Draft Genome Sequence of the Biocontrol and Plant Growth-Promoting Rhizobacterium Pseudomonas fluorescens strain UM270

The Pseudomonas fluorescens strain UM270 was isolated form the rhizosphere of wild Medicago spp. A previous work has shown that this pseudomonad isolate was able to produce diverse diffusible and volatile compounds involved in plant protection and growth promotion. Here, we present the draft genome sequence of the rhizobacterium P. fluorescens strain UM270. The sequence covers 6,047,974 bp of a single chromosome, with 62.66 % G + C content and no plasmids. Genome annotations predicted 5,509 genes, 5,396 coding genes, 59 RNA genes and 110 pseudogenes. Genome sequence analysis revealed the presence of genes involved in biological control and plant-growth promoting activities. We anticipate that the P. fluorescens strain UM270 genome will contribute insights about bacterial plant protection and beneficial properties through genomic comparisons among fluorescent pseudomonads.


Introduction
Plant pathogens cause diverse crop plant diseases resulting in drastic economic losses around the world. An alternative to the use of chemicals to control plant pathogens is the employment of eco-friendly bacterial agents [1,2]. An ideal bacterial biocontrol agent would be one with the additional capacity to directly stimulate plant growth [3]. Here, we report the draft genome sequence of the novel strain Pseudomonas fluorescens strain UM270. This strain was previously isolated and characterized for its excellent capacities for biocontrol of phytopathogens and plant growth promotion [4].
In a previous report, our group showed that the P. fluorescens strain UM270, among other three pseudomonad strains, was the best in promoting the growth of Medicago truncatula Gaertn. plants by significantly increasing biomass and chlorophyll content. During confrontation assays, strain UM270 inhibited the growth of agro-economically important fungal phytopathogens such as Botrytis cinerea, Rhizoctonia solani, Diaporthe phaseolorum, and Colletotrichum lindemuthianum [4]. In biocontrol experiments, the strain UM270 protected M. truncatula plants from B. cinerea infection, reducing general stem disease symptoms, root browning and necrosis [4].

Organism Information
Classification and features P. fluorescens strain UM270 is a Gram-negative, nonsporulating, motile, rod-shaped bacterium belonging to the Order Pseudomonadales and the Family Pseudomonadaceae (Fig. 1). The strain exhibits the general and common features of a Pseudomonas species phenotype (Table 1) [6].

Genome sequencing information
Genome project history The P. fluorescens strain UM270 was selected among other pseudomonads for its higher ability to control fungal pathogens and protect Medicago truncatula Gaertn. from B. cinerea infection [4], for being highly competitive, an excellent root and rhizosphere colonizer of maize plants and for inducing ISR in plants (Rojas-Solis and Santoyo, Unpublished results). A high-quality draft sequence of the genome has been deposited at DDBJ/ EMBL/GenBank. A summary of the project information is shown in Table 2.

Growth conditions and genomic DNA preparation
From a single colony culture the P. fluorescens strain UM270 was inoculated on 50 ml of King's B medium [10], grown overnight at 28°C with in agitation (250 rpm). One milliliter of the culture was serially diluted to be analyzed further. We confirmed the morphology and antibiotic-resistance phenotype of the strain. From the culture, 20 ml were taken to isolate the genomic DNA by using the Wizard® Genomic DNA Purification Kit following manufacture's instructions (Promega). DNA samples were subjected to an additional purification step with the same Wizard® Fig. 3 Graphical map of the P. fluorescens strain UM270. Numbers represent Megabases (Mb). From outside to the center: Genes on forward strand (blue), Genes on reverse strand (red), RNA genes (rRNAs black color, tRNAs red color) G + C% (green and gray), G + C skew (purple and yellow). To display the P. fluorescens strain UM270 draft genome we ran a blastn comparison using the contigs as query, against the genome sequence of P. fluorescens strain Pf0-1 as target reference. We then used these results to order the contigs following the matching coordinates of the reference genome. Contigs not matching the reference genome were ordered from largest to smallest and appended to the contigs matching the genome of reference. The ordered contigs were joined with 50 bp of "N" to draw this figure using the DNA plotter software Genomic DNA Purification Kit (Promega). The quality and quantity of the final DNA sample were evaluated by agarose gel electrophoresis and by using a NanoDrop 1000 Spectrophotometer (Thermo Scientific).

