Open Access

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

  • Julie E. Hernández-Salmerón1,
  • Rocio Hernández-León1,
  • Ma. Del Carmen Orozco-Mosqueda1,
  • Eduardo Valencia-Cantero1,
  • Gabriel Moreno-Hagelsieb2 and
  • Gustavo Santoyo1Email authorView ORCID ID profile
Standards in Genomic Sciences201611:5

https://doi.org/10.1186/s40793-015-0123-9

Received: 27 May 2015

Accepted: 9 September 2015

Published: 13 January 2016

Abstract

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.

Keywords

Pseudomonas fluorescens BiocontrolPGPR

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].

Importantly, the strain UM270 exerted these activities through the emission of either diffusible compounds (such as phenazines, cyanogens, 1-aminocyclopropane-1-carboxylate deaminase, siderophores, proteases and indole-3-acetic acid) or volatiles (like dimethyl disulfide and dimethylhexadecylamine) [4], revealing that the strain UM270 contains direct and indirect mechanisms to promote plant growth [5].

Organism Information

Classification and features

P. fluorescens strain UM270 is a Gram-negative, non-sporulating, 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].
Fig. 1

Images of P. fluorescens strain UM270 using scanning electron microscopy (left and right) and phase-contrast (center)

Table 1

Classification and general features of Pseudomonas fluorescens strain UM270

MIGS ID

Property

Term

Evidence codea

 

Current classification

Domain Bacteria

TAS [14]

Phylum Proteobacteria

TAS [15]

Class Gammaproteobacteria

TAS [16, 17]

Order Pseudomonadales

TAS [18, 19]

Family Pseudomonadaceae

TAS [18, 20]

Genus Pseudomonas

TAS [18, 21]

Species Pseudomonas fluorescens

TAS [18, 22]

Strain UM270

TAS [4]

 

Gram stain

Negative

TAS [6]

 

Cell shape

Rod-shaped

TAS [6]

 

Motility

Motile

NAS [6]

 

Sporulation

None

NAS

 

Temperature range

Mesophilic

IDA

 

pH range; Optimum

6-8.5;7-8

IDA

 

Optimum temperature

28 °C

IDA

 

Carbon source

Heterotroph

IDA, [6]

 

Energy source

Chemoorganotroph

NAS

MIGS-6

Habitat

Rhizospheric soil

TAS [4]

MIGS-6.3

Salinity

NaCl 1-4 %

IDA

MIGS-22

Oxygen Requirement

Aerobic

IDA

MIGS-15

Biotic relationship

Medicago spp. root associated

TAS [4]

MIGS-14

Pathogenicity

Non-pathogenic

TAS [4]

MIGS-4

Geographic location

Morelia, México

TAS [4]

MIGS-5

Sample collection

March, 2012

NAS

MIGS-4.1

Latitude

19° 46’ 6” N

TAS [4]

MIGS-4.2

Longitude

101° 11’ 22” W

TAS [4]

MIGS-4.3

Depth

10-20 cm

NAS

MIGS-4.4

Altitude

1800 M.A.S.L.

NAS

aEvidence 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

The UM270 strain was isolated from the rhizosphere of Medicago spp. located in an agricultural field in Morelia, Michoacán, México. As mentioned above, this bacterium was further characterized and found to produce several diffusible and volatile compounds involved in biocontrol against several fungal pathogens, particularly effective against the grey mold disease caused by Botrytis cinerea [4]. Recent work in our lab has demonstrated that this strain is highly competitive and an efficient root and rhizosphere colonizer, as well as an inducer of ISR (Induced systemic resistance) in plants [Rojas-Solis and Santoyo, Unpublished results]. The Minimum Information about the Genome Sequence of P. fluorescens strain UM270 is summarized in Table 1. Its phylogenetic position is shown in Fig. 2, where the 16S rRNA gene of P. fluorescens strain UM270 is 99 % similar to that of P. fluorescens strain Pf0-1 [79].
Fig. 2

Phylogenetic tree showing the close relationship of P. fluorescens strain UM270 with P. fluorescens Pf0-1, as well as with other Pseudomonas species based on aligned sequences of the 16S rRNA gene. Phylogenetic analyses were performed using SeaView and edited in iTol. The tree was built using the maximum likelihood method. Bootstrap analysis (1000 replicates) was performed to assess the support of the clusters. E. coli was used as an outgroup

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.
Table 2

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

High-quality draft (Full genome representation)

MIGS-28

Libraries used

3 libraries of 400–450 bp, 600 bp and 1,000 bp.

