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

Abstract

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).

Keywords:

motilenon-sporulatingmesophileGram-negativefree livingplant-associatedchemoorganotrophicEnterobacteriaceaeCSP 2010

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-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]

MIGS ID

     Property

     Term

      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

      IDA

     Gram stain

     negative

      IDA

     Cell shape

     Rod-shaped

      IDA

     Motility

     Motile

      IDA

     Sporulation

     Non-sporulating

      IDA

     Temperature range

     Mesophilic

      IDA

     Optimum temperature

     28°C

      IDA

     Carbon source

     Glucose, mannitol, sucrose, arabinose, cellobiose

      IDA

     Energy metabolism

     Chemoorganotrophic

      NAS

     Terminal electron receptor

     --

MIGS-6

     Habitat

     Rapeseed roots

      NAS

MIGS-6.3

     Salinity

     Medium

      IDA

MIGS-22

     Oxygen

     Facultative

      IDA

MIGS-15

     Biotic relationship

     Free living

      NAS

MIGS-14

     Pathogenicity

     Non-pathogenic

      IDA

     Biosafety level

     1+

      TAS [24]

MIGS-4

     Geographic location

     Uppsala, Sweden

      NAS

MIGS-5

     Sample collection time

     Summer 1998

      NAS

MIGS-4.1

     Latitude

     59.8

      NAS

MIGS-4.2

     Longitude

     17.65

      NAS

MIGS-4.3

     Depth

     0.1 m

      NAS

MIGS-4.4

     Altitude

     24-25 m

      NAS

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.

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 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

MIGS ID

     Property

     Term

MIGS-31

     Finishing quality

     Finished

MIGS-28

     Libraries used

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

MIGS-29

     Sequencing platforms

     Illumina GAii, 454 GS FLX Titanium

MIGS-31.2

     Sequencing coverage

     323.5 × Illumina; 8.8 × pyrosequencing

MIGS-30

     Assemblers

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

MIGS-32

     Gene calling method

     Prodigal 1.4, GenePRIMP

     NCBI project ID

     60457

     INSDC ID

     CP002773

     Genbank Date of Release

     October 12, 2011

     GOLD ID

     Gc01772

MIGS-13

     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

Attribute

    Value

     % of totala

Genome size (bp)

    5,442,880

     100.00%

DNA coding region (bp)

    4,739,233

     87.07%

DNA G+C content (bp)

    3,045,898

     55.96%

Total genesa

    5,139

     100.00%

RNA genes

    113

     2.19%

rRNA operons

    7

Protein-coding genes

    4,952

     96.36%

Pseudo genes

    75

     1.46%

Genes in paralog clusters

    124

     2.4%

Genes assigned to COGs

    3,807

     74.08%

Genes assigned in Pfam domains

    4,185

     81.43%

Genes with signal peptides

    677

     13.17%

Genes with transmembrane helices

    1,227

     23.87%

CRISPR repeats

    1

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

Code

     Value

    %agea

      Description

J

     201

    4.27

      Translation, ribosomal structure and biogenesis

A

     1

    0.02

      RNA processing and modification

K

     481

    10.22

      Transcription

L

     160

    3.40

      DNA replication, recombination and repair

B

     1

    0.02

      Chromatin structure and dynamics

D

     37

    0.79

      Cell division and chromosome partitioning

Y

     0

    0.00

      Nuclear structure

V

     64

    1.36

      Defense mechanisms

T

     187

    3.97

      Signal transduction mechanisms

M

     265

    5.63

      Cell envelope biogenesis, Outer membrane

N

     94

    2.00

      Cell motility and secretion

Z

     0

    0.00

      Cytoskeleton

W

     0

    0.00

      Extracellular structure

U

     116

    2.47

      Intracellular trafficking and secretion

O

     153

    3.25

      Posttranslational modification, protein turnover, chaperones

C

     272

    5.78

      Energy production and conversion

G

     424

    9.01

      Carbohydrate transport and metabolism

E

     470

    9.99

      Amino acid transport and metabolism

F

     106

    2.25

      Nucleotide transport and metabolism

H

     185

    3.93

      Coenzyme metabolism

I

     135

    2.87

      Lipid metabolism

P

     285

    6.06

      Inorganic ion transport and metabolism

Q

     133

    2.83

      Secondary metabolites biosynthesis, transport and catabolism

R

     537

    11.41

      General function prediction only

S

     398

    8.46

      Function unknown

-

     917

    17.85

      Not in COG

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

Declarations

Acknowledgements

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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