Isolation and characterization of Micromonospora phage PhiHAU8 and development into a phasmid

Abstract

PhiHAU8, a temperate Micromonospora phage, which is capable of infecting Micromonospora sp. strains 40027 and A-M-01, was isolated. The PhiHAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca. 42.5 kb) with double-stranded DNA and cohesive ends. PhiHAU8 was most stable at 4 degrees C in Difco nutrient broth within a pH range of 6 to 12. PhiHAU8 plaque formation on Micromonospora sp. strain 40027 was optimal with 32 mM Ca(2+) and 30 mM Mg(2+). A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a lambda cosmid vector in Escherichia coli and as a phage in Micromonospora sp. strain 40027 was constructed. Pulsed-field gel electrophoresis of AseI-digested total DNA showed that PhiHAU8 DNA integrates into the 500-kb AseI fragment of Micromonospora sp. strain 40027.

FIG. 1.

Electron micrograph of ΦHAU8 negatively stained with uranyl acetate. Dimensions are indicated in nanometers.

FIG. 2.

Southern hybridization with ³²P-labeled ΦHAU8 (lane A) as the probe to prove that ΦHAU8 integrated into the Micromonospora sp. strain 40027 chromosome in lysogenized strain LXH8. All samples were digested with AseI and separated by PFGE. Electrophoresis was performed for 20 h at 6 V/cm with a 20- to 80-s switch time at an included angle of 120°. The lanes labeled with lowercase letters are autoradiographs of the lanes labeled with the corresponding uppercase letters. A clear size difference (500 versus 540 kb) for the AseI fragments detected between strains 40027 (lane B) and LXH8 (lane C) was further proved by a hybridization signal detected in lane c (corresponding to lane C) but not in lane b (corresponding to lane B). AseI fragments of S. coelicolor M145 (lane D) were used as standards; the sizes (in kilobases) are indicated on the right.

FIG. 3.

ΦHAU8 DNA has cos ends. The gel on the left shows a ladder of multimers after ligation was observed in DNA of ΦHAU8 (lane B) by PFGE, which is similar to the DNA of ΦHAU3 (lane A) used for comparison, as well as a size standard. The sizes of DNA fragments (in kilobases) are indicated on the left and right. Electrophoresis was performed for 15.5 h at 6 V/cm with a 5- to 60-s switch time at an included angle of 120°. In the gel on the right, BamHI-digested ΦHAU8 DNA samples that were heated at 70°C (lane D) and were not heated (lane C) before loading were run in parallel. Clearly, one of the two comigrating bands (which had a twofold intensity and is indicated by the solid arrow) in lane C is a sum of the 1.3-kb fragment (corresponding to fragment J1 in Fig. 4) and the 0.7-kb fragment (corresponding to fragment J2 in Fig. 4) in lane D, which are indicated by open arrows. Lambda DNA digested with HindIII (lane E) and a 1-kb ladder (lane F) were used as size standards; the sizes (in kilobases) are indicated on the right.

FIG. 4.

BamHI restriction maps of ΦHAU8 and its phasmid derivative, pJTU120. The DNA region dispensable for plaque formation and lysogeny in Micromonospora sp. strain 40027 is shaded. The circular plasmid below ΦHAU8 is the cosmid part (pHZ1358) of the ΦHAU8-derived phasmid, pJTU120. tsr, thiostrepton resistance gene, selectable in Micromonospora sp. strain 40027; bla, β-lactamase gene, selectable in E. coli. cos at the ΦHAU8 termini indicates cohesive ends at the ends of fragments J1 and J2, and cos in circular plasmid pHZ1358 was derived from E. coil phage λ. The sizes of fragments in ΦHAU8 (in kilobases) are indicated. ori/pIJ101, rep/pIJ101, and sti/pIJ101 (5, 8) derived from the multicopy Streptomyces plasmid pIJ101 are not functional in Micromonospora sp. strain 40027 and are thus not described in detail. The presence of the restriction sites indicated in pHZ1358 other than the BamHI sites was not examined in ΦHAU8.