The origin and direction of replication of the chromosome of Escherichia coli B-r.

Abstract

In the resting state between replication cycles, the chromosome of E. coli is a simple circular unit (probably a single molecule of double-stranded DNA about 1. mm in. length).' E. coli grown slowly in minimal medium possesses chromosomes which replicate by the continuous linear motion of a single Y-shaped growing point that progresses along the molecule at approximately constant speed. Numerous attempts have been made to determine the direction of this replication process along the genetic map and the origin from which it begins, but contradictory results have been reported. Some investigators2-5 find that the origin is located at the integration site of the F factor in Hfr strains, but there is disagreement about the direction of replication. No unique origin for Fstrains is found by some authors. 2, 4 Other investigators6-8 have found a single origin for several Hfr and Fstrains at a position corresponding to the 7-8 o'clock region of the genetic map.'I Evidence has been presented for a unique, though undetermined, origin for FFstrains, 10 as well as evidence for the noninvolvement of the F factor in determining the origin of replication.1' These differences may reflect differences among the strains tested or they may arise from technical differences among the methods used. The present experiments were done to locate the origin and direction of replication in E. coli B/r by techniques chosen in an attempt to avoid some of the artifacts of earlier methods. Materials and Alethods.-Bacterial strain: E. coli B/r (ATCC 12407) was used throughout. Media: Cells were routinely grown on. a minimal salts medium that consisted of two parts which were mixed shortly before use. The first solution contained 2.0 gm (NH4)2SO4, 6.0 gm Na2HPO4, 3.0 gm KH2PO4, 3.9 gm NaCl, anid 0.011 gm Na2SO4 dissolved in deionized H20; the second part contained 0.2 gm MgCl2, 0.1 gm CaCl2, and 0.0005 gm FeCl3 7H20 dissolved in 800 ml deionized H20. The carbon source was either glucose (0.2%) or glycerol (0.2%), depending upon the experiment. Experimental Procedures.-Two different techniques were used to determine when a particular gene replicates during the division cycle of the cell. In technique A, newly divided cells were separated physically from an exponenitially growing culture in order to obtain a synchronized culture. Samples taken at various times from this synchronized culture were used to measure the inducibility of several enzymes as a function of cell age. Inducibility is defined as the amount of enzyme synthesized in a fixed, short period of induction. The synchrony procedure used12 13 permits one to collect the newly divided cells that have been eluted from a membrane filter to which a growing population of bacteria have been affixed. When necessary, cells were concentrated by collecting newly divided cells in chilled flasks for an hour, filtering the chilled cells, and resuspending in a desired volume of warm conditioned medium, i.e., medium that had sustained growth to about 3 X 107 cells/ml and had been filtered to remove the bacteria. Aliquots were taken from synchronously growing cultures and added to tubes containing appropriate inducers. After 10 min of induction, chloramphenicol was added to a final concentration of 100 ,ug/ml to inhibit fuirther protein synthesis and the samples were chilled until assayed.