Programed 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
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-specffic recombination. The first is an l1-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 nitS 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 nijS gene on the chromosome. Although the nipD 11-kb and nifS 55-kb rearrangements both occurred under normnal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 is accompanied by two developmentally regulated genome rearrangements (8,9,14). Unlike most DNA rearrangements in procaryotic organisms, such as the movement of transposable elements and phase variation inversions which occur at very low frequencies (33), rearrangement of the heterocyst genome is tightly regulated by an environmentally induced developmental sequence.Heterocysts are highly specialized, terminally differentiated cells which reduce atmospheric dinitrogen to ammonia and then export the fixed nitrogen to neighboring vegetative cells in the form of glutamine (reviewed in references 12 and 37). Differentiation of a vegetative cell into a heterocyst is repressed either by the presence of an external source of combined nitrogen such as ammonia or by the close proximity of neighboring heterocysts in the filament. In the absence of a combined nitrogen source, approximately 10% of the cells along a filament form heterocysts at a relatively constant interval, producing a simple one-dimensional pattern of development.Global changes in gene expression occur during heterocyst differentiation (5, 22). Most notably, nitrogen fixation (nif) genes are induced an...
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 coil 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.
Summary 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 xisl 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 hetero‐cysts. The 3.2 kb deletion was complemented by the two overlapping genes xisH and xisl. Interestingly, extra copies of xlsHI 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 xisHl‐induced excision of the fdxN element produced strains lacking the element and demonstrates that the 55 kb element contains no essential genes. xisH and xisl do not show similarity to any known genes.
The 3' region of the Anabaena variabilis nifD gene contains an li-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 ni/D gene and a free circularized element which was neither amplified nor degraded. The Anabaena strain 7120 xisA gene is located at the ni/K-proximal end of the ni/D 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 xis4 gene. A. variabilis did not contain the equivalent of the Anabaena strain 7120 fdxN 55-kilobase-pair element.
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