Cultivation notes for Pseudomonas strains

Genetic instability of Pseudomonas strains during prolonged cultivation

from Joyce Loper, USDA-ARS Horticultural Crops Research Lab

Many strains of Pseudomonas  fluorescens, exhibit some genetic instability if they are grown for several days in culture.  Fortunately, variants of the strain that arise in culture can be detected by carefully observing the morphologies of colonies grown on agar media.  On Luria-Bertani (LB) medium, colonies of "variants" are larger than the "wildtype" colonies and have orange pigmentation.  On King's medium B, colonies of "variants" are larger and more fluorescent than "wildtype" colonies.  Colony sectoring and the presence of multiple colony morphologies in cultures of Pseudomonas spp. have perplexed scientists for decades, but it is now clear that most of the colonies with these characteristics have mutations in the gacA (encodes a transcriptional response regulator) or gacS (encodes a membrane-bound sensor kinase) genes. These mutants typically lack extracellular protease production, which can be seen as a cleared zone around bacterial colonies on BBL Litmus Milk agar.
The genetic instability phenomenon is described in the paper of Duffy and Défago (2000).

Reference

Duffy B.K. and Défago G. 2000. Controlling instability in gacS-gacA regulatory genes during inoculant production of Pseudomonas fluorescens biocontrol strains. Appl. Environ. Microbiol. 66: 3142-3150.

Hydrogen Cyanide Mutant hcnABC Pseudomonas fluorescens strain CHA77 (CCOS 546)

Hydrogen cyanide (HCN) is produced from glycine. The complete hydrogen cyanide synthase genes hcnABC from Pseudomonas fluorescens is published under GenBank accession No. AF053760. The mutant strain CHA77 carries a chromosomal deletion of the hcnABC gene with partial deletion of hcnA, hcnC and complete deletion of hcnB. The tetracycline resistance determinant used for selection was removed in strain CHA77 (Laville et al., 1998).

To verify the absence of the hcnB gene in P. fluorescens CCOS 546 (CHA77) a PCR based test system was developped:
Using the primer-set hcnA-fw and hcnC-rv, a 3.4 kb sized fragment of the whole hcnABC gene casette is amplified when a HCN-producing P. fluorescens strain like CCOS 2 (CHA0) is tested. Only a 0.55 kb sized fragment is obtained when the mutant strain CCOS 546 (CHA77) is tested, confirming the deletion of hcnB.

Adenine Auxotrophy of Pseudomonas fluorescens CHA15 (CCOS 525)

From Dieter Haas (personal communication) and from the publication of Heeb et al, 2000. MPMI 13:232-37.

Pseudomonas fluorescens strain CHA15 is an adenine-auxotroph mutant derived from strain CHA0 (CCOS 2). The mutagenesis was performed with EMS (ethyl methane sulfonate).

The adenine-auxotroph strain is grown aerobically at 30°C in succinate minimal medium composed of (g per liter): K₂HPO₄, 6.0; KH₂PO₄, 3.0; (NH₄)SO₄, 1.0, MgSO₄.7H₂O, 0.2; succinic acid, 4.0 (Meyer, J.M. and Abdallah, M.A. 1978, J. Gen. Microbiol. 107:319-28).

The adenine auxotrophic phenotype is tested as follows:

0.1 ml of an over-night culture of CHA15 is plated onto the surface of succinate minimal agar medium. A sterile filter-paper disk, impregnated with 50 µl 50 mM adenine is placed onto the surface of the minimal agar plate, which has been inoculated with CHA15 cells. After incubation at 30°C, CHA15 auxotroph colonies will grow around the filter disc. No growth of CHA15 colonies occurs in zones without concentrations of adenine.

CCOS published strains like Pseudomonas fluorescens are available as actively growing cultures or as cryo cultures. We look forward to receiving your order and please contact us for further details.