Pseudomonas fluorescens soil project

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Classification

Classification
Kingdom: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Pseudomonadales
Family: Pseudomonadaceae
Genus: Pseudomonas
Species: P. fluorescens

Species

NCBI: Taxonomy

Pseudomonas fluorescens

Habitat Information

The soil organism was collected in the front yard of an Austin, TX home on January 26, 2018.
Soil was a little moist
Picked up on a day that had 83% humidity
Zero rainfall
Calm wind
51℉ air temperature.

Pseudomonas fluorescens is mainly found in plants, soil, and water surfaces.

Description and Significance

Pseudomonas fluorescens are gram-negative bacilli shaped bacteria. It grows best in temperatures that are 25-30℃. Certain strains of Pseudomonas fluorescens have been found to help stop plant disease by protecting the root and seed from fungal infection[1]. Other strains contribute to plant growth. Due to P. fluorescens having different flagella it has different strains which cause it to be in different environments including the bloodstream[2].

Genome Structure

P. fluorescens’ genome is composed of a single, circular chromosome with a median length of 6,300,000 base pairs. Guanine and Cytosine make up 60.3% of the nucleotides found in its DNA (its G/C ratio) [3].

L1 Forward sequence:
TACTGGGCGTNNGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGC TCAACCTGGGAACTGCATTCAAAACTGACAAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATG CGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAG CAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCG CAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAA GCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGATG GATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC GTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAG GAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTT GCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCG GAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGG AGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTTACCACGGTGTGATTCATGACTGGGGTGAAGTC

L2 Reverse sequence:
CCTCCCNANNGTTAGACTAGCTACTTCTGGTGCAACCCACTCCCATGGTGT GACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCACG CAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTCTGT ACCGACCATTGTAGCACGTGTGTAGCCCAGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTG TCACCGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAAC CCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAATGTTCCCGAAGGCACCAATCCATCTCTGGA AAGTTCATTGGATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGC CCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAG AGCTCAAGGCTCCCAACGGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGC TTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCG CTACACAGGAAATTCCACCACCCTCTACCATACTCTAGCTTGTCAGTTTTGAATGCAGTTCCCNGNTGAGCCCGGGGCTT TCACATCCAACTTAACAAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCTGTATTACCG CG

L1_7-16S-rRNA Forward genetic sequence:
TCGGATTACTGGGCGTNNGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCA ACCTGGGAACTGCATTCAAAACTGACAAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGT AGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAA ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCGCAG CTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCG GTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGATGGAT TGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTA ACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAA GGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCC AAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAA TCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGT GGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTTACCACGGTGTGATTCATGACTGGGGTGAA

L1_7-16S-rRNA Reverse genetic sequence:
GCGGNGTACTCNNCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAGAGCTCAAGGCTCCCAAC GGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTC AGTATCAGTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCA CCACCCTCTACCATACTCTAGCTTGTCAGTTTTGAATGCAGTTCCCAGGTTGAGCCCGGGGCTTTCACATCCAACTTAAC AAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGA GTTAGCCGGTGCTTATTCTGTCGGTAACGTCAAAACACTAACGTATTAGGTTAATGCCCTTCCTCCCAACTTAAAGTGCT TTACAATCCGAAGACCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTG CCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTTG GTGAGCCATTACCTCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTC CCGTAGGACGTATGCGGTATTAGCGTCCCTTTCGAGACGTTGTCCCCCACTACCAGGCAGATTCCTAGGCATTACTCACC CGTCCGCCGCTGAATTCAGGAGCAAGCTCCTGTCATCCGCTCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTCAATCT GAGCAGAA

Cell Structure, Metabolism and Life Cycle

Cell Structure

P. fluorescens are small-to-medium sized Gram-negative, rod-shaped bacilli. They are often found with multiple flagella in a lophotrichous arrangement. These many flagella, along with its ability to generate a biofilm, make P. fluorescens a great colonizer on various different surfaces and in different hosts and able to easily adapt to its environment[4]. One particularly prominent role of this biofilm is to serve as a protective agents to plants against parasitic fungi. Less is known about how P. fluorescens’ structure allows it to bind to mammalian cells, however it has been known to adhere to red blood cells in humans, which is one reason it is believed that, when found as a pathogenic agent in humans (which is very rare), it is almost always in the bloodstream. This organism follows a similar life cycle pattern found with other biofilm generating species, as discussed in “Life Cycle” [5].

Metabolism
P. fluorescens is well-known for having an extensive variety of metabolic capabilities, which allows it to live in so many different environments such as on the surfaces of plants, in soil, in the rhizosphere, and even in the bloodstream of humans and other animals[2].

P. fluorescens is a obligate aerobe, however, it has a unique ability to use nitrate (NO3) instead of atmospheric oxygen (O2) as its final electron acceptor in the Electron Transport Chain due to its ability to produce the enzyme nitrate reductase [6].

A unique metabolic feature of P. fluorescens is that it secretes a fluorescent pigment, pyoverdine, which imparts fluorescent properties to the organism under UV light, which is what led to its name. Pyoverdine is a high-affinity iron-chelating molecule that is essential for the organism’s acquisition of iron from the environment and used for bacterial growth. [7]

See more in “Physiology” for biochemical tests conducted in class.

