Complete Genome Sequence of Burkholderia rhizoxinica, an Endosymbiont of Rhizopus microsporus

Abstract

Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic fungus Rhizopus microsporus. The vertically transmitted endosymbiont not only delivers the antimitotic macrolide rhizoxin to its host but is also essential for vegetative spore formation of the fungus. To shed light on the genetic equipment of this model organism, we sequenced the whole genome of B. rhizoxinica HKI 0454, thus providing the first genomic insight into an intracellular mutualist of a fungal species. The 3.75-Mb genome consists of a chromosome and two strain-specific plasmids. The primary metabolism appears to be specialized for the uptake of fungal metabolites. Besides the rhizoxin biosynthesis gene cluster, there are 14 loci coding for nonribosomal peptide synthetase (NRPS) assembly lines, which represent novel targets for genomic mining of cryptic natural products. Furthermore, the endosymbionts are equipped with a repertoire of virulence-related factors, which can now be studied to elucidate molecular mechanisms underlying bacterial-fungal interaction.

Rice seedling blight is a severe plant disease affecting Asian agriculture (4, 8). The causative agent is the phytopathogenic zygomycete Rhizopus microsporus, which employs an antimitotic agent, rhizoxin, as a virulence factor (10, 13). We have found that rhizoxin is produced not by the fungus but by symbiotic bacteria residing within the fungal cytosol (7, 8, 11). The vertically transmitted endosymbionts are essential for vegetative spore formation of the fungus (9). Up to now, we have identified eight related Burkholderia-Rhizopus associations (3) from five continents (Africa, Asia, Australia, Europe, and North America), and it appears that the fungal-bacterial interaction has undergone a parasitism-mutualism shift during evolution (12). Here, we present the genome of the endofungal type strain Burkholderia rhizoxinica HKI 0454 (6). Genomic DNA was sequenced to an 8-fold coverage by the Sanger technology. The Arachne 3 software (1) allowed for correct assembly of paired reads derived from a combination of a shotgun library (2.8-kb insert size) and a cosmid library (36-kb insert size). The remaining gaps were closed by sequencing on cosmid templates or combinatorial PCR. Regions of low sequence quality or coverage were resequenced using cosmid templates to a minimum of 2-fold coverage. The three circular contigs obtained were submitted to the ERGO (Integrated Genomics, Chicago, IL) annotation platform for automated annotation and pathway prediction. Selected loci were reannotated manually.

The genome of B. rhizoxinica is a tripartite genome with a total size of 3,750,139 bp and an overall G+C content of 60.7%. The three circular replicons include a 2.75-Mbp chromosome, an 822,304-bp megaplasmid (pBRH01), and a 172,525-bp plasmid (pBRH02). The whole genome consists of 3,878 open reading frames, 2,437 (62.8%) of which were assigned a biological function.

B. rhizoxinica lacks common genes for sugar importers. Instead, genes coding for importers for amino acids, citrate, and malate suggest uptake of fungal metabolites. The rhizoxin biosynthesis gene cluster (RBRH_02584 to RBRH_02572) (7) is located on the chromosome. Additionally, B. rhizoxinica harbors 14 nonribosomal peptide synthetase (NRPS) gene clusters, whose corresponding peptide products are unidentified.

B. rhizoxinica possesses a type II secretion pathway (RBRH_00771 to RBRH_00782). An encoded type III secretion system (RBRH_03541 to RBRH_03014) has already been shown to play a crucial role for the establishment of the symbiosis (2). A putative type IV secretion system is encoded on the 172-kb plasmid pBRH02 (RBRH_00673 to RBRH_00645). A lipopolysaccharide biosynthesis gene cluster (RBRH_01441 to RBRH_01472) that is critical for the formation of surface determinants was identified. The O-antigen has been shown to be important for the stability of the symbiosis (5).

The whole genome of B. rhizoxinica represents the first reported genome sequence of a bacterial endosymbiont of a fungus. It grants insights into the genetic potential of endofungal bacteria and delivers an excellent basis for further investigations of bacterium-fungus interactions.

Nucleotide sequence accession numbers.

The B. rhizoxinica genome has been deposited in the EMBL database under the following accession numbers: FR687359 (B. rhizoxinica HKI 0454, complete genome), FR687360 (B. rhizoxinica HKI 0454 plasmid pBRH01, complete sequence), and FR687361 (B. rhizoxinica HKI 0454 plasmid pBRH02, complete sequence).