Genome Analysis of Agrobacterium tumefaciens: Construction of Physical Maps for Linear and Circular Chromosomal DNAs, Determination of Copy Number Ratio and Mapping of Chromosomal Virulence Genes

Abstract

The phytopathogenic bacterium Agrobacterium tumefaciens is unique in that it possesses both linear and circular DNA chromosomes in addition to a plant-tumor-inducing (Ti) plasmid. We analyzed the two chromosomal DNA molecules in strain MAFF301001, whose Ti plasmid has already been sequenced completely. Physical maps of the chromosomal DNAs were constructed by Southern hybridization experiments using Pme I and Swa I fragments and short fragments bridging the Swa I fragments with special care to avoid any missing fragment. Hybridization with 16S rDNA probe showed one rDNA locus on the linear chromosome and two loci on the circular chromosome. For this bacterium to be pathogenic, not only Ti plasmid but also chromosomal genes are required. The chromosomal virulence (chv) genes (chvA, chvB, chvD, chvE, chvG, chvH, and chvI) and the chromosomal genes affecting the virulence [acvB, pgm(exoC), glgP, miaA, and ros] were successfully mapped onto 5 different regions in the chromosomal physical maps. These chv genes and the chromosomal genes affecting the virulence other than pgm and glgP were found on the circular chromosome, whereas the pgm and glgP genes were located on the linear chromosome. In contrast to the large terminal inverted repeats of Streptomyces linear chromosomal DNA, no hybridization signal was detected between left and right terminal fragments of the linear A. tumefaciens chromosome. Quantitative analysis of DNA fragments indicated that the copy numbers of the two chromosomal DNAs and the Ti plasmid are identical.

Physical and gene maps of Agrobacterium biovar 2 strains and their relationship to biovar 1 chromosomes

“…This report, together with that of Jumas-Bilak et al (1998), showed that rDNA sequences are only found on the chromosome in biovar 2 strains, whereas rDNA loci are located on both chromosomes in biovar 1 (Goodner et al, 2001;Wood et al, 2001;Suzuki et al, 2001).…”

“…Electrophoresis was carried out using a contour-clamped homogeneous electric field (CHEF) electrode as previously described by Suzuki et al (2001). Sizes of DNA fragments were calculated based on migration rate in PFGE, with Saccharomyces cerevisiae chromosomal DNA used as a molecular mass standard.…”

“…Autoradiographic detection was carried out using BAS-III imaging plates (Fuji Photo Film) with BAS2000 (Fuji Photo Film) and STORM (Amersham-Pharmacia) image analysers as recommended by the manufacturers. Other techniques were performed according to Suzuki et al (2001) and Sambrook & Russell (2001).…”

“…The physical and genetic map of MAFF301001 was prepared earlier by Suzuki et al (2001). For more reliable comparison between the two biovars, we also briefly characterized chromosomal DNA of another biovar 1 strain.…”

Developmental and environmental regulatory pathways in alpha-proteobacteria

“…In both cases, the circular chromosome has an origin of replication resembling that of C. crescentus, whereas the linear chromosome of A. tumefaciens and the (mega)plasmids carry a plasmid-like replication system of the repABC family -in which RepA and RepB are responsible for partitioning and RepC is required for the initiation of replication (121)(122)(123). In A. tumefaciens, all replicons are duplicated at the same time during the cell cycle and preferentially localize at, or near, the cell pole, although their positions do not overlap (19,121,124,125). Similarly, in S. meliloti the replication origins of all three replicons preferentially localize at the cell pole, with a higher polar bias for the chromosome origin (124).…”