The epiphytic bacterium Erwinia amylovora causes fire blight, a devastating disease of rosaceous plants such as apple and pear. The closely related but nonpathogenic bacterium Pantoea agglomerans (formerly E. herbicola), which usually accompanies E. amylovora in the wild, produces a family of antibiotics that inhibit E. amylovora in a poorly understood strain-dependent fashion. The numerous members of this antibiotic family constitute a source of biocontrol agents for fire blight, a pool of new and potentially useful antibiotics against human bacterial pathogens, and a model of nature's ability to produce a "library" of biologically active molecules. The complexity of antibiotic production, especially the multiple antibiotics produced by a given strain and low production in liquid culture, has hampered chemical investigations. We have taken a genomic approach to deconvolute the multiple antibiotics and to address the production problems by a particular strain. Heterologous expression of a genomic DNA library from P. agglomerans in Escherichia coli provides access to the small-molecule antibiotics. Since both P. agglomerans and E. coli are members of the widely distributed Enterobacteriacea, E. coli should be a suitable host for heterologous expression of P. agglomerans genes that encode antibiotics. Herein we report the isolation and structure determination of pantocin A, a new peptidederived antibiotic, and its molecular target.