4.8 Selected Research Ammlications of StreamlinedeGenome E. coli 694.8.2 Mobile Genetic Elements, Mutations, and Evolution
Bacterial genomes are usually loaded with a great number of ISs of many types.
The evolutionary forces driving their accumulation and their general impact on
adaptive evolution of the host are unknown. IS-free MDS42 provides a unique
opportunity to investigate the initial spread and evolutionary impact of ISs. By
introducing a single IS1 element into the genome MDS42, its impact could be
analyzed in laboratory evolutionary experiments. Although the IS element
increased the mutational supply and contributed to adaptation, another mutator
gene (mutS), frequently found in natural isolates, had a much greater impact on
the evolution of the cell. Moreover, mutS cells outcompeted IS-carrying cells,
limiting their spread. This work showed that the initial spread of IS elements
might depend on the presence of other mutator mechanisms in the population,
hence demonstrating the evolutionary conflict between different mutation-gen-
erating mechanisms [104].
Mobile element-free strains were also used in synthetic biology studies. To
improve the stability of synthetic genetic circuits, bidirectional (overlapping
forward and backward) promoters were designed to couple transcription of a
target nonessential gene to the transcription of an essential gene. The evolu-
tionary half-life of the gene of interest increased 4–10 times, and the circuit
was more stable in the IS-free MDS42 than in MG1655. However, eventually
point mutations, insertions/deletions and recombination occurred even in
MDS42, demonstrating the need for further stabilization of synthetic con-
structs [105].
4.8.3 Gene Function and Network Regulation
MDS42 proved to be especially useful in transcriptional studies elucidating the
physiological role and the molecular mechanisms of the rho-dependent tran-
scription termination system [54]. Rho silences foreign DNA, repressing
prophages and other horizontally acquired portions of the genome, but this
function becomes less important in MDS42 that lacks prophages and many hori-
zontally transferred regions. As a consequence, MDS42 shows 10^4 times lower
sensitivity to the Rho-inhibitor bicyclomycin than the ancestor MG1655.
Moreover, Rho cofactors NusA and NusG, normally essential in E. coli, become
dispensable in MDS42.
Reduced-genome strains were used to identify the genes required for biofilm
development [106]. They found new genes, some of them being cryptic in
MG1655 but expressed in the reduced-genome mutant, discovered by this
approach. In addition, by means of the deletion strains, a new repressor was
identified for starvation-sensing protein RspA [107].
The relationship between the genomic and environmental contributions to the
transcriptome was analyzed by comparing the transcriptomes of MG1655 and
MDS42 grown in regular and transient heat-shock conditions. Results suggest a
cross-talk guiding transcriptional reorganization in E. coli in response to both
genetic and environmental disturbances [108].