DNA Replication of Induced Prophage in Haemophilus influenzae

Abstract

DNA synthesis during transition from the lysogenic state to the lytic cycle and throughout the latter has been studied in Haemophilus influenzae BC200 (HP1c1). Following exposure to ultraviolet light, there is a 30-min delay in DNA synthesis after which there is a rapidly increasing rate of phage DNA synthesis. The phage genome is replicated without extensive utilization of segments or of breakdown products of the bacterial chromosome. The mode of phage DNA replication was investigated by zonal sedimentation of labeled DNA in 5 to 20% neutral and alkaline sucrose gradients. Tritiated thymidine, incorporated during a 2-min pulse given at 38 min, chases rapidly into DNA, sedimenting like linear DNA of approximately 2 × 10 8 daltons, and then, at the expense of label in this peak, chases into slower-sedimenting phage DNA (2 × 10 7 daltons). The fast-sedimenting, rapidly labeled DNA satisfies certain criteria for being a concatenated replicative intermediate. Observations in the electron microscope revealed linear concatemers in the faster-sedimenting material and circular phage-sized DNA in the slower-sedimenting DNA. When induced cells are gently lysed with lysozyme and Brij 58 to maintain DNA-membrane associations and sedimented in neutral sucrose over a cesium chloride shelf, the concatemer is found with the cell-membrane-wall complex. Membrane-associated label chases to membrane-free material sedimenting like deproteinized HP1c1 DNA. When membrane-associated DNA from the cesium chloride shelf is deproteinized and resedimented in neutral sucrose, the sedimentation profile reveals that sedimentation rates of labeled DNA from this complex are indicative of sizes ranging from 2 × 10 8 daltons down to phage-sized pieces of 2 to 3 × 10 7 daltons. A model is presented which places HP1c1-DNA replication on the cell membrane where a concatemer of phage DNA is synthesized and subsequently degraded to phage-equivalent DNA. Phage-equivalent DNA is then either released from the membrane for packaging or is packaged while still membrane associated. Thus, the cell membrane is not only the site of DNA replication during which phage DNA is synthesized in multiple phage-equivalent concatemers but it is also the site at which these concatemers are selectively reduced to phage-sized pieces.