Observations on the formation of clones containing araB-lacZ cistron fusions

Abstract

Casadaban (1976) developed a technique for isolating E. coli clones containing fusions of the amino terminal-encoding portion of any cistron with the carboxy terminal-encoding portion of lacZ. The technique utilizes prophage Mu homology to bring the two cistrons into proximity. I have followed the appearance over time of colonies containing araB-lacZ fusions from a strain where the beginning of the araB cistron is connected to lacZ by an intact Mucts62 prophage. Cultures of the starting strain grown on a variety of media have fewer than 2 in 10(10) cells capable of forming colonies within three days after plating on selective arabinose-lactose medium. At 32 degrees C, there is a delay of between 4 and 19 days before the first colony appears. The kinetics of colony appearance over the next two to four weeks then shows a rapid increase in the number of new colonies emerging per day followed by a decline. The pattern of colonial emergence and the final numbers of fusion colonies obtained are not grossly affected by reducing the number of cells plated over five orders of magnitude. Fusion colonies sometimes show a clustered pattern when they first emerge. Inoculation of pre-existing fusion clones at specific locations on the arabinose-lactose selection plates seeded with the starting strain leads to the formation of inhibitory zones where no fusion colonies appear. Selection plates contain many microcolonies and papillae which do not proliferate into scoreable colonies but nonetheless contain cells capable of growth when replated on the same selective medium. Up to 39% of all plated cells are capable of producing fusion clones. The kinetics of fusion colony appearance can be altered by environmental and genetic manipulations. Partial derepression of the Mucts prophage at 37 degrees accelerates the appearance of colonies but also reduces the final yield. Addition of limiting concentrations of glucose to the selective medium also accelerates the appearance of colonies in a specific fashion: enrichments below the level required for maximum acceleration produce a biphasic kinetics with two waves of fusion clone emergence separated by an eight-day interval. Infection with Muc+pAp phage produces dilysogens that have almost completely lost the ability to produce fusions. Infection with MuctsAampAp phage produces strains that are reduced in phage production and have delayed kinetics of fusion clone emergence. The implications of these observations for theories of hereditary change in bacteria are discussed.