Frankia sequences exhibiting RNA polymerase promoter activity

Frankia are Gram-positive, filamentous bacteria capable of fixing atmospher ic dinitrogen either in the free-living state or in symbiosis with a variet y of woody plants. Only a few Frankia genes have been sequenced and gene ex pression is not well characterized. To isolate a segment of Frankia DNA tha t functions as an RNA polymerase promoter, fragments of Frankia strain Ar15 genomic DNA were cloned upstream of a promoterless, Vibrio harveyi luxAB c assette. Constructs were screened for luminescence in E. coil and positive clones assayed for in vitro transcription activity with a partially purifie d Frankia RNA polymerase extract. Primer extension analysis of in vitro tra nscripts produced from one clone, GLO7, identified two major transcription start sites, TSP-1 and TSP-2, 52 bp apart. Deletion analysis then localized sequences essential for promoter activity. The upstream promoter region, G LO7p1, contains sequences resembling the -35 element of a Streptomyces prom oter and the -35 and -10 elements of the canonical E. coil promoter. Also w ithin this region are two pentamers identical to sequences near the 5' end of the Frankia strain Cpl1 glutamine synthetase gene. The second promoter, GLO7p2, contains a putative NtrC binding site at -145 and a possible sigma (N)-RNA polymerase recognition sequence at -14 suggesting that GLO7p2 may b e a nitrogen-regulated promoter. An in vivo transcript representing an ORF of 498 aa starting 64 bp downstream of the distal transcription start, TSP- 1, was detected by RT-PCR. This supports the conclusion that this DNA fragm ent has promoter activity in vivo as well as in vitro.