ORFs were encoded on the plus strand of RF DNA and showed no significant homology with any known phage genes, except ORF 5, which showed 60% identity with the gene VIII product of the Thermus filamentous phage PH75. All the ORFs were similar to predicted genes annotated in the Thermus aquaticus Y51MC23 and Meiothermus timidus DSM 17022 genomes at the amino acid sequence level. This is the first report of the whole genome structure and DNA multiplication of a filamentous T. thermophilus phage within its host cell.

Keywords: Inoviridae; Thermus thermophilus; filamentous phage; hyperthermophilic phage; replicative form.

Figure 1

Morphology of phage φOH3. Shown are transmission electron micrographs of inovirus φOH3 negatively stained with 2% (wt/vol) potassium phosphotungstate (pH 7.0). The scale bar is 500 nm.

Figure 2

Heat, pH and salt stability of φOH3. Surviving fractions (P/P0) are plotted, where P0 is the φOH3 initial titer, and P is the mean titer from triplicate assays after incubation for 1 h at the indicated temperature (A), 24 h at the indicated pH (B), or 24 h at the indicated NaCl concentration (C).

Figure 3

Propagation of φOH3 in T. thermophilus HB8. (A) One-step growth curve of φOH3. (B) Agarose gel electrophoresis of phage φOH3 genomic DNA. The φOH3 genome was extracted with alkaline lysis after the indicated cultivation periods. Double stranded RF DNA and ssDNA were distinguished by acrydine orange staining. Corresponding estimated copy numbers of pTT8 (Takayama et al., 2004), RF DNA, and ssDNA of φOH3 are shown under the gels.

Figure 4

Genetic map of φOH3 RF DNA. Predicted ORFs are indicated by arrows. Arrow colors show putative gene functions as follows: replication, red; structure, green; assembly, yellow. Since the conserved domain of Zot was detected in the ORF8 product, ORF8 might function for phage assembly.

Figure 5

Southern blot confirming the plus strand of phage φOH3. Lanes: M, lambda -Hind III marker; 1, φOH3 ssDNA; 2, φOH3 RF DNA. (A) RNA probe-1, which was transcribed by T7 RNA polymerase (antisense strand of ORF2). (B) RNA probe-2, which were transcribed by T3 RNA polymerase (sense strand of ORF2). Bands corresponding to ss DNA or RF DNA are indicted with arrows.

Figure 6

Comparison of the amino acid sequences of ORF5 product (φOH3), 1hgv (PH75), Pf3_2 (Pf3), and g8p (M13). Identical and similar amino acid residues are indicated in red and blue, respectively. Hyphens represent gaps in the alignment.

Figure 7

SDS-PAGE of proteins from purified φOH3 virions staining with Coomassie brilliant blue R250. The numbers indicate the bands excised for mass spectrometric analysis (Supplemental Tables 1, 2). The left lane contains protein molecular mass markers (kDa). Red colored and underlined characters indicate matched peptides and domain of DNA translocase FtsK (PRK10263).

Figure 8

Comparison of the genome organization of phage φOH3 with that of the phage Pf3, B5, M13, and T. aquaticus Y51MC23 genomes. Open reading frames with similar functions are indicated using the same color scheme used in Figure 4. White arrows show regions encoding hypothetical proteins with unknown functions.