Regulation of a Pseudomonas syringae locus, estA, that confers fitness on leaves.
D. SPIELBAUER* and G. A. BEATTIE. Iowa State University, Ames, IA.

Epiphytic bacteria are bacteria that can establish and maintain populations on leaf surfaces. The estA locus in the common epiphyte Pseudomonas syringae has been demonstrated to contribute to the fitness of this species on leaves. Although estA mutants can establish populations on leaves, they cannot maintain those populations when the leaves are exposed to dry conditions. The estA locus contains a 3.8-kb gene whose sequence at the amino acid level shows homology to the sequence of three putative Escherichia coli proteins of unknown function. To better understand the role of the estA locus in epiphytic fitness, we examined how estA is regulated. We predicted that estA is involved in stress tolerance, since inactivation of the locus resulted in decreased survival on leaves under stressful (dry and hot) conditions but not under wet conditions, and because an estA::Tn5 mutant was greatly reduced in its ability to tolerate high salt concentrations in culture. To measure estA expression, we constructed a transcriptional fusion by inserting a luxAB cassette from Vibrio harveyi into estA and integrating it into the P. syringae chromosome by marker exchange. To monitor any changes in luminescence that may occur due to direct effects an luciferase activity, a control fusion between the nptll promoter of Tn5 and luxAB was also constructed and introduced on a plasmid into P. syringae. We found that estA expression was not detectably influenced by the application of hyper-osmotic shock, hypo- osmotic shock, oxidative shock, nor by growth phase, growth in the presence of plant extracts, or growth in rich versus minimal media. Additionally, we found no evidence to support differential regulation on wet versus dry leaves.