Life Cycle

P. fluorescens follows a typical “biofilm” life cycle in that generally proceeds as follows:
1. Attachment: planktonic cells adhere to a surface and become sessile
2. Growth: cells exude exoenzymes and proteins to create a protective biofilm in which to flourish and grow.
3. Detachment: individual cells or clusters of cells will detach from the biofilm in order to move and colonize new surfaces/hosts

Physiology and Pathogenesis

Physiology

Gelatin Hydrolysis: Negative
DNA Hydrolysis: Negative
Lipid Hydrolysis: Positive
Phenol Red Broth: No fermentation
Starch Hydrolysis: Negative
Casein Hydrolysis: Positive
Methyl Red: Negative
Voges-Proskauer: Negative
Citrate: Positive
SIM: Negative
Nitrate Reduction: Positive
Urea Hydrolysis: Negative
Triple Sugar Iron: No fermentation, does not reduce sulfur
Decarboxylation: Arginine is positive, lysine and ornithine are negative
Phenylalanine: Negative
Oxidase: Positive
EMB Agar: Positive
HE Agar: Negative
Catalase: Positive
Blood Agar: Positive
Mannitol Salts Agar: Negative
PEA Agar: Negative
Bile Esculin: Negative
6.5% Salt Tolerance: Negative
Kirby-Bauer Antimicrobial Susceptibility Test for disinfectants: Kirby-Bauer Antimicrobial Susceptibility Tests for antibiotics: sensitive to several antibiotics [8]

Pathophysiology
Although P. fluorescens itself is largely considered non-pathogenic, it contains a number of metabolic abilities to allow it to thrive in mammalian hosts, including, but not limited to:
Production of bioactive secondary metabolites
- P. fluorescens produces a long list of secondary metabolites that allow it to successfully compete with other, similar organisms, such as phenazine, hydrogen cyanide, 2,4-diacetylphloroglucinol (DAPG), rhizoxin, and pyoluteorin. [4]
Production of biofilms
As aforementioned, one of the key structural components of P. fluorescens is its ability to produce biofilms.
Type III secretions
Type III secretion systems (T3SSs) are molecular, needle-like complexes that inject cellular products into the cells of its host/surface, known as effectors. The most common T3SS in P. fluorescens is the Hrp1 family[9]. These “hypersensitive response” secretion systems trigger a hypersensitive response in resistant plants, but leads to infection in vulnerable plants. Less is known about T3SSs involved in this organism’s infections in mammals, but different strains have been found to adhere to human Red Blood Cells, as well as human glial cells in culture. [10]

References

1. Ramette A, Moënne-Loccoz Y, Défago G, Prevalence of fluorescent pseudomonads producing antifungal phloroglucinols and/or hydrogen cyanide in soils naturally suppressive or conducive to tobacco black root rot. FEMS Microbiol Ecol. 2003 May 1; 44(1):35-43.
2. Gibaud M, Martin-Dupont P, Dominguez M, Laurentjoye P, Chassaing B, Leng B. Pseudomonas fluorescens septicemia following transfusion of contaminated blood. Presse Med. 1984 Nov 24
3. Hernández-Salmerón JE, et al. Draft Genome Sequence of the Biocontrol and Plant Growth-Promoting Rhizobacterium Pseudomonas fluorescens strain UM270. Stand Genomic Sci 2016
4. Scales BS, Dickson RP, LiPuma JJ, Huffnagle GB. 2014. Microbiology, genomics, and clinical significance of the Pseudomonas fluorescens species complex, an unappreciated colonizer of humans. Clin Microbiol Rev 27:927–948. doi:10.1128/CMR.00044-14.
5. Baum MM, Kainović A, O'Keeffe T, Pandita R, McDonald K, Wu S, Webster P. Characterization of structures in biofilms formed by a Pseudomonas fluorescens isolated from soil. BMC Microbiol. 2009 May 21; 9():103
6. Ghiglione JF, Gourbiere F, Potier P, Philippot L, Lensi R. Role of respiratory nitrate reductase in ability of Pseudomonas fluorescens YT101 to colonize the rhizosphere of maize. Appl Environ Microbiol. 2000;66(9):4012–4016. Doi: 10.1128/AEM.66.9.4012-4016.2000
7. Hohnadel D, Meyer JM. Specificity of pyoverdine-mediated iron uptake among fluorescent Pseudomonas strains. J Bacteriol. 1988 Oct; 170(10):4865-73.
8. Adebusuyi AA, Foght JM. An alternative physiological role for the EmhABC efflux pump in Pseudomonas fluorescens cLP6a. BMC Microbiol. 2011;11:252. doi: 10.1186/1471-2180-11-252. [Online.]
9. Preston GM, Bertrand N, Rainey PB. Type III secretion in plant growth-promoting Pseudomonas fluorescens SBW25. Mol Microbiol. 2001 Sep; 41(5):999-1014.
10. Chapalain A, Rossignol G, Lesouhaitier O, Merieau A, Gruffaz C, Guerillon J, Meyer JM, Orange N, Feuilloley MG. Comparative study of 7 fluorescent pseudomonad clinical isolates.Can J Microbiol. 2008 Jan; 54(1):19-27.

Author

Page authored by Patrick Lawrence and Leah Carrizales, students of Prof. Kristine Hollingsworth at Austin Community College.