MIGS 29

Sequencing platforms

Illumina MiSeq

MIGS 31.2

Fold coverage

45.0 ×

MIGS 30

Assemblers

Newbler v. 2.9

MIGS 32

Gene calling method

NCBI Prokaryotic Genome, Annotation Pipeline

 

Locus Tag

RL74

 

Genbank ID

JXNZ00000000

 

GenBank Date of Release

2014-12-09

 

GOLD ID

Gb0118948

 

BIOPROJECT

PRJNA269735

MIGS 13

Source Material Identifier

UM270

 

Project relevance

Agriculture, Plant-Bacteria Interaction, Biocontrol

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® 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 Table 4 presents the number of genes associated with the COG functional categories.
Table 3

Genome statistics

Attribute

Value

% of total

Genome size (bp)

6,047,974

100.00

DNA coding (bp)

5,284,158

87.00

DNA G + C (bp)

3,772,331

62.00

DNA scaffolds

524

100.00

Total genes

5,509

100.00

Protein coding genes

5,396

98.00

RNA genes

59

-

Pseudo genes

110

1.90

Genes in internal clusters

NA

-

Genes with function prediction

4,490

82.00

Genes assigned to COGs

3,821

68.00

Genes with Pfam domains

4,297

78.00

Genes with signal peptides

5

0.09

Genes with transmembrane helices

30

0.50

CRISPR repeats

0

-

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

Table 4

Number of genes associated with the 25 general COG functional categories

Code

Value

% of totala

Description

J

159

2.94

Translation, ribosomal structure and biogenesis

A

0

0.00

RNA processing and modification

K

342

6.33

Transcription

L

117

2.16

Replication, recombination and repair

B

3

0.00

Chromatin structure and dynamics

D

32

0.59

Cell cycle control, cell division, chromosome partitioning

Y

0

0.00

Nuclear structure

V

55

1.01

Defense mechanisms

T

216

4.00

Signal transduction mechanisms

M

212

3.92

Cell wall/membrane biogenesis

N

142

2.63

Cell motility

Z

0

0.00

Cytoskeleton

W

0

0.00

Extracellular structures

U

55

1.01

Intracellular trafficking and secretion

O

150

2.77

Posttranslational modification, protein turnover, chaperones

C

244

4.52

Energy production and conversion

G

190

3.52

Carbohydrate transport and metabolism

E

434

8.04

Amino acid transport and metabolism

F

78

1.44

Nucleotide transport and metabolism

H

143

2.65

Coenzyme transport and metabolism

I

185

3.42

Lipid transport and metabolism

P

226

4.18

Inorganic ion transport and metabolism

Q

67

1.24

Secondary metabolites biosynthesis, transport and catabolism

R

364

6.74

General function prediction only

S

372

6.89

Function unknown

-

1,610

29.83

Not in COGs

aThe total is based on the total number of protein coding genes in the annotated genome

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.

Declarations

Acknowledgements

We thank CONACYT-México (Project Number: 169346) and CIC-UMSNH (2014–2015) for financial support to our research projects. JEH-S and RH-L received scholarships from CONACYT-México. JEH-S acknowledges a scholarship from the Government of Canada under The Emerging Leaders in the Americas Program (ELAP). This paper is a requirement to obtain the Ph.D. of JEH-S.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Instituto de Investigaciones Químico Biológicas, Universidad Michoacana de San Nicolás de Hidalgo
(2)
Department of Biology, Wilfrid Laurier University

