Site-Specific Recombination in the Cyanobacterium Anabaena sp. Strain PCC 7120 Catalyzed by the Integrase of Coliphage HK022

Abstract

The integrase (Int) of the -like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP ؋ attB and attL ؋ attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate P glnA -attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter P glnA . The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli.The site-specific recombination mechanism of the temperate coliphage HK022 is similar, if not identical, to that of phage . To lysogenize its Escherichia coli host, circular phage DNA recombines via a site-specific recombination reaction between the nonidentical attP and attB sites of the phage and host genomes, respectively. This reaction results in the integrated prophage that is flanked by the recombinant attL and attR sites, which are the sites for phage excision. The phage-encoded site-specific integrase (Int), which belongs to the Tyr recombinase family, catalyzes phage integration and excision. Both reactions require the presence of the host-encoded DNA-bending heterodimeric accessory protein integration host factor (IHF); excision requires in addition the phage-encoded excisionase (Xis), whose activity can be partially replaced by the host-encoded FIS (factor for inversion stimulation). The bacterial attB consists of a 21-base-pair (bp) core sequence (BOBЈ) that is composed of 7 central bp, defined as the overlap O, which is identical in all four att sites and serves as the site of DNA exchange. O is flanked by two 7-bp inverted repeats (B and BЈ) that serve as weak binding sites for Int. The phage attP site is composed of a similar core (COCЈ) that is flanked by two long arms (P and PЈ of 142 and 85 bp, respectively) carrying tight binding sites for Int and for the accessory proteins (IHF, Xis, and FIS). The accessory proteins, bound to their sites on the arms of attP, regulate and facilitate arm-bound Int monomers access to the core, the site of the reaction. Subsequently, this proteo-DNA complex (the intasome) captures attB to execute the recombination. Each of the recombinant sites attL (BOPЈ) and attR (POBЈ) that flank the integrated prophage is composed of...

Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase

“…The reverse prophage excision reaction occurs between the attL and attR sites and restores the original attP and attB 13 . We have previously applied Int for genome manipulation in plants, cyanobacteria and human cells and have shown that the accessory proteins that are required in the E. coli host are dispensable in the eukaryotic milieu 14 15 16 17 18 . In the present study we developed a cancer specific binary cell expression system that is based on the hTERT promoter.…”

Genomic Insights into the Taxonomy and Metabolism of the Cyanobacterium Pannus brasiliensis CCIBt3594

“…Bacteriophages play a crucial role in the evolution and ecology of the Cyanobacteria group, serving not only as a means of population control but also facilitating the transfer of genes between species [59]. The integrase gene is responsible for phage integration and excision of the host's chromosome [60,61]. The presence of this gene may suggest the potential ability of the prophage region identified in the genome to establish lysogenic relationships with P. brasiliensis CCIBt3594, with further analyses required to elucidate this relationship.…”