Expression of the Anabaena sp. strain PCC 7120 xisA gene from a heterologous promoter results in excision of the nifD element

Abstract

An 11-kilobase-pair element interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. The nifD element normally excises only from the chromosomes of cells that differentiate into nitrogen-fixing heterocysts. The xisA gene contained within the element is required for the excision. Shuttle vectors containing the Escherichia coli tac consensus promoter fused to various 5' deletions of the xisA gene were constructed and conjugated into Anabaena sp. strain PCC 7120 cells. Some of the expression plasmids resulted in excision of the nifD element in a high proportion of vegetative cells. Excision of the element required deletion of an xisA 5' regulatory region which presumably blocks expression in Anabaena sp. strain PCC 7120 vegetative cells but not in E. coli. Strains lacking the nifD element grew normally in medium containing a source of combined nitrogen and showed normal growth and heterocyst development in medium lacking combined nitrogen. The xisA gene was shown to be the only Anabaena gene required for the proper rearrangement in E. coli of a plasmid containing the borders of the nifD element.

A sequence-specific DNA-binding factor (VF1) from Anabaena sp. strain PCC 7120 vegetative cells binds to three adjacent sites in the xisA upstream region

“…1) because this was the smallest xisA fragment that did not allow expression of the xisA gene when conjugated to vegetative cells of Anabaena sp. strain PCC 7120 (6). Crude extracts of vegetative cells appeared to contain binding activity for a 230-bp XbaI-ScaI fragment from pAM249 that contains the xisA upstream region.…”

“…Our identification of a regulatory region upstream of the xisA gene (6) prompted experiments designed to detect trans-acting DNAbinding factors in Anabaena sp. strain PCC 7120 vegetative cells.…”

“…The presence of the xisA gene is sufficient to cause rearrangement of a substrate plasmid in E. coli (6). It is likely that the xisA gene is developmentally regulated because its activity, seen as the excision of the niJD element, is expressed only during heterocyst differentiation (10), but 15,000 x g for 5 min.…”

“…1). The XbaI and BamHI sites are in the plasmid vector, and their position relative to the Scal site varies depending on the extent of the deletion (6 (14). The samples were heated to 90°C for 3 min and loaded onto 10% sequencing gels.…”

A preliminary analysis of the gene all0012 of Anabaena PCC 7120

“…Isolation of chromosomal DNA of Anabaena sp. strain PCC 7120 and gene cloning Total chromosomal DNA was prepared as described by Brusca et al (1990), but with slight modification to the method for cell disruption. The protocol was as follows: 7 mL of cells grown in the mid-log phase of growth (OD 730 approximately 0.6) were harvested from culture by centrifugation (10,000 × g for 5 min).…”