Characterization of genes involved in tabtoxinine-beta-lactam and lysine biosynthesis by Pseudomonas syringae pv. tabaci strain PTBR2.024
Liu, Lixia
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https://hdl.handle.net/2142/20655
Description
Title
Characterization of genes involved in tabtoxinine-beta-lactam and lysine biosynthesis by Pseudomonas syringae pv. tabaci strain PTBR2.024
Author(s)
Liu, Lixia
Issue Date
1996
Doctoral Committee Chair(s)
Shaw, Paul D.
Department of Study
Crop Sciences
Discipline
Crop Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Microbiology
Language
eng
Abstract
The relationship between the biosynthesis of tabtoxinine-$\beta$-lactam $\rm(T\beta L)$ and lysine by Pseudomonas syringae pv. tabaci strain BR2.024 (PTBR2.024) was investigated. A Tn5 mutant of PTBR2.024 showing diaminopimelate auxotrophy and $\rm T\beta L$-deficiency was isolated. Complementation results indicate that a region containing single open reading frame (ORF) is able to complement both mutant phenotypes. The deduced amino acid sequence of the ORF has a significant sequence homology to known dapB gene products, a lysine biosynthetic enzyme of bacteria. Furthermore, the ORF is able to complement an E. coli dapB mutant. The ORF was designated dapB. These results suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate in lysine and $\rm T\beta L$ biosynthesis.
A gene with a predicted product having an amino acid sequence homologous to known dapD gene products, another lysine biosynthetic enzyme in bacteria, was also identified. Complementation tests, in vitro transcription/translation analysis and enzymatic assays indicate that the dapD-like gene is able to encode a product with THDPA N-succinyltransferase (THDPA-ST) activity in E. coli. However, no THDPA-ST activity was detected in PTBR2.024 and a mutation in the dapD-like gene has no effect on lysine biosynthesis of strain PTBR2.024. These results indicate that the dapD-like gene is not involved in lysine biosynthesis in PTBR2.024. The mutation, however, caused a 50 percent reduction in $\rm T\beta L$ production. This suggests that the dapD-like gene may be involved in $\rm T\beta L$ biosynthesis. The dapD-like gene was named tabB.
Enzymatic assay results indicate that PTBR2.024 uses an acetyl pathway for lysine biosynthesis. This is the first report of a Gram-negative bacterium utilizing that pathway.
An gene, ORF$\rm\sb{L}$, able to complement the dapD mutant $\beta$274 also was isolated from the PTBR2.024 genomic library. A mutant with an insertion in ORF$\rm\sb{L}$ produced as much T$\beta$L as parent strain. However, the mutant had reduced growth rate in minimal medium. The growth rate was restored by addition of lysine in minimal medium. Complementation analyses indicate that the ORF$\rm\sb{L}$ mutant phenotype was due to a polar effect. This suggests that the ORF$\rm\sb{L}$ may not be involved in either lysine or tabtoxin biosynthesis.
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