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-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 []. The tree was inferred under the maximum likelihood criterion [MEGA5, ] 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% []. Lineages with type strain genome sequences registered in GOLD [] 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.