Sakaguchi et al. 10.1073/pnas.0505503102. |
Fig. 5. Genome comparison of c-st and a Bacillus subtilis temperate phage, SPb. The cluster III region (A)and the cluster II region (B)of the SPb genome (Upper) were compared with analogous regions of the c-st genome (Lower). Horizontal arrows represent ORFs on each phage genome, and ORF numbers or gene products are indicated above or below each arrow. ORFs conserved in both phages are indicated by black arrows, and homologous regions are connected by gray shading. Striped ORFs represent transposase genes on insertion sequence (IS) elements found in the c-st genome. Four c-st ORFs, whose gene products have been identified in the c-st phage particle by our preliminary N-terminal amino acid sequencing analyses, are marked with asterisks.
Fig. 6. Electron micrographs of purified phage c-st negatively contrasted with uranyl acetate. The c-st phage was propagated on strain (C)-AO2 in LYG medium (1% lactalbumin/2% yeast extract/0.5% glucose/0.15% cysteine hydrochloride, pH 7.2). After removing cell debris by centrifugation at 11,000 × g for 20 min at 4°C, phage particles were recovered by polyethylene glycol precipitation and resuspended in PBS. The suspension was placed on carbon-coated grids and negatively contrasted with 2% uranylacetate dihydrate (Merck). Electron micrographs were taken at magnifications of ×20,000 (A), ×30,000 (B), ×50,000 (C) on a transmission electron microscope (H-7100, Hitachi) operated at 75 kV. The virus head is 81.0 ± 8.3 nm wide and 91.0 ± 10.1 nm long. The phage tail has an overall length of 375.8 ± 50.9 nm. The tail core is 9.3 ± 2.4 nm wide, surrounded by a sheath 22.9 ± 2.4 nm wide. A tail-tip fiber is observed at the extremity of the tail. The average lengths were determined by tracing with a calibrated map measure from 35 virions. Because the sheath of c-st is highly fragile, most of the observed virions contained partially disrupted sheath structures (B and C), and only a small number of virions contained intact sheaths. All of the previously published electron micrographs of BoNTX phages exhibited such partially disrupted virions, and such structures were sometimes described as "contracted sheath." However, intact and partially disrupted sheaths show no difference in width. The length of partially disrupted sheaths is also highly variable. Thus, it is unlikely that the sheath of c-st is contractile. Although the tail part covered by sheath is rather straight, the uncovered parts were flexible. These morphological features suggest that the c-st phage is an atypical siphovirus. It is not known whether the phage particle with partially disrupted sheath is infective or not. The c-st phage is phylogenetically related to SPb, a temperate phage of B. subtilis (see Fig. 5). SPb is also a siphovirus containing a long tail of about 358 nm, but the tail morphology is different [Warner, E. D., Kitos, G. A., Romano, M. P. and Hempgill, H. E. (1977) Can. J. Microbiol. 23, 45–51].
Fig. 7. Structures of four IS elements identified on the c-st genome. The c-st genome harbored a total of 12 copies of IS elements. They were classified into seven types, and all were previously unidentified IS elements belonging to the IS200/605 family, which lacks terminal inverted repeats. By comparing the IS-inserted regions in c-st with analogous unoccupied regions in other BoNTX/C1 or D phages (c-468, c-203, d-1873, c-6813, d-4947, and d-sa), structures of four IS elements were defined. (A) The data for ISCbt1. (B) The data for ISCbt2. (C) The data for ISCbt3. (D) The data for ISCbt4. ISCbt1, ISCbt2, and ISCbt3 are inserted site-specifically, without target-site duplication. The preferable target sequences of each element, indicated by underlines, were identified by comparing the multiple insertion sites in c-st and other BoNTX phages. Inverted or direct repeat sequences identified in each IS element are indicated by horizontal arrows.
Fig. 8. The genome structure of the BoNTX/D gene-flanking region in a BoNTX/D phage, d-1873. DNA sequences of upstream and downstream regions of the BoNTX/D gene cluster of d-1873 were determined and compared with the analogous regions of c-st. The DNA sequence for the BoNTX gene cluster of d-1873 has been determined by our group [Nakajima, H., Inoue, K., Ideda, T., Fujinaga, U., Sunagawa, H., Takeshi, K., Ohyama, T., Watanabe, T. & Oguma, K. (1998) Microbiol. Immunol. 42, 599–605] (accession no. AB012112). Horizontal arrows represent ORFs on each phage genomes, and ORF numbers or gene products in c-st are indicated. ORFs conserved in both phages are indicated by black arrows and ORFs unique to each phage by open arrows. Regions with significant DNA sequence similarities are connected by gray shading, and percentile sequence identities observed between homologous regions are indicated. The deletion of CST094 in d-1873 is most likely to have been generated by homologous recombination between the direct repeat sequences, each located upstream and downstream of CST094.