Genome sequencing and assembly
Genomic DNA samples of P. fluorescens strain UM270 were sent to a sequencing service at the LANGEBIO-Irapuato, México. Genome sequencing was performed using a MiSeq Sequencer (Illumina, Inc.) generating three paired-end libraries (400-450 bp, 600 bp and 1,000 bp, respectively) with a coverage of approximately 45×. The P. fluorescens strain UM270 draft genome we ran a blastn comparison using the contigs as query, against the genome sequence of P. fluorescens Pf0-1 as target reference. To order the contigs we followed the matching coordinates of the reference genome. Project information is shown in Table 2.

Genome annotation
Genome annotation was carried out with RAST [11] and the Prokaryotic Genome Annotation Pipeline tools [12]. Statistics for the genome assembly were calculated using software Newbler v2.9 (Roche) and are shown in Table 2. This Whole Genome Shotgun sequence project has been deposited at DDBJ/EMBL/GenBank under accession JXNZ00000000. The version described in this paper is version JXNZ00000000.

Genome Properties
The total length of the assembled sequences obtained was 6,047,974 bp belonging to one chromosome, with a G + C content of 62.66 %. The sequenced fragments of the genome are predicted to contain 5,509 genes, consisting of 5,396 coding sequences, 59 RNA genes, 110 pseudogenes and 14 frameshifted genes. Genome statistics are in Table 3 and a graphical map is represented in Fig. 3. The The total is based on the total number of protein coding genes in the annotated genome Table 4 presents the number of genes associated with the COG functional categories.

Insights from the genome sequence
The draft genome sequence reported here covers its full genome and at first analysis reveals the presence of multiple genes participating in the synthesis of diffusible metabolites and volatile organic compounds produced by P. fluorescens strain UM270. Some of this antimicrobial arsenal includes compounds like phenazine (phzFABCD), pyocyanin (pcnCDE), pyoverdine (pvdPD), 2,4-diacetylphloroglucinol (phlACBD) and the volatile hydrogen cyanide (hcnCB), important for the biological control of several plant diseases caused by phytopathogenic fungi, oomycetes, and bacteria [2]. Other plant-bacteria communication genes detected in the strain UM270 genome are acdS and iaaMH, encoding for an ACC deaminase (1-aminocyclopropane-1-carboxylate) protein and IAA (indole-3-acetic acid) biosynthesis. The synergistic interaction of ACC deaminase and both plant and bacterial auxin, IAA, is relevant for the optimal functioning of PGPR to directly promote plant growth and also protect plants against environmental stresses, and bacterial and fungal pathogens [5]. Other genes such as pcdQ, which codes for an Acyl-homoserine lactone acylase, important for bacterial communication and biofilm formation, were detected, as well as Secretion Systems Type II to VI and orthologs of the toxin-antitoxin loci vapBC-1 and vapXD. These last determinants are important for survival, competence and colonization of the rhizosphere and root systems [13].

Conclusions
The strain UM270 was selected for genome sequencing due to its biocontrol and plant growth promoting properties [4]. The plant beneficial mechanisms exerted by this rhizobacterium involved direct and indirect mechanisms. Here, the draft genome sequence of the P. fluorescens strain UM270 revealed further genetic elements involved in plant-bacterial communication, as well as in rhizosphere competence and colonization. We anticipate that the genome of P. fluorescens strain UM270 will contribute to new insights about biocontrol and plant beneficial activities through genomic comparisons among available complete genomes of pseudomonad strains.