M. P. Nuti

Summary: A mutant of A. tumefaciens strain B6S3, carrying the R factor RP4, was able to transfer its TI plasmid to various avirulent Agrobacterium strains and to a strain of Rhizobium. Strains carrying the TI(B6S3) plasmid were selected by their ability to utilize octopine. The isolates were able to induce tumours and exclude phage AP1. The tumours induced on Kalanchoë daigremontiana were rough and contained octopine. It was concluded that octopine utilization, phage exclusion, induction of rough tumours and synthesis of octopine in the tumours are determined by the TI(B6S3) plasmid. Tumorigenicity was determined this plasmid both in Agro-bacteria and in Rhizobium, suggesting that all the genes necessary for tumour induction are plasmid-borne.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.

Summary Large plasmids were detected in Rhizobium species by sedimentation analysis of lysates on alkaline sucrose gradients and by dye buoyant density centrifugation. Mitomycin C treatment did not increase the amount of plasmid DNA in the bacteria. Renaturation kinetics were used to confirm that these large DNA molecules had the molecular complexity of plasmids and to estimate their molecular weights. These ranged from 0·7 × 108 to 4·0 × 108 daltons. The maximum yield of isolated plasmid DNA relative to chromosomal DNA was 3·8%.

Genes encoding beta-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORF1 were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB- Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB- strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORF1 with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.