Abstract
Pseudomonas aureofaciens PGS12 produces three phenazine antibiotics, in addition to siderophores, hydrogen cyanide, pyrrolnitrin, and indoleacetic acid. Tn5-259.7 transposon mutagenesis was carried out to identify and clone a chromosomal locus involved in phenazine biosynthesis. Three classes of mutants were obtained: mutants deficient in phenazine production (Phz-), mutants deficient in hydrogen cyanide production (HCN-), and mutants deficient in the production of both compounds. EcoRI DNA fragments that contained the transposon and flanking regions were cloned from three mutants with single-transposon insertions, one from each phenotypic class. Phenazine and hydrogen cyanide production was restored by complementation of Phz- or HCN- mutants with selected cosmids from a PGS12 genomic library. No cosmids that complemented the doubly deficient Phz-HCN- mutant were obtained. A promoterless ice nucleation reporter gene was inserted in a phenazine biosynthetic locus by Tn3-spice transposon mutagenesis of a cosmid which complemented a phenazine-minus mutant. Reporter gene fusions that expressed the ice nucleation phenotype and no longer complemented phenazine production were introduced into the PGS12 chromosome by marker exchange. The expression of this locus was then monitored under different culture conditions. Expression decreased at pH levels below 7, and it was not affected by iron. Shikimic acid and phenylalanine favored higher expression levels. Expression was reduced in media with low substrate concentrations, indicating the importance of nutrient availability.
Full text
PDF![2931](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3f07/201745/51069a58db2b/aem00025-0275.png)
![2932](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3f07/201745/b734613abfd1/aem00025-0276.png)
![2933](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3f07/201745/066cc0d069c0/aem00025-0277.png)
![2934](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3f07/201745/455b798a9d0d/aem00025-0278.png)
![2935](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3f07/201745/95c27d458823/aem00025-0279.png)
![2936](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3f07/201745/2f9229ece653/aem00025-0280.png)
![2937](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3f07/201745/63be54ad3700/aem00025-0281.png)
![2938](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3f07/201745/4b45c6bbb8c7/aem00025-0282.png)
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Broadbent P., Baker K. F., Waterworth Y. Bacteria and actinomycetes antagonistic to fungal root pathogens in Australian soils. Aust J Biol Sci. 1971 Oct;24(5):925–944. doi: 10.1071/bi9710925. [DOI] [PubMed] [Google Scholar]
- Castric K. F., Castric P. A. Method for rapid detection of cyanogenic bacteria. Appl Environ Microbiol. 1983 Feb;45(2):701–702. doi: 10.1128/aem.45.2.701-702.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chesney R. H., Scott J. R., Vapnek D. Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli. J Mol Biol. 1979 May 15;130(2):161–173. doi: 10.1016/0022-2836(79)90424-8. [DOI] [PubMed] [Google Scholar]
- Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- James D. W., Jr, Gutterson N. I. Multiple antibiotics produced by Pseudomonas fluorescens HV37a and their differential regulation by glucose. Appl Environ Microbiol. 1986 Nov;52(5):1183–1189. doi: 10.1128/aem.52.5.1183-1189.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- KING E. O., WARD M. K., RANEY D. E. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954 Aug;44(2):301–307. [PubMed] [Google Scholar]
- Korth H. Carbon source regulation of the phenazine-alpha-carboxylic acid synthesis in Pseudomonas aureofaciens. Arch Mikrobiol. 1973;92(2):175–177. doi: 10.1007/BF00425015. [DOI] [PubMed] [Google Scholar]
- Korth H. Mixed carbon source effect in the phenazine-alpha-carboxylic acid synthesis and the aromatic pathway in Pseudomonas spp. Arch Microbiol. 1974 May 16;97(3):245–252. doi: 10.1007/BF00403064. [DOI] [PubMed] [Google Scholar]
- Lindgren P. B., Frederick R., Govindarajan A. G., Panopoulos N. J., Staskawicz B. J., Lindow S. E. An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola. EMBO J. 1989 May;8(5):1291–1301. doi: 10.1002/j.1460-2075.1989.tb03508.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mazzola M., Cook R. J., Thomashow L. S., Weller D. M., Pierson L. S., 3rd Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats. Appl Environ Microbiol. 1992 Aug;58(8):2616–2624. doi: 10.1128/aem.58.8.2616-2624.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Orser C., Staskawicz B. J., Panopoulos N. J., Dahlbeck D., Lindow S. E. Cloning and expression of bacterial ice nucleation genes in Escherichia coli. J Bacteriol. 1985 Oct;164(1):359–366. doi: 10.1128/jb.164.1.359-366.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peet R. C., Lindgren P. B., Willis D. K., Panopoulos N. J. Identification and cloning of genes involved in phaseolotoxin production by Pseudomonas syringae pv. "phaseolicola". J Bacteriol. 1986 Jun;166(3):1096–1105. doi: 10.1128/jb.166.3.1096-1105.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rahme L. G., Mindrinos M. N., Panopoulos N. J. Genetic and transcriptional organization of the hrp cluster of Pseudomonas syringae pv. phaseolicola. J Bacteriol. 1991 Jan;173(2):575–586. doi: 10.1128/jb.173.2.575-586.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schroth M. N., Hancock J. G. Disease-suppressive soil and root-colonizing bacteria. Science. 1982 Jun 25;216(4553):1376–1381. doi: 10.1126/science.216.4553.1376. [DOI] [PubMed] [Google Scholar]
- Shanahan P., O'sullivan D. J., Simpson P., Glennon J. D., O'gara F. Isolation of 2,4-diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production. Appl Environ Microbiol. 1992 Jan;58(1):353–358. doi: 10.1128/aem.58.1.353-358.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stachel S. E., An G., Flores C., Nester E. W. A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J. 1985 Apr;4(4):891–898. doi: 10.1002/j.1460-2075.1985.tb03715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomashow L. S., Weller D. M., Bonsall R. F., Pierson L. S. Production of the antibiotic phenazine-1-carboxylic Acid by fluorescent pseudomonas species in the rhizosphere of wheat. Appl Environ Microbiol. 1990 Apr;56(4):908–912. doi: 10.1128/aem.56.4.908-912.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomashow L. S., Weller D. M. Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici. J Bacteriol. 1988 Aug;170(8):3499–3508. doi: 10.1128/jb.170.8.3499-3508.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Turner J. M., Messenger A. J. Occurrence, biochemistry and physiology of phenazine pigment production. Adv Microb Physiol. 1986;27:211–275. doi: 10.1016/s0065-2911(08)60306-9. [DOI] [PubMed] [Google Scholar]
- Wolber P. K., Deininger C. A., Southworth M. W., Vandekerckhove J., van Montagu M., Warren G. J. Identification and purification of a bacterial ice-nucleation protein. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7256–7260. doi: 10.1073/pnas.83.19.7256. [DOI] [PMC free article] [PubMed] [Google Scholar]