Letter to the Editor Heterologous Gene Expression in an Escherichia coli Population Under Starvation Stress Conditions Renato Fani, 1 Romina Gallo, 1 Silvia Fancelli, 1 Elena Mori, 1 Elena Tamburini, 1 Antonio Lazcano 2 1 Dipartimento di Biologia Animale e Genetica, Universita ` degli Studi di Firenze, via Romana 17, I-50125 Firenze, Italy 2 Facultad de Ciencias, Universidad Nacional Auto ´noma de Me ´xico, Me ´xico 04510, D.F., Me ´xico Received: 30 October 1997 / Accepted: 15 January 1998 Abstract. A novel system to study the evolution of transcription signals in heterologous systems under se- lective starvation conditions is described. It is based on the plasmid-mediated transfer of his biosynthetic genes from Azospirillum brasilense into a heterologous Esch- erichia coli mutant population lacking histidine biosyn- thetic ability. We show that under highly selective stress- ful conditions, genetic changes in the donor plasmid lead to mutated sequences that are efficiently recognized as promoters by the E. coli RNA polymerase. Key words: Starvation conditions — Promoter- creating mutations — Heterologous gene transfer — Metabolic evolution — Adaptive mutations Introduction ‘‘Directed evolution’’ experiments in which populations of microorganisms are challenged under stress condi- tions with novel substrates have shown that the develop- ment of new catabolic activities is often due to the re- cruitment of preexisting enzymes following regulatory mutations (Lin et al. 1976; Mortlock and Gallo 1992; Hall and Hauer 1993). As demonstrated by the rapid interspecies dispersal of biodegradative and antibiotic- resistance genes, the capture of xenologous sequences carried by plasmids of different host ranges can also lead to the accretion of catabolic abilities. As suggested by the incongruencies between deep phylogenies based on dif- ferent macromolecules, which have been explained by horizontal gene transfer (Gogarten et al. 1996) and even cell fusion events (Sogin 1991; Golding and Gupta 1995), the acquisition of metabolic activities from donor cells to heterologous recipients may have taken place since Archaean times. How can the newly acquired genes be brought into the preexisting regulatory system of the host organism? Al- though it is reasonable to assume that mutational adjust- ment of preexisting promoter sequences would take place, this possibility has not been addressed. This issue can be analyzed under laboratory conditions, by trans- ferring genes for a given metabolic route from a donor organism into a heterologous recipient lacking that path- way, whose transcriptional apparatus does not recognize the regulatory signals of the donor DNA. Reports of the stress-induced enhancement of spontaneous mutation rates (Cairns et al. 1988; Hall 1990; Foster 1993) suggest that under starvation conditions, recognition by the host of the transcription signals of the donor DNA can be adjusted to the host transcriptional milieu (Fig. 1). Here we present experimental evidence that when the histidine biosynthetic genes are transferred from a donor bacte- rium to a heterologous His - strain initially unable to Correspondence to: R. Fani; e-mail: r fani@dbag.unifi.it J Mol Evol (1998) 47:363–368 © Springer-Verlag New York Inc. 1998