Organization and Expression of the Genes on pAgK84 That Encode Production of Agrocin 84. Chang-Lin Wang. Department of Plant Pathology; University of Illinois at Urbana/Champaign, Urbana 61801 U.S.A. Stephen K. Farrand(1,2), and Ingyu Hwang(1). (1)Department of Plant Pathology and (2) department of Microbiology, University of Illinois at Urbana/Champaign, Urbana 61801 U.S.A. MPMI 7:472-481. Accepted 14 April 1994. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1994.

Agrocin 84 biosynthesis genes located in a 21-kb segment of pAgK84 were characterized by mutagenesis with Tn3HoHol and complementation analysis. Three overlapping fragments of the 21-kb segment, cloned into pRK415 or pLAFR6, were mutagenized with Tn3HoHol, and 94 independent insertions were mapped and oriented. A series of merodiploid strains, each containing a Tn5 insertion in pAgK84 affecting agrocin 84 biosynthesis, and a clone containing a Tn3HoIIol insertion that blocks antibiotic production in homogenotes were constructed to determine the number of complementation groups involved in agrocin 84 biosynthesis. Five complementation groups were identified and named agnA through agnE. Analysis of lacZ fusions formed by the Tn3HoHol inserts indicated that all of the loci except agnD are transcribed in an anticlockwise direction. Insertions carried on clones and insertions marker-exchanged into pAgK84 had similar patterns of expression. The five agn loci were not expressed at significant levels in Escherichia coli DH5o( grown in minimal or rich media. Levels of expression in Agrobacterium tumefaciens NT1 differed for each region; agnA was expressed at relatively high levels, agnC and agnE at intermediate levels, and agnB and agnD at very low levels. Similar patterns of expression were observed in minimal media, regardless of the carbon source, and at neutral and acidic pHs. Expression levels were lower in cells grown in rich medium. The level of expression of each agn locus was not affected by the presence of other agn genes or by the presence or absence of the large nopaline plasmid pAtK84b, present in strain K84. Nor were the levels of expression influenced by the addition of opines or root exudates to the culture media. AH five agn loci were expressed at all growth stages, and expression reached maximum levels during late exponential growth. The agn loci were expressed in planta, and the patterns of expression were similar to those seen in bacteria grown in vitro. The presence of pAtK84b did not affect agn expression in planta.