Single colony of CRS05-R5 was inoculated into 5 ml NB (Nutrient Broth, BD, USA) at 30°C with 180 rpm vigorous shaking. 2.5 ml of culture broth was used to isolate the genomic DNA. DNA was extracted by Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). The quality of purified genomic DNA was tested by using NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, MA, USA) and Qubit 2.0 fluorometer (Life Technologies, MA, USA), respectively.

Whole genome sequencing of CRS05-R5 was carried out by using PacBio RS II platform. Six hundred Megabytes raw data was obtained with 100X coverage. After quality control, genome assembly was de novo assembled using HGAP assembly protocol, which is available with the SMRT Analysis packages and accessed through the SMRT Analysis Portal version 2.1. Genome annotation was later done by using RAST annotation system (Overbeek et al., 2014). The completeness of the assembled genome was tested by CheckM with default parameters (Parks et al., 2015). In addition, GO and COG programs were used to do further functional analysis of all annotated ORFs (Ashburner et al., 2000; Tatusov et al., 2000). Specifically, since antagonistic bacteria, especially fluorescent pseudomonads, usually compete with other microorganisms by using secondary metabolism (Haas and Défago, 2005), genes, which are predicted to be involved in secondary metabolism were compared with DoBISCUIT database (Ichikawa et al., 2013). The circular genome map of CRS05-R5 including all predicted ORFs with COG functional assignments, rRNA, tRNA, G+C content, and GC skew information were generated using Circos (Krzywinski et al., 2009), as shown in Figure S2.

Genome comparison among CRS05-R5 and other fully sequenced Pseudomonas genomes were carried out by using ANI (average nucleotide identity) and AF (alignment fraction), which were calculated by ANIcalculator (Varghese et al., 2015), and Circos (Krzywinski et al., 2009). In order to find out the unique genes in CRS05-R5, all the fully sequenced Pseudomonas genomes were downloaded from NCBI (58 genomes). The protein sequences from CRS05-R5 were compared with the protein sequences from these 58 genomes with 40% identity as cutoff. The protein sequences which cannot find any homologs in these 58 genomes were classified as unique genes.

This strain has been deposited in CGMCC with deposit number 1.15630. This genome sequencing project has been deposited at DDBJ/EMBL/GenBank under the Accession Number CP015852. The BioProject designation for this project is PRJNA322127.

The total size of the genome is 5,991,224 bp and has a G+C content of 60.6%. The completeness and contamination of this genome is 99.86% and 0.05% after running the CheckM. These results indicate the high quality of assembled genome. A total of 5352 CDSs were predicted. Of these, 3832 could be assigned to a COG number. The most abundant COG category was “General function prediction only” (581 proteins) followed by “Signal transduction mechanisms” (547 proteins), “Amino acid transport and metabolism” (539 proteins), “Transcription” (448 proteins), and “Function unknown” (302 proteins). In addition, 82 RNAs including rRNA and tRNA were identified. All the genomic information was shown in Table 1.

Biosurfactants are amphiphilic compounds produced by microorganisms (Mulligan, 2005). This compound, which is produced by Pseudomonas spp., has been widely used in biocontrol (Debode et al., 2007). In CRS05-R5 genome, more than 800 genes are predicted to be involved in secondary metabolism by comparing with DoBISCUIT database (Ichikawa et al., 2013). Interestingly, we found one gene cluster, which is annotated as cyclic lipopeptides (CLPs), exists in CRS05-R5 (arfABC). CLP is a kind of biosurfactant, and has been proved to be important in antagonistic Pseudomonas sp. (Raaijmakers et al., 2006). This information indicates that CRS05-R5 can be used as biocontrol agent.

Strikingly, we found even the ANI value between CRS05-R5 and P. koreensis D26 is only 92.5% (Figure S3). Also, the highest AF value is only 74.2% (Table S1). Indeed, 631 CDSs were predicted to be unique genes existing in CRS05-R5 genome. These results indicate the huge genome diversity among Pseudomonas strains, which is consistent with previous findings (Loper et al., 2012). Circos result found many unique genes in CRS05-R5 (Figure 1). To better understand the features of these genes, Pseudomonas database was used to deeply annotate these genes (Winsor et al., 2011). Except for a lot of hypothetical proteins, we found one gene cluster encoding fimbrial associated proteins only existing in CRS05-R5 (A8L59_09240–A8L59_09310). These genes have been reported to be critical for the initial stage of biofilm development (Wei and Ma, 2013). Except for this gene cluster, we also found three rhs genes exist as the unique genes in CRS05-R5 (A8L59_00750, A8L59_09890, and A8L59_11585). These genes have been found to be linked to the second type VI secretion cluster in P. aeruginosa (Jones et al., 2014). Also, these genes have been reported to mediate intercellular competition (Koskiniemi et al., 2013). More strikingly, one rhs gene is located in a unique gene cluster (A8L59_11555–A8L59_11600). Although, most of the genes are hypothetical proteins, we may infer that this cluster maybe related with secretions. Indeed, by searching with TMHMM Server v. 2.0, which is a transmembrane helices prediction database (http://www.cbs.dtu.dk/services/TMHMM/), we found that 3 genes (A8L59_11560, A8L59_11565, and A8L59_11600) have predicted transmembrane helices. Since biofilm and intercellular competition are very important for bacterial survival and adaptation, we infer that these genes may confer some fitness advantages for CRS05-R5 on the root of Oryza sativa. Also, we found one unique gene cluster (A8L59_18500–A8L59_18575), which encodes wbpL and other glycosyl transferase. These genes have been confirmed to be important in lipopolysaccharide formation (Rocchetta et al., 1998). These compounds have been proved to be important in plant roots colonization (Duijff et al., 1997) as well as induction of systemic resistance against some plant pathogens (Leeman et al., 1995). All these results strongly indicate the biocontrol potential of CRS05-R5 strain.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.