1887

Abstract

are Gram-positive, filamentous bacteria capable of fixing atmospheric dinitrogen either in the free-living state or in symbiosis with a variety of woody plants. Only a few genes have been sequenced and gene expression is not well characterized. To isolate a segment of DNA that functions as an RNA polymerase promoter, fragments of strain ArI5 genomic DNA were cloned upstream of a promoterless, cassette. Constructs were screened for luminescence in and positive clones assayed for transcription activity with a partially purified RNA polymerase extract. Primer extension analysis of transcripts 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, GLO7p1, contains sequences resembling the −35 element of a promoter and the −35 and −10 elements of the canonical promoter. Also within this region are two pentamers identical to sequences near the 5′ end of the strain CpI1 glutamine synthetase gene. The second promoter, GLO7p2, contains a putative NtrC binding site at −145 and a possible σ-RNA polymerase recognition sequence at −14 suggesting that GLO7p2 may be a nitrogen-regulated promoter. An 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 fragment has promoter activity as well as .

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2001-02-01
2020-01-25
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