Increased Fitness of Pseudomonas fluorescens Pf0-1 Leucine Auxotrophs in Soil

Center for Adaptation Genetics and Drug Resistance, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA

^* To whom correspondence should be addressed. Email: Stuart.Levy{at}tufts.edu.

	Abstract

The annotation process of a newly sequenced bacterial genome is largely based on algorithms derived from databases of previously defined RNA and protein-coding gene structures. This process generally excludes the possibility that the two strands of a given stretch of DNA can each harbor a gene in an overlapping manner. While the presence of such structures in eukaryotic genomes is considered to be relatively common, their counterparts in prokaryotic genomes are just beginning to be recognized. Application of an in vivo expression technology (IVET) previously identified twenty two discrete genetic loci in Pseudomonas fluorescens Pf0-1 that were specifically activated in the soil environment, of which ten were present in an antisense orientation to previously annotated genes. This observation led to the hypothesis that the physiological role of overlapping genetic structures may be relevant to growth conditions outside artificial laboratory media. Here, we examined the role of one of the overlapping gene pairs, iiv19 and leuA2, in soil. Although iiv19 was previously demonstrated to be preferentially activated in the soil environment, its absence did not alter P. fluorescens ability to colonize or survive in soil. Surprisingly, the absence of the leuA2 gene conferred a fitness advantage in the soil environment when leucine was supplied exogenously. This effect was determined to be independent of the iiv19 gene, and further analyses revealed that amino acid antagonism was the underlying mechanism behind the observed fitness advantage in soil. Our findings provide a potential mechanism for the frequent occurrence of auxotrophic mutants of Pseudomonas spp. in the lungs of Cystic Fibrosis patients.