Transcription of foreign DNA in Escherichia coli.
Propagation of heterologous DNA in E. coli host cells is central to molecular biology. DNA constructs are often engineered for expression of recombinant protein in E. coli, but the extent of incidental transcription arising from natural regulatory sequences in cloned DNA remains underexplored. Here, we have used programmable microarrays and RT-PCR to measure, comprehensively, the transcription of H. influenzae, P. aeruginosa, and human DNA propagating in E. coli as bacterial artificial chromosomes. We find evidence that at least half of all H. influenzae genes are transcribed in E. coli. Highly transcribed genes are principally involved in energy metabolism, and their proximal promoter regions are significantly enriched with E. coli sigma(70) (also known as RpoD) binding sites. H. influenzae genes acquired from an ancient bacteriophage Mu insertion are also highly transcribed. Compared with H. influenzae, a smaller proportion of P. aeruginosa genes are transcribed in E. coli, and in E. coli there is punctuated transcription of human DNA. The presence of foreign DNA in E. coli disturbs the host transcriptional profile, with expression of the E. coli phage shock protein operon and the flagellar gene cluster being particularly strongly up-regulated. While cross-species transcriptional activation is expected to be enabling for horizontal gene transfer in bacteria, incidental expression of toxic genes can be problematic for DNA cloning. Ongoing characterization of cross-expression will help inform the design of biosynthetic gene clusters and synthetic microbial genomes.
Warren
René L
RL
BC Cancer Agency, Canada's Michael Smith Genome Sciences Centre, Vancouver, British Columbia V5Z 4S6, Canada.
Freeman
John D
JD
Levesque
Roger C
RC
Smailus
Duane E
DE
Flibotte
Stephane
S
Holt
Robert A
RA
D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D020869:Gene Expression Profiling; D022761:Gene Transfer, Horizontal; D005798:Genes, Bacterial; D005818:Genetic Engineering; D006193:Haemophilus influenzae / Q000235:genetics; D006801:Humans; D005810:Multigene Family; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012808:Sigma Factor / Q000235:genetics; D013045:Species Specificity; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases