Letting Escherichia coli teach me about genome engineering

Abstract

A career of following unplanned observations has serendipitously led to a deep appreciation of the capacity that bacterial cells have for restructuring their genomes in a biologically responsive manner. Routine characterization of spontaneous mutations in the gal operon guided the discovery that bacteria transpose DNA segments into new genome sites. A failed project to fuse lambda sequences to a lacZ reporter ultimately made it possible to demonstrate how readily Escherichia coli generated rearrangements necessary for in vivo cloning of chromosomal fragments into phage genomes. Thinking about the molecular mechanism of IS1 and phage Mu transposition unexpectedly clarified how transposable elements mediate large-scale rearrangements of the bacterial genome. Following up on lab lore about long delays needed to obtain Mu-mediated lacZ protein fusions revealed a striking connection between physiological stress and activation of DNA rearrangement functions. Examining the fate of Mudlac DNA in sectored colonies showed that these same functions are subject to developmental control, like controlling elements in maize. All these experiences confirmed Barbara McClintock's view that cells frequently respond to stimuli by restructuring their genomes and provided novel insights into the natural genetic engineering processes involved in evolution.

Figure 1.—

The gal and trp regions of the E. coli chromosome. The dashed line indicates >400 kb between the two operons. This distance makes galU mutations easy to separate from gal operon mutations that inactivate galK expression. The positions of four strongly polar insertion mutations are indicated by the vertical arrows. The figure is based on the deletion mapping of Shapiro and Adhya (1969).

Figure 2.—

The results of CsCl equilibrium density-gradient centrifugation of a mixture of λ-phages (λ 857, plaque-forming phage; λ dg⁺, phage carrying the wild-type gal operon; λ dg_S114⁻, phage carrying an insertion mutation). The fractions on the left contain the densest particles. From Shapiro (1969) with permission.

Figure 3.—

A molecular model for IS element and phage Mu transposition and replication. The long rectangles indicate the two strands of the transposable element (TE). The sets of three squares indicate the strands of the duplicated target sequence. Arrowheads indicate 3′ hydroxyl ends, and solid circles indicate 5′ phosphate ends. Although the initial strand cleavages are indicated as occurring simultaneously before ligation at step I, we now know that TE termini are first cleaved in the transposasome complex to liberate 3′ hydroxyl groups, which then proceed to attack the target duplex and induce trans-esterification reactions to produce the strand-transfer product illustrated following step II. The open rectangles and boxes after step III represent newly replicated DNA. Many bacterial and the majority of eukaryotic transposons undergo a double-strand cleavage before attacking the target duplex to undergo nonreplicative transposition. This figure is reproduced from Shapiro (1979) with permission.

Figure 4.—

IS- and Mu-mediated DNA rearrangements. The panels illustrate replicon fusion or co-integration (top), deletion formation (middle), and inversion (bottom). These rearrangements occur when the final reciprocal exchange step IV of complete transposition illustrated in Figure 3 fails to occur. The open rectangle containing an arrow represents the transposable element. Note how it becomes duplicated in each process. In the bottom two panels, the small boxes represent the target sequence that is duplicated to form a TSD. Further rearrangements are possible after the ones illustrated here. For example, the BC circle excised in deletion formation can fuse with a target site to create an inserted segment flanked by direct repeats of the transposable element. This process would create a transposon carrying the BC segment. Reproduced and corrected from Shapiro (1979) with permission.

Figure 5.—

Steps in the activation and execution of Mu-mediated araB–lacZ fusions. The process begins with Mu prophage derepression, which can occur either by aerobic starvation or by a temperature shift. Under starvation conditions, derepression involves ClpPX and Lon proteases and RpoS σ-factor. Expression of MuA transposase leads to formation of a super-twisted transposasome complex with the prophage termini. If this transposasome binds a site downstream of the U118 lacZ ochre mutation as illustrated, strand transfer will produce the branched strand transfer complex (STC) structure illustrated at the middle of the figure. Degradation of the bound transposase by ClpPX protease permits further processing of the STC. During active growth, this would produce a small inversion flanked by the replicated prophage. Under starvation conditions, an araB–lacZ translational fusion results, frequently containing at least one rearranged Mu terminus. Phosphodiester- and strand-specific details are in Shapiro and Leach (1990) and Maenhaut-Michel et al. (1997). Adapted from Shapiro (1997) with permission.