References

  1. Adesemoye AO, Torbert HA, Kloepper JW. Plant growth-promoting rhizobacteria allow reduced application rates of chemical fertilizers. Microb Ecol. 2009;58:921–9. doi:10.1007/s00248-009-9531-y.PubMedView ArticleGoogle Scholar
  2. Santoyo G, Orozco-Mosqueda Ma Del C, Govindappa M. Mechanisms of biocontrol and plant growth-promoting activity in soil bacterial species of Bacillus and Pseudomonas: a review. Biocon Sci Technol. 2012;22:855–72. doi:10.1080/09583157.2012.694413.View ArticleGoogle Scholar
  3. Compant S, Duffy B, Nowak J, Clement C, Barka EA. Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Appl Environ Microbiol. 2005;71:4951–9. doi:10.1128/AEM.71.9.4951-4959.2005.PubMedPubMed CentralView ArticleGoogle Scholar
  4. Hernández-León R, Rojas-Solís D, Contreras-Pérez M, Orozco-Mosqueda Ma Del C, Macías-Rodríguez LI, Reyes-de La Cruz H, et al. Antifungal and plant-growth promoting effects of diffusible and volatile sulfur-containing compounds produced by Pseudomonas fluorescens strains. Biol Control. 2014;81:83–92. doi:10.1016/j.biocontrol.2014.11.011.View ArticleGoogle Scholar
  5. Glick BR. Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiol Res. 2014;169(1):30–9.PubMedView ArticleGoogle Scholar
  6. Palleroni NJ. Pseudomonadaceae. In: Krieg NR, Holt JG, editors. Bergey’s Manual of Systematic Bacteriology. Baltimore: The Williams and Wilkins Co; 1984. p. 141–99.Google Scholar
  7. Anzai Y, Kim H, Park JY, Wakabayashi H, Oyaizu H. Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Intl J Sys Evol Microbiol. 2000;50:1563–89.View ArticleGoogle Scholar
  8. Silby MW, Cerdeño-Tárraga AM, Vernikos GS, Giddens SR, Jackson RW, Preston GM, et al. Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens. Genome Biol. 2009;10:R51. doi:10.1186/gb-2009-10-5-r51.PubMedPubMed CentralView ArticleGoogle Scholar
  9. Loper JE, Hassan KA, Mavrodi DV, Davis EW, Lim CK, Shaffer BT, et al. Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions. PLoS Genet. 2012;8(7):e1002784.PubMedPubMed CentralView ArticleGoogle Scholar
  10. King EO, Ward M, Raney D. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954;44:301–7.PubMedGoogle Scholar
  11. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 2008;9:75. doi:10.1186/1471-2164-9-75.PubMedPubMed CentralView ArticleGoogle Scholar
  12. NCBI Prokaryotic Genome Annotation Pipeline. www.ncbi.nlm.nih.gov/genome/annotation_prok/
  13. Annette A. Angus and Ann M Hirsch: Biofilm Formation in the Rhizosphere: Multispecies Interactions and Implications for Plant Growth. Molecular Microbial Ecology of the Rhizosphere, Volume 2, First Edition. Edited by Frans J. de Bruijn. U S A: John Wiley & Sons, Inc.; 2013.Google Scholar
  14. Woese CR. Towards a natural system of organisms: Proposal for the domains Archea, Bacteria and Eucarya. Proc Natl Acad Sci U S A. 1990;87:4576–9.PubMedPubMed CentralView ArticleGoogle Scholar
  15. Garrity GM, Bell JA, Lilburn T: Phylum XIV. Proteobacteria phyl. nov. Bergey’s Manual of Systematic Bacteriology 2005, 2, Part B: 1.Google Scholar
  16. Garrity GM, Bell JA, Lilburn T: Class III. Gammaproteobacteria class. nov. Bergey’s Manual of Systematic Bacteriology 2005, 2, Part B: 1.Google Scholar
  17. List Editor: Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 106. International Journal of Systematic and Evolutionary Microbiology 2005, 55: 2235–2238.Google Scholar
  18. Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980;30:225–420.View ArticleGoogle Scholar
  19. Orla-Jensen S. The main lines of the natural bacterial system. J Bacteriol. 1921;6:263–73.PubMedPubMed CentralGoogle Scholar
  20. Winslow CEA, Broadhurst J, Buchanan RE, Krumwiede C, Rogers LA, Smith GH. The Families and Genera of the Bacteria: Preliminary Report of the Committee of the Society of American Bacteriologists on Characterization and Classification of Bacterial Types. J Bacteriol. 1917;2:505–66.PubMedPubMed CentralGoogle Scholar
  21. Migula W. Über ein neues System der Bakterien. Arbeiten aus dem Bakteriologischen Institut der Technischen Hochschule zu Karlsruhe. 1894;1:235–8.Google Scholar
  22. Migula W. Bacteriaceae (Stabchenbacterien). Die Natürlichen Pflanzenfamilien. 1895;I:20–30.Google Scholar

Copyright

© Hernández-Salmerón et al. 2015