Excessive Promoters as Silencers of Genes Horizontally Acquired by Escherichia coli

Horizontally acquired genes are usually transcriptionally inactive, although most of them are associated with genomic loci enriched with promoter-like sequences forming “promoter islands”. We hypothesized that lateral DNA transfer provokes local mutagenesis, leading to accumulation of AT base pairs and, as a result, RNA polymerase binding sites that suppress the transcription of foreign genes due to promoter multiplicity or interaction with a specific silencer H-NS. Error-prone mutagenesis was implemented for the “promoter island” of a foreign gene appY and the promoter cluster of an inherent gene dps. Derivatives with changed transcriptional activity were selected using a reporter plasmid pET28_eGFP. Only one cycle of mutagenesis with negative selection suppressed the activity of the main dps promoter to the background level due to a single substitution in its -10 element, while positive selection gave a nucleotide sequence with improved -35 element. The same suppression for appY was achieved by three cycles, while 8-fold transcription activation required nine iterations of mutagenesis with positive selection. In both cases, the number of potential start points decreased and quite an ordinary regulatory region with only one dominant promoter was obtained at the end of positive selection. Excessive promoters therefore may give a negative impact on transcription. Efficiency of H-NS binding remained virtually unchanged in all mutant constructs. Thus, we suggest that RNA polymerase itself can operate as a repressor on promoter-dense regions of alien genes.