Transfer of a genetic marker from a megaplasmid of Anabaena sp. strain PCC 7120 to a megaplasmid of a different Anabaena strain

Abstract

The 410-kb a megaplasmid of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was found to bear the nucA gene that encodes a sugar-nonspecific nuclease. That gene was mutated by insertion of a cassette that confers resistance to neomycin. The resulting strain, AMP2, was mated with a streptomycinresistant derivative of Anabaena sp. strain PCC 7118, a strain that does not form heterocysts. Cells 25 ,ug/ml in solid medium. Chlorophyll a was determined in methanolic extracts (13). Escherichia coli DH5ot, used for all plasmid constructions, was grown in Luria broth as described previously (14).Preparation and manipulation of DNA. Total DNA from cyanobacteria was isolated as described by Cai and Wolk (6).DNA of high molecular weight from Anabaena sp. strains PCC 7120 and PCC 7118 and their derivatives was prepared and cut with restriction endonucleases, and the resulting plasmids were subjected to pulsed homogeneous orthogonal field gel electrophoresis (PHOGE) (2) as described by Kuritz et al. (11). Bands with a size of less than 50 kb were measured by using HindIll digests of coliphage lambda DNA as standards in pulsed-field gels (11).Plasmid isolation from E. coli, transformation of E. coli, restriction, and ligation with T4 DNA ligase were performed by standard procedures (1,14). DNA fragments were purified from agarose gels with the GeneClean kit (Bio 101, Inc.). The nucA-containing hybridization probes were either plasmid pRLA1, which carries 12 kb of strain PCC 7120 genomic DNA including the nucA gene and flanking sequences, or a 473-bp HindIII-BstNI fragment internal to nucA, isolated from pCSAM29 (16). The neomycin phosphotransferase (npt) probe used was cassette C.K3 (7)

Mobility of Plasmids

“…Given that cyanobacterial cells are among the most abundant on earth and that cyanobacteria genomes have many plasmids, uncovering how plasmids spread in this clade should become a priority. Indeed, the 408-kb Anabaena MOB V plasmid pCC7120␣ was reported to be transmissible (103), although the exact mechanism of transmission was not investigated. As mentioned above, the VirB4 analogue of MPF I is less similar to VirB4 than to VirD4.…”

Homologous regions of the Salmonella enteritidis virulence plasmid and the chromosome of Salmonella typhi encode thiol: disulphide oxidoreductases belonging to the DsbA thioredoxin family

“…While this issue is difficult to address without knowing the extent of paralogous nifH sequences within the cyanobacteria, the clustering of the cyanobacterial nitrogenase sequences indicates that if lateral transfer occurred, it happened early in the evolution of cyanobacteria and does not explain the haphazard distribution of nitrogenase within cyanobacterial taxonomic Subsections I to 111. There has been little evidence yet of lateral transfer among the cyanobacteria (Castenholz, 1992;Muro-Pastor et al, 1994), but the use of 16s rRNA and genes such as nifH may help to identify whether such events have occurred. A recent analysis of a limited number of sequences of nifH, n i p and nifK genes from species representing proteobacteria, cyanobacteria and Gram-positive bacteria could not resolve the issue of horizontal versus vertical descent (Hirsch et al, 1995).…”

Plasmid Conjugation from Proteobacteria as Evidence for the Origin of Xenologous Genes in Cyanobacteria

“…strain PCC 7002 (3), and Synechocystis sp. strain PCC 6803 (4), while conjugative transfer among Anabaena strains was also reported (5). Although no experimental evidence for transduction has been reported, several marine phages that contain photosynthetic genes have been detected (6,7).…”