Complete genome sequencing of Agrobacterium sp. H13-3, the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a circular and a linear chromosome and an accessory plasmid but lacking a tumor-inducing Ti-plasmid
Introduction
Rhizobium lupini H13-3, a soil bacterium isolated from the rhizophere of Lupinus luteus, was first described and used for genetic transformation by Balassa (1957) and Gabor (1965). Rhizobia are an agronomically important group of α-Proteobacteria that colonize the roots of legumes for symbiotic nitrogen fixation. However, R. lupini H13-3 is unable to nodulate Lupinus under laboratory conditions.
R. lupini H13-3 and the related nitrogen-fixing symbiont, Sinorhizobium meliloti, have been established as prototypes for studying novel features of flagellum structure, motility and chemotaxis that distinguish the motile and sensory systems of these and other Rhizobiaceae from those of the Enterobacteriaceae (Krupski et al., 1985, Schmitt, 2002, Schmitt et al., 1974). Unlike the ‘plain’ flagella of enteric bacteria, the flagellar filaments of these soil bacteria have a ‘complex’ structure with three prominent undulations winding around the right-handed helical filament, thus providing rigidity and forceful propulsion to the bacterium swimming through viscous media (Götz et al., 1982, Pleier and Schmitt, 1991, Trachtenberg et al., 1987). Whereas enterobacteria and related motile bacteria change their swimming direction by switching the sense of flagellar rotation, R. lupini H13-3 and S. meliloti rotate their complex flagella exclusively clockwise and direct the swimming path by changing the rotary speed of individual flagella (Götz and Schmitt, 1987, Scharf, 2002, Szurmant and Ordal, 2004). Moreover, the chemosensory signalling chain to the flagellar motor is different. Signal transduction involves two types of interacting response regulators and a specific relay of protein phosphorylations, a new feature pertaining to all α-Proteobacteria tested so far (Sourjik and Schmitt, 1996, Sourjik and Schmitt, 1998).
The importance of S. meliloti and R. lupini H13-3 as model bacteria for studying the molecular mechanisms of motility and chemotaxis in a large group of bacteria demands their exact taxonomic and phylogenetic assignment within the family Rhizobiaceae, which comprises among other genera Rhizobium, Agrobacterium and Sinorhizobium. S. meliloti has been phylogenetically positioned by 16S rDNA and genome sequence analysis (Galibert et al., 2001, Slater et al., 2009). However, the species R. lupini is poorly characterized and reliable phylogenetic analyses have not been performed (Brunner et al., 2005).
A relationship between R. lupini H13-3 and A. tumefaciens C58 was suggested previously, when the fla genes that encode the protein subunits of the flagellar filaments were sequenced and compared to corresponding genes of other species. Four genes, namely flaABCD, were mapped on the main chromosomes of S. meliloti (Sourjik et al., 1998) and A. tumefaciens (Deakin et al., 1999). In S. meliloti, these genes map in sequential order, whereas in A. tumefaciens a large inversion with a break point within the flaABCD gene cluster separates flaABC from flaD placing them 20 kb apart at the far ends of the inverted portion. Interestingly, the R. lupini H13-3 fla gene region exhibits the same gene arrangement as seen in A. tumefaciens C58, except for a deletion of flaC at the translocation breakpoint (Scharf et al., 2001).
The complete genome sequence of R. lupini H13-3 is presented here. The resulting tripartite genome structure and phylogenetic analyses identified this strain as non-pathogenic Agrobacterium strain belonging to the genomic species G1 of the biovar I cluster. Because the epithet ‘tumefaciens’ has become solidly associated with the designation of pathogenic Agrobacterium spp. with tumorigenic activity, the non-pathogenic strain is named Agrobacterium sp. H13-3, a designation that will be used throughout the text.
Section snippets
Origin and growth of Agrobacterium sp. H13-3
Agrobacterium sp. H13-3 (formerly R. lupini H13-3) was isolated from the rhizosphere of L. luteus in Hungary (Balassa, 1957, Gabor, 1965). The aerobic, heterotrophic strain was grown in TYC broth (0.5% tryptone, 0.3% yeast extract, and 0.1% CaCl2) at 30 °C.
454 pyrosequencing of the Agrobacterium sp. H13-3 genome and genome assembly
Genomic DNA of Agrobacterium sp. H13-3 for 454 pyrosequencing was isolated as described previously (Kuhn et al., 2008). To confirm the identity of the DNA, a 16S rDNA-specific region was amplified and sequenced. BLASTn analysis revealed that
Directed sequencing of the Agrobacterium sp. H13-3 genome
The Agrobacterium sp. H13-3 (formerly R. lupini H13-3) genome was de novo sequenced by applying a strategy combining 454-pyrosequencing on the Genome Sequencer FLX platform and Sanger-sequencing of PCR-amplicons. One 454-sequencing run resulted in 500,415 reads with a total of about 110 million bases sequence information. This amount of sequenced bases led to a ca. 20-fold coverage of the Agrobacterium sp. H13-3 genome as calculated on basis of the genome size of the completely sequenced
Acknowledgements
The bioinformatics support of the Bioinformatics Resource Facility (BRF) at the Center for Biotechnology (CeBiTec, Bielefeld University) is gratefully acknowledged. We thank Irene Krahn for excellent technical assistance.
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