Genome Sequence of the Biocontrol Agent Pantoea vagans Strain C9-1

Abstract

Pantoea vagans is a Gram-negative enterobacterial plant epiphyte of a broad range of plants. Here we report the 4.89-Mb genome sequence of P. vagans strain C9-1 (formerly Pantoea agglomerans), which is commercially registered for biological control of fire blight, a disease of pear and apple trees caused by Erwinia amylovora.

Pantoea vagans (syn. Pantoea agglomerans, Erwinia herbicola) (10), is a common plant epiphyte. Strain C9-1, isolated from apple (Malus × domestica ‘Jonathan’) (Michigan) (5), is registered as BlightBan C9-1 (Nufarms America Inc., Burr Ridge, IL) for biocontrol of fire blight caused by the related enterobacterium Erwinia amylovora. Applied during bloom, C9-1 provides effective disease control, similar to oxytetracycline and slightly less disease control than streptomycin treatment provides (6). As for most biocontrol agents, growth and efficacy vary among locations and years (6). We sequenced the complete genome of P. vagans C9-1 as a step toward elucidating genetic factors that modulate performance reliability.

Genomic DNA, isolated using the Wizard genomic DNA purification kit (Promega, Madison, WI) was whole-genome shot-gun sequenced by 454 Life Sciences (Branford, CT) with four runs on a GS-20 sequencer. This resulted in 1,224,924 high-quality filtered reads with an average read length of 97 bp and coverage equivalent to 25 times. Quality filtered sequences were assembled in silico using the 454 Newbler assembler giving 207 contigs, 43 contigs larger than 500 bp. Gap closure and repetitive element sequencing were achieved by PCR walking using the Sanger method on a 3130XL sequencer (Applied Biosystems, Rotkreuz, Switzerland). Final assembly and sequence manipulations were done using the Lasergene package v8.1 (DNASTAR, Madison, WI).

The P. vagans C9-1 genome has a 4,025-Mb circular chromosome and three circular plasmids (pPag1 [167,983 bp], pPag2 [165,692 bp], and pPag3 [529,676 bp]). A total of 4,619 coding sequences were predicted (8) using GLIMMER (11) and CRITICA (1). Putative functions of protein-encoding genes were automatically assigned using the GenDB annotation pipeline (9) and manually optimized. The chromosome harbors 7 rRNA operons and 78 tRNAs over the chromosome.

The P. vagans C9-1 genome has four large regions containing phage-related genes. Bordering regions could not be identified explicitly (e.g., one phage region is flanked by a tRNA^Met [AttL] and an AttR region, while others were on the other side of AttR). Transposon-related genes were largely absent, but small genomic islands were observed. Only plasmid pPag2 contains remnants of transposases and recombinases, including an inactivated ISEhe3 and ISEhe4, related to those on pPATH_pag in P. agglomerans pv. gypsophilae 824-1 (4).

The P. vagans C9-1 genome contains a repertoire of carbohydrate metabolic pathways identified using the KEGG databases (7). Important biosynthetic genes for antibacterial metabolites (i.e., pantocin A and dapdiamide E) and epiphytic fitness genes (e.g., autoinducer 1 [AI-1] quorum-sensing genes, indoleacetic acid, and carotenoid biosynthesis) were identified. No known virulence determinants of enterobacteria (e.g., type III secretion systems, effectors, toxins, and pectolytic enzymes) were found in the C9-1 genome (2, 3, 12-14). The genome sequence of P. vagans C9-1 provides a foundation for biosafety assessment of biological control strains introduced into agricultural environments and identification of genes and regulatory cascades that may impact the efficacy of this and related bacterial antagonists used for plant disease control.

Nucleotide sequence accession numbers.

The P. vagans C9-1 chromosome and plasmid pPag1, pPag2, and pPag3 sequences have been deposited in GenBank under accession numbers CP002206 (chromosome), CP001893 (pPag1), CP001894 (pPag2), and CP001895 (pPag3).