ORGANIZATION AND EXPRESSION OF THE GENES ON PAGK84 THAT ENCODE PRODUCTION OF AGROCIN-84

Agrocin 84 biosynthesis genes located in a 21-kb segment of pAgK84 wer e characterized by mutagenesis with Tn3HoHo1 and complementation analy sis. Three overlapping fragments of the 21-kb segment, cloned into pRK 415 or pLAFR6, were mutagenized with Tn3HoHo1, and 94 independent inse rtions were mapped and oriented. A series of merodiploid strains, each containing a Tn5 insertion in pAgK84 affecting agrocin 84 biosynthesi s, and a clone containing a Tn3HoHo1 insertion that blocks antibiotic production in homogenotes were constructed to determine the number of complementation groups involved in agrocin 84 biosynthesis. Five compl ementation groups were identified and named agnA through agnE. Analysi s of lacZ fusions formed by the Tn3HoHo1 inserts indicated that all of the loci except agnD are transcribed in an anticlockwise direction. I nsertions carried on clones and insertions marker-exchanged into pAgK8 4 had similar patterns of expression. The five agn loci were not expre ssed at significant levels in Escherichia coil DH5 alpha grown in mini mal or rich media. Levels of expression in Agrobacterium tumefaciens N T1 differed for each region; agnA was expressed at relatively high lev els, agnC and agnE at intermediate levels, and agnB and agnD at very l ow levels, Similar patterns of expression were observed in minimal med ia, regardless of the carbon source, and at neutral and acidic pHs. Ex pression levels were lower in cells grown in rich medium. The level of expression of each agn locus was not affected by the presence of othe r agn genes or by the presence or absence of the large nopaline plasmi d pAtR84b, present in strain K84. Nor were the levels of expression in fluenced by the addition of opines or root exudates to the culture med ia. All five agn loci were expressed at all growth stages, and express ion reached maximum levels during late exponential growth. The ago loc i were expressed in planta, and the patterns of expression were simila r to those seen in bacteria grown in vitro. The presence of pAtK84b di d not affect agn expression in planta.