DNA Synthesis and Gene Expression in Bacillus subtilis Infected with Wild-Type and Hypermodification-Defective Bacteriophage SP10

Abstract

A hypermodified base (Y-Thy) replaces 20%o of the thymine (Thy) in mature DNA of Bacillus subtilis phage SP10. Two noncomplementing hypermodificationdefective (hmd) mutants are described. At 30°C, hmd phage carried out a normal program, but at temperatures of .37TC, the infection process was nonproductive. When cells were infected at 37°C with hmd phage, DNA synthesis started at its usual time (12 mmin), proceeded at about half the normal rate for 6 to 8 min, and then stopped or declined manyfold. All, or nearly all, of the DNA made under hmd conditions consisted of fully hypermodified parental DNA strands H-bonded to unhypermodifled nascent strands. The reduced levels of DNA synthesis on September 25, 2020 by guest http://jvi.asm.org/ Downloaded from ROLE OF Y-Thy IN SP10 DEVELOPMENT 637 seems to be required for proper replication of the phage genome as well as cleavage of concatenates to unit-size DNA. MATERIALS AND METHODS Phage and bacteria. SP10 hmdl and SP10 hmd2 are heat-sensitive mutants defective in hypermodification (see below and reference 39). SP10 hmd+ is the same clear-plaque variant used in all previous studies (29, 30). B. subtilis W23 was the host in all experiments. Mutagenesis. Cells infected with SP10 hmd+ at 30°C were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine as described by Kahan (20). Screening for hmd mutants. Survivors of mutagenesis were plated at 30°C. Plaques were picked with sterile toothpicks and scored for their ability to plate at 42°C. Lysates of heat-sensitive mutants were prepared at 30°C and used to infect cells at 42°C in the presence of 6-(p-hydroxyphenylazo)uracil, an inhibitor of bacterial DNA polymerase III (5, 32) that has no remarkable effect on SP10 development (29, 30). The cells were labeled with [6-3H]uracil (10 uCi/ml) for 15 min. Cellular DNA was isolated (6) and acid hydrolyzed to a mixture of purine bases and pyrimidine deoxynucleosides (36). The digests were chromatographed in one direction on unmodified cellulose thin layers (10), using deoxythymidine (dThd) and Y-dThd as optical markers. Spots corresponding to the optical markers were eluted (10, 29), and the amount of label present was determined. In those cases where no label was recovered in the Y-dThd spot, a sample of [3H]DNA was enzymatically degraded to mononucleotides (36, 39) that were fractionated by two-dimensional chromatography on unmodified cellulose thin layers (7). Of the 184 single-plaque isolates tested to date, only 2 generated DNA at 42°C which was totally devoid of Y-dThd. The bases replacing Y-Thy in SP10 hmdl and SPO hmnd2 DNAs, under nonpermissive conditions, are 5-(hydroxymethyl-O-pyrophosphoryl)uracil and

Polymer-level synthesis of oxopyrimidine deoxynucleotides by Bacillus subtilis phage SP10: characterization of modification-defective mutants

“…(i) Isopycnic centrifugation. Preliminary evidence obtained during the mutant search (20) showed that no mutant-specific DNA made at 42°C had a normal oxopyrimidine content, and as a result, each should have an atypical buoyant density relative to wt (20,21). Accordingly, mutant phage containing [32P]DNA were prepared at 30°C and used to infect cells at 42°C.…”

“…Identically treated controls were infected with wt. DNA was extracted (2) and banded in neutral CsCl gradients (16,20,21).…”

“…Heat-sensitive hypermodification-defective (hmd) mutants were described recently (20,21). At nonpermissive temperatures, hmd-1 synthesized small amounts of DNA that contained normal levels of dTMP and an amount of PPOMedUMP equivalent to the now-missing YdTMP.…”

Resistance of bacteriophage H1 to restriction and modification by Bacillus subtilis R

“…Several other studies have likewise demonstrated an inhibitory effect of HMU and other substituted pyrimidines on restriction (3,4,18,27,33). How HMU, which is not contained within the GGCC sequence, affects the interaction between BsuR and Hi DNA is not clear.…”

DNA synthesis in Pseudomonas acidovorans infected with mutants of bacteriophage phi W-14 defective in the synthesis of alpha-putrescinylthymine

“…As with 4W-14 DNA, thymine and Y-Thy are formed from hmUra at the polynucleotide level (18). Normal levels of Y-Thy appear to be required for the replication and maturation of SP10 DNA; although not required absolutely for late gene expression, reduction of the level of Y-Thy in the DNA results in a reduced expression of the late genes (19). The glucosylation of hydroxymethylcytosine in T4 DNA is analogous to the conversion of hmUra to Y-Thy in SP10 and to putThy in XW-14.…”