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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGY AND MANAGEMENT OF SOILBORNE DISEASES OF HORTICULTURAL CROPS Title: Arbitrary PCR for Rapid Mapping of Tn5 Insertions in Pyoverdine Genes of Pseudomonas fluorescens Pf-5

Authors
item Hartney, Sierra - OREGON STATE UNIVERSITY
item Loper, Joyce

Submitted to: American Society for Microbiology Conference
Publication Type: Abstract Only
Publication Acceptance Date: June 8, 2007
Publication Date: August 26, 2007
Citation: Hartney, S., Loper, J.E. 2007. Arbitrary PCR for Rapid Mapping of Tn5 Insertions in Pyoverdine Genes of Pseudomonas fluorescens Pf-5. American Society for Microbiology Conference on Pseudomonas. p. 87.

Technical Abstract: A collection of 13 transposon mutants deficient in pyoverdine production was analyzed using an arbitrary polymerase chain reaction (PCR) approach to map the sites of Tn5 insertions in the genome of Pseudomonas fluorescens Pf-5. The arbitrary PCR method involved two rounds of reactions, with the first using one degenerate primer and one primer specific for Tn5. The degenerate primer was designed to contain a seven nucleotide sequence on the 3’ end that occurs commonly in the pyoverdine biosynthetic region of Pf-5. The 182-kb region encompassing the three pyoverdine gene clusters in the Pf-5 genome (Paulsen et al. 2005) was evaluated using the compseq software program (European Molecular Biology Open Software Suite) to identify a seven nucleotide sequence that occurs 350 times (at an average interval of approximately 600 bp) in this region. The random primer was composed (from 3’ to 5’) of these seven nucleotides, 10 random nucleotides, and a previously-described 20-nucleotide sequence (Das et al. 2005). For the second round of PCR, nested primers were used to amplify PCR products from the first round. To date, the Tn5 insertions of nine mutants have been mapped. Five Tn5 insertions are in three non-ribosomal peptide synthetase genes: three are in PFL_4189 (76% identical to pvdL of P. aeruginosa PA01); one is in PFL_4094 (58% identical to pvdD); and one is in PFL_4093 (58% identical to pvdI) . Three insertions are in PFL_2902, which is 55% identical to pvdQ of P. aeruginosa PA01. Whereas pvdQ is located in a pyoverdine gene cluster in P. aeruginosa PA01, PFL_2902 is not clustered with other known pyoverdine biosynthesis genes in the Pf-5 genome. One of the Tn5 insertions resulting in loss of pyoverdine production by Pf-5 was localized to a gene not previously known to be involved in pyoverdine biosynthesis by Pseudomonas spp. Characterization of this mutant is a subject of ongoing studies. Arbitrary PCR provided an efficient approach to map sites of Tn5 insertions in the genome of P. fluorescens Pf-5.

Last Modified: 10/20/2014
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