Replication of Bacteriophage M 13: Inhibition of Single-Strand DNA Synthesis by Rifampicin

Abstract

Synthesis of single-stranded DNA of bacteriophage M 13 is blocked by rifampicin. This inhibition is not observed in an Escherichia coli mutant possessing an RNA polymerase resistant to rifampicin. Since rifampicin stops single-strand synthesis faster than chloramphenicol, inhibition by rifampicin does not seem to result from a transcriptional block of protein synthesis, particularly in view of the long half-life of the messenger RNA for M 13. It is, therefore, concluded that the interaction of rifampicin with RNA polymerase directly affects single-strand DNA synthesis. Possible mechanisms are discussed.The DNA of the filamentous phage M 13 replicates in three stages (1, 2). First, the infecting single-stranded DNA is converted by host enzymes into a double-stranded form (SS --RF). Early in the infection the replicative forms multiply to establish a pool of RF molecules (RF --RF). Later, double-strand synthesis ceases almost completely, and the RF molecules serve as precursors for single-strand synthesis (RF SS). Single-stranded viral DNA is formed by an asymmetric replication process in which the viral strand of the RF is displaced as a new one is synthesized (3).These three stages of phage DNA repliction show differences in their sensitivity towards antibiotics. Inhibition of protein synthesis by chloramphenicol has no effect on the SS --RF conversion (4). Ongoing RF replication is also rather insensitive to chloramphenicol, and continues at a slowly decreasing rate after all protein synthesis has stopped (5). If chloramphenicol is added late in the infection, an immediate switch back from single-strand synthesis to RF replication is observed (6). However, the conversion of single-stranded parental DNA into the replicative form, as well as the multiplication of the double-stranded forms, are rapidly inhibited by rifampicin (5), a specific inhibitor of RNA polymerase.Since it has been shown in this lab that the specific messenger RNA for phage M 13 has an exceptionally long halflife (ref. 7 and unpublished data), we expected that blockage of transcription with rifampicin should have a considerably delayed effect on single-strand synthesis and phage maturation, as compared to chloramphenicol treatment. However, after we added rifampicin to M 13-infected cells late in infection, a rapid halt of phage release was observed (Fig. 1). Following up this observation, we found, as shown in this paper, that addition of rifampicin results in an immediate stop of single-strand synthesis. Since no inhibition was observed in an Escherichia coli mutant with an RNA polymerase resistant to rifampicin, we conclude that rifampicin stops M 13 single-strand synthesis by interaction with RNA polymerase. METHODS AND RESULTSThe effect of rifampicin and chloramphenicol on DNA synthesis in uninfected cells and in M 13-infected cells was measured 2 hr after infection, a time when single-strand synthesis is predominant, by exposure of aliquots of the treated and untreated cultures to [3H thymidine for 1 min. As shown in Fig. 2A, ch...

F deoxyribonucleic acid transferred to recipient cells in the presence of rifampin

“…It has been reported that rif'ampin, an inhibitor of ribonucleic acid (RNA) polymerase, blocks the conversion of M13 single-strand DNA to the replicative form (2,35,38,39), but fails to block the synthesis of' X174 replicative form (38). It has been f'urther reported that a transcriptional step is directly involved in DNA replication of' various replicons: phage X (7,15,32), T7 (10), colEl (3,4,30,31), F (17,18), R64-11 (8), plasmid of E. coli 15 (22), plasmid of' Salmonella pullorum ( 16), E. coli (12, 13, 19, 21; T. Saitoh and S. Hiraga, accepted for publication), and Bacillus subtilis (20,23).…”

Influence of Protein and Ribonucleic Acid Synthesis on the Replication of the Bacteriocinogenic Factor Clo DF13 in Escherichia coli Cells and Minicells

“…Whereas inhibition of protein synthesis by chloramphenicol (CAP) blocks the replication of chromosomal DNA and DNA of transmissible plasmids (2), de novo protein on October 9, 2020 by guest http://jb.asm.org/ Downloaded from VELTKAMP, BARENDSEN, AND NIJKAMP synthesis appeared not to be required for the replication of the non-transmissible plasmid Col El (6), minicircular DNA of E. coli 15 (27), or Clo DF13 (this report). RNA synthesis is a prerequisite for the replication of chromosomal DNA and for DNA of transmissible as well as non-transmissible plasmids (3,5,7,8,12,20,24,26,27,33,35). It has been shown that rifampinsensitive RNA synthesis is required for the initiation of chromosomal DNA replication at the fixed origin (26).…”