Open Access

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

  • Saraswoti Neupane
  • , Nils Högberg
  • , Sadhna Alström
  • , Susan Lucas
  • , James Han
  • , Alla Lapidus
  • , Jan-Fang Cheng
  • , David Bruce,
  • , Lynne Goodwin,
  • , Sam Pitluck
  • , Lin Peters
  • , Galina Ovchinnikova
  • , Megan Lu,
  • , Cliff Han,
  • , John C. Detter,
  • , Roxanne Tapia,
  • , Anne Fiebig
  • , Miriam Land,
  • , Loren Hauser,
  • , Nikos C. Kyrpides
  • , Natalia Ivanova
  • , Ioanna Pagani
  • , Hans-Peter Klenk
  • , Tanja Woyke
  • and Roger D. Finlay

DOI: 10.4056/sigs.2595762

Received: 05 March 2012

Published: 19 March 2012


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).


motilenon-sporulatingmesophileGram-negativefree livingplant-associatedchemoorganotrophicEnterobacteriaceaeCSP 2010


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-7T from a DSM4540 culture using the GGDC web server [12].

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.

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 [26-28]. 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].

Figure 2

Scanning electron micrograph of S. plymuthica AS9

Table 1

Classification and general features of S. plymuthica AS9 according to the MIGS recommendations [13]




      Evidence codea

     Current classification

Domain Bacteria

      TAS [14]

Phylum Proteobacteria

      TAS [15]

Class Gammaproteobacteria

      TAS [15,16]

Order “Enterobacteriales

      TAS [17]

Family Enterobacteriaceae

      TAS [18-20]

Genus Serratia

      TAS [18,21,22]

Species Serratia plymuthica

      TAS [18,23]

     Strain AS9


     Gram stain



     Cell shape









     Temperature range



     Optimum temperature



     Carbon source

     Glucose, mannitol, sucrose, arabinose, cellobiose


     Energy metabolism



     Terminal electron receptor




     Rapeseed roots











     Biotic relationship

     Free living






     Biosafety level


      TAS [24]


     Geographic location

     Uppsala, Sweden



     Sample collection time

     Summer 1998












     0.1 m




     24-25 m


a) Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., 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.


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 C16:0 (24.13%), C16:1ω7c (19.41%), C18:1ω7c (18.76%), C14:0 (5.24%) along with other minor fatty acid components. Previously it has been shown that Serratia spp. contain a mixture of C14:0, C16:0, C16:1 and C18:1+2 fatty acids of which 50-80% of the total was C14:0 and other were less than 3% each [29]. This is consistent with the fact that the C14:0 3OH is characteristic of the family Enterobacteriaceae.

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.

Table 2

Genome sequencing project information





     Finishing quality



     Libraries used

     Three libraries: one 454 standard library, one 454 PE library (12.5 kb insert size), one Illumina library


     Sequencing platforms

     Illumina GAii, 454 GS FLX Titanium


     Sequencing coverage

     323.5 × Illumina; 8.8 × pyrosequencing



     Velvet v. 0.7.63, Newbler v. 2.3 pre-release, phrap version SPS – 4.24


     Gene calling method

     Prodigal 1.4, GenePRIMP

     NCBI project ID




     Genbank Date of Release

     October 12, 2011

     GOLD ID



     Source material identifier

     CCUG 61396

     Project relevance

     Biocontrol, Agricultural

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]. The sequence data from Illumina GAii (1,790.7 Mb) were assembled with Velvet [33] and the consensus sequence computationally shredded into 1.5 kb overlapping fake reads. The sequencing data from 454 pyrosequencing (102.2 Mb) were assembled with Newbler (Roche). The initial draft assembly contained 41 contigs in one scaffold and consensus sequences were computationally shredded into 2 kb overlapping fake reads. The 454 Newbler consensus reads, the Illumina velvet consensus reads and the read pairs in the 454 paired end library were integrated using a software phrap (High Performance Software, LLC) [34]. Possible mis-assemblies were corrected with gapResolution [30], Dupfinisher [35], or by sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI). The gaps between contigs were closed by editing in the software Consed [36-38], by PCR and by Bubble PCR (J.-F. Chang, unpublished) primer walks. Thirty seven additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The sequence reads from Illumina were used to correct potential base errors and increase consensus quality using the software Polisher, developed at JGI [39]. The final assembly is based on 47.3 Mb of 454 draft data which provides an average 8.8× coverage of the genome and 1,746.8 Mb of Illumina draft data which provides an average 323.5× coverage of the genome.

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) non-redundant database, Uniport, TIGR-Fam, Pfam, 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.

Figure 3

Graphical circular map of the chromosome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 3

Genome statistics



     % of totala

Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Total genesa



RNA genes



rRNA operons


Protein-coding genes



Pseudo genes



Genes in paralog clusters



Genes assigned to COGs



Genes assigned in Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats


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.

Table 4

Number of genes associated with the 25 general COG functional categories








      Translation, ribosomal structure and biogenesis




      RNA processing and modification








      DNA replication, recombination and repair




      Chromatin structure and dynamics




      Cell division and chromosome partitioning




      Nuclear structure




      Defense mechanisms




      Signal transduction mechanisms




      Cell envelope biogenesis, Outer membrane




      Cell motility and secretion








      Extracellular structure




      Intracellular trafficking and secretion




      Posttranslational modification, protein turnover, chaperones




      Energy production and conversion




      Carbohydrate transport and metabolism




      Amino acid transport and metabolism




      Nucleotide transport and metabolism




      Coenzyme metabolism




      Lipid metabolism




      Inorganic ion transport and metabolism




      Secondary metabolites biosynthesis, transport and catabolism




      General function prediction only




      Function unknown




      Not in COG

a) The total is based on the total number of protein coding genes in the annotated genome.



We would like to gratefully acknowledge the help of Elke Lang for providing cell pastes of reference material and Evelyne-Marie Brambilla for extraction of DNA for digital DNA-DNA hybridizations with the reference strains (both at DSMZ). The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.


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