Reiterated DNA sequences in Rhizobium and Agrobacterium spp

Abstract

Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295: [384][385][386][387][388][389] 1982], has DNA reiteration been reported as a common genomic feature. (1,13,14,17,23,24,30,39,49), but it has been demonstrated as a common genomic feature only in a few cases. For instance, Sapienza and Doolittle (42) showed that DNA reiteration is a common feature in the genome of the archaebacteria Halobacterium halobium and Halobacterium volcanii.We have previously reported that nitrogen fixation genes (nif genes) are reiterated in Rhizobium phaseoli (33,34) MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used are listed in Table 1.Molecular cloning. To prepare bacterial gene libraries, total DNA from each strain was digested with EcoRI restriction endonuclease and the fragments were cloned in the EcoRI site of pBR329 by using Escherichia coli MC1061 as a recipient for transformation.Filter blot hybridization. Total DNA digested with EcoRI restriction endonuclease was subjected to electrophoresis in a 1% agarose gel and blotted onto nitrocellulose by the method of Southern (48). Recombinant plasmids were labeled with 32P by nick translation (36) at high specific activity (108 cpm/,Lg of DNA) and used as probes for hybridization.For each lane, 2 x 106 cpm of probe DNA was used. The blots were prehybridized for 2 h at 65TC in 5 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 0.2% Ficoll (Pharmacia Fine Chemicals, Piscataway, N.J.), 0.2% bovine serum albumin, 0.2% polyvinylpirrolidone, 0.1 M phosphates (pH 6.7), and 0.05 mg of salmon sperm DNA per ml. The blots were then hybridized for 16 h at 65°C in the same solution with the labeled probe. The filters were washed once with 2 x SSC containing 0.01% sodium dodecyl sulfate for 10 min at room temperature and three times with 0.1 x SSC containing 0.1% sodium dodecyl sulfate for 30 min at 50°C. Autoradiography in the presence of intensifying screens was carried out at -70°C for 24 h.Transfer of R. phaseoli plasmids to A. tumefaciens. R. phaseoli CFN-42 and CFN-285 were randomly mutagenized with TnS-mob (45). Different Kmr derivatives of each strain were mated with the plasmidless A. tumefaciens GM19023 in triparental matings by using pJB3JI as a helper plasmid for mobilization. Agrobacterium transconjugants were analyzed for plasmid profiles by the Eckhardt procedure (12), and strains carrying each different plasmid were selected. A

Amplification and deletion of a nod-nif region in the symbiotic plasmid of Rhizobium phaseoli

“…For Rhizobium phaseoli, the symbiont of the common bean (Phaseolus vulgaris L.), we have estimated the presence of about 700 reiterated elements, belonging to 200 different families (9). This high degree of reiteration is not restricted to R. phaseoli; other members of the Rhizobiaceae family, including the closely related bacteria Agrobacterium tumefaciens, possess a large amount of reiterated DNA (9,24).…”

The genomes of the family Rhizobiaceae: size, stability, and rarely cutting restriction endonucleases

“…Genomic rearrangements are common in some Rhizobiaceae, notably in R. leguminosarum biovar phaseoli (13,39,40). Furthermore, repeated DNA sequences exist in B. japonicum (20), Sinorhizobium fredii (26,31), and other Rhizobiaceae (12 [7,15]), yet it still leaves questions unresolved. Electron microscopy (EM) has been used to measure contour lengths of R. meliloti megaplasmids (4,5 (10,16), suggesting that these replicons may harbor genes that are essential to the bacterium's survival.…”

Rhizobial plasmids — replication, structure and biological role

“…Rhe, more than 200 reiterated DNA families could be found, indicating that intra-genomic rearrangements might indeed be frequently occurring events [90,125], resulting in structural complexity of rhizobial replicons [36,88,126]. In turn, the rearrangements in complex rhizobial genomes might enhance the adaptive potential of the bacterium, allowing the reassortment of essential, nonessential, and redundant functions to contend with challenging environments [36].…”