Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences

Abstract

Use of the sacB gene (J. L. Ried and A. Collmer, Gene 57:239-246, 1987) provides a simple, effective, positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium. This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form. By the use of this technique, we mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.

Isolation and characterization of multiple adenylate cyclase genes from the cyanobacterium Anabaena sp. strain PCC 7120

“…The sensitivity to sucrose provided by the sacB vector pRL271, which does not bear any cyanobacterial origin of replication, allows for the positive selection of double recombinations in Anabaena sp. (8). pRL271, which includes inactivated cya genes, was introduced into Anabaena strain PCC 7120 by conjugation.…”

“…The cya genes were inactivated by sacB-mediated positive selection for double recombinations (8). A SmaI fragment containing the omega Sp r -Sm r cassette from pDW9 (19) was inserted into the NaeI site of p665SK to produce p665SK⍀, the ScaI site of pQ3SK to produce pQ3SK⍀, the SnaBI site of p667SK to produce p667SK⍀, and the HpaI site of pQ6-2SK to produce pQ6-2SK⍀.…”

Hfq Is Required for Optimal Nitrate Assimilation in the Cyanobacterium Anabaena sp. Strain PCC 7120

“…HATF08550) set successively on top of solid medium (BG11) supplemented with 5% LB medium (incubated for 24 h), medium not supplemented (incubated for 24 h), and medium supplemented with neomycin (20 g/ml) and incubated until colonies appeared. Double recombinants were selected by their ability to grow in a sucrose-containing medium (8). The genomic structures of the exconjugants were confirmed by PCR and Southern blot analyses.…”

Comparison of conserved structural and regulatory domains within divergent 16S rRNA–23S rRNA spacer sequences of cyanobacteria The GenBank accession numbers for the sequences reported in this paper are AF180968 and AF180969 for ITS-L and ITS-S, respectively.

“…PCR amplifications were performed with DNA of Nostoc PCC 7120 and Synechocystis PCC 6803 isolated by the mini extraction method (Cai & Wolk, 1990) the position of each primer are indicated in Fig. 1.…”