Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria.

Abstract

Wild-type cyanobacteria of the genus Anabaena are capable of oxygenic photosynthesis, differentiation of cells called heterocysts at semiregular intervals along the cyanobacterial filaments, and aerobic nitrogen fixation by the heterocysts. To foster analysis of the physiological processes characteristic of these cyanobacteria, we have constructed a family of shuttle vectors capable of replication and selection in Escherichia coli and, in unaltered form, in several strains of Anabaena. Highly efficient conjugative transfer of these vectors from E. coli to Anabaena is dependent upon the presence of broad host-range plasmid RP-4 and of helper plasmids. The shuttle vectors contain portions of plasmid pBR322 required for replication and mobilization, with sites for Anabaena restriction enzymes deleted; cyanobacterial replicon pDUl, which lacks such sites; and determinants for resistance to chloramphenicol, streptomycin, neomycin, and erythromycin.Many filamentous cyanobacteria fix dinitrogen aerobically within specialized cells called heterocysts that differentiate at semiregular intervals along the filaments. All cyanobacteria are capable of oxygenic photosynthesis. Genetic methods usable for study of nitrogen fixation, differentiation, pattern formation, and photosynthesis in these organisms have long been sought.Several unicellular cyanobacteria can be transformed by DNA in the growth medium (1-3). Shuttle vectors, plasmids able to replicate in Escherichia coli and in an alternative host, have been constructed for two such strains, Anacystis nidulans strain R2 and Agmenellum quadruplicatum ). An Anacystis gene cloned in a shuttle vector in E. coli was returned to the cyanobacterium by transformation (7). To date, no reproducible transformation system is known for filamentous cyanobacteria.Conjugation provides an alternative approach to transfer of cloned DNA. RP-4 and related plasmids can transfer themselves or derivatives of themselves into a wide range of We report the construction of a hybrid between pBR322 and plasmid pDU1 (16) from the filamentous cyanobacterium Nostoc. Because restriction endonucleases present in strains of cyanobacteria (17) apparently reduce retention of DNA transferred into those cyanobacteria (4, 18), the hybrid plasmid was restructured to eliminate sites for restriction enzymes present in several strains of Anabaena. Additional antibiotic-resistance determinants were added, lest an organism be unable to use any one such determinant. The derivatives of the hybrid plasmid proved to be shuttle vectors, capable of RP-4-mediated transfer into several strains of Anabaena and of replication in those strains.MATERIALS AND METHODS Anabaena sp. PCC 7120, Anabaena sp. U. Leningrad strain 458 (PCC 7118), Anabaena sp. U. Tokyo M-131, and Nostoc sp. PCC 7524 were grown with nitrate as described (19). Anacystis nidulans strain R2 was grown in medium BG-11 (20).E. coli was grown in L broth, supplemented as appropriate with none, one, or two of the following: 25 jig of chloramphenicol (Cm) per...

Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

“…The IncP transfer apparatus is known to build conjugative bridges between a huge variety of organisms (Grahn et al, 2000;Thomas and Nielsen, 2005). Shuttle vectors for gene transfer from Proteobacteria to distantly related recipients such as Cyanobacteria (Wolk et al, 1984) or Grampositive bacteria and yeast (Heinemann and Sprague, 1989;Samuels et al, 2000) have, indeed, been built using the RP4 transfer system, an IncP-1a subgroup plasmid. Although the wide transfer Figure 4 Phylogenetic tree showing all identified transconjugant OTUs for three different plasmids (pKJK5, RP4 and pIPO2tet) from the same donor (P. putida).…”

Cyanobacterial defense mechanisms against foreign DNA transfer and their impact on genetic engineering

“…Strategies to circumvent the RM barrier include either the use of helper plasmids carrying cognate MTases for the REases encoded by the cyanobacteria (Thiel and Poo, 1989;Elhai et al, 1997) or deletion of the restriction sites from the foreign DNA (Wolk et al, 1984). Another alternative is the engineering of mutant strains that are devoid of REases, but this solution still requires cyanobacteria strains that are transformable (Iwai et al, 2004).…”

“…Many cyanobacteria carry plasmids, whose replicons can be used for engineering shuttle vectors for E. coli and cyanobacteria. Such vectors have been designed from Synechocystis, Synechococcus and Anabaena/Nostoc plasmids (Wolk et al, 1984, Golden and Sherman 1984, Buzby et al, 1985, Buikema and Haselkorn 1991, Summers et al, 1995. Among those, plasmids based on the pDU1 replicon from Nostoc sp.…”

“…DNA can also be transferred by electroporation to unicellular and fi lamentous cyanobacteria (Thiel andPoo 1989, Mermet-Bouvier et al, 1993). The transfer of circular DNA from E. coli to a diverse range of host bacteria via conjugation (Wolk et al, 1984) is the preferred method for gene transfer into cyanobacteria.…”

Molecular cloning and characterization of the recA gene from the cyanobacterium Synechococcus sp. strain PCC 7002

“…These s take assimilation of a single strand of DNA into a DNA duplex, advantage of the ability of a number of cyai tetia to and mediate polarized branch migration have all been demundergo DNA-mediated transformation (35) oi Sserve as onstrated in vitro (38). Its regulatory and direct biochemical recipients in conjugation involving E. coli do lls (49).…”