Complete genome sequence of the rapeseed plant-growth promoting Serratia plymuthica strain AS9

Serratia plymuthica are plant-associated, plant beneficial species belonging to the family Enterobacteriaceae. The members of the genus Serratia are ubiquitous in nature and their life style varies from endophytic to free-living. S. plymuthica AS9 is of special interest for its ability to inhibit fungal pathogens of rapeseed and to promote plant growth. The genome of S. plymuthica AS9 comprises a 5,442,880 bp long circular chromosome that consists of 4,952 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome is part of the project entitled “Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens” awarded through the 2010 DOE-JGI Community Sequencing Program (CSP2010).


Introduction
The genus Serratia belongs to a group of Gammaproteobacteria, commonly found in soil, water, plants, insects and humans [1]. The genus includes antagonists of soil borne pathogens of different plant species, plant growth promoters and insect pathogens, as well as opportunistic human pathogens. The most common human pathogen in this genus is Serratia marcescens which causes nosocomial infections in humans, while other species are harmless. In agriculture, S. plymuthica is successfully used for control of many soil borne fungal pathogens of different crops (e.g. strawberry, rapeseed) [2,3], while S. proteamaculans promotes the growth of poplar trees [4]. S.plymuthica AS9 (= CCUG 61396) was isolated from field samples of rapeseed roots in Uppsala, Sweden. Our interest in S. plymuthica AS9 is attributed to its ability to stimulate rapeseed plant growth, to inhibit soil borne fungal pathogens and to increase oilseed production. Here we present a description of the complete genome sequencing of S. plymuthica AS9 and its annotation.

Classification and features
The bacterial strain AS9 was previously considered a member of the family Enterobacteriaceae [5]. Recently, comparison of 16S rRNA gene sequences with the most recent databases from GenBank using NCBI BLAST [6] under default settings showed that S. plymuthica AS9 shares 99% similarity with many Serratia species including S. plymuthica (AJ233433) and Serratia proteamaculans (CP000826.1). When considering high-scoring segment pairs (HSPs) from the best 250 hits, the most frequent matches were with various Serratia species (17.2% with maximum identity of 97-100%) with S. plymuthica (5.2% with maximum identity of 97-99%), S. proteamaculans (4.8% with maximum identity of 97-99%), S. marcescens (4.8% with maximum identity of 96-97%) and various Rahnella species. (7% with maximum identity of 97-98%). Figure 1 shows the phylogenetic relationship of S. plymuthica AS9 with other species within the genus Serratia in a 16S rRNA based tree. The tree shows its close relationship with the type strain of S. plymuthica, which was confirmed by digital DNA-DNA hybridization values [11] above 70% with the (unpublished) draft genome sequence of the S. plymuthica type strain Breed K-7 T from a DSM4540 culture using the GGDC web server [12]. S. plymuthica AS9 is a Gram-negative, rod shaped, motile bacterium, 1-2 µm long and 0.5-0.7 µm wide ( Figure 2 and Table 1) . It forms red to pink colored colonies 1-2 mm in diameter on tryptic soy agar and potato dextrose agar. The color of the bacterium is the result of its production of the red pigment, prodigiosin, but the colony color or production of pigment depends on the ingredients, pH of the medium and the incubation temperature [26][27][28]. S. plymuthica is a facultative anaerobe, grows between 4 °C and 40 °C and within the pH range 4 -10. It can utilize a wide range of carbon sources and also has chitinolytic, proteolytic, cellulolytic, and phospholytic activity [5].

Chemotaxonomy
The whole cell lipid pattern of S. plymuthica AS9 contains a mixture of saturated and unsaturated fatty acids. The main fatty acids in AS9 strain comprise C 16:0 (24.13%), C 16:1ω7c (19.41%), C 18:1ω7c (18.76%), C 14:0 (5.24%) along with other minor fatty acid components. Previously it has been shown that Serratia spp. contain a mixture of C 14:0 , C 16:0 , C 16:1 and C 18:1+2 fatty acids of which 50-80% of the total was C 14:0 and other were less than 3% each [29]. This is consistent with the fact that the C 14:0 3OH is characteristic of the family Enterobacteriaceae. Figure 1. Phylogenetic tree highlighting the position of S. plymuthica AS9 in relation to other species within the genus Serratia, which is based on 1,479 characters of the 16S rRNA gene sequence aligned in ClustalW2 [7]. The tree was inferred under the maximum likelihood criterion [MEGA5,8] and rooted with Yersinia pseudotuberculosis (a member of the family Enterobacteriaceae). The branches are scaled in terms of the expected number of substitutions per site. The numbers above branches are support values from 1,000 bootstrap replicates if larger than 60% [9]. Lineages with type strain genome sequences registered in GOLD [10] are shown in blue. Standards in Genomic Sciences  , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [25]. If the evidence code is IDA, then the property was observed by one of the authors, or an expert mentioned in the acknowledgements.

Genome sequencing information
S. plymuthica AS9, one of the strains isolated from rapeseed roots and rhizosphere soils was selected for sequencing on the basis of its ability to promote rapeseed growth and inhibit soil borne fungal pathogens. The genome project is deposited in the Genomes On Line Databases [10] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2 and its association with MIGS identifiers.

Growth conditions and DNA isolation
S. plymuthica AS9 was grown in Luria Broth (LB) medium at 28°C for 12 hours (cells were in the early stationary phase) and the DNA was isolated using a standard CTAB protocol for bacterial genomic DNA isolation which is available at JGI [30].

Genome sequencing and assembly
The genome of strain AS9 was sequenced using a combination of Illumina [31] and 454 sequencing platforms [32]. The details of library construction and sequencing are available at the JGI website [30].

Genome annotation
Genes were identified using Prodigal [40] as part of the genome annotation pipeline at Oak Ridge National Laboratory (ORNL), Oak Ridge, TN, USA, followed by a round of manual curation using the JGI GenPRIMP pipeline [41]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, Uniport, TIGR-Fam, Pfam, Standards in Genomic Sciences PRIAM, KEGG, COG and InterPro databases. The tRNAScanSE tool [42] was used to find tRNA genes. Additional gene prediction analysis and functional annotation were performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [43].

Genome properties
The S. plymuthica AS9 genome includes a single circular chromosome of 5,442,880 bp with 55.96% GC content. The genome had 5,139 predicted genes of which 4,952 were assigned as protein-coding genes, 113 RNA genes and 75 pseudogenes [ Figure 3]. The majority of protein coding genes (87.42%) was assigned as a putative function while those remaining were annotated as hypothetical proteins [ Table 3]. The distribution into COG functional categories is presented in Table 4.  a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome. a) The total is based on the total number of protein coding genes in the annotated genome.