Cell-type specificity of the Anabaena fdxN-element rearrangement requires xisH and xisI.

The fdxN element, along with two other DNA elements, is excised from the chromosome during heterocyst differentiation in Anabaena sp. strain PCC 7120. Previous work showed that rearrangement of the fdxN element requires the xisF gene, which encodes a site-specific recombinase, and suggested that at least one other heterocyst-specific factor is involved. Here we report that the xisH and xisI genes are necessary for the heterocyst-specific excision of the fdxN element. Deletion of a 3.2 kb region downstream of the xisF gene blocked the fdxN-element rearrangement in heterocysts. The 3.2 kb deletion was complemented by the two overlapping genes xisH and xisI. Interestingly, extra copies of xisHI on a replicating plasmid resulted in the xisF-dependent excision of the fdxN element in vegetative cells. Therefore, xisHI are involved in the control of cell-type specificity of the fdxN rearrangement. The xisHI genes had no effect on the two other DNA rearrangements. The xisHI-induced excision of the fdxN element produced strains lacking the element and demonstrates that the 55 kb element contains no essential genes. xisH and xisI do not show similarity to any known genes.

Two heterocyst-specific DNA rearrangements of nif operons in Anabaena cylindrica and Nostoc sp. strain Mac.

Two site-specific DNA rearrangements occur during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120: the deletion of an 11 kb element from within the nifD gene and the deletion of a 55 kb element from within the fdxN gene. Three Nostoc and six Anabaena strains were screened for the presence of the nifD and fdxN elements by Southern hybridization with Anabaena PCC 7120 DNA probes. Eight of the nine strains contained DNA sequences that were similar to the nifD element. Three strains, Nostoc sp. strain Mac, Anabaena cylindrica and Anabaena sp. strain M131, also showed significant similarity to portions of the 55 kb fdxN element. Anabaena sp. strain CA lacked both the nifD and fdxN elements. Southern analysis of vegetative cell and heterocyst DNA from A. cylindrica and a Fox+ revertant of Nostoc Mac (isolate R2) showed rearrangement of the nifD and fdxN elements in heterocysts. We found no RFLPs between Anabaena M131 and Anabaena PCC 7120 suggesting that strain M131 is a Het- derivative of strain PCC 7120.

Programmed DNA rearrangement of a cyanobacterial hupL gene in heterocysts.

Programmed DNA rearrangements that occur during cellular differentiation are uncommon and have been described in only two prokaryotic organisms. Here, we identify the developmentally regulated rearrangement of a hydrogenase gene in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120. Heterocysts are terminally differentiated cells specialized for nitrogen fixation. Late during heterocyst differentiation, a 10.5-kb DNA element is excised from within the hupL gene by site-specific recombination between 16-bp direct repeats that flank the element. The predicted HupL polypeptide is homologous to the large subunit of [NiFe] uptake hydrogenases. hupL is expressed similarly to the nitrogen-fixation genes; hupL message was detected only during the late stages of heterocyst development. An open reading frame, named xisC, identified near one end of the hupL DNA element is presumed to encode the element's site-specific recombinase. The predicted XisC polypeptide is homologous with the Anabaena sp. strain PCC 7120 site-specific recombinase XisA. Neither XisC nor XisA shows sequence similarity to other proteins, suggesting that they represent a different class of site-specific recombinase.

Anabaena xisF gene encodes a developmentally regulated site-specific recombinase.

Two DNA elements are excised from the chromosome during Anabaena heterocyst differentiation. We have identified the gene xisF which encodes the site-specific recombinase responsible for the excision of a 55-kb element from within the fdxN gene. The cloned xisF gene is sufficient to cause site-specific rearrangement of an artificial substrate in Escherichia coli. Inactivation of xisF in the Anabaena chromosome prevents excision of the fdxN element and growth in nitrogen-deficient medium but does not alter the development of heterocysts. Forced transcription of xisF in vegetative cells did not result in excision of the fdxN element, suggesting that other factors may be involved in cell-type specificity. The predicted XisF protein shows significant similarity to the Bacillus subtilis SpoIVCA recombinase.

Excision of an 11-kilobase-pair DNA element from within the nifD gene in anabaena variabilis heterocysts.

The 3' region of the Anabaena variabilis nifD gene contains an 11-kilobase-pair element which is excised from the chromosome during heterocyst differentiation. We have sequenced the recombination sites which border the element in vegetative cells and the rearranged heterocyst sequences. In vegetative cells, the element was flanked by 11-base-pair direct repeats which were identical to the repeats present at the ends of the nifD element in Anabaena sp. strain PCC 7120 (Anabaena strain 7120). Although Anabaena strain 7120 and A. variabilis are quite distinct in many ways, the overall sequence similarity between the two strains for the regions sequenced was 96%. Like the Anabaena strain 7120 element, the A. variabilis element was excised in heterocysts to produce a functional nifD gene and a free circularized element which was neither amplified nor degraded. The Anabaena strain 7120 xisA gene is located at the nifK-proximal end of the nifD element and is required for excision of the element in heterocysts. The A. variabilis element also contained an xisA gene which could complement a defective Anabaena strain 7120 xisA gene. A. variabilis did not contain the equivalent of the Anabaena strain 7120 fdxN 55-kilobase-pair element.

Deletion of a 55-kilobase-pair DNA element from the chromosome during heterocyst differentiation of Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 produces terminally differentiated heterocysts in response to a lack of combined nitrogen. Heterocysts are found approximately every 10th cell along the filament and are morphologically and biochemically specialized for nitrogen fixation. At least two DNA rearrangements occur during heterocyst differentiation in Anabaena sp. strain PCC 7120, both the result of developmentally regulated site-specific recombination. The first is an 11-kilobase-pair (kb) deletion from within the 3' end of the nifD gene. The second rearrangement occurs near the nifS gene but has not been completely characterized. The DNA sequences found at the recombination sites for each of the two rearrangements show no similarity to each other. To determine the topology of the rearrangement near the nifS gene, cosmid libraries of vegetative-cell genomic DNA were constructed and used to clone the region of the chromosome involved in the rearrangement. Cosmid clones which spanned the DNA separating the two recombination sites that define the ends of the element were obtained. The restriction map of this region of the chromosome showed that the rearrangement was the deletion of a 55-kb DNA element from the heterocyst chromosome. The excised DNA was neither degraded nor amplified, and its function, if any, is unknown. The 55-kb element was not detectably transcribed in either vegetative cells or heterocysts. The deletion resulted in placement of the rbcLS operon about 10 kb from the nifS gene on the chromosome. Although the nifD 11-kb and nifS 55-kb rearrangements both occurred under normal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.