Overlapping Protein-Encoding Genes in Pseudomonas fluorescens Pf0-1

Abstract

The annotated genome sequences of prokaryotes seldom include overlapping genes encoded opposite each other by the same stretch of DNA. However, antisense transcription is becoming recognized as a widespread phenomenon in eukaryotes, and examples have been linked to important biological processes. Pseudomonas fluorescens inhabits aquatic and terrestrial environments, and can be regarded as an environmental generalist. The genetic basis for this ecological success is not well understood. In a previous search for soil-induced genes in P. fluorescens Pf0-1, ten antisense genes were discovered. These were termed ‘cryptic’ genes, as they had escaped detection by gene-hunting algorithms, and lacked easily recognizable promoters. In this communication, we designate such genes as ‘non-predicted’ or ‘hidden’. Using reverse transcription PCR, we show that at each of six non-predicted gene loci chosen for study, transcription occurs from both ‘sense’ and ‘antisense’ DNA strands. Further, at least one of these hidden antisense genes, iiv14, encodes a protein, as does the sense transcript, both identified by poly-histidine tags on the C-terminus of the proteins. Mutational and complementation studies showed that this novel antisense gene was important for efficient colonization of soil, and multiple copies in the wildtype host improved the speed of soil colonization. Introduction of a stop codon early in the gene eliminated complementation, further implicating the protein in colonization of soil. We therefore designate iiv14 “cosA”. These data suggest that, as is the case with eukaryotes, some bacterial genomes are more densely coded than currently recognized. Sequenced bacterial genomes provide a vast resource for research fields such as pathogenesis, drug discovery, and microbial ecology. Once sequenced, the genes within a genome are predicted using computational and manual methods. An assumption underlying both approaches is that any given length of DNA encodes only a single gene. This concept has been challenged by findings in eukaryotic genomes, and in bacterial plasmids and viruses where it is known that some stretches of DNA specify both ‘sense’ and ‘antisense’ RNA molecules. In prokaryotic cells there is little information regarding the potential of the genome to code two genes within the same stretch of DNA. We show that in the bacterium Pseudomonas fluorescens Pf0-1, both strands of DNA are transcribed at six locations in the genome, and that at one of these locations (iiv14), two different proteins are specified by the same piece of DNA. At the iiv14 locus, we demonstrate that the newly identified gene (antisense to the predicted gene) functions to promote colonization of soil, and name this gene cosA. Our findings indicate that bacterial genomes have more genes than currently thought, and important genes that have escaped detection occupy the same stretch of DNA as known genes.