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.

Figure 1. Transcription, organization, and translation of annotated and non-predicted antisense genes.

A. RT-PCR, using gene-specific primers, of six pairs of annotated and antisense genes. At each locus, transcription from both DNA strands was detected. In all cases, negative controls which excluded reverse transcriptase from the reactions showed that contaminating DNA was not present. Pfl numbers refer to predicted Pf0-1 genes, (GenBank). The iiv numbers are as described . B. Organization of the iiv14 locus. Solid arrows indicate predicted open reading frames for annotated (Pfl ORFS) and cryptic (iiv14) genes. C. Western blot of purified iiv14-His protein. Lane 1: His-tagged iiv14 protein (arrow). Lane 2: control extract from Pf0-1(pME6000), showing no non-specific detection of untagged proteins. D. Western blot of His-tagged Pfl_0939. Crude extracts were prepared from Pf0-1 with and without Pfl_0939-His. Lane 1: His-tagged Pfl_0939 protein (arrow). Lane 2: extract from Pf0-1(pME6000) showing no non-specific detection of proteins similar in size to Pfl_0939. There is a non-specific background band at 115 kDa in both. Approximate sizes of molecular weight standards (Invitrogen BenchMark Pre-stained Protein Ladder) used in the SDS-PAGE are shown in panels C and D.

Figure 2. Soil colonization experiments.

A. Growth of Pf0-1 wild-type and iiv14 mutant strains (Δiiv14) in sterile soil, inoculated from laboratory culture. Data points represent fold increases from individual experiments, horizontal lines represent median values. 0–1, population increase between inoculation and day 1. The population increase of mutant Δiiv14 is significantly lower than that of Pf0-1 (p<0.001); 1–2, the population increase over the second day. The mutant Δiiv14 recovers from the colonization defect, and increases significantly more than Pf0-1 (p<0.005), which had already neared its maximum. B. Growth of Pf0-1 and the iiv14 mutant in sterile soil, after prior growth in soil. Both strains were grown in separate soil samples for seven days. Populated soil was then mixed with fresh sterile soil to achieve dilution in the range of 1∶1000–1∶10000, and water was added to achieve approximately 50% water holding capacity. Populations in the fresh soil were monitored as described above. 0–1, population increase between inoculation and day 1. Over this period, Pf0-1 colonized the soil significantly better than the mutant Δiiv14 (p<0.005); 1–2, the population increase over the second day. The increases are not significantly different. Pf0-1 is represented by circles, while the iiv14 mutant is indicated by triangles.

Figure 3. Restoring and enhancing soil colonization with multicopy clones of iiv14.

A. Organization of iiv14, Pfl_0939, Pfl_0940, and Pfl_0941 in the Pf0-1 genome. Horizontal lines below indicate the cloned regions in each complementation construct. Vertical dotted lines show the boundaries of the iiv14 ORF. The arrow indicates the location of the iiv14 transcription start site. From this it can be seen that the only clones possessing the full length iiv14 sequence are pME14CF1 and pME14CF2. B. Fold increase in population of the iiv14 mutant (Δiiv14) bearing complementing plasmids pME14CF1 and pME14CF2, compared to mutant and wild-type strains harboring vector pME6000, and compared to the iiv14 mutant harboring complementation plasmids with a stop codon replacing codon 17 of the gene, after two days growth in soil. The mutant Δiiv14 carrying complementing plasmids colonize significantly better than the mutant carrying the plasmid vector alone (pCF1, p<0.0005; pCF2, p<0.005). The mutant harboring plasmids carrying the nonsense codon (indicated by *) did not colonize significantly better than the mutant carrying the vector alone, and colonized significantly less than Pf0-1 harboring the vector (p<0.01). C. Effect of multiple copies of both iiv14 gene clones in Pf0-1 on soil colonization, relative to Pf0-1(pME6000). Population increases over 24 and 48 hours, are shown. The greater colonization shown by Pf0-1 carrying pCF1 or pCF2 was significant over the 48 hour colonization period (columns 4–6; p<0.05). In panels B and C, data points represent fold increases from individual experiments, horizontal lines represent median values. Pf0-1 and the iiv14 mutant are represented by circles and triangles, respectively. Inverted triangles represent strains in which the plasmid carries the stop codon at codon 17 of iiv14. Filled symbols show strains carrying pME6000 (vector), open symbols indicate carriage of complementing plasmids: pCF1 = pME14CF1; pCF2 = pME14CF2; stars show plasmids carrying stop codons at codon 17.

Figure 4. Soil competition experiments.

Pf0-1 and the cosA mutant strains marked with Sm^r (Pf0-1) or Km^r (ΔcosA) carried on miniTn7 were each diluted to contain approximately 10⁴ cfu/mL. Soil was inoculated with 1mL of a 50∶50 mix of each. Populations were monitored daily by cfu counting. Data shown are the average cfu/0.5 g soil, from four independent experiments. Error bars represent the standard error of the mean.